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 is-sued a contract for the testing of a dozen chemicals,most of them known to be carcinogenic in animals,in pairwise combinations. Those tests, which involved918 pairwise tests and over 14,500 laboratory rats, pro-duced no good evidence for any synergistic interac-tions in which exposure to two chemicals resulted in anumber of tumors greater than the number producedby exposure to either chemical alone. A number of testsresulted in antagonism, in which the number of tu-mors in animals exposed to a pair of carcinogens wasless than the number seen after exposure to either car-cinogen by itself. More generally, the results indicatethat synergism is an unlikely consequence when ani-mals are exposed to pairs of chemicals, even when bothchemicals are carcinogens. C© 2002 Elsevier Science (USA)

Key Words: carcinogenicity; synergy; antagonism;pairwise testing; SRI; NCI.

SRI, however, had a copy of the report and made it

BACKGROUND

In 1974, the National Cancer Institute (NCI) issueda contract to The Stanford Research Institute of MenloPark, California (SRI), to test the effects of exposinglaboratory rats to mixtures of paired carcinogenic sub-stances. A number of scientists are aware of the SRIproject, and some know of the results in a general way,but none had a complete copy of the report, “CombinedEffects of Chemical Carcinogens and Other Chemicals,”dated May 1978 (SRI International, 1978) in the fall of2001. The NCI found no reference to the report in itsfiles when a request for the report was made. A Freedomof Information Act (FOIA) request to the Department ofHealth and Human Services for a copy of the report andfor copies of “any government agencies’ and/or advisorypanels’ reviews of the report” resulted in a letter stat-ing that “no records responsive to your request werelocated” (Frangipane, 2001).

1
Correspondence may be addressed to the author at 6404 E.Halbert Road, Bethesda, MD 20817-5423. E-mail: [email protected].
38

available upon request from an official of the NationalInstitutes of Health (NIH), the original governmentcontracting agency. The full text of the SRI report isavailable at http://www.isrtp.org.

TEST DESIGN

SRI obtained male and female Fischer 344 rats fromSimonsen Laboratories, Inc. (Gilroy, CA), quarantinedthe animals, culled them on the basis of slow growthand disease, and housed the animals three to a cage.The animals were assigned to control or test groupssubsequent to their being assigned to cages and wereprovided lab chow and water ad libitum.

Subchronic feeding tests were conducted on groupsof 15 male and 15 females to determine the maximumtolerated doses (MTD) for each of the test substances.Based on information in the literature and the resultsof the subchronic tests, the SRI set dose levels expectedto produce tumors in 20 to 80% of the exposed animals.

SRI scientists tested 12 chemicals, which were di-vided among four groups (see Table 1).

� Group 1 chemicals had different target organs;� Group 2 chemicals were liver carcinogens;� Group 3 chemicals had different target organs and

1-thiouracil was “well known for its endocrine activity”;� Group 4 chemicals were known to have toxic effects

on the nervous system and were tested for possible ner-vous system carcinogenicity. When none was found tocause nervous system tumors, the liver was assigned asthe target organ.

One of the chemicals, aflatoxin (AF), was included inthree of the four groups (2, 3, and 4); and N-butanol-N-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 7that were known as carcinogens at the time the testswere made. Of the remaining 5, 3—the trisodiumsalt of nitrilotriacetic acid (NTA), lasiocarpine (LAS),and Aroclor 1254 (AR)—were carcinogenic in the SRItests (see Table 2). Two chemicals—dieldrin (D) and

3 0273-2300/02 $35.00C© 2002 Elsevier Science (USA)

All rights reserved.

