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The low pH Syrian hamster embryo (SHE) cell transformation assay: A revitalized role in carcinogen prediction
Fundamental and Molecular Mechanisms of Mutagenesis
ELSEVIER
Mutation
Research 356 (1996) 5-9
The low pH Syrian hamster embryo ( SHE) cell transformation assay: A revitalized role in carcinogen prediction Marilyn J. Aardema
*,
Robert J. Isfort, Edward D. Thompson, Robert A. LeBoeuf
The Procter and Gamble Co., P.O. Box 538707, Cincinnati, OH 45253-8707.
USA
Abstract A series of publications of the results of National Toxicology Program (NTP) studies (Tennant et al. (1987) Science, 236. 933-941; Haseman et al. (1990) J. Am. Stat. Assoc., 85, 964-971; Shelby et al. (1993) Environ. Mol. Mutagen., 21, 160- 179) show that the commonly used short-term genotoxicity tests are less predictive of rodent carcinogenicity than once thought. These results have fueled a great deal of debate in the field of genetic toxicology regarding appropriate strategies for assessing the potential carcinogenicity of chemicals. The debate has continued in the recent discussion of harmonized genotoxicity test strategies (Ashby (1993) Mutation Res., 298, 291-295 and Ashby (1994) 308, 113-114; Madle (1993) Mutation Res., 300, 73-76 and Madle (1994) 308, 11 l-l 12; Zeiger (1994) Mutation Res., 304, 309-314) since the underlying problem still has not been resolved. The underlying problem is the fact that the current short-term genotoxicity tests in any combination do not provide both the necessary high sensitivity and high specificity needed for accurate rodent carcinogen detection. In this discussion, we describe the utility of the newly revised Syrian hamster embryo (SHE) cell transformation assay alone and in combination with the Salmonella mutation assay for improved accuracy of screening of rodent carcinogens relative to standard short-term genotoxicity tests. The accompanying papers provide details of improved methodologies for the conduct of the SHE cell transformation assay and an extensive review of the databases which support our conclusion that the SHE cell transformation assay provides an improved prediction of rodent bioassay results relative to other in vitro genotoxicity test batteries. Keywords:
Syrian hamster embryo: Cell transformation;
Carcinogen
1. Introduction Short-term tests for determining the potential carcinogenicity of chemicals have been in use for over 20 years. Results in the mid-1970s indicated a high degree of concordance (around 90%) between results from short-term tests and rodent carcinogenicity (McCann et al., 1975; Purchase et al., 1976). In contrast, more recent results with more diverse
* Corresponding 1087.
author.
Elsevier Science B.V. SSDZ 0027.5107(95)00196-4
Tel.:
513-627-2647:
Fax:
513-627-
detection
chemical classes, indicate that individual short-term tests or combinations of short-term tests have a much lower concordance (around 60%) with rodent carcinogenicity (Tennant et al., 1987; Haseman et al., 1990). It is clear that while the use of a battery of assays greatly increases the number of rodent carcinogens detected (increased sensitivity), the number of noncarcinogens that are positive also increases proportionately (decreased specificity). The net result is that there is no significant change in the overall concordance by the use of a battery of assays compared to the use of any one assay.
