Title 40—Protection of Environment--Volume 1
PART 799—IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE TESTING REQUIREMENTS
§ 799.9510   TSCA bacterial reverse mutation test.
(a) Scope. This section is intended to meet the testing requirements under section 4 of TSCA.
(1) The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.
(2) Point mutations are the cause of many human genetic diseases and there is substantial evidence that point mutations in oncogenes and tumor suppressor genes of somatic cells are involved in tumor formation in humans and experimental animals. The bacterial reverse mutation test is rapid, inexpensive and relatively easy to perform. Many of the test strains have several features that make them more sensitive for the detection of mutations, including responsive DNA sequences at the reversion sites, increased cell permeability to large molecules and elimination of DNA repair systems or enhancement of error-prone DNA repair processes. The specificity of the test strains can provide some useful information on the types of mutations that are induced by genotoxic agents. A very large data base of results for a wide variety of structures is available for bacterial reverse mutation tests and well-established methodologies have been developed for testing chemicals with different physico-chemical properties, including volatile compounds.
(b) Source. The source material used in developing this TSCA test guideline are the OECD replacement guidelines for 471 and 472 (February 1997). This source is available at the address in paragraph (g) of this section.
(c) Definitions. The following definitions apply to this section:
A reverse mutation test in either Salmonella typhimurium or Escherichia coli detects mutation in an amino-acid requiring strain (histidine or tryptophan, respectively) to produce a strain independent of an outside supply of amino-acid.
Base pair substitution mutagens are agents that cause a base change in DNA. In a reversion test this change may occur at the site of the original mutation, or at a second site in the bacterial genome.
Frameshift mutagens are agents that cause the addition or deletion of one or more base pairs in the DNA, thus changing the reading frame in the RNA
(d) Initial considerations. (1) The bacterial reverse mutation test utilizes prokaryotic cells, which differ from mammalian cells in such factors as uptake, metabolism, chromosome structure and DNA repair processes. Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. In vitro metabolic activation systems cannot mimic entirely the mammalian in vivo conditions. The test therefore does not provide direct information on the mutagenic and carcinogenic potency of a substance in mammals.
(2) The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity. An extensive data base has demonstrated that many chemicals that are positive in this test also exhibit mutagenic activity in other tests. There are examples of mutagenic agents which are not detected by this test; reasons for these shortcomings can be ascribed to the specific nature of the endpoint detected, differences in metabolic activation, or differences in bioavailability. On the other hand, factors which enhance the sensitivity of the bacterial reverse mutation test can lead to an overestimation of mutagenic activity.
(3) The bacterial reverse mutation test may not be appropriate for the evaluation of certain classes of chemicals, for example highly bactericidal compounds (e.g. certain antibiotics) and those which are thought (or known) to interfere specifically with the mammalian cell replication system (e.g. some topoisomerase inhibitors and some nucleoside analogues). In such cases, mammalian mutation tests may be more appropriate.
(4) Although many compounds that are positive in this test are mammalian carcinogens, the correlation is not absolute. It is dependent on chemical class and there are carcinogens that are not detected by this test because they act through other, non-genotoxic mechanisms or mechanisms absent in bacterial cells.
(e) Test method —(1) Principle. (i) Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. In the plate incorporation method, these suspensions are mixed with an overlay agar and plated immediately onto minimal medium. In the preincubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium. For both techniques, after 2 or 3 days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.
(ii) Several procedures for performing the bacterial reverse mutation test have been described. Among those commonly used are the plate incorporation method, the preincubation method, the fluctuation method, and the suspension method. Suggestions for modifications for the testing of gases or vapors are described in the reference in paragraph (g)(12) of this section.
(iii) The procedures described in this section pertain primarily to the plate incorporation and preincubation methods. Either of them is acceptable for conducting experiments both with and without metabolic activation. Some compounds may be detected more efficiently using the preincubation method. These compounds belong to chemical classes that include short chain aliphatic nitrosamines, divalent metals, aldehydes, azo-dyes and diazo compounds, pyrollizidine alkaloids, allyl compounds and nitro compounds. It is also recognized that certain classes of mutagens are not always detected using standard procedures such as the plate incorporation method or preincubation method. These should be regarded as “special cases” and it is strongly recommended that alternative procedures should be used for their detection. The following “special cases” could be identified (together with examples of procedures that could be used for their detection): azo-dyes and diazo compounds (alterative procedures are described in the references in paragraphs (g)(3), (g)(5), (g)(6), and (g)(13) of this section), gases and volatile chemicals (alterative procedures are described in the references in paragraphs (g)(12), (g)(14), (g)(15), and (g)(16) of this section), and glycosides (alterative procedures are described in the references in paragraphs (g)(17) and (g)(18) of this section). A deviation from the standard procedure needs to be scientifically justified.
(2) Description —(i) Preparations —(A) Bacteria. ( 1 ) Fresh cultures of bacteria should be grown up to the late exponential or early stationary phase of growth (approximately 10 9 cells per ml). Cultures in late stationary phase should not be used. The cultures used in the experiment shall contain a high titre of viable bacteria. The titre may be demonstrated either from historical control data on growth curves, or in each assay through the determination of viable cell numbers by a plating experiment.
( 2 ) The culture temperature shall be 37 °C.
( 3 ) At least five strains of bacteria should be used. These should include four strains of S. typhimurium (TA1535; TA1537 or TA97a or TA97; TA98; and TA100) that have been shown to be reliable and reproducibly responsive between laboratories. These four S. typhimurium strains have GC base pairs at the primary reversion site and it is known that they may not detect certain oxidizing mutagens, cross-linking agents, and hydrazines. Such substances may be detected by E. coli WP2 strains or S. typhimurium TA102 (see reference in paragraph (g)(19) of this section) which have an AT base pair at the primary reversion site. Therefore the recommended combination of strains is:
( i ) S. typhimurium TA1535.
( ii ) S. typhimurium TA1537 or TA97 or TA97a.
( iii ) S. typhimurium TA98.
( iv ) S. typhimurium TA100.
( v ) E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. In order to detect cross-linking mutagens it may be preferable to include TA102 or to add a DNA repair-proficient strain of E.coli [e.g. E.coli WP2 or E.coli WP2 (pKM101).]
( 4 ) Established procedures for stock culture preparation, marker verification and storage should be used. The amino-acid requirement for growth should be demonstrated for each frozen stock culture preparation (histidine for S. typhimurium strains, and tryptophan for E. coli strains). Other phenotypic characteristics should be similarly checked, namely: the presence or absence of R-factor plasmids where appropriate [i.e. ampicillin resistance in strains TA98, TA100 and TA97a or TA97, WP2 uvrA and WP2 uvrA (pKM101), and ampicillin = tetracycline resistance in strain TA102]; the presence of characteristic mutations (i.e. rfa mutation in S. typhimurium through sensitivity to crystal violet, and uvrA mutation in E. coli or uvrB mutation in S. typhimurium, through sensitivity to ultra-violet light). The strains should also yield spontaneous revertant colony plate counts within the frequency ranges expected from the laboratory's historical control data and preferably within the range reported in the literature.
(B) Medium. An appropriate minimal agar (e.g. containing Vogel-Bonner minimal medium E and glucose) and an overlay agar containing histidine and biotin or tryptophan, to allow for a few cell divisions, shall be used. The procedures described in the references under paragraphs (g)(1), (g)(2), and (g)(9) of this section may be used for this analysis.
(C) Metabolic activation. Bacteria shall be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (the system described in the references under paragraphs (g)(1) and (g)(2) of this section may be used) or a combination of phenobarbitone and β-naphthoflavone (the system described in the references under paragraphs (g)(18), (g)(20), and (g)(21) of this section may be used). The post-mitochondrial fraction is usually used at concentrations in the range from 5 to 30% v/v in the S9-mix. The choice and condition of a metabolic activation system may depend upon the class of chemical being tested. In some cases it may be appropriate to utilize more than one concentration of post-mitochondrial fraction. For azo-dyes and diazo-compounds, using a reductive metabolic activation system may be more appropriate (the system described in the references under paragraphs (g)(6) and (g)(13) of this section may be used).
(D) Test substance/preparation. Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if appropriate prior to treatment of the bacteria. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations should be employed unless stability data demonstrate the acceptability of storage.
(ii) Test conditions —(A) Solvent/vehicle. The solvent/vehicle should not be suspected of chemical reaction with the test substance and shall be compatible with the survival of the bacteria and the S9 activity (for further information see the reference in paragraph (g)(22) of this section). If other than well-known solvent/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents used be free of water.
(B) Exposure concentrations. ( 1 ) Amongst the criteria to be taken into consideration when determining the highest amount of test substance to be used are cytotoxicity and solubility in the final treatment mixture. It may be useful to determine toxicity and insolubility in a preliminary experiment. Cytotoxicity may be detected by a reduction in the number of revertant colonies, a clearing or diminution of the background lawn, or the degree of survival of treated cultures. The cytotoxicity of a substance may be altered in the presence of metabolic activation systems. Insolubility should be assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye. The recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 µl/plate. For non-cytotoxic substances that are not soluble at 5 mg/plate or 5 µl/plate, one or more concentrations tested should be insoluble in the final treatment mixture. Test substances that are cytotoxic already below 5mg/plate or 5 µl/plate should be tested up to a cytotoxic concentration. The precipitate should not interfere with the scoring.
( 2 ) At least five different analyzable concentrations of the test substance shall be used with approximately half log (i.e. √10) intervals between test points for an initial experiment. Smaller intervals may be appropriate when a concentration-response is being investigated.
( 3 ) Testing above the concentration of 5 mg/plate or 5 µl/plate may be considered when evaluating substances containing substantial amounts of potentially mutagenic impurities.
(C) Controls. ( 1 ) Concurrent strain-specific positive and negative (solvent or vehicle) controls, both with and without metabolic activation, shall be included in each assay. Positive control concentrations that demonstrate the effective performance of each assay should be selected.
( 2 )( i ) For assays employing a metabolic activation system, the positive control reference substance(s) should be selected on the basis of the type of bacteria strains used. The following chemicals are examples of suitable positive controls for assays with metabolic activation:
( ii ) 2-Aminoanthracene should not be used as the sole indicator of the efficacy of the S9-mix. If 2-aminoanthracene is used, each batch of S9 should also be characterized with a mutagen that requires metabolic activation by microsomal enzymes, e.g., benzo(a)pyrene, dimethylbenzanthracene.
( 3 ) For assays performed without metabolic activation system, examples of strain-specific positive controls are:
( 4 ) Other appropriate positive control reference substances may be used. The use of chemical class-related positive control chemicals may be considered, when available.
( 5 ) Negative controls, consisting of solvent or vehicle alone, without test substance, and otherwise treated in the same way as the treatment groups, shall be included. In addition, untreated controls should also be used unless there are historical control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent.
(3) Procedure —(i) Treatment with test substance. (A) For the plate incorporation method, without metabolic activation, usually 0.05 ml or 0.1 ml of the test solutions, 0.1 ml of fresh bacterial culture (containing approximately 10 8 viable cells) and 0.5 ml of sterile buffer are mixed with 2.0 ml of overlay agar. For the assay with metabolic activation, usually 0.5 ml of metabolic activation mixture containing an adequate amount of post-mitochondrial fraction (in the range from 5 to 30% v/v in the metabolic activation mixture) are mixed with the overlay agar (2.0 ml), together with the bacteria and test substance/test solution. The contents of each tube are mixed and poured over the surface of a minimal agar plate. The overlay agar is allowed to solidify before incubation.
(B) For the preincubation method the test substance/test solution is preincubated with the test strain (containing approximately 10 8 viable cells) and sterile buffer or the metabolic activation system (0.5 ml) usually for 20 min. or more at 30-37 °C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. Usually, 0.05 or 0.1 ml of test substance/test solution, 0.1 ml of bacteria, and 0.5 ml of S9-mix or sterile buffer, are mixed with 2.0 ml of overlay agar. Tubes should be aerated during pre-incubation by using a shaker.
(C) For an adequate estimate of variation, triplicate plating should be used at each dose level. The use of duplicate plating is acceptable when scientifically justified. The occasional loss of a plate does not necessarily invalidate the assay.
(D) Gaseous or volatile substances should be tested by appropriate methods, such as in sealed vessels (methods described in the references under paragraphs (g)(12), (g)(14), (g)(15), and (g)(16) of this section may be used).
(ii) Incubation. All plates in a given assay shall be incubated at 37 °C for 48-72 hrs. After the incubation period, the number of revertant colonies per plate is counted.
(f) Data and reporting —(1) Treatment of results. (i) Data shall be presented as the number of revertant colonies per plate. The number of revertant colonies on both negative (solvent control, and untreated control if used) and positive control plates shall also be given.
(ii) Individual plate counts, the mean number of revertant colonies per plate and the standard deviation shall be presented for the test substance and positive and negative (untreated and/or solvent) controls.
(iii) There is no requirement for verification of a clear positive response. Equivocal results shall be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing, the method of treatment (plate incorporation or liquid preincubation), and metabolic activation conditions.
(2) Evaluation and interpretation of results. (i) There are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
(ii) A test substance for which the results do not meet the criteria described under paragraph (f)(2)(i) of this section is considered non-mutagenic in this test
(iii) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.
(iv) Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested species.
(3) Test report. The test report shall include the following information:
(i) Test substance:
(A) Identification data and CAS no., if known.
(B) Physical nature and purity.
(C) Physicochemical properties relevant to the conduct of the study.
(D) Stability of the test substance, if known.
(ii) Solvent/vehicle:
(A) Justification for choice of solvent/vehicle.
(B) Solubility and stability of the test substance in solvent/vehicle, if known.
(iii) Strains:
(A) Strains used.
(B) Number of cells per culture.
(C) Strain characteristics.
(iv) Test conditions:
(A) Amount of test substance per plate (mg/plate or ml/plate) with rationale for selection of dose and number of plates per concentration.
(B) Media used.
(C) Type and composition of metabolic activation system, including acceptability criteria.
(D) Treatment procedures.
(v) Results:
(A) Signs of toxicity.
(B) Signs of precipitation.
(C) Individual plate counts.
(D) The mean number of revertant colonies per plate and standard deviation.
(E) Dose-response relationship, where possible.
(F) Statistical analyses, if any.
(G) Concurrent negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations.
(H) Historical negative (solvent/vehicle) and positive control data, with e.g. ranges, means and standard deviations.
(vi) Discussion of the results.
(vii) Conclusion.
(g) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting Carcinogens and Mutagens With the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research. 31, 347-364 (1975).
(2) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research. 113, 173-215 (1983).
(3) Gatehouse, D., Haworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcion, C., Nohmi, T., Venitt, S., and Zeiger, E. Recommendations for the Performance of Bacterial Mutation Assays. Mutation Research. 312, 217-233 (1994).
(4) Kier, L.D., Brusick, D.J., Auletta, A.E., Von Halle, E.S., Brown, M.M., Simmon, V.F., Dunkel, V., McCann, J., Mortelmans, K., Prival, M., Rao, T.K., and Ray V. The Salmonella Typhimurium/Mammalian Microsomal Assay: A Report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research. 168, 69-240 (1986).
(5) Yahagi, T., Degawa, M., Seino, Y.Y., Matsushima, T., Nagao, M., Sugimura, T., and Hashimoto, Y. Mutagenicity of Carcinogen Azo Dyes and Their Derivatives. Cancer Letters, 1. 91-96 (1975).
(6) Matsushima, M., Sugimura, T., Nagao, M., Yahagi, T., Shirai, A., and Sawamura, M. Factors Modulating Mutagenicity Microbial Tests. Eds. Norpoth, K.H. and Garner, R.C. Short-Term Test Systems for Detecting Carcinogens (Springer, Berlin-Heidelberg-New York, 1980) pp. 273-285.
(7) Gatehouse, D.G., Rowland, I.R., Wilcox, P., Callender, R.D., and Foster, R. Bacterial Mutation Assays. Ed. Kirkland, D.J. Basic Mutagenicity Tests. UKEMS Part 1 Revised (Cambridge University Press, 1990) pp. 13-61.
