Processed meat consumption is linked to colorectal cancer (CRC) risk, with nitrite/nitrate additives suspected as contributors via N-nitroso-compound (NOC) formation. Nitrite salts prevent pathogenic bacteria growth and contribute to meat quality. Previous research showed that cooked meat without nitrite limited CRC promotion in a rodent model, but also increased lipid peroxidation. This study explores the effects of sodium nitrite reduction or removal, and replacement strategies (using antioxidants and natural compounds) on endogenous reactions, the colon ecosystem, CRC promotion in rats, and Listeria monocytogenes growth in cooked ham. The aim was to provide insights for food regulation and nitrite reduction/replacement strategies in the meat industry.

Epidemiological and experimental evidence strongly suggests a link between processed meat consumption and increased CRC risk. Meta-analyses confirm this association. Studies using CRC animal models have demonstrated that cooked ham and sausages promote colon preneoplastic lesions. The role of nitrite/nitrate salts and related nitrogen species is debated, with the ingestion of these salts under conditions leading to endogenous nitrosation considered "probably carcinogenic." NOCs, particularly N-nitrosodimethylamine (NDMA), are highly carcinogenic. Nitrite and nitrate salts are added to processed meats to inhibit bacterial growth, extend shelf life, and improve organoleptic qualities. Previous studies indicated that a nitrite-free diet limited CRC promotion but also increased lipid peroxidation. Further research highlighted a link between nitrosylated heme iron and CRC development in rats. This study builds upon this existing knowledge.

The study used a cooked ham model produced with varying levels of sodium nitrite (120 mg/kg, 90 mg/kg, and 0 mg/kg) and three alternatives: vegetable stock, yeast extract, and polyphenol-rich extract. **Microbiological Risk Assessment:** *Listeria monocytogenes* growth was monitored in sliced cooked ham samples over time under different storage conditions. **Biochemical Characterization:** Nitrosylation, nitrosation, and lipid peroxidation were assessed in cooked ham samples. Various parameters such as total heme iron, nitrosylated iron, TBARS (thiobarbituric acid reactive substances), residual nitrite and nitrate, and total non-volatile N-nitrosamines were measured. **Colon Ecosystem Analysis:** Male F344 rats were given diets containing different ham samples for approximately 100 days after azoxymethane treatment. Fecal and urinary biomarkers of lipid peroxidation and NOC formation were analyzed. Fecal microbiota composition was assessed by 16S rRNA gene amplicon sequencing. **Colorectal Carcinogenesis Assessment:** The number of preneoplastic lesions (mucin-depleted foci, MDF) in the rat colons was determined. Cytotoxicity and genotoxicity of fecal water samples were also tested on colon epithelial cells. **Statistical Analyses:** ANOVA and non-parametric tests (Kruskal-Wallis, Dunn's mean comparison test) were used to compare the data.

Compared to the reference level (120 mg/kg nitrite), nitrite reduction (90 mg/kg) similarly decreased preneoplastic lesions in rats but was more effective in inhibiting *Listeria monocytogenes* growth. Nitrite removal strongly reduced nitrosylated iron and increased lipid peroxidation. Reduction or removal of nitrite resulted in a dose-dependent decrease in fecal nitroso compounds. The removal increased luminal lipid peroxidation and urinary DHN-MA. Microbiota alterations were observed only in the nitrite removal group. None of the three nitrite alternatives tested showed significant advantages over the reference level: vegetable stock was comparable due to nitrate presence; yeast extract led to increased luminal peroxidation; polyphenol-rich extract showed a downward trend in preneoplastic lesions but contained nitrosyl iron in feces. The alternatives were less effective than sodium nitrite in reducing *L. monocytogenes* growth, except for the vegetable stock.

This study confirms that nitrite removal decreases colorectal carcinogenesis promotion. Nitrite reduction also had a protective effect. The similar effect of nitrite removal and reduction on preneoplastic lesions highlights that while nitrite reduction is effective in mitigating carcinogenic risk, it still warrants further investigation to fully eliminate such risk. The increased lipid peroxidation with nitrite removal, offsetting the benefit of reduced NOCs, highlights the need to explore antioxidant strategies to further enhance the protective effects of nitrite reduction or removal. The alternative nitrite strategies showed varying efficacies. Vegetable stock, containing nitrate, was comparable to the reference level. Other alternatives proved less effective at controlling *Listeria monocytogenes* growth compared to the reference level.

Nitrite reduction offers a short-term strategy to decrease nitrite exposure and potentially colorectal cancer risk, while maintaining control over Listeria monocytogenes growth. Further research should focus on combining nitrite reduction/removal with antioxidants to mitigate increased lipid peroxidation. Comprehensive evaluations of alternatives are needed before market introduction.

The study was conducted on a rat model, which may not fully translate to humans. The alternatives tested represent a small subset of available options. The use of a specific cooked ham model limits the generalizability of the findings to other processed meat types.