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Article

Comprehensive In Vitro Safety Assessment of Acorus calamus Rhizome Oil Using OECD-Compliant New Approach Methods: Classification as a GHS Category 1B Sensitiser and Category 2 Irritant

by
Karishma R. Desai
1,*,
Jay R. Ranade
1,
Rajendra M. Nagane
1,
Manish V. Patel
1,
Abhay D. Deshpande
1,
Clive S. Roper
2 and
Gireesh Babu Kantli
3,*
1
Department of Toxicology (Mutagenicity), Jai Research Foundation, Valsad 396105, Gujarat, India
2
Roper Toxicology Consulting Limited, Edinburgh EH3 6AD, UK
3
Department of Life Sciences, Parul Institute of Applied Sciences, Parul University, Vadodata 391760, Gujarat, India
*
Authors to whom correspondence should be addressed.
Toxics 2025, 13(12), 1006; https://doi.org/10.3390/toxics13121006
Submission received: 16 October 2025 / Revised: 13 November 2025 / Accepted: 14 November 2025 / Published: 21 November 2025
(This article belongs to the Special Issue Applicability of New Approach Methodologies (NAMs) to Skin Sensitization: Insights from Industry and Regulatory Perspectives)
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Highlights

What are the main findings?
  1. Acorus calamus rhizome oil was comprehensively evaluated using OECD-compliant new approach methods (NAMs) for skin irritation, corrosion and skin sensitisation.
  2. The hydro-distilled oil from the A. calamus rhizome was classified as a GHS Category 1B skin sensitiser and a Category 2 skin irritant but exhibited a non-corrosive profile.
  3. Mechanistic evidence was obtained across all three key events of the skin sensitisation adverse outcome pathway (AOP): protein reactivity (DPRA), keratinocyte activation (KeratinoSens™), and dendritic cell activation (h-CLAT).
  4. Chemical analysis revealed high β-asarone content (40.75%), a known toxic constituent.
  5. Risk modelling using the SARA-ICE models and SCCS default exposure parameters provided safe-use concentration limits for topical applications.
What is the implication of the main finding?
  1. These findings fill an important data gap in the dermal safety profile of A. calamus rhizome oil, enabling NAM-based risk assessments for its use in cosmetics and topical formulations.
  2. This study also highlights the regulatory utility of NAMs in botanical safety evaluation, supporting animal-free hazard identification as well as potency categorization.
  3. The derived safe-use concentrations offer practical guidance, particularly for leave-on products such as face and hand creams, where sensitisation risk is crucial.
  4. This work sets a precedent for chemotype-specific safety assessments of plant-derived ingredients, promoting consumer protection and regulatory compliance.

Abstract

Background: Acorus calamus (sweet flag) is widely used in traditional medicine, yet its dermal safety profile remains insufficiently defined under modern regulatory standards. Objective: To comprehensively evaluate the skin irritation, corrosion, and sensitisation potential of A. calamus rhizome oil using new approach methodologies’ (NAMs) test batteries under GLP conditions. Results: The A. calamus rhizome oil was predicted as a Category 2 skin irritant, non-corrosive and GHS Category 1B skin sensitiser. Chemical analysis revealed β-asarone as the major constituent (~40.75%). The reconstructed human epidermis models established reversible irritation without corrosion. Mechanistic concordance across the Direct Peptide Reactivity Assay, KeratinoSens™, and Human Cell Line Activation Test showed activation of the three key events of the skin sensitisation adverse outcome pathway. Using the “2-out-of-3” Defined Approach with the KE 3/1 sequential strategy allowed for hazard classification into GHS Category 1B. Quantitative risk modelling using SARA-ICE models and SCCS parameters yielded conservative safe-use concentrations ranging from 0.13 to 0.78% (w/w) for leave-on products and up to 7.46% (w/w) for rinse-off formulations. Conclusions: The combined evidence from the NAM-based assays showed that A. calamus rhizome oil is a moderate sensitiser and irritant but not corrosive, providing critical data for risk assessment and regulatory decision-making, which was previously unknown. The SARA-ICE PoD-derived safe-use concentrations provide guidance for cosmetic formulators to ensure consumer safety, particularly in leave-on applications such as face and hand creams, where sensitisation risk is highest. This study demonstrates the utility of NAMs for botanical safety assessment and regulatory decision-making.
Keywords: A. calamus; rhizome oil; skin irritation; skin corrosion; skin sensitisation; non-animal methods; new approach methodologies (NAMs) A. calamus; rhizome oil; skin irritation; skin corrosion; skin sensitisation; non-animal methods; new approach methodologies (NAMs)

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MDPI and ACS Style

Desai, K.R.; Ranade, J.R.; Nagane, R.M.; Patel, M.V.; Deshpande, A.D.; Roper, C.S.; Kantli, G.B. Comprehensive In Vitro Safety Assessment of Acorus calamus Rhizome Oil Using OECD-Compliant New Approach Methods: Classification as a GHS Category 1B Sensitiser and Category 2 Irritant. Toxics 2025, 13, 1006. https://doi.org/10.3390/toxics13121006

AMA Style

Desai KR, Ranade JR, Nagane RM, Patel MV, Deshpande AD, Roper CS, Kantli GB. Comprehensive In Vitro Safety Assessment of Acorus calamus Rhizome Oil Using OECD-Compliant New Approach Methods: Classification as a GHS Category 1B Sensitiser and Category 2 Irritant. Toxics. 2025; 13(12):1006. https://doi.org/10.3390/toxics13121006

Chicago/Turabian Style

Desai, Karishma R., Jay R. Ranade, Rajendra M. Nagane, Manish V. Patel, Abhay D. Deshpande, Clive S. Roper, and Gireesh Babu Kantli. 2025. "Comprehensive In Vitro Safety Assessment of Acorus calamus Rhizome Oil Using OECD-Compliant New Approach Methods: Classification as a GHS Category 1B Sensitiser and Category 2 Irritant" Toxics 13, no. 12: 1006. https://doi.org/10.3390/toxics13121006

APA Style

Desai, K. R., Ranade, J. R., Nagane, R. M., Patel, M. V., Deshpande, A. D., Roper, C. S., & Kantli, G. B. (2025). Comprehensive In Vitro Safety Assessment of Acorus calamus Rhizome Oil Using OECD-Compliant New Approach Methods: Classification as a GHS Category 1B Sensitiser and Category 2 Irritant. Toxics, 13(12), 1006. https://doi.org/10.3390/toxics13121006

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