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Workshop on the practical application of the mammalian spot test in routine mutagenicity testing of drugs and other chemicals
Mutation Research, 97 (1982) 155-161 Elsevier Biomedical Press
155
Meeting Report
Workshop on the practical application of the mammalian spot test in routine mutagenicity testing of drugs and other chemicals held at the Central Institute of Genetics and Research in Cultivated Plants of the Academy of Sciences of the GDR, 4325 Gatersleben, German Democratic Republic, 28-30 April, 1981
R. Braun a, L.B. Russell b and J. Sch6neich
a
a Zentralinstitutfiir Genetik und Kulturpflanzenforschungder A d W der DDR, 4325 Gatersleben (German Democratic Republic), h Biology Division, Oak Ridge National Laborato~. , Oak Ridge, TN 37830 (U.S.A.)
(Received 24 August 1981) (Accepted 21 September 1981)
Introduction
The mammalian spot test, developed in radiation mutagenesis (Russell and Major, 1957), is now being used in several laboratories for chemical mutagenesis experiments. To date, this assay has been performed largely with reference mutagens, but the testing of chemicals with unknown genetic properties will undoubtedly increase in ttie future. A generally agreed-upon method should be available so that different laboratories may arrive at comparable and acceptable results. On the other hand, the method should allow as much flexibility as possible in order to guarantee the highest sensitivity for genetic toxicology testing. The main goal of this workshop was the exchange of experiences among investigators working with this test, and the formulation of recommendations concerning certain details in the procedure. Then sources of inter-laboratory variations and misinterpretation of results were also discussed. A summary of the recent GENETOX report on the spot test (Russell et al., 1981) was presented. Since the genetic basis of the test, its development, and the published literature are all reviewed in the GENE-TOX paper, the major focus of the Gatersleben Workshop was on the formulation of practical recommendations based on the working experience of several laboratories. These recommendations are summarized here with respect to: treatment procedure, spot classification, statistical evaluation, and interpretation of the results. The recommendations in no way conflict with the protocol outlined in the GENE-TOX report, but concern some more detailed aspects of the procedure. 0 i 65-1161/82/0000-0000/$02.75 © Elsevier Biomedical Press
156
I. Treatment In the mouse spot test, a target-cell population, the melanoblasts of embryos in utero, are exposed to the chemical to be tested. Mutation induction is monitored post-natally be checking the fur of the young mice for coloured spots that result from expression of a recessive gene involved in coat-colour determination. Embryos heterozygous for several coat-colour genes on a homozygous a/a (non-agouti) background can be obtained by crosses of C57BL X T stock or T stock × HT. Either strain combination is acceptable.
A. Time of treatment Some laboratories have designated embryonic age on the basis of time elapsed from conception, others by considering the plug day as day 1, and still others by using yet different schemes. To avoid confusion, the use of a common designation for stage of pregnancy is recommended, namely, the first of those enumerated. By this scheme, the afternoon of the plug day is considered as day 0.5 of pregnancy. If some other timing scheme is applied, or if reverse light cycles are used, all pertinent information concerning plug day in relation to treatment day(s) should be given in each publication. Criteria for choosing the optimal stage at which a mutagen should reach the embryo have been outlined in the past. Agents that are known to reach the embryo immediately on application, such as ionizing radiations, can reveal inherent sensitivity patterns. Such experiments, done in the C57BL X T cross, show maximal sensitivity to be on day 10.25, when about 200 melanoblasts are estimated to be present. Similar experiments have not yet been performed for the H T X T cross. Unknown chemical agents may require time between application to the pregnant female and maximal concentration in the embryo, and may therefore have to be applied before the stage of inherent maximal sensitivity. Most data obtained so far with the C57BL X T cross come from treatment on day 10.25 of pregnancy (Oak Ridge, Gatersleben), or 24 h earlier (other European laboratories). For the T X H T cross (as used in Dublin and Neuherberg), injections on days 7.25-9.25 (using the above-recommended designation) have resulted in the highest spot frequencies. B. Dosage High exposures should be applied; but dose selection is limited by embryotoxicity, as revealed by reduction of average litter size, abortion (or resorption) of whole litters, or poor post-natal survival. A preliminary study is needed to determine optimal doses. In the toxic dose range, selective elimination of embryos with mutations cannot be ruled out. Therefore, in addition to dose(s) that produce some toxicity, at least one clearly non-embryotoxic dose should be included. Because the purpose of the test is detection of genotoxicity, rather than risk assessment, generation of dose-response curves is not required. The usefulness of multiple-exposure dosing has been demonstrated for some compounds, whereas for other compounds there was no advantage in giving multiple exposures as compared with a single application. When compounds with unknown
157 genetic properties are tested, pharmacokinetic parameters should be taken into account in determining whether or not multiple application might increase the concentration of the agent in the target cells at the optimal stage. If information on this point does not exist, a single exposure should be attempted first, and multiple exposures only if this first attempt fails to yield a positive result. If multiple exposures are chosen, suggested stages are days 8.25, 9.25 and 10.25 for the C57BL X T cross, and days 7.25, 8.25 and 9.25 for the T X H T cross.
