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Reflections on the declining ability of the Salmonella assay to detect rodent carcinogens as positive
Mutation Research, 205 (1988) 51-58 Elsevier
51
MTR 04436
Reflections on the declining ability of the Salmonella assay to detect rodent carcinogens as positive J. Ashby and I.F.H. Purchase Imperial Chemical Industries PLC, Central Toxicology Laboratory, Macclesfield, Cheshire SK10 4T7 (Great Britain)
(Received 17 March 1987) (Accepted 1 May 1987)
Keywords: Salmonella assay; Declining ability; Rodent carcinogens; Non-genotoxic
carcinogens; Tumour-promoting
agents.
Summary It is suggested that urgent attempts should be made to define and gain general agreement for the existence of two classes of animal carcinogen, those which are genotoxic and those which are not. In the absence of such a step, attempts to validate in vivo genotoxicity assays, and to derive a meaningful structure-activity database for chemical carcinogenesis, will be frustrated. These suggestions are supported by the preliminary findings of a detailed analysis of the carcinogen database accrued by the United States National Toxicology Program. The possibility that many non-genotoxic carcinogens should be regarded as tumour-promoting agents is considered.
Discussion Genotoxic carcinogens
A decade ago there seemed to be ample confirmation of the fact that the Salmonella mutation assay was capable of detecting as positive about 90% of the animal carcinogens known at the time, while maintaining an equally high specificity for non-carcinogens. The low (- 10%) incidence of ‘false’ positive responses was deemed acceptable given that the available cancer bioassays on such mutagenic non-carcinogens were often less than definitive. The low (- 10%) incidence of carcinogens that remained undetected was perceived as a
Correspondence: Dr. J. Ashby, Imperial Chemical Industries PLC, Central Toxicology Laboratory, Macclesfield, Cheshire SK10 4T7 (Great Britain).
cause for concern, and that led to several collaborative studies whose aim was to discern a generally acceptable in vitro assay with which to complement the Salmonella test. At that time (1976) it was appreciated that a small minority of carcinogens may have elicited tumours in animals via their protracted disturbance of normal body homeostasis, as opposed to via their direct interaction with DNA; however, there seemed to be few such agents and they were consequently deemed to present a negligible problem. Examples of these presumed nongenotoxic (epigenetic) carcinogens were provided at that time by thiourea (rat thyroid) and DDT (mouse liver). A decade later it fell to Zeiger and Tennant (1986) of the United States National Toxicology Program (NTP) to confirm with data a growing perception that the Salmonella assay was not as efficient at detecting carcinogens as had once been
0165-1218/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
52
thought. In particular, they demonstrated a reduced sensitivity (53%) for this assay to 130 carcinogens and equivocal carcinogens defined by the NTP, together with a reduced specificity of 71% to 80 non-carcinogens. More recently, Tennant et al. (1987) have concluded that the employment of additional mammalian cell genotoxicity assays as complements to the Salmonella assay does little to increase the overall detection rate for NTP carcinogens. The situation appears bleak the standard in vitro genotoxicity assays appear to offer little more than a random sensitivity to the NTP rodent carcinogens, or put another way, the earlier and seemingly minor problem of nongenotoxic carcinogens seems to have reappeared in force in the developing NTP carcinogen bioassay program. The failing fortunes of in vitro genotoxicity assays appear in stark contrast to the progress which has been made in molecular biology over the past few years, the data from most of which studies appear to enhance the credibility of the somatic mutation theory of carcinogenesis. It is suggested here that the declining sensitivity of the Salmonella assay to carcinogens is the direct result of a change in the design and sensitivity of the rodent cancer bioassay protocols, rather than of a change in the intrinsic sensitivity of the assay itself. In fact, rather than decreasing, the sensitivity to mutagens of the Salmonella assay protocol has increased over the past few years. The cancer bioassay protocols employed by the NTP involve the lifetime administration of chemicals to mice and rats at the maximum tolerated dose level (MTD). Further, these studies are accompanied by extensive pathological assessment of the treated animals and are assessed using statistical models in cases where a clear carcinogenic effect is not observed. It is suggested that such exhaustive protocols, while enabling the detection of weak genotoxic carcinogenic effects, also offer an optimised environment in which to observe chemical-induced modulations (promotion) of spontaneous tumour incidences. Agents in the latter category, when classified as carcinogens, are essentially indistinguishable from genotoxic carcinogens in the absence of ancillary information. Therefore, if an amplification of the earlier class of non-genotoxic carcinogen has taken place in the NTP program over the past decade, the
only way to appreciate this change will be to employ classical structure-activity relationships (SA), coupled to knowledge of the activity of the test agent as a genotoxin. This, of course, represents one of the purest cases of cyclic logic, but it is felt to be worthy of consideration given the magnitude of the current problems faced by in vitro genotoxicity assays. In fact, it is initially difficult to understand why the detractors of in vitro genotoxicity assays have not made more capital out of the present poor performance of the Salmonella assay, for it would require little literary skill to use the carcinogen predictivity figures published by Zeiger and Tennant (1986) to destroy the fundamental premise that carcinogens are mutagens (McCann et al., 1975). The fact that this has not happened probably indicates a shared perception that something other than the Salmonella assay has changed. The need to understand the present situation is made the more important by the fact that efforts to study and predict new tumour-promoting agents may currently be being hampered by unqualified use of the word carcinogen. Thus, it is suggested later in this article that many of the non-genotoxic NTP ‘carcinogens’ may in fact be tumour-promoting agents, and that as such their classification as ‘carcinogens’ presents the dual problem of forcing genetic toxicologists to doubt the validity of their assays, and of depriving workers in tumour-promotion of chemicals which could perhaps act as their primary reference agents for study. A further danger resides in our current failure to distinguish genotoxic from non-genotoxic carcinogens, that is, that many investigators are currently attempting to evaluate the value of short-term in vivo genotoxicity assays - mainly as a means to discern which new in vitro genotoxins are likely to also be mutagenic/carcinogenic in vivo. As a part of such evaluations it is legitimate to ask questions about the general sensitivity of these in vivo assays to carcinogens before they are employed to qualify predictions of potential activity for a chemical based on observations made in vitro. Yet, if one were to attempt a validation of any of these in vivo genotoxicity assays against the NTP carcinogen database, one would as surely devalue them as has recently occurred with the in vitro assays. One therefore either has to accept that geno-