the practical consequences of the use of the most common in vitro genotoxicity tests to assess the carcinogenic potential of chemicals. First, the consequences of using only the Salmonella assay to assess the carcinogenic potential of chemicals are examined. The effect of adding the in vitro cytogenetic assay as a second assay and. finally, adding the LS 17XY mouse lymphoma assay to the assessment as a third assay is also examined. The results of this exercise for a population of chemicals consisting of 60 carcinogens and 40 noncarcinogens are shown in Table I. This is the same proportion of carcinogens and noncarcinogens as in the NTP database which consisted of 67 (60%) rodent carcinogens and 47 (40%) rodent noncarcinogens (Haseman et al.. 1990). Relative to conducting no short-term testing and developing all 100 chemicals for commerce, the use of the Salmonella assay reduces the number of carcinogens developed by about half from 60 to 31 (column B), if one adopts a decision rule that a positive result prevents further development of a chemical. Use of additional assays clearly leads to a further reduction in the number of carcinogens developed; the greatest reduction in the number of carcinogens from 60 (no testing) to 13 is achieved with the use of all three assays if one continues to use the decision rule that a positive result in one or more of the assays stops further development of the chemical. Although many carcinogens are detected with this test battery, it is important to note that a number of carcinogens still go undetected. These
It can be argued that the goal of short-term grnotoxicity testing is not detection of rodent carcinogens, rather the goal is detection of human carcinogens. While this is true. in practice, because chemcals can not be tested in humans, the rodent bioassay (conducted by current or modified procedures) is now and for the foreseeable future. the standard by which regulatory. industrial, and academic scientists assess potential human carcinogenicity of chemicals. Based on this. the results of the rodent bioassay directly impact the development of most industrial chemicals and pharmaceuticals unless the results arc proven not to be relevant. The number of chemicals for which results from the rodent bioassay have been concluded by a regulatory body not to be relevant to humans, however. are few (e.g.. EPA. 199 1: Flamm and Lehman-McKeeman. I991 1 and have required years of research, which is usually not compatible with the timeline for the development of new products. With the significant impact that results from the rodent bioassay will continue to have on commerce. improved methods for predicting the outcome of rodent carcinogenicity studies are needed.
2. Impact of using in vitro genotoxicity detect rodent carcinogens
assays to
An exercise with a hypothetical group of 100 chemicals being developed for commerce illustrates
Test strategy
Rodent
Rodent
Rodent
Carcinogens
Carcinogrn\
Noncarcinogens
Noncarcinogens
Developed
Not developed ”
Developed
Not developed h
Rodent
No testing
60
0
30
0
SAL
31
29
36
4
27
3s
2x
I’
I?
47
13
77
Sen = 48%. spcc = Y I (it
(’
SAL + CHO Sen = 64%. Spec = 7 I’k ’ SAL + CHO + ML Sen = 79%. Spec = 32% ,’
Abbreviatwns: SAL. Sulrrrorwll~r r~~~l~iw~uriu~?~ mutation asaay: CHO.
m \ itro chromowme
aberration asuy in Chinese hamster ovary ceil\;
ML, L5 178Y mouse lymphtrma TK k mutation assay. ” Sensitivity and specificity value\ from Haseman et aI. (IWO). ” DeciGon rule i\ that any pwitive test result prevent\ further de\rlopment
cutthe chemical.
M.J. Aardema et al./Mutation
have been classified as ‘nongenotoxic’ carcinogens for which different methods are required for detection. As shown in Table 1, the use of a battery of tests to increase the detection of carcinogens clearly has tradeoffs. As indicated in Columns C and D of Table 1, the use of multiple tests produced false-positive results for a substantial number of noncarcinogens. In this example, development of 27/40 (68%) of the noncarcinogens would have been discontinued due to false-positive results in one or more of the three assays (Column El. These 27 noncarcinogens represent lost product opportunities and consumer benefits. This example may appear overly simple in that the decision to discontinue development of a chemical is not always based solely on positive in vitro genotoxicity test results. However, in reality, the investment of time and money required to determine the relevance of positive results from in vitro assay(s) is a critical factor in the decision to discontinue development of chemicals, some of which may have provided significant benefit to society.