C. Route of application Because the initial aim of the spot test is detection of genotoxicity, the route of administration should be chosen so as to give the highest concentration of the compound at the target cells, rather than to copy the manner by which humans might be exposed. For this reason, the past practice of intraperitoneal injections may be continued unless pharmacokinetic considerations dictate otherwise. The solvent volume should not exceed 0.5 ml per animal for aqueous solutions (physiological saline, or buffer), and should be significantly lower when non-polar solvents are used.
II. Classification
A. Classes of spots Spots resulting from expression of recessives (RS), spots resulting from cell killing (WMVS), and spots due to misdifferentiation (MDS) are distinguishable by location and colour, and must be recorded separately. Recessive spots (RS) are randomly located and generally non-white (various shades of gray, tan, or brown); however, near-white RS can be produced if the linked genes p and c ch are simultaneously uncovered by deletion. Most RSs are diffuse (and diffuseness is not reduced by the s allele in the heterozygous condition, as claimed by one author). Laboratories that have no prior experience with the spot test should, in their first publications, describe RS locations, but description of individual RS colours is not meaningful, because gross visual identification is difficult and cannot give an accurate indication of genotypes. WMVS are white, and are in or very near (within 5 mm of) the midventral line. Occasionally, non-white spots, too, may occur near the midventral region and should then be recorded as RS, provided the non-white nature is certain. Spots made up of agouti-like, or phaeomelanin-containing, hairs that are extensions of, or otherwise associated with, regions which can normally contain such hairs are classified as MDS. These regions are mammae, genitalia, throat, axillary and inguinal areas. Such spots may also occur on the mid-forehead. B. Microscopical examination of spots Microscopical examination is not recommended as a routine procedure. Occasionally, however, such examination can be helpful in the making of decisions between an RS and an MDS classification. In the latter case, phaeomelanin present in the
50 i.p. 75 i.p. 20 i.p. 60 i.p. 3X20 i.p. 75 i.p.
450 i.p.
100 i.p.
EMS
Natulan
Dose (mg/kg)
ENU
Compound
Iso-tris
HBSS
Phosphate buffer Phosphate buffer Saline Saline Buffer Saline
Solvent
8.25
7.25
10.25 10.25 10.25 10.25 7.25-8.25-9.25 8.25
Stage
TXHT
T×HT
C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J T× HT
Cross
x T ×T xT x T ×T
10.9
14.9
18.0 19.2 5.5 18.4 28.8 11.7
RS
34.6
10.3
29.7 42.4 0.4 7.9 11.6 16.5
WMVS
n.c.
n.c.
22.1 33.6 n.c. n.c. n.c. 3.9
Morphol. abnorm.
Percentage of animals with
Mahon and Dawson
M a h o n and Dawson
Russell, L.B. Russell, L.B. Braun et al. Braun et al. Lang, R. Neuh~,user-Klaus, A.
