Biological Evaluation of Medical
Devices
Part 3: Tests for Genotoxicity, Carcinogenicity and Reproductive Toxicity
International Standard ISO 10993-3 / Technical Committee ISO/TC
194 27oct98
COMMITTEE DRAFT
ISO/CD 10993-3.2
Contents
- Foreword
- Introduction
- 1 Scope
- 2 Nonnative references
- 3 Definitions
- 4 Genotoxicity tests
- 5 Carcinogenicity tests
- 6 Reproductive toxicity tests
- 7 Test report
- Annex A - Flow chart for genotoxicity testing -
- Annex B - Bibliography -
- Annex C - Cell Transformation Systems -
- Annex D - Carcinogenicity tests performed as implantation tests -
- Annex E - Rationale of tests systems -
ISO (the International Organization for Standardization) a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and nongovernmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.
International Standard ISO 10993-3 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices.
This second edition cancels and replaces the first edition (ISO 10993-3:1993), which has been technically revised.
ISO 10993 consists of the following parts, under the general title Biological evaluation of medical evices:- Part 1: Evaluation and testing
- Part 2: Animal welfare requirements
- Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity
- Part 4: Selection of tests for interactions with blood
- Part 5: Tests for cytotoxicity: in vitro methods
- Part 6: Tests for local effects after implantation
- Part 7: Ethylene oxide sterilization residuals
- Part 9: Framework for the identification and quantification of potential degradation products
- Part 10: Tests for irritation and sensitization
- Part 11: Tests for systemic toxicity
- Part 12: Sample preparation and reference materials
- Part 13: Identification and quantification of degradation products from polymers
- Part 14: Identification and quantification of degradation products from ceramics
- Part 15: Identification and quantification of degradation products from metals and alloys
- Part 16: Toxicokinetic study design for degradation products and leachables
- Part 18: Material characterisation
Future parts will deal with other relevant aspects of biological testing.
Annexes A, B. C, D and E of this part of ISO 10993 are for information only.
The basis for biocompatibility evaluation of medical devices is often empirical and driven by the relevant concerns for human safety. Not all test methods for the assessment of Genotoxicity, carcinogenicity or reproductive toxicity are equally well developed, nor is their validity well established for the testing of medical devices.
Significant issues in test sample size and preparation, scientific understanding of disease processes and test validation can be cited as limitations of available methods. For example the biological significance of solid state carcinogenesis is poorly understood. It is expected that ongoing scientific and medical advances will alter our understanding and approaches to these important toxicity test methods. At the time the document was prepared, the test methods proposed were those most acceptable. Sound scientific alternatives to the proposed testing should be acceptable insofar as they address relevant matters of safety assessment.
In the selection of tests needed to evaluate a particular device, there is no substitute for a careful assessment of expected human uses and potential interactions of the device with various biological systems. These considerations will be particularly important in such areas as reproductive and developmental toxicology.
This part of ISO 10993 presents test methods for the detection of specific biological hazards, and therefore maximum test sensitivity is required. The interpretation of findings and implications for human health effects are beyond the scope of this part of ISO 10993. Because of the multitude of possible outcomes and the importance of such factors as extent of exposure, species differences and mechanical or physical considerations, risk assessment has to be performed on a case-by-case basis.
This part of ISO 10993 specifies tests for the following
biological aspects:
- genotoxicity,
- carcinogenicity, and
- reproductive and developmental toxicity.
These are relevant in the biological evaluation of some categories of medical devices. Guidance on selection of tests is provided in ISO 10993-1. Where the need for the evaluation of the potential for Genotoxicity, carcinogenicity or reproductive toxicity has been identified, they should be evaluated in accordance with this part of ISO 10993.
Most tests included in this part of ISO 10993 refer to Guidelines for Testing of Chemicals, prepared by the Organisation for Economic Co-operation and Development (OECD). Reference to these OECD test guidelines is made by the term "OECD" followed by the appropriate test number(s).
The following international documents contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication, the editions indicated were valid. All documents are subject to revision, and parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Standards.
