Title 40—Protection of Environment--Volume 1
PART 798—HEALTH EFFECTS TESTING GUIDELINES
§ 798.5275   Sex-linked recessive lethal test in drosophila melanogaster.
(a) Purpose. The sex-linked recessive lethal (SLRL) test using Drosophila melanogaster detects the occurrence of mutations, both point mutations and small deletions, in the germ line of the insect. This test is a forward mutation assay capable of screening for mutations at about 800 loci on the X-chromosome. This represents about 80 percent of all X-chromosome loci. The X-chromosome represents approximately one-fifth of the entire haploid genome.
(b) Definitions. (1) Lethal mutation is a change in the genome which, when expressed, causes death to the carrier.
(2) Recessive mutation is a change in the genome which is expressed in the homozygous or hemizygous condition.
(3) Sex-Linked genes are present on the sex (X or Y) chromosomes. Sex-linked genes in the context of this guideline refer only to those located on the X-chromosome.
(c) Reference substances. These may include, but need not be limited to, ethyl methanesulfonate or N-nitroso-dimethylamine.
(d) Test method —(1) Principle. Mutations in the X-chromosome of D. melanogaster are phenotypically expressed in males carrying the mutant gene. When the mutation is lethal in the hemizygous condition, its presence is inferred from the absence of one class of male offspring out of the two that are normally produced by a heterozygous female. The SLRL test takes advantage of these facts by means of specially marked and arranged chromosomes.
(2) Description. Wild-type males are treated and mated to appropriate females. Female offspring are mated individually to their brothers, and in the next generation the progeny from each separate dose are scored for phenotypically wild-type males. Absence of these males indicates that a sex-linked recessive lethal mutation has occurred in a germ cell of the P 1 male.
(3) Drosophila stocks. Males of a well-defined wild type stock and females of the Muller-5 stock may be used. Other appropriately marked female stocks with multiple inverted X-chromosomes may also be used.
(4) Control groups —(i) Concurrent controls. Concurrent positive and negative (vehicle) controls shall be included in each experiment.
(ii) Positive controls. Examples of positive controls include ethyl methanesulfonate and N-nitroso-dimethylamine.
(iii) Other positive controls. Other positive control reference substances may be used.
(iv) Negative controls. Negative (vehicle) controls shall be included. The size of the negative (vehicle) control group shall be determined by the availability of appropriate laboratory historical control data.
(5) Test chemicals —(i) Vehicle. Test chemicals should be dissolved in water. Compounds which are insoluble in water may be dissolved or suspended in appropriate vehicles (e.g., a mixture of ethanol and Tween-60 or 80) and then diluted in water or saline prior to administration. Dimethylsulfoxide should br avoided as a vehicle.
(ii) Dose levels. For the initial assessment of mutagenicity, it is sufficient to test a single dose of the test substance for screening purposes. This dose should be the maximum tolerated dose, or that which produces some indication of toxicity, or shall be the highest dose attainable. For dose-response purposes, at least three additional dose levels should be used.
(iii) Route of administration. Exposure may be oral, by injection or by exposure to gases or vapors. Feeding of the test compound may be done in sugar solution. When necessary, substances may be dissolved in 0.7 percent NaCl solution and injected into the thorax or abdomen.
(e) Test performance —(1) Treatment and mating. Wild-type males (3 to 5 days old) shall be treated with the test substance and mated individually to an appropriate number of virgin females from the Muller-5 stock or females from another appropriately marked (with multiply-inverted X-chromosomes) stock. The females shall be replaced with fresh virgins every 2 to 3 days to cover the entire germ cell cycle. The offspring of these females are scored for lethal effects corresponding to the effects on mature sperm, mid or late stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment.
(2) F 1 1 matings. Heterozygous F 1 females from the above crosses shall be allowed to mate individually (i.e., one female per vial) with their brothers. In the F 2 generation, each culture shall be scored for the absence of wild-type males. If a culture appears to have arisen from an F 1 female carrying a lethal in the parental X-chromosome (i.e., no males with the treated chromosome are observed), daughters of that female with the same genotype shall be tested to ascertain if the lethality is repeated in the next generation.
(3) Number of matings. (i) The test should be designed with a predetermined sensitivity and power. The number of flies in each group should reflect these defined parameters. The spontaneous mutant frequency observed in the appropriate control group will strongly influence the number of treated chromosomes that must be analysed to detect substances which show mutation rates close to those of the controls.
(ii) Test results should be confirmed in a separate experiment.
(f) Data and report —(1) Treatment of results. Data shall be tabulated to show the number of chromosomes tested, the number of nonfertile males and the number of lethal chromosomes at each exposure concentration and for each mating period for each male treated. Numbers of clusters of different size per male shall be reported.
(2) Statistical evaluation. Data shall be evaluated by appropriate statistical techniques.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of sex-lined recessive lethals. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of sex-linked recessive lethals or a statistically significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the SLRL test in D. melanogaster indicate that under the test conditions the test agent causes mutations in germ cells of this insect.
(ii) Negative results indicate that under the test conditions the test substance is not mutagenic in D. melanogaster.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information shall be reported.
(i) Drosophila stock used in the assay, age of insects, number of males treated, number of sterile males, number of F 2 cultures established, number of F 2 cultures without progeny.
(ii) Test chemical vehicle, treatment and sampling schedule, exposure levels, toxicity data, negative (vehicle) and positive controls, if appropriate.
(iii) Criteria for scoring lethals.
(iv) Number of chromosomes tested, number of chromosomes scored, number of chromosomes carrying a lethal mutation.
(v) Historical control data, if available.
(vi) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Sobels, F.H., Vogel, E. “The capacity of Drosophila for detecting relevant genetic damage,” Mutation Research 41:95-106 (1976).
(2) Wurgler F.E., Sobels F.H., Vogel E. “ Drosophila as assay system for detecting genetic changes,” Handbook of mutagenicity test procedures. Eds. Kilbey, B.J., Legator, M., Nichols, W., Ramel, C., (Amsterdam: Elsevier/North Holland Biomedical Press, 1977) pp. 335-373.
§ 798.5300   Detection of gene mutations in somatic cells in culture.
(a) Purpose. Mammalian cell culture systems may be used to detect mutations induced by chemical substances. Widely used cell lines include L5178Y mouse lymphoma cells and the CHO and V-79 lines of Chinese hamster cells. In these cell lines the most commonly used systems measure mutation at the thymidine kinase (TK), hypoxanthine-guanine-phosphoribosyl transferase (HPRT) and Na = /K = ATPase loci. The TK and HPRT mutational systems detect base pair mutations, frameshift mutations, and small deletions; the Na = /K = ATPase system detects base pair mutations only.
(b) Definitions. (1) A forward mutation assay detects a gene mutation from the parental type to the mutant form which gives rise to a change in an enzymatic or functional protein.
(2) Base pair mutagens are agents which cause a base change in the DNA.
(3) Frameshift mutagens are agents which cause the addition or deletion of single or multiple base pairs in the DNA molecule.
(4) Phenotypic expression time is a period during which unaltered gene products are depleted from newly mutated cells.
(c) Reference substances. These may include, but need not be limited to, ethyl methanesulfonate, N-nitroso-dimethylamine, 2-acetylaminofluorene, 7,12-dimethylbenzanthracene or hycanthone.
(d) Test method —(1) Principle. Cells are exposed to 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. Cells deficient in thymidine kinase (TK) due to the forward mutation TK = → TK − are resistant to the cytotoxic effects of pyrimidine analogues such as bromodeoxyuridine (BrdU), fluorodeoxyuridine (FdU) or trifluorothymidine (TFT). The deficiency of the “salvage” enzyme thymidine kinase means that these antimetabolites are not incorporated into cellular nucleotides and the nucleotides needed for cellular metabolism are obtained solely from de novo synthesis. However, in the presence of thymidine kinase, BrdU, FdU or TFT are incorporated into the nucleotides, resulting in inhibition of cellular metabolism and cytotoxicity. Thus mutant cells are able to proliferate in the presence of BrdU, FdU or TFT whereas normal cells, which contain thymidine kinase, are not. Similarly cells deficient in HPRT are selected by resistance to 8-azaguanine (AG) or 6-thioguanine (TG) and cells with altered Na = /K = ATPase are selected by resistance to ouabain.
(2) Description. Cells in suspension or monolayer culture are exposed to the test substance, both with and without metabolic activation, for a defined period of time. Cytotoxicity is determined by measuring the colony forming ability or growth rate of the cultures after the treatment period. The treated cultures are maintained in growth medium for a sufficient period of time—characteristic of each selected locus—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. After a suitable incubation time, cell colonies are counted. The number of mutant colonies in selective medium is adjusted by the number of colonies in nonselective medium to derive the mutant frequency.
(3) Cells —(i) Type of cells used in the assay. A variety of cell lines are available for use in this assay including subclones of L5178Y, CHO cells or V-79 cells. Cell types used in this assay should have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a low spontaneous mutation frequency. Cells should be checked for Mycoplasma contamination and may be periodically checked for karyotype stability.
(ii) Cell growth and maintenance. Appropriate culture media and incubation conditions (culture vessels, CO 2 concentrations, temperature and humidity) shall be used.
(4) Metabolic activation. Cells shall be exposed to test substance both in the presence and absence of an appropriate metabolic activation system.
(5) Control groups. Positive and negative (untreated and/or vehicle) controls shall be included in each experiment. When metabolic activation is used, the positive control substance shall be known to require such activation.
(6) Test chemicals —(i) Vehicle. Test substances may be prepared in culture media or dissolved or suspended in appropriate vehicles prior to treatment of the cells. The final concentration of the vehicle shall not interfere with cell viability or growth rate. Treatment vessels should be chosen to ensure that there is no visible interaction, such as etching, between the solvent, the test chemical, and the vessel.
(ii) Exposure concentrations. (A) 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 number of cells per culture is based on the expected background mutant frequency; a general guide is to use a number which is 10 times the inverse of this frequency.
(B) Several concentrations (usually at least 4) of the test substance shall be used. Generally, these shall yield a concentration-related toxic effect. The highest concentration shall produce a low level of survival (approximately 10 percent), and the survival in the lowest concentration shall approximate the negative control. Cytotoxicity shall be determined after treatment with the test substance both in the presence and in the absence of an exogenous metabolic activation system. Relatively insoluble substances should be tested up to their limit of solubility under culture conditions. For freely-soluble nontoxic substances the highest concentration used should be determined on a case-by-case basis.
(e) Test performance. (1) Cells shall be exposed to the test substance both with and without exogenous metabolic activation. Exposure shall be for a suitable period of time, in most cases 1 to 5 hours is effective; exposure time may be extended over one or more cell cycles.
(2) At the end of the exposure period, cells shall be washed and cultured to determine viability and to allow for expression of the mutant phenotype.
(3) At the end of the expression period, which shall be sufficient to allow near optimal phenotypic expression of induced mutants, cells should be grown in medium with and without selective agent(s) for determination of number of mutants and cloning efficiency, respectively.
(4) Results shall be confirmed in an independent experiment. When appropriate, a single positive response should be confirmed by testing over a narrow range of concentrations.
(f) Data and report —(1) Treatment of results. Data shall be presented in tabular form. Individual colony counts for the treated and control groups shall be presented for both mutation induction and survival. Survival and cloning efficiencies shall be given as a percentage of the controls. Mutant frequency shall be expressed as number of mutants per number of surviving cells.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant concentration-related increase in the mutant frequency. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically significant concentration-related increase in the mutant frequency or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results for an in vitro mammalian cell gene mutation test indicate that, under the test conditions, a substance induces gene mutations in the cultured mammalian cells used.
(ii) Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(i) Cell type used, number of cell cultures, methods used for maintenance of cell cultures.
(ii) Rationale for selection of concentrations and number of cultures.
(iii) Test conditions: composition of media, CO 2 concentration, concentration of test substance, vehicle, incubation temperature, incubation time, duration of treatment, cell density during treatment, type of metabolic activation system, positive and negative controls, length of expression period (including number of cells seeded and subculture and feeding schedules, if appropriate), selective agent(s).
(iv) Methods used to enumerate numbers of viable and mutant cells.
(v) Dose-response relationship, where possible.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Amacher, D.E., Paillet, S.C., Ray, V. “Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to genetic toxicology testing,” Mutation Research, 64:391-406 (1979).
(2) Amacher, D.E., Paillet, S.C., Turner, G.N., Ray, V.A. Salsburg, V.A. “Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test validation and interpretation,” Mutation Research, 72:447-474 (1980).
(3) Bradley, M.O., Bhuyan B., Francis, M.C., Langenback, R., Peterson, A., Huberman, E. “Mutagenesis by chemical agents in V-79 Chinese hamster cells: a review and analysis of the literature: a report of the Gene-Tox Program,” Mutation Research, 87:81-142 (1981).
(4) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., Brown, M.M. “Validation and characterization of the L5178Y TK = / − mouse lymphoma mutagen assay system,” Mutation Research, 59:61-108 (1979).
(5) Clive, D., Spector, J.F.S. “Laboratory procedures for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells,” Mutation Research, 31:17-29 (1975).
(6) Hsie, A.W., Casciano, D.A., Couch, D.B., Krahn, D.F., O'Neill, J.P., Whitfield, B.L. “The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals: a report of the U.S. EPA's Gene-Tox Program,” Mutation Research, 86:193-214 (1981).
