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Antagonism—No Synergism—in Pairwise Tests of Carcinogens in Rats
Regulatory Toxicology and Pharmacology 35, 383–392 (2002) doi:10.1006/rtph.2002.1546
Antagonism—No Synergism—in Pairwise Tests of Carcinogens in Rats Michael Gough1 Cato Institute, Washington, DC Received January 6, 2002
In the mid-1970s, the National Cancer Institute issued a contract for the testing of a dozen chemicals, most of them known to be carcinogenic in animals, in pairwise combinations. Those tests, which involved 918 pairwise tests and over 14,500 laboratory rats, produced no good evidence for any synergistic interactions in which exposure to two chemicals resulted in a number of tumors greater than the number produced by exposure to either chemical alone. A number of tests resulted in antagonism, in which the number of tumors in animals exposed to a pair of carcinogens was less than the number seen after exposure to either carcinogen by itself. More generally, the results indicate that synergism is an unlikely consequence when animals are exposed to pairs of chemicals, even when both chemicals are carcinogens. C 2002 Elsevier Science (USA) Key Words: carcinogenicity; synergy; antagonism; pairwise testing; SRI; NCI.
BACKGROUND In 1974, the National Cancer Institute (NCI) issued a contract to The Stanford Research Institute of Menlo Park, California (SRI), to test the effects of exposing laboratory rats to mixtures of paired carcinogenic substances. A number of scientists are aware of the SRI project, and some know of the results in a general way, but none had a complete copy of the report, “Combined Effects of Chemical Carcinogens and Other Chemicals,” dated May 1978 (SRI International, 1978) in the fall of 2001. The NCI found no reference to the report in its files when a request for the report was made. A Freedom of Information Act (FOIA) request to the Department of Health and Human Services for a copy of the report and for copies of “any government agencies’ and/or advisory panels’ reviews of the report” resulted in a letter stating that “no records responsive to your request were located” (Frangipane, 2001).
1
Correspondence may be addressed to the author at 6404 E. Halbert Road, Bethesda, MD 20817-5423. E-mail: mgough@ bellatlantic.net.
SRI, however, had a copy of the report and made it available upon request from an official of the National Institutes of Health (NIH), the original government contracting agency. The full text of the SRI report is available at http://www.isrtp.org. TEST DESIGN SRI obtained male and female Fischer 344 rats from Simonsen Laboratories, Inc. (Gilroy, CA), quarantined the animals, culled them on the basis of slow growth and disease, and housed the animals three to a cage. The animals were assigned to control or test groups subsequent to their being assigned to cages and were provided lab chow and water ad libitum. Subchronic feeding tests were conducted on groups of 15 male and 15 females to determine the maximum tolerated doses (MTD) for each of the test substances. Based on information in the literature and the results of the subchronic tests, the SRI set dose levels expected to produce tumors in 20 to 80% of the exposed animals. SRI scientists tested 12 chemicals, which were divided among four groups (see Table 1).
r Group 1 chemicals had different target organs; r Group 2 chemicals were liver carcinogens; r Group 3 chemicals had different target organs and 1-thiouracil was “well known for its endocrine activity”; r Group 4 chemicals were known to have toxic effects on the nervous system and were tested for possible nervous system carcinogenicity. When none was found to cause nervous system tumors, the liver was assigned as the target organ. One of the chemicals, aflatoxin (AF), was included in three of the four groups (2, 3, and 4); and N-butanolN-butylnitrosamine (NBBN) and dipentylnitrosamine (DPN) were included in two groups (1 and 3 and 1 and 2, respectively). As shown in Table 1, the 12 chemicals included 7 that were known as carcinogens at the time the tests were made. Of the remaining 5, 3—the trisodium salt of nitrilotriacetic acid (NTA), lasiocarpine (LAS), and Aroclor 1254 (AR)—were carcinogenic in the SRI tests (see Table 2). Two chemicals—dieldrin (D) and
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MICHAEL GOUGH
TABLE 1 Chemicals Tested in SRI Experiments Dose in diet (ppm) Group
Chemical name
Abbr.
C/Ua
1
N-Methyl-N -nitro-N-nitrosoguanidine N-Butanol-N-butylnitrosaminec Nitrilotriacetic acid, trisodium salt Dipentylnitrosaminec
MNNG NBBN NTA DPN
C C U C
Sto Sin Bla Kid Liv
2
Aflatoxin B1 c Cycad flour d Lasiocarpine Dipentylnitrosaminec
AF CY LAS DPN
C C U C
3
Aflatoxin B1 c N-Butanol-N-butylnitrosaminec Lead (II) acetate, trihydrate 2-Thiouracil
AF NBBN LA TH
4e
Aflatoxin B1 c Dieldrin Hexachlorophene Aroclor 1254
AF D HEX AR
Target
organb
Low
Mid
High
20 30 200 50
40 60 2000 150
80 120 20000 450
Liv Liv Liv Liv
0.005 2000 7 50
0.015 4000 15 150
0.045 8000 30 450
C C C U
Liv Bla Kid Thy
0.005 30 500 83
0.015 60 2000 250
0.045 120 8000 750
C U U U
Liv Liv Liv Liv
0.005 2 17 25
0.015 10 50 50
0.045 50 150 100
Source: SRI International (1978), Table 1, p. 21. carcinogen; U, carcinogenicity unknown at time of test. b Sto, stomach; Sin, small intestine; Bla, bladder; Kid, kidney or ureter; Liv, liver; Thy, thyroid. c Replicate chemical that appears in more than one group. d Contained 2% cycasin.
a C,
hexachlorophene (HEX)—were not carcinogenic in the tests (see Table 2). In tests for carcinogenicity, a single chemical or two chemicals were incorporated into lab chow, and tests were made on groups of 24 males and 24 females. At death or sacrifice, all control and test animals were subjected to gross examinations for tumors, and each tumor was examined microscopically. Each chemical was tested at three different levels in the diet (see Table 1), and it was tested pairwise with the other three chemicals in that group at low, middle, and high doses. For example, in group 1: r N-methyl-N -nitro-N-nitrosoguanidine (MNNG) was tested at low, middle, and high doses, and each of those doses was tested in combination with NBBN at low, middle, and high doses; with the trisodium salt of NTA at low, middle, and high doses; and with DPN at low, middle, and high doses. r NBBN was tested at low, middle, and high doses, and each of those doses was tested in combination with NTA at low, middle, and high doses and with DPN at low, middle, and high doses. r NTP was tested at low, middle, and high doses, and each of those doses was tested in combination with DPN at low, middle, and high doses. r DPN was tested at low, middle, and high doses. For each group of four chemicals, the SRI scientists reported the results of feeding each of the four chemicals at three doses to both sexes (24 results = 4 chem-
icals × 3 dose levels × 2 sexes) and results of testing 96 paired diets (96 = 4! combinations for each chemical × 4 chemicals × 3 doses × 2 sexes). [There was one additional paired diet result because two end points—liver and kidney tumors—were reported for the paired feeding of cycad flour (CY) and the other group 2 chemicals.] SRI scientists reported tumor incidence as the number of animals in each group of 24 that developed tumors. In addition, they reported the median survival time (MST) for each group, with a maximum of 104 weeks when surviving animals were sacrificed. In all, SRI used 606 groups of 24 animals and a total of 14,556 animals in its tests. RESULTS SRI and NCI scientists were interested in the possibility of carcinogenic synergy in laboratory rats simultaneously exposed to two carcinogens. The majority of tests, however, could not detect such effects—if they occurred. Although the SRI scientists had designed their experiments so that feeding of a single chemical was expected to cause tumors in no more than 80% of the exposed animals, the low dose, in many cases, caused tumors in 19 or more of the 24 animals (≥80%; see Table 2). Such high tumor rates eliminated any opportunity to detect synergy in those tests. Nevertheless, the SRI scientists allude to one possible case of
385
PAIRWISE COMBINATIONS OF CARCINOGENIC CHEMICALS
TABLE 2 Tested Chemicals and Tumor Rates Rats with tumors at dose
MST (weeks) at dose
Chemical
Target organa
Sex
0
Low
Mid
High
0
Low
Mid
High
Group 1 MNNG
Sto; Sin
M F M F M F M F
0 0 0 0 0 0 2 5
19 8 19 23 0 0 21 16
19 19 23 23 0 1 23 24
23 20 23 24 11 6 24 23
104 104 104 104 104 104 104 104
104 104 104 101 104 104 104 101
97 103 102 92 97 103 77 96
84 95 83 75 92 95 54 76
M F M F M F M F M F
2 5 2 5 2 5 2 5 0 0
5 13 19 20 18 24 23 22 5 6
18 19 18 20 24 23 24 24 12 16
20 22 9 7 19 18 24 24 6 3
104 104 104 104 104 104 104 104 104 104
104 104 91 86 104 102 104 104 91 86
104 104 64 68 95 78 79 100 64 68
104 104 36 33 77 48 53 78 36 33
M F M F M F M F
2 5 0 0 0 0 0 1
6 6 24 20 1 0 8 4
14 19 24 21 8 2 20 17
21 20 21 22 21 11 9 23
104 104 104 104 104 104 104 104
104 104 104 103 104 104 104 104
104 104 101 98 104 104 102 104
104 104 86 78 104 104 72 104
M F M F M F M F
2 5 2 5 2 5 2 5
4 10 1 2 5 4 20 18
19 12 0 1 2 9 17 21
21 22 4 3 5 9 18 23
104 104 104 104 104 104 104 104
104 104 104 104 104 104 104 104
104 104 104 104 104 104 104 104
104 104 104 104 104 104 104 104
NBBNb
Bla
NTA
Kid
DPNb
Liv
Group 2 AFb
Liv
CYc
Liv
LAS
Liv
DPNb
Liv
CYc
Kid
Group 3 AFb
Liv
NBBNb
Bla
LA
Kid
TH
Thy
Group 4 AFb
Liv
D
Liv
HEX
Liv
AR
Liv
Source: SRI International (1978), Tables 4, 9, 15, 20; pp. 24, 29, 35, 40. bladder; Kid, kidney or ureter; Liv, liver; Sto, stomach; Sin, small intestine. b >1 test done on this chemical because it was included in >1 group of chemicals. c Two entries for this chemical because it had two different target organs.
