FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
19,207-2 13 ( 1992)
An Examination of the Association between Maximum-Tolerated Dose and Carcinogenicity in 326 Long-Term Studies in Rats and Mice J. K. HASEMAN*,’
AND S. K. SEILKOP~
*Division qj’Bi0metr.v and Risk Assessment. National Institute qf Environmental Health Sciences, P.O. BO.Y12233. Research Triangle Park, North Carolina 27709: and tilnalytical Sciences, Inc., Alston Technical Park. 100 Capitola Drive, Suite 106. Durham, North Carolina 27713
Received July 17,1991;accepted February24, 1992 An Examination of the Association betweenMaximum-Tolerated Dose and Carcinogenicity in 326 Long-Term Studies in Rats and Mice. HASEMAN, J. K., AND SEILKOP, S. K. (1992). Fundam. Appl. Toxicol. 19, 207-213. The associationbetween rodent carcinogenicity and maximum-tolerateddose(MTD) wasevaluatedin 326 long-term carcinogenicity studiesin mice and rats. Others investigating this associationhave focusedprimarily on positive studies,but our investigation consideredall experimental outcomes. We found that chemicals with low MTDs were somewhatmore likely to be rodent carcinogensthan chemicalswith high MTDs, but this associationwaslimited primarily to gavagestudies.Overall, the MTD was not a reliable predictor of whether or not a chemical would be a rodent carcinogen. Our investigation confirms that comparisonsof carcinogenic potenciesbasedonly on positive
studies may result in artifactually elevated estimates of the underlying associationbetweenchemical toxicity and rodent carcinogenicity and thus may alsoinflate the estimatedinterspecies correlation in carcinogenic response.Nevertheless,the results
of our study are consistent with the frequently cited 75% concordancein carcinogenicity outcomebetweenrats and mice. This
concordance is quite high, particularly since 80% is approximately the maximum level of observableinterspeciesconcordanceachievablefor a set of chemicalswith relatively low carcinogenic potency, becauseof the variability in observedtumor responsesthat can induce false negative or false positive outcomes in either of the two species.Thus, the underlying qualitative interspeciescorrelation in carcinogenicresponsebetween rats and mice may be greater than is commonly recognized. 0 1992 Society of Toxicology.
During the past two decades the National Cancer Institute (NCI) and the National Toxicology Program (NTP) have designed, carried out, and evaluated approximately 400 longterm rodent carcinogenicity studies (Haseman et al., 1987; Huff and Haseman, 1991; Huff et al., 1991). The majority of these studies involve four sex-species experiments: male and female rats and mice. ’ To whom
correspondence
should
be addressed.
The experimental designs of these studies are similar: within each sex-species experiment there are generally two dosed groups and a control; the more recent NTP studies include a third dosed group. There are usually 50 animals per group, although certain of the earlier NC1 studies used fewer (10-25) control animals. Most of the studies are of 2year duration, which includes the majority of the animal’s natural life span. At the conclusion of the study, all surviving animals are killed, and approximately 30 different organs sites are examined for possible carcinogenic effects. The top dose utilized in these experiments is the “maximum-tolerated dose” or MTD. The MTD is a dose determined from the results of shorter duration studies that is estimated to produce some minimal toxic effects in a longterm study (e.g., a small reduction in body weight), but should not shorten an animal’s life span or unduly compromise normal well-being except for chemically induced carcinogenicity (International Life Sciences Institute, 1984; Haseman, 1985). The lower doses are generally chosen to be some fraction of the MTD (e.g., f and $). For feeding studies, there is a practical limiting upper dose of 50,000 ppm (i.e., 5% of the diet), since higher doses might well produce nutritional deficiencies. Previous evaluations of the NCI/NTP and other large databases have consistently estimated that the qualitative concordance in carcinogenicity outcome between rats and mice is approximately 75% (Haseman and Huff, 1987; Gold et al.. 1989). That is, approximately 75% ofthe studies involving these two species agree in either their positive or negative carcinogenicity outcome for both species, The evaluation of quantitative interspecies correlation in carcinogenic response has been more problematic. Most of these analyses use some function of the median tumorigenic dose, or TD50 (Peto et al., 1984), as the measure of carcinogenic potency. Initial indications showed that the carcinogenic potencies in rats and mice were highly correlated (Crouch and Wilson, 1979; Crouch, 1983) but other investigators (Bernstein et al., 1985) maintained that some portion of this correlation was an “artifact” resulting basically from two other associations.
207 Copyright All tights
0272-0590/92 $5.00 0 1992 by the Society of Toxicology. of reproduction in any form reserved.
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First, it has been demonstrated that estimated TDSOs are selected and evaluated 326 studies: 223 feeding studies and 103 oral gavage studies that were adequately evaluated in at least one of the four sex-species virtually always constrained to be within a 30-fold factor of groups. The remaining studies utilized other routes of administration (e.g.. the MTD, the top dose used in the long-term study (Bernstein inhalation, skin paint, drinking water) that did not occur in sufficient numbers et al., 1985; Crouch et al., 1987; Reith and Starr, 1989). for meaningful evaluation. Moreover, the range of MTDs (and TDSOs) for a given set For each of the 326 studies the top dose employed in the study, i.e., the of chemicals may be quite large: as much as five or six orders estimated MTD. was evaluated in each sex-species group. For feeding studies units were ppm: for gavage studies the dosage units were mg/kg/day. of magnitude. Under these conditions MTDs and TDSOs dosage In the gavage studies the doses were adjusted when necessary to take into within a species will necessarily be highly correlated. account differences in the frequency of dosing: generally the chemical was Second, the MTDs employed in long-term studies are administered five times/week, but certain studies utilized dosing schedules generally quite similar (and are often identical) for rats and of two, three, or seven times per week. The carcinogenicity outcomes used in our evaluation were those reported mice. The logical consequence of these two strong associaby the NCI/NTP in its Technical Report series and summarized in various tions (i.e., MTD vs TD50 within a species: MTDs across publications (Griesemer and Cueto, 1980; Haseman et al.. 1987; Huff and species) is that for any group of chemicals carcinogenic to Haseman, I99 I). An experiment was considered “positive” if the chemical both rats and mice, the TDSOs in the two species will also being evaluated was considered to be carcinogenic or (using the more recent be highly (and to some extent artifactually) correlated. If NTP terminology) showed “clear evidence” or “some evidence” of carcieither of these two correlations is reduced, then the corre- nogenic activity (Huff et al., 1991). Other possible experimental outcomes sponding interspecies correlation in carcinogenic response were “equivocal” (for uncertain findings), “negative” (for no carcinogenic effects),and “inadequate” (for experiments that because of major limitations will also be reduced. could not be meaningfully evaluated for