§ 798.5375   In vitro mammalian cytogenetics.
(a) Purpose. The in vitro cytogenetics test is a mutagenicity test system for the detection of chromosomal aberrations in cultured mammalian cells. Chromosomal aberrations may be either structural or numerical. However, because cytogenetic assays are usually designed to analyse cells at their first post-treatment mitosis and numerical aberrations require at least one cell division to be visualized, this type of aberration is generally not observed in a routine cytogenetics assay. Structural aberrations may be of two types, chromosome or chromatid.
(b) Definitions. (1) Chromosome-type aberrations are changes which result from damage expressed in both sister chromatids at the same time.
(2) Chromatid-type aberrations are damage expressed as breakage of single chromatids or breakage and/or reunion between chromatids.
(c) Reference substances. Not applicable.
(d) Test method —(1) Principle. In vitro cytogenetics assays may employ cultures of established cell lines, cell strains or primary cell cultures. Cell cultures are exposed to the test substance both with and without metabolic activation. Following exposure of cell cultures to test substances, they are treated with a spindle inhibitor (e.g., colchicine or Colcemid#) to arrest cells in a metaphase-like stage of mitosis (c-metaphase). Cells are then harvested and chromosome preparations made. Preparations are stained and metaphase cells are analyzed for chromosomal aberrations.
(2) Description. Cell cultures are exposed to test compounds and harvested at various intervals after treatment. Prior to harvesting, cells are treated with a spindle inhibitor (e.g., colchicine or Colcemid#) to accumulate cells in c-metaphase. Chromosome preparations from cells are made, stained and scored for chromosomal aberrations.
(3) Cells —(i) Type of cells used in the assay. There are a variety of cell lines or primary cell cultures, including human cells, which may be used in the assay. Established cell lines and strains should be checked for Mycoplasma contamination and may be periodically checked for karyotype stability.
(ii) Cell growth and maintenance. Appropriate culture media, and incubation conditions (culture vessels CO 2 concentrations, temperature and humidity) shall be used.
(4) Metabolic activation. Cells shall be exposed to test substance both in the presence and absence of an appropriate metabolic activation system.
(5) Control groups. Positive and negative (untreated and/or vehicle) controls both with and without metabolic activation shall be included in each experiment. When metabolic activation is used, the positive control substance shall be known to require such activation.
(6) Test chemicals —(i) Vehicle. Test substances may be prepared in culture media or dissolved or suspended in appropriate vehicles prior to treatment of the cells. Final concentration of the vehicle shall not interfere with cell viability or growth rate. Treatment vessels should be chosen to ensure that there is no visible interaction, such as etching, between the solvent, the test chemical, and the vessel.
(ii) Exposure concentrations. Multiple concentrations of the test substance over a range adequate to define the response should be tested. Generally the highest test substance concentrations tested with and without metabolic activation should show evidence of cytotoxicity or reduced mitotic activity. Relatively insoluble substances should be tested up to the limit of solubility. For freely soluble nontoxic chemicals, the upper test chemical concentration should be determined on a case by case basis.
(e) Test performance —(1) Established cell lines and strains. Prior to use in the assay, cells should be generated from stock cultures, seeded in culture vessels at the appropriate density and incubated at 37 °C.
(2) Human lymphocyte cultures. Heparinized or acid-citrate-dextrose whole blood should be added to culture medium containing a mitogen, e.g., phytohemagglutinin (PHA) and incubated at 37 °C. White cells sedimented by gravity (buffy coat) or lymphocytes which have been purified on a density gradient may also be utilized.
(3) Treatment with test substance. For established cell lines and strains, cells in the exponential phase of growth shall be treated with test substances in the presence and absence of an exogenous metabolic activation system. Mitogen-stimulated human lymphocyte cultures may be treated with the test substance in a similar manner.
(4) Number of cultures. At least two independent cultures shall be used for each experimental point.
(5) Culture harvest time. (i) For established cell lines and strains, multiple harvest times are recommended. However, for screening purposes, a single harvest time may be appropriate. If the test chemical changes the cell cycle length, the fixation intervals should be changed accordingly. If a single harvest time is selected, supporting data for the harvest time should be presented in such a study.
(ii) For human lymphocyte cultures, the substance to be tested may be added to the cultures at various times after mitogen stimulation so that there is a single harvest time after the initiation of the cell culture. Alternatively, a single treatment may be followed by multiple harvest times. Harvest time should be extended for those chemicals which induce an apparent cell cycle delay. Because the population of human lymphocytes is only partially synchronized, a single treatment, at, or close to, the time when metaphase stages first appear in the culture will include cells in all phases of the division cycle. Therefore, a single harvest at the time of second mitosis may be carried out for screening purposes.
(iii) Cell cultures shall be treated with a spindle inhibitor, (e.g., colchicine or Colcemid ®), 1 or 2 hours prior to harvesting. Each culture shall be harvested and processed separately for the preparation of chromosomes.
(6) Chromosome preparation. Chromosome preparation involves hypotonic treatment of the cells, fixation and staining.
(7) Analysis. Slides shall be coded before analysis. In human lymphocytes, only cells containing 46 centromeres shall be analyzed. In established cell lines and strains, only metaphases containing ±2 centromeres of the modal number shall be analyzed. Uniform criteria for scoring aberrations shall be used.
(8) Confirmatory tests. When appropriate, a single positive response shall be confirmed by testing over a narrow range of concentrations.
(f) Data and report —(1) Treatment of results. Data shall be presented in a tabular form. Different types of structural chromosomal aberrations shall be listed with their numbers and frequencies for experimental and control groups. Data should be evaluated by appropriate statistical methods. Gaps or achromatic lesions are recorded separately and not included in the total aberration frequency.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of structural chromosomal aberrations. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of structural chromosomal aberrations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the in vitro cytogenetics assay indicate that under the test conditions the test substance induces chromosomal aberrations in cultured mammalian somatic cells.
(ii) Negative results indicate that under the test conditions the test substance does not induce chromosomal aberrations in cultured mammalian somatic cells.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(i) Cells used, density and passage number at time of treatment, number of cell cultures.
(ii) Methods used for maintenance of cell cultures including medium, temperature and CO 2 concentration.
(iii) Test chemical vehicle, concentration and rationale for the selection of the concentrations used in the assay, duration of treatment.
(iv) Details of both the protocol used to prepare the metabolic activation system and of its use in the assay.
(v) Identity of spindle inhibitor, its concentration and duration of treatment.
(vi) Date of cell harvest.
(vii) Positive and negative controls.
(viii) Methods used for preparation of slides for microscopic examination.
(ix) Number of metaphases analysed.
(x) Mitotic index where applicable.
(xi) Criteria for scoring aberrations.
(xii) Type and number of aberrations, given separately for each treated and control culture, total number of aberrations per group; frequency distribution of number of chromosomes in established cell lines and strains.
(xiii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted.
(1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test,” Mutation Research, 31:347-364 (1975).
(2) Evans, H.J. “Cytological methods for detecting chemical mutagens,” Chemical mutagens, principles and methods for their detection, Vol. 4, Ed. A. Hollaender (New York, London: Plenum Press, 1976) pp. 1-29.
(3) Howard, P.N., Bloom, A.D., Krooth, R.S. “Chromosomal aberrations induced by N-methyl-N′-nitro-N-nitrosoguanidine in mammalian cells,” In Vitro 7:359-365 (1972).
(4) Ishidate, M. Jr., Odashima, S. “Chromosome tests with 134 compounds on Chinese hamster cells in vitro: A screening for chemical carcinogens,” Mutation Research, 48:337-354 (1975).
(5) Preston, R.J., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V., Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J.S., “Mammalian in vivo and in vitro cytogenetic assays: A report of the Gene-tox Program,” Mutation Research, 87:143-188 (1981).
§ 798.5385   In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.
(a) Purpose. The in vivo bone marrow cytogenetic test is a mutagenicity test for the detection of structural chromosomal aberrations. Chromosomal aberrations are generally evaluated in first post-treatment mitoses. With the majority of chemical mutagens, induced aberrations are of the chromatid type but chromosome type aberrations also occur.
(b) Definitions. (1) Chromosome-type aberrations are changes which result from damage expressed in both sister chromatids at the same time.
(2) Chromatid-type aberrations are damage expressed as breakage of single chromatids or breakage and/or reunion between chromatids.
(c) Reference substances. Not applicable.
(d) Test method —(1) Principle. Animals are exposed to test chemicals by appropriate routes and are sacrificed at sequential intervals. Chromosome preparations are made from bone marrow cells. The stained preparations are examined and metaphase cells are scored for chromosomal aberrations.
(2) Description. The method employs bone marrow of laboratory rodents which have been exposed to test chemicals. Prior to sacrifice, animals are further treated with a spindle inhibitor, (e.g., colchicine or Colcemid ®) to arrest the cells in c-metaphase. Chromosome preparations from the cells are stained and scored for chromosomal aberrations.
(3) Animal selection —(i) Species and strain. Any appropriate mammalian species may be used. Examples of commonly used rodent species are rats, mice, and hamsters.
(ii) Age. Healthy young adult animals shall be used.
(iii) Number and sex. At least five female and five male animals per experimental and control group shall be used. Thus, 10 animals would be sacrificed per time per group treated with the test compound if several test times after treatment are included in the experimental schedule. The use of a single sex or smaller number of animals should be justified.
(iv) Assignment to groups. Animals shall be randomized and assigned to treatment and control groups.
(4) Control groups —(1) Concurrent controls. (i) Concurrent positive and negative (vehicle) controls shall be included in the assay.
(ii) Positive controls. A single dose positive control showing a significant response at any one time point is adequate. A compound known to produce chromosomal aberrations in vivo shall be employed as the positive control.
(5) Test chemicals —(i) Vehicle. When possible, test chemicals shall be dissolved in isotonic saline or distilled water. Water insoluble chemicals may be dissolved or suspended in appropriate vehicles. The vehicles used shall neither interfere with the test chemical nor produce toxic effects. Fresh preparations of the test compound should be employed.
(ii) Dose levels. For an initial assessment, one dose of the test substance may be used, the dose being the maximum tolerated dose (to a maximum of 5,000 mg/kg) or that producing some indication of cytotoxicity (e.g., partial inhibition of mitosis) or shall be the highest dose attainable (to a maximum of 5,000 mg/kg). Additional dose levels may be used. For determination of dose-response, at least three dose levels should be used.
(iii) Route of administration. The usual routes are oral or by intraperitoneal injection. Other routes may be appropriate.
(iv) Treatment schedule. In general, test substances should be administered once only. However, based on toxicological information a repeated treatment schedule may be employed.
(e) Test performance —(1) Generally the test may be performed in two assays. (i) Animals should be treated with the test substance once at the selected dose(s). Samples should be taken at three times after treatment. For rodents, the central sampling interval is 24 hours. Since cell cycle kinetics can be influenced by the test substance, one earlier and one later sampling interval adequately spaced within the range of 6 to 48 hours shall be applied. Where the additional dose levels are tested in a subsequent experiment, samples shall be taken at the predetermined most sensitive interval or, if this is not established, at the central sampling time. If the most sensitive interval is known and documented with data, only this one time point shall be sampled.
(ii) If a repeated treatment schedule is used at the selected dose(s), samples shall be taken 6 and 24 hours after the last treatment; other sampling times may be used if justified. Where the additional dose levels are tested in a subsequent experiment, samples shall be taken at the predetermined most sensitive interval or, if this is not established, at 6 hours after the last treatment.
(2) Administration of spindle inhibitor. Prior to sacrifice, animals shall be injected IP with an appropriate dose of a spindle inhibitor (e.g., colchicine or Colcemid ®) to arrest cells in c-metaphase.
(3) Preparation of slides. Immediately after sacrifice, the bone marrow shall be obtained, exposed to hypotonic solution, and fixed. The cells shall then be spread on slides and stained. Chromosome preparations shall be made following standard procedures.
(4) Analysis. The number of cells to be analyzed per animal should be based upon the number of animals used, the negative control frequency, the predetermined sensitivity, and the power chosen for the test. Slides shall be coded before microscopic analysis.
(f) Data and report —(1) Treatment of results. Data should be presented in tabular form for both cells and animals. Different types of structural chromosomal aberrations should be listed with their numbers and a mean frequency per cell for each animal in all treated and control groups. Gaps (achromatic lesions) should be recorded separately and not included in the total abberration frequency. Differences among animals within each group should be considered before making comparisons between treated and control groups.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of structual chromosomal aberrations or abnormal metaphase figures. Another criterion may be based upon detection of a reproducible and statistically significant positive response for a least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of chromosomal aberrations or abnormal metaphase figures or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the in vivo bone marrow cytogenetics assay indicate that under the test conditions the test substance induces chromosomal aberrations in the bone marrow of the test species.
(ii) Negative results indicate that under the test conditions, the test substance does not induce chromosomal aberrations in the bone marrow of the test species.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(i) Species, strain, age, weight, number and sex of animals in each treatment and control group.
(ii) Test chemical vehicle, dose levels used, rationale for dose selection.
(iii) Route of administration, treatment and sampling schedules, toxicity data, negative and positive controls.
(iv) Identity of spindle-inhibitor, its concentration and duration of treatment.
(v) Details of the protocol used for chromosome preparation, number of cells scored per animal, type and number of aberrations given separately for each treated and control animal.