a Bla,
synergy, which will be discussed below. In addition the authors dismissed three other possible synergistic effects and did not mention a fifth example in which pairwise exposure resulted in a lengthened MST and a higher tumor number. The SRI test results also present opportunities to look for antagonism in which simultaneous exposures to two carcinogens result in a reduction of tumor numbers. The SRI report notes several examples of antagonism, many of which were accompanied or perhaps caused by life span shortening. Examples of antagonisms that occurred in the absence of life span shortening will be discussed below.
Synergy No statistical analysis of the SRI data has been undertaken (or, at least, it has not been made available; see Discussion). In this paper, both “synergy” and “antagonism” are judgment calls, and an increase of more than 50% in tumor number was necessary to invoke a suggestion of synergy. Five possible examples of synergy (identified by bold numbers in Tables 3–5) are discussed below. Possible synergistic effect between NTA and DPN. As shown in Table 3, the high dose of NTA caused 11 kidney or ureter tumors in male rats. The simultaneous
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MICHAEL GOUGH
TABLE 3 Number of Kidney or Ureter Tumors in Rats Fed NTP and Combinations of NTP and DPN Number of rats with tumors Sex
DPN dose
M
F
NTA dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 0
0 2 1 0
0 0 0 0
0 Low Mid High
0 1 0 0
0 0 1 0
1 0 1 0
NTA dose:
0
Low
Mid
High
11 17 6 0
104 104 77 54
104 104 102 54
104 104 84 56
92 79 70 51
6 6 3 1
104 104 94 76
104 104 104 83
104 104 98 79
103 104 94 63
Source: SRI International (1978), Table 7, p. 27.
feeding of the low dose of DPN along with the highdose NTA increased the number of tumors to 17. The increase is consistent with a synergistic interaction, and the SRI scientists cautiously interpreted the increase: Although low-level DPN appear to increase the incidence of target (kidney and ureter) tumors somewhat in high-level (NTA) males, there was no corresponding increase in incidence in females even though they survived longer and examination of food consumption data showed that they accumulated nearly as much NTA (SRI International, 1978, pp. 13–24).
Possible synergy between MNNG and the other three Group 1 chemicals—NBBN, NTA, and DPN. The low dose of MNNG caused 8 stomach or small intestine tumors in female rats. The addition of the low dose of any of the three other Group 1 chemicals, NBBN, NTA, or DPN, increased the tumor numbers to 14, 12, and 13, respectively (Table 4). The number of tumors in male rats exposed to the low dose of MNNG was higher, 19, and exposures to the other three Group 1 chemicals had no effect on that number. The SRI authors dismiss the number of tumors in the female animals exposed only to MNNG as a possible “underestimate, since
TABLE 4 Number of Stomach and /or Small Intestine Tumors in Rats Fed MNNG and MNNG in Combination with NBBN, NTA, and DPN
Sex
Second chemical dose
Number of rats with tumors MNNG dose:
0
Low
Mid
High
MST (weeks) MNNG dose:
0
Low
Mid
High
M
0 Low NBBN Mid NBBN High NBBN Low NTA Mid NTA High NTA Low DPN Mid DPN High DPN
0 0 1 0 0 0 1 1 0 0
19 19 21 5 19 18 1 20 2 0
19 22 20 14 23 22 1 22 18 4
23 20 20 17 24 22 14 22 19 4
104 104 102 83 104 104 92 104 77 54
104 104 98 77 104 104 88 104 84 50
97 97 94 75 100 104 84 95 82 54
84 82 76 78 80 86 81 75 74 50
F
0 Low NBBN Mid NBBN High NBBN Low NTA Mid NTA High NTA Low DPN Mid DPN High DPN
0 0 0 0 0 0 1 0 0 0
8 14 6 1 12 9 0 13 10 0
19 19 16 2 19 18 3 19 19 18
20 19 20 13 23 20 8 21 23 18
104 101 92 75 104 104 104 104 94 76
104 104 92 78 104 104 104 104 98 80
103 104 88 73 102 104 102 95 97 80
95 88 80 72 97 90 102 86 88 77
Source: SRI International (1978), Table 5, p. 25.
387
PAIRWISE COMBINATIONS OF CARCINOGENIC CHEMICALS
TABLE 5 Number of Thyroid Tumors in Rats Fed TH and Combinations of TH and LA Number of rats with tumors Sex
LA dose
M
F
TH dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 0
8 6 14 2
20 23 19 15
0 Low Mid High
1 0 0 1
4 3 3 3
17 20 10 13
TH dose:
0
Low
Mid
High
9 16 19 18
104 104 104 104
104 104 104 99
104 104 104 101
72 84 93 97
23 21 23 22
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
Source: SRI International (1978), Table 19, p. 39.
TABLE 6 NTA-Caused Reductions in the Number of Tumors Induced by MNNG, NBBN, or DPN MNNG-caused stomach and small intestine tumors Number of rats with tumors Sex
NTA dose
M
F
MNNG dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 1
19 19 18 1
19 23 22 1
0 Low Mid High
0 0 0 0
8 12 6 1
19 19 16 2
MNNG dose:
0
Low
Mid
High
23 24 22 14
104 104 104 104
104 104 104 88
97 100 104 84
84 80 86 81
20 23 20 13
104 104 104 104
104 104 104 104
103 102 104 102
95 97 90 102
Source: SRI International (1978), Table 5, p. 25. NBBN-caused bladder tumors Number of rats with tumors Sex
NTA dose
M
F
NBBN dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 2
19 24 23 8
23 23 24 14
0 Low Mid High
0 0 0 4
23 21 19 13
23 21 23 12
NBBN dose:
0
Low
Mid
High
22 24 24 19
104 104 104 92
104 104 104 100
102 104 104 96
83 84 88 86
24 21 24 19
104 104 104 104
101 104 104 104
92 99 97 104
75 74 88 104
Source: SRI International (1978), Table 6, p. 26. DPN-caused liver tumors Number of rats with tumors Sex
NTA dose
M
F
DPN dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
2 4 1 4
21 22 24 9
23 23 24 23
0 Low Mid High
5 6 7 10
16 9 13 18
24 24 24 24
Source: SRI International (1978), Table 8, p. 28.