53
toxicity assays are of little value for the prediction of animal carcinogenicity, or one has to look in detail at the NTP carcinogen database, and to speculate regarding its true nature and status. A detailed reanalysis of the NTP carcinogen database (- 300 chemicals) was recently undertaken in cooperation with Dr. Raymond Tennant of the NTP. The results of that study will be published in a special issue of Mutation Research, but in the meantime, some of its main conclusions are discussed here as they enable some of the rather diffuse concepts discussed above to be formalized. Our analysis was based on the bringing together of three separate studies. The first was to collect together chemicals which had been evaluated for carcinogenicity in Fischer 344 rats and B6C3Fl mice in studies deemed acceptable by the NTP peer review group (222 chemicals). Conclusions of carcinogenicity were accepted and the supporting tumour database tabulated. The carcinogens thus identified were segregated into six groups according to their level of carcinogenicity. The highest level was accorded to agents found carcinogenic to both species and producing tumours at two or more sites. The lowest categories were agents only found to be active at a single site in a single sex of a single species, and agents deemed to give only equivocal evidence of carcinogenicity. 88 non-carcinogens were also identified. The second study involved classifying the resultant 222 agents according to structure-activity (SA) relationships established for carcinogens prior to 1976, i.e., according to whether their chemical structure indicated genotoxicity or not. This was a relatively uncomplicated exercise based upon recently published criteria (Ashby, 1985). Finally, Dr. Errol Zeiger of the NTP made available to us the Salmonella mutation assay data which he had for most of these agents. It is not intended to present the results of that tridentine study here, rather, the following points are selected as relevant to the present debate: (1) The overall sensitivity of the Salmonella assay to the 109 unequivocal carcinogens was 56%, and its specificity for the non-carcinogens was 69%. These figures reflect those published earlier by Zeiger and Tennant (1986) and confirm the need for the present discussion. (2) The level of concordance between the
Salmonella assay and SA was consistently high (85-988 agreement across the several levels of carcinogenic response). This suggests that the electrophilic theory of carcinogenesis advanced by the Miller’s, and the ability of the Salmonella assay to discern electrophiles as positive, remain unchanged for the NTP database. (3) Trans-species carcinogens (e.g. TRIS, Fig. 1) were detected by the Salmonella assay in 37 of 51 cases (73%), and there was 92% concordance with SA for these compounds. The figure of 73% sensitivity for the Salmonella assay for these agents would probably have been higher had all of the experiments been conducted according to the more sensitive pre-incubation test protocol. Whatever, the data indicate a much more encouraging performance for this assay than is seen across the whole database. (4) The overall specificity of the Salmonella assay (69%) reflects the fact that 26 of 84 (31%) non-carcinogens were mutagenic in vitro, 21 of which were also classed as positive based upon structural considerations. This would seem to confirm that in vitro genotoxicity assays are intrinsically over-sensitive, as befits their role as screening tests. But of much greater significance is the fact that a similar proportion of Salmonella mutagens were observed for the classes of carcinogen with low levels of carcinogenicity, yet in those cases it is regarded as the ‘sensitivity’
Br
Salmonella
+ve
P/d rat
ive
P Id mouse
+ve
Kidney, forestomach, mouse B
NTP
liver, lung
rat
Fig. 1. NTP mutagenicity and carcinogenicity data for tris(2,3dibromopropyl)phosphate (TRIS).
54
figure for the Salmonella assay to these ‘carcinogens’, as opposed to the incidence (96) of false positive predictions for the non-carcinogens. For example, for the 20 chemicals recorded as selectively carcinogenic to a single tissue of a single species, only 8 were classified as having a potentially electrophilic structure, and 7 of these 8 comprised the only agents from among the 20 (35%) which were mutagenic to Salmonella. One construction to place on these figures is that the Salmonella assay only predicted 35% of the carcinogens of this class. An alternative construction is that as a class, these agents had the same proportion of in vitro mutagens as possessed by the class of non-carcinogens, and that these were 20 selective ‘carcinogens’ all of which had modified the tumour incidence in the test animals by a non-genotoxic mechanism. Two examples are provided to illustrate these concepts. In Fig. 2 the data for the non-mutagenic rat thyroid carcinogen DETU are shown. The critical question presented is ‘should one penalise the Salmonella assay for failing to detect as positive this carcinogen rather, should not its non-mutagenicity, taken together with its non-electrophilicity, alert to an alternative mechanism of carcinogenic action? Similar questions could be asked regarding the profile seen for Dicofol, one of the chemicals present in the species/ sex/ site-specific group of carcinogens (Fig. 3). If one collects together the 20 equivocal
DICOFOL
Hepatocellular
Salmonella
-ve
p/a
-ve
rat
9 mouse
-ve
d mouse
+ve
cart.
*SA
Fig. 3. NTP mutagenicity See legend to Fig. 2.
and carcinogenicity
data for Dicofol.
carcinogens, the 20 which affected only a single site of a single species, and the further 20 which only affected a single site of a single sex of a single species of animal, then a group of 60 compounds is formed. The performance of the Salmonella assay for these 60 agents does not differ significantly from its performance with the 84 noncarcinogens for which Salmonella data were available (Fig. 4). The fact that structural considera100
r
A
HNYCINH I I it
it
AlI carcinogens
DETU
(109)
2-species/2-5,,e carcinqlens
(54)
Thyroid,
Salmonella
-ve
p Id mouse
-ve
O/d
+ve
rat
NTP
foil. cell. cart. foil. cell. aden.
?/?%!k
z
Fig. 2. NTP mutagenicity and carcinogenicity data for N, N’diethylthiourea (DETU). TBA = tumour-bearing animals in control (C), low (L) and high (H) dose levels.
NO”-CWC
(e-4)
l-sPecles/ +e~“!“ocak+
I-see “owcarc
(142)
Fig. 4. Abstract of data being prepared for publication (Ashby and Tennant, 1987). For 109 NTP carcinogens and 4 noncarcinogens, the carcinogenicity data for which is adequate in both F344 rats and B6C3Fl mice, the Salmonella assay appears to have a sensitivity of 56% and a specificity of 69%. However, for the 54 2-species multiple-site carcinogens, an enhanced sensitivity was evident, while the proportion of Salmonella mutagens among the selective carcinogens, equivocal carcinogens and non-carcinogens (142 compounds) was essentially the same as for the non-carcinogens, suggesting an alternative mechanism of carcinogenic action.