carcinogenicity). Most investigators agree that the correlation between rat Comparisons of MTDs were made between positive and negative carciand mouse MTDs is quite high. However, evaluating the nogenicity studies for each sex-species group. Equivocal or uncertain outcorrelation between carcinogenicity and MTD within a spe- comes were considered negative: inadequate outcomes were excluded. Comparisons were also made for each species by combining results for males cies is somewhat more difficult. A number of investigators and females (i.e., analyses based on zero, one, or two sex-specific positive (e.g., Zeise et al., 1984, 1986; Gold et al., 1986; Crouch et experiments; MTDs were averaged across sexes)and also for those chemicals al., 1987: Rieth and Starr, 1989; Goodman et al., 1991; evaluated adequately in all four sex-species groups (analyses based on zero, Shlyakhter et al., 1992) have carried out analyses to deter- one. two, three, or four positive experiments; MTDs averaged across the mine the degree to which the interspecies correlation in car- four groups). Feeding and gavage studies were evaluated separately, since it cinogenicity is artifactually constrained. At present, no con- is difficult to equate the single daily bolus dose received by the gavage route of administration with the more diffuse chemical exposure received in feeding sensus has been reached on this matter. Moreover, these studies. evaluations have limited their attention primarily to those Pairwise comparisons were made by Mann-Whitney U tests. Dose-rechemicals showing carcinogenic effects in both species. sponse trends were assessedby Jonckheere’s test. Evaluations of paired comHowever, a comprehensive evaluation of interspecies cor- parison data were carried out by Wilcoxon signed-rank tests. Dichotomous relation should take into account all experimental out- response variables were evaluated by Fisher’s exact test. and analysesinvolving the pooling of 2 X 2 tables were carried out by Mantel-Haenszel tests. All comes-negative as well as positive. Our paper addresses of these procedures are described by Hollander and Wolfe (1973) and/or by this issue by examining the strength of the correlation be- Cart et al. ( 1986). Two-sided tests were used for all statistical procedures. tween carcinogenicity and MTD for all chemicals evaluated for carcinogenicity in a series of long-term rodent studies. RESULTS There is another reason to examine this association: Haseman et al. (1990) noted that in 114 NTP long-term studies, Table 1 summarizes the comparisons of MTDs for each the estimated MTD appeared to be correlated with carci- sex-species group. There are a number of interesting findings nogenicity outcome. These authors speculated that the es- from this table that do not relate directly to the association timated MTD of a chemical might be as good or better a of carcinogenicity and MTD. For example, for each of the predictor of rodent carcinogenicity than certain short-term four sex-species groups, chemicals evaluated by the gavage (genetic toxicity) assays that they also evaluated. However, route are more likely to be carcinogenic than are chemicals they concluded that “more work is needed before definitive evaluated by the feed route. Overall, 26% (2 18/847) of the conclusions can be reached.” The current paper addresses feeding experiments are positive compared with 4 1% ( 159/ this issue by evaluating the association between MTD and 384) of the gavage experiments. carcinogenicity in a database of long-term rodent studies There are no statistically significant (p < 0.05) differences approximately three times larger than the previous evalua- among the four sex-species groups with regard to the protion. portion of studies showing carcinogenic effects for either gavage or feeding studies. However, for both routes of adMETHODS ministration male rats and female mice appear to be slightly more likely to show carcinogenic effects than the other two The initial database that we considered consisted of 379 long-term chemical sex-species groups, a finding consistent with previous obcarcinogenicity studies carried out in rats and/or mice by the NC1 or the NTP (Huff and Haseman, 199 1; Huff et al.. 199 1). From this database we servations (Haseman et al., 1987; Huff et al., 1991).
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AND CARCINOGENICITY
209
TABLE 1 Comparison of Maximum Tolerated Doses (MTDs) for Positive and Negative Feed and Gavage Rodent Carcinogenicity Studies Positive carcinogenicity studies Median MTD
Mean MTD
SD
N
Median MTD
MTD
SD
59 51 50 58
3000 2500 2500 2500
6763 5906 6079 4984
10.499 9,580 9,028 6,352
157 166 156 I50
3000 3000 4000 4915
9,22 I 9,232 10,252 10,317
14,332 13,912 15.414 15,312
46 32 37 44
90 100 185 175
181 151 439 300
227 187 898 371
50 65 57 53
125 122 245 254
474 364 538 704
842 491 800 1,056
N
Feed studies Male rats Female rats Male mice Female mice Gavage studies Male rats Female rats* Male mice Female mice*
Negative carcinogenicity studies
-
Note. MTD dosage units for feeding studies are ppm and for gavage studies are mg/kg/day. N. number of studies; SD, standard deviation. * Marginally significant (p = 0.043 for female rats;p = 0.052 for female mice) difference in MTD between positive and negative studies (Mann-Whitney U test).
A comparison of MTDs among sex-species groups permits an evaluation of relative sensitivity to chemically related toxicity. Within a species there are no significant differences between males and females. However, rats appear to be more sensitive than mice to chemically related toxicity, in that the MTDs on average are significantly (p < 0.001) lower (see Table 1). Although an inspection of Tables 1 and 2 suggests that this difference in species sensitivity is limited primarily to gavage studies, the corresponding effect in feeding studies becomes more apparent when one considers that comparisons of ppm doses across species do not take food consumption and body weight differences into account. NTP
studies have consistently shown that when equivalent ppm doses in feeding studies are converted to mg/kg, the resulting doses in mg/kg are approximately 2-3 times higher for mice than for rats (see also Table 1 of Gold et al., 1984). Thus, the species sensitivity difference in MTD is evident for both routes of administration if comparisons are made on a mg/ kg basis. Table 1 indicates a consistent, but relatively weak, association between chemical toxicity and carcinogenicity. There is a suggestion in all four sex-species groups and for both routes of administration that positive studies have lower MTDs (i.e., higher toxicities) than negative studies. However, the strongest statistical evidence of an effect is only marginal
TABLE 2 Association between Maximum Tolerated Dose (MTD) and Number of Positive Carcinogenicity Experiments for Rats and Mice Gavage studies*
Feeding studies No. positive experiments
N
Median MTD
Mean MTD
SD
N
Median MTD
Mean MTD
SD
Rats
0 (both sexes -) 1 (one sex +) 2 (both sexes +)
148 25 41
3000 3000 2200
9,464 7,067 6,142
14,336 9,509 10,471
45 24 27
130 90 92
453 222 151
684 276 175
Mice
0 (both sexes -) 1 (one sex +) 2 (both sexes +)
138 26 41
4566 2500 2450
10,831 6,121 5.129
15,883 8,477 6.612
47 13 33
239 160 188
614 603 322
874 1346 440
Note MTD dosage units for feeding studies are ppm and for gavage studies are mg/kg/day. N. number of studies; SD. standard deviation. * p = 0.047 trend for rats and p = 0.07 1 trend for mice (Jonckheere’s test), i.e., MTD decreases as the number of positive experiments increases. The corresponding trends for feeding studies are not statistically significant.