ISO 10993-1: 1997 Biological evaluation of medical devices - Part 1: Selection of tests.ISO 10993-2:1992 Biological evaluation of medical devices - Part 2: Animal welfare requirements.
ISO 10993-6:1994 Biological evaluation of medical devices - Part 6: Tests for local effects after implantation.
ISO 10993-12: 1996 Biological evaluation of medical devices - Part 12: Sample preparation and reference materials.
2.3 OECD Guidelines for Testing of Chemicals Selected assays
2.3.1.1 In vitro genotoxicity testsOECD 471 Genetic Toxicology: Salmonella typhitnurium, Reverse Mutation Assay.
OECD 472 Genetic Toxicology: Escherichia coli, Reverse Mutation Assay.
OECD 473 Genetic Toxicology: In vitro Mammalian Cytogenetic Test.
OECD 476 Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test.
OECD 479 Genetic Toxicology: In vitro Sister Chromatid Exchange Assay in Mammalian Cells.
OECD 480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay.
OECD 481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay.
OECD 482 Genetic Toxicology: DNA Damage and Repair/Unscheduled DNA Synthesis in Mammalian Cells In vitro.
2.3.1.2 In vivo genotoxicity tests
OECD 474 Genetic Toxicology: Micronucleus Test.
OECD 475 Genetic Toxicology: In vivo Mammalian Bone Marrow Cytogenetic Test - Chromosomal Analysis.
OECD 478 Genetic Toxicology: Rodent Dominant Lethal Test.
OECD 483 Genetic Toxicology: Mammalian Germ-Cell Cytogenetic Assay.
OECD 484 Genetic Toxicology: Mouse Spot Test.
OECD 485 Genetic Toxicology: Mouse Heritable Translocation Assay.
OECD 486 Genetic Toxicology: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In vivo.
OECD 451 Carcinogenicity Studies.
OECD 453 Combined Chronic Toxicity/Carcinogenicity Studies.
2.5 Reproductive toxicity tests
OECD 414 Teratogenicity.
OECD 415 One-Generation Reproduction Toxicity Study.
OECD 421 Reproduction/Developmental Toxicity Screening Test
For the purposes of this part of ISO 10993, the definitions given in
ISO 10993-1, ISO 10993-12 and the following definitions apply.
3.1 genotoxicity test: Test that applies mammalian or non-mammalian cells, bacteria, yeasts or fungi to determine whether gene mutations, changes in chromosome structure, or other DNA or gene changes are caused by the test samples.
NOTE Tests on whole animals may also address these endpoints.
3.2 carcinogenicity test: Test to determine the tumorigenic potential of devices, materials, and/or extracts to either a single or multiple exposures over a period of the total life-span of the test animal.
NOTE These tests may be designed to examine both chronic toxicity and tumorigenicity in a single experimental study.
3.3 reproductive and developmental toxicity test: Test to evaluate the potential effects of test samples on reproductive function, embryonic development (teratogenicity), and prenatal and early postnatal development.
NOTE To avoid unnecessary morbidity in animals on a long-term test, preliminary testing may be necessary.
3.4 maximum tolerable dose (MTD): Maximum amount of dose that a test animal can tolerate without any adverse physical effects.
3.5 energy-depositing device: Device intended to exert its therapeutic or diagnostic effect by the delivery of electromagnetic radiation, ionic radiation or ultrasound.
NOTE This does not include devices that deliver simple electrical current, such as electrocautery devices, pacemakers or functional electrical stimulators.
4.1.1 When the genetic toxicity of a medical device has to be experimentally assessed, a series of in vitro tests shall be used. This series shall include at least three assays. At least two of these should use mammalian cells as a target.
4.1.2 In vivo testing shall only be carried out, when indicated by the results of in vitro tests, in accordance with subclause 4.1 of ISO 10993-2.