55
tions support the low overall assay sensitivity observed (37%) indicates that these 60 agents are not just weak examples of the trans.-species/multi-site carcinogens discussed in (3) above. Of course, the present analysis leads to the definition of provocative examples which lie across the borders of the two proposed classes of carcinogen. One such is shown in Fig. 5, 4acetamido-2-ethoxyaniline (AEA). This agent has a structure which is consistent with its mutagenicity of Salmonella, yet it is uniquely and weakly carcinogenic to the thyroid of the female B6C3Fl mouse. At present there are no data to indicate if this represents a highly selective genotoxic carcinogenic response, or an example of a nongenotoxic endocrine carcinogen which happens to be, in common with 26 of 84 NTP non-carcinogens, mutagenic to Salmonella. In summary, it is speculated that the NTP database of 109 chemicals adequately tested in 2 species and concluded as unequivocal carcinogens contains a significant sub-group (40; 37%) of non-genotoxic carcinogens. The performance of the Salmonella assay is acceptable when testing the trans-species/multi-site carcinogens, and is no more sensitive to the 40 selective and the 20 equivocal carcinogens than it is to the noncarcinogens. Non-genotoxic agents
carcinogens / tumour-promoting
Evaluation of the 60 selective or equivocal NTP HNAc
NH, 63 AEA Salmonella 0 Id
Thyroid,
+ve
rat
-ve
P mouse
-ve
d mouse
+ve
folk cell. cart.
NTP I
C,
1 C,
0
0
Fig. 5. NTP mutagenicity and acetamido-2-ethoxyaniline (AM).
1 Low 0
1 High 1 16%TBA
carcinogenicity data for 4See legend to Fig. 2.
carcinogens for tumour-promoting properties in those species/ sexes/ tissues in which tumours were observed may prove to be more rewarding than their repetitive testing in increasingly esoteric, and increasingly unreliable in vitro genotoxicity assays. Certainly, such agents should temporarily be excluded from consideration when extending the already impressive database supporting the validity of certain of the available in vivo genotoxicity assays. Attempts to study the possible tumour promoting properties of these 60 chemicals should take account of two factors: (i) That as with genotoxicity assays, in vitro assays for promotional activities may be over-sensitive, i.e., they may involve the generation of a significant incidence of false positive responses. Thus, for a chemical to interrupt intercellular communication among cultured cells may not mean it will elicit a similar effect in vivo. (ii) The selective carcinogenic (promotional?) effects observed for the above 60 agents may be uniquely associated with enhancement of the ‘spontaneous’ tumour incidences in the appropriate tissues of the species/ strains/ sexes of the animals used in the NTP cancer bioassay program. The promotional influence may be sufficient to reveal ‘spontaneous’ tumours in tissues normally not observed to carry control tumours - initiated cells may nonetheless be present (cf. Fig. 2). Rosenkranz et al. (1986) have acknowledged the need for separate computer databases for carcinogens and Salmonella mutagens. That problem is probably due to two separate factors: (i) The hypersensitivity of in vitro assays, as illustrated by the results of the above-mentioned study, i.e., a significant proportion of noncarcinogens will be genotoxic in vitro yet be nongenotoxic in vivo and non-carcinogenic (genotoxic carcinogenesis). (ii) The possibly incorrect classification of a range of tumour-producing agents as carcinogens. The second of these two factors is potentially the most serious as it will eventually lead to a corruption of the carcinogenicity databases by benign structures such as thiourea and Dicofol (Fig. 6). These concerns endorse further the need to define and agree two distinct classes of chem-
56
Testing strategies
CARCINOGENS L
NON-GENOTOXIC
GENOTOXIC
S/A A 4
k SIA 0
4
I+
S/A C CI
k SIA D I+
CI
Fig. 6. Stylized illustration of the effect upon structure-activity (S/A) relationships in chemical carcinogenesis of segregating genotoxic from non-genotoxic carcinogens. S/A A represents the relationships established by - 1976, and these are generally consistent with those observed in genotoxicity assays (S/A B). If a diverse set of new S/A relationships exist for non-genotoxic carcinogens (as seems likely, S/A D), then this will confuse S/A relationships for all ‘carcinogens’ (S/A C; cf., Rosenkranz et al., 1985; Ashby, 1985).
