Comparative Assessment of Physicochemical and Cytotoxic Characterisation, in Khaini and Filtered Khaini Smokeless Tobacco Products

Comparative Assessment of Physicochemical and Cytotoxic Characterisation, in Khaini and Filtered Khaini Smokeless Tobacco Products | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Assessment of Physicochemical and Cytotoxic Characterisation, in Khaini and Filtered Khaini Smokeless Tobacco Products Arun Kumar Sundaragopal, Senthil Murugappan, Sharath Asokan, Shyam Sivasamy, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8793685/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Objectives Smokeless tobacco (SLT) products such as Cool Lip, marketed in India in filter-pouch form and resembling Swedish snus, are widely promoted as reduced-risk alternatives to traditional SLT such as Khaini(HANS). This study aims to systematically characterize the physicochemical, chemical, and biological toxicity profile of Cool Lip in comparison to a conventional SLT brand, Hans, to provide evidence on their health risks. Study Design Comprehensive analyses were performed to assess physical properties (pH, moisture, particle size using SEM imaging), chemical constituents (nicotine, TSNA, heavy metals, PAHs, alkalis, pesticides, and microplastic contamination), and microbiological load. Cytotoxicity was evaluated via MTT assay and apoptosis by AO staining in primary human gingival fibroblasts exposed to product extracts. Results Cool Lip exhibited a finer, more porous, and fragmented microstructure than Hans, which can accelerate chemical dissolution and absorption. Both products contained high levels of carcinogenic TSNAs and PAHs, and demonstrated higher concentrations of NNN, NNK, and nicotine, while Cool Lip had a substantially greater benzo[a]pyrene burden. Cytotoxicity and apoptosis were dose-dependent for both, with Hans consistently causing greater cellular toxicity. Conclusions Despite being marketed as a harm-reduced alternative, Cool Lip exhibits comparable or even greater toxicological risks relative to established Indian SLT products. The findings emphasize the urgent need for regulatory scrutiny and consumer education to counter misleading safety perceptions. Cool Lip smokeless tobacco TSNA cytotoxicity Indian SLT products Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 Apr, 2026 Reviews received at journal 31 Mar, 2026 Reviews received at journal 28 Mar, 2026 Reviews received at journal 27 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviews received at journal 14 Mar, 2026 Reviewers agreed at journal 11 Mar, 2026 Reviewers invited by journal 02 Mar, 2026 Editor invited by journal 02 Mar, 2026 Editor assigned by journal 16 Feb, 2026 Submission checks completed at journal 16 Feb, 2026 First submitted to journal 16 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8793685","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":599507389,"identity":"54ced773-3f61-4bd5-8d59-68dca69fb028","order_by":0,"name":"Arun Kumar Sundaragopal","email":"","orcid":"","institution":"Madha Dental College and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Arun","middleName":"Kumar","lastName":"Sundaragopal","suffix":""},{"id":599507393,"identity":"47837cef-68d5-4fae-a299-5f8eca61892f","order_by":1,"name":"Senthil Murugappan","email":"","orcid":"","institution":"Sri Balaji Vidyapeeth 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This study aims to systematically characterize the physicochemical, chemical, and biological toxicity profile of Cool Lip in comparison to a conventional SLT brand, Hans, to provide evidence on their health risks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComprehensive analyses were performed to assess physical properties (pH, moisture, particle size using SEM imaging), chemical constituents (nicotine, TSNA, heavy metals, PAHs, alkalis, pesticides, and microplastic contamination), and microbiological load. Cytotoxicity was evaluated via MTT assay and apoptosis by AO staining in primary human gingival fibroblasts exposed to product extracts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003cbr\u003e\n \u003c/strong\u003eCool Lip exhibited a finer, more porous, and fragmented microstructure than Hans, which can accelerate chemical dissolution and absorption. Both products contained high levels of carcinogenic TSNAs and PAHs, and demonstrated higher concentrations of NNN, NNK, and nicotine, while Cool Lip had a substantially greater benzo[a]pyrene burden. Cytotoxicity and apoptosis were dose-dependent for both, with Hans consistently causing greater cellular toxicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003cbr\u003e\n \u003c/strong\u003eDespite being marketed as a harm-reduced alternative, Cool Lip exhibits comparable or even greater toxicological risks relative to established Indian SLT products. The findings emphasize the urgent need for regulatory scrutiny and consumer education to counter misleading safety perceptions.\u003c/p\u003e","manuscriptTitle":"Comparative Assessment of Physicochemical and Cytotoxic Characterisation, in Khaini and Filtered Khaini Smokeless Tobacco Products","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-05 09:43:13","doi":"10.21203/rs.3.rs-8793685/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-02T17:27:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-31T08:03:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-29T00:25:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-27T08:58:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79536950945618377674985720322234951417","date":"2026-03-23T17:00:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20059313881661545196866613081784029766","date":"2026-03-18T17:18:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36988585089936340340509381437237926490","date":"2026-03-17T08:40:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-14T19:54:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170544473849776464453753829385889008068","date":"2026-03-11T17:17:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-02T17:31:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-02T08:41:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-16T08:39:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-16T08:25:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Public Health","date":"2026-02-16T08:21:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-public-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Public Health](https://link.springer.com/journal/12982)","snPcode":"12982","submissionUrl":"https://submission.springernature.com/new-submission/12982/3","title":"Discover Public Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6a89d83-871b-4d54-a906-d01d37516782","owner":[],"postedDate":"March 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T17:08:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-05 09:43:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8793685","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8793685","identity":"rs-8793685","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}