4.1.3 ISO 10993-1 indicates circumstances where the potential for genotoxicity is a relevant hazard for consideration in an overall biological safety evaluation (see ISO 109931, table 1). Testing for genotoxicity, however, will not be necessary for medical devices made only from materials, and components thereof, known to show no genotoxicity. Testing for genotoxicity is indicated where a review of the composition of the materials reveals the possible presence in the final device of compounds that might interact with genetic material. In such circumstances, the genotoxic potential of suspect chemical components should be assessed, bearing in mind the potential for synergy, in preference to carrying out genotoxicity tests on the material or device as a whole.
4.2.1 Sample preparation shall be in accordance with ISO 10993-12. Tests shall be performed either on extracts, concentrated extracts or the dissolved material using appropriate media.
4.2.2 The choice of solvents should be justified based on consideration of maximally extracting the material or device under conditions that mimic final use. The rationale shall be documented.
4.2.3 Where relevant, two appropriate extractants shall be used, one of which is a polar solvent, the second a non-polar solvent or other relevant extraction liquid or liquid appropriate to the nature and use of the device, both of which are reasonably compatible with the test system.
NOTE: If extracts are concentrated care should be taken that this does not change their chemical characteristics.
4.3.1 General
4.3.1 In vitro genotoxicity tests
4.3.1.1 Test methods for in vitro genotoxicity tests shall be chosen from the following OECD Guidelines for testing of chemicals: OECD 471, OECD 472, OECD 473, OECD 476, OECD 479, and OECD 482.
NOTE A number of materials can influence the assay e.g. antibiotics and antiseptics. It may be necessary to consider this in the design and selection of tests. Where this is relevant it should be documented.
4.3.1.1 The first choice should be OECD 471, OECD 473 and OECD 476.
4.3.1.2 The second choice should be OECD 472 (instead of OECD 471), OECD 479 (instead of OECD 476) or OECD 482.
4.3.2 In vivo genotoxicity testsIf scientifically indicated or in vitro test results indicate potential genotoxicity, then in vivo genotoxicity tests shall be undertaken. Test methods shall be chosen from the OECD Guidelines for testing of chemicals: OECD 474, OECD 475, OECD 478, OECD 483, OECD 484, OECD 485 and OECD 486.
NOTE Recently, transgenic animal test systems are being developed for genotoxicity testing. These tests may prove valuable for implant testing but their use had not been validated at the time of publication of this International Standard. References on test systems employing transgenic animals are given in B.1.
4.4.1 Genotoxicity testing shall begin with the following three in vitro endpoints: 1. a bacterial assay for gene mutations (e.g. according to OECD 471); 2. a test for gene mutations in mammalian cells (e.g. according to OECD 476); 3. a test for clastogenicity in mammalian cells (e.g. according to OECD 473).
4.4.2 This can be acchieved by combination of OECD 471, OECD 473, and OECD 476, or by combination of OECD 471 and the mouse Iymphoma assay incorporating colony number and size determination in order to cover both endpoints.
4.4.3 The performance of only one single test is not sufficient for detecting all genotoxic mechanism.
4.4.4 When the results of all tests are negative, further testing is not necessary.
4.4.5 In the event of positive in vitro results, either the lack of in vivo mutagenic potential should be established on the evaluation of biological safety or the evaluation of biological safety should be based on the assumption that the compound is mutagenic.
4.4.6 If either or both of the in vitro tests is positive, and further clarification of mutagenic potential is needed, an in vivo test using somatic cells shall be performed. The in vivo test shall be chosen on the basis of the most appropriate target organ:a) metaphase analysis in rodent bone marrow according to OECD 475 or
b) micronucleus test in rodents according to OECD 474 or
c) test to investigate unscheduled DNA synthesis with mammalian liver cells according to OECD 486.
4.4.7 Other test systems concerning genotoxicity can be performed in order to obtain additional information. The rationale for the choices made shall be justified and documented.
5.1.1 Carcinogenicity tests shall be considered when indicated in ISO
10993-1. A decision to undertake carcinogenicity testing shall be justified on
the basis of an assessment of the risk of carcinogenesis arising from the use of
the medical device.