ical carcinogen. Tennant has suggested the terms inductive and adaptive carcinogens to describe these two classes, the latter of which terms would replace the excess of terms currently in use e.g., non-genotoxic/ epigenetic/ hormonal/ ambivalent, etc., carcinogen and tumour-promoting agent. It is suggested to be unlikely that any single assay, be it conducted in vitro or in vivo, will be sufficient to detect all non-genotoxic carcinogens. Attempts to anticipate such agents would seem to be best made in the whole mammal, and as such they could form part of the normal evaluation of a chemical for general toxicity. Thus, it may be, for example, that the most relevant alert to the rodent ‘carcinogenicity’ of the following non-mutagens may be provided by the forms of toxicity they are known to elicit (shown in parenthesis): Sodium saccharin Thiourea, AEA, DETU, etc Nitrotriacetic
acid
Diethylhexylphthalate Butylated hydroxyanisole Dicofol, TCDD
and analogs
(rat bladder hyperplasia) (hyperplasia of both c-cells and follicular cells in the thyroid) (heavy metal ion disturbances in the kidney and bladder) (disturbances in lipid metabolism in the liver) (selective toxicity to the rodent stomach) (interruption of cell-cell corn munication in the liver)
The above considerations impinge upon the design of test batteries, in particular, on the proposals made on that subject a year ago (Ashby, 1986). First, in relation to the need for in vitro assays to act as a complement to the Salmonella assay; second, in relation to false positive in vitro responses. The need for a reliable complementary assay remains as real yet as small as it has been for the past five years - a few genotoxic (electrophilic) carcinogens and in vivo mutagens remain undetected by the Salmonella assay, however advanced the test protocol employed. These include benzene, hexamethylphosphoramide, urethane and, perhaps the best example, as recently emphasized by Gatehouse and Tweats (1986) procarbazine. These agents require detection as genotoxins in vitro and no in vitro battery can be regarded as effective until its sensitivity to them is established. The imminent danger is that the performance of complementary assays will be judged against the 60 NTP non-genotoxic ‘carcinogens’ referred to above. As has been suggested earlier, no genotoxicity assay is likely to detect these agents, and if agents such as these are employed as standards, the important need for an efficient complementary assay for genotoxins may be devalued. If that happens, it will do a great disservice to the science. Second, the incidence of Salmonella mutagens which fail to prove carcinogenic (and non-mutagenic in vivo where evaluated) is too high ( - 30%) for sole reliance to be placed on in vitro assays in any screening strategy. This emphasises a critical role for in vivo genotoxicity assays, but they, like in vitro complementary assays, could easily be wrongly discredited by their failure to detect as positive ‘carcinogens’ such as diethylthiourea (Fig. 2) and Dicofol (Fig. 3). The continuing validation of in vivo genotoxicity assays should be, at least initially, undertaken with significant genotoxic carcinogens such as Tris (Fig. 1). This article is built on the implicit assumption that trans-species/multi-tissue/genotoxic carcinogens are of maximum potential hazard to man, while selective and non-genotoxic carcinogens (tumour-promoting agents?) present a neglible hazard in isolation. This is partially supported by the fact that agents in the latter category already
51
1988
-220
STT ,” vitro
-90 non-carcinogens
NTP carcinogens
I
I I
I
I
This is the source of the current debate about m
vitroassays
Anticipated outcome Of 1” YWO STT
Fig. 7. Summary (stylized) of the situation apparent in 1983 concerning the activity of ‘classical’ carcinogens in in vitro and in viva genotoxicity assays. Most carcinogens (about 60 studied) were potentially electroptic in structure and genotoxic in vitro. Some non-mutagenic (epigenetic?) carcinogens were known and some non-carcinogens were genotoxic in vitro. Some of the latter responses may have been due to artefacts of the test or its protocol (that remains true in 1987). In 1983 the data available suggested that the combination of two in vivo genotoxicity assays would isolate the carcinogens as positive, thus their role in testing. Shaded areas represent positive responses - the scale of these histograms is purely illustrative and is not precise.
have limited evidence to suggest that their carcinogenicity in one species is not predictive of their activity in a second. It may be that repeated exposure of humans to a range of tumour-promoting agents could increase the spontaneous cancer incidence, and generalisations should be considered carefully should they involve the assumption, for example, that the non-genotoxic carcinogen TCDD presents a negligible human hazard. Nonetheless, it remains true that an individual is unlikely to discern initially his exposure to a carcinogenic dose-level of benzidine, yet might become alerted by the consequences to health of exposure to sub-carcinogenic doses of thiourea or chloroform, etc. The Salmonella assay, used as part of a small battery of in vitro and in vivo genotoxicity assays, continues to alert to carcinogens such as benzidine - the failure of such a battery to alert to Dicofol and diethylthiourea may eventually be seen as an advantage.
t Pure GenotoxicEpigenetics, active in vitro epigenetics
Non-carcinogens
carcinogens
STT m YWO
x.
:
s
C
Fig. 8. Summary (stylized) of the situation which may prevail in 1988 if the NTP carcinogens are fully evaluated for genotoxicity in viva. The present debate concerning the value of short-term tests (SIT) in vitro is evident and exaggerated as compared to in 1983 (Fig. 7). Acquisition of in viva genotoxicity data will lead to a large number of carcinogens being found to be inactive. These may be the epigenetic carcinogens referred to herein. Whatever, selection of chemicals from group A or group B will enable in viva genotoxicity assays to be ‘validated’ or devalued. Selection from groups A or B could be based on structural alerts and genotoxic activities observed in vitro. Nonetheless, the original aim (Ashby, 1983) would have been fulfilled - the isolation of genotoxic carcinogens as a maximum hazard sub-group. Assays for tumour promotion may be required to detect group B agents. Shaded areas represent positive activity and the scale is not precise.
Conclusions After a decade of continuing attempts to force genotoxicity assays to detect all rodent carcinogens as positive, it has become clear that this is not to be. It is suggested that future validation studies of genotoxicity assays should take account of changes taking place in perceptions of the word carcinogen. When a unique role for rodent genotoxicity assays in the detection of possible human mutagens and carcinogens was initially proposed
58
(Ashby, 1983), the relevant database appeared as shown in Fig. 7. Were a corresponding in vivo genotoxicity database for the NTP carcinogens to be acquired (say by 1988) it would probably appear as shown in Fig. 8. Hopefully, by that time we will have agreed a name for the non-genotoxic carcinogens which will prevent them from being used to ‘validate’ (undermine?) genotoxicity assays. If we have not, then it will have to be accepted that genotoxicity assays have little to offer for the prediction of rodent carcinogenicity, irrespective of how well they may define potential and/or actual rodent mutagens. References Ashby, J. (1983) The unique role of rodents in the detection of possible human carcinogens and mutagens, Mutation Res., 115, 117-213. Ashby, J. (1985) Fundamental structural alerts to potential
carcinogenicity or non-carcinogenicity, Environ. Mutagen., 17, 919-921. Ashby, J. (1986) The prospects for a simplified and internationally harmonized approach to the detection of possible human carcinogens and mutagens, Mutagenesis, 1, 3-16. Ashby, J., and R.W. Temrant (1987) Categorization of 223 chemicals evaluated by carcinogenicity in rats and mice according to structural criteria, mutagenicity to Salmonella and level of carcinogenicity, Mutation Res., in press. Gatehouse, D.G., and D.J. Tweats (1986) Letter to the Editor, Mutagenesis, 1, 307-309. McCann, J., 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. (U.S.A.), 72, 5135-5139; 73, 950-954. Rosenkranz, H.S., C.S. Mitchell and G. KJopman (1985) Artificial intelligence and Bayesian decision theory in the prediction of chemical carcinogens, Mutation Res., 150, l-11. Tennant et al. (1987) in preparation. Zeiger, E., and R.W. Tennant (1986) Mutagenesis, carcinogenesis: expectations, correlations and relations, Genet. Toxicol. Environ. Chemical, 8, 75-84.