(vi) Mitotic index, where applicable.
(vii) Criteria for scoring aberrations.
(viii) Number and frequency of aberrant cells per animal in each treatment and control groups.
(ix) Total number of aberrations per group.
(x) Number of cells with aberrations per group.
(xi) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Adler, I.D., Ramarao, G., Epstein, S.S. “In vivo cytogenetic effects of trimethyl-phosphate and of TEPA on bone marrow cells of male rats,” Mutation Research, 13:263-273 (1971).
(2) Evans, H.J. “Cytological methods for detecting chemical mutagens,” Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4. Ed. A. Hollaender (New York and London: Plenum Press, 1976) pp. 1-29.
(3) Kilian, J.D., Moreland, F.E. Benge, M.C., Legator, M.S., Whorton, E.B. Jr. “A collaborative study to measure intralaboratory variation with the in vivo bone morrow metaphase procedure,” Handbook of mutagenicity test procedures. Eds. Kilby, B.J., Legator, M. Nichols, C., Ramel, D., (Amsterdam: Elsevier/North Holland Biomedical Press, 1977) 243-260.
(4) Preston, J.R., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V. Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J. “Mammalian in vivo and vitro cytogenetics assays: Report of the Gene-Tox Program,” Mutation Research, 87:143-188 (1981).
§ 798.5395   In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay.
(a) Purpose. The micronucleus test is a mammalian in vivo test which detects damage of the chromosomes or mitotic apparatus by chemicals. Polychromatic erythrocytes in the bone marrow of rodents are used in this assay. When the erythroblast develops into an erythrocyte the main nucleus is extruded and may leave a micronucleus in the cytoplasm. The visualization of micronuclei is facilitated in these cells because they lack a nucleus. Micronuclei form under normal conditions. The assay is based on an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow of treated animals.
(b) Definition. Micronuclei are small particles consisting of acentric fragments of chromosomes or entire chromosomes, which lag behind at anaphase of cell division. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm.
(c) Reference substances. Not applicable.
(d) Test method —(1) Principle. (i) Animals are exposed to test substance by an appropriate route. They are sacrificed, the bone marrow extracted and smear preparations made and stained. Polychromatic erythrocytes are scored for micronuclei under the microscope.
(ii) Micronuclei may also be detected in other test systems:
(A) Tissue culture.
(B) Plants.
(C) Blood smears.
(D) Fetal tissues.
(E) Meiotic cells.
(F) Hepatic cells.
(iii) The present guideline is based on the mammalian bone marrow assay.
(2) Description. The method employs bone marrow of laboratory mammals which are exposed to test substances.
(3) Animal selection —(i) Species and strain. Mice are recommended. However, any appropriate mammalian species may be used.
(ii) Age. Young adult animals shall be used.
(iii) Number and sex. At least five female and five male animals per experimental and control group shall be used. Thus, 10 animals would be sacrificed per time per group if several test times after treatment were included in the experimental schedule. The use of a single sex or a smaller number of animals should be justified.
(iv) Assignment to groups. Animals shall be randomized and assigned to treatment and control groups.
(4) Control groups —(i) Concurrent controls. Concurrent positive and negative (vehicle) controls shall be included in each assay.
(ii) Positive controls. A compound known to produce micronuclei in vivo shall be employed as the positive control.
(5) Test chemicals —(i) Vehicle. When appropriate for the route of administration, solid and liquid test substances should be dissolved or suspended in distilled water or isotonic saline. Water insoluble chemicals may be dissolved or suspended in appropriate vehicles. The vehicle used shall neither interfere with the test compound nor produce toxic effects. Fresh preparations of the test compound should be employed.
(ii) Dose levels. For an initial assessment, one dose of the test substance may be used, the dose being the maximum tolerated dose (to a maximum of 5,000 mg/kg) or that producing some indication of cytotoxicity, e.g., a change in the ratio of polychromatic to normochromatic erythrocytes. Additional dose levels may be used. For determination of dose response, at least three dose levels shall be used.
(iii) Route of administration. The usual routes of administration are IP or oral. Other routes may be appropriate.
(iv) Treatment schedule. Test substances should generally be administered only once. However, based upon toxicological information a repeated treatment schedule may be employed.
(e) Test performance —(1) Treatment and sampling times. (i) Animals shall be treated with the test substance once at the highest tolerated dose. Sampling times should coincide with the maximum responses of the assay which varies with the test substance. Therefore, using the highest dose, bone marrow samples should be taken at least three times, starting not earlier than 12 hours after treatment, with appropriate intervals following the first sample but not extending beyond 72 hours. When other doses are used sampling shall be at the maximum sensitive period, or, if that is not known, approximately 24 hours after treatment. Other appropriate sampling times may be used in addition. If the most sensitive interval is known and documented with data, only this one time point need be sampled.
(ii) If a repeated treatment schedule is used, samples shall be taken at least three times, starting not earlier than 12 hours after the last treatment and at appropriate intervals following the first sample, but not extending beyond 72 hours.
(iii) Bone marrow shall be obtained immediately after sacrifice. Cells shall be prepared, put on slides, spread as a smear and stained.
(2) Analysis. Slides shall be coded before microscopic analysis. At least 1,000 polychromatic erythrocytes per animal shall be scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes should be determined for each animal by counting a total of 200 erythrocytes. To ensure consistency with OECD and other guidelines, 1,000 polychromatic erythrocytes are recommended. Additional information may be obtained by scoring normochromatic erythrocytes for micronuclei.
(f) Data and report —(1) Treatment of results. Criteria for scoring micronuclei shall be given. Individual data shall be presented in a tabular form including positive and negative (vehicle) controls and experimental groups. The number of polychromatic erythrocytes scored, the number of micronucleated polychromatic erythrocytes, the percentage of micronucleated cells, the number of micronucleated normochromatic erythrocytes, and, if applicable, the percentage of micronucleated erythrocytes and the ratio of normochromatic to polychromatic erythrocytes shall be listed separately for each experimental and control animal. Absolute numbers shall be included if percentages are reported.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive response, one of which is a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) The results of the micronucleus test provide information on the ability of a chemical to induce micronuclei in polychromatic erythrocytes of the test species under the conditions of the test. This damage may have been the result of chromosomal damage or damage to the mitotic apparatus.
(ii) Negative results indicate that under the test conditions the test substance does not produce micronuclei in the bone marrow of the test species.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J, the following specific information shall be reported:
(i) Species, strain, age, weight, number and sex of animals in each treatment and control group.
(ii) Test chemical vehicle, dose levels used, rationale for dose selection.
(iii) Rationale for and description of treatment and sampling schedules, toxicity data, negative and positive controls.
(iv) Details of the protocol used for slide preparation.
(v) Criteria for identifying micronucleated erythrocytes.
(vi) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Cihak, R. “Evaluation of benzidine by the micronucleus test,” Mutation Research, 67: 383-384 (1979).
(2) Cole, R.J., Taylor, N., Cole, J., Arlett, C.F. “Short-term tests for transplacentally active carcinogens. 1. Micronucleus formation in fetal and maternal mouse erythroblasts,” Mutation Research, 80: 141-157 (1981).
(3) Kliesch, U., Danford, N., Adler, I.D. “Micronucleus test and bone-marrow chromosome analysis. A comparison of 2 methods in vivo for evaluating chemically induced chromosomal alterations,” Mutation Research, 80: 321-332 (1981).
(4) Matter, B., Schmid, W. “Trenimon-induced chromosomal damage in bone-marrow cells of six mammalian species, evaluated by the micronucleus test,” Mutation Research, 12: 417-425 (1971).
(5) Schmid, W. “The micronucleus test,” Mutation Research, 31:9-15 (1975).
(6) Schmid, W. “The micronucleus test for cytogenetic analysis,” Chemical Mutagens, Principles and Methods for their Detection. Vol. 4 Hollaender A, (Ed. A ed. (New York and London: Plenum Press, (1976) pp. 31-53.
(7) Heddle, J.A., Hite, M., Kurkhart, B., Mavournin, K., MacGregor, J.T., Newell, G.W., Salamone, M.F. “The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program,” Mutation Research, 123: 61-118 (1983).
§ 798.5450   Rodent dominant lethal assay.
(a) Purpose. Dominant lethal (DL) effects cause embryonic or fetal death. Induction of a dominant lethal event after exposure to a chemical substance indicates that the substance has affected germinal tissue of the test species. Dominant lethals are generally accepted to be the result of chromosomal damage (structural and numerical anomalies) but gene mutations and toxic effects cannot be excluded.
(b) Definition. A dominant lethal mutation is one occurring in a germ cell which does not cause dysfunction of the gamete, but which is lethal to the fertilized egg or developing embryo.
(c) Reference substances. These may include, but need not be limited to, triethylenemelamine, cyclophosphamide or ethyl methanesulfonate.
(d) Test method —(1) Principle. Generally, male animals are exposed to the test substance and mated to untreated virgin females. The various germ cell stages can be tested separately by the use of sequential mating intervals. The females are sacrificed after an appropriate period of time and the contents of the uteri are examined to determine the numbers of implants and live and dead embryos. The calculation of the dominant lethal effect is based on comparison of the live implants per female in the treated group to the live implants per female in the control group. The increase of dead implants per female in the treated group over the dead implants per female in the control group reflects the post-implantation loss. The post-implantation loss is calculated by determining the ratio of dead to total implants from the treated group compared to the ratio of dead to total implants from the control group. Pre-implantation loss can be estimated on the basis of corpora lutea counts or by comparing the total implants per female in treated and control groups.
(2) Description. (i) Several treatment schedules are available. The most widely used requires single administration of the test substance. Other treatment schedules, such as treatment on five consecutive days, may be used if justified by the investigator.
(ii) Individual males are mated sequentially to virgin females at appropriate intervals. The number of matings following treatment is governed by the treatment schedule and should ensure that germ cell maturation is adequately covered. Females are sacrificed in the second half of pregnancy and the uterine contents examined to determine the total number of implants and the number of live and dead embryos.
(3) Animal selection —(i) Species. Rats or mice are generally used as the test species. Strains with low background dominant lethality, high pregnancy frequency and high implant numbers are recommended.
(ii) Age. Healthy, sexually mature animals shall be used.
(iii) Number. An adequate number of animals shall be used taking into account the spontaneous variation of the biological characteristics being evaluated. The number chosen should be based on the predetermined sensitivity of detection and power of significance. For example, in a typical experiment, the number of males in each group shall be sufficient to provide between 30 and 50 pregnant females per mating interval.
(iv) Assignment to groups. Animals shall be randomized and assigned to treatment and control groups.
(4) Control groups —(i) Concurrent controls. Generally concurrent positive and negative (vehicle) controls shall be included in each experiment. When acceptable positive control results are available from experiments conducted recently (within the last 12 months) in the same laboratory these results can be used instead of a concurrent positive control.
(ii) Positive controls. Positive control substances shall be used at a dose which demonstrates the test sensitivity.
(5) Test chemicals —(i) Vehicle. When possible, test substances shall be dissolved or suspended in isotonic saline or distilled water. Water-insoluble chemicals may be dissolved or suspended in appropriate vehicles. The vehicle used shall neither interfere with the test chemical nor produce toxic effects. Fresh preparations of the test chemical should be employed.
(ii) Dose levels. Normally, three dose levels shall be used. The highest dose shall produce signs of toxicity (e.g., slightly reduced fertility and slightly reduced body weight). However, in an initial assessment of dominant lethality a single high dose may be sufficient. Nontoxic substances shall be tested at 5g/kg or, if this is not practicable, then as the highest dose attainable.
(iii) Route of administration. The usual routes of administration are oral or by IP injection. Other routes may be appropriate.
(e) Test performance. (1) Individual males are mated sequentially at appropriate predetermined intervals to one or two virgin females. Females should be left with the males for at least the duration of one estrus cycle or alternatively until mating has occurred as determined by the presence of sperm in the vagina or by the presence of a vaginal plug.
(2) The number of matings following treatment should be governed by the treatment schedule and should ensure that germ cell maturation is adequately covered.
(3) Females should be sacrificed in the second half of pregnancy and uterine contents examined to determine the number of implants and live and dead embryos. The ovaries may be examined to determine the number of corpora lutea.
(f) Data and report —(1) Treatment of results. Data shall be tabulated to show the number of males, the number of pregnant females, and the number of nonpregnant females. Results of each mating, including the identity of each male and female, shall be reported individually. For each female, the dose level and week of mating and the frequencies of live implants and of dead implants shall be enumerated. If the data are recorded as early and late deaths, the tables shall make that clear. If preimplantation loss is estimated, it shall be reported. Preimplantation loss can be calculated as the difference between the number of corpora lutea and the number of implants or as a reduction in the average number of implants per female in comparison with control matings.
(2) Statistical evaluation. Data shall be evaluated by appropriate statistical methods. Differences among animals within the control and treatment groups shall be considered before making comparisons between treated and control groups.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of dominant lethals. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of dominant lethals or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) A positive DL assay suggests that under the test conditions the test substance may be genotoxic in the germ cells of the treated sex of the test species.
(ii) A negative result suggests that under the conditions of the test the test substance may not be genotoxic in the germ cells of the treated sex of the test species.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(i) Species, strain, age and weights of animals used, number of animals of each sex in experimental and control groups.
