ISO and USP Standards for Antimicrobial Effectiveness Testing: What You Need to Know

From toothbrushes to tongue scrapers, oral care products tackle one of the most microbiologically active environments in the human body, with conditions where diverse microbial species thrive. This makes antimicrobial effectiveness testing essential, and proves to be a unique challenge for product developers: proving their antimicrobial claims while satisfying strict regulatory standards.

USP and ISO standards are procedural benchmarks that help ensure that a developer’s results are consistent and repeatable. Despite their shared goals, these standards differ in how they are applied and what they are intended to achieve. Knowing their key differences is crucial to ensure that products comply with relevant regulations and meet consumer expectations.

Microbial Challenges of the Oral Environment

The environment inside of the mouth, plus the steady flow of nutrients, make it one of the most dynamic microbial environments in the human body (second only to the gut). This creates several challenges for oral product developers.

Biofilms

Biofilms are structured communities of microorganisms that adhere to surfaces like teeth and gums. Once established, biofilms are notoriously resistant to antibiotics, disinfectants, and even the immune system, making them difficult to eliminate.

Oral Microbiota

In addition to their structural resilience, the oral microbiota is incredibly diverse. From common species like Streptococcus mutans to opportunistic pathogens such as Candida albicans, this ecosystem presents a moving target for antimicrobial efficacy.

For developers, these complexities have far-reaching implications regarding antimicrobial effectiveness testing. Accurate shelf-life predictions and microbial stability require test conditions that provide reproducible results while still modeling the real world.

USP Standards

The United States Pharmacopeia (USP) is a scientific nonprofit organization. Its mandate is to set standards for the strength, purity, and quality of medicines, dietary supplements, and food ingredients. For oral care product developers, understanding and applying USP standards is essential to ensure microbiological safety.

USP testing methods are designed for the pharmaceutical industry, but they are highly relevant for oral care products. Key USP guidelines specific to antimicrobial effectiveness testing in oral care include:

• USP <51> Antimicrobial Effectiveness Test (AET) – This standard evaluates the effectiveness of antimicrobial preservatives in products such as oral care formulations and pharmaceuticals. A product is inoculated with five common microorganisms and microbial reduction is monitored over 28 days. This test is critical for oral care formulations because the results will show how well the product can resist microbial growth during storage and use, as well as maintain its safety and efficacy throughout its shelf life.
• USP <61> Microbial Enumeration Tests – This standard quantifies the number of aerobic bacteria and fungi in a product. Oral care products must have low levels of this contamination to ensure consumer safety. This standard quantifies the total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) in the product, confirming that microbial levels are within acceptable limits for oral use.
• USP <62> Tests for Specified Microorganisms – This standard detects the presence or absence of specific objectionable microorganisms, such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella species. This test ensures that oral products are free from pathogens that could pose health risks to consumers if introduced into the oral cavity.

ISO Standards

The International Organization for Standardization (ISO) is composed of representatives from various national standards organizations. Applying ISO standards helps oral care product developers validate the antimicrobial efficacy and sterility of their products, especially when introducing innovative materials or devices.

The ISO guidelines specific to antimicrobial efficacy testing in oral care include:

• ISO 22196 Measurement of Antibacterial Activity on Plastics and Other Non-Porous Surfaces – Many oral care products use plastic or non-porous materials. This standard allows manufacturers to compare the efficacy of antimicrobial surfaces. It involves inoculating treated and untreated surfaces with bacteria. After 24 hours, the number of viable bacteria is assessed and evaluated to determine the microbial reduction attributed to the antimicrobial treatment.
• ISO 20743 Textiles: Determination of Antibacterial Activity –This standardapplies to various textile items and is useful when developing textile-based oral devices, such as antimicrobial gauze. It specifies three inoculation methods—absorption, transfer, and printing—to evaluate antibacterial efficacy. Manufacturers compare the effectiveness of antibacterial treatments on textiles by either the “colony plate count” method or the “ATP luminescence” method.
• ISO 11737-1 Sterilization of Health Care Products: Microbiological Methods (Part 1: Determination of a population of microorganisms on products) – This standard helps manufacturers measure how many live microorganisms (bioburden) are present on oral care products, raw materials, packaging, or components before they are sterilized. Determining the initial microbial load establishes a baseline before sterilization, to help ensure that sterilization methods are effective.
• ISO 11737-2 Sterilization of Health Care Products: Microbiological Methods (Part 2: Tests of sterility performed in the definition, validation, and maintenance of a sterilization process) – This standard guides how to conduct sterility tests on medical devices that have been exposed to reduced sterilization doses, rather than full-strength sterilization. Lower doses are used during product development to test whether a sterilization process is effective and helps validate if oral care products are ready to use.

Leveraging iFyber’s Expertise in Antimicrobial Effectiveness Testing

Given the complexity of oral microbiology and regulatory requirements, expert guidance is invaluable. A contract research organization like iFyber can offer a strategic advantage. With experience in antimicrobial assay development and protocol optimization, iFyber helps clients navigate the intricacies of oral microbiological testing. By tailoring assays to mimic real-world oral conditions and generating submission-ready data to support antimicrobial claims, iFyber helps product developers build confidence in their results while ensuring alignment with USP and ISO standards.

iFyber supports oral care product developers with antimicrobial effectiveness testing and regulatory strategy. Through custom assay development, the team creates tailored models that simulate realistic intra-oral conditions. Their collaborative approach begins by aligning testing strategies with regulatory expectations. With a strong understanding of ISO and USP requirements, iFyber ensures that developers can move forward with confidence and clarity in product performance and compliance.

To learn more about iFyber’s collaborative approach, and how our antimicrobial efficacy and biocompatibility assays allow us to help developers align with regulatory expectations, please click here to contact us.