51
MTR 04436
Reflections on the declining ability of the Salmonella assay to detect rodent carcinogens as positive J. Ashby and I.F.H. Purchase Imperial Chemical Industries PLC, Central Toxicology Laboratory, Macclesfield, Cheshire SK10 4T7 (Great Britain)
(Received 17 March 1987) (Accepted 1 May 1987)
Keywords: Salmonella assay; Declining ability; Rodent carcinogens; Non-genotoxic
carcinogens; Tumour-promoting
agents.
Summary It is suggested that urgent attempts should be made to define and gain general agreement for the existence of two classes of animal carcinogen, those which are genotoxic and those which are not. In the absence of such a step, attempts to validate in vivo genotoxicity assays, and to derive a meaningful structure-activity database for chemical carcinogenesis, will be frustrated. These suggestions are supported by the preliminary findings of a detailed analysis of the carcinogen database accrued by the United States National Toxicology Program. The possibility that many non-genotoxic carcinogens should be regarded as tumour-promoting agents is considered.
Discussion Genotoxic carcinogens
A decade ago there seemed to be ample confirmation of the fact that the Salmonella mutation assay was capable of detecting as positive about 90% of the animal carcinogens known at the time, while maintaining an equally high specificity for non-carcinogens. The low (- 10%) incidence of ‘false’ positive responses was deemed acceptable given that the available cancer bioassays on such mutagenic non-carcinogens were often less than definitive. The low (- 10%) incidence of carcinogens that remained undetected was perceived as a
Correspondence: Dr. J. Ashby, Imperial Chemical Industries PLC, Central Toxicology Laboratory, Macclesfield, Cheshire SK10 4T7 (Great Britain).
cause for concern, and that led to several collaborative studies whose aim was to discern a generally acceptable in vitro assay with which to complement the Salmonella test. At that time (1976) it was appreciated that a small minority of carcinogens may have elicited tumours in animals via their protracted disturbance of normal body homeostasis, as opposed to via their direct interaction with DNA; however, there seemed to be few such agents and they were consequently deemed to present a negligible problem. Examples of these presumed nongenotoxic (epigenetic) carcinogens were provided at that time by thiourea (rat thyroid) and DDT (mouse liver). A decade later it fell to Zeiger and Tennant (1986) of the United States National Toxicology Program (NTP) to confirm with data a growing perception that the Salmonella assay was not as efficient at detecting carcinogens as had once been
0165-1218/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
52
thought. In particular, they demonstrated a reduced sensitivity (53%) for this assay to 130 carcinogens and equivocal carcinogens defined by the NTP, together with a reduced specificity of 71% to 80 non-carcinogens. More recently, Tennant et al. (1987) have concluded that the employment of additional mammalian cell genotoxicity assays as complements to the Salmonella assay does little to increase the overall detection rate for NTP carcinogens. The situation appears bleak the standard in vitro genotoxicity assays appear to offer little more than a random sensitivity to the NTP rodent carcinogens, or put another way, the earlier and seemingly minor problem of nongenotoxic carcinogens seems to have reappeared in force in the developing NTP carcinogen bioassay program. The failing fortunes of in vitro genotoxicity assays appear in stark contrast to the progress which has been made in molecular biology over the past few years, the data from most of which studies appear to enhance the credibility of the somatic mutation theory of carcinogenesis. It is suggested here that the declining sensitivity of the Salmonella assay to carcinogens is the direct result of a change in the design and sensitivity of the rodent cancer bioassay protocols, rather than of a change in the intrinsic sensitivity of the assay itself. In fact, rather than decreasing, the sensitivity to mutagens of the Salmonella assay protocol has increased over the past few years. The cancer bioassay protocols employed by the NTP involve the lifetime administration of chemicals to mice and rats at the maximum tolerated dose level (MTD). Further, these studies are accompanied by extensive pathological assessment of the treated animals and are assessed using statistical models in cases where a clear carcinogenic effect is not observed. It is suggested that such exhaustive protocols, while enabling the detection of weak genotoxic carcinogenic effects, also offer an optimised environment in which to observe chemical-induced modulations (promotion) of spontaneous tumour incidences. Agents in the latter category, when classified as carcinogens, are essentially indistinguishable from genotoxic carcinogens in the absence of ancillary information. Therefore, if an amplification of the earlier class of non-genotoxic carcinogen has taken place in the NTP program over the past decade, the
only way to appreciate this change will be to employ classical structure-activity relationships (SA), coupled to knowledge of the activity of the test agent as a genotoxin. This, of course, represents one of the purest cases of cyclic logic, but it is felt to be worthy of consideration given the magnitude of the current problems faced by in vitro genotoxicity assays. In fact, it is initially difficult to understand why the detractors of in vitro genotoxicity assays have not made more capital out of the present poor performance of the Salmonella assay, for it would require little literary skill to use the carcinogen predictivity figures published by Zeiger and Tennant (1986) to destroy the fundamental premise that carcinogens are mutagens (McCann et al., 1975). The fact that this has not happened probably indicates a shared perception that something other than the Salmonella assay has changed. The need to understand the present situation is made the more important by the fact that efforts to study and predict new tumour-promoting agents may currently be being hampered by unqualified use of the word carcinogen. Thus, it is suggested later in this article that many of the non-genotoxic NTP ‘carcinogens’ may in fact be tumour-promoting agents, and that as such their classification as ‘carcinogens’ presents the dual problem of forcing genetic toxicologists to doubt the validity of their assays, and of depriving workers in tumour-promotion of chemicals which could perhaps act as their primary reference agents for study. A further danger resides in our current failure to distinguish genotoxic from non-genotoxic carcinogens, that is, that many investigators are currently attempting to evaluate the value of short-term in vivo genotoxicity assays - mainly as a means to discern which new in vitro genotoxins are likely to also be mutagenic/carcinogenic in vivo. As a part of such evaluations it is legitimate to ask questions about the general sensitivity of these in vivo assays to carcinogens before they are employed to qualify predictions of potential activity for a chemical based on observations made in vitro. Yet, if one were to attempt a validation of any of these in vivo genotoxicity assays against the NTP carcinogen database, one would as surely devalue them as has recently occurred with the in vitro assays. One therefore either has to accept that geno-