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and involves gavage studies with female rats (p = 0.043) and female mice (p = 0.052). One reason for the high mean MTD values in the negative feeding studies in Table 1 is the relatively high frequency of negative studies utilizing a top dose of 50,000 ppm. As noted earlier, it is NCI/NTP policy to employ this as the upper limit in dosed feed studies, even if it might not strictly be an MTD, because higher levels of chemical in the diet could produce nutritional deficiencies. Even though these experiments constitute a relatively small proportion (7%) of the feeding studies considered in our evaluation, it is interesting that almost all chemicals evaluated at these levels (14, 13, 17, and 16 chemicals for male rats, female rats, male mice, and female mice, respectively) were not carcinogenic. Only two chemicals evaluated at 50,000 ppm were found to be carcinogenic: Decabromodiphenyl oxide (male and female rats) and C.I. Vat Yellow 4 (male mice). Since it is possible that certain “negative studies” evaluated at 50,000 ppm would have been positive had higher doses been employed, a case could be made for excluding these studies from consideration. If this is done, then the mean MTD levels for positive and negative feeding studies shown in Table 1 would be virtually the same. This further supports the finding that the association between carcinogenicity outcome and MTD seems to be limited primarily to the gavage studies. Table 2 evaluates the data by species (combining sexes) and produces results similar to those reported in Table 1. There is marginal statistical evidence (p = 0.047 for rats; p = 0.072 for mice) of a positive association between MTD and the number of positive studies for the gavage route of administration (Table 2). However, the corresponding trends in the feeding studies are not statistically significant. Table 3 provides the corresponding comparisons for the 197 feeding studies and 87 gavage studies with adequate experiments in all four sex-species groups. For gavage studies there is a highly significant (a < 0.01) association between the number of positive experiments and mean MTD (av-
eraged over sexes and species). The corresponding trend for feeding studies is not significant. Gold et al. ( 1989) reported that chemicals toxic at relatively low doses are more accurate predictors of carcinogenicity in the second species than are chemicals that are toxic only at relatively high doses. Piegorsch et al. ( 199 1) found a similar result for concordance, i.e., interspecies correlation in carcinogenic outcome is greater for low MTD chemicals than for high MTD chemicals. The current database shows similar trends, but only for the gavage studies, as can be seen in Table 4. For example, there is a clear (p < 0.05) gradient in interspecies concordance as gavage MTD increases: concordance is quite good (77%) for the “more toxic” (lower MTD) chemicals, but poor (46%) for the less toxic chemicals. Feeding studies do not show this same trend. In fact, due in part to the negative results consistently obtained in 50,000 ppm feeding studies (noted above), the overall concordance in carcinogenic response for this route of administration is actually slightly higher for “less toxic” chemicals than for more toxic chemicals (Table 4). Interspecies concordance is slightly, but not significantly, higher in feeding studies than in gavage studies. Overall interspecies concordance is 73%, consistent with other estimates (Haseman and Huff, 1987; Gold et a/., 1989; Huff and Haseman, 199 1). One final analysis was carried out: an evaluation analogous to that conducted by Haseman et al. (1990). who defined a “toxic” chemical to be a chemical whose mean MTD was less than 4000 ppm (feeding studies) or less than 300 mg/ kg/day (gavage studies). These doses provide approximately equivalent exposure when food consumption and body weights are taken into account (Haseman and Clark, 1990). Under these conditions (and also utilizing a small number of inhalation and dosed water studies) these authors found that the concordance between this dichotomized MTD variable and carcinogenicity was 65% (72/ 111). Adopting these same definitions for the current database of studies adequately evaluated in male and female rats and
TABLE 3 Comparison of MTDs for Rodent Carcinogenicity Studies Showing 0, 1, 2, 3, or 4 Positive Experiments for Rats and Mice Feeding studies
Gavage studies*
No. positive experiments
N
Median MTD
Mean MTD
SD
N
Median MTD
Mean MTD
SD
0 I 2 3 4
112 22 31 15 17
4000 2614 3725 2175 2750
11.083 5,682 7,317 5,385 4,46 1
15.889 6,441 11.737 7,687 4.300
27 14 22 10 14
176 400 200 80 100
510 681 342 120 124
649 995 362 151 112
Note. MTD dosage units for feeding studies are ppm and for gavage studies are mg/kg/day. N. number of studies; SD, standard deviation. * p -C0.01 trend (Jonckheere’s test), i.e., MTD decreases as the number of positive experiments increases. The corresponding trend for feeding studies is not statistically significant.
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211
AND CARCINOGENICITY
TABLE 4 Interspecies Concordance in Carcinogenic Response as a Function of Mean MTD Dose in 87 Gavage and 197 Feeding Studies Carcinogenicity (rats/mice) ++
+-
-t
Total
Interspecies concordance
Mean gavage MTD study (mg/kg)* O-I 10 Ill-299 300+
15 12 2
3 5 8
4 4 I
8 8 11
30 29 28
77% (23/30) 69% (20/29) 46% (13128)
Total
29
16
15
27
87
64% (56187)
Mean feeding study MTD (ppm)* o-1499 1500-7499 7500+
11 18 9
9 6 7
9 9 7
36 33 43
65 66 66
72% (47165) 77% (5 l/66) 79% (52166)
Total
38
22
25
112
197
76% (150/197)
Feed and gavage studies
67
38
40
139
284
73% (206/284)
* MTD ranges chosen to divide the data into three approximately equal-sized groups.
mice, for feeding studies 46% (47/ 102) of the toxic chemicals were found to be carcinogens compared with 40% (38/95) of the “nontoxic” chemicals. For gavage studies 73% (43/ 59) of the toxic chemicals were carcinogens compared with 61% (17/28) of the nontoxic chemicals. Thus, the overall concordance between toxicity and carcinogenicity is 56% (158/284), which is not significantly different from the association expected by chance alone. When individual sexspecies groups were considered, this association was similar, ranging from 5 1% (female rats) to 56% (female mice). Thus, although some association between MTD and carcinogenic outcome appears to exist, particularly for gavage experiments (see Table 3) the magnitude of the MTD does not appear to be a reliable predictor of whether or not a chemical will be a rodent carcinogen. DISCUSSION It is recognized that the MTD employed in a given longterm study is only one of several possible measures of chemical toxicity. Experience has shown that the MTD is generally estimated reasonably well, and even in the instances in which the MTD is not accurately estimated, it is seldom “missed” by more than a factor of two or four in either direction. Given the six orders of magnitude difference in MTDs from study to study and the large number of chemicals evaluated, it is unlikely that slight inaccuracies in the estimation of MTDs would affect the results of this study. The frequency of statistically significant tumor effects detected at the MTD and at lower doses has been studied by Hoe1 et al. (1988). These authors found that in 99 NTP carcinogenicity studies, more than half of the positive experiments (73/127, 57%) had statistically significant (a < 0.05)
chemically related tumor increases for at least one dose below the MTD. They also found that a high proportion of the remaining experiments (42/54,78%) had tumor rates in the lower dosed groups that were elevated, but not significantly. Thus, less than 10% ( 12/ 127) of the experiments had evidence of carcinogenicity limited to the MTD. On a chemical basis, only three of the 99 chemicals considered by Hoe1 et al. (1988) showed elevated tumor rates limited to the MTD: melamine, propylene oxide, and butyl benyl phthalate. These results are more definitive than a similar evaluation carried out earlier by Haseman (1985) based on a much smaller group of chemicals, which are included as a subset of the chemicals evaluated by Hoe1 et al. ( 1988). These data suggest that there are relatively few instances in which the MTD produces unique effects that are not also observed (with reduced incidence) at lower dose levels. The reasons for the differences observed between feed and gavage studies in our investigation are not always apparent. The increased frequency of rodent carcinogens observed in gavage studies relative to feeding studies has been reported previously (Haseman et al., 1984) and may be related to the disproportionate number of halogenated hydrocarbons (which tend to be liver carcinogens) evaluated by the gavage route. Further, the gavage route of administration is generally utilized for chemicals that are volatile, unpalatable, unstable, and/or otherwise unsuitable for administration in the diet, which perhaps increases the a priori likelihood that such chemicals will subsequently show carcinogenic effects in rodents. It is less clear why gavage studies apparently show a stronger correlation between MTD and carcinogenicity outcome than feeding studies (Table 3) or why relatively nontoxic chemicals evaluated bv Ravage apparently show little or no