(ii) Test substance, vehicle used, dose levels and rationale for dosage selection, negative (vehicle) and positive controls, experimental observations, including signs of toxicity.
(iii) Route and duration of exposure.
(iv) Mating schedule.
(v) Methods used to determine that mating has occurred (where applicable).
(vi) Criteria for scoring dominant lethals including the number of early and late embryonic deaths.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Brewen, J.G., Payne, H.S., Jones, K.P., Preston, R.J. “Studies on chemically induced dominant lethality. I. The cytogenetic basis of MMS-induced dominant lethality in post-meiotic germ cells” Mutation Research, 33:239-250 (1975).
(2) Ehling, U.H., Machemer, L., Buselmaier, E., Dycka, D., Frohberg, H., Kratochvilova, J., Lang, R., Lorke, D., Muller, D., Pheh, J., Rohrborn, G., Roll, R., Schulze-Schencking, M., Wiemann, H. “Standard protocol for the dominant lethal test on male mice. Set up by the Work Group “Dominant lethal mutations of the ad hoc Committee Chemogenetics,” Archives of Toxicology, 39:173-185 (1978).
§ 798.5460   Rodent heritable translocation assays.
(a) Purpose. This test detects transmitted chromosomal damage which manifests as balanced reciprocal translocations in progeny descended from parental males treated with chemical mutagens.
(b) Definitions. (1) A heritable translocation is one in which distal segments of nonhomologous chromosomes are involved in a reciprocal exchange.
(2) Diakinesis and metaphase I are stages of meiotic prophase scored cytologically for the presence of multivalent chromosome association characteristic of translocation carriers.
(c) Reference substances. Not applicable.
(d) Test method —(1) Principle. When a balanced reciprocal translocation is induced in a parental male germ cell, the resulting progeny is translocation heterozygote.
(i) Basis for fertility screening. Male translocation heterozygotes may be completely sterile. This class consists of two types of translocations:
(A) Translocations between non-homologous chromosomes in which at least one of the breaks occurs close to one end of a chromosome.
(B) Those that carry multiple translocations. The majority of male translocation heterozygotes are semisterile—they carry one or (rarely) two translocations. The degree of semisterility is dependent upon the proportions of balanced and unbalanced (duplication-deficiency) gametes produced in the ejaculate as a function of meiotic segregation. Balanced and unbalanced sperm are equally capable of fertilizing an egg. Balanced sperm lead to viable progeny. Unbalanced sperm result in early embryonic lethality.
(ii) Basis for cytological screening. The great majority of male translocation heterozygotes can be identified cytologically through analysis of diakinesis metaphase I spermatocytes. Translocation heterozygotes are characterized by the presence of multivalent chromosome association such as a ring or chain of four chromosomes held together by chiasmata in paired homologous regions. Some translocation carriers can be identified by the presence of extra long and/or extra short chromosomes in spermatogonial and somatic cell metaphase preparations.
(2) Description. Essentially, two methods have been used to screen for translocation heterozygosity; one method uses a mating sequence to identify sterile and semisterile males followed by cytological examination of suspect male individuals; the other method deletes the mating sequence altogether and all F 1 male progeny are examined cytologically for presence of translocation. In the former approach, the mating sequence serves as a screen which eliminates most fully fertile animals for cytological confirmation as translocation heterozygotes.
(3) Animal selection —(i) Species. The mouse is the species generally used, and is recommended.
(ii) Age. Healthy sexually mature animals shall be used.
(iii) Number. (A) The number of male animals necessary is determined by the following factors:
( 1 ) The use of either historical or concurrent controls.
( 2 ) The power of the test.
( 3 ) The minimal rate of induction required.
( 4 ) Whether positive controls are used.
( 5 ) The level of significance desired.
(B) [Reserved]
(iv) Assignment to groups. Animals shall be randomized and assigned to treatment and control groups.
(4) Control groups —(i) Concurrent controls. No concurrent positive or negative (vehicle) controls are recommended as routine parts of the heritable translocation assay. However, investigators not experienced in performing translocation testing shall include a substance known to produce translocations in the assay as a positive control reference chemical.
(ii) Historical controls. At the present time, historical control data must be used in tests for significance. When statistically reliable historical controls are not available, negative (vehicle) controls shall be used.
(5) Test chemicals —(i) Vehicle. When appropriate for the route of administration, solid and liquid test substances should be dissolved or suspended in distilled water or isotonic saline. Water-insoluble chemicals may be dissolved or suspended in appropriate vehicles. The vehicle used shall neither interfere with the test chemical nor produce toxic effects. Fresh preparations of the test chemical should be employed.
(ii) Dose levels. At least two dose levels shall be used. The highest dose level shall result in toxic effects (which shall not produce an incidence of fatalities which would prevent a meaningful evaluation) or shall be the highest dose attainable or 5g/kg body weight.
(iii) Route of administration. Acceptable routes of administration include oral, inhalation, admixture with food or water, and IP or IV injection.
(e) Test performance —(1) Treatment and mating. The animals shall be dosed with the test substances 7 days per week over a period of 35 days. After treatment, each male shall be caged with 2 untreated females for a period of 1 week. At the end of 1 week, females shall be separated from males and caged individually. When females give birth, the day of birth, litter size, and sex of progeny shall be recorded. All male progeny should be weaned, and all female progeny should be discarded.
(2) Testing for translocation heterozygosity. When males are sexually mature, testing for translocation heterozygosity shall begin. One of two methods shall be used; the first method involves mating, determining those F 1 progeny which are sterile or semisterile and subsequent cytological analysis of suspect progeny; the other method does not involve mating and determining sterility or semisterility; all progeny are examined cytologically.
(i) Determination of sterility or semisterility —(A) Conventional method. Females are mated, usually three females for each male, and each female is killed at midpregnancy. Living and dead implantations are counted. Criteria for determining normal and semisterile males are usually established for each new strain because the number of dead implantations varies considerably among strains.
(B) Sequential method. Males to be tested are caged individually with females and the majority of the presumably normal males are identified on the basis of a predetermined size of 1 or 2 litters. Breeding pens are examined daily on weekdays beginning 18 days after pairing. Young are discarded immediately after they are scored. Males that sire a litter whose size is the same as or greater than the minimum set for a translocation-free condition are discarded with their litter. If the litter size is smaller than the predetermined number, a second litter is produced with the same rule applying. Males that cannot be classified as normal after production of a second litter are tested further by the conventional method or by cytological confirmation of translocation.
(ii) Cytological analysis. For cytological analysis of suspected semisteriles, the air-drying technique is used. Observation of at least 2 diakinesis-metaphase 1 cells with mutivalent association constitutes the required evidence for the presence of a translocation. Sterile males are examined by one of two methods, those with testes of normal size and sperm in the epididymis are examined by the same techniques used for semisteriles. Animals with small testes are examined by squash preparations or, alternatively, by examination of mitotic metaphase preparations. If squash preparations do not yield diakinesis-metaphase 1 cells, analysis of spermatogonia or bone marrow for the presence of unusually long or short chromosomes should be performed.
(f) Data and report —(1) Treatment of results. (i) Data shall be presented in tabular form and shall include the number of animals at risk, the germ cell stage treated, the number of partial steriles and semisteriles (if the fertility test is used), the number of cytogenetically confirmed translocation heterozygotes (if the fertility test is used, report the number of confirmed steriles and confirmed partial steriles), the translocation rate, and either the standard error of the rate or the upper 95 percent confidence limit on the rate.
(ii) These data shall be presented for both treated and control groups. Historical or concurrent controls shall be specified, as well as the randomization procedure used for concurrent controls.
(2) Statistical evaluation. Data shall be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of heritable translocations. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of heritable translocations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the heritable translocation assay indicate that under the test conditions the test substance causes heritable chromosomal damage in the test species.
(ii) Negative results indicate that under the test conditions the test substance does not cause heritable chromosomal damage in the test species.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J, the following specific information shall be reported:
(i) Species, strain, age, weight and number of animals of each sex in each group.
(ii) Test chemical vehicle, route and schedule of administration, toxicity data.
(iii) Dosing regimen, doses tested and rationale for dosage selection.
(iv) Mating schedule, number of females mated to each male.
(v) The use of historical or concurrent controls.
(vi) Screening procedure including the decision criteria used and the method by which they were determined.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Generoso, W.M., Bishop, J.B., Goslee, D.G., Newell, G.W., Sheu, G-J, von Halle, E. “Heritable translocation test in mice,” Mutation Research, 76:191-215 (1980).
(2) [Reserved]
§ 798.5500   Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”
(a) Purpose. Bacterial DNA damage or repair tests measure DNA damage which is expressed as differential cell killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient strains. These tests do not measure mutagenic events per se. They are used as an indication of the interaction of a chemical with genetic material implying the potential for genotoxicity.
(b) Definition. Test for differential growth inhibition of repair proficient and repair deficient bacteria measure differences in chemically induced cell killing between wild-type strains with full repair capacity and mutant strains deficient in one or more of the enzymes which govern repair of damaged DNA.
(c) Reference substances. These may include, but need not be limited to, chloramphenicol or methyl methanesulfonate.
(d) Test method —(1) Principle. The tests detect agents that interact with cellular DNA to produce growth inhibition or killing. This interaction is recognized by specific cellular repair systems. The assays are based upon the use of paired bacterial strains that differ by the presence of absence of specific DNA repair genes. The response is expressed in the preferential inhibition of growth or the preferential killing of the DNA repair deficient strain since it is incapable of removing certain chemical lesions from its DNA.
(2) Description. Several methods for performing the test have been described. Those described here are:
(i) Tests performed on solid medium (diffusion tests).
(ii) Tests performed in liquid culture (suspension tests).
(3) Strain selection —(i) Designation. At the present time, Escherichia coli polA (W3110/p3478) or Bacillus subtilis rec (H17/M45) pairs are recommended. Other pairs may be utilized when appropriate.
(ii) Preparation and storage. Stock culture preparation and storage, growth requirements, method of strain identification and demonstration of appropriate phenotypic requirements should be performed using good microbiological techniques and should be documented.
(4) Bacterial growth. Good microbiological techniques should be used to grow fresh cultures of bacteria. The phase of growth and cell density should be documented and should be adequate for the experimental design.
(5) Metabolic activation. Bacteria should 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 postmitochondrial fraction prepared from the livers of rodents treated with enzyme inducing agents. The use of other species, tissues or techniques may also be appropriate.
(6) Control groups —(i) Concurrent controls. Concurrent positive, negative, and vehicle controls should be included in each assay.
(ii) Negative controls. The negative control should show nonpreferential growth inhibition (i.e., should affect both strains equally). Chloramphenicol is an example of a negative control.
(iii) Genotype specific controls. Examples of genotype specific positive controls are methyl methanesulfonate for polA strains and mitomycin C for rec strains.
(iv) Positive controls to ensure the efficacy of the activation system. The positive control reference substance for tests including a metabolic activation system should be selected on the basis of the type of activation system used in the test.
(v) Other positive controls. Other positive control reference substances may be used.
(7) Test chemicals —(i) Vehicle. Test chemicals and positive and negative control reference substances should be dissolved in an appropriate vehicle and then further diluted in vehicle for use in the assay.
(ii) Exposure concentrations. The test should initially be performed over a broad range of concentrations. Among the criteria to be taken into consideration for determining the upper limits of test chemical concentration are cytotoxicity and solubility. Cytotoxicity of the test chemical may be altered in the presence of metabolic activation systems. For freely soluble nontoxic chemicals, the upper test chemical concentration should be determined on a case by case basis. Because results are expressed as diameters of zones of growth inhibition in the diffusion test, it is most important that the amounts of chemical on the disc (or in the wells) are exact replicates. When appropriate, a positive response should be confirmed by testing over a narrow range of concentrations.
(e) Test performance —(1) Diffusion assay —(i) Disc diffusion assays. Disc diffusion assays. may be performed in two ways:
(A) A single strain of bacteria may be added to an agar overlay or spread on the surface of the agar and the test chemical placed on a filter disc on the surface of the agar or;
(B) DNA repair proficient and DNA repair deficient bacteria may be streaked in a line on the surface of the agar of the same plate and a disc saturated with test chemical placed on the surface of the agar in contact with the streaks.
(ii) Well diffusion assays. In well diffusion assays, bacteria may be either added to the agar overlay or spread onto the surface of the agar. A solution of the test chemical is then placed into a well in the agar.
(2) Suspension assays. (i) A bacterial suspension may be exposed to the test chemical and the number of surviving bacteria determined (as colony-forming units) either as a function of time of treatment or as a function of the concentration of test agent.
(ii) Nonturbid suspensions of bacteria may be exposed to serial dilutions of the test agent and a minimal inhibitory concentration for each strain determined, as evidenced by the presence or absence of visible growth after a period of incubation.
(iii) Paired bacterial suspensions (usually with some initial turbidity) may be treated with a single dose of the chemical. Positive results are indicated by a differential inhibition in the rate of increase of turbidity of the paired cultures.
(3) Number of cultures. When using a plate diffusion procedure, at least two independent plates should be used at each dilution. In liquid suspension assays, at least two independent specimens for determination of the number of viable cells should be plated.
(4) Incubation conditions. All plates in a given test should be incubated for the same time period. This incubation period should be for 18 to 24 hrs at 37 °C.