53
toxicity assays are of little value for the prediction of animal carcinogenicity, or one has to look in detail at the NTP carcinogen database, and to speculate regarding its true nature and status. A detailed reanalysis of the NTP carcinogen database (- 300 chemicals) was recently undertaken in cooperation with Dr. Raymond Tennant of the NTP. The results of that study will be published in a special issue of Mutation Research, but in the meantime, some of its main conclusions are discussed here as they enable some of the rather diffuse concepts discussed above to be formalized. Our analysis was based on the bringing together of three separate studies. The first was to collect together chemicals which had been evaluated for carcinogenicity in Fischer 344 rats and B6C3Fl mice in studies deemed acceptable by the NTP peer review group (222 chemicals). Conclusions of carcinogenicity were accepted and the supporting tumour database tabulated. The carcinogens thus identified were segregated into six groups according to their level of carcinogenicity. The highest level was accorded to agents found carcinogenic to both species and producing tumours at two or more sites. The lowest categories were agents only found to be active at a single site in a single sex of a single species, and agents deemed to give only equivocal evidence of carcinogenicity. 88 non-carcinogens were also identified. The second study involved classifying the resultant 222 agents according to structure-activity (SA) relationships established for carcinogens prior to 1976, i.e., according to whether their chemical structure indicated genotoxicity or not. This was a relatively uncomplicated exercise based upon recently published criteria (Ashby, 1985). Finally, Dr. Errol Zeiger of the NTP made available to us the Salmonella mutation assay data which he had for most of these agents. It is not intended to present the results of that tridentine study here, rather, the following points are selected as relevant to the present debate: (1) The overall sensitivity of the Salmonella assay to the 109 unequivocal carcinogens was 56%, and its specificity for the non-carcinogens was 69%. These figures reflect those published earlier by Zeiger and Tennant (1986) and confirm the need for the present discussion. (2) The level of concordance between the
Salmonella assay and SA was consistently high (85-988 agreement across the several levels of carcinogenic response). This suggests that the electrophilic theory of carcinogenesis advanced by the Miller’s, and the ability of the Salmonella assay to discern electrophiles as positive, remain unchanged for the NTP database. (3) Trans-species carcinogens (e.g. TRIS, Fig. 1) were detected by the Salmonella assay in 37 of 51 cases (73%), and there was 92% concordance with SA for these compounds. The figure of 73% sensitivity for the Salmonella assay for these agents would probably have been higher had all of the experiments been conducted according to the more sensitive pre-incubation test protocol. Whatever, the data indicate a much more encouraging performance for this assay than is seen across the whole database. (4) The overall specificity of the Salmonella assay (69%) reflects the fact that 26 of 84 (31%) non-carcinogens were mutagenic in vitro, 21 of which were also classed as positive based upon structural considerations. This would seem to confirm that in vitro genotoxicity assays are intrinsically over-sensitive, as befits their role as screening tests. But of much greater significance is the fact that a similar proportion of Salmonella mutagens were observed for the classes of carcinogen with low levels of carcinogenicity, yet in those cases it is regarded as the ‘sensitivity’
Br
Salmonella
+ve
P/d rat
ive
P Id mouse
+ve
Kidney, forestomach, mouse B
NTP
liver, lung
rat
Fig. 1. NTP mutagenicity and carcinogenicity data for tris(2,3dibromopropyl)phosphate (TRIS).
54
figure for the Salmonella assay to these ‘carcinogens’, as opposed to the incidence (96) of false positive predictions for the non-carcinogens. For example, for the 20 chemicals recorded as selectively carcinogenic to a single tissue of a single species, only 8 were classified as having a potentially electrophilic structure, and 7 of these 8 comprised the only agents from among the 20 (35%) which were mutagenic to Salmonella. One construction to place on these figures is that the Salmonella assay only predicted 35% of the carcinogens of this class. An alternative construction is that as a class, these agents had the same proportion of in vitro mutagens as possessed by the class of non-carcinogens, and that these were 20 selective ‘carcinogens’ all of which had modified the tumour incidence in the test animals by a non-genotoxic mechanism. Two examples are provided to illustrate these concepts. In Fig. 2 the data for the non-mutagenic rat thyroid carcinogen DETU are shown. The critical question presented is ‘should one penalise the Salmonella assay for failing to detect as positive this carcinogen rather, should not its non-mutagenicity, taken together with its non-electrophilicity, alert to an alternative mechanism of carcinogenic action? Similar questions could be asked regarding the profile seen for Dicofol, one of the chemicals present in the species/ sex/ site-specific group of carcinogens (Fig. 3). If one collects together the 20 equivocal
DICOFOL
Hepatocellular
Salmonella
-ve
p/a
-ve
rat
9 mouse
-ve
d mouse
+ve
cart.
*SA
Fig. 3. NTP mutagenicity See legend to Fig. 2.
and carcinogenicity
data for Dicofol.
carcinogens, the 20 which affected only a single site of a single species, and the further 20 which only affected a single site of a single sex of a single species of animal, then a group of 60 compounds is formed. The performance of the Salmonella assay for these 60 agents does not differ significantly from its performance with the 84 noncarcinogens for which Salmonella data were available (Fig. 4). The fact that structural considera100
r
A
HNYCINH I I it
it
AlI carcinogens
DETU
(109)
2-species/2-5,,e carcinqlens
(54)
Thyroid,
Salmonella
-ve
p Id mouse
-ve
O/d
+ve
rat
NTP
foil. cell. cart. foil. cell. aden.
?/?%!k
z
Fig. 2. NTP mutagenicity and carcinogenicity data for N, N’diethylthiourea (DETU). TBA = tumour-bearing animals in control (C), low (L) and high (H) dose levels.
NO”-CWC
(e-4)
l-sPecles/ +e~“!“ocak+
I-see “owcarc
(142)
Fig. 4. Abstract of data being prepared for publication (Ashby and Tennant, 1987). For 109 NTP carcinogens and 4 noncarcinogens, the carcinogenicity data for which is adequate in both F344 rats and B6C3Fl mice, the Salmonella assay appears to have a sensitivity of 56% and a specificity of 69%. However, for the 54 2-species multiple-site carcinogens, an enhanced sensitivity was evident, while the proportion of Salmonella mutagens among the selective carcinogens, equivocal carcinogens and non-carcinogens (142 compounds) was essentially the same as for the non-carcinogens, suggesting an alternative mechanism of carcinogenic action.
