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A Practical Guide to ISO 10993-3: Genotoxicity

Medical Device & Diagnostic Industry

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An MD&DI October 1998 Column

ISO 10993

The growing concern that medical devices can contribute to changes in DNA structure is addressed in the standard on genotoxicity. Note: this article is part of an ongoing series on ISO 10993. The previous installment covered design of subchronic and chronic
systemic toxicity tests

Although measures of a medical product's biocompatibility have largely been reported in terms of irritation, sensitization, and systemic toxicity, there is growing concern that devices, their components, or material extracts also may exert genotoxic effects. Thus, any attempt to assess the safety of a device intended for intimate body contact or permanent implantation would be incomplete without testing for the presence of toxins that exert an effect on the genetic material of cells. In its set of harmonized standards for the biological evaluation of medical devices, the International Organization for Standardization (ISO) has outlined the need for such genotoxicity testing in ISO 10993-3: "Tests for Genotoxicity, Carcinogenicity, and Reproductive Toxicity."


Although the human body is extremely complex, its relationship to a genotoxin can be described in fairly simple terms. In any living organism, the smallest unit capable of independent existence is the cell, and each cell has specific functions that it must maintain to sustain life. Instructions for these activities are encoded within genes, or sections of DNA, devoted to each specific function. A thread consisting of many genes strung together with large amounts of DNA is called a chromosome, each of which is divided into two chromatids that come together at a junction known as the centromere. An alteration in any part of this DNA structure that results in permanent inheritable changes in cell function is called a mutation, and the agents that cause such mutations are known as genotoxic agents or genotoxins.

There are three major types of genotoxic effects: gene mutations, chromosomal aberrations, and DNA effects. Because no single in vitro assay is capable of detecting all three types, a battery of tests is recommended. Gene mutation and chromosomal aberration tests detect actual lesions in the DNA molecule, while DNA effects tests detect events that may lead to cell damage. In vitro tests in each category can be conducted using microorganisms or mammalian cells. Although ISO 10993-3 states that in vivo evaluations are required only if scientifically warranted or if the results of in vitro assays indicate a need for further testing, the International Conference on Harmonization document for pharmaceuticals recommends the inclusion of an in vivo model in the battery of genotoxicity tests.


As do several other sections of the international standards, ISO 10993-3 refers readers to the Organization for Economic Cooperation and Development (OECD) guidelines for specific test methods. Because most biomaterials are insoluble and the OECD methods are designed to test soluble chemicals, the tests must be modified to accommodate the evaluation of fluid extracts.

The United States Pharmacopoeia (USP) has established standard preparation methods for materials testing that can be used for genotoxicity testing, and ISO 10993-12 "Sample Preparation and Reference Materials" also describes standard methods for the preparation of extracts of device materials. Selection of the appropriate extraction vehicle varies with the test system of choice. For example, bacterial test systems are frequently exposed to 0.9% sodium chloride solution and either ethanol or dimethyl sulfoxide extracts. Since in vitro mammalian test systems require media that can support cell growth, the nutrient medium used for culture is often employed as the extraction vehicle. In vivo test models frequently employ the standard aqueous and nonaqueous USP extraction fluids that are capable of extracting both water-soluble (polar) and lipid-soluble (nonpolar) chemicals.

Gene Mutation Tests. Mutations affecting a small portion of the DNA molecule, including frameshifts and base-pair substitutions, are referred to as point mutations. The test most commonly used to detect such gene mutations is the Ames bacterial reverse mutation assay, which utilizes histidine-dependent Salmonella typhimurium strains as the test organisms. S9 active rat liver microsomes are incorporated into a portion of the test organisms to simulate whole-animal exposure. Following exposure to the fluid extract from the test article, the organisms are plated in triplicate onto histidine-free growth nutrient agar and incubated for a specified period. The colonies are then enumerated and these data are compared to counts obtained for negative and positive control conditions. Since the unreverted test strains will not grow without histidine, any further growth indicates that exposure to a genotoxic agent has caused point mutations that have produced bacterial strains that no longer require histidine. Other bacterial strains such as Escherichia coli, with specific amino acid requirements, may be incorporated into this test design.