(8) Aeschbacher, H.U., Wolleb, U., and Porchet, L.J. Liquid Preincubation Mutagenicity Test for Foods. Food Safety. 8, 167-177 (1987).
(9) Green, M.H.L., Muriel, W.J., and Bridges, B.A. Use of a Simplified Fluctuation Test to Detect Low Levels of Mutagens. Mutation Research. 38, 33-42 (1976).
(10) Hubbard, S.A., Green, M.H.L., Gatehouse, D., and J.W. Bridges. The Fluctuation Test in Bacteria. 2nd Edition. Ed. Kilbey, B.J., Legator, M., Nichols, W., and Ramel C. Handbook of Mutagenicity Test Procedures (Elsevier, Amsterdam-New York-Oxford, 1984) pp. 141-161.
(11) Thompson, E.D. and Melampy, P.J. An Examination of the Quantitative Suspension Assay for Mutagenesis With Strains of Salmonella Typhimurium. Environmental Mutagenesis. 3, 453-465 (1981).
(12) Araki, A., Noguchi, T., Kato, F., and T. Matsushima. Improved Method for Mutagenicity Testing of Gaseous Compounds by Using a Gas Sampling Bag. Mutation Research. 307, 335-344 (1994).
(13) Prival, M.J., Bell, S.J., Mitchell, V.D., Reipert, M.D., and Vaughn, V.L. Mutagenicity of Benzidine and Benzidine-Congener Dyes and Selected Monoazo Dyes in a Modified Salmonella Assay. Mutation Research. 136, 33-47 (1984).
(14) Zeiger, E., Anderson, B. E., Haworth, S, Lawlor, T., and Mortelmans, K. Salmonella Mutagenicity Tests. V. Results from the Testing of 311 Chemicals. Environ. Mol. Mutagen. 19, 2-141 (1992).
(15) Simmon, V., Kauhanen, K., and Tardiff, R.G. Mutagenic Activity of Chemicals Identified in Drinking Water. Ed. Scott, D., Bridges, B., and Sobels, F. Progress in Genetic Toxicology (Elsevier, Amsterdam, 1977) pp. 249-258.
(16) Hughes, T.J., Simmons, D.M., Monteith, I.G., and Claxton, L.D. Vaporization Technique to Measure Mutagenic Activity of Volatile Organic Chemicals in the Ames/Salmonella Assay. Environmental Mutagenesis. 9, 421-441 (1987).
(17) Matsushima, T., Matsumoto, A., Shirai, M., Sawamura, M., and Sugimura, T. Mutagenicity of the Naturally Occurring Carcinogen Cycasin and Synthetic Methylazoxy Methane Conjugates in Salmonella Typhimurium. Cancer Research. 39, 3780-3782 (1979).
(18) Tamura, G., Gold, C., Ferro-Luzzi, A., and Ames. B.N. Fecalase: A Model for Activation of Dietary Glycosides to Mutagens by Intestinal Flora. Proc. National Academy of Science. (USA, 1980) 77, 4961-4965.
(19) Wilcox, P., Naidoo, A., Wedd, D. J., and Gatehouse, D. G. Comparison of Salmonella Typhimurium TA 102 With Escherichia Coli WP2 Tester Strains. Mutagenesis. 5, 285-291 (1990).
(20) Matsushima, T., Sawamura, M., Hara, K., and Sugimura, T. A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. Ed. F.J. de Serres et al. In Vitro Metabolic Activation in Mutagenesis Testing. (Elsevier, North Holland, 1976) pp. 85-88.
(21) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M., and Wolf, R.C. Alternatives to Aroclor 1254-Induced S9 in In Vitro Genotoxicity Assays. Mutagenesis. 7, 175-177 (1992).
(22) Maron, D., Katzenellenbogen, J., and Ames, B.N. Compatibility of Organic Solvents With the Salmonella/Microsome Test. Mutation Research. 88, 343-350 (1981).
(23) Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E., and Zeiger, E. Guide for the Salmonella Typhimurium/Mammalian Microsome Tests for Bacterial Mutagenicity. Mutation Research. 189, 83-91 (1987).
(24) Mahon, G.A.T., Green, M.H.L., Middleton, B., Mitchell, I., Robinson, W.D., and Tweats, D.J. Analysis of Data from Microbial Colony Assays. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing Part II. Ed. Kirkland, D.J. Statistical Evaluation of Mutagenicity Test Data (Cambridge University Press, 1989) pp. 28-65.
§ 799.9530   TSCA in vitro mammalian cell gene mutation test.
(a) Scope. This section is intended to meet the testing requirements under section 4 of TSCA. The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. Suitable cell lines include L5178Y mouse lymphoma cells, the CHO, AS52 and V79 lines of Chinese hamster cells, and TK6 human lymphoblastoid cells under paragraph (g)(1) of this section. In these cell lines the most commonly-used genetic endpoints measure mutation at thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HPRT), and a transgene of xanthine-guanine phosphoribosyl transferase (XPRT). The TK, HPRT and XPRT mutation tests detect different spectra of genetic events. The autosomal location of TK and XPRT may allow the detection of genetic events (e.g. large deletions) not detected at the HPRT locus on X-chromosomes (For a discussion see the references in paragraphs (g)(2), (g)(3), (g)(4),(g)(5), and (g)(6) of this section).
(b) Source. The source material used in developing this TSCA test guideline is the OECD guideline 476 (February 1997). This source is available at the address in paragraph (g) of this section.
(c) Definitions. The following definitions apply to this section:
Base pair substitution mutagens are substances which cause substitution of one or several base pairs in the DNA.
Forward mutation is a gene mutation from the parental type to the mutant form which gives rise to an alteration or a loss of the enzymatic activity or the function of the encoded protein.
Frameshift mutagens are substances which cause the addition or deletion of single or multiple base pairs in the DNA molecule.
Mutant frequency is the number of mutant cells observed divided by the number of viable cells.
Phenotypic expression time is a period during which unaltered gene products are depleted from newly mutated cells.
Relative suspension growth is an increase in cell number over the expression period relative to the negative control.
Relative total growth is an increase in cell number over time compared to a control population of cells; calculated as the product of suspension growth relative to the negative control times cloning efficiency relative to negative control.
Survival is the cloning efficiency of the treated cells when plated at the end of the treatment period; survival is usually expressed in relation to the survival of the control cell population.
Viability is the cloning efficiency of the treated cells at the time of plating in selective conditions after the expression period.
(d) Initial considerations. (1) In the in vitro mammalian cell gene mutation test, cultures of established cell lines or cell strains can be used. The cells used are selected on the basis of growth ability in culture and stability of the spontaneous mutation frequency. Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This metabolic activation system cannot mimic entirely the mammalian in vivo conditions. Care should be taken to avoid conditions which would lead to results not reflecting intrinsic mutagenicity. Positive results which do not reflect intrinsic mutagenicity may arise from changes in pH, osmolality or high levels of cytotoxicity.
(2) This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are carcinogens that are not detected by this test because they appear to act through other, non-genotoxic mechanisms or mechanisms absent in bacterial cells.
(e) Test method —(1) Principle. (i) Cells deficient in thymidine kinase (TK) due to the mutation TK = / − -≤TK − / − are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). Thymidine kinase proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not. Similarly, cells deficient in HPRT or XPRT are selected by resistance to 6-thioguanine (TG) or 8-azaguanine (AG). The properties of the test substance should be considered carefully if a base analogue or a compound related to the selective agent is tested in any of the mammalian cell gene mutation tests. For example, any suspected selective toxicity by the test substance for mutant and non-mutant cells should be investigated. Thus, performance of the selection system/agent shall be confirmed when testing chemicals structurally related to the selective agent.
(ii) Cells in suspension or monolayer culture shall be exposed to the test substance, both with and without metabolic activation, for a suitable period of time and subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The treated cultures shall be maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies shall be counted. The mutant frequency is derived from the number of mutant colonies in selective medium and the number of colonies in non-selective medium.
(2) Description —(i) Preparations —(A) Cells. ( 1 ) A variety of cell types are available for use in this test including subclones of L5178Y, CHO, CHO-AS52, V79, or TK6 cells. Cell types used in this test should have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant frequency. Cells should be checked for mycoplasma contamination and should not be used if contaminated.
( 2 ) The test should be designed to have a predetermined sensitivity and power. The number of cells, cultures, and concentrations of test substance used should reflect these defined parameters. The parameters discussed in the reference under paragraph (g)(13) of this section may be used. The minimal number of viable cells surviving treatment and used at each stage in the test should be based on the spontaneous mutation frequency. A general guide is to use a cell number which is at least ten times the inverse of the spontaneous mutation frequency. However, it is recommended to utilize at least 10 6 cells. Adequate historical data on the cell system used should be available to indicate consistent performance of the test.
(B) Media and culture conditions. Appropriate culture media and incubation conditions (culture vessels, temperature, CO 2 concentration and humidity) shall be used. Media should be chosen according to the selective systems and cell type used in the test. It is particularly important that culture conditions should be chosen that ensure optimal growth of cells during the expression period and colony forming ability of both mutant and non-mutant cells.
(C) Preparation of cultures. Cells are propagated from stock cultures, seeded in culture medium and incubated at 37 °C. Prior to use in this test, cultures may need to be cleansed of pre-existing mutant cells.
(D) Metabolic activation. Cells shall be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a co-factor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 or a combination of phenobarbitone and β-naphthoflavone. The post-mitochondrial fraction is usually used at concentrations in the range from 1-10% v/v in the final test medium. The choice and condition of a metabolic activation system may depend upon the class of chemical being tested. In some cases it may be appropriate to utilize more than one concentration of post-mitochondrial fraction. A number of developments, including the construction of genetically engineered cell lines expressing specific activating enzymes, may provide the potential for endogenous activation. The choice of the cell lines used should be scientifically justified (e.g. by the relevance of the cytochrome P450 isoenzyme to the metabolism of the test substance).
(E) Test substance/preparations. Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if appropriate prior to treatment of the cells. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations should be employed unless stability data demonstrate the acceptability of storage.
(ii) Test conditions —(A) Solvent/vehicle. The solvent/vehicle shall not be suspected of chemical reaction with the test substance and shall be compatible with the survival of the cells and the S9 activity. If other than well-known solvent/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents used should be free of water. Water can be removed by adding a molecular sieve.
(B) Exposure concentrations. ( 1 ) Among the criteria to be considered when determining the highest concentration are cytotoxicity and solubility in the test system and changes in pH or osmolality.
( 2 ) Cytotoxicity should be determined with and without metabolic activation in the main experiment using an appropriate indicator of cell integrity and growth, such as relative cloning efficiency (survival) or relative total growth. It may be useful to determine cytotoxicity and solubility in a preliminary experiment.
( 3 ) At least four analyzable concentrations shall be used. Where there is cytotoxicity, these concentrations shall cover a range from the maximum to little or no toxicity; this will usually mean that the concentration levels should be separated by no more than a factor between 2 and √10. If the maximum concentration is based on cytotoxicity then it shall result in approximately 10-20% but not less than 10% relative survival (relative cloning efficiency) or relative total growth. For relatively non-cytotoxic compounds the maximum concentration should be 5 mg/ml, 5 µl/ml, or 0.01 M, whichever is the lowest.
( 4 ) Relatively insoluble substances should be tested up to or beyond their limit of solubility under culture conditions. Evidence of insolubility should be determined in the final treatment medium to which cells are exposed. It may be useful to assess solubility at the beginning and end of the treatment, as solubility can change during the course of exposure in the test system due to presence of cells, S9, serum etc. Insolubility can be detected by using the unaided eye. The precipitate should not interfere with the scoring.
(C) Controls. ( 1 ) Concurrent positive and negative (solvent or vehicle) controls both with and without metabolic activation shall be included in each experiment. When metabolic activation is used the positive control chemical shall be one that requires activation to give a mutagenic response.
( 2 ) Examples of positive control substances include:
( 3 ) Other appropriate positive control reference substances may be used, e.g., if a laboratory has a historical data base on 5-Bromo 2′-deoxyuridine [CAS No. 59-14-3], this reference substance could be used as well. The use of chemical class-related positive control chemicals may be considered, when available.
( 4 ) Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same way as the treatment groups shall be included. In addition, untreated controls should also be used unless there are historical control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent.
(3) Procedure —(i) Treatment with test substance. (A) Proliferating cells shall be exposed to the test substance both with and without metabolic activation. Exposure shall be for a suitable period of time (usually 3 to 6 hrs is effective). Exposure time may be extended over one or more cell cycles.
(B) Either duplicate or single treated cultures may be used at each concentration tested. When single cultures are used, the number of concentrations should be increased to ensure an adequate number of cultures for analysis (e.g. at least eight analyzsable concentrations). Duplicate negative (solvent) control cultures should be used.
(C) Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels. Methods described in the references under paragraphs (g)(20) and (g)(21) of this section may be used.
(ii) Measurement of survival, viability, and mutant frequency. (A) At the end of the exposure period, cells shall be washed and cultured to determine survival and to allow for expression of the mutant phenotype. Measurement of cytotoxicity by determining the relative cloning efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period.
(B) Each locus has a defined minimum time requirement to allow near optimal phenotypic expression of newly induced mutants (HPRT and XPRT require at least 6-8 days, and TK at least 2 days). Cells are grown in medium with and without selective agent(s) for determination of numbers of mutants and cloning efficiency, respectively. The measurement of viability (used to calculate mutant frequency) is initiated at the end of the expression time by plating in non-selective medium.
(C) If the test substance is positive in the L5178Y TK = / − test, colony sizing should be performed on at least one of the test cultures (the highest positive concentration) and on the negative and positive controls. If the test substance is negative in the L5178Y TK = / − test, colony sizing should be performed on the negative and positive controls. In studies using TK6TK = / − , colony sizing may also be performed.
(f) Data and reporting —(1) Treatment of results. (i) Data shall include cytotoxicity and viability determination, colony counts and mutant frequencies for the treated and control cultures. In the case of a positive response in the L5178Y TK = / − test, colonies are scored using the criteria of small and large colonies on at least one concentration of the test substance (highest positive concentration) and on the negative and positive control. The molecular and cytogenetic nature of both large and small colony mutants has been explored in detail and is discussed in the references under paragraphs (g)(22) and (g)(23) of this section. In the TK = / − test, colonies are scored using the criteria of normal growth (large) and slow growth (small) colonies (a scoring system similar to the one described in the reference under paragraph (g)(24) of this section may be used). Mutant cells that have suffered the most extensive genetic damage have prolonged doubling times and thus form small colonies. This damage typically ranges in scale from the losses of the entire gene to karyotypically visible chromosome aberrations. The induction of small colony mutants has been associated with chemicals that induce gross chromosome aberrations. Less seriously affected mutant cells grow at rates similar to the parental cells and form large colonies.
(ii) Survival (relative cloning efficiencies) or relative total growth shall be given. Mutant frequency shall be expressed as number of mutant cells per number of surviving cells.
(iii) Individual culture data shall be provided. Additionally, all data shall be summarized in tabular form.
(iv) There is no requirement for verification of a clear positive response. Equivocal results shall be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments for either equivocal or negative results. Study parameters that might be modified include the concentration spacing, and the metabolic activation conditions.
(2) Evaluation and interpretation of results. (i) There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response.
(ii) A test substance, for which the results do not meet the criteria described in paragraph (f)(2)(i) of this section is considered non-mutagenic in this system.
(iii) Although most studies will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.
(iv) Positive results for an in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells used. A positive concentration-response that is reproducible is most meaningful. Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.
(3) Test report. The test report shall include the following information:
(i) Test substance:
(A) Identification data and CAS no., if known.
(B) Physical nature and purity.
(C) Physicochemical properties relevant to the conduct of the study.
(D) Stability of the test substance.
(ii) Solvent/vehicle:
(A) Justification for choice of vehicle/solvent.
(B) Solubility and stability of the test substance in solvent/vehicle, if known.