55
tions support the low overall assay sensitivity observed (37%) indicates that these 60 agents are not just weak examples of the trans.-species/multi-site carcinogens discussed in (3) above. Of course, the present analysis leads to the definition of provocative examples which lie across the borders of the two proposed classes of carcinogen. One such is shown in Fig. 5, 4acetamido-2-ethoxyaniline (AEA). This agent has a structure which is consistent with its mutagenicity of Salmonella, yet it is uniquely and weakly carcinogenic to the thyroid of the female B6C3Fl mouse. At present there are no data to indicate if this represents a highly selective genotoxic carcinogenic response, or an example of a nongenotoxic endocrine carcinogen which happens to be, in common with 26 of 84 NTP non-carcinogens, mutagenic to Salmonella. In summary, it is speculated that the NTP database of 109 chemicals adequately tested in 2 species and concluded as unequivocal carcinogens contains a significant sub-group (40; 37%) of non-genotoxic carcinogens. The performance of the Salmonella assay is acceptable when testing the trans-species/multi-site carcinogens, and is no more sensitive to the 40 selective and the 20 equivocal carcinogens than it is to the noncarcinogens. Non-genotoxic agents
carcinogens / tumour-promoting
Evaluation of the 60 selective or equivocal NTP HNAc
NH, 63 AEA Salmonella 0 Id
Thyroid,
+ve
rat
-ve
P mouse
-ve
d mouse
+ve
folk cell. cart.
NTP I
C,
1 C,
0
0
Fig. 5. NTP mutagenicity and acetamido-2-ethoxyaniline (AM).
1 Low 0
1 High 1 16%TBA
carcinogenicity data for 4See legend to Fig. 2.
carcinogens for tumour-promoting properties in those species/ sexes/ tissues in which tumours were observed may prove to be more rewarding than their repetitive testing in increasingly esoteric, and increasingly unreliable in vitro genotoxicity assays. Certainly, such agents should temporarily be excluded from consideration when extending the already impressive database supporting the validity of certain of the available in vivo genotoxicity assays. Attempts to study the possible tumour promoting properties of these 60 chemicals should take account of two factors: (i) That as with genotoxicity assays, in vitro assays for promotional activities may be over-sensitive, i.e., they may involve the generation of a significant incidence of false positive responses. Thus, for a chemical to interrupt intercellular communication among cultured cells may not mean it will elicit a similar effect in vivo. (ii) The selective carcinogenic (promotional?) effects observed for the above 60 agents may be uniquely associated with enhancement of the ‘spontaneous’ tumour incidences in the appropriate tissues of the species/ strains/ sexes of the animals used in the NTP cancer bioassay program. The promotional influence may be sufficient to reveal ‘spontaneous’ tumours in tissues normally not observed to carry control tumours - initiated cells may nonetheless be present (cf. Fig. 2). Rosenkranz et al. (1986) have acknowledged the need for separate computer databases for carcinogens and Salmonella mutagens. That problem is probably due to two separate factors: (i) The hypersensitivity of in vitro assays, as illustrated by the results of the above-mentioned study, i.e., a significant proportion of noncarcinogens will be genotoxic in vitro yet be nongenotoxic in vivo and non-carcinogenic (genotoxic carcinogenesis). (ii) The possibly incorrect classification of a range of tumour-producing agents as carcinogens. The second of these two factors is potentially the most serious as it will eventually lead to a corruption of the carcinogenicity databases by benign structures such as thiourea and Dicofol (Fig. 6). These concerns endorse further the need to define and agree two distinct classes of chem-
56
Testing strategies
CARCINOGENS L
NON-GENOTOXIC
GENOTOXIC
S/A A 4
k SIA 0
4
I+
S/A C CI
k SIA D I+
CI
Fig. 6. Stylized illustration of the effect upon structure-activity (S/A) relationships in chemical carcinogenesis of segregating genotoxic from non-genotoxic carcinogens. S/A A represents the relationships established by - 1976, and these are generally consistent with those observed in genotoxicity assays (S/A B). If a diverse set of new S/A relationships exist for non-genotoxic carcinogens (as seems likely, S/A D), then this will confuse S/A relationships for all ‘carcinogens’ (S/A C; cf., Rosenkranz et al., 1985; Ashby, 1985).