DPN dose:
0
Low
Mid
High
24 24 23 22
104 104 104 92
104 104 104 79
77 102 84 70
54 56 56 51
23 24 24 24
104 104 104 104
104 104 104 104
96 102 98 94
76 83 79 63
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MICHAEL GOUGH
administration of the low MNNG with low NBBN, low NTA and low DPN resulted in higher incidences (12–14 animals)” (SRI International, 1978, p. 13). While the explanation may be correct, there is nothing to corroborate it. Equally, it is possible that all three of the other Group 1 chemicals acted synergistically with NBBN. Increasing MST and increasing tumor number in male rats exposed to high-dose 2-thiouracil (TH) and lead (II) acetate trihydrate (LA). The dose response for TH-caused thyroid tumors in male rats indicates that the high dose is so toxic that it decreases the MST (Table 5). The reduced life span in male rats is accompanied by a reduction in tumor number perhaps because the shorter life spans are not sufficient to allow the development of tumors. All three doses of LA increase the MST in male rats exposed to the high dose of TH, and the number of tumors seen in the TH + LA-exposed males exceed the number following exposure only to TH. The increase in MST coupled with the increase in tumor number can be interpreted as opposite in effect from the more frequently observed phenomenon in which decreased MSTs are accompanied by reduced tumor numbers. Increasing doses of TH caused increasing numbers of tumors in female rats, and there was no possibility to observe synergism in female rats, if it occurs. The high dose of TH did not shorten life spans in female rats, making it impossible to see if the addition of LA increased the life span of TH-exposed females. Antagonism In contrast to the absence of the word “increase” from the SRI “Summary and Conclusions” (SRI International, 1978, pp. 19–20), the words “reduce” and “reductions” are used several times to describe the number of tumors observed in animals fed two carcinogens. In some cases, the tumor reduction went hand in hand
with shortened MST and is of little interest. In 20 cases, however, the feeding of two carcinogens halved or nearly halved the number of tumors observed when either carcinogen was fed singly and had no effect on MST. Those cases are identified by bold numbers in Tables 6–11 and are discussed below. NTA reduces tumor rates from exposures to MNNG, NBBN, and DPN. High doses of NTA reduced the number of stomach and small intestine tumors caused by MNNG, the number of bladder cancers caused by NBBN, and the number of kidney or ureter cancers caused by DPN. The antagonistic effect of NTA on MNNG-, NBBN-, and DPN-caused tumors, in the absence of any effect on the MST, was limited to female rats. In female rats, the high dose of NTA reduced tumor numbers at all levels of MNNG exposure and had no effect on MST (Table 6). In male rats, the addition of the high dose to NTA to the food reduced tumor number at all doses of MNNG but decreased the MST seen at low and mid doses of MNNG. High doses of NTA also reduced the number of bladder cancers caused by NBBN in both male and female rats. The reduction in tumor number in males was accompanied by reductions in MST. In females, the combination of high-dose NTA and low or middle doses of NBBN reduced the tumor number and, at the same time, lengthened MST (Table 6). NTA had a similar effect on DPN-caused liver tumors in rats. As shown in Table 6, the high dose of NTA reduced the number of liver tumors in male rats exposed to low-dose DPN, but that reduction was accompanied by a shortened MST. In female rats, the low dose of NTA reduced the number of tumors caused by the low level of DPN and did not affect MST (Table 6). NBBN reduces tumor rates from exposures to NTA. The high dose of NBBN reduced the number of kidney and/or ureter tumors in female rats simultaneously
TABLE 7 NBBN-Caused Reductions in the Number of Tumors Induced by NTA NTA-caused kidney or ureter tumors Number of rats with tumors Sex
NBBN dose
M
F
NTA dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 0
0 1 0 0
0 1 1 0
0 Low Mid High
0 0 0 0
0 0 0 0
1 0 0 1
Source: SRI International (1978), Table 7, p. 27.
NTA dose:
0
Low
Mid
High
11 6 9 11
104 104 104 83
104 104 104 84
104 100 104 88
92 100 96 86
6 8 3 0
104 104 92 75
104 104 99 74
103 104 97 88
95 104 104 104
389
PAIRWISE COMBINATIONS OF CARCINOGENIC CHEMICALS
TABLE 8 AF-Caused Reductions in the Number of Tumors Induced by LA LA-caused kidney tumors Number of rats with tumors Sex
AF
M
F
LA dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 1 0
1 0 1 0
8 9 12 11
0 Low Mid High
0 0 0 0
0 0 0 0
2 2 3 1
LA dose:
0
Low
Mid
High
21 18 19 19
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
11 11 14 5
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
Source: SRI International (1978), Table 18, p. 38.
exposed to the high dose of NTA. Remarkably, the low, middle, and high dose of NBBN increased MST in all female rats exposed to the high dose of NTA. In male rats, NBBN did not affect NTA-caused tumor induction and high-dose NBBN-shortened MSTs (Table 7). AF reduces tumor rate from exposure to LA. Highdose AF reduced the number of tumors associated with the high dose of LA and did not affect MST in female
rats (Table 8). The addition of AF to the diet of male rats fed LA had no effect. TH reduces tumor rates from exposure to AF and LA. TH antagonized tumor induction by AF in both male and female rats (Table 9). In males, the reduction in tumors associated with high-dose TH was accompanied by severe reductions in MST. In females, the reduction in tumors associated with the middle dose of TH occurred
TABLE 9 TH-Caused Reductions in the Number of Tumors Induced by AF and LA AF-caused liver tumors Number of rats with tumors Sex
TH dose
M
F
AF dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
2 1 2 0
6 5 3 0
14 19 11 0
0 Low Mid High
5 3 1 0
6 3 3 0
19 14 5 2
AF dose:
0
Low
Mid
High
21 21 23 0
104 102 104 72
104 104 104 48
104 100 104 46
104 100 104 47
20 15 3 3
104 104 104 104
104 104 104 87
104 104 104 85
104 104 104 98
Source: SRI International (1978), Table 16, p. 36. LA-caused kidney tumors Number of rats with tumors Sex
TH dose
M
F
LA dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 0
1 1 0 0
8 8 5 0
0 Low Mid High
0 0 0 0
0 1 0 0
2 2 1 0
Source: SRI International (1978), Table 18, p. 38.
LA dose:
0
Low
Mid
High
21 18 22 14
104 102 104 72
104 104 104 84
104 104 104 93
104 99 101 97
11 4 10 5
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
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MICHAEL GOUGH
TABLE 10 D-Caused Reductions in the Number of Tumors Induced by AF AF-caused liver tumors Number of rats with tumors Sex
D dose
M
F
AF dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
2 1 0 4
4 2 5 8
19 19 16 17
0 Low Mid High
5 2 1 3
10 13 3 5
12 11 7 10
AF dose:
0
Low
Mid
High
21 21 24 21
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
22 21 21 19
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
Source: SRI International (1978), Table 21, p. 41.
in the absence of any effect on MST. At the high dose of TH, both tumor number and MST were reduced. Low- and high-dose TH were associated with lower tumor numbers in female rats fed high-dose LA, with no effect on MST (Table 9), but the dose response to TH in rats fed high-level LA is not simple. The number of tumors at middle-dose TH and high-dose LA (10) is higher than the numbers seen at low and high-dose TH and high-dose LA (4 and 5). The simpler explanation for the observed numbers is that the result at middledose LA is higher than would be expected on repeated testing than that the observations at both low- and high-level LA are lower than would be found on repeated testing. The text of the SRI report that discusses the results of feeding LA and TH to rats states, “Pairing of LA with TH tended to reduce kidney tumor incidences somewhat, and there was some associated reduction in lifespan” (SRI International, 1978, p. 17). That statement does not, however, describe the results for female rats, in which there was no shortening of MST (Table 9).
D reduces tumor rate from exposure to AF. D, in the SRI tests, “did not induce liver tumors to any discernible extent in either sex” (SRI International, 1978, p. 17). The addition of the middle dose of D to female rats exposed to the low and middle dose of AF reduced the number of tumors with no effect on MST (Table 10), and the addition of the high dose of D reduced the number of tumors caused by the low dose. D did not affect tumor number or MST in male rats. HEX reduces tumor rate from exposure to AF. HEX, like D in the SRI tests, was not carcinogenic; “Liver tumor incidences in females receiving mid and high levels of HEX were slightly higher than the median control value, but not appreciably so; consequently HEX does not appear to be a liver carcinogen” (SRI International, 1978, pp. 17–18). All doses of HEX reduced the number of tumors caused by low-dose AF in female rats with no effect on MST, and no dose of HEX affected tumor number or MST in males (Table 11).