4. Utility of the Syrian hamster embryo (SHE) cell transformation assay As described in the papers that follow, the SHE cell transformation assay provides both the high sensitivity and high specificity needed to identify rodent carcinogens and noncarcinogens. The SHE assay as conducted according to the modifications described by LeBoeuf (LeBoeuf and Kerckaert, 1986, 1987; LeBoeuf et al., 1989) reduces or eliminates many of the previous difficulties with the conduct of the SHE cell assay. Under these conditions, the assay has an overall concordance of 85%, a sensitivity of
As discussed above, the decision to drop a chemical from development usually is not made based on in vitro genotoxicity test results alone. For many chemicals, efforts to determine the relevance of positive results from in vitro assays are employed. Perhaps the most definitive approach is to determine the
of the use of the SHE cell transformation
7
mechanism of the in vitro genotoxic effect (e.g., contaminant, acidic pH, osmolality. inhibition of DNA repair/synthesis, direct DNA interaction) and thereby the in vivo relevance. This can require both considerable time and money. The most common approach to determine the relevance of positive results from in vitro assays is to conduct an in vivo assay(s). Again, however, this approach requires time, money, and also utilizes animals. These considerations in combination with the low sensitivity of the commonly used in vivo micronucleus assay for determining rodent carcinogens (overall sensitivity = 28%, calculated from the data reported by Shelby et al., 1993) conducted via a rigorous protocol (three i.p. injections, dosages to near lethality, sensitive statistical methods) limits the utility of this approach. Ideally, one would like to avoid false positive results in the first place.
3. Follow-up methods to determine the relevance of positive results from in vitro assays
Table 2 Consequences
Research 356 (1996) 5-9
assay for 100 chemicals:
60 carcinogens,
40 noncarcinogens
A
B
C
D
E
Test strategy
Rodent Carcinogens Developed
Rodent Carcinogens Not developed (positive test results)
Rodent Noncarcinogens Developed
Rodent Noncarcinogens Not developed (positive test results)
No testing SAL + CHO + ML Sen = 79% Spec = 32% ’ SHE Sen = 87% Snec = 83% h
60 13 8
0 41 52
40 13 33
0 27 7
Abbreviations: SAL, SalmoneUa tyhimurium mutation assay; CHO, in vitro chromosome aberration assay in Chinese hamster ovary cells; ML, L5 178Y mouse lymphoma TK + mutation assay: SHE, Syrian hamster embryo cell transformation assay. a Sensitivity and specificity values from Haseman et al. (1990). b Sensitivity and specificity values from LeBoeuf et al., this issue.
8
M.J. Acrrdemtr ct cd. /Mutation
87%. and a specificity of 83% which is consistent with a larger historical database of over 200 chemicals (LeBoeuf et al., Isfort et al., accompanying papers). The consequences of screening the same 100 chemicals from Table 1 with the low pH SHE cell transformation assay are shown in Table 2. It is clear from the data in Table 2 that the SHE cell transformation assay detects more rodent carcinogens (52/60) than the battery of Salmonella, in vitro chromosome aberrations, and mouse lymphoma tests (47/60). The greatest differences is the decrease in false-positive results for noncarcinogens in the SHE cell assay compared to the standard three test battery. In the example in Table 2. 33/40 (83%) of the noncarcinogens are correctly identified compared to only 13/40 (32%) with the three test battery. Thus. the use of the SHE cell assay provides both of the desired attributes, high sensitivity and high specificity. for accurate detection of rodent carcinogens and noncarcinogens. Since our database at pH 6.70 is not large enough to definitively address whether the low pH SHE cell transformation assay detects all Salmonella-positive and since Salmonella-positive rodent carcinogens, carcinogens represent more of the two-species, multicite rodent carcinogens suspected of being more relevant to humans (Tennant, 19931, it would be prudent to include both the SHE cell transformation and Salmonella assays in routine screening at this time. The sensitivity of the use of both the SHE cell transformation and Salmonella assays for identification of rodent carcinogens is 87% (same as the SHE cell assay alone), but the specificity is decreased to 61% (LeBoeuf et al., accompanying paper). This battery still provides a significant improvement over the current three test battery (specificity 32%). It will be necessary to increase the number of Salmonellapositive carcinogens tested in the SHE assay at pH 6.70 to determine whether both assays are needed in the future. An interesting and somewhat surprising finding from this data set is the high sensitivity observed for such a diverse group of chemicals. some of which have both tissue and species specificity for rodent carcinogenicity. A possible explanation for this is the fact that since the cell isolate is embryo derived. multiple cell types and cell lineages at different
Resrarch
356
CI9961
5-9
stages in the differentiation process are represented as discussed in detail in the LeBoeuf et al. paper (this volume). Additionally, stem cells and/or nonterminally differentiated cells are present in the cell population (LeBoeuf et al, this volume), cells which may represent a common target for neoplastic transformation across multiple tissue types. As such, this complex combination of multiple differentiated and undifferentiated cell types within the SHE cell population should increase the probability that a relevant target cell type for a particular carcinogen is present in the culture. Finally it is should be emphasized that results from the SHE assay. as with any other short-term test used to predict carcinogenic potential, indicate a potential for activity in the rodent bioassay, but do not speak to the relative human risk of such an activity under anticipated human exposure conditions. Similar to results from the rodent bioassay, additional information such as metabolism and pharmacokinetic data, mechanistic understanding and exposure data is necessary to assess accurately the carcinogenic risk from chemical exposure to humans.