(f) Data and report —(1) Treatment of results —(i) Diffusion assays. Results should be expressed in diameters of zones of growth inhibition in millimeters or as areas derived therefrom as mm 2 . Dose-response data, if available, should be presented using the same units.
(ii) Liquid suspension assays. (A) Survival data can be presented as dose responses, preferably as percentage of survivors or fractional survival of each strain or as a relative survival (ratio) of the two strains.
(B) Results can also be expressed as the concentrations required to effect a predetermined survival rate (e.g., D 37 , the dose permitting 37 percent survival). These data are derived from the survival curve. The concentration should be expressed as weight per volume, as moles, or as molarity.
(C) Similarly, results can be expressed as minimal inhibitory concentration or as minimal lethal dose. The former is determined by the absence of visible growth in liquid medium and the latter is determined by plating dilutions onto semisolid media.
(iii) In all tests, concentrations must be given as the final concentrations during the treatment. Raw data, prior to transformation, should be provided. These should include actual quantities measured, e.g., neat numbers. For measurement of diffusion, the diameters of the discs and/or well should be indicated and the measurements should indicate whether the diameter of the discs and/or well was subtracted. Moreover, mention should be made as to whether the test chemical gave a sharp, diffuse, or double-zone of growth inhibition. If it is the latter, the investigator should indicate whether the inner or the outer zone was measured.
(iv) Viability data should be given as the actual plate counts with an indication of the dilution used and the volume plated or as derived titers (cells per ml). Transformed data alone in the absence of experimental data are not acceptable (i.e, ratios, differences, survival fraction).
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related preferential inhibition or killing of the repair deficient strain. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related preferential inhibition or killing of the repair deficient strain or a statistically significant and reproducible positive response at any one of the test points is considered not to interact with the genetic material of the organisms used in assay.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. DNA damage tests in bacteria do not measure DNA repair per se nor do they measure mutations. They measure DNA damage which is expressed as cell killing or growth inhibition. A positive result in a DNA damage test in the absence of a positive result in another system is difficult to evaluate in the absence of a better data base.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information should be reported:
(i) Bacterial strains used.
(ii) Phase of bacterial cell growth at time of use in the assay.
(iii) Media composition.
(iv) Details of both the protocol used to prepare the metabolic activation system and its use in the assay.
(v) Treatment protocol, including doses used and rationale for dose selection, positive and negative controls.
(vi) Method used for determination of degree of cell kill.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the Salmonella /mammalian-microsome mutagenicity test,” Mutation Research, 31:347-364 (1975).
(2) Kada, T., Sadie, Y., Tutikawa, K. “In vitro and host-mediated “rec-assay” procedures for screening chemical mutagens; and phloxine, a mutagenic red dye detected,” Mutation Research, 16:165-174 (1972).
(3) Leifer, Z., Kada, T., Mandel, M., Zeiger, E., Stafford, R., Rosenkranz, H.S. “An evaluation of bacterial DNA repair tests for predicting genotoxicity and carcinogenicity: A report of the U.S. EPA's Gene-Tox Program,” Mutation Research, 87:211-297 (1981).
(4) Slater, E.E., Anderson, M.D., Rosenkranz, H.S. “Rapid detection of mutagens and carcinogens.” Cancer Research, 31:970-973 (1971).
§ 798.5955   Heritable translocation test in drosophila melanogaster.
(a) Purpose. The heritable translocation test in Drosophila measures the induction of chromosomal translocations in germ cells of insects. Stocks carrying genetic markers on two or more chromosomes are used to follow the assortment of chromosomes in meiosis. The F 1 male progeny of treated parents are individually mated to females and the F 2 progeny phenotypes are scored. The observed spectrum of phenotypes is used to determine the presence or absence of a translocation. This is usually indicated by a lack of independent assortment of genes on different chromosomes.
(b) Definitions —(1) Chromosome mutations are chromosomal changes resulting from breakage and reunion of chromosomes. Chromosomal mutations are also produced through nondisjunction of chromosomes during cell division.
(2) Reciprocal translocations are chromosomal translocations resulting from reciprocal exchanges between two or more chromosomes.
(3) Heritable translocations are reciprocal translocations transmitted from parent to the succeeding progeny.
(c) Reference substances. These may include, but need not be limited to, ethyl methanesulfonate or N-dimethyl-nitrosamine.
(d) Test method —(1) Principle. The method is based on the principle that balanced reciprocal chromosomal translocations can be induced by chemicals in the germ cells of treated flies and that these translocations are detected in the F 2 progeny using genetic markers (mutations). Different mutations may be used as genetic markers and two or more of the four chromosomes may be genetically marked for inclusion in this test.
(2) Description. Wild-type males are treated with chemical and bred with females of known genetic markers. The F 1 males are collected and individually bred with virgin females of the female parental stock. The resulting F 2 progeny are scored. Putative translocation carriers are confirmed with an F 3 cross.
(i) Illustrative example. The following example serves to illustrate the method. Males carrying genes for red eye color on chromosomes II and III are bred with females of white eye color carrying alleles for brown (bw) on the second chromosome and scarlet (st) and pink (pp) on the third chromosome. The F 1 male progeny are bred with virgin females of the female parental stock and the resulting F 2 progeny are examined for eye color phenotypes. If there is no translocation in the F 1 male, then the resulting F 2 progeny will have four eye color phenotypes: red, white, orange, and brown. If the F 1 male carries a translocation between chromosomes II and III, only red and white eye phenotypes are obtained in the F 2 generation. This happens because the F 1 translocation heterozygote produces two balanced (carrying either the parental or the translocated configuration of markers) and two unbalanced gametes. The unbalanced gametes (carrying one normal and one translocated chromosome) are unable to develop into normal individuals in the F 2 generation.
(ii) [Reserved]
(3) Drosophila stocks. Wild-type males and females of the genotype bw:st:pp (white eyes) may be used in the heritable translocation test. Other appropriately marked Drosophila stocks may also be used.
(4) Control groups. (i) Concurrent positive and negative (vehicle) controls should be included in each experiment.
(ii) Negative (vehicle) controls should be included. The size of the negative (vehicle) control group should be determined by the availability of appropriate laboratory historical control data.
(iii) If the historical control data are of sufficient numbers, concurrent controls may not be necessary.
(5) Test chemicals —(i) Vehicle. Test chemicals should be dissolved in water. Compounds which are insoluble in water may be dissolved or suspended in appropriate vehicles (e.g., a mixture of ethanol and Tween-60 or 80), and then diluted in water or saline prior to administration. Dimethylsulfoxide should be avoided as a vehicle.
(ii) Dose levels. For the initial assessment of mutagenicity, it may be sufficient to test a single dose of the test substance. This dose should be the maximum tolerated dose or that which produces some indication of toxicity. If the test is being used to verify mutagenic activity, at least two additional exposure levels should be used.
(iii) Route of administration. Exposure may be oral, by injection or by exposure to gases or vapours. Feeding of the test compound may be done in sugar solution. When necessary, substances may be dissolved in 0.7 percent NaCl solution and injected into the thorax or abdomen.
(e) Test performance —(1) P1 mating. (i) In the primary screen of a chemical, it is enough to sample one germ cell stage, either mature sperm or spermatids (for indirect acting mutagens). Other stages may be sampled if needed, i.e., when mature germ cells give a positive result and data from earlier germ cells are needed for the purpose of risk assessment. Thus, the treated males may be mated only once for a period of 3 days to sample sperm or transferred every 2 to 3 days to cover the entire germ cell cycle.
(ii) Mass matings may be performed because the control rate for translocations in the available literature is very low (near 0) and clustered events are extremely rare. Mated females may be aged for 2 weeks in order to recover an enhanced incidence of translocation due to the storage effect. The females are then allowed to lay eggs and F 1 males are collected for test mating.
(2) F 1 mating. F 1 males should be bred with virgin females of the parental female stock. Since each F 1 male represents one treated gamete of the male parent, the F 1 males have to be mated individually to virgin females. Each F 1 male should be mated to three females to ensure sufficient progeny.
(3) Scoring the F 2 generation. F 2 cultures (each representing 1 F 1 male tested) should be scored for the presence or absence of phenotype variations (linkage of markers) from the expected types. The test should be designed with a predetermined sensitivity and power. The number of flies in each group should reflect these defined parameters. The spontaneous mutant frequency observed in the appropriate control group will strongly influence the number of treated chromosomes that must be analyzed to detect substances which show mutation rates close to those of the controls. A positive test should be confirmed by F 3 mating trials.
(4) Number of replicate experiments. Replicate experiments are usually performed for each dose of the compound tested. If a chemical is a potent inducer of translocations, one experiment may be sufficient. Otherwise two or three replicate experiments should be done.
(f) Data and report —(1) Treatment of results. Data should be tabulated to show the number of translocations and the number of fertile F 1 males at each exposure for each germ cell stage sampled.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of heritable translocations. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of heritable translocations or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the heritable translocation test in Drosophila indicate that under the test conditions the test substance causes chromosome damage in germ cells of this insect.
(ii) Negative results indicate that under the test conditions the test substance does not cause chromosomal damage in D. melanogaster.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J, the following specific information should be reported:
(i) Drosophila stock used in the assay, age of insects, number of males treated, number of F 2 cultures established, number of replicate experiments.
(ii) Test chemical vehicle, treatment and mating schedule, exposure levels, toxicity data, dose and route of exposure.
(iii) Positive and negative (vehicle) controls.
(iv) Historical control data, if available.
(v) Number of chromosomes scored.
(vi) Criteria for scoring mutant chromosomes.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Wurgler, F.E., Sobels, F.H., Vogel, E. “ Drosophila as assay system for detecting genetic changes,” Handbook of mutagenicity test procedures. Eds. Kilby, B.J., Legator, M., Nichols, W., Ramel, C. (Amsterdam: Elsevier/North Holland Biomedical Press, 1979) pp. 335-374.
(2) [Reserved]
§ 798.6050   Functional observational battery.
(a) Purpose. In the assessment and evaluation of the potential human health effects of substances, it may be necessary to test for neurotoxic effects. Substances that have been observed to cause neurotoxic signs (e.g., convulsions, tremors, ataxia) in other toxicity tests, as well as those having a structural similarity to known neurotoxicants, should be evaluated for neurotoxicity. The functional observational battery is a noninvasive procedure designed to detect gross functional deficits in young adults resulting from exposure to chemicals and to better quantify neurotoxic effects detected in other studies. This battery of tests is not intended to provide a detailed evaluation of neurotoxicity. It is designed to be used in conjunction with neuropathologic evaluation and/or general toxicity testing. Additional functional tests may be necessary to assess completely the neurotoxic potential of a chemical.
(b) Definitions. (1) Neurotoxicity is any adverse effect on the structure or function of the central and/or peripheral nervous system related to exposure to a chemical substance.
(2) A toxic effect is an adverse change in the structure or function of an experimental animal as a result of exposure to a chemical substance.
(c) Principle of the test method. The material is administered by an appropriate route to laboratory rodents. The animals are observed under carefully standardized conditions with sufficient frequency to ensure the detection of behavioral and/or neurologic abnormalities, if present. Various functions that could be affected by neurotoxicants are assessed during each observation period.
(d) Test procedures —(1) Animal selection —(i) Species and strain. The laboratory rat or mouse is recommended. Although information will generally be lacking, whenever possible the choice of species should take into consideration such factors as the comparative metabolism of the chemical and species sensitivity to the toxic effects of the test substance, as evidenced by the results of other studies. The potential for combined studies should also be considered. Standard strains should be used.
(ii) Age. Young adult animals (at least 42 days old for the rat or mouse) shall be used.
(iii) Sex. (A) Equal numbers of animals of each sex are required for each dose level.
(B) The females shall be nulliparous and nonpregnant.
(2) Number of animals. At least eight animals of each sex should be used at each dose level and should be designated for behavioral testing. If interim sacrifices are planned, the number should be increased by the number of animals scheduled to be sacrificed before the end of the study. Animals shall be randomly assigned to treatment and control groups.
(3) Control groups. (i) A concurrent (“sham” exposure or 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.
(ii) Concurrent or historic data from the laboratory performing the testing shall provide evidence of the ability of the procedures used to detect major neurotoxic endpoints such as limb weakness or paralysis (e.g., acrylamide), CNS stimulation (e.g., β, β′-iminodiproprionitrile) autonomatic signs (e.g., physostigmine).
(iii) A satellite group may be treated with the high dose level for the duration of exposure and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate duration, normally not less than 28 days.
(4) Dose levels and dose selection. At least 3 doses, equally spaced on a log scale (e.g., 1/2 log units) over a range of at least 1 log unit shall be used in addition to a zero dose or vehicle administration. The data should be sufficient to produce a dose-effect curve.
(i) The highest dose shall produce (A) clear behavioral effects or (B) life-threatening toxicity.
(ii) The data from the lower doses must show either (A) graded dose-dependent effects at 2 dose levels or (B) no effects at 2 dose levels, respectively.
(5) Duration and frequency of exposure. The duration and frequency of exposure will be specified in the test rule.
(6) Route of exposure. The test substance shall be administered by the route specified in the test rule. This route will usually be the one most closely approximating the expected route of human exposure. The exposure potocol shall conform to that outlined in the appropriate acute or subchronic toxicity study guideline under subpart B or subpart C of this part.
(7) Combined protocol. Subjects used for other toxicity studies may be used if none of the requirements of either study are violated by the combination.