TABLE 11 HEX-Caused Reductions in the Number of Tumors Induced by AF AF-caused liver tumors Number of rats with tumors Sex
HEX dose
M
F
AF dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
2 2 2 5
4 4 3 3
19 20 19 18
0 Low Mid High
5 4 9 9
10 4 3 2
12 10 14 8
Source: SRI International (1978), Table 21, p. 41.
AF dose:
0
Low
Mid
High
21 23 19 20
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
22 22 24 19
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
PAIRWISE COMBINATIONS OF CARCINOGENIC CHEMICALS
Low doses of AF produced different numbers of tumors in male rats (4) and female rats (10). The disparity makes it reasonable to question the apparent reductions in tumor numbers in female rats following exposures to D and HEX. For instance, if the number of tumors at the low dose of AF in females was spuriously high, the apparent reductions could represent a regression to the means. That explanation seems less likely because the number of tumors reported in Tables 10 and 11 in female rats exposed to the low dose of AF by itself is consistent with the numbers of tumors (13, 6, and 10) determined in three different tests of low dose AF (Table 2). The number of tumors associated with the low dose of AF in male rats (4) reported in Tables 10 and 11 is consistent with the results (5, 6, and 4) reported for three tests of the low dose in male rats (Table 2). If, however, the lowest number of tumors reported in female rats exposed to the low dose of AF (6) is more representative of the effect of that dose level, the apparent antagonism between D and AF and between HEX and AF would disappear.
DISCUSSION The authors of the SRI report note increases and decreases in tumor number and state that a more quantitative analysis of the results was being prepared by a group at the University of California at Los Angeles. It has not been possible to locate that analysis, if it was prepared. In the absence of such an analysis or repeated testing, any discussion of the results is limited necessarily to “eye-balling” the reported numbers of tumors and lengths of MSTs. The first comment about the results of the SRI report is that it contains nothing to suggest that synergism, even between known carcinogens, is common or striking. Indeed, the SRI scientists dismissed four of the five examples of possible synergism. In the case of low-level DPN appearing to cause an increase in male rats exposed to high-level NTA (Table 3), the SRI scientists pointed to the absence of any such effect in females fed high-level NTA and do not discuss the increase beyond that. NBBN, NTA, and DPN increased the number of tumors in female rates exposed to low-dose MNNG (Table 4). The SRI scientists did not interpret the result as suggesting synergy. Quite the contrary, they suggest that the increase seen in all three cases where a second chemical was fed indicates that the number of tumors seen in the females exposed only to MNNG was an “underestimate.” Give the limited information available, the four cases where tumor numbers increased with the addition of a second chemical could be interpreted as either synergy or the result of fluctuations in the numbers of tumors produced in the tests. Depending on which interpreta-
391
tion is correct, there is either no or very scant evidence for synergy. The fifth possible example of synergy is more perplexing. The highest tested dose of TH is toxic to both male and female rats (Table 5). The addition of LA to the diet of male rats fed the highest TH dose increased their MST and the number of thyroid tumors. In this case, increasing the MST—a good thing—led to an increase in cancer number—a bad thing, as could be expected regardless of synergism. The result is a parallel to the changes in human life expectancy and cancer risk in the past century in this country, in which increasing life spans have resulted in populations with a larger proportion of older individuals who are more likely to develop cancer. The interpretation of the possible cases of antagonism is subject to the same limitations as for possible cases of synergism. In particular, tumor number fluctuations could result in spurious conclusions. Any reported test result of pairwise testing might represent a “low” number or an “underestimate” of the number of tumors that would be seen upon repeated tests and produce an apparent antagonism, when none was present. Still, the evidence that NTA reduces tumor numbers resulting from feeding MNNG and NBBN is strengthened because it does not depend on a difference seen at a single dose level of MNNG or NBBN. The results in Table 6 show that high-dose NTA reduced the number of tumors caused by high-dose MNNG and had little effect on MST. This is the only unequivocal example of antagonism of carcinogenic effects reported in male rats in the SRI results. High-dose NTA also caused reductions in the number of tumors in female rats exposed to both low- and middle-dose MNNG (Table 6). The NTA had no effect on MST in the low-dose MNNG group or in the middledose MNNG group (103 to 102 weeks), and lengthened the MST in the high-dose MNNG from 95 to 102. High-dose NTA reduced the number of tumors in female rats exposed to low- and middle-dose NBBN. In those cases, the addition of NTA to the diets of the animals increased their MSTs, while reducing the tumor numbers. More than one combination of D and AF and of HEX and AF resulted in numbers of tumors less than those caused by AF alone. Both middle- and high-dose D caused a reduction in tumors in female rats exposed to low-dose AF, and all doses of HEX caused a reduction in the number of tumors from low-dose AF. Neither AF, D, nor HEX nor any paired combination of those chemicals affects MSTs in males or females (Tables 10 and 11). An example of the more typical result that indicates antagonism is shown on Table 6. The low-dose DPN– low-dose NTA combination caused 9 tumors compared to 16 tumors observed in the female rats exposed to low-dose DPN only. The evidence, here, is a difference in
392
MICHAEL GOUGH
the two numbers of a pair of numbers, and fluctuation in either or both might account for an apparent difference when no actual difference exists. Although only the high-dose NBBN reduced the tumor number in female rats exposed to high-dose NTA, all NBBN doses extended the MST of female rats exposed to high-dose NTA (Table 7). This effect on life span supports the idea of antagonistic interactions between the two chemicals that reduce the toxic effects of NTA. There are three other examples of antagonism between pairs of chemicals: High-dose AF reduced the tumors observed in female rats exposed to high-dose LA (Table 8) and did not affect MST. High-dose TH reduced the number of tumors seen in female rats exposed to high-dose AF without affecting MST, and both low- and high-dose TH reduced the number of tumors resulting from exposure to high-dose LA (Table 8). The SRI report noted many of these reductions in the “Results” (SRI International, 1978, pp. 12–18) and “Summary and Conclusions” (SRI International, 1978, pp. 19–20) sections, apparently accepting them as “real” and not the results of fluctuations or other artifacts. The report did not, however, use the words “antagonism” or “antagonistic.” In contrast to the discussion of reductions in the “Summary and Conclusions” section, there is no mention of increases. The SRI tests are one example of how testing of pairs of chemicals can be done, and examination of the mechanics of those tests would provide guidance for additional or better tests. Such tests, costly and timeconsuming as they could be, could inform the many discussions that are concerned about synergism. Needless to say, additional tests would be useful only to the extent that the results are published. The SRI results, inexplicably buried for almost a quarter century, provide experimental evidence that synergism may be less likely than antagonism in mixed exposures
to known carcinogenic substances, but they have not been published or publicized. CONCLUSIONS Testing of a dozen chemicals in pairwise combinations produced no convincing evidence for synergistic carcinogen interactions. Four of the five possible synergistic interactions that were observed may be explained by fluctuations in the numbers of tumors observed in the tests, and one could be the effect of lengthened life span. Also observed were 20 possible cases of antagonism in which combined exposures to two chemicals resulted in fewer tumors than exposure to either chemical alone. Antagonism to generalized toxic effects were also apparent in several tests when the addition of a second chemical increased the life span of the test animals without a concomitant increase in tumors. Most significantly, perhaps, given public concern about synergies, the tests described here, done under contract to the NCI in the mid-1970s, produced no good evidence of synergy. By contrast, the same tests produced several examples of antagonism, in which exposure to two chemicals resulted in fewer cancers than exposure to either chemical by itself. REFERENCES Frangipane, N. (2001). Freedom of Information Act Coordinator, NCI. Letter to Michael Gough, in reference to NCI 02–008, FOIA Case No. 27066, November 6, 2001. SRI International (1978). Combined Effects of Chemical Carcinogens and Other Chemicals: Final Report, May 1978, D. C. L. Jones, prepared for Bioassay Operations Program, Tracor Jitco, Inc., Contract 74-23-106002, SRI Project LSD-3478. Approved: W. A. Skinner, Executive Director, Life Sciences Division. [Photocopied typescript, iv + 44 pages]
Antagonism—No Synergism—in Pairwise Tests of Carcinogens in Rats Michael Gough1 Cato Institute, Washington, DC Received January 6, 2002
In the mid-1970s, the National Cancer Institute issued a contract for the testing of a dozen chemicals, most of them known to be carcinogenic in animals, in pairwise combinations. Those tests, which involved 918 pairwise tests and over 14,500 laboratory rats, produced no good evidence for any synergistic interactions in which exposure to two chemicals resulted in a number of tumors greater than the number produced by exposure to either chemical alone. A number of tests resulted in antagonism, in which the number of tumors in animals exposed to a pair of carcinogens was less than the number seen after exposure to either carcinogen by itself. More generally, the results indicate that synergism is an unlikely consequence when animals are exposed to pairs of chemicals, even when both chemicals are carcinogens. C 2002 Elsevier Science (USA) Key Words: carcinogenicity; synergy; antagonism; pairwise testing; SRI; NCI.