5.1.2 Situations where the need for carcinogenicity testing should be considered
may include the following:
-
a) resorbable materials and devices for which the resorption time is greater than 30 days, unless there are significant and adequate data on toxicokinetics or human use or exposure;
-
b) materials and devices where positive results have been obtained in genetic toxicity testing in both mammalian cells and in vivo;
-
c) materials and devices introduced in the body and/or its cavities with a permanent or cumulative contact of 30 days or longer, except when significant and adequate human use history is available.
NOTE: Carcinogenicity testing is not required where the biological risk assessment of the device makes proper provision for the fact that the risk of carcinogenicity has not been ruled out.
5.1.3 There are suitable cell transformation systems which may be used for carcinogenicity rescreening. Cell transformation tests have so far not been described in International Standards. Additional information on cell transformation test systems is given in annex C.
5.2.1 Sample preparation shall be in accordance with ISO 10993-12. Whenever
possible the device shall be tested in its "ready-to-use" form.
5.2.2 Otherwise a suitably formed implant in accordance with ISO 10993-6 shall be made of the test material, with appropriate consideration of potential solid state carcinogenicity (Oppenheimer effect, see annex B.3, [15]. The weight, size or dose should exceed the expected clinical exposure, on an mg/kg body weight basis.
5.3.1 Carcinogenicity tests for medical devices should be performed as implantation tests (see Annex D).
5.3.2 Prior to implantation, consideration shall be given to the clinical use of the medical device in selecting the implant site.
5.3.3 Carcinogenicity tests shall be performed in accordance with OECD 451 or OECD 453 after suitable modifications for implantable materials.
5.3.4 Where implantation does not represent the most appropriate route of exposure, scientifically justified alternatives should be considered. The rationale for this shall be documented.
5.3.5 When testing of an extract is considered relevant the carcinogenicity tests shall be performed in accordance with OECD 451 or OECD 453.
5.3.6 Tissues evaluated shall include relevant tissues from the list indicated in OECD 451 or OECD 453, as well as the implantation site and adjacent tissues.
5.4.1 When, according to ISO 10993-1, a chronic toxicity and carcinogenicity study shall be considered, and it is determined that testing is necessary, testing should be carried out according to OECD 453.
5.4.2 When, according to ISO 10993-1, only a carcinogenicity study shall be considered, and it is determined that testing is necessary, testing should be carried out according to OECD 451.
5.4.3 One animal species is sufficient for testing of medical devices. The choice of this species shall be justified and documented.
6.1.1 Reproductive toxicity tests should normally be considered for the following medical devices:
-
a) intrauterine devices (IUDs), or any other long-term contact devices likely to come into direct contact with reproductive tissues or the embryo/foe/us;
- b) energy-depositing devices;
- c) resorbable or leachable materials and devices.
6.1.2 There is no need for the testing of resorbable devices or devices containing leachable moieties where there is adequate and reassuring data from absorption, metabolism, distribution and on the reproductive toxicity of all components identified in extracts of materials or devices.
6.2.1 Sample preparation shall be in accordance with ISO 10993- 12.
6.2.2 In the case of energy-depositing devices, whole-body exposure of the animals should be appropriate. A multiple of the dose to be expected in humans should be applied.
6.2.3 When possible, lUDs, resorbable devices or devices containing leachable moieties shall be tested in their "ready-to-use" form. Otherwise a suitably formed implant shall be made of the test material.
6.2.4 The maximum tolerable dose (MTD) of a material or device should be applied. Where possible this dose should be expressed as a multiple of the worst case human exposure (in milligrams per kilogram).
6.3.1 Assessment of effects on this first generation (F1) should be made according to absorption- kinetic data and OECD 414, OECD 415 and OECD 421. As the OECD guidelines were not intended for implantable devices the following modifications shall be considered:
- - dose (in the case of energy-depositing devices),
- - route of application,
- - exposure time (elevated blood levels during organogenesis when possible).
6.3.2 If information derived from other tests indicates potential effects on the male reproduction system, then appropriate tests for male reproductive toxicity shall be conducted.
NOTE Recently, in vitro reproductive test systems have been developed. They may be useful as a prescreening test method for reproductive toxicity. References to in vitro reproductive test systems are included in annex B.4.