Another useful in vitro method for detecting gene mutations in a mammalian system is the mouse lymphoma assay. In this test, L5178Y mouse lymphoma cells are exposed to test extracts in the presence and absence of exogenous metabolic activation. Following incubation, the cultures are cloned in restrictive media for the mutant phenotype. Mutations are measured at the thymidine kinase locus to detect base-pair mutations, frameshift mutations, and small deletions. Since mutant colonies exhibit a characteristic size distribution frequency, colony measurements can be used to distinguish the type of genetic effect.

Chromosomal Aberration Tests. Either in vitro or in vivo methods can be used for chromosomal aberration testing. These tests detect chromosomal damage induced after one cellular division; structural changes in the chromosomes are evaluated while cells are in the metaphase stage of division. The in vitro model employs Chinese hamster ovary cells. As with other in vitro methods, the test system is evaluated in the presence and absence of exogenous metabolic activation. Most aberrations can be identified as either chromosomal or chromatid type. Gaps, breaks, and exchanges are other examples of observable aberrations (Figure 1).

Figure 1. Chinese hamster ovary cells (100x magnification) following exposure to a known genotoxic agent (the positive control) in the in vitro chromosomal aberration test model.

DNA Effects Tests. Genotoxins can induce DNA damage by any of several mechanisms. Among the methods for evaluating such effects is the mouse bone marrow micronucleus test. This in vivo assay detects damage to the chromosomes or the mitotic apparatus of immature red blood cells found in bone marrow. During cell division, undamaged chromosomes give rise to normal daughter nuclei, but if the chromosomes are broken or the mitotic apparatus of the cell is damaged, chromosome fragments may be incorporated in secondary nuclei instead of into the main nucleus. Secondary nuclei are much smaller than the main nucleus and are referred to as micronuclei. When erythroblasts develop into polychromatic erythrocytes (PCEs), the main nucleus is extruded but any micronuclei that are present remain behind. Thus, an increase in the number of micronucleated PCEs in animals treated with the test article extract is an indication of the presence of a genotoxin (Figure 2).

Figure 2. Murine erythrocytes following exposure to a known genotoxic agent. The micronucleus is distinguished by the small, differentially stained segment of nuclear material.

The sister chromatid exchange test is also used to evaluate DNA effects. In this assay, metabolically active and inactive mammalian cells are exposed to the test extract and the differentially stained chromatids are then examined to see if segments of DNA have been reciprocated or exchanged between the sister chromatids. Evidence of such exchanges appears as striations or a banding effect. An increase in the number of exchanges observed in test cultures is an indication of genetic toxicity. Currently, it appears that the sister chromatid exchange test is losing favor both in the United States and internationally. Some of the experts involved in the development of this ISO standard favor the use of the mouse lymphoma assay as the only mammalian cell test needed.


Classical in vitro and in vivo tests can be used to evaluate the genotoxicity of medical device materials. In all cases, adverse or equivocal findings warrant further investigation. Confirmation testing by dose-response relationship is the standard course of action. In addition, a presumptive positive finding in an in vitro assay can be confirmed by conducting an alternative in vivo model.

Acceptable results from a battery of genotoxicity tests will not only go a long way toward ensuring the safety of a proposed biomaterial; in some cases such data can justify not pursuing in vivo carcinogenicity studies, particularly if there is existing information about the lack of genotoxicity of the material in question.

Continue to the next article in this series, hemocompatibility.

Gina M. Johnson is the manager of toxicology at NAMSA's Irvine, CA, division, and Paul J. Upman, PhD, is a senior scientist, and Richard F. Wallin, DVM, PhD, is president of NAMSA (Northwood, OH).

Copyright ©1998 Medical Device & Diagnostic Industry

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