BACKGROUND In 1974, the National Cancer Institute (NCI) issued a contract to The Stanford Research Institute of Menlo Park, California (SRI), to test the effects of exposing laboratory rats to mixtures of paired carcinogenic substances. A number of scientists are aware of the SRI project, and some know of the results in a general way, but none had a complete copy of the report, “Combined Effects of Chemical Carcinogens and Other Chemicals,” dated May 1978 (SRI International, 1978) in the fall of 2001. The NCI found no reference to the report in its files when a request for the report was made. A Freedom of Information Act (FOIA) request to the Department of Health and Human Services for a copy of the report and for copies of “any government agencies’ and/or advisory panels’ reviews of the report” resulted in a letter stating that “no records responsive to your request were located” (Frangipane, 2001).
1
Correspondence may be addressed to the author at 6404 E. Halbert Road, Bethesda, MD 20817-5423. E-mail: mgough@ bellatlantic.net.
SRI, however, had a copy of the report and made it available upon request from an official of the National Institutes of Health (NIH), the original government contracting agency. The full text of the SRI report is available at http://www.isrtp.org. TEST DESIGN SRI obtained male and female Fischer 344 rats from Simonsen Laboratories, Inc. (Gilroy, CA), quarantined the animals, culled them on the basis of slow growth and disease, and housed the animals three to a cage. The animals were assigned to control or test groups subsequent to their being assigned to cages and were provided lab chow and water ad libitum. Subchronic feeding tests were conducted on groups of 15 male and 15 females to determine the maximum tolerated doses (MTD) for each of the test substances. Based on information in the literature and the results of the subchronic tests, the SRI set dose levels expected to produce tumors in 20 to 80% of the exposed animals. SRI scientists tested 12 chemicals, which were divided among four groups (see Table 1).
r Group 1 chemicals had different target organs; r Group 2 chemicals were liver carcinogens; r Group 3 chemicals had different target organs and 1-thiouracil was “well known for its endocrine activity”; r Group 4 chemicals were known to have toxic effects on the nervous system and were tested for possible nervous system carcinogenicity. When none was found to cause nervous system tumors, the liver was assigned as the target organ. One of the chemicals, aflatoxin (AF), was included in three of the four groups (2, 3, and 4); and N-butanolN-butylnitrosamine (NBBN) and dipentylnitrosamine (DPN) were included in two groups (1 and 3 and 1 and 2, respectively). As shown in Table 1, the 12 chemicals included 7 that were known as carcinogens at the time the tests were made. Of the remaining 5, 3—the trisodium salt of nitrilotriacetic acid (NTA), lasiocarpine (LAS), and Aroclor 1254 (AR)—were carcinogenic in the SRI tests (see Table 2). Two chemicals—dieldrin (D) and
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MICHAEL GOUGH
TABLE 1 Chemicals Tested in SRI Experiments Dose in diet (ppm) Group
Chemical name
Abbr.
C/Ua
1
N-Methyl-N -nitro-N-nitrosoguanidine N-Butanol-N-butylnitrosaminec Nitrilotriacetic acid, trisodium salt Dipentylnitrosaminec
MNNG NBBN NTA DPN
C C U C
Sto Sin Bla Kid Liv
2
Aflatoxin B1 c Cycad flour d Lasiocarpine Dipentylnitrosaminec
AF CY LAS DPN
C C U C
3
Aflatoxin B1 c N-Butanol-N-butylnitrosaminec Lead (II) acetate, trihydrate 2-Thiouracil
AF NBBN LA TH
4e
Aflatoxin B1 c Dieldrin Hexachlorophene Aroclor 1254
AF D HEX AR
Target
organb
Low
Mid
High
20 30 200 50
40 60 2000 150
80 120 20000 450
Liv Liv Liv Liv
0.005 2000 7 50
0.015 4000 15 150
0.045 8000 30 450
C C C U
Liv Bla Kid Thy
0.005 30 500 83
0.015 60 2000 250
0.045 120 8000 750
C U U U
Liv Liv Liv Liv
0.005 2 17 25
0.015 10 50 50
0.045 50 150 100
Source: SRI International (1978), Table 1, p. 21. carcinogen; U, carcinogenicity unknown at time of test. b Sto, stomach; Sin, small intestine; Bla, bladder; Kid, kidney or ureter; Liv, liver; Thy, thyroid. c Replicate chemical that appears in more than one group. d Contained 2% cycasin.
a C,
hexachlorophene (HEX)—were not carcinogenic in the tests (see Table 2). In tests for carcinogenicity, a single chemical or two chemicals were incorporated into lab chow, and tests were made on groups of 24 males and 24 females. At death or sacrifice, all control and test animals were subjected to gross examinations for tumors, and each tumor was examined microscopically. Each chemical was tested at three different levels in the diet (see Table 1), and it was tested pairwise with the other three chemicals in that group at low, middle, and high doses. For example, in group 1: r N-methyl-N -nitro-N-nitrosoguanidine (MNNG) was tested at low, middle, and high doses, and each of those doses was tested in combination with NBBN at low, middle, and high doses; with the trisodium salt of NTA at low, middle, and high doses; and with DPN at low, middle, and high doses. r NBBN was tested at low, middle, and high doses, and each of those doses was tested in combination with NTA at low, middle, and high doses and with DPN at low, middle, and high doses. r NTP was tested at low, middle, and high doses, and each of those doses was tested in combination with DPN at low, middle, and high doses. r DPN was tested at low, middle, and high doses. For each group of four chemicals, the SRI scientists reported the results of feeding each of the four chemicals at three doses to both sexes (24 results = 4 chem-
icals × 3 dose levels × 2 sexes) and results of testing 96 paired diets (96 = 4! combinations for each chemical × 4 chemicals × 3 doses × 2 sexes). [There was one additional paired diet result because two end points—liver and kidney tumors—were reported for the paired feeding of cycad flour (CY) and the other group 2 chemicals.] SRI scientists reported tumor incidence as the number of animals in each group of 24 that developed tumors. In addition, they reported the median survival time (MST) for each group, with a maximum of 104 weeks when surviving animals were sacrificed. In all, SRI used 606 groups of 24 animals and a total of 14,556 animals in its tests. RESULTS SRI and NCI scientists were interested in the possibility of carcinogenic synergy in laboratory rats simultaneously exposed to two carcinogens. The majority of tests, however, could not detect such effects—if they occurred. Although the SRI scientists had designed their experiments so that feeding of a single chemical was expected to cause tumors in no more than 80% of the exposed animals, the low dose, in many cases, caused tumors in 19 or more of the 24 animals (≥80%; see Table 2). Such high tumor rates eliminated any opportunity to detect synergy in those tests. Nevertheless, the SRI scientists allude to one possible case of
385
PAIRWISE COMBINATIONS OF CARCINOGENIC CHEMICALS
TABLE 2 Tested Chemicals and Tumor Rates Rats with tumors at dose
MST (weeks) at dose
Chemical
Target organa
Sex
0
Low
Mid
High
0
Low
Mid
High
Group 1 MNNG
Sto; Sin
M F M F M F M F
0 0 0 0 0 0 2 5
19 8 19 23 0 0 21 16
19 19 23 23 0 1 23 24
23 20 23 24 11 6 24 23
104 104 104 104 104 104 104 104
104 104 104 101 104 104 104 101
97 103 102 92 97 103 77 96
84 95 83 75 92 95 54 76
M F M F M F M F M F
2 5 2 5 2 5 2 5 0 0
5 13 19 20 18 24 23 22 5 6
18 19 18 20 24 23 24 24 12 16
20 22 9 7 19 18 24 24 6 3
104 104 104 104 104 104 104 104 104 104
104 104 91 86 104 102 104 104 91 86
104 104 64 68 95 78 79 100 64 68
104 104 36 33 77 48 53 78 36 33
M F M F M F M F
2 5 0 0 0 0 0 1
6 6 24 20 1 0 8 4
14 19 24 21 8 2 20 17
21 20 21 22 21 11 9 23
104 104 104 104 104 104 104 104
104 104 104 103 104 104 104 104
104 104 101 98 104 104 102 104
104 104 86 78 104 104 72 104
M F M F M F M F
2 5 2 5 2 5 2 5
4 10 1 2 5 4 20 18
19 12 0 1 2 9 17 21
21 22 4 3 5 9 18 23
104 104 104 104 104 104 104 104
104 104 104 104 104 104 104 104
104 104 104 104 104 104 104 104
104 104 104 104 104 104 104 104
NBBNb
Bla
NTA
Kid
DPNb
Liv
Group 2 AFb
Liv
CYc
Liv
LAS
Liv
DPNb
Liv
CYc
Kid
Group 3 AFb
Liv
NBBNb
Bla
LA
Kid
TH
Thy
Group 4 AFb
Liv
D
Liv
HEX
Liv
AR
Liv
Source: SRI International (1978), Tables 4, 9, 15, 20; pp. 24, 29, 35, 40. bladder; Kid, kidney or ureter; Liv, liver; Sto, stomach; Sin, small intestine. b >1 test done on this chemical because it was included in >1 group of chemicals. c Two entries for this chemical because it had two different target organs.