(8) Study conduct. (i) All animals in a given study should be observed carefully by trained technicians who are blind with respect to the animals' treatments. Standard procedures to minimize observer variability shall be followed. 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 inter-observer reliability is required. All animals should be observed prior to initiation of exposure. Subsequent observations should be made with sufficent frequency to ensure the detection of behavioral and/or neurologic abnormalities, if present. At minimum, observations at 1 hour, 6 hours, 24 hours, 7 days, and 14 days and monthly thereafter are recommended. In a subchronic study, subsequent to the first exposure all observations should be made before the daily exposure. The animals should 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 function should be used. The development of objective quantitative measures of the observational endpoints specified is encouraged.
(ii) The following is a minimal list of observations that shall be noted:
(A) Any unusual responses with respect to body position, activity level, coordination of movement, and gait.
(B) Any unusual or bizarre behavior including, but not limited to, headflicking, head searching, compulsive biting or licking, self-mutilation, circling, and walking backwards.
(C) The presence of:
( 1 ) Convulsions.
( 2 ) Tremors.
( 3 ) Increased levels of lacrimation and/or red-colored tears.
( 4 ) Increased levels of salivation.
( 5 ) Piloerection.
( 6 ) Pupillary dilation or constriction.
( 7 ) Unusual respiration (shallow, labored, dyspneic, gasping, and retching) and/or mouth breathing.
( 8 ) Diarrhea.
( 9 ) Excessive or diminished urination.
( 10 ) Vocalization.
(D) Forelimb/hindlimb grip strength. The procedure described by Meyer et al. (1979), under paragraph (f)(9) of this section is recommended.
(E) Sensory function. A simple assessment of sensory function (vision, audition, pain perception) shall be made. Marshall et al. (1971) under paragraph (f)(8) of this section have described a neurologic exam for this purpose; these procedures are also discussed by Deuel (1977), under paragraph (f)(4) of this section. Irwin (1968) under paragraph (f)(7) of this section described a number of reflex tests intended to detect gross sensory deficits, including the visual placing response, Preyer reflex, and tail pinch. Many procedures have been developed for assessing pain perception (e.g., Ankier, 1974 under paragraph (f)(1) of this section; D'Amour and Smith 1941 under paragraph (f)(3) of this section; Evans 1971 under paragraph (f)(6) of this section).
(e) Data reporting and evaluation. In addition to the reporting requirements specified under 40 CFR part 792 subpart J the final test report must include the following information.
(1) Description of system and test methods. (i) A detailed description of the procedures used to standardize observation, including the arena and operational definitions for scoring observations.
(ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. Historic data may be used if all aspects of the experimental protocol are the same, including personnel.
(2) Results. The following information must be arranged by test group dose level.
(i) In tabular form, data for each animal must 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).
(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 percentage of animals showing each abnormal sign at each observation time.
(D) The mean and standard deviation for each continuous endpoint at each observation time.
(3) Evaluation of data. The findings of a functional observational battery should be evaluated in the context of preceding and/or concurrent toxicity studies and any correlative 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 should include appropriate statistical analyses. Choice of analyses should consider tests appropriate to the experimental design and needed adjustments for multiple comparisons.
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) Ankier, S.I. “New hot plate tests to quantify antinociceptic and narcotic antagonist activities,” European Journal of Pharmacology, 27: 1-4 (1974).
(2) Coughenour, L.L., McLean, J.R. and Parker, R.B. “A new device for the rapid measurement of impaired motor function in mice,” Pharmacology, Biochemistry and Behavior, 6: 351-353 (1977).
(3) D'Amour, F.E., Smith, D.L. “A method for determining loss of pain sensation,” Journal of Pharmacology and Experimental Therapeutics, 72: 74-79 (1941).
(4) Deuel, R.K. “Determining sensory deficits in animals,” Methods in Psychobiology Ed. Myers R.D. (New York: Academic Press, 1977) pp. 99-125.
(5) Edwards, P.M., 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).
(6) Evans, W.O. “A new technique for the investigation of some analgesic drugs on reflexive behavior in the rat,” Psychopharmacologia, 2: 318-325 (1961).
(7) Irwin, S. “Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral and physiologic state of the mouse,” Psychopharmacologia, 13: 222-257 (1968).
(8) Marshall, J.F., Turner, B.H., Teitlbaum, P. “Sensory neglect produced by lateral hypothalamic damage,” Science, 174: 523-525 (1971).
(9) Meyer, O.A., Tilson, H.A., Byrd, W.C., Riley, M.T. “A method for the routine assessment of fore- and hindlimb grip strength of rats and mice,” Neurobehavioral Toxicology, 1: 233-236 (1979).
§ 798.6200   Motor activity.
(a) Purpose —(1) General. In the assessment and evaluation of the toxic characteristics of a substance, determination of the effects of administration of the substance on motor activity is useful when neurotoxicity is suspected.
(2) Acute Motor Activity Test. The purpose of the acute motor activity test is to examine changes in motor activity occurring over a range of acute exposure levels. These changes may then be evaluated in the context of changes occurring in other organ systems. This test is an initial step in determining the potential of a substance to produce acute neurotoxicity and may be used to screen members of a class of substances for known neurotoxicity, and/or to establish a dosage regimen prior to the initiation of subchronic neurotoxicity testing.
(3) Subchronic Motor Activity Test. The purpose of the subchronic motor activity test is to determine whether the repeated administration of a suspected neurotoxicant results in changes in motor activity. These changes may be evaluated in the context of changes occurring in other organ systems. This test is an initial step in determining the potential of a substance to produce subchronic neurotoxicity.
(b) Definitions. (1) Neurotoxicity is the adverse effect on the structure or function of the central and/or peripheral nervous system related to exposure to a chemical substance.
(2) Motor activity is any movement of the experimental animal.
(3) A toxic effect is an adverse change in the structure or function of an experimental animal as a result of exposure to a chemical substance.
(c) Principle of the test method. The test substance is administered to several groups of experimental animals, one dose being used per group. Measurements of motor activity are made. The exposure levels at which significant changes in motor activity are produced are compared to those levels which produce toxic effects not originating in the central and/or peripheral nervous system.
(d) Test procedures —(1) Animal selection —(i) Species and strain. Testing shall be performed in a laboratory rat or mouse. The choice of species should take into consideration such factors as the comparative metabolism of the chemical and species sensitivity to the toxic effects of the test substance, as evidenced by the results of other studies, the potential for combined studies, and the availability of other toxicity data for the species.
(ii) Age. Young adult animals (at least 42 days old for rat or mouse) should be used.
(iii) Sex. (A) Equal numbers of animals of each sex are required for each dose level for the motor activity test.
(B) The females shall be nulliparous and nonpregnant.
(2) Number of animals. Animals shall be randomly assigned to test and control groups. Each test or control group must be designed to contain a sufficient number of animals at the completion of the study to detect a 40 percent change in activity of the test groups relative to the control group with 90 percent power at the 5 percent level. For most designs, calculations can be made according to Dixon and Massey (1957) under paragraph (f)(1) of this section, Neter and Wasserman (1974) under paragraph (f)(5) of this section, Sokal and Rohlf (1969) under paragraph (f)(9) of this section, or Jensen (1972) under paragraph (f)(3) of this section.
(3) Control groups. (i) A concurrent control group is required. This group must be an untreated group, or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control group are required.
(ii) Positive control data are required to demonstrate the sensitivity and reliability of the activity measuring device and testing procedure. These data should demonstrate the ability to detect increases or decreases in activity and to generate a dose-effect curve or its equivalent using three values of the dose or equivalent independent variable. A single administration of the dose (or equivalent) is sufficient. It is recommended that chemical exposure be used to collect positive control data. Positive control data shall be collected at the time of the test study unless the laboratory can demonstrate the adequacy of historical data for this purpose.
(iii) A satellite group may be treated with the high dose level for 90 days and observed for reversibility, persistence or delayed occurrence of toxic effects for a post-treatment period of appropriate length, normally not less than 28 days.
(4) Dose levels and dose selection. At least 3 doses, equally spaced on a log scale (e.g., 1/2 log units) over a range of at least 1 log unit shall be used in addition to a zero dose or vehicle administration. The data should be sufficient to produce a dose-effect curve.
(i) The highest dose shall produce (A) clear effects on motor activity or (B) life-threatening toxicity.
(ii) The data from the lower doses must show either (A) graded dose-dependent effects at 2 dose levels or (B) no effects at 2 dose levels, respectively.
(5) Duration of testing. The duration of exposure will be specified in the test rule.
(6) Route of administration. The test substance shall be administered by the method specified in the test rule. This will usually be the route most closely approximating the route of human exposure. The exposure protocol shall conform to that outlined in the appropriate acute or subchronic toxicity study guideline.
(7) Combined protocol. The tests described herein may be combined with any other toxicity study, as long as none of the requirements of either are violated by the combination.
(8) Study conduct —(i) General. Motor activity must 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 decreases nor so high as to preclude increases. Each device shall be tested by standard procedure 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 percent of the session for most treatments and animals. All sessions should have the same duration. Treatment groups shall be counter-balanced 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 home cage or novel test cage and environmental distractions. Tests shall be executed by an appropriately trained individual.
(ii) Acute. Testing shall be timed to include the time of peak signs.
(iii) Subchronic. All animals shall be tested prior to initiation of exposure and at 30 ±2, 60 ±2 and 90 ±2 days during the exposure period. Testing shall occur prior to the daily exposure. Animals shall be weighed on each test day and at least once weekly during the exposure period.
(e) Data reporting and evaluation. In addition to the reporting requirements specified under 40 CFR part 792, subpart J the final test report must include the following information:
(1) Description of system and test methods. (i) Positive control data from the laboratory performing the test which demonstrate the sensitivity of the procedure being used.
(ii) Procedures for calibrating and assuring the equivalence of devices and balancing treatment groups.
(2) Results. The following information must be arranged by test group (dose level).
(i) In tabular form, data must be provided showing for each animal:
(A) Its identification number.
(B) Body weight, total session activity counts, and intrasession subtotals for each date measured.
(ii) Group summary data should also be reported.
(3) Evaluation of data. An evaluation of the test results (including statistical analysis comparing total activity counts at the end of exposure of treatment vs control animals must be made and supplied. This submission must include dose-effect curves for motor activity expressed as activity counts.
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) Dixon, W.J., Massey, E.J. Introduction to Statistical Analysis 2nd Ed. (New York: McGraw-Hill, 1957).
(2) Finger, F.W. “Measuring behavioral activity,” Methods in Psychobiology Vol. 2. Ed. R.D. Myers (New York: Academic, 1972) pp. 1-19.
(3) Jensen, D.R. “Some simultaneous multivariate procedures using Hotelling's T 2 Statistics,” Biometrics, 28:39-53 (1972).
(4) 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).
(5) Neter, J. and Wasserman, W. Applied Linear Statistical Models. Homewood, Richard D. Irwin, Inc., 1974.
(6) Reiter, L.E. “Use of activity measures in behavioral toxicology,” Environmental Health Perspectives, 26:9-20 (1978).
(7) Reiter, L.W. and MacPhail, R.C. “Motor Activity: A survey of methods with potential use in toxicity testing,” Neurobehavioral Toxicology, 1: Suppl. 1, 53-66 (1979).
(8) Robbins, T.W. “A critique of the methods available for the measurement of spontaneous motor activity,” Handbook of Psychopharmacology. Vol. 7. Eds. Iversen, L.L., Iversen, D.S., Snyder, S.H. (New York: Plenum, 1977) pp. 37-82.
(9) Sokal, R.P. and Rohlf, E.J. Biometry. (San Francisco: W.H. Freeman and Co., 1969).
§ 798.6400   Neuropathology.
(a) Purpose. The techniques in this guideline are designed to develop data on morphologic changes in the nervous system for chemical substances and mixtures subject to such testing under the Toxic Substances Control Act. The data will detect and characterize morphologic changes, if and when they occur, and determine a no-effect level for such changes. Neuropathological evaluation should be complemented by other neurotoxicity studies, e.g. behavioral and neurophysiological studies. Neuropathological evaluation may be done following acute, subchronic or chronic exposure.
(b) Definition. Neurotoxicity or a neurotoxic effect is an adverse change in the structure or function of the nervous system following exposure to a chemical agent.
(c) 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 sacrificed and tissues in the nervous system are examined grossly and prepared for microscopic examination. Starting with the highest dosage level, tissues are examined under the light microscope for morphologic changes, until a no effect level is determined. In cases where light microscopy has revealed neuropathology, the no effect level may be confirmed by electron microscopy.
(d) Test procedure —(1) Animal selection —(i) Species and strain. Testing shall be performed in the species being used in other tests for neurotoxicity. This will generally be the laboratory rat. The choice of species shall take into consideration such factors as the comparative metabolism of the chemical and species sensitivity to the toxic effects of the test substance, as evidenced by the results of other studies, the potential for combined studies, and the availability of other toxicity data for the species.
(ii) Age. Animals shall be young adults (150-200 gm for rats) at the start of exposure.
(iii) Sex. Both sexes shall be used unless it is demonstrated that one sex is refractory to the effects.
(2) Number of animals. A minimum of six animals per group shall be used. The tissues from each animal shall be examined separately. It is recomse (iv)mended that ten animals per group be used.
(3) Control groups. (i) A concurrent control group(s) is (are) required. This group must be an untreated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the vehicle used has a known or potential toxic property, both untreated and vehicle control groups are required.