(iii) Cells:
(A) Type and source of cells.
(B) Number of cell cultures.
(C) Number of cell passages, if applicable.
(D) Methods for maintenance of cell cultures, if applicable.
(E) Absence of mycoplasma.
(iv) Test conditions:
(A) Rationale for selection of concentrations and number of cell cultures including e.g., cytotoxicity data and solubility limitations, if available.
(B) Composition of media, CO 2 concentration.
(C) Concentration of test substance.
(D) Volume of vehicle and test substance added.
(E) Incubation temperature.
(F) Incubation time.
(G) Duration of treatment.
(H) Cell density during treatment.
(I) Type and composition of metabolic activation system including acceptability criteria.
(J) Positive and negative controls.
(K) Length of expression period (including number of cells seeded, and subcultures and feeding schedules, if appropriate).
(L) Selective agent(s).
(M) Criteria for considering tests as positive, negative or equivocal.
(N) Methods used to enumerate numbers of viable and mutant cells.
(O) Definition of colonies of which size and type are considered (including criteria for “small” and “large” colonies, as appropriate).
(v) Results:
(A) Signs of toxicity.
(B) Signs of precipitation.
(C) Data on pH and osmolality during the exposure to the test substance, if determined.
(D) Colony size if scored for at least negative and positive controls.
(E) Laboratory's adequacy to detect small colony mutants with the L5178Y TK = / − system, where appropriate.
(F) Dose-response relationship, where possible.
(G) Statistical analyses, if any.
(H) Concurrent negative (solvent/vehicle) and positive control data.
(I) Historical negative (solvent/vehicle) and positive control data with ranges, means, and standard deviations.
(J) Mutant frequency.
(vi) Discussion of the results.
(vii) Conclusion.
(g) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Chu, E.H.Y. and Malling, H.V. Mammalian Cell Genetics. II. Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells In Vitro, Proc. National Academy Science (USA, 1968) 61, 1306-1312.
(2) Liber, H.L. and Thilly, W.G. Mutation Assay at the Thymidine Kinase Locus in Diploid Human Lymphoblasts. Mutation Research. 94, 467-485 (1982).
(3) Moore, M.M., Harrington-Brock, K., Doerr, C.L., and Dearfield, K.L. Differential Mutant Quantitation at the Mouse Lymphoma TK and CHO HGPRT Loci. Mutagenesis. 4, 394-403 (1989).
(4) Aaron, C.S. and Stankowski, Jr., L.F. Comparison of the AS52/XPRT and the CHO/HPRT Assays: Evaluation of Six Drug Candidates. Mutation Research. 223, 121-128 (1989).
(5) Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y., Stankowski, L., Theiss, J., and Thompson, E. Mammalian Cell Gene Mutation Assays Working Group Report. Report of the International Workshop on Standardization of Genotoxicity Test Procedures. Mutation Research. 312, 235-239 (1994).
(6) Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M., Brusick, D., Ashby, J., and Myhr, B.C. Genotoxicity Under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutation Research. 257, 147-204 (1991).
(7) Clive, D., McCuen, R., Spector, J.F.S., Piper, C., and Mavournin, K.H. Specific Gene Mutations in L5178Y Cells in Culture. A Report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research. 115, 225-251 (1983).
(8) Li, A.P., Gupta, R.S., Heflich, R.H., and Wasson, J. S. A Review and Analysis of the Chinese Hamster Ovary/Hypoxanthine Guanine Phosphoribosyl Transferase System to Determine the Mutagenicity of Chemical Agents: A Report of Phase III of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research. 196, 17-36 (1988).
(9) Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., Loveday, K.S., O'Neill, J.P., Riddle, J.C., Stankowski, Jr., L.F., and Yang, L.L. A Guide for the Performance of the Chinese Hamster Ovary Cell/Hypoxanthine-Guanine Phosphoribosyl Transferase Gene Mutation Assay. Mutation Research. 189, 135-141 (1987).
(10) Liber, H.L., Yandell, D.W., and Little, J.B. A Comparison of Mutation Induction at the tk and hprt Loci in Human Lymphoblastoid Cells; Quantitative Differences are Due to an Additional Class of Mutations at the Autosomal TK Locus. Mutation Research. 216, 9-17 (1989).
(11) Stankowski, L.F. Jr., Tindall, K.R., and Hsie, A.W. Quantitative and Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-Induced Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-Induced Mutation in AS52 Cells. Mutation Reseach. 160, 133-147 (1986).
(12) Turner, N.T., Batson, A.G., and Clive, D. Eds. Kilbey, B.J. et al. Procedures for the L5178Y/TK = / − >TK = / − Mouse Lymphoma Cell Mutagenicity Assay. Handbook of Mutagenicity Test Procedures (Elsevier Science Publishers, New York, 1984) pp. 239-268.
(13) Arlett, C.F., Smith, D.M., Clarke, G.M., Green, M.H.L., Cole, J., McGregor, D.B., and Asquith, J.C. Ed. Kirkland, D.J. Mammalian Cell Gene Mutation Assays Based Upon Colony Formation. Statistical Evaluation of Mutagenicity Test Data (Cambridge University Press, 1989) pp. 66-101.
(14) Abbondandolo, A., Bonatti, S., Corti, G., Fiorio, R., Loprieno, N., and Mazzaccaro, A. Induction of 6-Thioguanine-Resistant Mutants in V79 Chinese Hamster Cells by Mouse-Liver Microsome-Activated Dimethylnitrosamine. Mutation Research. 46, 365-373 (1977).
(15) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Reseach. 31, 347-364 (1975).
(16) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., and Brown M.M.M. Validation and Characterization of the L5178Y/TK = / − Mouse Lymphoma Mutagen Assay System. Mutation Reseach. 59, 61-108 (1979).
(17) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutation Reseach. 113, 173, 215 (1983).
(18) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M., and Wolf, R.C. Alternatives to Aroclor 1254-Induced S9 in In Vitro Genotoxicity Assays. Mutagenesis. 7, 175-177 (1992).
(19) Matsushima, T., Sawamura, M., Hara, K., and Sugimura, T. A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. (Eds.) de Serres, F.J., Fouts, J.R., Bend, J.R., and Philpot, R.M. In Vitro Metabolic Activation in Mutagenesis Testing (Elsevier, North-Holland, 1976) pp. 85-88.
(20) Krahn, D.F., Barsky, F.C., and McCooey, K.T. Eds. Tice, R.R., Costa, D.L., and Schaich, K.M. CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids. Genotoxic Effects of Airborne Agents (New York, Plenum, 1982) pp. 91-103.
(21) Zamora, P.O., Benson, J.M., Li, A.P., and Brooks, A.L. Evaluation of an Exposure System Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay. Environmental Mutagenesis. 5, 795-801 (1983).
(22) Applegate, M.L., Moore, M.M., Broder, C.B., Burrell, A., and Hozier, J.C. Molecular Dissection of Mutations at the Heterozygous Thymidine Kinase Locus in Mouse Lymphoma Cells. Proc. National Academy Science (USA, 1990) 87, 51-55.
(23) Moore, M.M., Clive, D., Hozier, J.C., Howard, B.E., Batson, A.G., Turner, N.T., and Sawyer, J. Analysis of Trifluorothymidine-Resistant (TFT r ) Mutants of L5178Y/TK = / − Mouse Lymphoma Cells. Mutation Research. 151, 161-174 (1985).
(24) Yandell, D.W., Dryja, T.P., and Little J.B. Molecular Genetic Analysis of Recessive Mutations at a Heterozygous Autosomal Locus in Human Cells. Mutation Research. 229, 89-102 (1990).
(25) Moore, M.M. and Doerr, C.L. Comparison of Chromosome Aberration Frequency and Small-Colony TK-Deficient Mutant Frequency in L5178Y/TK = / − 3.7.2C Mouse Lymphoma Cells. Mutagenesis. 5, 609-614 (1990).
§ 799.9537   TSCA in vitro mammalian chromosome aberration test.
(a) Scope —(1) Applicability. This section is intended to meet testing requirements under section 4 of the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this TSCA test guideline is the Office of Prevention, Pesticides, and Toxic Substances (OPPTS) harmonized test guideline 870.5375 (August 1998, final guidelines). The source is available at the address in paragraph (i) of this section.
(b) Purpose. (1) The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells (see paragraphs (i)(1), (i)(2), and (i)(3) of this section). Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome-type aberrations also occur. An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations. However, this guideline is not designed to measure numerical aberrations and is not routinely used for that purpose. Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations and related events causing alterations in oncogenes and tumour-suppressor genes of somatic cells are involved in cancer induction in humans and experimental animals.
(2) The in vitro chromosome aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. The cells used are selected on the basis of growth ability in culture, stability of the karyotype, chromosome number, chromosome diversity, and spontaneous frequency of chromosome aberrations.
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR Part 792—Good Laboratory Practice Standards apply to this test guideline. The following definitions also apply to this test guideline.
Chromatid-type aberration is structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids.
Chromosome-type aberration is structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.
Endoreduplication is a process in which after an S period of DNA replication, the nucleus does not go into mitosis but starts another S period. The result is chromosomes with 4, 8, 16,...chromatids.
Gap is an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the chromatid(s).
Mitotic index is the ratio of cells in metaphase divided by the total number of cells observed in a population of cells; an indication of the degree of proliferation of that population.
Numerical aberration is a change in the number of chromosomes from the normal number characteristic of the cells utilized.
Polyploidy is a multiple of the haploid chromosome number (n) other than the diploid number (i.e., 3n, 4n, and so on).
Structural aberration is a change in chromosome structure detectable by microscopic examination of the metaphase stage of cell division, observed as deletions and fragments, intrachanges, and interchanges.
(d) Initial considerations. (1) Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This metabolic activation system cannot mimic entirely the mammalian in vivo conditions. Care should be taken to avoid conditions which would lead to positive results which do not reflect intrinsic mutagenicity and may arise from changes in pH, osmolality, or high levels of cytotoxicity (the test techniques described in the references under paragraphs (i)(4) and (i)(5) of this section may be used).
(2) This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are carcinogens that are not detected by this test because they appear to act through mechanisms other than direct DNA damage.
(e) Principle of the test method. Cell cultures are exposed to the test substance both with and without metabolic activation. At predetermined intervals after exposure of cell cultures to the test substance, they are treated with a metaphase-arresting substance (e.g., Colcemid ® or colchicine), harvested, stained, and metaphase cells are analysed microscopically for the presence of chromosome aberrations.
(f) Description of the method —(1) Preparations —(i) Cells. A variety of cell lines, strains, or primary cell cultures, including human cells, may be used (e.g., Chinese hamster fibroblasts, human, or other mammalian peripheral blood lymphocytes).
(ii) Media and culture conditions. Appropriate culture media, and incubation conditions (culture vessels, CO 2 concentration, temperature and humidity) must be used in maintaining cultures. Established cell lines and strains must be checked routinely for stability in the modal chromosome number and the absence of Mycoplasma contamination and should not be used if contaminated. The normal cell-cycle time for the cells and culture conditions used should be known.
(iii) Preparation of cultures —(A) Established cell lines and strains. Cells are propagated from stock cultures, seeded in culture medium at a density such that the cultures will not reach confluency before the time of harvest, and incubated at 37 °C.
(B) Lymphocytes. Whole blood treated with an anti-coagulant (e.g., heparin) or separated lymphocytes obtained from healthy subjects are added to culture medium containing a mitogen (e.g., phytohemagglutinin) and incubated at 37 °C.
(iv) Metabolic activation. Cells must be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a co-factor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (the test techniques described in the references under paragraphs (i)(6), (i)(7), (8)(i), and (i)(9) of this section may be used), or a mixture of phenobarbitone and β-naphthoflavone (the test techniques described in the references under paragraphs (i)(10), (i)(11), and (i)(12) of this section may be used). The post-mitochondrial fraction is usually used at concentrations in the range from 1-10% v/v in the final test medium. The condition of a metabolic activation system may depend upon the class of chemical being tested. In some cases, it may be appropriate to utilize more than one concentration of post-mitochondrial fraction. A number of developments, including the construction of genetically engineered cell lines expressing specific activating enzymes, may provide the potential for endogenous activation. The choice of the cell lines used should be scientifically justified (e.g., by the relevance of the cytochrome P450 isoenzyme for the metabolism of the test substance).
(v) Test substance/preparation. Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted, if appropriate, prior to treatment of the cells. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.
(2) Test conditions —(i) Solvent/vehicle. The solvent/vehicle should not be suspected of chemical reaction with the test substance and must be compatible with the survival of the cells and the S9 activity. If other than well-known solvent/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents used should be free of water. Water can be removed by adding a molecular sieve.
(ii) Exposure concentrations. (A) Among the criteria to be considered when determining the highest concentration are cytotoxicity, solubility in the test system, and changes in pH or osmolality.
(B) Cytotoxicity should be determined with and without metabolic activation in the main experiment using an appropriate indication of cell integrity and growth, such as degree of confluency, viable cell counts, or mitotic index. It may be useful to determine cytotoxicity and solubility in a preliminary experiment.
(C) At least three analyzable concentrations should be used. Where cytotoxicity occurs, these concentrations should cover a range from the maximum to little or no toxicity; this will usually mean that the concentrations should be separated by no more than a factor between 2 and √10. At the time of harvesting, the highest concentration should show a significant reduction in degree of confluency, cell count or mitotic index, (all greater than 50%). The mitotic index is only an indirect measure of cytotoxic/cytostatic effects and depends on the time after treatment. However, the mitotic index is acceptable for suspension cultures in which other toxicity measurements may be cumbersome and impractical. Information on cell-cycle kinetics, such as average generation time (AGT), could be used as supplementary information. AGT, however, is an overall average that does not always reveal the existence of delayed subpopulations, and even slight increases in average generation time can be associated with very substantial delay in the time of optimal yield of aberrations. For relatively non-cytotoxic compounds the maximum concentration should be 5 µg/ml, 5mg/ml, or 0.01M, whichever is the lowest.
(D) For relatively insoluble substances that are not toxic at concentrations lower than the insoluble concentration, the highest dose used should be a concentration above the limit of solubility in the final culture medium at the end of the treatment period. In some cases (e.g., when toxicity occurs only at higher than the lowest insoluble concentration) it is advisable to test at more than one concentration with visible precipitation. It may be useful to assess solubility at the beginning and the end of the treatment, as solubility can change during the course of exposure in the test system due to presence of cells, S9, serum etc. Insolubility can be detected by using the unaided eye. The precipitate should not interfere with the scoring.
(iii) Controls. (A) Concurrent positive and negative (solvent or vehicle) controls both with and without metabolic activation must be included in each experiment. When metabolic activation is used, the positive control chemical must be the one that requires activation to give a mutagenic response.
(B) Positive controls must employ a known clastogen at exposure levels expected to give a reproducible and detectable increase over background which demonstrates the sensitivity of the test system. Positive control concentrations should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. Examples of positive-control substances include:
(C) Other appropriate positive control substances may be used. The use of chemical class-related positive-control chemicals may be considered, when available.
(D) Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same way as the treatment cultures, must be included for every harvest time. In addition, untreated controls should also be used unless there are historical-control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent.
(g) Procedure —(1) Treatment with test substance. (i) Proliferating cells are treated with the test substance in the presence and absence of a metabolic-activation system. Treatment of lymphocytes should commence at about 48 hours after mitogenic stimulation.
(ii) Duplicate cultures must be used at each concentration, and are strongly recommended for negative/solvent control cultures. Where minimal variation between duplicate cultures can be demonstrated (the test techniques described in the references under paragraphs (i)(13) and (i)(14) of this section may be used), from historical data, it may be acceptable for single cultures to be used at each concentration.
(iii) Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels (the test techniques described in the references under paragraphs (i)(15) and (i)(16) of this section may be used).