5. Conclusions In this discussion, we have demonstrated the advantages of the SHE cell transformation assay for identifying rodent carcinogens and noncarcinogens. The papers that follow describe a considerable data set which supports our conclusion that the SHE cell transformation assay provides an improved method for assessing rodent carcinogens compared with any combination of frequently used short-term genotoxicity tests. Based on this, we propose that the SHE cell transformation assay be considered for use in the routine safety assessment of chemicals.
References J. ( 1993) Precedents or possibilities: which should guide the harmonization of mutagenicity test protocols and carcinogen prediction strategies? Mutation Res., 29X. 291-295. Ashby, J. (1994) Propositions in genetic toxicology and their erasure, Mutation Res.. 308. 113~1 14. Risk Assessment Forum. US EPA (1991) Alpha 2u-globin: Association with chemically induced renal toxicity and neoplasia in Aahby,
M.J. Aardema et al. /Mutation the male rat. U.S. Environemntal Protection Agency, Washington, DC. Flamm, W.G. and L.D. Lehman-McKeeman (1991) The human relevance of the renal tumor-inducing potential of plimonene in male rats: Implications for risk assessment, Reg. Toxicol. Pharmacol., 13, 70-86. Haseman, J.K., E. Zeiger, M.D. Shelby, B.H. Margolin and R.W. Tennant (1990) Predicting rodent carcinogenicity from four in vitro genetic toxicity assays: an evaluation of 114 chemicals studied by the National Toxicology Program. J. Am. Stat. Assoc., 85, 964-971. LeBoeuf, R.A. and G.A. Kerckaert (1986) The induction of transformed-like morphology and enhanced growth in Syrian hamster embryo cells grown at acidic pH, Carcinogenesis, 7. 1431-1440. LeBoeuf, R.A. and G.A. Kerckaert (1987) Enhanced morphological transformation of early passage Syrian hamster embryo cells cultured in medium with a reduced bicarbonate concentration and pH, Carcinogenesis, 8, 689-697. LeBoeuf, R.A., G.K. Kerckaert, J.A. Poiley, and R. Raineri (1989) An interlaboratory comparison of enhanced morphological transformation of Syrian hamster embryo cells culture under conditions of reduced bicarbonate concentration and pH. Mutation Res., 222, 205-218. Madle, S. (1993) Problem of the harmonization of philosophies for genotoxicity testing. Mutation Res., 300, 73-76.