6.4 Test strategy
When testing is required this shall be according to OECD 414, OECD 415 and OECD 421.
7.1 The test report shall include at least the following
details
-
a) description of material and/or device including intended use (e.g. chemical composition, processing, conditioning and surface treatment);
- b) description and justification of test methods, test conditions, test materials, and test procedures;
- c) description of analytical methods including quantification limits;
- d) statement of compliance to appropriate good laboratory practices;
- e) test results including summary;
- f) statistical methods;
- g) interpretation and discussion of results.
7.2 Further details on the reporting, specified in the following OECD guidelines, shall be included in the test report, if applicable:
OECD 414, OECD 415, OECD 421, OECD 451, OECD 453, OECD 471, OECD 472, OECD 473, OECD 474, OECD 475, OECD 476, OECD 478, OECD 479, OECD 480, OECD 481, OECD 482, OECD 485, OECD 486.
(informative)
Flow chart for genotoxicity testing
(informative)
Bibliography
B.1 Literature on transgenic animals
[1] KOHLER, SW., PROVOST, GS., KRETZ, PL., DYCAICO, MI., SORGE, JA. And SHORT, JM. Development of a short-term in vitro mutagenesis assay: the effect of methylation on the recovery of a lambda phage shuttle vector from transgenic mice. Nucleic Acid Research. 1990, vol. 18, p. 3007-3013.
[2] SHORT, JM., KOHLER, SW. And PROVOST, GS. The use of lambda phage shuttle vectors in transgenic mice for development of a short term mutagenicity assay. In Mutation and the environment. Wiley-Liss: New York, 1990. P. 355-367.
B.2 Literature on cell transformation assays
[3] Advances in Modern Environment Toxicology, Vol. 1. Mammalian Cell Transformation by Chemical Carcinogens. N. Mishra, V. Dunkel, and M. Mehlman (eds). Senate Press: Princeton Junction (New Jersey, 08550), 1981.
[4] Transformation Assays of Established Cell Lines: Mechanisms and Application. T. Kakunaga and H. Yamasaki (eds). Proceedings of a Workshop Organized by IARC in Collaboration with the US National Cancer Institute and the US Environmental Protection Agency, Lyon 15-17 Feb. 1984. IARC Scientific Publication No. 67.
[5] BARRETT, JC., OHSHIMURA, M., TANAKA, N. And TSUTSUI, T. Genetic and Epigenetic Mechanisms of Presumed Nongenotoxic Carcinogens. In Banbury Report 25: Nongenotoxic Mechanisms in Carcinogenesis, 1987, p. 311-324.
[6] OSHIMURA, M., HESTERBERG, TOO., TSUTSUI, T. And BARRETT, JC. Correlation of Asbestos-induced Cytogenetic Effects with Cell Transformation of Syrian Hamster Embryo Cells in Culture. Cancer Res. Nov. 1984, vol. 44, p. 50175022.
[7] BARRETT, JC., OSHIMURA, M., TANAKA, N. And TSUTSUI, T. Role of Aneuploidy in Early and late Stages of Neoplastic Progression of Syrian Hamster Embryo Cells in Culture. In Aneuploidy. Wicki L. Dellargo, Peter E. Voytek and Alexander Hollaender (eds). Plenum Publishing, 1985.
[8] FITZGERALD, DJ. And YAMASAKI, H. Tumor promotion: models and assay systems. Teratogenesis Carcinog. Mutagen., 1990, vol. 10, No. 2, p. 89-102.
[9] KUROKI, T. And MATSUSHIMA, T. Performance of short-term tests for detection of human carcinogens. Mutagenesis, 1987, vol. 2, No. 1, p. 33-7.
[10] RAY, VA., KIER, LD., KANNAN, KL., HAAS, RT., AULETTA, AK., WASSOM, JS., NESNOW, S. And WATERS, MD. An approach to identifying specialized batteries of bioassays for specific classes of chemicals: class analysis using mutagenicity and carcinogenicity relationships and phylogenetic concordance and discordance patterns. 1. Composition and analysis of the overall data base. A report of phase II of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat Res, 1987, vol. 3, p. 197- 241.