a Bla,
synergy, which will be discussed below. In addition the authors dismissed three other possible synergistic effects and did not mention a fifth example in which pairwise exposure resulted in a lengthened MST and a higher tumor number. The SRI test results also present opportunities to look for antagonism in which simultaneous exposures to two carcinogens result in a reduction of tumor numbers. The SRI report notes several examples of antagonism, many of which were accompanied or perhaps caused by life span shortening. Examples of antagonisms that occurred in the absence of life span shortening will be discussed below.
Synergy No statistical analysis of the SRI data has been undertaken (or, at least, it has not been made available; see Discussion). In this paper, both “synergy” and “antagonism” are judgment calls, and an increase of more than 50% in tumor number was necessary to invoke a suggestion of synergy. Five possible examples of synergy (identified by bold numbers in Tables 3–5) are discussed below. Possible synergistic effect between NTA and DPN. As shown in Table 3, the high dose of NTA caused 11 kidney or ureter tumors in male rats. The simultaneous
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MICHAEL GOUGH
TABLE 3 Number of Kidney or Ureter Tumors in Rats Fed NTP and Combinations of NTP and DPN Number of rats with tumors Sex
DPN dose
M
F
NTA dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 0
0 2 1 0
0 0 0 0
0 Low Mid High
0 1 0 0
0 0 1 0
1 0 1 0
NTA dose:
0
Low
Mid
High
11 17 6 0
104 104 77 54
104 104 102 54
104 104 84 56
92 79 70 51
6 6 3 1
104 104 94 76
104 104 104 83
104 104 98 79
103 104 94 63
Source: SRI International (1978), Table 7, p. 27.
feeding of the low dose of DPN along with the highdose NTA increased the number of tumors to 17. The increase is consistent with a synergistic interaction, and the SRI scientists cautiously interpreted the increase: Although low-level DPN appear to increase the incidence of target (kidney and ureter) tumors somewhat in high-level (NTA) males, there was no corresponding increase in incidence in females even though they survived longer and examination of food consumption data showed that they accumulated nearly as much NTA (SRI International, 1978, pp. 13–24).
Possible synergy between MNNG and the other three Group 1 chemicals—NBBN, NTA, and DPN. The low dose of MNNG caused 8 stomach or small intestine tumors in female rats. The addition of the low dose of any of the three other Group 1 chemicals, NBBN, NTA, or DPN, increased the tumor numbers to 14, 12, and 13, respectively (Table 4). The number of tumors in male rats exposed to the low dose of MNNG was higher, 19, and exposures to the other three Group 1 chemicals had no effect on that number. The SRI authors dismiss the number of tumors in the female animals exposed only to MNNG as a possible “underestimate, since
TABLE 4 Number of Stomach and /or Small Intestine Tumors in Rats Fed MNNG and MNNG in Combination with NBBN, NTA, and DPN
Sex
Second chemical dose
Number of rats with tumors MNNG dose:
0
Low
Mid
High
MST (weeks) MNNG dose:
0
Low
Mid
High
M
0 Low NBBN Mid NBBN High NBBN Low NTA Mid NTA High NTA Low DPN Mid DPN High DPN
0 0 1 0 0 0 1 1 0 0
19 19 21 5 19 18 1 20 2 0
19 22 20 14 23 22 1 22 18 4
23 20 20 17 24 22 14 22 19 4
104 104 102 83 104 104 92 104 77 54
104 104 98 77 104 104 88 104 84 50
97 97 94 75 100 104 84 95 82 54
84 82 76 78 80 86 81 75 74 50
F
0 Low NBBN Mid NBBN High NBBN Low NTA Mid NTA High NTA Low DPN Mid DPN High DPN
0 0 0 0 0 0 1 0 0 0
8 14 6 1 12 9 0 13 10 0
19 19 16 2 19 18 3 19 19 18
20 19 20 13 23 20 8 21 23 18
104 101 92 75 104 104 104 104 94 76
104 104 92 78 104 104 104 104 98 80
103 104 88 73 102 104 102 95 97 80
95 88 80 72 97 90 102 86 88 77
Source: SRI International (1978), Table 5, p. 25.
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PAIRWISE COMBINATIONS OF CARCINOGENIC CHEMICALS
TABLE 5 Number of Thyroid Tumors in Rats Fed TH and Combinations of TH and LA Number of rats with tumors Sex
LA dose
M
F
TH dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 0
8 6 14 2
20 23 19 15
0 Low Mid High
1 0 0 1
4 3 3 3
17 20 10 13
TH dose:
0
Low
Mid
High
9 16 19 18
104 104 104 104
104 104 104 99
104 104 104 101
72 84 93 97
23 21 23 22
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
Source: SRI International (1978), Table 19, p. 39.
TABLE 6 NTA-Caused Reductions in the Number of Tumors Induced by MNNG, NBBN, or DPN MNNG-caused stomach and small intestine tumors Number of rats with tumors Sex
NTA dose
M
F
MNNG dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 1
19 19 18 1
19 23 22 1
0 Low Mid High
0 0 0 0
8 12 6 1
19 19 16 2
MNNG dose:
0
Low
Mid
High
23 24 22 14
104 104 104 104
104 104 104 88
97 100 104 84
84 80 86 81
20 23 20 13
104 104 104 104
104 104 104 104
103 102 104 102
95 97 90 102
Source: SRI International (1978), Table 5, p. 25. NBBN-caused bladder tumors Number of rats with tumors Sex
NTA dose
M
F
NBBN dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 2
19 24 23 8
23 23 24 14
0 Low Mid High
0 0 0 4
23 21 19 13
23 21 23 12
NBBN dose:
0
Low
Mid
High
22 24 24 19
104 104 104 92
104 104 104 100
102 104 104 96
83 84 88 86
24 21 24 19
104 104 104 104
101 104 104 104
92 99 97 104
75 74 88 104
Source: SRI International (1978), Table 6, p. 26. DPN-caused liver tumors Number of rats with tumors Sex
NTA dose
M
F
DPN dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
2 4 1 4
21 22 24 9
23 23 24 23
0 Low Mid High
5 6 7 10
16 9 13 18
24 24 24 24
Source: SRI International (1978), Table 8, p. 28.