212
HASEMAN
interspecies correlation in carcinogenic response (Table 4). These matters require further investigation. Crouch et al. (1987) list carcinogenic potencies and MTDs for 2 13 chemicals evaluated for carcinogenicity in female mice by the NC1 or NTP, reporting that 108 were positive (i.e., had calculated carcinogenic potencies) and 105 were negative. While this database includes many of the same studies used in our evaluation, there were also some notable differences. For example, Crouch et al. calculated carcinogenic potencies for more than 30 chemicals considered negative by the NCI/NTP; they also combined various routes of administration. The underlying model assumed by these authors may be written p = 1 - (1 - a)exp{ -bd/( 1 - a)}, where p is the probability of tumor occurrence, u is the background tumor rate, d is the dose, and b is the measure of carcinogenic potency (Crouch and Wilson, 1979; Crouch, 1983). Note that as the background tumor rate approaches zero, the carcinogenic potency approaches ln(2)/TD50. Although Crouch, Wilson, and Zeise ( 1987) found a strong correlation between MTD and carcinogenic potency in the positive studies, we found that this correlation was greatly reduced when negative studies were included in the evaluation. For example, the 108 positive and 105 negative studies did not differ significantly with regard to the MTD employed in these experiments. This finding is consistent with our own results, particularly considering that the Crouch/Wilson/ Zeise database consists primarily of feeding studies, and the association found in the NCI/NTP database was limited primarily to gavage studies. Further, the correlation between log( l/MTD) and log(carcinogenic potency) for positive studies in Crouch/ Wilson/Zeise female mice database is quite impressive: r = 0.9 1. However, suppose the negative studies are also considered and the carcinogenic potency for these chemicals is set equal to some arbitrarily small number, say 0.00000 1. Then, the correlation between log( l/MTD) and log(carcinogenic potency), while still highly significant (p < 0.00 1), is reduced to only Y = 0.30. If alternative values are assumed for the carcinogenic potency of negative studies (e.g., 0.00001 or O.OOOOOOl), then the correlation will change slightly (e.g., r = 0.39 and r = 0.25 for the two cases noted), but the basic point remains: if negative studies are considered, the correlation between rodent carcinogenicity and MTD is considerably reduced. The best method for dealing with “carcinogenic potencies” in negative studies is not altogether clear. One approach is to select a tumor type showing a numerical increase and to compute a carcinogenic potency, even if the increase is not considered to be chemically related. Alternatively, it may be possible to calculate some confidence limit on the carcino-
AND SEILKOP
genie potency. The problem with both of these approaches is that such estimates of carcinogenic potency will almost certainly be constrained by the MTD and thus will lead to the same type of problem discussed above. For purposes of assessing interspecies correlation in carcinogenic response, we prefer instead to consider chemicals judged to be noncarcinogens to be in fact noncarcinogens, with (essentially) carcinogenic potencies of zero. Our evaluation of the NCI/NTP and Crouch/Wilson/Zeise databases suggests that while carcinogenic potency and MTD are highly correlated in positive studies, there is a much weaker (although still statistically significant) correlation between MTD and rodent carcinogenicity when all experimental outcomes are considered. Thus, overall interspecies correlation in carcinogenic outcome will be similarly reduced. While one should not over interpret the apparently high interspecies correlation in carcinogenic potency, it would be equally unwise to minimize the qualitative concordance in carcinogenic response between rats and mice, which has consistently been reported to be approximately 75%. This association is more impressive that might first appear: Piegorsch et al. (1992), assuming an underlying exponential (one-hit) model and the basic experimental design currently used by the NTP, evaluated the sensitivity of the bioassay for detecting low potency carcinogens. The database of Gold et al. (1984) was used to define the characteristics of typical “low potency” carcinogens. Piegorsch et al. (1992) demonstrated that for a set of such chemicals, the maximum level of observable concordance achievable between rats and mice is only approximately 80%. even if the underlying interspecies concordance in carcinogenic response for these chemicals is 100%. Higher concordance is difficult to achieve because of the variability in observed tumor responses that can occur by chance, resulting in some false negative or false positive outcomes in one of the two species. Thus, the underlying qualitative interspecies correlation in carcinogenic response between rats and mice may be greater than is commonly recognized. REFERENCES Bernstein.L.. Gold, L. S., Ames, B. N., Pike, M. C.. and Hoel. D. G. (1985). Some tautological aspects of the comparison of carcinogenic potency in rats and mice. Fzrndu?n. :1pp/. To.uicol. 5, 79-86. Crouch, E. A. C. (1983). Uncertainties in interspecies extrapolations of carcinogenicity. Envirorl. Health Perspect. 5, 32 l-327. Crouch,E.. and Wilson. R. (1979). Interspecies comparisons in carcinogenic potency. J. Toxicol. Owirorl. Health 5, 1095-l I 18. Crouch. E., Wilson. R.. and Zeise, L. (1987). Tautology or not tautology. J. Toxicol. Envirorl. Health. 20, I- 10. Cart. J. J.. Krewski. D.. Lee. P. N.. Tarone. R. E., and Wahrendorf, J. (1986). Statistical Methods in Cancer Research. Volume III. The Design and Ana/wis of’ Long-Term ,4nimal E.uperiment.s. International Agency for Research on Cancer, Lyon.
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Gold, L. S., Bernstein, L., Magaw, R., and Slone, T. H. (1989). Interspecies extrapolation in carcinogenesis: Prediction between rats and mice. Environ. Health Perspect. 81, 21 I-219. Gold, L. S., Sawyer, C. B., Magaw, R., Backman. G. M., De Veciana, M., Levinson, R., Hooper, N. K., Havender, W. R., Bernstein, L., Peto. R., Pike, M. C.. and Ames, B. N. (1984). Carcinogenic potency database of the standardized results of animal bioassays. Environ. Health Perspect. 58,9-3
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Gold, L. S.. Ward, J. M., Bernstein, L., and Stem, B. (1986). Association between carcinogenic potency and tumor pathology in rodent carcinogenesis bioassays. Fundam. Appl. Toxicol. 6, 677-690. Goodman, G., Shlyakhter. A., and Wilson, R. (199 I). The relationship between carcinogenic potency and maximum tolerated dose is similar for mutagens and nonmutagens. In Chemicall.v-Induced Cell Pro&ration: Implicationsfor Risk Assessment (B. E. Butterworth, T. J. Slaga, W. Farland, and M. McClain, Eds.), pp. 501-516. Wiley-Liss. New York. Griesemer, R. A., and Cueto, C. (1980). Toward a classification scheme for degrees of experimental evidence for the carcinogenicity of chemicals in animals. In Molecular and Cellular Aspects of Carcinogen Screening Testing (R. Montesano, H. Bartsch. and L. Tomatis, Eds.), pp. 259-28 I. IARC Scientific Publications, Lyon. Haseman, J. K. (1985). Issues in carcinogenicity testing: Dose selection. Fundam. Appl. To.uicol. 5, 66-B. Haseman. J. K.. and Clark, A. M. (1990). Carcinogenicity results for 1I4 laboratory animal studies used to assessthe predictivity of four in vitro genetic toxicity assaysfor rodent carcinogenicity. Environ. Mot. Mutagen. 16(Supp/ement 18). 15-3 1. Haseman, J. K., Crawford, D. D., Huff, J. E., Boorman, G. A., and McConnell, E. E. (I 984). Results from 86 two-year carcinogenicity studies conducted by the National Toxicology Program. J. Toxicol. Environ. Health 14, 621-639. Haseman. J. K., and Huff, J. E. (1987). Species correlation in long-term carcinogenicity studies. Cancer Lett. 37, 125- 132. Haseman, J. K., Huff. J. E., Zeiger, E., and McConnell, E. E. (1987). Comparative results of 327 chemical carcinogenicity studies. Environ. Health Perspect. 74, 229-235.