[11] DUNKEL, VD., SCHECHTMAN, LM., TU, AS., SIVAK, A., LUBET, RA. And CAMERON, TP. Interlaboratory evaluation of the C3H/lOT1/2 cell transformation assay. Environ. Mol. Mutagen., 1988, vol. 12, No. 1, p. 12-31.
[12] JONES, CA., HUBERMAN, E:, CALLAHAM, MF., TU, A., HALLOWEEN, W., PALLOTA, S., SIVAK, A., LUBET, RA., AVERY, MD., KOURI, RE., SPALDING, J. And TENNANT, RW. An interlaboratory evaluation of the Syrian hamster emryo cell transformation assay using eighteen coded chemicals. Toxicology in vitro, 1988, vol.2, No. 2, p. 103-116.
B.3 Literature on genotoxicity and carcinogenicity testing
[13] Department of Health. Guidelines for the testing of chemicals for mutagenicity. Londong: HMSO, 1989. (Report on Health and Social Security Subjects No. 35).
[14] Department of Health. Guidelines for the evaluation of chemicals for carcinogenicity. London: HMSO, 1992. (Report on Health and Social Security Subjects No. 42).
[15] OPPENHEIMER, BS., OPPENHEIMER, ET. And STOUT, AP. Sarcomas induced in rats by implanting cellophane. Proc. Soc. Exp. Biol. Med., 1948, vol. 67, No. 33.
[16] BRAND, KG., JOHNSON, KH and BUON, LC. Foreign Body, Tumorgenesis CRC Crit. Rev. In Toxicology, October 1976, p. 353.
[17] BRAND, L. And BRAND, KG. Testing of Implant Materials for Foreign Body Carcinogenesis. In Biomaterials, 1980, p. 819. G.D. Winter, D.F. Gibbons, H. Plenk Jr. (Eds). Advances in Biomaterials, vol. 3. New YorkL. J. Wiley, 1982.
[18] Biological Bases for Interspecies Extrapolation of Carcinogenicity Data. Hill TA., Wands, RC., Leukroth RW. Jr. (eds). (Prepared for the Center for Food Safety and Applied Nutrition, Food and Drug Administration, Department of Health and Human Services, Washington, D.C.) July 1986, Bethesda (MD): Life Science Research Office, Federation of American Societies for Experimental Biology.
[19] National Toxicology Program Report of the BTP Ad Hoc Panel on Chemical Carcinogenesis Testing and Evaluation, August 1984, Board of Scientific Counselors.
[20] ASTM F 1439-39 Standard guide for performance of lifetime bioassay for the tumorgenic potential of implant materials
[21] T. Tsuchiya And A. Nakamura, A New Hypothesis of Tumorigenesis Induced by Biomaterials: Inhibitory Potentials of Intercellular Communication Play an Important Role on the Tumor-Promotion Stage, Journal of long-Term Effects of Medical Implants, 5(4):233-242 (1995)
B.4 Literature on reproductive toxicity testing
[22] 1990 Guideline for toxicity studies of drugs manual, Chapter 4: Reproductive and developmental toxicity studies. First edition. Editorial Supervision by New Drugs Division, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Yakuji Nippo Ltd.
[23] Gabrielson, JL. and LARSSON, KS. Proposal for improving risk assessment in reproductive toxicology. Pharmacology & Toxicology, 1990, vol. 66, p. 10-17.
[24] NEUBERT, D., BLANKENBURG, G., CHAHOUD, I., FRANZ, G., HERKEN, R., KASTNER, M., KLUG, S., KROGER, I., KROWKE, R., LEWANDOWSKI, C., MERKER, HJ. And SCHULZ, T. Results of in vivo and in vitro Studies for Assessing Prenatal Toxicity. Environmental Health Perspectives, 1986, vol. 70, p. 89-103.