Research 3.56 (1996) 5-9
9
Madle, S. (1994) Strategies and philosophies of genotoxicity testing: highly sophisticated versus pragmatic regulatory approaches, Mutation Res., 308, 11 l-l 12 McCann, H.E., E. Choi, E. Yamasaki and B. N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. USA, 72, 5135-5139. Purchase, I.F.H., E. Longstaff, J. Ashby. J.A. Styles, D. Anderson, P.A. Lefevre, and F.R. Westwood (1976) Evaluation of six short-term tests for detecting chemical carcinogens and recommendations for their use, Nature, 264, 624-629. Shelby, M.D., G.L. Erexson, G.J. Hook and R.R. Tice (1993) Evaluation of a three-exposure mouse bone marrow micronucleus protocol: results with 49 chemicals, Environ. Mol. Mutagen.. 21, 160-179. Tennant, R.W., B.H. Margolin, M.D. Shelby, E. Zeiger, J.K. Haseman, J. Spalding, W. Caspary, M. Resnick, S. Stasiewicz, B. Anderson and R. Minor (1987) Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assay, Science, 236, 933-941. Tennant, R.W. (1993) Stratification of rodent carcinogenicity bioassay results to reflect human hazard, Mutation Res., 286. 111-118. Zeiger, E. (1994) Strategies and philosophies of genotoxicity testing: What is the question?, Mutation Res., 304, 309-314.
ELSEVIER
Mutation
Research 356 (1996) 5-9
The low pH Syrian hamster embryo ( SHE) cell transformation assay: A revitalized role in carcinogen prediction Marilyn J. Aardema
*,
Robert J. Isfort, Edward D. Thompson, Robert A. LeBoeuf
The Procter and Gamble Co., P.O. Box 538707, Cincinnati, OH 45253-8707.
USA
Abstract A series of publications of the results of National Toxicology Program (NTP) studies (Tennant et al. (1987) Science, 236. 933-941; Haseman et al. (1990) J. Am. Stat. Assoc., 85, 964-971; Shelby et al. (1993) Environ. Mol. Mutagen., 21, 160- 179) show that the commonly used short-term genotoxicity tests are less predictive of rodent carcinogenicity than once thought. These results have fueled a great deal of debate in the field of genetic toxicology regarding appropriate strategies for assessing the potential carcinogenicity of chemicals. The debate has continued in the recent discussion of harmonized genotoxicity test strategies (Ashby (1993) Mutation Res., 298, 291-295 and Ashby (1994) 308, 113-114; Madle (1993) Mutation Res., 300, 73-76 and Madle (1994) 308, 11 l-l 12; Zeiger (1994) Mutation Res., 304, 309-314) since the underlying problem still has not been resolved. The underlying problem is the fact that the current short-term genotoxicity tests in any combination do not provide both the necessary high sensitivity and high specificity needed for accurate rodent carcinogen detection. In this discussion, we describe the utility of the newly revised Syrian hamster embryo (SHE) cell transformation assay alone and in combination with the Salmonella mutation assay for improved accuracy of screening of rodent carcinogens relative to standard short-term genotoxicity tests. The accompanying papers provide details of improved methodologies for the conduct of the SHE cell transformation assay and an extensive review of the databases which support our conclusion that the SHE cell transformation assay provides an improved prediction of rodent bioassay results relative to other in vitro genotoxicity test batteries. Keywords:
Syrian hamster embryo: Cell transformation;
Carcinogen
1. Introduction Short-term tests for determining the potential carcinogenicity of chemicals have been in use for over 20 years. Results in the mid-1970s indicated a high degree of concordance (around 90%) between results from short-term tests and rodent carcinogenicity (McCann et al., 1975; Purchase et al., 1976). In contrast, more recent results with more diverse
* Corresponding 1087.
author.
Elsevier Science B.V. SSDZ 0027.5107(95)00196-4
Tel.:
513-627-2647:
Fax:
513-627-
detection
chemical classes, indicate that individual short-term tests or combinations of short-term tests have a much lower concordance (around 60%) with rodent carcinogenicity (Tennant et al., 1987; Haseman et al., 1990). It is clear that while the use of a battery of assays greatly increases the number of rodent carcinogens detected (increased sensitivity), the number of noncarcinogens that are positive also increases proportionately (decreased specificity). The net result is that there is no significant change in the overall concordance by the use of a battery of assays compared to the use of any one assay.