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Haseman. J. K.. Zeiger, E., Shelby, M. D., Margolin, B. H., and Tennant, R. W. (1990). Predicting rodent carcinogenicity from four in vitro genetic toxicity assays:An evaluation of 114 chemicals studies by the National Toxicology Program. J. Am. Stat. .4ssoc.85,964-97 1. Hoel, D. G., Haseman. J. K., Hogan, M. D., Huff, J.. and McConnell, E. E. ( 1988). The impact of toxicity on carcinogenicity studies: Implications for risk assessment. Carcinogenesis 9, 2045-2052. Hollander, M.. and Wolfe, D. A. (1973). Nonparametric Statistical Methods. Wiley, New York. Huff. J., and Haseman. J. (1991). Long-term chemical carcinogenesis experiments for identifying potential human cancer hazards. Collective data base of the National Cancer Institute and National Toxicology Program (1976-199 1). Envir~)n~nenfa~ Health Perspectives 96, 23-3 I. Huff, J., Haseman. J., and Rail, D. (1991). Scientific concepts. value, and significance of chemical carcinogenesis studies. In Annual Review aj’Pharmacology and To.yicologJa(A. K. Cho, T. F. Blaschke, H. H. Loh. and J. L. Way, Eds.). pp. 62 l-652. Annual Reviews, Inc.. Palo Alto, CA. International Life Sciences Institute (1984). The selection of doses in chronic toxicity/carcinogenicity studies. In Current Issues in To,xicolog.v (H. C. Grice, Ed.). pp. 6-49. Springer-Verlag, New York. Peto, R., Pike, M. C., Bernstein, L., Gold, L. S., and Ames, B. N. (1984). The TD50: A proposed general convention for the numerical description of the carcinogenic potency of chemicals in chronic-exposure animal experiments. Environ. Health Perspect. 58, 1-8. Piegorsch, W. W.. Carr, G. J., Portier, C. J.. and Hoel, D. G. (1992). Concordance of carcinogenic response between rodent species: Potency dependence and potential underestimation. Risk Anal. 12, I IS- 12 1. Rieth, J. P., and Starr, T. B. (1989). Experimental design constraints on carcinogenic potency estimates. J. Toxical. Environ. Health 27,287-296. Shlyakhter, A.. Goodman, G.. and Wilson, R. (1992). Monte Carlo simulation of rodent carcinogenicity bioassays. Risk Anal. 12, 73-82. Zeise, L., Wilson R.. and Crouch, E. (1984). Use ofacute toxicity to estimate carcinogenic risk. Risk Anal. 4, 187-199. Zeise. L., Crouch, E. A. C.. and Wilson, R. (1986). A possible relationship between toxicity and carcinogenicity. J. Am. Coil. To,xicol. 5, I37- I5 I.
AND
APPLIED
TOXICOLOGY
19,207-2 13 ( 1992)
An Examination of the Association between Maximum-Tolerated Dose and Carcinogenicity in 326 Long-Term Studies in Rats and Mice J. K. HASEMAN*,’
AND S. K. SEILKOP~
*Division qj’Bi0metr.v and Risk Assessment. National Institute qf Environmental Health Sciences, P.O. BO.Y12233. Research Triangle Park, North Carolina 27709: and tilnalytical Sciences, Inc., Alston Technical Park. 100 Capitola Drive, Suite 106. Durham, North Carolina 27713
Received July 17,1991;accepted February24, 1992 An Examination of the Association betweenMaximum-Tolerated Dose and Carcinogenicity in 326 Long-Term Studies in Rats and Mice. HASEMAN, J. K., AND SEILKOP, S. K. (1992). Fundam. Appl. Toxicol. 19, 207-213. The associationbetween rodent carcinogenicity and maximum-tolerateddose(MTD) wasevaluatedin 326 long-term carcinogenicity studiesin mice and rats. Others investigating this associationhave focusedprimarily on positive studies,but our investigation consideredall experimental outcomes. We found that chemicals with low MTDs were somewhatmore likely to be rodent carcinogensthan chemicalswith high MTDs, but this associationwaslimited primarily to gavagestudies.Overall, the MTD was not a reliable predictor of whether or not a chemical would be a rodent carcinogen. Our investigation confirms that comparisonsof carcinogenic potenciesbasedonly on positive
studies may result in artifactually elevated estimates of the underlying associationbetweenchemical toxicity and rodent carcinogenicity and thus may alsoinflate the estimatedinterspecies correlation in carcinogenic response.Nevertheless,the results
of our study are consistent with the frequently cited 75% concordancein carcinogenicity outcomebetweenrats and mice. This
concordance is quite high, particularly since 80% is approximately the maximum level of observableinterspeciesconcordanceachievablefor a set of chemicalswith relatively low carcinogenic potency, becauseof the variability in observedtumor responsesthat can induce false negative or false positive outcomes in either of the two species.Thus, the underlying qualitative interspeciescorrelation in carcinogenicresponsebetween rats and mice may be greater than is commonly recognized. 0 1992 Society of Toxicology.
During the past two decades the National Cancer Institute (NCI) and the National Toxicology Program (NTP) have designed, carried out, and evaluated approximately 400 longterm rodent carcinogenicity studies (Haseman et al., 1987; Huff and Haseman, 1991; Huff et al., 1991). The majority of these studies involve four sex-species experiments: male and female rats and mice. ’ To whom
correspondence
should
be addressed.
The experimental designs of these studies are similar: within each sex-species experiment there are generally two dosed groups and a control; the more recent NTP studies include a third dosed group. There are usually 50 animals per group, although certain of the earlier NC1 studies used fewer (10-25) control animals. Most of the studies are of 2year duration, which includes the majority of the animal’s natural life span. At the conclusion of the study, all surviving animals are killed, and approximately 30 different organs sites are examined for possible carcinogenic effects. The top dose utilized in these experiments is the “maximum-tolerated dose” or MTD. The MTD is a dose determined from the results of shorter duration studies that is estimated to produce some minimal toxic effects in a longterm study (e.g., a small reduction in body weight), but should not shorten an animal’s life span or unduly compromise normal well-being except for chemically induced carcinogenicity (International Life Sciences Institute, 1984; Haseman, 1985). The lower doses are generally chosen to be some fraction of the MTD (e.g., f and $). For feeding studies, there is a practical limiting upper dose of 50,000 ppm (i.e., 5% of the diet), since higher doses might well produce nutritional deficiencies. Previous evaluations of the NCI/NTP and other large databases have consistently estimated that the qualitative concordance in carcinogenicity outcome between rats and mice is approximately 75% (Haseman and Huff, 1987; Gold et al.. 1989). That is, approximately 75% ofthe studies involving these two species agree in either their positive or negative carcinogenicity outcome for both species, The evaluation of quantitative interspecies correlation in carcinogenic response has been more problematic. Most of these analyses use some function of the median tumorigenic dose, or TD50 (Peto et al., 1984), as the measure of carcinogenic potency. Initial indications showed that the carcinogenic potencies in rats and mice were highly correlated (Crouch and Wilson, 1979; Crouch, 1983) but other investigators (Bernstein et al., 1985) maintained that some portion of this correlation was an “artifact” resulting basically from two other associations.
207 Copyright All tights
0272-0590/92 $5.00 0 1992 by the Society of Toxicology. of reproduction in any form reserved.