(2) Culture harvest time. In the first experiment, cells should be exposed to the test substance both with and without metabolic activation for 3-6 hours, and sampled at a time equivalent to about 1.5 normal cell-cycle length after the beginning of treatment (the test techniques described in the references under paragraph (i)(12) of this section may be used). If this protocol gives negative results both with and without activation, an additional experiment without activation should be done, with continuous treatment until sampling at a time equivalent to about 1.5 normal cell-cycle lengths. Certain chemicals may be more readily detected by treatment/sampling times longer than 1.5 cycle lengths. Negative results with metabolic activation need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided.
(3) Chromosome preparation. Cell cultures must be treated with Colcemid ® or colchicine usually for 1 to 3 hours prior to harvesting. Each cell culture must be harvested and processed separately for the preparation of chromosomes. Chromosome preparation involves hypotonic treatment of the cells, fixation and staining.
(4) Analysis. (i) All slides, including those of positive and negative controls, must be independently coded before microscopic analysis. Since fixation procedures often result in the breakage of a proportion of metaphase cells with loss of chromosomes, the cells scored must therefore contain a number of centromeres equal to the modal number ±2 for all cell types. At least 200 well-spread metaphases should be scored per concentration and control equally divided amongst the duplicates, if applicable. This number can be reduced when high numbers of aberrations are observed.
(ii) Though the purpose of the test is to detect structural chromosome aberrations, it is important to record polyploidy and endoreduplication when these events are seen.
(h) Data and reporting —(1) Treatment of results. (i) The experimental unit is the cell, and therefore the percentage of cells with structural chromosome aberration(s) should be evaluated. Different types of structural chromosome aberrations must be listed with their numbers and frequencies for experimental and control cultures. Gaps are recorded separately and reported but generally not included in the total aberration frequency.
(ii) Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment(s) should also be recorded.
(iii) Individual culture data should be provided. Additionally, all data should be summarized in tabular form.
(iv) There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using modification of experimental conditions. The need to confirm negative results has been discussed in paragraph (g)(2) of this section. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing and the metabolic activation conditions.
(2) Evaluation and interpretation of results. (i) There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (see paragraphs (i)(3) and (i)(13) of this section). Statistical significance should not be the only determining factor for a positive response.
(ii) An increase in the number of polyploid cells may indicate that the test substance has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test substance has the potential to inhibit cell-cycle progression (the test techniques described in the references under paragraphs (i)(17) and (i)(18) of this section may be used).
(iii) A test substance for which the results do not meet the criteria in paragraphs (h)(2)(i) and (h)(2)(ii) of this section is considered nonmutagenic in this system.
(iv) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.
(v) Positive results from the in vitro chromosome aberration test indicate that the test substance induces structural chromosome aberrations in cultured mammalian somatic cells. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aberrations in cultured mammalian somatic cells.
(3) Test report. The test report must include the following information.
(i) Test substance.
(A) Identification data and CAS no., if known.
(B) Physical nature and purity.
(C) Physicochemical properties relevant to the conduct of the study.
(D) Stability of the test substance, if known.
(ii) Solvent/vehicle.
(A) Justification for choice of solvent/vehicle.
(B) Solubility and stability of the test substance in solvent/vehicle, if known.
(iii) Cells.
(A) Type and source of cells.
(B) Karyotype features and suitability of the cell type used.
(C) Absence of Mycoplasma, if applicable.
(D) Information on cell-cycle length.
(E) Sex of blood donors, whole blood or separated lymphocytes, mitogen used.
(F) Number of passages, if applicable.
(G) Methods for maintenance of cell cultures if applicable.
(H) Modal number of chromosomes.
(iv) Test conditions.
(A) Identity of metaphase arresting substance, its concentration and duration of cell exposure.
(B) Rationale for selection of concentrations and number of cultures including, e.g., cytotoxicity data and solubility limitations, if available.
(C) Composition of media, CO 2 concentration if applicable.
(D) Concentration of test substance.
(E) Volume of vehicle and test substance added.
(F) Incubation temperature.
(G) Incubation time.
(H) Duration of treatment.
(I) Cell density at seeding, if appropriate.
(J) Type and composition of metabolic activation system, including acceptability criteria.
(K) Positive and negative controls.
(L) Methods of slide preparation.
(M) Criteria for scoring aberrations.
(N) Number of metaphases analyzed.
(O) Methods for the measurements of toxicity.
(P) Criteria for considering studies as positive, negative or equivocal.
(v) Results.
(A) Signs of toxicity, e.g., degree of confluency, cell-cycle data, cell counts, mitotic index.
(B) Signs of precipitation.
(C) Data on pH and osmolality of the treatment medium, if determined.
(D) Definition for aberrations, including gaps.
(E) Number of cells with chromosome aberrations and type of chromosome aberrations given separately for each treated and control culture.
(F) Changes in ploidy if seen.
(G) Dose-response relationship, where possible.
(H) Statistical analyses, if any.
(I) Concurrent negative (solvent/vehicle) and positive control data.
(J) Historical negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations.
(vi) Discussion of the results.
(vii) Conclusion.
(i) References. For additional background information on this test guideline, the following references should be consulte. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Evans, H.J. Cytological Methods for Detecting Chemical Mutagens. Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. Ed. Plenum Press, New York and London, pp. 1-29 (1976).
(2) Ishidate, M. Jr. and Sofuni, T. The In Vitro Chromosomal Aberration Test Using Chinese Hamster Lung (CHL) Fibroblast Cells in Culture. Progress in Mutation Research, Vol. 5, Ashby, J. et al., Eds. Elsevier Science Publishers, Amsterdam-New York-Oxford, pp. 427-432 (1985).
(3) Galloway, S.M. et al. Chromosome aberration and sister chromatid exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals. Environmental and Molecular Mutagenesis 10 (suppl. 10), 1-175 (1987).
(4) Scott, D. et al. Genotoxicity under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutation Research 257, 147-204 (1991).
(5) Morita, T. et al. Clastogenicity of Low pH toVarious Cultured Mammalian Cells. Mutation Research 268, 297-305 (1992).
(6) Ames, B.N., McCann, J. and Yamasaki, E. Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian Microsome Mutagenicity Test. Mutation Research 31, 347-364 (1975).
(7) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research 113, 173-215 (1983).
(8) Natarajan, A.T. et al. Cytogenetic Effects of Mutagens/Carcinogens after Activation in a Microsomal System In Vitro, I. Induction of Chromosome Aberrations and Sister Chromatid Exchanges by Diethylnitrosamine (DEN) and Dimethylnitrosamine (DMN) in CHO Cells in the Presence of Rat-Liver Microsomes. Mutation Research 37, 83-90 (1976).
(9) Matsuoka, A., Hayashi, M. and Ishidate, M., Jr. Chromosomal Aberration Tests on 29 Chemicals Combined with S9 Mix In Vitro. Mutation Research 66, 277-290 (1979).
(10) Elliot, B.M. et al. Report of UK Environmental Mutagen Society Working Party. Alternatives to Aroclor 1254-induced S9 in In Vitro Genotoxicity Assays. Mutagenesis 7, 175-177 (1992).
(11) Matsushima, T. et al. A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. de Serres, F.J., Fouts, J.R., Bend, J.R. and Philpot, R.M. Eds. In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier, North-Holland, pp. 85-88 (1976).
(12) Galloway, S.M. et al. Report from Working Group on In Vitro Tests for Chromosomal Aberrations. Mutation Research 312, 241-261 (1994).
(13) Richardson, C. et al. Analysis of Data from In Vitro Cytogenetic Assays. Statistical Evaluation of Mutagenicity Test Data. Kirkland, D.J., Ed. Cambridge University Press, Cambridge, pp. 141-154 (1989).
(14) Soper, K.A. and Galloway S.M. Replicate Flasks are not Necessary for In Vitro Chromosome Aberration Assays in CHO Cells. Mutation Research 312, 139-149 (1994).
(15) Krahn, D.F., Barsky, F.C. and McCooey, K.T. CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids. Tice, R.R., Costa, D.L., Schaich, K.M. Eds. Genotoxic Effects of Airborne Agents. New York, Plenum, pp. 91-103 (1982).
(16) Zamora, P.O. et al. Evaluation of an Exposure System Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay. Environmental Mutagenesis 5, 795-801 (1983).
(17) Locke-Huhle, C. Endoreduplication in Chinese hamster cells during alpha-radiation induced G2 arrest. Mutation Research 119, 403-413 (1983).
(18) Huang, Y., Change, C. and Trosko, J.E. Aphidicolin—induced endoreduplication in Chinese hamster cells. Cancer Research 43, 1362-1364 (1983).
§ 799.9538   TSCA mammalian bone marrow chromosomal aberration test.
(a) Scope. This section is intended to meet the testing requirements under section 4 of TSCA. The mammalian bone marrow chromosomal aberration test is used for the detection of structural chromosome aberrations induced by test compounds in bone marrow cells of animals, usually rodents. Structural chromosome aberrations may be of two types, chromosome or chromatid. An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations. With the majority of chemical mutagens, induced aberrations are of the chromatid-type, but chromosome-type aberrations also occur. Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations and related events causing alterations in oncogenes and tumor suppressor genes are involved in cancer in humans and experimental systems.
(b) Source. The source material used in developing this TSCA test guideline is the OECD guideline 475 (February 1997). This source is available at the address in paragraph (g) of this section.
(c) Definitions. The following definitions apply to this section:
Chromatid-type aberration is structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids.
Chromosome-type aberration is structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.
Endoreduplication is a process in which after an S period of DNA replication, the nucleus does not go into mitosis but starts another S period. The result is chromosomes with 2,4,8,...chromatids.
Gap is an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the chromatids.
Numerical aberration is a change in the number of chromosomes from the normal number characteristic of the animals utilized.
Polyploidy is a multiple of the haploid chromosome number (n) other than the diploid number (i.e., 3n, 4n and so on).
Structural aberration is a change in chromosome structure detectable by microscopic examination of the metaphase stage of cell division, observed as deletions and fragments, intrachanges or interchanges.
(d) Initial considerations. (1) Rodents are routinely used in this test. Bone marrow is the target tissue in this test, since it is a highly vascularised tissue, and it contains a population of rapidly cycling cells that can be readily isolated and processed. Other species and target tissues are not the subject of this section.
(2) This chromosome aberration test is especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes although these may vary among species and among tissues. An in vivo test is also useful for further investigation of a mutagenic effect detected by an in vitro test.
(3) If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test.
(e) Test method —(1) Principle. Animals are exposed to the test substance by an appropriate route of exposure and are sacrificed at appropriate times after treatment. Prior to sacrifice, animals are treated with a metaphase-arresting agent (e.g., colchicine or Colcemid ®). Chromosome preparations are then made from the bone marrow cells and stained, and metaphase cells are analyzed for chromosome aberrations.
(2) Description —(i) Preparations —(A) Selection of animal species. Rats, mice and Chinese hamsters are commonly used, although any appropriate mammalian species may be used. Commonly used laboratory strains of young healthy adult animals should be employed. At the commencement of the study, the weight variation of animals should be minimal and not exceed ±20% of the mean weight of each sex.
(B) Housing and feeding conditions. The temperature in the experimental animal room should be 22 °C ±3 °C). Although the relative humidity should be at least 30% and preferably not exceed 70% other than during room cleaning, the aim should be 50-60%. Lighting should be artificial, the sequence being 12 hrs light, 12 hrs dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this method. Animals may be housed individually, or be caged in small groups of the same sex.
(C) Preparation of the animals. Healthy young adult animals shall be randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimized. The animals are identified uniquely. The animals are acclimated to the laboratory conditions for at least 5 days.
(D) Preparation of doses. Solid test substances shall be dissolved or suspended in appropriate solvents or vehicles and diluted, as appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted prior to dosing. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.
(ii) Test conditions —(A) Solvent/vehicle. The solvent/vehicle shall not produce toxic effects at the dose levels used, and shall not be suspected of chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported with data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle should be considered first.
(B) Controls. ( 1 ) Concurrent positive and negative (solvent/vehicle) controls shall be included for each sex in each test. Except for treatment with the test substance, animals in the control groups should be handled in an identical manner to the animals in the treated groups.
( 2 ) Positive controls shall produce structural chromosome aberrations in vivo at exposure levels expected to give a detectable increase over background. Positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. It is acceptable that the positive control be administered by a route different from the test substance and sampled at only a single time. The use of chemical class related positive control chemicals may be considered, when available. Examples of positive control substances include:
( 3 ) Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same way as the treatment groups, shall be included for every sampling time, unless acceptable inter-animal variability and frequencies of cells with chromosome aberrations are available from historical control data. If single sampling is applied for negative controls, the most appropriate time is the first sampling time. In the absence of historical or published control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent/vehicle, untreated animals should be used.
(3) Procedure —(i) Number and sex of animals. Each treated and control group shall include at least 5 analyzable animals per sex. If at the time of the study there are data available from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences in toxicity between sexes, then testing in a single sex will be sufficient. Where human exposure to chemicals may be sex-specific, as for example with some pharmaceutical agents, the test should be performed with animals of the appropriate sex.
(ii) Treatment schedule. (A) Test substances are preferably administered as a single treatment. Test substances may also be administered as a split dose, i.e. two treatments on the same day separated by no more than a few hrs, to facilitate administering a large volume of material. Other dose regimens should be scientifically justified.
(B) Samples shall be taken at two separate times following treatment on one day. For rodents, the first sampling interval is 1.5 normal cell cycle length (the latter being normally 12-18 hr) following treatment. Since the time required for uptake and metabolism of the test substance as well as its effect on cell cycle kinetics can affect the optimum time for chromosome aberration detection, a later sample collection 24 hr after the first sample time is recommended. If dose regimens of more than one day are used, one sampling time at 1.5 normal cell cycle lengths after the final treatment should be used.
(C) Prior to sacrifice, animals shall be injected intraperitoneally with an appropriate dose of a metaphase arresting agent (e.g. Colcemid ® or colchicine). Animals are sampled at an appropriate interval thereafter. For mice this interval is approximately 3-5 hrs; for Chinese hamsters this interval is approximately 4-5 hrs. Cells shall be harvested from the bone marrow and analyzed from chromosome aberrations.
(iii) Dose levels. If a range finding study is performed because there are no suitable data available, it shall be performed in the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study (an approach to dose selection is presented in the reference under paragraph (g)(5) of this section). If there is toxicity, three dose levels shall be used for the first sampling time. These dose levels shall cover a range from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some indication of toxicity in the bone marrow (e.g. greater than 50% reduction in mitotic index).
(iv) Limit test. If a test at one dose level of at least 2,000 mg/kg body weight using a single treatment, or as two treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected based on data from structurally related compounds, then a full study using three dose levels may not be considered necessary. For studies of a longer duration, the limit dose is 2,000 mg/kg/body weight/day for treatment up to 14 days, and 1,000 mg/kg/body weight/day for treatment longer than 14 days. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.
(v) Administration of doses. The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be administered by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 ml/100g body weight. The use of volumes higher than these must be justified. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimized by adjusting the concentration to ensure a constant volume at all dose levels.
(vi) Chromosome preparation. Immediately after sacrifice, bone marrow shall be obtained, exposed to hypotonic solution and fixed. The cells shall be then spread on slides and stained.
(vii) Analysis. (A) The mitotic index should be determined as a measure of cytotoxicity in at least 1,000 cells per animal for all treated animals (including positive controls) and untreated negative control animals.
(B) At least 100 cells should be analyzed for each animal. This number could be reduced when high numbers of aberrations are observed. All slides, including those of positive and negative controls, shall be independently coded before microscopic analysis. Since slide preparation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, the cells scored should therefore contain a number of centromeres equal to the number 2n ±2.
(f) Data and reporting —(1) Treatment of results. Individual animal data shall be presented in tabular form. The experimental unit is the animal. For each animal the number of cells scored, the number of aberrations per cell and the percentage of cells with structural chromosome aberration(s) shall be evaluated. Different types of structural chromosome aberrations shall be listed with their numbers and frequencies for treated and control groups. Gaps shall be recorded separately and reported but generally not included in the total aberration frequency. If there is no evidence for a difference in response between the sexes, the data may be combined for statistical analysis.