Reference
RESULTS O B T A I N E D W I T H R E F E R E N C E M U T A G E N S IN T H E M A M M A L I A N SPOT TEST IN D I F F E R E N T L A B O R A T O R I E S ( C O M P I L E D F R O M MOSTLY U N P U B L I S H E D D A T A OF T H E P A R T I C I P A N T S )
TABLE I
O¢
159 hair will fluoresce at wave-lengths of < 500 A. It remains to be determined, however, that none of the recessive phenotypes can mimic such fluorescence. For microscopic studies, hairs are placed on a dry slide and embedded in Eukitt. C. Size accepted as a spot Each laboratory should clearly state its criteria for spot classification, and should be consistent in applying them. Because an RS must be clearly distinguishable from the background of black fur, an assembly of only 2 or 3 non-white hairs should not be classified as RS. In the case of WMVS, however, very small spots are easily identifiable. Some laboratories include these in the WMVS count, others do not. As long as the procedure is consistent and clearly described, it does not matter which course is chosen. D. Time for fur examination Scoring for spots should be done in a 'blind' study when mice are 12-14 days old, i.e. at a time when the fur is short and dense. A second observation is done at the age of 25-35 days. Litter size is recorded at birth and at the time of both checks, yielding information on peri-natal and post-natal survival in control and experimental groups. Furthermore, any morphological malformations affecting feet, tail, head or eyes should be monitored at birth. These parameters provide some information about possible teratological activity of the compound tested. E. Other spots Besides expression of the recessive genes under study, dominant mutations can, theoretically, also occur. Therefore, agouti-type spots in body regions not associated with areas mentioned in IIA should be counted as mutational events and combined with the RS category. As mentioned in IIA, non-ro.idventral white spots should be counted as RS, since they can represent c ch p deletions; and clearly non-white near-midventral spots should also be counted as RS.
!II. Statistical evaluation and design of the spot test A. Controls Each sp0t-test experiment should be done with an appropriate negative control, i.e., a solvent control. A laboratory-historical control for each solvent should be accumulated. If the concurrent solvent control of at least 150 observations does not differ significantly from the historical control for that solvent, it may be added to the latter for statistical comparison with the current experimental group (see IIIB). The pooling of controls from experiments with different solvents is not recommended, since experience has shown that corn-oil or olive-oil control can yield RS frequencies that are significantly different from results in untreated animals. A positive control is not necessary for each experiment, but laboratories that have had no prior experience with the spot test should experiment first with strongly positive compounds. A number of such compounds was recommended by the workshop, and results from different laboratories are shown in Table 1.
160
B. Statistics The concurrent solvent control, or, if permissible (see IliA), the combined concurrent and historical solvent controls, should be compared with the treated group with respect to RS frequencies, using Fisher's exact test (one-tailed) at the 5% level of significance. By establishing certain criteria, non-positive results can be designated as either negative or inconclusive. Thus, the GENE-TOX Work Group on the Mouse Spot Test designates a result as negative if (a) the frequency in the experimental group is not significantly higher than that of the appropriate control, and (b) the induced (i.e., experimental minus control) RS frequency is less than 4 times the historical control RS frequency. Non-positive results not meeting these criteria are designated as inconclusive. For details of calculation, the GENE-TOX report should be consulted (Russell et al., 1981). The investigator may wish to choose less stringent criteria for distinguishing between negative and inconclusive, i.e., a factor greater than 4 in (b). If so, this should be clearly stated. C. Sample size An illustrative calculation based on the G E N E - T O X criteria (see IIIB), which uses combined published saline controls from Oak Ridge, Schering, and Gatersleben for the C57BL X T and NMRI X DBA crosses (3 RS in 784 = 0.38%), indicates that samples of > 155 are needed for the' finding of negative results. In a sample of 156-226, for example, a finding of 0 RS is negative, and a finding of 4 or more RS is positive; but there are several results that are inconclusive according to the above criteria, namely 1, 2 or 3 RS. Clearly positive results can, of course, be obtained with smaller sample sizes. The Gatersleben workshop recommended that, in routine testing, there should be 200-300 offspring scored in experimental groups. If the average litter size is about 5, this requires 40-60 litters; but since only about 2 / 3 of C57BL females with plugs produce litters, 60-90 females must be treated with the unknown agent. If this agent produces toxic effects, the number of females must be increased. (If productivity is greater than assumed above, the number could, of course, be decreased.) In the case of the T × H T cross, the productivity of females is better than in the C57BL X T cross, but the control RS frequencies appear to be higher (7 RS in 406 = 1.72% in the G E N E - T O X tabulation), requiring larger samples to obtain significantly positive results.