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First, it has been demonstrated that estimated TDSOs are selected and evaluated 326 studies: 223 feeding studies and 103 oral gavage studies that were adequately evaluated in at least one of the four sex-species virtually always constrained to be within a 30-fold factor of groups. The remaining studies utilized other routes of administration (e.g.. the MTD, the top dose used in the long-term study (Bernstein inhalation, skin paint, drinking water) that did not occur in sufficient numbers et al., 1985; Crouch et al., 1987; Reith and Starr, 1989). for meaningful evaluation. Moreover, the range of MTDs (and TDSOs) for a given set For each of the 326 studies the top dose employed in the study, i.e., the of chemicals may be quite large: as much as five or six orders estimated MTD. was evaluated in each sex-species group. For feeding studies units were ppm: for gavage studies the dosage units were mg/kg/day. of magnitude. Under these conditions MTDs and TDSOs dosage In the gavage studies the doses were adjusted when necessary to take into within a species will necessarily be highly correlated. account differences in the frequency of dosing: generally the chemical was Second, the MTDs employed in long-term studies are administered five times/week, but certain studies utilized dosing schedules generally quite similar (and are often identical) for rats and of two, three, or seven times per week. The carcinogenicity outcomes used in our evaluation were those reported mice. The logical consequence of these two strong associaby the NCI/NTP in its Technical Report series and summarized in various tions (i.e., MTD vs TD50 within a species: MTDs across publications (Griesemer and Cueto, 1980; Haseman et al.. 1987; Huff and species) is that for any group of chemicals carcinogenic to Haseman, I99 I). An experiment was considered “positive” if the chemical both rats and mice, the TDSOs in the two species will also being evaluated was considered to be carcinogenic or (using the more recent be highly (and to some extent artifactually) correlated. If NTP terminology) showed “clear evidence” or “some evidence” of carcieither of these two correlations is reduced, then the corre- nogenic activity (Huff et al., 1991). Other possible experimental outcomes sponding interspecies correlation in carcinogenic response were “equivocal” (for uncertain findings), “negative” (for no carcinogenic effects),and “inadequate” (for experiments that because of major limitations will also be reduced. could not be meaningfully evaluated for carcinogenicity). Most investigators agree that the correlation between rat Comparisons of MTDs were made between positive and negative carciand mouse MTDs is quite high. However, evaluating the nogenicity studies for each sex-species group. Equivocal or uncertain outcorrelation between carcinogenicity and MTD within a spe- comes were considered negative: inadequate outcomes were excluded. Comparisons were also made for each species by combining results for males cies is somewhat more difficult. A number of investigators and females (i.e., analyses based on zero, one, or two sex-specific positive (e.g., Zeise et al., 1984, 1986; Gold et al., 1986; Crouch et experiments; MTDs were averaged across sexes)and also for those chemicals al., 1987: Rieth and Starr, 1989; Goodman et al., 1991; evaluated adequately in all four sex-species groups (analyses based on zero, Shlyakhter et al., 1992) have carried out analyses to deter- one. two, three, or four positive experiments; MTDs averaged across the mine the degree to which the interspecies correlation in car- four groups). Feeding and gavage studies were evaluated separately, since it cinogenicity is artifactually constrained. At present, no con- is difficult to equate the single daily bolus dose received by the gavage route of administration with the more diffuse chemical exposure received in feeding sensus has been reached on this matter. Moreover, these studies. evaluations have limited their attention primarily to those Pairwise comparisons were made by Mann-Whitney U tests. Dose-rechemicals showing carcinogenic effects in both species. sponse trends were assessedby Jonckheere’s test. Evaluations of paired comHowever, a comprehensive evaluation of interspecies cor- parison data were carried out by Wilcoxon signed-rank tests. Dichotomous relation should take into account all experimental out- response variables were evaluated by Fisher’s exact test. and analysesinvolving the pooling of 2 X 2 tables were carried out by Mantel-Haenszel tests. All comes-negative as well as positive. Our paper addresses of these procedures are described by Hollander and Wolfe (1973) and/or by this issue by examining the strength of the correlation be- Cart et al. ( 1986). Two-sided tests were used for all statistical procedures. tween carcinogenicity and MTD for all chemicals evaluated for carcinogenicity in a series of long-term rodent studies. RESULTS There is another reason to examine this association: Haseman et al. (1990) noted that in 114 NTP long-term studies, Table 1 summarizes the comparisons of MTDs for each the estimated MTD appeared to be correlated with carci- sex-species group. There are a number of interesting findings nogenicity outcome. These authors speculated that the es- from this table that do not relate directly to the association timated MTD of a chemical might be as good or better a of carcinogenicity and MTD. For example, for each of the predictor of rodent carcinogenicity than certain short-term four sex-species groups, chemicals evaluated by the gavage (genetic toxicity) assays that they also evaluated. However, route are more likely to be carcinogenic than are chemicals they concluded that “more work is needed before definitive evaluated by the feed route. Overall, 26% (2 18/847) of the conclusions can be reached.” The current paper addresses feeding experiments are positive compared with 4 1% ( 159/ this issue by evaluating the association between MTD and 384) of the gavage experiments. carcinogenicity in a database of long-term rodent studies There are no statistically significant (p < 0.05) differences approximately three times larger than the previous evalua- among the four sex-species groups with regard to the protion. portion of studies showing carcinogenic effects for either gavage or feeding studies. However, for both routes of adMETHODS ministration male rats and female mice appear to be slightly more likely to show carcinogenic effects than the other two The initial database that we considered consisted of 379 long-term chemical sex-species groups, a finding consistent with previous obcarcinogenicity studies carried out in rats and/or mice by the NC1 or the NTP (Huff and Haseman, 199 1; Huff et al.. 199 1). From this database we servations (Haseman et al., 1987; Huff et al., 1991).
ASSOCIATION
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209
TABLE 1 Comparison of Maximum Tolerated Doses (MTDs) for Positive and Negative Feed and Gavage Rodent Carcinogenicity Studies Positive carcinogenicity studies Median MTD
Mean MTD
SD
N
Median MTD
MTD
SD
59 51 50 58
3000 2500 2500 2500
6763 5906 6079 4984
10.499 9,580 9,028 6,352
157 166 156 I50
3000 3000 4000 4915
9,22 I 9,232 10,252 10,317
14,332 13,912 15.414 15,312
46 32 37 44
90 100 185 175
181 151 439 300
227 187 898 371
50 65 57 53
125 122 245 254
474 364 538 704
842 491 800 1,056
N
Feed studies Male rats Female rats Male mice Female mice Gavage studies Male rats Female rats* Male mice Female mice*
Negative carcinogenicity studies
-
Note. MTD dosage units for feeding studies are ppm and for gavage studies are mg/kg/day. N. number of studies; SD, standard deviation. * Marginally significant (p = 0.043 for female rats;p = 0.052 for female mice) difference in MTD between positive and negative studies (Mann-Whitney U test).