[25] SADLER, TOO., HORTON, WE. And WARNER, CW. Whole Embryo Culture: A Screening Technique for Teratogens? Teratogenesis, Carcinogenesis, and Mutagenesis, 1982, vol. 2, p. 243-253.
[26] In vitro Methods in Developmental Toxicology: Use in Defining Mechanisms and Risk Parameters. GL. Kimmel and DM. Kochhar (eds.). Boca Raton (Florida): CRC Press, 1990.
[27] In vitro Embryotoxicity and Teratogenicity Tests.
F. Homburger and AH. Goldberg (eds.). Concepts in Toxicology, vol. 3. Basel:
KARGER, 1985.
[28] BRENT, RL. Predicting Teratogenic and Reproductive Risks in Humans from Exposure to Various Environmental Agents Using In vitro Techniques and In vivo Animal Studies. Cong. Anom., 1988, vol. 28 (Suppl.), S41-S55.
[29] TSUCHIYA, T., NAKAMURA, A., IIO, T. And TAKAHASI, A. Species Differences between Rats and Mice in the Teratogenic Action of Ethylenethiourea: In vivo/In vitro Tests and Teratogenic Activity of Sera Using an Embryonic Cell Differentiation System. Toxicology and Applied Pharmacology, 1991, vol. 109, p. 16.
[30] TSUCHIYA, T., BURGIN, H., TSUCHIYA, M., WINTERNITZ, P. and KISTLER, A. Embryolethality of new herbicides is not detected by the micromass teratogen tests. Arch. Toxicol, 1991, vol. 65, p. 145-149.
[31] KISTLER, A., TSUCHIYA, T., TSUCHIYA, M. And KLAUS, M.
Teratogenicity of arotinoids (retinoids) in vivo and in vitro. Arch.
Toxicol., 1990, vol. 64, p. 616-622.
[32] TSUCHYIA, T., TAKAHASHI, A., ASADA, S., TAKAKUBO, F., OHSUMI, YAMASHITA, N. And ETO, K. Comparative Studies of Embryotoxic Action of Ethylenethiourea in Rat Whole Embryo and Embryonic Cell Culture. Teratology, 1991, vol. 43, p. 319-324.
[33]Report of the in vitro teratology task force, Organized by the Devision of Toxicology, Office of Toxicological Sciences, Center for Food Safety and Applied Nutrition, Food and Drug Administration. Environmental Health Perspectives, 1987, vol. 72, p. 200235.
[34] BASS, R., ULBRICH, B., HILDEBRANDT, AG., WEISSINGER, J.,
DOI, O., BAEDER, C., EUMERO, S., HARADA, Y., LEHMANN, H., MANSON, J., NEUBERT,
D., OMORI, Y., PALMER, A. SULLIVAN, F., TAKAYAMA, S. And TANIMUTA, T. Draft
guideline on detection of toxicity to reproduction for medical products. Adverse
Drug React. Toxicol. Rev., 1991, vol. 9, No. 3, p. 127-141.
[35] BROWN et al., Screening chemicals for reproductive toxcity: the current
approaches the report and recommendations of an ECVAM/EST workshop (ECVAM
Workshop 12), ATLA 23, 868-882, 1995.
[36] SPIELMANN, R., Reproduction and development, Environmental Health
Perspective, 1998, in press
(informative)
Cell Transformation Systems
Cell transformation systems may be used for carcinogenicity prescreening.
Guidance is given by the Official Journal of the European Communities L 133/73 from 30.5.1988 for In vitro-cell transformation test. References on cell transformation test systems are given in annex B.2.
There is also some evidence that two-step cell transformation assays can detect carcinogens which are non-genotoxic, but it is at this time not possible to conclude that all nongenotoxic carcinogens can be detected by cell transformation assays. Therefore, carcinogenicity tests have to be performed as lifetime studies in vivo on at least one appropriate rodent species.
(informative)
Carcinogenicity tests performed as implantation tests
Carcinogenicity tests for medical devices are generally performed as implantation tests.