DPN dose:
0
Low
Mid
High
24 24 23 22
104 104 104 92
104 104 104 79
77 102 84 70
54 56 56 51
23 24 24 24
104 104 104 104
104 104 104 104
96 102 98 94
76 83 79 63
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MICHAEL GOUGH
administration of the low MNNG with low NBBN, low NTA and low DPN resulted in higher incidences (12–14 animals)” (SRI International, 1978, p. 13). While the explanation may be correct, there is nothing to corroborate it. Equally, it is possible that all three of the other Group 1 chemicals acted synergistically with NBBN. Increasing MST and increasing tumor number in male rats exposed to high-dose 2-thiouracil (TH) and lead (II) acetate trihydrate (LA). The dose response for TH-caused thyroid tumors in male rats indicates that the high dose is so toxic that it decreases the MST (Table 5). The reduced life span in male rats is accompanied by a reduction in tumor number perhaps because the shorter life spans are not sufficient to allow the development of tumors. All three doses of LA increase the MST in male rats exposed to the high dose of TH, and the number of tumors seen in the TH + LA-exposed males exceed the number following exposure only to TH. The increase in MST coupled with the increase in tumor number can be interpreted as opposite in effect from the more frequently observed phenomenon in which decreased MSTs are accompanied by reduced tumor numbers. Increasing doses of TH caused increasing numbers of tumors in female rats, and there was no possibility to observe synergism in female rats, if it occurs. The high dose of TH did not shorten life spans in female rats, making it impossible to see if the addition of LA increased the life span of TH-exposed females. Antagonism In contrast to the absence of the word “increase” from the SRI “Summary and Conclusions” (SRI International, 1978, pp. 19–20), the words “reduce” and “reductions” are used several times to describe the number of tumors observed in animals fed two carcinogens. In some cases, the tumor reduction went hand in hand
with shortened MST and is of little interest. In 20 cases, however, the feeding of two carcinogens halved or nearly halved the number of tumors observed when either carcinogen was fed singly and had no effect on MST. Those cases are identified by bold numbers in Tables 6–11 and are discussed below. NTA reduces tumor rates from exposures to MNNG, NBBN, and DPN. High doses of NTA reduced the number of stomach and small intestine tumors caused by MNNG, the number of bladder cancers caused by NBBN, and the number of kidney or ureter cancers caused by DPN. The antagonistic effect of NTA on MNNG-, NBBN-, and DPN-caused tumors, in the absence of any effect on the MST, was limited to female rats. In female rats, the high dose of NTA reduced tumor numbers at all levels of MNNG exposure and had no effect on MST (Table 6). In male rats, the addition of the high dose to NTA to the food reduced tumor number at all doses of MNNG but decreased the MST seen at low and mid doses of MNNG. High doses of NTA also reduced the number of bladder cancers caused by NBBN in both male and female rats. The reduction in tumor number in males was accompanied by reductions in MST. In females, the combination of high-dose NTA and low or middle doses of NBBN reduced the tumor number and, at the same time, lengthened MST (Table 6). NTA had a similar effect on DPN-caused liver tumors in rats. As shown in Table 6, the high dose of NTA reduced the number of liver tumors in male rats exposed to low-dose DPN, but that reduction was accompanied by a shortened MST. In female rats, the low dose of NTA reduced the number of tumors caused by the low level of DPN and did not affect MST (Table 6). NBBN reduces tumor rates from exposures to NTA. The high dose of NBBN reduced the number of kidney and/or ureter tumors in female rats simultaneously
TABLE 7 NBBN-Caused Reductions in the Number of Tumors Induced by NTA NTA-caused kidney or ureter tumors Number of rats with tumors Sex
NBBN dose
M
F
NTA dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 0
0 1 0 0
0 1 1 0
0 Low Mid High
0 0 0 0
0 0 0 0
1 0 0 1
Source: SRI International (1978), Table 7, p. 27.
NTA dose:
0
Low
Mid
High
11 6 9 11
104 104 104 83
104 104 104 84
104 100 104 88
92 100 96 86
6 8 3 0
104 104 92 75
104 104 99 74
103 104 97 88
95 104 104 104
389
PAIRWISE COMBINATIONS OF CARCINOGENIC CHEMICALS
TABLE 8 AF-Caused Reductions in the Number of Tumors Induced by LA LA-caused kidney tumors Number of rats with tumors Sex
AF
M
F
LA dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 1 0
1 0 1 0
8 9 12 11
0 Low Mid High
0 0 0 0
0 0 0 0
2 2 3 1
LA dose:
0
Low
Mid
High
21 18 19 19
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
11 11 14 5
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
Source: SRI International (1978), Table 18, p. 38.
exposed to the high dose of NTA. Remarkably, the low, middle, and high dose of NBBN increased MST in all female rats exposed to the high dose of NTA. In male rats, NBBN did not affect NTA-caused tumor induction and high-dose NBBN-shortened MSTs (Table 7). AF reduces tumor rate from exposure to LA. Highdose AF reduced the number of tumors associated with the high dose of LA and did not affect MST in female
rats (Table 8). The addition of AF to the diet of male rats fed LA had no effect. TH reduces tumor rates from exposure to AF and LA. TH antagonized tumor induction by AF in both male and female rats (Table 9). In males, the reduction in tumors associated with high-dose TH was accompanied by severe reductions in MST. In females, the reduction in tumors associated with the middle dose of TH occurred
TABLE 9 TH-Caused Reductions in the Number of Tumors Induced by AF and LA AF-caused liver tumors Number of rats with tumors Sex
TH dose
M
F
AF dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
2 1 2 0
6 5 3 0
14 19 11 0
0 Low Mid High
5 3 1 0
6 3 3 0
19 14 5 2
AF dose:
0
Low
Mid
High
21 21 23 0
104 102 104 72
104 104 104 48
104 100 104 46
104 100 104 47
20 15 3 3
104 104 104 104
104 104 104 87
104 104 104 85
104 104 104 98
Source: SRI International (1978), Table 16, p. 36. LA-caused kidney tumors Number of rats with tumors Sex
TH dose
M
F
LA dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
0 0 0 0
1 1 0 0
8 8 5 0
0 Low Mid High
0 0 0 0
0 1 0 0
2 2 1 0
Source: SRI International (1978), Table 18, p. 38.
LA dose:
0
Low
Mid
High
21 18 22 14
104 102 104 72
104 104 104 84
104 104 104 93
104 99 101 97
11 4 10 5
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
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TABLE 10 D-Caused Reductions in the Number of Tumors Induced by AF AF-caused liver tumors Number of rats with tumors Sex
D dose
M
F
AF dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
2 1 0 4
4 2 5 8
19 19 16 17
0 Low Mid High
5 2 1 3
10 13 3 5
12 11 7 10
AF dose:
0
Low
Mid
High
21 21 24 21
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
22 21 21 19
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
Source: SRI International (1978), Table 21, p. 41.
in the absence of any effect on MST. At the high dose of TH, both tumor number and MST were reduced. Low- and high-dose TH were associated with lower tumor numbers in female rats fed high-dose LA, with no effect on MST (Table 9), but the dose response to TH in rats fed high-level LA is not simple. The number of tumors at middle-dose TH and high-dose LA (10) is higher than the numbers seen at low and high-dose TH and high-dose LA (4 and 5). The simpler explanation for the observed numbers is that the result at middledose LA is higher than would be expected on repeated testing than that the observations at both low- and high-level LA are lower than would be found on repeated testing. The text of the SRI report that discusses the results of feeding LA and TH to rats states, “Pairing of LA with TH tended to reduce kidney tumor incidences somewhat, and there was some associated reduction in lifespan” (SRI International, 1978, p. 17). That statement does not, however, describe the results for female rats, in which there was no shortening of MST (Table 9).
D reduces tumor rate from exposure to AF. D, in the SRI tests, “did not induce liver tumors to any discernible extent in either sex” (SRI International, 1978, p. 17). The addition of the middle dose of D to female rats exposed to the low and middle dose of AF reduced the number of tumors with no effect on MST (Table 10), and the addition of the high dose of D reduced the number of tumors caused by the low dose. D did not affect tumor number or MST in male rats. HEX reduces tumor rate from exposure to AF. HEX, like D in the SRI tests, was not carcinogenic; “Liver tumor incidences in females receiving mid and high levels of HEX were slightly higher than the median control value, but not appreciably so; consequently HEX does not appear to be a liver carcinogen” (SRI International, 1978, pp. 17–18). All doses of HEX reduced the number of tumors caused by low-dose AF in female rats with no effect on MST, and no dose of HEX affected tumor number or MST in males (Table 11).