A comparison of MTDs among sex-species groups permits an evaluation of relative sensitivity to chemically related toxicity. Within a species there are no significant differences between males and females. However, rats appear to be more sensitive than mice to chemically related toxicity, in that the MTDs on average are significantly (p < 0.001) lower (see Table 1). Although an inspection of Tables 1 and 2 suggests that this difference in species sensitivity is limited primarily to gavage studies, the corresponding effect in feeding studies becomes more apparent when one considers that comparisons of ppm doses across species do not take food consumption and body weight differences into account. NTP
studies have consistently shown that when equivalent ppm doses in feeding studies are converted to mg/kg, the resulting doses in mg/kg are approximately 2-3 times higher for mice than for rats (see also Table 1 of Gold et al., 1984). Thus, the species sensitivity difference in MTD is evident for both routes of administration if comparisons are made on a mg/ kg basis. Table 1 indicates a consistent, but relatively weak, association between chemical toxicity and carcinogenicity. There is a suggestion in all four sex-species groups and for both routes of administration that positive studies have lower MTDs (i.e., higher toxicities) than negative studies. However, the strongest statistical evidence of an effect is only marginal
TABLE 2 Association between Maximum Tolerated Dose (MTD) and Number of Positive Carcinogenicity Experiments for Rats and Mice Gavage studies*
Feeding studies No. positive experiments
N
Median MTD
Mean MTD
SD
N
Median MTD
Mean MTD
SD
Rats
0 (both sexes -) 1 (one sex +) 2 (both sexes +)
148 25 41
3000 3000 2200
9,464 7,067 6,142
14,336 9,509 10,471
45 24 27
130 90 92
453 222 151
684 276 175
Mice
0 (both sexes -) 1 (one sex +) 2 (both sexes +)
138 26 41
4566 2500 2450
10,831 6,121 5.129
15,883 8,477 6.612
47 13 33
239 160 188
614 603 322
874 1346 440
Note MTD dosage units for feeding studies are ppm and for gavage studies are mg/kg/day. N. number of studies; SD. standard deviation. * p = 0.047 trend for rats and p = 0.07 1 trend for mice (Jonckheere’s test), i.e., MTD decreases as the number of positive experiments increases. The corresponding trends for feeding studies are not statistically significant.
210
HASEMAN
AND SEILKOP
and involves gavage studies with female rats (p = 0.043) and female mice (p = 0.052). One reason for the high mean MTD values in the negative feeding studies in Table 1 is the relatively high frequency of negative studies utilizing a top dose of 50,000 ppm. As noted earlier, it is NCI/NTP policy to employ this as the upper limit in dosed feed studies, even if it might not strictly be an MTD, because higher levels of chemical in the diet could produce nutritional deficiencies. Even though these experiments constitute a relatively small proportion (7%) of the feeding studies considered in our evaluation, it is interesting that almost all chemicals evaluated at these levels (14, 13, 17, and 16 chemicals for male rats, female rats, male mice, and female mice, respectively) were not carcinogenic. Only two chemicals evaluated at 50,000 ppm were found to be carcinogenic: Decabromodiphenyl oxide (male and female rats) and C.I. Vat Yellow 4 (male mice). Since it is possible that certain “negative studies” evaluated at 50,000 ppm would have been positive had higher doses been employed, a case could be made for excluding these studies from consideration. If this is done, then the mean MTD levels for positive and negative feeding studies shown in Table 1 would be virtually the same. This further supports the finding that the association between carcinogenicity outcome and MTD seems to be limited primarily to the gavage studies. Table 2 evaluates the data by species (combining sexes) and produces results similar to those reported in Table 1. There is marginal statistical evidence (p = 0.047 for rats; p = 0.072 for mice) of a positive association between MTD and the number of positive studies for the gavage route of administration (Table 2). However, the corresponding trends in the feeding studies are not statistically significant. Table 3 provides the corresponding comparisons for the 197 feeding studies and 87 gavage studies with adequate experiments in all four sex-species groups. For gavage studies there is a highly significant (a < 0.01) association between the number of positive experiments and mean MTD (av-
eraged over sexes and species). The corresponding trend for feeding studies is not significant. Gold et al. ( 1989) reported that chemicals toxic at relatively low doses are more accurate predictors of carcinogenicity in the second species than are chemicals that are toxic only at relatively high doses. Piegorsch et al. ( 199 1) found a similar result for concordance, i.e., interspecies correlation in carcinogenic outcome is greater for low MTD chemicals than for high MTD chemicals. The current database shows similar trends, but only for the gavage studies, as can be seen in Table 4. For example, there is a clear (p < 0.05) gradient in interspecies concordance as gavage MTD increases: concordance is quite good (77%) for the “more toxic” (lower MTD) chemicals, but poor (46%) for the less toxic chemicals. Feeding studies do not show this same trend. In fact, due in part to the negative results consistently obtained in 50,000 ppm feeding studies (noted above), the overall concordance in carcinogenic response for this route of administration is actually slightly higher for “less toxic” chemicals than for more toxic chemicals (Table 4). Interspecies concordance is slightly, but not significantly, higher in feeding studies than in gavage studies. Overall interspecies concordance is 73%, consistent with other estimates (Haseman and Huff, 1987; Gold et a/., 1989; Huff and Haseman, 199 1). One final analysis was carried out: an evaluation analogous to that conducted by Haseman et al. (1990). who defined a “toxic” chemical to be a chemical whose mean MTD was less than 4000 ppm (feeding studies) or less than 300 mg/ kg/day (gavage studies). These doses provide approximately equivalent exposure when food consumption and body weights are taken into account (Haseman and Clark, 1990). Under these conditions (and also utilizing a small number of inhalation and dosed water studies) these authors found that the concordance between this dichotomized MTD variable and carcinogenicity was 65% (72/ 111). Adopting these same definitions for the current database of studies adequately evaluated in male and female rats and
TABLE 3 Comparison of MTDs for Rodent Carcinogenicity Studies Showing 0, 1, 2, 3, or 4 Positive Experiments for Rats and Mice Feeding studies
Gavage studies*
No. positive experiments
N
Median MTD
Mean MTD
SD
N
Median MTD
Mean MTD
SD
0 I 2 3 4
112 22 31 15 17
4000 2614 3725 2175 2750
11.083 5,682 7,317 5,385 4,46 1
15.889 6,441 11.737 7,687 4.300
27 14 22 10 14
176 400 200 80 100
510 681 342 120 124
649 995 362 151 112
Note. MTD dosage units for feeding studies are ppm and for gavage studies are mg/kg/day. N. number of studies; SD, standard deviation. * p -C0.01 trend (Jonckheere’s test), i.e., MTD decreases as the number of positive experiments increases. The corresponding trend for feeding studies is not statistically significant.
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211
AND CARCINOGENICITY
TABLE 4 Interspecies Concordance in Carcinogenic Response as a Function of Mean MTD Dose in 87 Gavage and 197 Feeding Studies Carcinogenicity (rats/mice) ++
+-
-t
Total
Interspecies concordance
Mean gavage MTD study (mg/kg)* O-I 10 Ill-299 300+
15 12 2
3 5 8
4 4 I
8 8 11
30 29 28
77% (23/30) 69% (20/29) 46% (13128)
Total
29
16
15
27
87
64% (56187)
Mean feeding study MTD (ppm)* o-1499 1500-7499 7500+
11 18 9
9 6 7
9 9 7
36 33 43
65 66 66
72% (47165) 77% (5 l/66) 79% (52166)
Total
38
22
25
112
197
76% (150/197)
Feed and gavage studies
67
38
40
139
284
73% (206/284)
* MTD ranges chosen to divide the data into three approximately equal-sized groups.