the practical consequences of the use of the most common in vitro genotoxicity tests to assess the carcinogenic potential of chemicals. First, the consequences of using only the Salmonella assay to assess the carcinogenic potential of chemicals are examined. The effect of adding the in vitro cytogenetic assay as a second assay and. finally, adding the LS 17XY mouse lymphoma assay to the assessment as a third assay is also examined. The results of this exercise for a population of chemicals consisting of 60 carcinogens and 40 noncarcinogens are shown in Table I. This is the same proportion of carcinogens and noncarcinogens as in the NTP database which consisted of 67 (60%) rodent carcinogens and 47 (40%) rodent noncarcinogens (Haseman et al.. 1990). Relative to conducting no short-term testing and developing all 100 chemicals for commerce, the use of the Salmonella assay reduces the number of carcinogens developed by about half from 60 to 31 (column B), if one adopts a decision rule that a positive result prevents further development of a chemical. Use of additional assays clearly leads to a further reduction in the number of carcinogens developed; the greatest reduction in the number of carcinogens from 60 (no testing) to 13 is achieved with the use of all three assays if one continues to use the decision rule that a positive result in one or more of the assays stops further development of the chemical. Although many carcinogens are detected with this test battery, it is important to note that a number of carcinogens still go undetected. These
It can be argued that the goal of short-term grnotoxicity testing is not detection of rodent carcinogens, rather the goal is detection of human carcinogens. While this is true. in practice, because chemcals can not be tested in humans, the rodent bioassay (conducted by current or modified procedures) is now and for the foreseeable future. the standard by which regulatory. industrial, and academic scientists assess potential human carcinogenicity of chemicals. Based on this. the results of the rodent bioassay directly impact the development of most industrial chemicals and pharmaceuticals unless the results arc proven not to be relevant. The number of chemicals for which results from the rodent bioassay have been concluded by a regulatory body not to be relevant to humans, however. are few (e.g.. EPA. 199 1: Flamm and Lehman-McKeeman. I991 1 and have required years of research, which is usually not compatible with the timeline for the development of new products. With the significant impact that results from the rodent bioassay will continue to have on commerce. improved methods for predicting the outcome of rodent carcinogenicity studies are needed.
2. Impact of using in vitro genotoxicity detect rodent carcinogens
assays to
An exercise with a hypothetical group of 100 chemicals being developed for commerce illustrates
Test strategy
Rodent
Rodent
Rodent
Carcinogens
Carcinogrn\
Noncarcinogens
Noncarcinogens
Developed
Not developed ”
Developed
Not developed h
Rodent
No testing
60
0
30
0
SAL
31
29
36
4
27
3s
2x
I’
I?
47
13
77
Sen = 48%. spcc = Y I (it
(’
SAL + CHO Sen = 64%. Spec = 7 I’k ’ SAL + CHO + ML Sen = 79%. Spec = 32% ,’
Abbreviatwns: SAL. Sulrrrorwll~r r~~~l~iw~uriu~?~ mutation asaay: CHO.
m \ itro chromowme
aberration asuy in Chinese hamster ovary ceil\;
ML, L5 178Y mouse lymphtrma TK k mutation assay. ” Sensitivity and specificity value\ from Haseman et aI. (IWO). ” DeciGon rule i\ that any pwitive test result prevent\ further de\rlopment
cutthe chemical.
M.J. Aardema et al./Mutation
have been classified as ‘nongenotoxic’ carcinogens for which different methods are required for detection. As shown in Table 1, the use of a battery of tests to increase the detection of carcinogens clearly has tradeoffs. As indicated in Columns C and D of Table 1, the use of multiple tests produced false-positive results for a substantial number of noncarcinogens. In this example, development of 27/40 (68%) of the noncarcinogens would have been discontinued due to false-positive results in one or more of the three assays (Column El. These 27 noncarcinogens represent lost product opportunities and consumer benefits. This example may appear overly simple in that the decision to discontinue development of a chemical is not always based solely on positive in vitro genotoxicity test results. However, in reality, the investment of time and money required to determine the relevance of positive results from in vitro assay(s) is a critical factor in the decision to discontinue development of chemicals, some of which may have provided significant benefit to society.