(2) Evaluation and interpretation of results. (i) There are several criteria for determining a positive result, such as a dose-related increase in the relative number of cells with chromosome aberrations or a clear increase in the number of cells with aberrations in a single dose group at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (some statistical methods are described in the reference under paragraph (g)(6) of this section). Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.
(ii) An increase in polyploidy may indicate that the test substance has the potential to induce numerical chromosome aberrations. An increase in endoreduplication may indicate that the test substance has the potential to inhibit cell cycle progression. This phenomenon is described in the references under paragraphs (g)(7) and (g)(8) of this section.
(iii) A test substance for which the results do not meet the criteria described in paragraph (f)(2)(i) of this section is considered non-mutagenic in this test.
(iv) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgment about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of experiments performed.
(v) Positive results from the in vivo chromosome aberration test indicate that a substance induces chromosome aberrations in the bone marrow of the species tested. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aberrations in the bone marrow of the species tested.
(vi) The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g., systemic toxicity) should be discussed.
(3) Test report. The test report shall include the following information:
(i) Test substance:
(A) Identification data and CAS No., if known.
(B) Physical nature and purity.
(C) Physicochemical properties relevant to the conduct of the study.
(D) Stability of the test substance, if known.
(ii) Solvent/vehicle:
(A) Justification for choice of vehicle.
(B) Solubility and stability of the test substance in solvent/vehicle, if known.
(iii) Test animals:
(A) Species/strain used.
(B) Number, age and sex of animals.
(C) Source, housing conditions, diet, etc.
(D) Individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group.
(iv) Test conditions:
(A) Positive and negative (vehicle/solvent) controls.
(B) Data from range-finding study, if conducted.
(C) Rationale for dose level selection.
(D) Details of test substance preparation.
(E) Details of the administration of the test substance.
(F) Rationale for route of administration.
(G) Methods for verifying that the test substance reached the general circulation or target tissue, if applicable.
(H) Conversion from diet/drinking water test substance concentration parts per million (ppm) to the actual dose (mg/kg body weight/day), if applicable.
(I) Details of food and water quality.
(J) Detailed description of treatment and sampling schedules.
(K) Methods for measurement of toxicity.
(L) Identity of metaphase arresting substance, its concentration and duration of treatment.
(M) Methods of slide preparation.
(N) Criteria for scoring aberrations.
(O) Number of cells analyzed per animal.
(P) Criteria for considering studies as positive, negative or equivocal.
(v) Results:
(A) Signs of toxicity.
(B) Mitotic index.
(C) Type and number of aberrations, given separately for each animal.
(D) Total number of aberrations per group with means and standard deviations.
(E) Number of cells with aberrations per group with means and standard deviations.
(F) Changes in ploidy, if seen.
(G) Dose-response relationship, where possible.
(H) Statistical analyses, if any.
(I) Concurrent negative control data.
(J) Historical negative control data with ranges, means and standard deviations.
(K) Concurrent positive control data.
(vi) Discussion of the results.
(vii) Conclusion.
(g) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Adler, I.D. Eds. S. Venitt and J.M. Parry. Cytogenetic Tests in Mammals. Mutagenicity Testing: A Practical Approach. (IRL Press, Oxford, Washington DC, 1984) pp. 275-306.
(2) Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, A.F., and Shelby, M. Mammalian In Vivo Cytogenetic Assays: Analysis of Chromosome Aberrations in Bone Marrow Cells. Mutation Research. 189, 157-165 (1987).
(3) Richold, M., Chandley, A., Ashby, J., Gatehouse, D.G., Bootman, J., and Henderson, L. Ed. D.J. Kirkland. In Vivo Cytogenetic Assays. Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report. Part I revised. (Cambridge University Press, Cambridge, NY, Port Chester, Melbourne, Sydney, 1990) pp. 115-141.
(4) Tice, R.R., Hayashi, M., MacGregor, J.T., Anderson, D., Blakey, D.H., Holden, H.E., Kirsch-Volders, M., Oleson Jr., F.B., Pacchierotti, F., Preston, R.J., Romagna, F., Shimada, H., Sutou, S., and Vannier, B. Report from the Working Group on the In Vivo Mammalian Bone Marrow Chromosomal Aberration Test. Mutation Research. 312, 305-312 (1994).
(5) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D. J., and Richold, M. Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose Setting in In Vivo Mutagenicity Assays. Mutagenesis. 7, 313-319 (1992).
(6) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, J.R.K. Ed. Kirkland,D. J. Statistical Analysis of In Vivo Cytogenetic Assays. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report Part III. Statistical Evaluation of Mutagenicity Test Data (Cambridge University Press, Cambridge, 1989) pp. 184-232.
(7) Locke-Huhle, C. Endoreduplication in Chinese Hamster Cells During Alpha-Radiation Induced G2 Arrest. Mutation Research. 119, 403-413 (1983).
(8) Huang, Y., Change, C., and Trosko, J. E. Aphidicolin-Induced Endoreduplication in Chinese Hamster Cells. Cancer Research. 43, 1362-1364 (1983).
§ 799.9539   TSCA mammalian erythrocyte micronucleus test.
(a) Scope. This section is intended to meet the testing requirements under section 4 of TSCA.
(1) The mammalian erythrocyte micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents.
(2) The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes.
(3) When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualization of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage.
(b) Source. The source material used in developing this TSCA test guideline is the OECD guideline 474 (February 1997). This source is available at the address in paragraph (g) of this section.
(c) Definitions. The following definitions apply to this section:
Centromere (kinetochore) is a region of a chromosome with which spindle fibers are associated during cell division, allowing orderly movement of daughter chromosomes to the poles of the daughter cells.
Micronuclei are small nuclei, separate from and additional to the main nuclei of cells, produced during telophase of mitosis (meiosis) by lagging chromosome fragments or whole chromosomes.
Normochromatic erythrocyte is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by stains selective for ribosomes.
Polychromatic erythrocyte is an immature erythrocyte, in an intermediate stage of development, that still contains ribosomes and therefore can be distinguished from mature, normochromatic erythrocytes by stains selective for ribosomes.
(d) Initial considerations. (1) The bone marrow of rodents is routinely used in this test since polychromatic erythrocytes are produced in that tissue. The measurement of micronucleated immature (polychromatic) erythrocytes in peripheral blood is equally acceptable in any species in which the inability of the spleen to remove micronucleated erythrocytes has been demonstrated, or which has shown an adequate sensitivity to detect agents that cause structural or numerical chromosome aberrations. Micronuclei can be distinguished by a number of criteria. These include identification of the presence or absence of a kinetochore or centromeric DNA in the micronuclei. The frequency of micronucleated immature (polychromatic) erythrocytes is the principal endpoint. The number of mature (normochromatic) erythrocytes in the peripheral blood that contain micronuclei among a given number of mature erythrocytes can also be used as the endpoint of the assay when animals are treated continuously for 4 weeks or more. This mammalian in vivo micronucleus test is especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes although these may vary among species, among tissues and among genetic endpoints. An in vivo assay is also useful for further investigation of a mutagenic effect detected by an in vitro system.
(2) If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test.
(e) Test method —(1) Principle. Animals are exposed to the test substance by an appropriate route. If bone marrow is used, the animals are sacrificed at appropriate times after treatment, the bone marrow extracted, and preparations made and stained (test techniques described in the references under paragraphs (g)(1), (g)(2), and (g)(3) of this section may be used). When peripheral blood is used, the blood is collected at appropriate times after treatment and smear preparations are made and stained (the test techniques described in the references under paragraphs (g)(3), (g)(4), (g)(5), and (g)(6) of this section may be used). For studies with peripheral blood, as little time as possible should elapse between the last exposure and cell harvest. Preparations are analyzed for the presence of micronuclei.
(2) Description —(i) Preparations —(A) Selection of animal species. Mice or rats are recommended if bone marrow is used, although any appropriate mammalian species may be used. When peripheral blood is used, mice are recommended. However, any appropriate mammalian species may be used provided it is a species in which the spleen does not remove micronucleated erythrocytes or a species which has shown an adequate sensitivity to detect agents that cause structural or numerical chromosome aberrations. Commonly used laboratory strains of young healthy animals should be employed. At the commencement of the study, the weight variation of animals should be minimal and not exceed ±20% of the mean weight of each sex.
(B) Housing and feeding conditions. The temperature in the experimental animal room should be 22 °C ±3 °C). Although the relative humidity should be at least 30% and preferably not exceed 70% other than during room cleaning, the aim should be 50-60%. Lighting should be artificial, the sequence being 12 hrs light, 12 hrs dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this route. Animals may be housed individually, or caged in small groups of the same sex.
(C) Preparation of the animals. Healthy young adult animals shall be randomly assigned to the control and treatment groups. The animals are identified uniquely. The animals are acclimated to the laboratory conditions for at least 5 days. Cages should be arranged in such a way that possible effects due to cage placement are minimized.
(D) Preparation of doses. Solid test substances shall be dissolved or suspended in appropriate solvents or vehicles and diluted, if appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted prior to dosing. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.
(ii) Test conditions —(A) Solvent/vehicle. The solvent/vehicle shall not produce toxic effects at the dose levels used, and shall not be suspected of chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported with reference data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle should be considered first.
(B) Controls. ( 1 ) Concurrent positive and negative (solvent/vehicle) controls shall be included for each sex in each test. Except for treatment with the test substance, animals in the control groups should be handled in an identical manner to animals of the treatment groups.
( 2 ) Positive controls shall produce micronuclei in vivo at exposure levels expected to give a detectable increase over background. Positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. It is acceptable that the positive control be administered by a route different from the test substance and sampled at only a single time. In addition, the use of chemical class-related positive control chemicals may be considered, when available. Examples of positive control substances include:
( 3 ) Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same way as the treatment groups shall be included for every sampling time, unless acceptable inter-animal variability and frequencies of cells with micronuclei are demonstrated by historical control data. If single sampling is applied for negative controls, the most appropriate time is the first sampling time. In addition, untreated controls should also be used unless there are historical or published control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent/vehicle.
( 4 ) If peripheral blood is used, a pre-treatment sample may also be acceptable as a concurrent negative control, but only in the short peripheral blood studies (e.g., one to three treatment(s)) when the resulting data are in the expected range for the historical control.
(3) Procedure —(i) Number and sex of animals. Each treated and control group shall include at least 5 analyzable animals per sex (techniques described in the reference under paragraph (g)(7) of this section may be used). If at the time of the study there are data available from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences between sexes in toxicity, then testing in a single sex will be sufficient. Where human exposure to chemicals may be sex-specific, as for example with some pharmaceutical agents, the test should be performed with animals of the appropriate sex.
(ii) Treatment schedule. (A) No standard treatment schedule (i.e. one, two, or more treatments at 24 h intervals) can be recommended. The samples from extended dose regimens are acceptable as long as a positive effect has been demonstrated for this study or, for a negative study, as long as toxicity has been demonstrated or the limit dose has been used, and dosing continued until the time of sampling. Test substances may also be administered as a split dose, i.e., two treatments on the same day separated by no more than a few hrs, to facilitate administering a large volume of material.
(B) The test may be performed in two ways:
( 1 ) Animals shall be treated with the test substance once. Samples of bone marrow shall be taken at least twice, starting not earlier than 24 hrs after treatment, but not extending beyond 48 hrs after treatment with appropriate interval(s) between samples. The use of sampling times earlier than 24 hrs after treatment should be justified. Samples of peripheral blood shall be taken at least twice, starting not earlier than 36 hrs after treatment, with appropriate intervals following the first sample, but not extending beyond 72 hrs. When a positive response is recognized at one sampling time, additional sampling is not required.
( 2 ) If two or more daily treatments are used (e.g. two or more treatments at 24 hr intervals), samples shall be collected once between 18 and 24 hrs following the final treatment for the bone marrow and once between 36 and 48 hrs following the final treatment for the peripheral blood (techniques described in the reference under paragraph (g)(8) of this section may be used).
(C) Other sampling times may be used in addition, when relevant.
(iii) Dose levels. If a range finding study is performed because there are no suitable data available, it shall be performed in the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study (guidance on dose setting is provided in the reference in paragraph (g)(9) of this section). If there is toxicity, three dose levels shall be used for the first sampling time. These dose levels shall cover a range from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some indication of toxicity in the bone marrow (e.g. a reduction in the proportion of immature erythrocytes among total erythrocytes in the bone marrow or peripheral blood).
(iv) Limit test. If a test at one dose level of at least 2,000 mg/kg body weight using a single treatment, or as two treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. For studies of a longer duration, the limit dose is 2,000 mg/kg/body weight/day for treatment up to 14 days, and 1,000 mg/kg/body weight/day for treatment longer than 14 days. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.
(v) Administration of doses. The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be administered by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 ml/100g body weight. The use of volumes higher than these must be justified. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimized by adjusting the concentration to ensure a constant volume at all dose levels.
(vi) Bone marrow/blood preparation. Bone marrow cells shall be obtained from the femurs or tibias immediately following sacrifice. Cells shall be removed from femurs or tibias, prepared and stained using established methods. Peripheral blood is obtained from the tail vein or other appropriate blood vessel. Blood cells are immediately stained supravitally (the test techniques described in the references under paragraphs (g)(4), (g)(5), and (g)(6) of this section may be used) or smear preparations are made and then stained. The use of a DNA specific stain (e.g. acridine orange (techniques described in the reference under paragraph (g)(10) of this section may be used) or Hoechst 33258 plus pyronin-Y) can eliminate some of the artifacts associated with using a non-DNA specific stain. This advantage does not preclude the use of conventional stains (e.g., Giemsa). Additional systems (e.g. cellulose columns to remove nucleated cells (the test techniques described in the references under paragraph (g)(12) of this section may be used)) can also be used provided that these systems have been shown to adequately work for micronucleus preparation in the laboratory.
(vii) Analysis. The proportion of immature among total (immature = mature) erythrocytes is determined for each animal by counting a total of at least 200 erythrocytes for bone marrow and 1,000 erythrocytes for peripheral blood (techniques described in the reference under paragraph (g)(13) of this section maybe used). All slides, including those of positive and negative controls, shall be independently coded before microscopic analysis. At least 2,000 immature erythrocytes per animal shall be scored for the incidence of micronucleated immature erythrocytes. Additional information may be obtained by scoring mature erythrocytes for micronuclei. When analyzing slides, the proportion of immature erythrocytes among total erythrocytes should not be less than 20% of the control value. When animals are treated continuously for 4 weeks or more, at least 2,000 mature erythrocytes per animal can also be scored for the incidence of micronuclei. Systems for automated analysis (image analysis) and cell suspensions (flow cytometry) are acceptable alternatives to manual evaluation if appropriately justified and validated.
(f) Data and reporting —(1) Treatment of results. Individual animal data shall be presented in tabular form. The experimental unit is the animal. The number of immature erythrocytes scored, the number of micronucleated immature erythrocytes, and the number of immature among total erythrocytes shall be listed separately for each animal analyzed. When animals are treated continuously for 4 weeks or more, the data on mature erythrocytes should also be given if it is collected. The proportion of immature among total erythrocytes and, if considered applicable, the percentage of micronucleated erythrocytes shall be given for each animal. If there is no evidence for a difference in response between the sexes, the data from both sexes may be combined for statistical analysis.
(2) Evaluation and interpretation of results. (i) There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (the test techniques described in the references paragraphs (g)(14) and (g)(15) of this section may be used). Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.
(ii) A test substance for which the results do not meet the criteria in paragraph (f)(2)(i) of this section is considered non-mutagenic in this test.
(iii) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results, may remain equivocal or questionable regardless of the number of times the experiment is repeated. Positive results in the micronucleus test indicate that a substance induces micronuclei which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species. Negative results indicate that, under the test conditions, the test substance does not produce micronuclei in the immature erythrocytes of the test species.
(iv) The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g. systemic toxicity) should be discussed.
(3) Test report. The test report shall include the following information:
(i) Test substance:
(A) Identification data and CAS no., if known.
(B) Physical nature and purity.