While a single maximum implantable dose group (MID) may be sufficient, two doses including the maximum implantable dose (MID) and a fraction thereof (usually one half of the MID) are recommended. The negative control group will generally receive a comparable shape and form of a clinically acceptable material or reference control material whose lack of carcinogenic potential is documented.
In carcinogenicity testing on rodents, the maximum implantable dose (MID) of a material or device should be applied. Where possible, this dose should be expressed as a multiple of the worst case human exposure in milligrams per kilogram.
There will ordinarily be two dose levels, the maximum implantable dose (MID), and a fraction thereof (usually one half of the MID). The negative controls will generally include polyethylene implants or other materials whose lack of carcinogenic potential is documented in a comparable form and shape.
In carcinogenicity testing on rodents, the maximum implantable dose (MID) of a material or device should be applied. Where possible, this dose should be expressed as multiple of the worst case human exposure in milligrams per kilogram.
(informative)
Rationale of test systems
The primary function of genotoxicity testing is to investigate, using test cells or organisms, the potential of products to induce mutations in man that may be transmitted via the germ cells to future generations. Scientific data generally support the hypotheses that DNA damage in somatic cells is a critical event in the initiation of cancer. Such damage can result in mutations and tests to detect mutagenic activity may also identify chemicals that have the potential to lead to carcinogenesis. Thus, some of the tests are useful for the investigation of putative carcinogenic activity.
While in classical toxicology testing several pertinent parameters or endpoints can be observed within one experimental design, the same is not true for genetic toxicology. The diversity of the genetic endpoints usually precludes the detection of more than one of them in a single assay system.
Approximately fifteen different assays are referred to in the test guidelines. The selection of the most appropriate of these to meet a particular requirement is governed by a number of factors. These include the type of genetic change it is required to detect, or the metabolic capability of the test system.
It must be emphasized that there is no international agreement on the best combination of tests for a particular purpose, though there have been attempts to harmonise the selection of the most appropriate assays. It may also be helpful to note that there are other mutagenicity tests in use or in development, which, although without an OECD Guideline, may also be useful.
Chemicals that interact with DNA produce lesions that, after the influrence of various repair processes, may lead to genetic changes at the gene level, e.g. gene or point mutations, small deletions, mitotic recombination, or various microscopically chromosome visible chromosome changes, and assys are available to investigate each of these events.
Current short-term tests cannot, of course, mimic all the stages in the carcinogenic process and are frequently assumed to detect only the event leading to the initiation phase, i.e. the ability to induce a mutagenic or clastogenic DNA lesion. The main value of these procedures, therefore, leis in their ability to identify substances that may, under certain exposure conditions, either cause cancer by a predominantly genotoxic mechanism or induce the initial phase of the carcinogenic process. It is apparent, from the complexity of the carcinogenic process compared with the relative simplicity of short term assays, that, although they provide useful qualitative information, considerable caution is required in their interpretation in terms of carcinogenic activity.
Since no single assay has proved capable of detecting mammalian mutagens and carcinogens with an acceptable level of precision and reproducibility, it is usual scientific practice to apply these assays in batteries . Initial information on the mutagenicitity of a substance can be obtained using assays that measure gene mutations and chromosomal damage. Because separate procedures are required to investigate these endpoints, a battery of assays is needed.
The objective of a long-term carcinogenicity study is to observe test animals, for a major portion of their life span, for the development of neoplastic lesions, during or after exposure to various doses of a test substance by an appropriate route. Such an assay requires careful planning and documentation of the experimental design, a high quality of pathology and unbiased statistical analysis.
E.3 Reproductive toxicity tests
Reproductive toxicity tests covers the areas of reproduction, fertility and teratogenicity. It has been found that many substances can affect fertility and reproduction, often in an insidious manner without other sign of toxicity. Fertility can be affected in males and females and effects can range from slightly decreased reproductive capability to complete sterility.
Teratogenicity deals with the adverse effects of a substance on the developing embryo and foetus. Reproductive toxicity is important as it has an important bearing on the health of mankind. Testing techniques are developing and the concept of combined tests, covering all aspects of reproductive toxicology, appears promising.