4. Utility of the Syrian hamster embryo (SHE) cell transformation assay As described in the papers that follow, the SHE cell transformation assay provides both the high sensitivity and high specificity needed to identify rodent carcinogens and noncarcinogens. The SHE assay as conducted according to the modifications described by LeBoeuf (LeBoeuf and Kerckaert, 1986, 1987; LeBoeuf et al., 1989) reduces or eliminates many of the previous difficulties with the conduct of the SHE cell assay. Under these conditions, the assay has an overall concordance of 85%, a sensitivity of
As discussed above, the decision to drop a chemical from development usually is not made based on in vitro genotoxicity test results alone. For many chemicals, efforts to determine the relevance of positive results from in vitro assays are employed. Perhaps the most definitive approach is to determine the
of the use of the SHE cell transformation
7
mechanism of the in vitro genotoxic effect (e.g., contaminant, acidic pH, osmolality. inhibition of DNA repair/synthesis, direct DNA interaction) and thereby the in vivo relevance. This can require both considerable time and money. The most common approach to determine the relevance of positive results from in vitro assays is to conduct an in vivo assay(s). Again, however, this approach requires time, money, and also utilizes animals. These considerations in combination with the low sensitivity of the commonly used in vivo micronucleus assay for determining rodent carcinogens (overall sensitivity = 28%, calculated from the data reported by Shelby et al., 1993) conducted via a rigorous protocol (three i.p. injections, dosages to near lethality, sensitive statistical methods) limits the utility of this approach. Ideally, one would like to avoid false positive results in the first place.
3. Follow-up methods to determine the relevance of positive results from in vitro assays
Table 2 Consequences
Research 356 (1996) 5-9
assay for 100 chemicals:
60 carcinogens,
40 noncarcinogens
A
B
C
D
E
Test strategy
Rodent Carcinogens Developed
Rodent Carcinogens Not developed (positive test results)
Rodent Noncarcinogens Developed
Rodent Noncarcinogens Not developed (positive test results)
No testing SAL + CHO + ML Sen = 79% Spec = 32% ’ SHE Sen = 87% Snec = 83% h
60 13 8
0 41 52
40 13 33
0 27 7
Abbreviations: SAL, SalmoneUa tyhimurium mutation assay; CHO, in vitro chromosome aberration assay in Chinese hamster ovary cells; ML, L5 178Y mouse lymphoma TK + mutation assay: SHE, Syrian hamster embryo cell transformation assay. a Sensitivity and specificity values from Haseman et al. (1990). b Sensitivity and specificity values from LeBoeuf et al., this issue.
8
M.J. Acrrdemtr ct cd. /Mutation
87%. and a specificity of 83% which is consistent with a larger historical database of over 200 chemicals (LeBoeuf et al., Isfort et al., accompanying papers). The consequences of screening the same 100 chemicals from Table 1 with the low pH SHE cell transformation assay are shown in Table 2. It is clear from the data in Table 2 that the SHE cell transformation assay detects more rodent carcinogens (52/60) than the battery of Salmonella, in vitro chromosome aberrations, and mouse lymphoma tests (47/60). The greatest differences is the decrease in false-positive results for noncarcinogens in the SHE cell assay compared to the standard three test battery. In the example in Table 2. 33/40 (83%) of the noncarcinogens are correctly identified compared to only 13/40 (32%) with the three test battery. Thus. the use of the SHE cell assay provides both of the desired attributes, high sensitivity and high specificity. for accurate detection of rodent carcinogens and noncarcinogens. Since our database at pH 6.70 is not large enough to definitively address whether the low pH SHE cell transformation assay detects all Salmonella-positive and since Salmonella-positive rodent carcinogens, carcinogens represent more of the two-species, multicite rodent carcinogens suspected of being more relevant to humans (Tennant, 19931, it would be prudent to include both the SHE cell transformation and Salmonella assays in routine screening at this time. The sensitivity of the use of both the SHE cell transformation and Salmonella assays for identification of rodent carcinogens is 87% (same as the