(C) Physiochemical properties relevant to the conduct of the study.
(D) Stability of the test substance, if known.
(ii) Solvent/vehicle:
(A) Justification for choice of vehicle.
(B) Solubility and stability of the test substance in the solvent/vehicle, if known.
(iii) Test animals:
(A) Species/strain used.
(B) Number, age, and sex of animals.
(C) Source, housing conditions, diet, etc.
(D) Individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group.
(iv) Test conditions:
(A) Positive and negative (vehicle/solvent) control data.
(B) Data from range-finding study, if conducted.
(C) Rationale for dose level selection.
(D) Details of test substance preparation.
(E) Details of the administration of the test substance.
(F) Rationale for route of administration.
(G) Methods for verifying that the test substance reached the general circulation or target tissue, if applicable.
(H) Conversion from diet/drinking water test substance concentration parts per million (ppm) to the actual dose (mg/kg body weight/day), if applicable.
(I) Details of food and water quality.
(J) Detailed description of treatment and sampling schedules.
(K) Methods of slide preparation.
(L) Methods for measurement of toxicity.
(M) Criteria for scoring micronucleated immature erythrocytes.
(N) Number of cells analyzed per animal.
(O) Criteria for considering studies as positive, negative or equivocal.
(v) Results:
(A) Signs of toxicity.
(B) Proportion of immature erythrocytes among total erythrocytes.
(C) Number of micronucleated immature erythrocytes, given separately for each animal.
(D) Mean = ±standard deviation of micronucleated immature erythrocytes per group.
(E) Dose-response relationship, where possible.
(F) Statistical analyses and method applied.
(G) Concurrent and historical negative control data.
(H) Concurrent positive control data.
(vi) Discussion of the results.
(vii) Conclusion.
(g) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Heddle, J.A. A Rapid In Vivo Test for Chromosomal Damage. Mutation Research. 18, 187-190 (1973).
(2) Schmid, W. The Micronucleus Test. Mutation Research. 31, 9-15 (1975).
(3) Mavournin, K.H., Blakey, D.H., Cimino, M.C., Salamone, M.F., and Heddle, J.A. The In Vivo Micronucleus Assay in Mammalian Bone Marrow and Peripheral Blood. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research. 239, 29-80 (1990).
(4) Hayashi, M., Morita, T., Kodama, Y., Sofuni, T., and Ishidate, Jr., M. The Micronucleus Assay with Mouse Peripheral Blood Reticulocytes Using Acridine Orange-Coated Slides. Mutation Research. 245, 245-249 (1990).
(5) The Collaborative Study Group for the Micronucleus Test (1992). Micronucleus Test with Mouse Peripheral Blood Erythrocytes by Acridine Orange Supravital Staining: The Summary Report of the 5th Collaborative Study by CSGMT/JEMS. MMS. Mutation Research. 278, 83-98.
(6) The Collaborative Study Group for the Micronucleus Test (CSGMT/JEMMS.MMS, The Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan) Protocol recommended for the short-term mouse peripheral blood micronucleus test. Mutagenesis. 10, 153-159 (1995).
(7) Hayashi, M., Tice, R.R., MacGregor, J.T., Anderson, D., Blakey, D.H., Kirsch-Volders, M., Oleson, Jr. F.B., Pacchierotti, F., Romagna, F., Shimada, H., Sutou, S., and Vannier, B. In Vivo Rodent Erythrocyte Micronucleus Assay. Mutation Research. 312, 293-304 (1994).
(8) Higashikuni, N. and Sutou, S. An optimal, generalized sampling time of 30 = /- 6 h after double dosing in the mouse peripheral blood micronucleus test. Mutagenesis. 10, 313-319 (1995).
(9) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D. J., and Richold, M. Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose Setting in In Vivo Mutagenicity Assays. Mutagenesis. 7, 313-319 (1992).
(10) Hayashi, M., Sofuni, T., and Ishidate, Jr., M. An Application of Acridine Orange Fluorescent Staining to the Micronucleus Test. Mutation Research. 120, 241-247 (1983).
(11) MacGregor, J.T., Wehr, C.M., and Langlois, R.G. A Simple Fluorescent Staining Procedure for Micronuclei and RNA in Erythrocytes Using Hoechst 33258 and Pyronin Y. Mutation Research. 120, 269-275 (1983).
(12) Romagna, F. and Staniforth, C.D. The automated bone marrow micronucleus test. Mutation Research. 213, 91-104 (1989).
(13) Gollapudi, B. and McFadden, L.G. Sample size for the estimation of polychromatic to normochromatic eruthrocyte ratio in the bone marrow micronucleus test. Mutation Research. 347, 97-99 (1995).
(14) Richold, M., Ashby, J., Bootman, J., Chandley, A., Gatehouse, D.G., and Henderson, L. Ed. Kirkland, D.J. In Vivo Cytogenetics Assays. Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report. Part I revised (Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, 1990) pp. 115-141.
(15) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, J.R.K. Ed. D.J. Kirkland. Statistical Analysis of In Vivo Cytogenetic Assays. Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing, Report, Part III. (Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, 1989) pp. 184-232.
(16) Heddle, J.A., Salamone, M.F., Hite, M., Kirkhart, B., Mavournin, K., MacGregor, J.G., and Newell, G.W. The Induction of Micronuclei as a Measure of Genotoxicity. Mutation Research. 123: 61-118 (1983).
(17) MacGregor, J.T., Heddle, J.A., Hite, M., Margolin, G.H., Ramel C., Salamone, M.F., Tice, R.R., and Wild, D. Guidelines for the Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes. Mutation Research. 189: 103-112 (1987).
(18) MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.E. (1990). The In Vivo Erythrocyte Micronucleus Test: Measurement at Steady State Increases Assay Efficiency and Permits Integration with Toxicity Studies. Fundamental Applied Toxicology. 14: 513-522.
(19) MacGregor, J.T., Schlegel, R. Choy, W.N., and Wehr, C.M. Eds. Hayes, A.W., Schnell, R.C., and Miya, T.S. Micronuclei in Circulating Erythrocytes: A Rapid Screen for Chromosomal Damage During Routine Toxicity Testing in Mice. Developments in Science and Practice of Toxicology (Elsevier, Amsterdam, 1983) pp. 555-558.
§ 799.9620   TSCA neurotoxicity screening battery.
(a) Scope. This section is intended to meet the testing requirements under section 4 of TSCA. This neurotoxicity screening battery consists of a functional observational battery, motor activity, and neuropathology. The functional observational battery consists of noninvasive procedures designed to detect gross functional deficits in animals and to better quantify behavioral or neurological effects detected in other studies. The motor activity test uses an automated device that measures the level of activity of an individual animal. The neuropathological techniques are designed to provide data to detect and characterize histopathological changes in the central and peripheral nervous system. This battery is designed to be used in conjunction with general toxicity studies and changes should be evaluated in the context of both the concordance between functional neurological and neuropatholgical effects, and with respect to any other toxicological effects seen. This test battery is not intended to provide a complete evaluation of neurotoxicity, and additional functional and morphological evaluation may be necessary to assess completely the neurotoxic potential of a chemical.
(b) Source. The source material used in developing this TSCA test guideline is the OPPTS harmonized test guideline 870.6200 (June 1996 Public Draft). This source is available at the address in paragraph (g) of this section.
(c) Definitions. The following definitions apply to this section.
ED is effective dose.
Motor activity is any movement of the experimental animal.
Neurotoxicity is any adverse effect on the structure or function of the nervous system related to exposure to a chemical substance.
Toxic effect is an adverse change in the structure or function of an experimental animal as a result of exposure to a chemical substance.
(d) Principle of the test method. The test substance is administered to several groups of experimental animals, one dose being used per group. The animals are observed under carefully standardized conditions with sufficient frequency to ensure the detection and quantification of behavioral and/or neurologic abnormalities, if present. Various functions that could be affected by neurotoxicants are assessed during each observation period. Measurements of motor activity of individual animals are made in an automated device. The animals are perfused and tissue samples from the nervous system are prepared for microscopic examination. The exposure levels at which significant neurotoxic effects are produced are compared to one another and to those levels that produce other toxic effects.
(e) Test procedures —(1) Animal selection —(i) Species. In general, the laboratory rat should be used. Under some circumstances, other species, such as the mouse or the dog, may be more appropriate, although not all of the battery may be adaptable to other species.
(ii) Age. Young adults (at least 42 days old for rats) shall be used.
(iii) Sex. Both males and females shall be used. Females shall be nulliparous and nonpregnant.
(2) Number of animals. At least 10 males and 10 females should be used in each dose and control group for behavioral testing. At least five males and five females should be used in each dose and control group for terminal neuropathology. If interim neuropathological evaluations are planned, the number should be increased by the number of animals scheduled to be perfused before the end of the study. Animals shall be randomly assigned to treatment and control groups.
(3) Control groups. (i) A concurrent (vehicle) control group is required. Subjects shall be treated in the same way as for an exposure group except that administration of the test substance is omitted. If the vehicle used has known or potential toxic properties, both untreated or saline treated and vehicle control groups are required.
(ii) Positive control data from the laboratory performing the testing shall provide evidence of the ability of the observational methods used to detect major neurotoxic endpoints including limb weakness or paralysis, tremor, and autonomic signs. Positive control data are also required to demonstrate the sensitivity and reliability of the activity-measuring device and testing procedures. These data should demonstrate the ability to detect chemically induced increases and decreases in activity. Positive control groups exhibiting central nervous system pathology and peripheral nervous system pathology are also required. Separate groups for peripheral and central neuropathology are acceptable (e.g. acrylamide and trimethyl tin). Permanently injurious substances need not be used for the behavioral tests. Historical data may be used if the essential aspects of the experimental procedure remain the same. Periodic updating of positive control data is recommended. New positive control data should also be collected when personnel or some other critical element in the testing laboratory has changed.
(4) Dose level and dose selection. At least three doses shall be used in addition to the vehicle control group. The data should be sufficient to produce a dose-effect curve. The Agency strongly encourage the use of equally spaced doses and a rationale for dose selection that will maximally support detection of dose-effect relations. For acute studies, dose selection may be made relative to the establishment of a benchmark dose (BD). That is, doses may be specified as successive fractions, e.g. 0.5, 0.25, ...n of the BD. The BD itself may be estimated as the highest nonlethal dose as determined in a preliminary range-finding lethality study. A variety of test methodologies may be used for this purpose, and the method chosen may influence subsequent dose selection. The goal is to use a dose level that is sufficient to be judged a limit dose, or clearly toxic.
(i) Acute studies. The high dose need not be greater than 2 g/kg. Otherwise, the high dose should result in significant neurotoxic effects or other clearly toxic effects, but not result in an incidence of fatalities that would preclude a meaningful evaluation of the data. This dose may be estimated by a BD procedure as described under paragraph (e)(4) of this section, with the middle and low dose levels chosen as fractions of the BD dose. The lowest dose should produce minimal effect, e.g. an ED10, or alternatively, no effects.
(ii) Subchronic and chronic studies. The high dose need not be greater than 1 g/kg. Otherwise, the high dose level should result in significant neurotoxic effects or other clearly toxic effects, but not produce an incidence of fatalities that would prevent a meaningful evaluation of the data. The middle and low doses should be fractions of the high dose. The lowest dose should produce minimal effects, e.g. an ED10, or alternatively, no effects.
(5) Route of exposure. Selection of route may be based on several criteria including, the most likely route of human exposure, bioavailability, the likelihood of observing effects, practical difficulties, and the likelihood of producing nonspecific effects. For many materials, it should be recognized that more than one route of exposure may be important and that these criteria may conflict with one another. Initially only one route is required for screening for neurotoxicity. The route that best meets these criteria should be selected. Dietary feeding will generally be acceptable for repeated exposures studies.
(6) Combined protocol. The tests described in this screening battery may be combined with any other toxicity study, as long as none of the requirements of either are violated by the combination.
(7) Study conduct —(i) Time of testing. All animals shall be weighed on each test day and at least weekly during the exposure period.
(A) Acute studies. At a minimum, for acute studies observations and activity testing shall be made before the initiation of exposure, at the estimated time of peak effect within 8 hrs of dosing, and at 7 and 14 days after dosing. Estimation of times of peak effect may be made by dosing pairs of rats across a range of doses and making regular observations of gait and arousal.
(B) Subchronic and chronic studies. In a subchronic study, at a minimum, observations and activity measurements shall be made before the initiation of exposure and before the daily exposure, or for feeding studies at the same time of day, during the 4th, 8th, and 13th weeks of exposure. In chronic studies, at a minimum, observations and activity measurements shall be made before the initiation of exposure and before the daily exposure, or for feeding studies at the same time of day, every 3 months.
(ii) Functional observational battery —(A) General conduct. All animals in a given study shall be observed carefully by trained observers who are unaware of the animals' treatment, using standardized procedures to minimize observer variability. Where possible, it is advisable that the same observer be used to evaluate the animals in a given study. If this is not possible, some demonstration of interobserver reliability is required. The animals shall be removed from the home cage to a standard arena for observation. Effort should be made to ensure that variations in the test conditions are minimal and are not systematically related to treatment. Among the variables that can affect behavior are sound level, temperature, humidity, lighting, odors, time of day, and environmental distractions. Explicit, operationally defined scales for each measure of the battery are to be used. The development of objective quantitative measures of the observational end-points specified is encouraged. Examples of observational procedures using defined protocols may be found in the references under paragraphs (g)(5), (g)(6), and (g)(9) of this section. The functional observational battery shall include a thorough description of the subject's appearance, behavior, and functional integrity. This shall be assessed through observations in the home cage and while the rat is moving freely in an open field, and through manipulative tests. Testing should proceed from the least to the most interactive with the subject. Scoring criteria, or explicitly defined scales, should be developed for those measures which involve subjective ranking.
(B) List of measures. The functional observational battery shall include the following list of measures:
( 1 ) Assessment of signs of autonomic function, including but not limited to:
( i ) Ranking of the degree of lacrimation and salivation, with a range of severity scores from none to severe.
( ii ) Presence or absence of piloerection and exophthalmus.
( iii ) Ranking or count of urination and defecation, including polyuria and diarrhea. This is most easily conducted during the open field assessment.
( iv ) Pupillary function such as constriction of the pupil in response to light or a measure of pupil size.
( v ) Degree of palpebral closure, e.g., ptosis.
( 2 ) Description, incidence, and severity of any convulsions, tremors, or abnormal motor movements, both in the home cage and the open field.
( 3 ) Ranking of the subject's reactivity to general stimuli such as removal from the cage or handling, with a range of severity scores from no reaction to hyperreactivity.
( 4 ) Ranking of the subject's general level of activity during observations of the unperturbed subject in the open field, with a range of severity scores from unresponsive to hyperactive.
( 5 ) Descriptions and incidence of posture and gait abnormalities observed in the home cage and open field.
( 6 ) Ranking of any gait abnormalities, with a range of severity scores from none to severe.
( 7 ) Forelimb and hindlimb grip strength measured using an objective procedure (the procedure described in the reference under paragraph (g)(8) of this section may be used).
( 8 ) Quantitative measure of landing foot splay (the procedure described in the reference under paragraph (g)(3) of this section may be used).
( 9 ) Sensorimotor responses to stimuli of different modalities will be used to detect gross sensory deficits. Pain perception may be assessed by a ranking or measure of the reaction to a tail-pinch, tail-flick, or hot-plate. The response to a sudden sound, e.g., click or snap, may be used to assess audition.
( 10 ) Body weight.
( 11 ) Description and incidence of any unusual or abnormal behaviors, excessive or repetitive actions (stereotypies), emaciation, dehydration, hypotonia or hypertonia, altered fur appearance, red or crusty deposits around the eyes, nose, or mouth, and any other observations that may facilitate interpretation of the data.
(C) Additional measures. Other measures may also be included and the development and validation of new tests is encouraged. Further information on the neurobehavioral integrity of the subject may be provided by:
( 1 ) Count of rearing activity on the open field.
( 2 ) Ranking of righting ability.
