What Is HPRT Test?

The HPRT (Hypoxanthine-Guanine Phosphoribosyltransferase) test, as described in ISO 10993-3, is an in vitro mammalian cell gene mutation assay used to evaluate the mutagenic potential of medical devices and their constituents. This test specifically assesses the ability of a test substance to induce gene mutations at the HPRT locus in cultured mammalian cells, typically using Chinese hamster ovary (CHO) or lung (V79) cells.

The HPRT gene test plays a crucial role in the purine salvage pathway, and mutations at this locus confer resistance to selective agents such as 6-thioguanine. In accordance with ISO 10993-3, which outlines tests for genotoxicity, carcinogenicity, and reproductive toxicity, the HPRT assay provides essential data on genetic safety by determining whether a material has the potential to cause heritable genetic changes. The results of this assay contribute to the overall biological risk assessment of medical devices, supporting their safe use in clinical applications.

HPRT Test Principal

Purpose: Evaluates mutagenic potential of medical device materials using in vitro mammalian cells.

Target Gene: HPRT gene locus in CHO or V79 cells.

Principle: Uses 6-thioguanine for positive selection—only mutant cells (HPRT-deficient) survive.

Procedure:

• Cells exposed to test extracts.
• Cultured to allow mutation expression.
• Grown in 6-thioguanine to isolate mutant colonies.

Detects: Point mutations, frameshifts, small deletions/insertions.

Outcome: Mutant frequency compared with controls to assess mutagenicity.

Relevance: Part of the genotoxicity testing under ISO 10993-3 for medical device safety.

For more information, click Genotoxicity Testing.

HPRT Assay Procedure

Below is a revised, formal description of the HPRT (Hypoxanthine‑Guanine Phosphoribosyltransferase) test procedure, grounded in the principles of ISO 10993‑3 and informed by Eurofins’ HPRT assay services—but completely rephrased and reformatted:

Purpose and Regulatory Context

The HPRT assay is an in vitro mammalian-cell gene mutation test, used to assess whether a test article or leachable can induce point mutations at the HPRT gene. Under ISO 10993‑3, this assay addresses key genotoxicity concerns linked to medical device materials. It is typically conducted alongside bacterial (e.g. Ames) and chromosomal damage assays to form a complete genotoxicity evaluation under OECD guidelines (notably OECD 476).

Cell System and Principle

The assay employs mammalian cell lines such as CHO‑K1, V79, or L5178Y cells, all bearing a functional HPRT gene located on the X chromosome.

The principle is positive selection: only cells in which the HPRT gene is disrupted (i.e. mutants) survive in culture containing 6‑thioguanine, a toxic purine analog. Such mutant colonies indicate gene-inactivating events.

Test Method Summary

 Seeding and Treatment

Cells are seeded at a suitable density (e.g. ~2 × 10⁶ per flask or dish) in complete culture medium prior to treatment.

Exposure may occur in both absence and presence of metabolic activation (e.g. S9 mix) to simulate in vivo biotransformation processes.

 Expression Period

After exposure, cells are washed and subcultured into fresh medium for a defined expression period (typically around 7–9 days), during which any induced mutations become phenotypically manifest.

 Cloning and Selection

Cells are plated in parallel:

Selective medium with 6‑thioguanine to identify HPRT‑deficient mutants.

Non‑selective medium to determine cloning efficiency or overall viability.

Typically multiple concentrations (at least four analyzable levels) are tested in replicates to enable cytotoxicity assessment and dose–response evaluation.

Colony Counting and Mutant Frequency

After suitable incubation, colonies under each condition are counted. The mutant frequency is calculated by normalizing the number of colonies in selective medium to those in non‑selective control cultures, adjusting for plating efficiency.

Data Interpretation and Acceptance Criteria

The test includes negative (vehicle) and positive control cultures to ensure assay validity. Historical performance ranges are used for comparison.

Cytotoxicity limits are observed to confirm that the test substance concentration does not exceed acceptable survival thresholds (usually not less than ~10–20 % relative total growth or survival)

A statistically significant or concentration‑dependent increase in mutant frequency over control indicates a positive genotoxic response.

 Significance in ISO 10993‑3 Context

The HPRT assay is a recognized component of the genotoxicity testing battery under ISO 10993‑3, addressing the gene mutation endpoint. It complements bacterial mutation tests and chromosomal assays for a comprehensive hazard profile.

If the HPRT assay (alongside other in vitro tests) yields negative results, in most cases further in vivo genotoxicity testing may be waived, per ISO‑recommended strategies.

 Outline of a Typical HPRT Test Protocol

Cell Seeding: ~2 × 10⁶ cells per flask, standard culture medium.

Treatment Phase: Exposure to test article (multiple concentrations), ± metabolic activation.

Expression Phase: Subculture and grow for ~7–9 days to allow mutation fixation.

Selection: Plate cells in selective medium (6‑thioguanine) and in parallel non‑selective medium.

Incubation & Colony Formation

Counting & Analysis: Determine mutant frequency and compare against controls.

Evaluation: Incorporate cytotoxicity data, verify control acceptability, assess dose-response and statistical significance.

The HPRT gene mutation assay is a sensitive, quantitative in vitro method aligned with ISO 10993‑3 (and OECD 476) for assessing point mutations at the gene level. It provides scientifically robust evidence for evaluating potential genotoxic risk from device materials or extracts. Its role in the genotoxicity testing strategy is pivotal, often informing whether further testing—including in vivo studies—is warranted.

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Why Choose International Nikopharmad Laboratory for The HPRT Test?

ILAC Accreditation and ISO/IEC 17025 Certification

International Nikopharmad Laboratory is proudly accredited under ISO/IEC 17025 and recognized by ILAC (International Laboratory Accreditation Cooperation)—a strong validation of our technical proficiency, methodological precision, and adherence to globally recognized quality systems. These credentials ensure that our genotoxicity testing data is not only scientifically robust but also fully acceptable for regulatory submissions to health authorities such as the FDA, EMA, and PMDA.

Expertise, Infrastructure, and Technical Excellence

With a strong foundation in pharmaceutical and medical device safety evaluation, Nikopharmad operates advanced platforms for both bacterial and mammalian cell-based assays. Our highly skilled team of molecular biologists and toxicologists apply best industry practices and modern statistical tools to ensure biological relevance, analytical precision, and regulatory readiness. All procedures are executed under rigorous quality control, using fully validated SOPs and GLP-aligned workflows.

Competitive Pricing with No Compromise on Quality

We understand the financial pressures of product development. That’s why we offer competitive pricing across all our genotoxicity testing services—delivering exceptional value without sacrificing scientific quality or regulatory integrity. Our cost-effective solutions help you stay within budget while meeting international standards.

Timely Turnaround and Project Agility

Time-to-market is critical. Our streamlined workflows, dedicated project managers, and automation-enhanced systems allow us to deliver high-quality data with rapid turnaround—accelerating your development pipeline and helping you avoid regulatory delays.

Confidentiality and Data Integrity

Your proprietary information is treated with the utmost confidentiality. We operate under strict non-disclosure agreements (NDAs) and enforce strong data integrity protocols to safeguard your intellectual property throughout the testing process.

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Partner with Nikopharmad for Trusted Genotoxicity Testing

When you choose International Nikopharmad Laboratory, you gain more than a testing service—you gain a strategic partner. Our internationally accredited methods, scientific rigor, operational efficiency, and cost-effective pricing make us the ideal choice for advancing your product safely and successfully through regulatory pathways.

Trust Nikopharmad to deliver reliable genotoxicity data—on time, on budget, and to the highest global standards.