mice, for feeding studies 46% (47/ 102) of the toxic chemicals were found to be carcinogens compared with 40% (38/95) of the “nontoxic” chemicals. For gavage studies 73% (43/ 59) of the toxic chemicals were carcinogens compared with 61% (17/28) of the nontoxic chemicals. Thus, the overall concordance between toxicity and carcinogenicity is 56% (158/284), which is not significantly different from the association expected by chance alone. When individual sexspecies groups were considered, this association was similar, ranging from 5 1% (female rats) to 56% (female mice). Thus, although some association between MTD and carcinogenic outcome appears to exist, particularly for gavage experiments (see Table 3) the magnitude of the MTD does not appear to be a reliable predictor of whether or not a chemical will be a rodent carcinogen. DISCUSSION It is recognized that the MTD employed in a given longterm study is only one of several possible measures of chemical toxicity. Experience has shown that the MTD is generally estimated reasonably well, and even in the instances in which the MTD is not accurately estimated, it is seldom “missed” by more than a factor of two or four in either direction. Given the six orders of magnitude difference in MTDs from study to study and the large number of chemicals evaluated, it is unlikely that slight inaccuracies in the estimation of MTDs would affect the results of this study. The frequency of statistically significant tumor effects detected at the MTD and at lower doses has been studied by Hoe1 et al. (1988). These authors found that in 99 NTP carcinogenicity studies, more than half of the positive experiments (73/127, 57%) had statistically significant (a < 0.05)
chemically related tumor increases for at least one dose below the MTD. They also found that a high proportion of the remaining experiments (42/54,78%) had tumor rates in the lower dosed groups that were elevated, but not significantly. Thus, less than 10% ( 12/ 127) of the experiments had evidence of carcinogenicity limited to the MTD. On a chemical basis, only three of the 99 chemicals considered by Hoe1 et al. (1988) showed elevated tumor rates limited to the MTD: melamine, propylene oxide, and butyl benyl phthalate. These results are more definitive than a similar evaluation carried out earlier by Haseman (1985) based on a much smaller group of chemicals, which are included as a subset of the chemicals evaluated by Hoe1 et al. ( 1988). These data suggest that there are relatively few instances in which the MTD produces unique effects that are not also observed (with reduced incidence) at lower dose levels. The reasons for the differences observed between feed and gavage studies in our investigation are not always apparent. The increased frequency of rodent carcinogens observed in gavage studies relative to feeding studies has been reported previously (Haseman et al., 1984) and may be related to the disproportionate number of halogenated hydrocarbons (which tend to be liver carcinogens) evaluated by the gavage route. Further, the gavage route of administration is generally utilized for chemicals that are volatile, unpalatable, unstable, and/or otherwise unsuitable for administration in the diet, which perhaps increases the a priori likelihood that such chemicals will subsequently show carcinogenic effects in rodents. It is less clear why gavage studies apparently show a stronger correlation between MTD and carcinogenicity outcome than feeding studies (Table 3) or why relatively nontoxic chemicals evaluated bv Ravage apparently show little or no
212
HASEMAN
interspecies correlation in carcinogenic response (Table 4). These matters require further investigation. Crouch et al. (1987) list carcinogenic potencies and MTDs for 2 13 chemicals evaluated for carcinogenicity in female mice by the NC1 or NTP, reporting that 108 were positive (i.e., had calculated carcinogenic potencies) and 105 were negative. While this database includes many of the same studies used in our evaluation, there were also some notable differences. For example, Crouch et al. calculated carcinogenic potencies for more than 30 chemicals considered negative by the NCI/NTP; they also combined various routes of administration. The underlying model assumed by these authors may be written p = 1 - (1 - a)exp{ -bd/( 1 - a)}, where p is the probability of tumor occurrence, u is the background tumor rate, d is the dose, and b is the measure of carcinogenic potency (Crouch and Wilson, 1979; Crouch, 1983). Note that as the background tumor rate approaches zero, the carcinogenic potency approaches ln(2)/TD50. Although Crouch, Wilson, and Zeise ( 1987) found a strong correlation between MTD and carcinogenic potency in the positive studies, we found that this correlation was greatly reduced when negative studies were included in the evaluation. For example, the 108 positive and 105 negative studies did not differ significantly with regard to the MTD employed in these experiments. This finding is consistent with our own results, particularly considering that the Crouch/Wilson/ Zeise database consists primarily of feeding studies, and the association found in the NCI/NTP database was limited primarily to gavage studies. Further, the correlation between log( l/MTD) and log(carcinogenic potency) for positive studies in Crouch/ Wilson/Zeise female mice database is quite impressive: r = 0.9 1. However, suppose the negative studies are also considered and the carcinogenic potency for these chemicals is set equal to some arbitrarily small number, say 0.00000 1. Then, the correlation between log( l/MTD) and log(carcinogenic potency), while still highly significant (p < 0.00 1), is reduced to only Y = 0.30. If alternative values are assumed for the carcinogenic potency of negative studies (e.g., 0.00001 or O.OOOOOOl), then the correlation will change slightly (e.g., r = 0.39 and r = 0.25 for the two cases noted), but the basic point remains: if negative studies are considered, the correlation between rodent carcinogenicity and MTD is considerably reduced. The best method for dealing with “carcinogenic potencies” in negative studies is not altogether clear. One approach is to select a tumor type showing a numerical increase and to compute a carcinogenic potency, even if the increase is not considered to be chemically related. Alternatively, it may be possible to calculate some confidence limit on the carcino-
AND SEILKOP
genie potency. The problem with both of these approaches is that such estimates of carcinogenic potency will almost certainly be constrained by the MTD and thus will lead to the same type of problem discussed above. For purposes of assessing interspecies correlation in carcinogenic response, we prefer instead to consider chemicals judged to be noncarcinogens to be in fact noncarcinogens, with (essentially) carcinogenic potencies of zero. Our evaluation of the NCI/NTP and Crouch/Wilson/Zeise databases suggests that while carcinogenic potency and MTD are highly correlated in positive studies, there is a much weaker (although still statistically significant) correlation between MTD and rodent carcinogenicity when all experimental outcomes are considered. Thus, overall interspecies correlation in carcinogenic outcome will be similarly reduced. While one should not over interpret the apparently high interspecies correlation in carcinogenic potency, it would be equally unwise to minimize the qualitative concordance in carcinogenic response between rats and mice, which has consistently been reported to be approximately 75%. This association is more impressive that might first appear: Piegorsch et al. (1992), assuming an underlying exponential (one-hit) model and the basic experimental design currently used by the NTP, evaluated the sensitivity of the bioassay for detecting low potency carcinogens. The database of Gold et al. (1984) was used to define the characteristics of typical “low potency” carcinogens. Piegorsch et al. (1992) demonstrated that for a set of such chemicals, the maximum level of observable concordance achievable between rats and mice is only approximately 80%. even if the underlying interspecies concordance in carcinogenic response for these chemicals is 100%. Higher concordance is difficult to achieve because of the variability in observed tumor responses that can occur by chance, resulting in some false negative or false positive outcomes in one of the two species. Thus, the underlying qualitative interspecies correlation in carcinogenic response between rats and mice may be greater than is commonly recognized. REFERENCES Bernstein.L.. Gold, L. S., Ames, B. N., Pike, M. C.. and Hoel. D. G. (1985). Some tautological aspects of the comparison of carcinogenic potency in rats and mice. Fzrndu?n. :1pp/. To.uicol. 5, 79-86. Crouch, E. A. C. (1983). Uncertainties in interspecies extrapolations of carcinogenicity. Envirorl. Health Perspect. 5, 32 l-327. Crouch,E.. and Wilson. R. (1979). Interspecies comparisons in carcinogenic potency. J. Toxicol. Owirorl. Health 5, 1095-l I 18. Crouch. E., Wilson. R.. and Zeise, L. (1987). Tautology or not tautology. J. Toxicol. Envirorl. Health. 20, I- 10. Cart. J. J.. Krewski. D.. Lee. P. N.. Tarone. R. E., and Wahrendorf, J. (1986). Statistical Methods in Cancer Research. Volume III. The Design and Ana/wis of’ Long-Term ,4nimal E.uperiment.s. International Agency for Research on Cancer, Lyon.
ASSOCIATION
BETWEEN
MTD
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