( 3 ) Body temperature.
( 4 ) Excessive or spontaneous vocalizations.
( 5 ) Alterations in rate and ease of respiration, e.g., rales or dyspnea.
( 6 ) Sensorimotor responses to visual or proprioceptive stimuli.
(iii) Motor activity. Motor activity shall be monitored by an automated activity recording apparatus. The device used must be capable of detecting both increases and decreases in activity, i.e., baseline activity as measured by the device must not be so low as to preclude detection of decreases nor so high as to preclude detection of increases in activity. Each device shall be tested by standard procedures to ensure, to the extent possible, reliability of operation across devices and across days for any one device. In addition, treatment groups must be balanced across devices. Each animal shall be tested individually. The test session shall be long enough for motor activity to approach asymptotic levels by the last 20% of the session for nontreated control animals. All sessions shall have the same duration. Treatment groups shall be counterbalanced across test times. Effort should be made to ensure that variations in the test conditions are minimal and are not systematically related to treatment. Among the variables which can affect motor activity are sound level, size and shape of the test cage, temperature, relative humidity, lighting conditions, odors, use of the home cage or a novel test cage, and environmental distractions.
(iv) Neuropathology: Collection, processing and examination of tissue samples. To provide for adequate sampling as well as optimal preservation of cellular integrity for the detection of neuropathological alterations, tissue shall be prepared for histological analysis using in situ perfusion and paraffin and/or plastic embedding procedures. Paraffin embedding is acceptable for tissue samples from the central nervous system. Plastic embedding of tissue samples from the central nervous system is encouraged, when feasible. Plastic embedding is required for tissue samples from the peripheral nervous system. Subject to professional judgment and the type of neuropathological alterations observed, it is recommended that additional methods, such as glial fibrillary acidic protein (GFAP) immunohistochemistry and/or methods known as Bodian's or Bielchowsky's silver methods be used in conjunction with more standard stains to determine the lowest dose level at which neuropathological alterations are observed. When new or existing data provide evidence of structural alterations it is recommended that the GFAP immunoassay also be considered. A description of this technique can be found in the reference under paragraph (g)(10) of this section.
(A) Fixation and processing of tissue. The nervous system shall be fixed by in situ perfusion with an appropriate aldehyde fixative. Any gross abnormalities should be noted. Tissue samples taken should adequately represent all major regions of the nervous system. The tissue samples should be postfixed and processed according to standardized published histological protocols (protocols described in the references under paragraphs (g)(1), (g)(2), or (g)(11) of this section may be used). Tissue blocks and slides should be appropriately identified when stored. Histological sections should be stained for hematoxylin and eosin (H&E), or a comparable stain according to standard published protocols (some of these protocols are described in the references under paragraphs (g)(1) and (g)(11) of this section).
(B) Qualitative examination. Representative histological sections from the tissue samples should be examined microscopically by an appropriately trained pathologist for evidence of neuropathological alterations. The nervous system shall be thoroughly examined for evidence of any treatment-related neuropathological alterations. Particular attention should be paid to regions known to be sensitive to neurotoxic insult or those regions likely to be affected based on the results of functional tests. Such treatment-related neuropathological alterations should be clearly distinguished from artifacts resulting from influences other than exposure to the test substance. A stepwise examination of tissue samples is recommended. In such a stepwise examination, sections from the high dose group are first compared with those of the control group. If no neuropathological alterations are observed in samples from the high dose group, subsequent analysis is not required. If neuropathological alterations are observed in samples from the high dose group, samples from the intermediate and low dose groups are then examined sequentially.
(C) Subjective diagnosis. If any evidence of neuropathological alterations is found in the qualitative examination, then a subjective diagnosis shall be performed for the purpose of evaluating dose-response relationships. All regions of the nervous system exhibiting any evidence of neuropathological changes should be included in this analysis. Sections from all dose groups from each region will be coded and examined in randomized order without knowledge of the code. The frequency of each type and severity of each lesion will be recorded. After all samples from all dose groups including all regions have been rated, the code will be broken and statistical analysis performed to evaluate dose-response relationships. For each type of dose-related lesion observed, examples of different degrees of severity should be described. Photomicrographs of typical examples of treatment-related regions are recommended to augment these descriptions. These examples will also serve to illustrate a rating scale, such as 1=, 2=, and 3= for the degree of severity ranging from very slight to very extensive.
(f) Data reporting and evaluation. The final test report shall include the following information:
(1) Description of equipment and test methods. A description of the general design of the experiment and any equipment used shall be provided. This shall include a short justification explaining any decisions involving professional judgment.
(i) A detailed description of the procedures used to standardize observations, including the arena and scoring criteria.
(ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. Historical data may be used if all essential aspects of the experimental protocol are the same. Historical control data can be critical in the interpretation of study findings. The Agency encourages submission of such data to facilitate the rapid and complete review of the significance of effects seen.
(2) Results. The following information shall be arranged by test group dose level.
(i) In tabular form, data for each animal shall be provided showing:
(A) Its identification number.
(B) Its body weight and score on each sign at each observation time, the time and cause of death (if appropriate), total session activity counts, and intrasession subtotals for each day measured.
(ii) Summary data for each group must include:
(A) The number of animals at the start of the test.
(B) The number of animals showing each observation score at each observation time.
(C) The mean and standard deviation for each continuous endpoint at each observation time.
(D) Results of statistical analyses for each measure, where appropriate.
(iii) All neuropathological observations shall be recorded and arranged by test groups. This data may be presented in the following recommended format:
(A) Description of lesions for each animal. For each animal, data must be submitted showing its identification (animal number, sex, treatment, dose, and duration), a list of structures examined as well as the locations, nature, frequency, and severity of lesions. Inclusion of photomicrographs is strongly recommended for demonstrating typical examples of the type and severity of the neuropathological alterations observed. Any diagnoses derived from neurological signs and lesions including naturally occurring diseases or conditions, should be recorded.
(B) Counts and incidence of neuropathological alterations by test group. Data should be tabulated to show:
( 1 ) The number of animals used in each group and the number of animals in which any lesion was found.
( 2 ) The number of animals affected by each different type of lesion, the locations, frequency, and average grade of each type of lesion.
(3) Evaluation of data. The findings from the screening battery should be evaluated in the context of preceding and/or concurrent toxicity studies and any correlated functional and histopathological findings. The evaluation shall include the relationship between the doses of the test substance and the presence or absence, incidence and severity, of any neurotoxic effects. The evaluation shall include appropriate statistical analyses, for example, parametric tests for continuous data and nonparametric tests for the remainder. Choice of analyses should consider tests appropriate to the experimental design, including repeated measures. There may be many acceptable ways to analyze data.
(g) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Bennet, H.S. et al. Science and art in the preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains. Stain Technology. 51:71-97 (1976).
(2) Di Sant Agnese, P.A. and De Mesy Jensen, K. Dibasic staining of large epoxy sections and application to surgical pathology. American Journal of Clinical Pathology. 81:25-29 (1984).
(3) Edwards, P.M. and Parker, V.H. A simple, sensitive and objective method for early assessment of acrylamide neuropathy in rats. Toxicology and Applied Pharmacology. 40:589-591 (1977).
(4) Finger, F.W. Ed. Myers, R.D. Measuring Behavioral Activity. Vol. 2. Methods in Psychobiology (Academic, NY, 1972) pp.1-19.
(5) Gad, S. A neuromuscular screen for use in industrial toxicology. Journal of Toxicology and Environmental Health. 9:691-704 (1982).
(6) Irwin, S. Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral physiological state of the mouse. Psychopharmacologia. 13:222-257 (1968).
(7) Kinnard, E.J. and Watzman, N. Techniques utilized in the evaluation of psychotropic drugs on animals activity. Journal of Pharmaceutical Sciences. 55:995-1012 (1966).
(8) Meyer, O.A. et al. A method for the routine assessment of fore- and hindlimb grip strength of rats and mice. Neurobehavioral Toxicology. 1:233-236 (1979).
(9) Moser V.C. et al. Comparison of chlordimeform and carbaryl using a functional observational battery. Fundamental and Applied Toxicology. 11:189-206 (1988).
(10) O'Callaghan, J.P. Quantification of glial fibrillary acidic protein: Comparison of slot-immunobinding assays with a novel sandwich ELISA. Neurotoxicology and Teratology. 13:275-281 (1991).
(11) Pender, M.P. A simple method for high resolution light microscopy of nervous tissue. Journal of Neuroscience Methods. 15:213-218 (1985).
(12) Reiter, L.W. Use of activity measures in behavioral toxicology. Environmental Health Perspectives. 26:9-20 (1978).
(13) Reiter, L.W. and MacPhail, R.C. Motor activity: A survey of methods with potential use in toxicity testing. Neurobehavorial Toxicology. 1—Supplement. 1:53-66 (1979).
(14) Robbins, T.W. Eds. Iversen, L.L., Iverson, D.S., and Snyder, S.H. A critique of the methods available for the measurement of spontaneous motor activity. Vol 7. Handbook of Psychopharmacology (Plenum, NY, 1977) pp. 37-82.
§ 799.9630   TSCA developmental neurotoxicity.
(a) Scope —(1) Applicability. This section is intended to meet the testing requirements under section 4 of the Toxic Substances Control Act (TSCA).
(2) Source. The source material used in developing this TSCA test guideline is the OPPTS harmonized test guideline 870.6300 (August 1998).
(b) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical substance or mixture (test substance), determination of the potential for developmental neurotoxicity is important. This study is designed to develop data on the potential functional and morphological hazards to the nervous system which may arise in the offspring from exposure of the mother during pregnancy and lactation.
(c) Principle of the test method. The test substance is administered to several groups of pregnant animals during gestation and early lactation, one dose level being used per group. Offspring are randomly selected from within litters for neurotoxicity evaluation. The evaluation includes observations to detect gross neurologic and behavioral abnormalities, determination of motor activity, response to auditory startle, assessment of learning, neuropathological evaluation, and brain weights. This protocol may be used as a separate study, as a followup to a standard developmental toxicity and/or adult neurotoxicity study, or as part of a two-generation reproduction study, with assessment of the offspring conducted on the second (F 2 ) generation.
(d) Test procedure —(1) Animal selection —(i) Species and strain. Testing must be performed in the rat. Because of its differences in timing of developmental events compared to strains that are more commonly tested in other developmental and reproductive toxicity studies, it is preferred that the Fischer 344 strain not be used. If a sponsor wishes to use the Fischer 344 rat or a mammalian species other than the rat, ample justification/reasoning for this selection must be provided.
(ii) Age. Young adult (nulliparous females) animals must be used.
(iii) Sex. Pregnant female animals must be used at each dose level.
(iv) Number of animals. (A) The objective is for a sufficient number of pregnant rats to be exposed to the test substance to ensure that an adequate number of offspring are produced for neurotoxicity evaluation. At least 20 litters are recommended at each dose level.
(B) On postnatal day 4, the size of each litter should be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four male and four females per litter. Whenever the number of pups of either sex prevents having four of each sex per litter, partial adjustment (for example, five males and three females) is permitted. Testing is not appropriate for litters of less than seven pups. Elimination of runts only is not appropriate. Individual pups should be identified uniquely after standardization of litters. A method that may be used for identification can be found under paragraph (f)(1) of this section.
(v) Assignment of animals for behavioral tests, brain weights, and neuropathological evaluations. After standardization of litters, one male or one female from each litter (total of 10 males and 10 females per dose group) must be randomly assigned to one of the following tests: Motor activity, auditory startle, and learning and memory, in weanling and adult animals. On postnatal day 11, either 1 male or 1 female pup from each litter (total of 10 males and 10 females per dose group) must be sacrificed. Brain weights must be measured in all of these pups and, of these pups, six per sex per dose must be selected for neuropathological evaluation. At the termination of the study, either 1 male or 1 female from each litter (total of 10 males and 10 females per dose group) must be sacrificed and brain weights must be measured. An additional group of six animals per sex per dose group (one male or one female per litter) must be sacrificed at the termination of the study for neuropathological evaluation.
(2) Control group. A concurrent control group is required. This group must be a sham-treated group or, if a vehicle is used in administering the test substance, a vehicle control group. The vehicle must neither be developmentally toxic nor have effects on reproduction. Animals in the control group must be handled in an identical manner to test group animals.
(3) Dose levels and dose selection. (i) At least three dose levels of the test substance plus a control group (vehicle control, if a vehicle is used) must be used.
(ii) If the test substance has been shown to be developmentally toxic either in a standard developmental toxicity study or in a pilot study, the highest dose level must be the maximum dose which will not induce in utero or neonatal death or malformations sufficient to preclude a meaningful evaluation of neurotoxicity.
(iii) If a standard developmental toxicity study has not been conducted, the highest dose level, unless limited by the physicochemical nature or biological properties of the substance, must induce some overt maternal toxicity, but must not result in a reduction in weight gain exceeding 20 percent during gestation and lactation.
(iv) The lowest dose should not produce any grossly observable evidence of either maternal or developmental neurotoxicity.
(v) The intermediate doses must be equally spaced between the highest and lowest doses used.
(4) Dosing period. Day 0 of gestation is the day on which a vaginal plug and/or sperm are observed. The dosing period must cover the period from day 6 of gestation through day 10 postnatally. Dosing should not occur on the day of parturition in those animals who have not completely delivered their offspring.
(5) Administration of the test substance. The test substance or vehicle must be administered orally. Other routes of administration may be acceptable, on a case-by-case basis, with ample justification/reasoning for this selection. The test substance or vehicle must be administered based on the most recent weight determination.
(6) Observation of dams. (i) A gross examination of the dams must be made at least once each day before daily treatment.
(ii) Ten dams per group must be observed outside the home cage at least twice during the gestational dosing period (days 6-21) and twice during the lactational dosing period (days 1-10) for signs of toxicity. The animals must be observed by trained technicians who are unaware of the animals' treatment, using standardized procedures to maximize interobserver reliability. Where possible, it is advisable that the same observer be used to evaluate the animals in a given study. If this is not possible, some demonstration of interobserver reliability is required.
(iii) During the treatment and observation periods under paragraph (d)(6)(ii) of this section, observations must include:
(A) Assessment of signs of autonomic function, including but not limited to:
( 1 ) Ranking of the degree of lacrimation and salivation, with a range of severity scores from none to severe.
( 2 ) Presence or absence of piloerection and exophthalmus.
( 3 ) Ranking or count of urination and defecation, including polyuria and diarrhea.
( 4 ) Pupillary function such as constriction of the pupil in response to light or a measure of pupil size.
( 5 ) Degree of palpebral closure, e.g., ptosis.
(B) Description, incidence, and severity of any convulsions, tremors, or abnormal movements.
(C) Description and incidence of posture and gait abnormalities.
(D) Description and incidence of any unusual or abnormal behaviors, excessive or repetitive actions (stereotypies), emaciation, dehydration, hypotonia or hypertonia, altered fur appearance, red or crusty deposits around the eyes, nose, or mouth, and any other observations that may facilitate interpretation of the data.
(iv) Signs of toxicity must be recorded as they are observed, including the time of onset, degree, and duration.
(v) Animals must be weighed at least weekly and on the day of delivery and postnatal days 11 and 21 (weaning) and such weights must be recorded.
(vi) The day of delivery of litters must be recorded and considered as postnatal day 0.
(7) Study conduct —(i) Observation of offspring. (A) All offspring must be examined cage-side at least daily for gross signs of mortality or morbidity.
(B) A total of 10 male offspring and 10 female offspring per dose group must be examined outside the cage for signs of toxicity on days 4, 11, 21, 35, 45, and 60. The offspring must be observed by trained technicians, who are unaware of the treatment being used, using standardized procedures to maximize interobserver reliability. Where possible, it is advisable that the same observer be used to evaluate the animals in a given study. If this is not possible, some demonstration of interobserver reliability is required. At a minimum, the end points outlined in paragraph (d)(6)(iii) of this section must be monitored as appropriate for the developmental stage being observed.
(C) Any gross signs of toxicity in the offspring must be recorded as they are observed, including the time of onset, degree, and duration.