(ii) A satellite group of animals may be treated with the high level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate length; normally not less than 28 days.
(4) Dose levels and dose selection. At least 3 doses, equally spaced on a log scale (e.g., 1/2 log units) over a range of at least 1 log unit shall be used in addition to a zero dose or vehicle administration. The data should be sufficient to produce a dose-effect curve.
(i) The highest dose shall produce (A) clear behavioral effects or (B) life-threatening toxicity.
(ii) The data from the lower doses must show either (A) graded dose-dependent effects at two dose levels or (B) no effects at two dose levels, respectively.
(5) Duration of testing. The exposure duration will be specified in the test rule. This will generally be 90 days exposure.
(6) Route of administration. The test substance shall be administered by a route specified in the test rule. This will generally be the route most closely approximating the route of human exposure. The exposure protocol shall conform to that outlined in the appropriate acute or subchronic toxicity guideline.
(7) Combined protocol. The tests described herein may be combined with any other toxicity study, as long as none of the requirements of either are violated by the combination.
(8) Study conduct —(i) Observation of animals. All toxicological (e.g., weight loss) and neurological signs (e.g., motor disturbance) shall be recorded frequently enough to observe any abnormality, and not less than weekly.
(ii) Sacrifice of animals —(A) General. The goal of the techniques outlined for sacrifice of animals and preparation of tissues is preservation of tissues morphology to simulate the living state of the cell.
(B) Perfusion technique. Animals shall be perfused in situ by a generally recognized technique. For fixation suitable for light or electronic microscopy, saline solution followed by buffered 2.5 percent glutaraldehyde or buffered 4.0 percent paraformaldehyde, is recommended. While some minor modifications or variations in procedures are used in different laboratories, a detailed and standard procedure for vascular perfusion may be found in the text by Zeman and Innes (1963) under paragraph (f)(7) of this section, Hayat (1970) under paragraph (f)(3) of this section, and by Spencer and Schaumburg (1980) under paragraph (f)(6) of this section. A more sophisticated technique is described by Palay and Chan-Palay (1974) under paragraph (f)(4) of this section.
(C) Removal of brain and cord. After perfusion, the bonystructure (cranium and vertebral column) shall be exposed. Animals shall then be stored in fixative-filled bags at 4 °C for 8-12 hours. The cranium and vertebral column shall be removed carefully by trained technicians without physical damage of the brain and cord. Detailed dissection procedures may be found in the text by Palay and Chan-Palay (1974) under paragraph (f)(4) of this section. After removal, simple measurement of the size (length and width) and weight of the whole brain (cerebrum, cerebellum, pons-medulla) shall be made. Any abnormal coloration or discoloration of the brain and cord shall also be noted and recorded.
(D) Sampling. Unless a given test rule specifies otherwise, cross-sections of the following areas shall be examined: The forebrain, the center of the cerebrum, the midbrain, the cerebellum and pons, and the medulla oblongata; the spinal cord at cervical and lumbar swelling (C 3 -C 6 and L 1 -L 4 ); Gasserian ganglia, dorsal root ganglia (C 3 -C 6 , L 1 -L 4 ), dorsal and ventral root fibers (C 3 -C 6 , L 1 -L 4 ), proximal sciatic nerve (mid-thigh and sciatic notch), sural nerve (at knee), and tibial nerve (at knee). Other sites and tissue elements (e.g., gastrocnemius muscle) should be examined if deemed necessary. Any observable gross changes shall be recorded.
(iii) Specimen storage. Tissue samples from both the central and peripheral nervous system shall be further immersion fixed and stored in appropriate fixative (e.g., 10 percent buffered formalin for light microscopy; 2.5 percent buffered gluteraldehyde or 4.0 percent buffered paraformaldehyde for electron microscopy) for future examination. The volume of fixative versus the volume of tissues in a specimen jar shall be no less than 25:1. All stored tissues shall be washed with buffer for at least 2 hours prior to further tissue processing.
(iv) Histopathology examination. (A) Fixation. Tissue specimens stored in 10 percent buffered formalin may be used for this purpose. All tissues must be immersion fixed in fixative for at least 48 hours prior to further tissue processing.
(B) Dehydration. All tissue specimens shall be washed for at least 1 hour with water or buffer, prior to dehydration. (A longer washing time is needed if the specimens have been stored in fixative for a prolonged period of time.) Dehydration can be performed with increasing concentration of graded ethanols up to absolute alcohol.
(C) Clearing and embedding. After dehydration, tissue specimens shall be cleared with xylene and embedded in paraffin or paraplast. Multiple tissue specimens (e.g. brain, cord, ganglia) may be embedded together in one single block for sectioning. All tissue blocks shall be labelled showing at least the experiment number, animal number, and specimens embedded.
(D) Sectioning. Tissue sections, 5 to 6 microns in thickness, shall be prepared from the tissue blocks and mounted on standard glass slides. It is recommended that several additional sections be made from each block at this time for possible future needs for special stainings. All tissue blocks and slides shall be filed and stored in properly labeled files or boxes.
(E) Histopathological techniques. Although the information available for a given chemical substance may dictate test-rule specific changes, the following general testing sequence is proposed for gathering histopathological data:
( 1 ) General staining. A general staining procedure shall be performed on all tissue specimens in the highest treatment group. Hematoxylin and eosin (H&E) shall be used for this purpose. The staining shall be differentiated properly to achieve bluish nuclei with pinkish background.
( 2 ) Special stains. Based on the results of the general staining, selected sites and cellular components shall be further evaluated by the use of specific techniques. If H&E screening does not provide such information, a battery of stains shall be used to assess the following components in all appropriate required samples: neuronal body (e.g., Einarson's gallocyanin), axon (e.g., Bodian), myelin sheath (e.g., Kluver's Luxol Fast Blue) and neurofibrils (e.g., Bielchosky). In addition, peripheral nerve fiber teasing shall be used. Detailed staining methodology is available in standard histotechnological manuals such as AFIP (1968) under paragraph (f)(1) of this section, Ralis et al. (1973) under paragraph (f)(5) of this section, and Chang (1979) under paragraph (f)(2) of this section. The nerve fiber teasing technique is discussed in Spencer and Schaumberg (1980) under paragraph (f)(6) of this section. A section of normal tissue shall be included in each staining to assure that adequate staining has occurred. Any changes shall be noted and representative photographs shall be taken. If a lesion(s) is observed, the special techniques shall be repeated in the next lower treatment group until no further lesion is detectable.
( 3 ) Alternative technique. If the anatomical locus of expected neuro-pathology is well-defined, epoxy-embedded sections stained with toluidine blue may be used for small sized tissue samples. This technique obviates the need for special stains for cellular components. Detailed methodology is available in Spencer and Schaumberg (1980) under paragraph (f)(6) of this section.
( 4 ) Electron microscopy. Based on the results of light microscopic evaluation, specific tissue sites which reveal a lesion(s) shall be further evaluated by electron microscopy in the highest treatment group which does not reveal any light microscopic lesion. If a lesion is observed, the next lower treatment group shall be evaluated until no significant lesion is found. Detailed methodology is available in Hayat (1970) under paragraph (f)(3) of this section.
(F) Examination —( 1 ) General. All stained microscopic slides shall be examined with a standard research microscope. Examples of cellular alterations (e.g., neuronal vacuolation, degeneration, and necrosis) and tissue changes (e.g., gliosis, leukocytic infiltration, and cystic formation) shall be recorded and photographed.
( 2 ) Electron microscopy. Since the size of the tissue samples that can be examined is very small, at least 3 to 4 tissue blocks from each sampling site must be examined. Tissue sections must be examined with a transmission electron microscope. Three main categories of structural changes must be considered:
( i ) Neuronal body. The shape and position of the nucleus and nucleolus as well as any change in the chromatin patterns shall be noted. Within the neuronal cytoplasm, cytoplasmic organelles such as mitochondria, lysosomes, neurotubules, neurofilaments, microfilaments, endoplasmic reticulum and polyribosomes (Nissl substance), Golgi complex, and secretory granules shall be examined.
( ii ) Neuronal processes. The structural integrity or alterations of dendrites, axons (myelinated and unmyelinated), myelin sheaths, and synapses shall be noted.
( iii ) Supporting cells. Attention must also be paid to the number and structural integrity of the neuroglial elements (oligodendrocytes, astrocytes, and microglia) of the central nervous system, and the Schwann cells, satellite cells, and capsule cells of the peripheral nervous system. Any changes in the endothelial cells and ependymal lining cells shall also be noted whenever possible. The nature, severity, and frequency of each type of lesion in each specimen must be recorded. Representative lesions must be photographed and labeled appropriately.
(e) Data collection, reporting, and evaluation. In addition to information meeting the requirements stated under 40 CFR part 792 subpart J, the following specific information shall be reported:
(1) Description of test system and test methods. A description of the general design of the experiment shall be provided. This shall include a short justification explaining any decisions where professional judgment is involved such as fixation technique and choice of stains.
(2) Results. All observations shall be recorded and arranged by test groups. This data may be presented in the following recommended format:
(i) Description of signs and lesions for each animal. For each animal, data must be submitted showing its identification (animal number, treatment, dose, duration), neurologic signs, location(s) nature of, frequency, and severity of lesion(s). A commonly-used scale such as 1 + , 2 + , 3 + , and 4 + for degree of severity ranging from very slight to extensive may be used. Any diagnoses derived from neurologic signs and lesions including naturally occurring diseases or conditions, should also be recorded.
(ii) Counts and incidence of lesions, by test group. Data shall be tabulated to show:
(A) The number of animals used in each group, the number of animals displaying specific neurologic signs, and the number of animals in which any lesion was found;
(B) The number of animals affected by each different type of lesion, the average grade of each type of lesion, and the frequency of each different type and/or location of lesion.
(iii) Evaluation of data. (A) An evaluation of the data based on gross necropsy findings and microscopic pathology observations shall be made and supplied. The evaluation shall include the relationship, if any, between the animal's exposure to the test substance and the frequency and severity of the lesions observed.
(B) The evaluation of dose-response, if existent, for various groups shall be given, and a description of statistical method must be presented. The evaluation of neuropathology data should include, where applicable, an assessment in conjunction with other neurotoxicity studies performed (eg. electrophysiological, behavioral, neurochemical).
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) AFIP. Manual of Histologic Staining Methods. (New York: McGraw-Hill (1968).
(2) Chang, L.W. A Color Atlas and Manual for Applied Histochemistry. (Springfield, IL: Charles C. Thomas, 1979).
(3) Hayat, M.A. “Vol. 1. Biological applications,” Principles and techniques of electron microscopy. (New York: Van Nostrand Reinhold, 1970)
(4) Palay S.L., Chan-Palay, V. Cerebellar Cortex: Cytology and Organization. (New York: Springer-Verlag, 1974).
(5) Ralis, H.M., Beesley, R.A., Ralis, Z.A. Techniques in Neurohistology. (London: Butterworths, 1973).
(6) Spencer, P.S., Schaumburg, H.H. (eds). Experimental and Clinical Neurotoxicology. (Baltimore: Williams and Wilkins, 1980).
(7) Zeman, W., JRM Innes, J.R.M. Craigie's Neuroanatomy of the Rat. (New York: Academic, 1963).
§ 798.6500   Schedule-controlled operant behavior.
(a) Purpose. (1) In the assessment and evaluation of the potential human health effects of substances, it may be necessary to test for functional neurotoxic effects. Substances that have been observed to produce neurotoxic signs in other toxicity studies (e.g. CNS depression or stimulation), as well as substances with a structural similarity to known neurotoxicants should be evaluated for these effects.
(2) This guideline defines procedures for conducting studies of schedule-controlled operant behavior, one way of evaluating functional neurotoxic effects (Dews, 1972 under paragraph (f)(1) of this section; NAS 1975, 1977, 1982 under paragraphs (f)(4), (5) and (6) of this section). Our purpose is to evaluate the effects of acute and repeated exposures on the rate and pattern of responding under schedules of reinforcement. Operant behavior tests may be used to evaluate many other aspects of behavior (Laties, 1978 under paragraph (f)(3) of this section). Additional tests may be necessary to completely assess the behavioral effects of any substance. Behavioral evaluation should be used in conjunction with neuropathologic evaluation and the evaluation of other toxic effects.
(b) Definitions —(1) Neurotoxicity. Neurotoxicity or a neurotoxic effect is an adverse change in the structure or function of the nervous system following exposure to a chemical agent. Behavioral toxicity is an adverse change in the functioning of the organism with respect to its environment following exposure to a chemical agent.
(2) Operant, operant behavior, operant conditioning. An operant is a class of behavioral responses which change or operates on the environment in the same way. Operant behavior is further distinguished as behavior which is modified by its consequences. Operant conditioning is the experimental procedure used to modify some class of behavior by reinforcement or punishment.
(3) Schedule of reinforcement. A schedule of reinforcement specifies the relation between behavioral responses and the delivery of reinforcers, such as food or water (Ferster and Skinner, 1957 under paragraph (f)(2) of this section). For example, a fixed ratio (FR) schedule requires a fixed number of responses to produce a reinforcer (e.g. FR 30). On a fixed interval (FI) schedule, the first response after a fixed period of time is reinforced (e.g. FI 5 minutes).