ical carcinogen. Tennant has suggested the terms inductive and adaptive carcinogens to describe these two classes, the latter of which terms would replace the excess of terms currently in use e.g., non-genotoxic/ epigenetic/ hormonal/ ambivalent, etc., carcinogen and tumour-promoting agent. It is suggested to be unlikely that any single assay, be it conducted in vitro or in vivo, will be sufficient to detect all non-genotoxic carcinogens. Attempts to anticipate such agents would seem to be best made in the whole mammal, and as such they could form part of the normal evaluation of a chemical for general toxicity. Thus, it may be, for example, that the most relevant alert to the rodent ‘carcinogenicity’ of the following non-mutagens may be provided by the forms of toxicity they are known to elicit (shown in parenthesis): Sodium saccharin Thiourea, AEA, DETU, etc Nitrotriacetic
acid
Diethylhexylphthalate Butylated hydroxyanisole Dicofol, TCDD
and analogs
(rat bladder hyperplasia) (hyperplasia of both c-cells and follicular cells in the thyroid) (heavy metal ion disturbances in the kidney and bladder) (disturbances in lipid metabolism in the liver) (selective toxicity to the rodent stomach) (interruption of cell-cell corn munication in the liver)
The above considerations impinge upon the design of test batteries, in particular, on the proposals made on that subject a year ago (Ashby, 1986). First, in relation to the need for in vitro assays to act as a complement to the Salmonella assay; second, in relation to false positive in vitro responses. The need for a reliable complementary assay remains as real yet as small as it has been for the past five years - a few genotoxic (electrophilic) carcinogens and in vivo mutagens remain undetected by the Salmonella assay, however advanced the test protocol employed. These include benzene, hexamethylphosphoramide, urethane and, perhaps the best example, as recently emphasized by Gatehouse and Tweats (1986) procarbazine. These agents require detection as genotoxins in vitro and no in vitro battery can be regarded as effective until its sensitivity to them is established. The imminent danger is that the performance of complementary assays will be judged against the 60 NTP non-genotoxic ‘carcinogens’ referred to above. As has been suggested earlier, no genotoxicity assay is likely to detect these agents, and if agents such as these are employed as standards, the important need for an efficient complementary assay for genotoxins may be devalued. If that happens, it will do a great disservice to the science. Second, the incidence of Salmonella mutagens which fail to prove carcinogenic (and non-mutagenic in vivo where evaluated) is too high ( - 30%) for sole reliance to be placed on in vitro assays in any screening strategy. This emphasises a critical role for in vivo genotoxicity assays, but they, like in vitro complementary assays, could easily be wrongly discredited by their failure to detect as positive ‘carcinogens’ such as diethylthiourea (Fig. 2) and Dicofol (Fig. 3). The continuing validation of in vivo genotoxicity assays should be, at least initially, undertaken with significant genotoxic carcinogens such as Tris (Fig. 1). This article is built on the implicit assumption that trans-species/multi-tissue/genotoxic carcinogens are of maximum potential hazard to man, while selective and non-genotoxic carcinogens (tumour-promoting agents?) present a neglible hazard in isolation. This is partially supported by the fact that agents in the latter category already
51
1988
-220
STT ,” vitro
-90 non-carcinogens
NTP carcinogens
I
I I
I
I
This is the source of the current debate about m
vitroassays
Anticipated outcome Of 1” YWO STT
Fig. 7. Summary (stylized) of the situation apparent in 1983 concerning the activity of ‘classical’ carcinogens in in vitro and in viva genotoxicity assays. Most carcinogens (about 60 studied) were potentially electroptic in structure and genotoxic in vitro. Some non-mutagenic (epigenetic?) carcinogens were known and some non-carcinogens were genotoxic in vitro. Some of the latter responses may have been due to artefacts of the test or its protocol (that remains true in 1987). In 1983 the data available suggested that the combination of two in vivo genotoxicity assays would isolate the carcinogens as positive, thus their role in testing. Shaded areas represent positive responses - the scale of these histograms is purely illustrative and is not precise.
have limited evidence to suggest that their carcinogenicity in one species is not predictive of their activity in a second. It may be that repeated exposure of humans to a range of tumour-promoting agents could increase the spontaneous cancer incidence, and generalisations should be considered carefully should they involve the assumption, for example, that the non-genotoxic carcinogen TCDD presents a negligible human hazard. Nonetheless, it remains true that an individual is unlikely to discern initially his exposure to a carcinogenic dose-level of benzidine, yet might become alerted by the consequences to health of exposure to sub-carcinogenic doses of thiourea or chloroform, etc. The Salmonella assay, used as part of a small battery of in vitro and in vivo genotoxicity assays, continues to alert to carcinogens such as benzidine - the failure of such a battery to alert to Dicofol and diethylthiourea may eventually be seen as an advantage.
t Pure GenotoxicEpigenetics, active in vitro epigenetics
Non-carcinogens
carcinogens
STT m YWO
x.
:
s
C
Fig. 8. Summary (stylized) of the situation which may prevail in 1988 if the NTP carcinogens are fully evaluated for genotoxicity in viva. The present debate concerning the value of short-term tests (SIT) in vitro is evident and exaggerated as compared to in 1983 (Fig. 7). Acquisition of in viva genotoxicity data will lead to a large number of carcinogens being found to be inactive. These may be the epigenetic carcinogens referred to herein. Whatever, selection of chemicals from group A or group B will enable in viva genotoxicity assays to be ‘validated’ or devalued. Selection from groups A or B could be based on structural alerts and genotoxic activities observed in vitro. Nonetheless, the original aim (Ashby, 1983) would have been fulfilled - the isolation of genotoxic carcinogens as a maximum hazard sub-group. Assays for tumour promotion may be required to detect group B agents. Shaded areas represent positive activity and the scale is not precise.
Conclusions After a decade of continuing attempts to force genotoxicity assays to detect all rodent carcinogens as positive, it has become clear that this is not to be. It is suggested that future validation studies of genotoxicity assays should take account of changes taking place in perceptions of the word carcinogen. When a unique role for rodent genotoxicity assays in the detection of possible human mutagens and carcinogens was initially proposed
58
(Ashby, 1983), the relevant database appeared as shown in Fig. 7. Were a corresponding in vivo genotoxicity database for the NTP carcinogens to be acquired (say by 1988) it would probably appear as shown in Fig. 8. Hopefully, by that time we will have agreed a name for the non-genotoxic carcinogens which will prevent them from being used to ‘validate’ (undermine?) genotoxicity assays. If we have not, then it will have to be accepted that genotoxicity assays have little to offer for the prediction of rodent carcinogenicity, irrespective of how well they may define potential and/or actual rodent mutagens. References Ashby, J. (1983) The unique role of rodents in the detection of possible human carcinogens and mutagens, Mutation Res., 115, 117-213. Ashby, J. (1985) Fundamental structural alerts to potential
carcinogenicity or non-carcinogenicity, Environ. Mutagen., 17, 919-921. Ashby, J. (1986) The prospects for a simplified and internationally harmonized approach to the detection of possible human carcinogens and mutagens, Mutagenesis, 1, 3-16. Ashby, J., and R.W. Temrant (1987) Categorization of 223 chemicals evaluated by carcinogenicity in rats and mice according to structural criteria, mutagenicity to Salmonella and level of carcinogenicity, Mutation Res., in press. Gatehouse, D.G., and D.J. Tweats (1986) Letter to the Editor, Mutagenesis, 1, 307-309. McCann, J., 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. (U.S.A.), 72, 5135-5139; 73, 950-954. Rosenkranz, H.S., C.S. Mitchell and G. KJopman (1985) Artificial intelligence and Bayesian decision theory in the prediction of chemical carcinogens, Mutation Res., 150, l-11. Tennant et al. (1987) in preparation. Zeiger, E., and R.W. Tennant (1986) Mutagenesis, carcinogenesis: expectations, correlations and relations, Genet. Toxicol. Environ. Chemical, 8, 75-84.