IV. Role of the spot test Although the spot test is relatively fast compared with some other whole-mammal tests, it requires more resources than do assays involving prokaryotes, lower eukaryotes and cell-culture systems. It will therefore probably be used more for the testing of selected compounds than for large-scale screening. Its major strengths are: (a) that it is a test in vivo; (b) that it monitors gene mutation end-points, as well as several types of chromosomal damage; and (c) that it provides ancillary information on cytotoxicity, embryotoxicity and teratogenicity of a compound.
161
A positive result will classify a chemical as a potential carcinogen; so far, all agents found positive in the spot test have either been carcinogens or not yet been tested for carcinogenicity. (No statement on specificity can as yet be made.) A positive spot-test result will also indicate the possibility that inherited damage could be produced, and such an indication should be followed up with germ-line mutagenicity studies. The spot test by itself is not designed to provide quantitative information on genetic risk, since it does not address such factors as transport of the agent to the gonads, specific germ-cell-type sensitivities, and local repair systems. A negative spot-test result must be qualified by the possibility of a placental barrier, unless there are clear indications that the agent or its active metabolites have reached the target cells. Even then, negative results should not be considered sufficient grounds in themselves for designating an agent non-genotoxic. It should also be noted that until large historical controls have been accumulated, most non-positive results will be inconclusive rather than clearly negative.
References Fahrig, R. (1978) The mammalian spot test: A sensitive in vivo method for the detection of genetic alterations in somatic cells of mice, in: A. Hollaender and F.J. de Serres (Eds.), Chemical Mutagens, Vol. 5, Plenum, New York, pp. 151 - 176. Russell, L.B., and M.H. Major (1957) Radiation-induced presumed somatic mutations in the house mouse, Genetics, 42, 161-175. Russell, L.B., P.B. Selby, E. von Halle, W. Sheridan and L. Valcovic (1981) Use of the mouse spot test in chemical mutagenesis; Interpretation of past data and recommendations for future work, Mutation Res., 86, 355-379.
155
Meeting Report
Workshop on the practical application of the mammalian spot test in routine mutagenicity testing of drugs and other chemicals held at the Central Institute of Genetics and Research in Cultivated Plants of the Academy of Sciences of the GDR, 4325 Gatersleben, German Democratic Republic, 28-30 April, 1981
R. Braun a, L.B. Russell b and J. Sch6neich
a
a Zentralinstitutfiir Genetik und Kulturpflanzenforschungder A d W der DDR, 4325 Gatersleben (German Democratic Republic), h Biology Division, Oak Ridge National Laborato~. , Oak Ridge, TN 37830 (U.S.A.)
(Received 24 August 1981) (Accepted 21 September 1981)
Introduction
The mammalian spot test, developed in radiation mutagenesis (Russell and Major, 1957), is now being used in several laboratories for chemical mutagenesis experiments. To date, this assay has been performed largely with reference mutagens, but the testing of chemicals with unknown genetic properties will undoubtedly increase in ttie future. A generally agreed-upon method should be available so that different laboratories may arrive at comparable and acceptable results. On the other hand, the method should allow as much flexibility as possible in order to guarantee the highest sensitivity for genetic toxicology testing. The main goal of this workshop was the exchange of experiences among investigators working with this test, and the formulation of recommendations concerning certain details in the procedure. Then sources of inter-laboratory variations and misinterpretation of results were also discussed. A summary of the recent GENETOX report on the spot test (Russell et al., 1981) was presented. Since the genetic basis of the test, its development, and the published literature are all reviewed in the GENE-TOX paper, the major focus of the Gatersleben Workshop was on the formulation of practical recommendations based on the working experience of several laboratories. These recommendations are summarized here with respect to: treatment procedure, spot classification, statistical evaluation, and interpretation of the results. The recommendations in no way conflict with the protocol outlined in the GENE-TOX report, but concern some more detailed aspects of the procedure. 0 i 65-1161/82/0000-0000/$02.75 © Elsevier Biomedical Press
156
I. Treatment In the mouse spot test, a target-cell population, the melanoblasts of embryos in utero, are exposed to the chemical to be tested. Mutation induction is monitored post-natally be checking the fur of the young mice for coloured spots that result from expression of a recessive gene involved in coat-colour determination. Embryos heterozygous for several coat-colour genes on a homozygous a/a (non-agouti) background can be obtained by crosses of C57BL X T stock or T stock × HT. Either strain combination is acceptable.