(c) Principle of the test method. Experimental animals are trained to perform under a schedule of reinforcement and measurements of their operant behavior are made. Several doses of the test substance are then administered according to the experimental design (between groups or within subjects) and the duration of exposure (acute or repeated). Measurements of the operant behavior are repeated. A descriptive and statistical evaluation of the data is made to evaluate the nature and extent of any changes in behavior in relation to exposures to the test substance. Comparisons are made between any exposures that influence the behavior and exposures that have neuropathological effects or effects on other targets of the chemical.
(d) Test procedures —(1) Experimental design. These test procedures may be used to evaluate the behavior of experimental animals receiving either acute or repeated exposures. For acute exposure studies, either within-subject or between groups, experimental designs may be used. For repeated exposure studies, between groups designs should be used, but within subject comparisons (pre-exposure and post-exposure) are recommended and encouraged.
(2) Animal selection —(i) Species. (A) For most studies, the laboratory mouse or rat is recommended. Standard strains should be used.
(B) Under some circumstances other species may be recommended.
(ii) Age. Experimental animals should be young adults. Rats or mice should be at least 14 and 6 weeks old, respectively, prior to exposure.
(iii) Sex. (A) Approximately equal numbers of male and female animals are required for each dose level and control group.
(B) Virgin females should be used.
(iv) Experimental history. Animals should be experimentally and chemically naive.
(3) Number of animals. Six to twelve animals should be exposed to each level of the test substance and/or control procedure. If post exposure effects are examined, a separate group, 6 to 12 additional animals not sacrificed for pathology, will required in subchronic studies.
(4) Control groups —(i) Untreated controls. A concurrent “sham” exposure or vehicle control group or session (according to the design of the study) is required. The subjects should be treated similarly except that administration of the test substance is omitted.
(ii) Positive controls. Positive control data is required to demonstrate that the experimental procedures, under the specific conditions in the testing laboratory, are sensitive to substances known to affect operant behavior. Both increases and decreases in response rate should be demonstrated. Data based on acute exposures will be adequate. Data should be collected according to the same experimental design as that proposed for the test substance. Historical data on the procedure collected in the same species and under the same conditions in the testing laboratory may be acceptable, but the presentation of concurrent control data is strongly encouraged since it provides evidence that the test has remained sensitive.
(5) Dose levels and dose selection. At least 3 doses, equally spaced over a log scale (e.g., 10, 30, 100), over a range of at least 1 log unit shall be used in addition to a zero dose or vehicle administration. The data should be sufficient to produce a dose-effect curve.
(i) The highest dose shall produce: (A) Clear behavioral effects; or (B) life-threatening toxicity.
(ii) The data from the lower doses must show either: (A) Graded dose-dependent effects at 2 dose levels; or (B) no effects at 2 dose levels, respectively.
(6) Duration of exposure. The duration and frequency of exposure will be specified in the test rule.
(7) Route of Administration. The route of administration will also be specified in the test rule and will usually be identical to one of the anticipated or actual routes of human exposure. For some chemicals, another route (e.g. parenteral) may be justified. The exposure protocol should conform to that outlined in the appropriate acute or subchronic toxicity study guideline under subpart B or subpart C of this part.
(8) Study conduct —(i) Apparatus. Behavioral responses and the delivery of reinforcers shall be controlled and monitored by automated equipment located so that its operation does not provide unintended cues or otherwise interfere with the ongoing behavior. Individual chambers should be sound attenuated to prevent disruptions of behavior by external noise. The response manipulanda, feeders, and any stimulus devices should be tested before each session; these devices should periodically be calibrated.
(ii) Chamber assignment. Concurrent treatment groups should be balanced across chambers. Each subject should be tested in the chamber to which it is initially assigned.
(iii) Deprivation and training. (A) If a nonpreferred positive reinforcer is used, all subjects should be deprived of food until they reach a fixed percentage (e.g. 80 to 90 percent, commonly) of their ad libitum body weight or for a fixed period (e.g., 18 hours) prior to training. Deprivation should be kept constant throughout the study.
(B) Subjects must be trained until they display demonstrable stability in performance across days prior to exposure. One simple and useful criterion is a minimum number of sessions on the schedule and no systematic trend during the 5 days before exposure.
(C) Cumulative records of cumulative responding over time for each animal should be presented to demonstrate that the pattern of responding is representative of that generated by the schedule of reinforcement.
(iv) Time, frequency, and duration of testing —(A) Time of testing. All experimental animals should be tested at the same time of day and with respect to the time of exposure. For acute studies, testing should be performed when effects are estimated to peak, usually shortly after exposure. For subchronic studies, subjects should be tested prior to daily exposure in order to assess cumulative effects.
(B) Frequency of testing. The maintenance of stable operant behavior normally will require regular and frequent (e.g., 5 days a week) testing sessions. Animals should be weighed on each test day.
(C) Duration of testing. ( 1 ) Experimental sessions should be long enough to reasonably see the effects of exposure, but brief enough to be practical. Under most circumstances, a session length of 30-40 minutes should be adequate.
( 2 ) If the nature or duration of effects following cessation of repeated exposure are a concern, animals from the high dose group should be tested following exposure for a suitable period of time.
(v) Schedule selection. The schedule of reinforcement chosen should generate response rates that may increase or decrease as a function of exposure. Many schedules of reinforcement can do this: a single schedule maintaining a moderate response rate; fixed-interval schedules, which engender a variety of response rates in each interval; or multiple schedules, where different components may maintain high and low response rates.
(e) Data reporting and evaluation. In addition to the reporting requirements specified under 40 CFR part 792, subpart J the final test report should contain the following information:
(1) Description of system, test methods, experimental design, and control data. (i) A description of the experimental chamber, programming equipment, data collection devices, and environmental conditions.
(ii) A description of the experimental design including counterbalancing procedures, and the stability criterion.
(iii) A description and statistical evaluation of positive control and other control data, including standard measures of central tendency, variability, coefficient of variation of response rates, and the slope of the dose-effect curve.
(2) Results. (i) Data for each animal should be arranged by test group in tabular form including the animal identification number, body weight, pre-exposure rate of responding, changes in response rate produced by the chemical, and group data for the same variables, including standard measures of central tendency, variability and coefficient of variation.
(ii) A description and statistical evaluation of the test results: With particular reference to the overall statistical procedures (e.g., parametric or nonparametric) dose-effect curve, and calculation of slope. Presentation of calculations is encouraged.
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) Dews, P.B. “Assessing the Effects of Drugs,” Methods in Psychobiology, Vol. 2, Ed., R.D. Myers (New York: Academic Press, 1972) 83-124.
(2) Ferster, C.B. Skinner, B.F. Schedules of Reinforcement. (New York: Appleton-Century-Crofts, 1957).
(3) Laties, V.G. “How Operant Conditioning can Contribute to Behavioral Toxicology,” Environmental Health Perspectives, 28: 29-35 (1978).
(4) National Academy of Science. Principles for Evaluating Chemicals in the Environment. (Washington, DC: National Academy of Sciences, 1975).
(5) National Academy of Science. Principles and Procedures for Evaluating the Toxicity of Household Substances. (Washington, DC: National Academy of Sciences, 1977).
(6) National Academy of Science. “Strategies to determine needs and priorities for toxicity testing,” Appendix 3B. Reference Protocol Guidelines For Neurobehavioral Toxicity Tests. 2: 123-129 (1982).
§ 798.6560   Subchronic delayed neuro-toxicity of organophosphorus substances.
(a) Purpose. In the assessment and evaluation of the toxic characteristics of organophosphorus substances the determination of subchronic delayed neurotoxicity may be carried out, usually after initial information on delayed neurotoxicity has been obtained by acute testing or by the demonstration of inhibition and aging of neurotoxic esterase in hen neural tissue. The subchronic delayed neurotoxicity test provides information on possible health hazards likely to arise from repeated exposures over a limited period of time. It will provide information on dose response and can provide an estimate of a non-effect level which can be of use for establishing safety criteria for exposure.
(b) Definitions. Subchronic delayed neurotoxicity is a prolonged, delayed-onset locomoter ataxia resulting from repeated daily administration of the test substance.
(c) Principle of the test method. Multiple dose levels of the test substance are administered orally to domestic hens ( Gallus gallus domesticus ) for 90 days. The animals are observed at least daily for behavioral abnormalities, locomotor ataxia and paralysis. Histopathological examination of selected neural tissues is undertaken at the termination of the test period.
(d) Test procedures —(1) Animal selection. The adult domestic laying hen, aged 8 to 14 months, is recommended. Standard size breeds and strains should be employed.
(2) Number of animals. Ten hens should be used for each treatment and control group.
(3) Control group —(i) General. A concurrent control group should be used. This group should be treated in a manner identical to the treated group, except that administration of the test substance is omitted.
(ii) Reference substances. If a positive control is used, a substance which is known to produce delayed neurotoxicity should be employed. Examples of such substances are triorthocresyl phosphate (TOCP) and leptophos.
(4) Housing and feeding conditions. Cages or enclosures which are large enough to permit free mobility of the hens and easy observation of gait should be used. Where the lighting is artificial, the sequence should be 12 hours light, 12 hours dark. Appropriate diets should be administered as well as an unlimited supply of drinking water.
(5) Dose levels. At least three dose levels should be used in addition to the control group(s). The highest dose level should result in toxic effects, preferably delayed neurotoxicity, but not produce an incidence of fatalities which would prevent a meaningful evaluation. The lowest dose level should not produce any evidence of toxicity.
(6) Route of administration. Oral dosing each day for at least 5 days per week should be carried out, preferably by gavage or administration of gelatine capsules.
(7) Study conduct —(i) General. Healthy young adult hens free from interfering viral diseases and medication and without abnormalities of gait should be acclimatized to the laboratory conditions for at least 5 days prior to randomization and assignment to treatment and control groups. The test or control substance should be administered and observations begun. All hens should be carefully observed at least once daily throughout the test period. Signs of toxicity should be recorded, including the time of onset, degree and duration. Observations should include, but not be limited to, behavioral abnormality, locomotor ataxia and paralysis. At least once a week the hens should be taken outside the cages and subjected to a period of forced motor activity, such as ladder climbing, in order to enhance the observation of minimal responses. The hens should be weighed weekly. Any moribund hens should be removed and sacrificed.
(ii) Pathology —(A) Gross necropsy. In the presence of clinical signs of delayed neurotoxicity useful information may be provided by gross necropsy.
(B) Histopathology. Tissues from all animals should be fixed in situ, using perfusion techniques. Sections should include medulla oblongata, spinal cord and peripheral nerves. The spinal cord sections should be taken from the upper cervical bulb, the mid-thoracic and lumbosacral regions. Sections of the proximal region of the tibial nerve and its branches and of the sciatic nerve should be taken. Sections should be stained with appropriate myelin and axon-specific stains. Microscopic examination should be carried out on all hens in the control and high-dose groups. Microscopic examination should also be carried out on hens in the low and intermediate dose groups when there is evidence of effects in the high-dose group.
(e) Data reporting and evaluation —(1) Test report. In addition to the reporting requirements specified under 40 CFR part 792, subpart J the final test report must include the following information:
(i) Toxic response data by group with a description of clinical manifestations of nervous system damage; where a grading system is used the criteria should be defined.
(ii) For each animal, time of death during the study or whether it survived to termination.
(iii) The day of observation of each abnormal sign and its subsequent course.
(iv) Body weight data.
(v) Necropsy findings for each animal, when performed.
(vi) A detailed description of all histopathological findings.
(vii) Statistical treatment of results, where appropriate.
(2) Treatment of results. (i) Data may be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions or effects, the types of lesions or effects and the percentage of animals displaying each type of lesion or effect.
(ii) All observed results should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods should be selected during the design of the study.
(3) Evaluation of results. The findings of a subchronic delayed neurotoxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the incidence and severity of observed neurotoxic effects and any other observed effects and histopathological findings in the treated and control groups. A properly conducted subchronic test should provide a satisfactory estimation of a no-effect level based on lack of clinical signs and histopathological changes.
(f) References. For additional background information on this test guideline the following references should be consulted:
(1) Abou-Donia, M.B. “Organophosphorus ester-induced delayed neurotoxicity” Annual Review of Pharmacology and Toxicology, 21:511-548 (1981).
(2) Abou-Donia, M.B., Pressing, S.H. “Delayed neurotoxicity from continuous low-dose oral administration of leptophos to hens.” Toxicology and Applied Pharmacology, 38:595-608 (1976).
(3) Baron, R.L. (ed). “Pesticide Induced Delayed Neurotoxicity,” Proceedings of a Conference, February 19-20, 1976, Washington, DC. U.S. Environmental Protection Agency. EPA Report No. 600/1-76-025, Washington, DC (1976).
(4) Cavanaugh, J.B. “Peripheral neuropathy caused by chemical agents” Critical Reviews of Toxicity, 2:365-417 CRC Press, Inc. (1973).
(5) Johannsen, F.R., Wright, P.L., Gordon, D.E., Levinskas, G.L., Radue, R.W., Graham, P.R. “Evaluation of delayed neurotoxicity and dose-response relationship of phosphate esters in the adult hen,” Toxicology and Applied Pharmacology, 41:291-304 (1977).
(6) Johnson, M.K. “Organophosphorus esters causing delayed neurotoxic effects: mechanism of action and structure/activity studies,” Archives of Toxicology, 34:259-288 (1975).