(ii) Developmental landmarks. Live pups must be counted and each pup within a litter must be weighed individually at birth or soon thereafter, and on postnatal days 4, 11, 17, and 21 and at least once every 2 weeks thereafter. The age of vaginal opening and preputial separation must be determined. General procedures for these determinations may be found in paragraphs (f)(1) and (f)(11) of this section.
(iii) Motor activity. Motor activity must be monitored specifically on postnatal days 13, 17, 21, and 60 (+ 2 days). Motor activity must be monitored by an automated activity recording apparatus. The device must be capable of detecting both increases and decreases in activity, (i.e., baseline activity as measured by the device must not be so low as to preclude detection of decreases nor so high as to preclude detection of increases in activity). Each device must be tested by standard procedures to ensure, to the extent possible, reliability of operation across devices and across days for any one device. In addition, treatment groups must be balanced across devices. Each animal must be tested individually. The test session must be long enough for motor activity to approach asymptotic levels by the last 20 percent of the session for nontreated control animals. All sessions must have the same duration. Treatment groups must be counter-balanced across test times. Activity counts must be collected in equal time periods of no greater than 10 minutes duration. Efforts must be made to ensure that variations in the test conditions are minimal and are not systematically related to treatment. Among the variables that can affect motor activity are sound level, size and shape of the test cage, temperature, relative humidity, light conditions, odors, use of home cage or novel test cage, and environmental distractions. Additional information on the conduct of a motor activity study may be obtained in § 799.9620.
(iv) Auditory startle test. An auditory startle habituation test should be performed on the offspring around the time of weaning and around day 60. Day of testing should be counterbalanced across treated and control groups. Details on the conduct of this testing may be obtained under paragraph (f)(1) of this section. In performing the auditory startle task, the mean response amplitude on each block of 10 trials (5 blocks of 10 trials per session on each day of testing) must be made. While use of prepulse inhibition is not a requirement, it is highly recommended. Details on the conduct of this test may be obtained in paragraph (f)(10) of this section.
(v) Learning and memory tests. A test of associative learning and memory should be conducted around the time of weaning and around day 60. Day of testing should be counterbalanced across treated and control groups. The same or separate tests may be used at these two stages of development. Some flexibility is allowed in the choice of tests for learning and memory in weanling and adult rats. However, the tests must be designed to fulfill two criteria. First, learning must be assessed either as a change across several repeated learning trials or sessions, or, in tests involving a single trial, with reference to a condition that controls for nonassociative effects of the training experience. Second, the tests must include some measure of memory (short-term or long-term) in addition to original learning (acquisition). If the tests of learning and memory reveal an effect of the test compound, it may be in the best interest of the sponsor to conduct additional tests to rule out alternative interpretations based on alterations in sensory, motivational, and/or motor capacities. In addition to the above two criteria, it is recommended that the test of learning and memory be chosen on the basis of its demonstrated sensitivity to the class of compound under investigation, if such information is available in the literature. In the absence of such information, examples of tests that could be made to meet the above criteria include: Delayed-matching-to-position, as described for the adult rat (see paragraph (f)(3) of this section) and for the infant rat (see paragraph (f)(9) of this section); olfactory conditioning, as described in paragraph (f)(13) of this section; and acquisition and retention of schedule-controlled behavior (see paragraphs (f)(4) and (f)(5) of this section). Additional tests for weanling rats are described under paragraphs (f)(20) and (f)(12) of this section, and for adult rats under paragraph (f)(16) of this section.
(vi) Neuropathology. Neuropathological evaluation must be conducted on animals on postnatal day 11 and at the termination of the study. At 11 days of age, one male or female pup must be removed from each litter such that equal numbers of male and female offspring are removed from all litters combined. Of these, six male and six female pups per dose group will be sacrificed for neuropathological analysis. The pups will be sacrificed by exposure to carbon dioxide and immediately thereafter the brains should be removed, weighed, and immersion-fixed in an appropriate aldehyde fixative. The remaining animals will be sacrificed in a similar manner and immediately thereafter their brains removed and weighed. At the termination of the study, one male or one female from each litter will be sacrificed by exposure to carbon dioxide and immediately thereafter the brain must be removed and weighed. In addition, six animals per sex per dose group (one male or female per litter) must be sacrificed at the termination of the study for neuropathological evaluation. Neuropathological analysis of animals sacrificed at the termination of the study must be performed in accordance with § 799.9620. Neuropathological evaluation of animals sacrificed on postnatal day 11 and at termination of the study must include a qualitative analysis and semiquantitative analysis as well as simple morphometrics.
(A) Fixation and processing of tissue samples for postnatal day 11 animals. Immediately following removal, the brain must be weighed and immersion fixed in an appropriate aldehyde fixative. The brains must be postfixed and processed according to standardized published histological protocols such as those discussed in references listed under paragraphs (f)(6), (f)(14), (f)(17), and (f)(21) of this section. Paraffin embedding is acceptable but plastic embedding is preferred and recommended. Tissue blocks and slides must be appropriately identified when stored. Histological sections must be stained for hematoxylin and eosin, or a similar stain according to standard published protocols such as those discussed in references listed under paragraphs (f)(2), (f)(18), and (f)(23) of this section. For animals sacrificed at the termination of the study, methods for fixation and processing of tissue samples are provided in § 799.9620(e)(7)(iv)(A).
(B) Qualitative analysis. The purposes of the qualitative examination are threefold—to identify regions within the nervous system exhibiting evidence of neuropathological alterations, to identify types of neuropathological alterations resulting from exposure to the test substance, and to determine the range of severity of the neuropathological alterations. Representative histological sections from the tissue samples should be examined microscopically by an appropriately trained pathologist for evidence of neuropathological alterations. The following stepwise procedure is recommended for the qualitative analysis. First, sections from the high dose group are compared with those of the control group. If no evidence of neuropathological alterations is found in animals of the high dose group, no further analysis is required. If evidence of neuropathological alterations are found in the high dose group, then animals from the intermediate and low dose group are examined. Subject to professional judgment and the kind of neuropathological alterations observed, it is recommended that additional methods such as Bodian's or Bielchowsky's silver methods and/or immunohistochemistry for glial fibrillary acid protein be used in conjunction with more standard stains to determine the lowest dose level at which neuropathological alterations are observed. Evaluations of postnatal day 11 pups is described in paragraphs (d)(7)(vi)(B)( 1 ) and (d)(7)(vi)(B)( 2 ) of this section. For animals sacrificed at the termination of the study, the regions to be examined and the types of alterations that must be assessed are identified in § 799.9620(e)(7)(iv)(B).
( 1 ) Regions to be examined. The brains should be examined for any evidence of treatment-related neuropathological alterations and adequate samples should be taken from all major brain regions (e.g., olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, midbrain (tectum, tegmentum, and cerebral peduncles), brainstem and cerebellum) to ensure a thorough examination.
( 2 ) Types of alterations. Guidance for neuropathological examination for indications of developmental insult to the brain can be found in paragraphs (f)(8) and (f)(22) of this section. In addition to more typical kinds of cellular alterations (e.g., neuronal vacuolation, degeneration, necrosis) and tissue changes (e.g., astrocytic proliferation, leukocytic infiltration, and cystic formation) particular emphasis should be paid to structural changes indicative of developmental insult including but not restricted to:
( i ) Gross changes in the size or shape of brain regions such as alterations in the size of the cerebral hemispheres or the normal pattern of foliation of the cerebellum.
( ii ) The death of neuronal precursors, abnormal proliferation, or abnormal migration, as indicated by pyknotic cells or ectopic neurons, or gross alterations in regions with active proliferative and migratory zones, alterations in transient developmental structures (e.g., the external germinal zone of the cerebellum, see paragraph (f)(15) of this section).
( iii ) Abnormal differentiation, while more apparent with special stains, may also be indicated by shrunken and malformed cell bodies.
( iv ) Evidence of hydrocephalus, in particular enlargement of the ventricles, stenosis of the cerebral aqueduct and general thinning of the cerebral hemispheres.
(C) Subjective diagnosis. If any evidence of neuropathological alterations is found in the qualitative examination, then a subjective diagnosis will be performed for the purpose of evaluating dose-response relationships. All regions of the brain exhibiting any evidence of neuropathological changes must be included in this analysis. Sections of each region from all dose groups will be coded as to treatment and examined in randomized order. The frequency of each type and the severity of each lesion will be recorded. After all sections from all dose groups including all regions have been rated, the code will be broken and statistical analyses performed to evaluate dose-response relationships. For each type of dose related lesion observed, examples of different ranges of severity must be described. The examples will serve to illustrate a rating scale, such as 1 + , 2 + , and 3 + for the degree of severity ranging from very slight to very extensive.
(D) Simple morphometric analysis. Since the disruption of developmental processes is sometimes more clearly reflected in the rate or extent of growth of particular brain regions, some form of morphometric analysis must be performed on postnatal day 11 and at the termination of the study to assess the structural development of the brain. At a minimum, this would consist of a reliable estimate of the thickness of major layers at representative locations within the neocortex, hippocampus, and cerebellum. For guidance on such measurements see Rodier and Gramann under paragraph (f)(19) of this section.
(e) Data collection, reporting, and evaluation. The following specific information must be reported:
(1) Description of test system and test methods. A description of the general design of the experiment should be provided. This must include:
(i) A detailed description of the procedures used to standardize observations and procedures as well as operational definitions for scoring observations.
(ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. These data do not have to be from studies using prenatal exposures. However, the laboratory must demonstrate competence in evaluation of effects in neonatal animals perinatally exposed to chemicals and establish test norms for the appropriate age group.
(iii) Procedures for calibrating and ensuring the equivalence of devices and the balancing of treatment groups in testing procedures.
(iv) A short justification explaining any decisions involving professional judgement.
(2) Results. The following information must be arranged by each treatment and control group:
(i) In tabular form, data for each animal must be provided showing:
(A) Its identification number and the litter from which it came.
(B) Its body weight and score on each developmental landmark at each observation time.
(C) Total session activity counts and intrasession subtotals on each day measured.
(D) Auditory startle response amplitude per session and intrasession amplitudes on each day measured.
(E) Appropriate data for each repeated trial (or session) showing acquisition and retention scores on the tests of learning and memory on each day measured.
(F) Time and cause of death (if appropriate); any neurological signs observed; a list of structures examined as well as the locations, nature, frequency, and extent of lesions; and brain weights.
(ii) The following data should also be provided, as appropriate:
(A) Inclusion of photomicrographs demonstrating typical examples of the type and extent of the neuropathological alterations observed is recommended.
(B) Any diagnoses derived from neurological signs and lesions, including naturally occurring diseases or conditions, should also be recorded.
(iii) Summary data for each treatment and control group must include:
(A) The number of animals at the start of the test.
(B) The body weight of the dams during gestation and lactation.
(C) Litter size and mean weight at birth.
(D) The number of animals showing each abnormal sign at each observation time.
(E) The percentage of animals showing each abnormal sign at each observation time.
(F) The mean and standard deviation for each continuous endpoint at each observation time. These will include body weight, motor activity counts, auditory startle responses, performance in learning and memory tests, regional brain weights and whole brain weights (both absolute and relative).
(G) The number of animals in which any lesion was found.
(H) The number of animals affected by each different type of lesion, the location, frequency and average grade of each type of lesion for each animal.
(I) The values of all morphometric measurements made for each animal listed by treatment group.
(3) Evaluation of data. An evaluation of test results must be made. The evaluation must include the relationship between the doses of the test substance and the presence or absence, incidence, and extent of any neurotoxic effect. The evaluation must include appropriate statistical analyses. The choice of analyses must consider tests appropriate to the experimental design and needed adjustments for multiple comparisons. The evaluation must include the relationship, if any, between observed neuropathological and behavioral alterations.
(f) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter, L.W., Sobotka, T.J., Tilson, H.A., and Nelson, B.K. Collaborative behavioral teratolgy study: Protocol design and testing procedures. Neurobehavioral Toxicology and Teratology 7:579-586 (1985).
(2) Bennett, H.S., Wyrick, A.D., Lee, S.W., and McNeil, J.H. Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains. Stain Technology 51:71-97 (1976).
(3) Bushnell, P.J. Effects of delay, intertrial interval, delay behavior and trimethyltin on spatial delayed response in rats. Neurotoxicology and Teratology 10:237-244 (1988).
(4) Campbell, B.A. and Haroutunian, V. Effects of age on long-term memory: Retention of fixed interval responding. Journal of Gerontology 36:338-341 (1981).
(5) Cory-Slechta, D.A., Weiss, B., and Cox, C. Delayed behavioral toxicity of lead with increasing exposure concentration. Toxicology and Applied Pharmacology 71:342-352 (1983).
(6) Di Sant Agnese, P. A. and De Mesy Jensen, K.L. Dibasic staining of large epoxy tissue sections and application to surgical pathology. American Journal of Clinical Pathology 81:25-29 (1984).
(7) U.S. Environmental Protection Agency. Neurotoxicity Screening Battery. In: Pesticide Assessment Guidelines, Subdivision F, Addendum 10. EPA 540/09-91-123. NTIS PB 91-154617 (1991).
(8) Friede, R. L. Developmental Neuropathology. Springer-Verlag, New York. pp. 1-23, 297-313, 326-351 (1975).
(9) Green, R.J. and Stanton, M.E. Differential ontogeny of working memory and reference memory in the rat. Behavioral Neuroscience 103:98-105 (1989).
(10) Ison, J.R. Reflex modification as an objective test for sensory processing following toxicant exposure. Neurobehavioral Toxicology and Teratology 6:437-445 (1984).
(11) Korenbrot, C.C., Huhtaniemi, I.T., and Weiner, R.I. Preputial separation as an external sign of pubertal development in the male rat. Biology of Reproduction 17:298-303 (1977).
(12) Krasnegor, N.A., Blass, E.M., Hofer, M.A., and Smotherman, W.P. (eds.) Perinatal Development: A Psychobiological Perspective. Academic Press, Orlando. pp.11-37, 145-167. (1987).
(13) Kucharski, D. and Spear, N.E. Conditioning of aversion to an odor paired with peripheral shock in the developing rat. Developmental Psychobiology 17:465-479 (1984).
(14) Luna, L. G. (editor). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. (Third Edition). McGraw-Hill, New York. pp. 1-31 (1968).
(15) Miale, I. L. and Sidman, R.L. An autoradiographic analysis of histogenesis in the mouse cerebellum. Experimental Neurology. 4:277-296 (1961).
(16) Miller, D.B. and Eckerman, D.A. Learning and memory measures. In: Neurobehavioral Toxicology, Z. Annau (ed). Johns Hopkins University Press, Baltimore. pp. 94-149 (1986).
(17) Pender, M.P. A simple method for high resolution light microscopy of nervous tissue. Journal of Neuroscience Methods. 15:213-218 (1985).
(18) Ralis, H.M., Beesley, R.A., and Ralis, Z.A. Techniques in Neurohistology. Butterworths, London. pp. 57-145 (1973).
(19) Rodier, P.M. and Gramann, W.J. Morphologic effects of interference with cell proliferation in the early fetal period. Neurobehavioral Toxicology 1:129-135 (1979).
(20) Spear, N.E. and Campbell, B.A. (eds.) Ontogeny of Learning and Memory. Erlbaum, New Jersey. pp. 101-133, 157-224 (1979).
(21) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H. Neuropathological methods for the detection of neurotoxic disease. In: Experimental and Clinical Neurotoxicology. Spencer, P.S. and Schaumburg, H.H. (eds.). Williams and Wilkins, Baltimore. pp. 743-757 (1980).
(22) Suzuki, K. Special vulnerabilities of the developing nervous system to toxic substances. In: Experimental and Clinical Neurotoxicology. Spencer, P.S. and Schaumburg, H.H. (eds.). Williams and Wilkins, Baltimore. pp. 48-61 (1980). (23) Luna, L.G. (ed.). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. (Third Edition). McGraw-Hill, New York. pp. 32-46 (1968).