A. Time of treatment Some laboratories have designated embryonic age on the basis of time elapsed from conception, others by considering the plug day as day 1, and still others by using yet different schemes. To avoid confusion, the use of a common designation for stage of pregnancy is recommended, namely, the first of those enumerated. By this scheme, the afternoon of the plug day is considered as day 0.5 of pregnancy. If some other timing scheme is applied, or if reverse light cycles are used, all pertinent information concerning plug day in relation to treatment day(s) should be given in each publication. Criteria for choosing the optimal stage at which a mutagen should reach the embryo have been outlined in the past. Agents that are known to reach the embryo immediately on application, such as ionizing radiations, can reveal inherent sensitivity patterns. Such experiments, done in the C57BL X T cross, show maximal sensitivity to be on day 10.25, when about 200 melanoblasts are estimated to be present. Similar experiments have not yet been performed for the H T X T cross. Unknown chemical agents may require time between application to the pregnant female and maximal concentration in the embryo, and may therefore have to be applied before the stage of inherent maximal sensitivity. Most data obtained so far with the C57BL X T cross come from treatment on day 10.25 of pregnancy (Oak Ridge, Gatersleben), or 24 h earlier (other European laboratories). For the T X H T cross (as used in Dublin and Neuherberg), injections on days 7.25-9.25 (using the above-recommended designation) have resulted in the highest spot frequencies. B. Dosage High exposures should be applied; but dose selection is limited by embryotoxicity, as revealed by reduction of average litter size, abortion (or resorption) of whole litters, or poor post-natal survival. A preliminary study is needed to determine optimal doses. In the toxic dose range, selective elimination of embryos with mutations cannot be ruled out. Therefore, in addition to dose(s) that produce some toxicity, at least one clearly non-embryotoxic dose should be included. Because the purpose of the test is detection of genotoxicity, rather than risk assessment, generation of dose-response curves is not required. The usefulness of multiple-exposure dosing has been demonstrated for some compounds, whereas for other compounds there was no advantage in giving multiple exposures as compared with a single application. When compounds with unknown
157 genetic properties are tested, pharmacokinetic parameters should be taken into account in determining whether or not multiple application might increase the concentration of the agent in the target cells at the optimal stage. If information on this point does not exist, a single exposure should be attempted first, and multiple exposures only if this first attempt fails to yield a positive result. If multiple exposures are chosen, suggested stages are days 8.25, 9.25 and 10.25 for the C57BL X T cross, and days 7.25, 8.25 and 9.25 for the T X H T cross.
C. Route of application Because the initial aim of the spot test is detection of genotoxicity, the route of administration should be chosen so as to give the highest concentration of the compound at the target cells, rather than to copy the manner by which humans might be exposed. For this reason, the past practice of intraperitoneal injections may be continued unless pharmacokinetic considerations dictate otherwise. The solvent volume should not exceed 0.5 ml per animal for aqueous solutions (physiological saline, or buffer), and should be significantly lower when non-polar solvents are used.