TABLE 11 HEX-Caused Reductions in the Number of Tumors Induced by AF AF-caused liver tumors Number of rats with tumors Sex
HEX dose
M
F
AF dose:
MST (weeks)
0
Low
Mid
High
0 Low Mid High
2 2 2 5
4 4 3 3
19 20 19 18
0 Low Mid High
5 4 9 9
10 4 3 2
12 10 14 8
Source: SRI International (1978), Table 21, p. 41.
AF dose:
0
Low
Mid
High
21 23 19 20
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
22 22 24 19
104 104 104 104
104 104 104 104
104 104 104 104
104 104 104 104
PAIRWISE COMBINATIONS OF CARCINOGENIC CHEMICALS
Low doses of AF produced different numbers of tumors in male rats (4) and female rats (10). The disparity makes it reasonable to question the apparent reductions in tumor numbers in female rats following exposures to D and HEX. For instance, if the number of tumors at the low dose of AF in females was spuriously high, the apparent reductions could represent a regression to the means. That explanation seems less likely because the number of tumors reported in Tables 10 and 11 in female rats exposed to the low dose of AF by itself is consistent with the numbers of tumors (13, 6, and 10) determined in three different tests of low dose AF (Table 2). The number of tumors associated with the low dose of AF in male rats (4) reported in Tables 10 and 11 is consistent with the results (5, 6, and 4) reported for three tests of the low dose in male rats (Table 2). If, however, the lowest number of tumors reported in female rats exposed to the low dose of AF (6) is more representative of the effect of that dose level, the apparent antagonism between D and AF and between HEX and AF would disappear.
DISCUSSION The authors of the SRI report note increases and decreases in tumor number and state that a more quantitative analysis of the results was being prepared by a group at the University of California at Los Angeles. It has not been possible to locate that analysis, if it was prepared. In the absence of such an analysis or repeated testing, any discussion of the results is limited necessarily to “eye-balling” the reported numbers of tumors and lengths of MSTs. The first comment about the results of the SRI report is that it contains nothing to suggest that synergism, even between known carcinogens, is common or striking. Indeed, the SRI scientists dismissed four of the five examples of possible synergism. In the case of low-level DPN appearing to cause an increase in male rats exposed to high-level NTA (Table 3), the SRI scientists pointed to the absence of any such effect in females fed high-level NTA and do not discuss the increase beyond that. NBBN, NTA, and DPN increased the number of tumors in female rates exposed to low-dose MNNG (Table 4). The SRI scientists did not interpret the result as suggesting synergy. Quite the contrary, they suggest that the increase seen in all three cases where a second chemical was fed indicates that the number of tumors seen in the females exposed only to MNNG was an “underestimate.” Give the limited information available, the four cases where tumor numbers increased with the addition of a second chemical could be interpreted as either synergy or the result of fluctuations in the numbers of tumors produced in the tests. Depending on which interpreta-
391
tion is correct, there is either no or very scant evidence for synergy. The fifth possible example of synergy is more perplexing. The highest tested dose of TH is toxic to both male and female rats (Table 5). The addition of LA to the diet of male rats fed the highest TH dose increased their MST and the number of thyroid tumors. In this case, increasing the MST—a good thing—led to an increase in cancer number—a bad thing, as could be expected regardless of synergism. The result is a parallel to the changes in human life expectancy and cancer risk in the past century in this country, in which increasing life spans have resulted in populations with a larger proportion of older individuals who are more likely to develop cancer. The interpretation of the possible cases of antagonism is subject to the same limitations as for possible cases of synergism. In particular, tumor number fluctuations could result in spurious conclusions. Any reported test result of pairwise testing might represent a “low” number or an “underestimate” of the number of tumors that would be seen upon repeated tests and produce an apparent antagonism, when none was present. Still, the evidence that NTA reduces tumor numbers resulting from feeding MNNG and NBBN is strengthened because it does not depend on a difference seen at a single dose level of MNNG or NBBN. The results in Table 6 show that high-dose NTA reduced the number of tumors caused by high-dose MNNG and had little effect on MST. This is the only unequivocal example of antagonism of carcinogenic effects reported in male rats in the SRI results. High-dose NTA also caused reductions in the number of tumors in female rats exposed to both low- and middle-dose MNNG (Table 6). The NTA had no effect on MST in the low-dose MNNG group or in the middledose MNNG group (103 to 102 weeks), and lengthened the MST in the high-dose MNNG from 95 to 102. High-dose NTA reduced the number of tumors in female rats exposed to low- and middle-dose NBBN. In those cases, the addition of NTA to the diets of the animals increased their MSTs, while reducing the tumor numbers. More than one combination of D and AF and of HEX and AF resulted in numbers of tumors less than those caused by AF alone. Both middle- and high-dose D caused a reduction in tumors in female rats exposed to low-dose AF, and all doses of HEX caused a reduction in the number of tumors from low-dose AF. Neither AF, D, nor HEX nor any paired combination of those chemicals affects MSTs in males or females (Tables 10 and 11). An example of the more typical result that indicates antagonism is shown on Table 6. The low-dose DPN– low-dose NTA combination caused 9 tumors compared to 16 tumors observed in the female rats exposed to low-dose DPN only. The evidence, here, is a difference in
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the two numbers of a pair of numbers, and fluctuation in either or both might account for an apparent difference when no actual difference exists. Although only the high-dose NBBN reduced the tumor number in female rats exposed to high-dose NTA, all NBBN doses extended the MST of female rats exposed to high-dose NTA (Table 7). This effect on life span supports the idea of antagonistic interactions between the two chemicals that reduce the toxic effects of NTA. There are three other examples of antagonism between pairs of chemicals: High-dose AF reduced the tumors observed in female rats exposed to high-dose LA (Table 8) and did not affect MST. High-dose TH reduced the number of tumors seen in female rats exposed to high-dose AF without affecting MST, and both low- and high-dose TH reduced the number of tumors resulting from exposure to high-dose LA (Table 8). The SRI report noted many of these reductions in the “Results” (SRI International, 1978, pp. 12–18) and “Summary and Conclusions” (SRI International, 1978, pp. 19–20) sections, apparently accepting them as “real” and not the results of fluctuations or other artifacts. The report did not, however, use the words “antagonism” or “antagonistic.” In contrast to the discussion of reductions in the “Summary and Conclusions” section, there is no mention of increases. The SRI tests are one example of how testing of pairs of chemicals can be done, and examination of the mechanics of those tests would provide guidance for additional or better tests. Such tests, costly and timeconsuming as they could be, could inform the many discussions that are concerned about synergism. Needless to say, additional tests would be useful only to the extent that the results are published. The SRI results, inexplicably buried for almost a quarter century, provide experimental evidence that synergism may be less likely than antagonism in mixed exposures
to known carcinogenic substances, but they have not been published or publicized. CONCLUSIONS Testing of a dozen chemicals in pairwise combinations produced no convincing evidence for synergistic carcinogen interactions. Four of the five possible synergistic interactions that were observed may be explained by fluctuations in the numbers of tumors observed in the tests, and one could be the effect of lengthened life span. Also observed were 20 possible cases of antagonism in which combined exposures to two chemicals resulted in fewer tumors than exposure to either chemical alone. Antagonism to generalized toxic effects were also apparent in several tests when the addition of a second chemical increased the life span of the test animals without a concomitant increase in tumors. Most significantly, perhaps, given public concern about synergies, the tests described here, done under contract to the NCI in the mid-1970s, produced no good evidence of synergy. By contrast, the same tests produced several examples of antagonism, in which exposure to two chemicals resulted in fewer cancers than exposure to either chemical by itself. REFERENCES Frangipane, N. (2001). Freedom of Information Act Coordinator, NCI. Letter to Michael Gough, in reference to NCI 02–008, FOIA Case No. 27066, November 6, 2001. SRI International (1978). Combined Effects of Chemical Carcinogens and Other Chemicals: Final Report, May 1978, D. C. L. Jones, prepared for Bioassay Operations Program, Tracor Jitco, Inc., Contract 74-23-106002, SRI Project LSD-3478. Approved: W. A. Skinner, Executive Director, Life Sciences Division. [Photocopied typescript, iv + 44 pages]