SHE cell assay alone), but the specificity is decreased to 61% (LeBoeuf et al., accompanying paper). This battery still provides a significant improvement over the current three test battery (specificity 32%). It will be necessary to increase the number of Salmonellapositive carcinogens tested in the SHE assay at pH 6.70 to determine whether both assays are needed in the future. An interesting and somewhat surprising finding from this data set is the high sensitivity observed for such a diverse group of chemicals. some of which have both tissue and species specificity for rodent carcinogenicity. A possible explanation for this is the fact that since the cell isolate is embryo derived. multiple cell types and cell lineages at different
Resrarch
356
CI9961
5-9
stages in the differentiation process are represented as discussed in detail in the LeBoeuf et al. paper (this volume). Additionally, stem cells and/or nonterminally differentiated cells are present in the cell population (LeBoeuf et al, this volume), cells which may represent a common target for neoplastic transformation across multiple tissue types. As such, this complex combination of multiple differentiated and undifferentiated cell types within the SHE cell population should increase the probability that a relevant target cell type for a particular carcinogen is present in the culture. Finally it is should be emphasized that results from the SHE assay. as with any other short-term test used to predict carcinogenic potential, indicate a potential for activity in the rodent bioassay, but do not speak to the relative human risk of such an activity under anticipated human exposure conditions. Similar to results from the rodent bioassay, additional information such as metabolism and pharmacokinetic data, mechanistic understanding and exposure data is necessary to assess accurately the carcinogenic risk from chemical exposure to humans.
5. Conclusions In this discussion, we have demonstrated the advantages of the SHE cell transformation assay for identifying rodent carcinogens and noncarcinogens. The papers that follow describe a considerable data set which supports our conclusion that the SHE cell transformation assay provides an improved method for assessing rodent carcinogens compared with any combination of frequently used short-term genotoxicity tests. Based on this, we propose that the SHE cell transformation assay be considered for use in the routine safety assessment of chemicals.
References J. ( 1993) Precedents or possibilities: which should guide the harmonization of mutagenicity test protocols and carcinogen prediction strategies? Mutation Res., 29X. 291-295. Ashby, J. (1994) Propositions in genetic toxicology and their erasure, Mutation Res.. 308. 113~1 14. Risk Assessment Forum. US EPA (1991) Alpha 2u-globin: Association with chemically induced renal toxicity and neoplasia in Aahby,
M.J. Aardema et al. /Mutation the male rat. U.S. Environemntal Protection Agency, Washington, DC. Flamm, W.G. and L.D. Lehman-McKeeman (1991) The human relevance of the renal tumor-inducing potential of plimonene in male rats: Implications for risk assessment, Reg. Toxicol. Pharmacol., 13, 70-86. Haseman, J.K., E. Zeiger, M.D. Shelby, B.H. Margolin and R.W. Tennant (1990) Predicting rodent carcinogenicity from four in vitro genetic toxicity assays: an evaluation of 114 chemicals studied by the National Toxicology Program. J. Am. Stat. Assoc., 85, 964-971. LeBoeuf, R.A. and G.A. Kerckaert (1986) The induction of transformed-like morphology and enhanced growth in Syrian hamster embryo cells grown at acidic pH, Carcinogenesis, 7. 1431-1440. LeBoeuf, R.A. and G.A. Kerckaert (1987) Enhanced morphological transformation of early passage Syrian hamster embryo cells cultured in medium with a reduced bicarbonate concentration and pH, Carcinogenesis, 8, 689-697. LeBoeuf, R.A., G.K. Kerckaert, J.A. Poiley, and R. Raineri (1989) An interlaboratory comparison of enhanced morphological transformation of Syrian hamster embryo cells culture under conditions of reduced bicarbonate concentration and pH. Mutation Res., 222, 205-218. Madle, S. (1993) Problem of the harmonization of philosophies for genotoxicity testing. Mutation Res., 300, 73-76.
Research 3.56 (1996) 5-9
9
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