II. Classification
A. Classes of spots Spots resulting from expression of recessives (RS), spots resulting from cell killing (WMVS), and spots due to misdifferentiation (MDS) are distinguishable by location and colour, and must be recorded separately. Recessive spots (RS) are randomly located and generally non-white (various shades of gray, tan, or brown); however, near-white RS can be produced if the linked genes p and c ch are simultaneously uncovered by deletion. Most RSs are diffuse (and diffuseness is not reduced by the s allele in the heterozygous condition, as claimed by one author). Laboratories that have no prior experience with the spot test should, in their first publications, describe RS locations, but description of individual RS colours is not meaningful, because gross visual identification is difficult and cannot give an accurate indication of genotypes. WMVS are white, and are in or very near (within 5 mm of) the midventral line. Occasionally, non-white spots, too, may occur near the midventral region and should then be recorded as RS, provided the non-white nature is certain. Spots made up of agouti-like, or phaeomelanin-containing, hairs that are extensions of, or otherwise associated with, regions which can normally contain such hairs are classified as MDS. These regions are mammae, genitalia, throat, axillary and inguinal areas. Such spots may also occur on the mid-forehead. B. Microscopical examination of spots Microscopical examination is not recommended as a routine procedure. Occasionally, however, such examination can be helpful in the making of decisions between an RS and an MDS classification. In the latter case, phaeomelanin present in the
50 i.p. 75 i.p. 20 i.p. 60 i.p. 3X20 i.p. 75 i.p.
450 i.p.
100 i.p.
EMS
Natulan
Dose (mg/kg)
ENU
Compound
Iso-tris
HBSS
Phosphate buffer Phosphate buffer Saline Saline Buffer Saline
Solvent
8.25
7.25
10.25 10.25 10.25 10.25 7.25-8.25-9.25 8.25
Stage
TXHT
T×HT
C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J T× HT
Cross
x T ×T xT x T ×T
10.9
14.9
18.0 19.2 5.5 18.4 28.8 11.7
RS
34.6
10.3
29.7 42.4 0.4 7.9 11.6 16.5
WMVS
n.c.
n.c.
22.1 33.6 n.c. n.c. n.c. 3.9
Morphol. abnorm.
Percentage of animals with
Mahon and Dawson
M a h o n and Dawson
Russell, L.B. Russell, L.B. Braun et al. Braun et al. Lang, R. Neuh~,user-Klaus, A.
Reference
RESULTS O B T A I N E D W I T H R E F E R E N C E M U T A G E N S IN T H E M A M M A L I A N SPOT TEST IN D I F F E R E N T L A B O R A T O R I E S ( C O M P I L E D F R O M MOSTLY U N P U B L I S H E D D A T A OF T H E P A R T I C I P A N T S )
TABLE I
O¢
159 hair will fluoresce at wave-lengths of < 500 A. It remains to be determined, however, that none of the recessive phenotypes can mimic such fluorescence. For microscopic studies, hairs are placed on a dry slide and embedded in Eukitt. C. Size accepted as a spot Each laboratory should clearly state its criteria for spot classification, and should be consistent in applying them. Because an RS must be clearly distinguishable from the background of black fur, an assembly of only 2 or 3 non-white hairs should not be classified as RS. In the case of WMVS, however, very small spots are easily identifiable. Some laboratories include these in the WMVS count, others do not. As long as the procedure is consistent and clearly described, it does not matter which course is chosen. D. Time for fur examination Scoring for spots should be done in a 'blind' study when mice are 12-14 days old, i.e. at a time when the fur is short and dense. A second observation is done at the age of 25-35 days. Litter size is recorded at birth and at the time of both checks, yielding information on peri-natal and post-natal survival in control and experimental groups. Furthermore, any morphological malformations affecting feet, tail, head or eyes should be monitored at birth. These parameters provide some information about possible teratological activity of the compound tested. E. Other spots Besides expression of the recessive genes under study, dominant mutations can, theoretically, also occur. Therefore, agouti-type spots in body regions not associated with areas mentioned in IIA should be counted as mutational events and combined with the RS category. As mentioned in IIA, non-ro.idventral white spots should be counted as RS, since they can represent c ch p deletions; and clearly non-white near-midventral spots should also be counted as RS.
!II. Statistical evaluation and design of the spot test A. Controls Each sp0t-test experiment should be done with an appropriate negative control, i.e., a solvent control. A laboratory-historical control for each solvent should be accumulated. If the concurrent solvent control of at least 150 observations does not differ significantly from the historical control for that solvent, it may be added to the latter for statistical comparison with the current experimental group (see IIIB). The pooling of controls from experiments with different solvents is not recommended, since experience has shown that corn-oil or olive-oil control can yield RS frequencies that are significantly different from results in untreated animals. A positive control is not necessary for each experiment, but laboratories that have had no prior experience with the spot test should experiment first with strongly positive compounds. A number of such compounds was recommended by the workshop, and results from different laboratories are shown in Table 1.
