I have a client requesting analysis of their DP for the presence of n-nitrososarcosine. The DP contains sarcosine as a precursor to the API, and they are concerned with the potential of forming the associated nitrosamine. I have not found a limit for this compound. Can I use the FDA NDSRI guidance and calculate an associated limit for it based on the predicted carcinogenic potency categorization?
Thanks,
Eric Hill
Hi,
Well, that compound have some level of Carcinogenic Data (how reliable to be determined). Lhasa Carcinogenicity Database - Study Information (lhasalimited.org)
If that do not works, by CPCA this compound ends in Category 4 = 1500 ng/day that makes sense considering the carboxilic acid there.
Indeed, Nitrososarcosine carcinogenicity data is described in the literature, so you might want to compare the CPCA exercise with the available data as part of the expert review (nitrososarcosine as weak mice liver / rat esophageal carcinogen, nitrososarcosine can decompose to NDMA under certain conditions) to confirm that CPCA is protective (in case CPCA is doable).
Some references:
Hi my self Hema, happy to know about this community as well as join in this huge knowledge gaining site.
as per New updated CPCA guideline N-nitroso sarcosine-limit is 1500 ng/day which under potency category-4 is fine.
May i know the Limit of N,N-dibenzyl nitrous amide as per New CPCA guideline?
This is a very peculiar case. If we apply the CPCA rules, this substance seems a very potent nitrosamine:
Count of hydrogen atoms on each α-carbon [2, 2] and corresponding α-Hydrogen Score is 1
Chains of â„5 consecutive non-hydrogen atoms (cyclic or acyclic) on both side,Individual Deactivating Feature Score +1
Aryl group bonded to α-carbon, Activating score -1
Score: 1
Potency Category 1 : AI = 18 ng/day
On the other hand, the substance was found negative in an old carcinogenicity study on rats (Druckery et al., 1967):
N-Nitrosodibenzylamine (Lhasa Carcinogenicity Database)
A summay of the corrent knowledge has been reported in the
EFSA - Risk assessment of N-nitrosamines in food
NDBzA is reported as unable to induce tumours by Druckery et al. (1967), that also reported problems with the solubility of the compound.
However, all genotoxicity studies and expected metabolism would predict carcinogenicity.
NDBzA is genotoxic, being positive in the Ames test and in several other assays, including the MutaMouse assay. In the latter system, the predominant type of NDBzA-induced mutations were transversions, mainly GC > TA. These may arise from unidentified DNA adducts with benzylation possibly being the primary mechanism. Interestingly, in the Ames test, NDBzA is positive not only in the usual TA100 and TA1535 strains (as the other N-NAs) (small adducts, point mutations), but also in strain TA98 (large adducts, frameshift mutations).
No metabolic data are available; however, given the above MutaMouse results and in analogy with results reported by Moschel et al. (1979), the ability to benzylate DNA can be hypothesised.
Regarding the estimation of TD50 by read across, a partially similar structure is the carcinogenic N-methyl-N-nitrosobenzamide (CAS 63412-06-6), with TD50rat = 3.23, for which a related type of DNA damage, DNA benzoylation, could be hypothesised. This agrees with the assignment to NDBzA of a
level of concern of LOW-MODERATE carcinogenic risk by Oncologic
If we consider the EFSA position, the TD50(rat) of 3,23 mg/kg/day will lead to a lifetime AI of 3.23 ”g/day (3230 ng/day).
I donât think that this read-across with N-methyl-N-nitrosobenzamide will be easily accepted by the authorities; on the other hand, the CPCA AI of 18 ng/day is very conservative.
Probably the truth is somewhere in the middle, but it is very hard decide whereâŠ
Increased carcinogenic potency methyl/benzyl compared to benzyl/benzyl is also a trend in the methyl/cyclohexyl and cyclohexyl/cyclohexyl pair.
I understand the design of CPCA takes into account the intrinsic lability of the (pseudo)benzylic C-H and thus its susceptibility to alpha-hydroxylation and not the further generation, stability and reactivity of a (pseudo)benzylic carbocation.
For the symmetric example N,N-dibenzylnitrosamine unavoidably both topics get to the surface due to its symmetric nature:
What is clear though is that the worst case is the methyl-benzyl combination (and not the benzyl-benzyl): benzyl effect boosts (speed-wise) getting access to the methyl carbocation compared to methyl-methyl
â Get the benzyl on side âAâ effect (activation), expulsion of benzaldehyde and generate a methyl diazohydroxide, subsequently leading to an alkylating species.
But what is clear is also the problem: mechanistic understanding on the benzyl/benzyl and its DNA benzylation and other metabolisation risks was in the early days always linked to the data of more potent analogues. Nonetheless, in later years further studies shed a little bit more light. Citing all the literature searching for mechanisms and metabolites would lead me too far, but an in vivo study with transgenic rodents is worth citing (and not in the EFSA report it seems): Jiao, J., Douglas, G. R., Gingerich, J. D., & Soper, L. M. (1997). Analysis of tissue-specific lacZ mutations induced by N-nitrosodibenzylamine in transgenic mice. Carcinogenesis (New York) , 18 (11), 2239â2245. Analysis of tissue-specific lacZ mutations induced by N-nitrosodibenzylamine in transgenic mice. | Carcinogenesis | Oxford Academic.
From a pragmatic perspective and supported by mechanistic data and mutagenicity studies, readacross with NDMA possibly could be justified as conservative, while having the advantage NDMA data suitability for surrogate use purposes is established and not requiring full characterization of DNA adducts and the link between mutagenicity and carcinogenicity. Ranking against NDMA (e.g. in the sense of dr. Raphael Nudelman presentation during 20 May 2024, FDA Fiscal Year (FY) 2024 Generic Drug Science and Research Initiatives Public Workshop) is possibly not appropriate/in scope of these efforts, considering that the in vivo carcinogenicity/in vivo mutagenicity correlation establishment project seems mostly focused on rodent carcinogens, whereas nitrosodibenzylamine was found to not induce tumors.
I believe the alpha-(pseudo)benzylic feature is one which requires interpretation/regards for the local similarity and is often leading to a conservative AI. Overall there are quite some compounds in the limit lists which get this CPCA score punishment probably unnecessarily. Also the reference compound data used for design of the feature often is linked with fairly high TD50 or if not more complicated mechanisms hidden behind the feature, cf. Determining Recommended Acceptable Intake Limits for N-Nitrosamine Impurities in Pharmaceuticals: Development and Application of the Carcinogenic Potency Categorization Approach (CPCA) - ScienceDirect. The activating features surely were designed from a precautionary and semi-theoretic perspective, to minimize AI overestimation risks.
For detailed comments by NITWG including discussion of nitrosodibenzylamine see also section 3.3.1. Aryl Group Bonded to α-Carbon (i.e., Benzylic and Pseudo-Benzylic) of https://www.sciencedirect.com/science/article/pii/S0273230024000813