160
B. Statistics The concurrent solvent control, or, if permissible (see IliA), the combined concurrent and historical solvent controls, should be compared with the treated group with respect to RS frequencies, using Fisher's exact test (one-tailed) at the 5% level of significance. By establishing certain criteria, non-positive results can be designated as either negative or inconclusive. Thus, the GENE-TOX Work Group on the Mouse Spot Test designates a result as negative if (a) the frequency in the experimental group is not significantly higher than that of the appropriate control, and (b) the induced (i.e., experimental minus control) RS frequency is less than 4 times the historical control RS frequency. Non-positive results not meeting these criteria are designated as inconclusive. For details of calculation, the GENE-TOX report should be consulted (Russell et al., 1981). The investigator may wish to choose less stringent criteria for distinguishing between negative and inconclusive, i.e., a factor greater than 4 in (b). If so, this should be clearly stated. C. Sample size An illustrative calculation based on the G E N E - T O X criteria (see IIIB), which uses combined published saline controls from Oak Ridge, Schering, and Gatersleben for the C57BL X T and NMRI X DBA crosses (3 RS in 784 = 0.38%), indicates that samples of > 155 are needed for the' finding of negative results. In a sample of 156-226, for example, a finding of 0 RS is negative, and a finding of 4 or more RS is positive; but there are several results that are inconclusive according to the above criteria, namely 1, 2 or 3 RS. Clearly positive results can, of course, be obtained with smaller sample sizes. The Gatersleben workshop recommended that, in routine testing, there should be 200-300 offspring scored in experimental groups. If the average litter size is about 5, this requires 40-60 litters; but since only about 2 / 3 of C57BL females with plugs produce litters, 60-90 females must be treated with the unknown agent. If this agent produces toxic effects, the number of females must be increased. (If productivity is greater than assumed above, the number could, of course, be decreased.) In the case of the T × H T cross, the productivity of females is better than in the C57BL X T cross, but the control RS frequencies appear to be higher (7 RS in 406 = 1.72% in the G E N E - T O X tabulation), requiring larger samples to obtain significantly positive results.
IV. Role of the spot test Although the spot test is relatively fast compared with some other whole-mammal tests, it requires more resources than do assays involving prokaryotes, lower eukaryotes and cell-culture systems. It will therefore probably be used more for the testing of selected compounds than for large-scale screening. Its major strengths are: (a) that it is a test in vivo; (b) that it monitors gene mutation end-points, as well as several types of chromosomal damage; and (c) that it provides ancillary information on cytotoxicity, embryotoxicity and teratogenicity of a compound.
161
A positive result will classify a chemical as a potential carcinogen; so far, all agents found positive in the spot test have either been carcinogens or not yet been tested for carcinogenicity. (No statement on specificity can as yet be made.) A positive spot-test result will also indicate the possibility that inherited damage could be produced, and such an indication should be followed up with germ-line mutagenicity studies. The spot test by itself is not designed to provide quantitative information on genetic risk, since it does not address such factors as transport of the agent to the gonads, specific germ-cell-type sensitivities, and local repair systems. A negative spot-test result must be qualified by the possibility of a placental barrier, unless there are clear indications that the agent or its active metabolites have reached the target cells. Even then, negative results should not be considered sufficient grounds in themselves for designating an agent non-genotoxic. It should also be noted that until large historical controls have been accumulated, most non-positive results will be inconclusive rather than clearly negative.
References Fahrig, R. (1978) The mammalian spot test: A sensitive in vivo method for the detection of genetic alterations in somatic cells of mice, in: A. Hollaender and F.J. de Serres (Eds.), Chemical Mutagens, Vol. 5, Plenum, New York, pp. 151 - 176. Russell, L.B., and M.H. Major (1957) Radiation-induced presumed somatic mutations in the house mouse, Genetics, 42, 161-175. Russell, L.B., P.B. Selby, E. von Halle, W. Sheridan and L. Valcovic (1981) Use of the mouse spot test in chemical mutagenesis; Interpretation of past data and recommendations for future work, Mutation Res., 86, 355-379.