384 MICHAEL GOUGH

TABLE 1Chemicals Tested in SRI Experiments

Dose in diet (ppm)

Group Chemical name Abbr. C/Ua Target organb Low Mid High

1 N-Methyl-N ′-nitro-N-nitrosoguanidine MNNG C Sto Sin 20 40 80N-Butanol-N-butylnitrosaminec NBBN C Bla 30 60 120Nitrilotriacetic acid, trisodium salt NTA U Kid 200 2000 20000Dipentylnitrosaminec DPN C Liv 50 150 450

2 Aflatoxin B1c AF C Liv 0.005 0.015 0.045

Cycad flour d CY C Liv 2000 4000 8000Lasiocarpine LAS U Liv 7 15 30Dipentylnitrosaminec DPN C Liv 50 150 450

3 Aflatoxin B1c AF C Liv 0.005 0.015 0.045

N-Butanol-N-butylnitrosaminec NBBN C Bla 30 60 120Lead (II) acetate, trihydrate LA C Kid 500 2000 80002-Thiouracil TH U Thy 83 250 750

4e Aflatoxin B1c AF C Liv 0.005 0.015 0.045

Dieldrin D U Liv 2 10 50Hexachlorophene HEX U Liv 17 50 150Aroclor 1254 AR U Liv 25 50 100

Source: SRI International (1978), Table 1, p. 21.a C, 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.
hexachlorophene (HEX)—were not carcinogenic in thetests (see Table 2).

In tests for carcinogenicity, a single chemical or twochemicals were incorporated into lab chow, and testswere made on groups of 24 males and 24 females. Atdeath or sacrifice, all control and test animals were sub-jected to gross examinations for tumors, and each tumorwas examined microscopically.

Each chemical was tested at three different levels inthe diet (see Table 1), and it was tested pairwise withthe other three chemicals in that group at low, middle,and high doses. For example, in group 1:

� N-methyl-N ′-nitro-N-nitrosoguanidine (MNNG)was tested at low, middle, and high doses, and each ofthose doses was tested in combination with NBBN atlow, middle, and high doses; with the trisodium salt ofNTA at low, middle, and high doses; and with DPN atlow, middle, and high doses.

� NBBN was tested at low, middle, and high doses,and each of those doses was tested in combination withNTA at low, middle, and high doses and with DPN atlow, middle, and high doses.

� NTP was tested at low, middle, and high doses, andeach of those doses was tested in combination with DPNat low, middle, and high doses.

� 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 chemi-
cals at three doses to both sexes (24 results = 4 chem-
icals × 3 dose levels × 2 sexes) and results of test-ing 96 paired diets (96 = 4! combinations for eachchemical × 4 chemicals × 3 doses × 2 sexes). [Therewas one additional paired diet result because two endpoints—liver and kidney tumors—were reported for thepaired feeding of cycad flour (CY) and the other group2 chemicals.]

SRI scientists reported tumor incidence as the num-ber of animals in each group of 24 that developedtumors. In addition, they reported the median sur-vival time (MST) for each group, with a maximum of104 weeks when surviving animals were sacrificed. Inall, SRI used 606 groups of 24 animals and a total of14,556 animals in its tests.

RESULTS

SRI and NCI scientists were interested in the pos-sibility of carcinogenic synergy in laboratory rats si-multaneously exposed to two carcinogens. The major-ity of tests, however, could not detect such effects—ifthey occurred. Although the SRI scientists had designedtheir experiments so that feeding of a single chemicalwas 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 op-portunity to detect synergy in those tests. Neverthe-less, the SRI scientists allude to one possible case of

PAIRWISE COMBINATIONS OF CARCINOGENIC CHEMICALS 385

TABLE 2Tested 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 1MNNG Sto; Sin M 0 19 19 23 104 104 97 84

F 0 8 19 20 104 104 103 95NBBNb Bla M 0 19 23 23 104 104 102 83

F 0 23 23 24 104 101 92 75NTA Kid M 0 0 0 11 104 104 97 92

F 0 0 1 6 104 104 103 95DPNb Liv M 2 21 23 24 104 104 77 54

F 5 16 24 23 104 101 96 76

Group 2AFb Liv M 2 5 18 20 104 104 104 104

F 5 13 19 22 104 104 104 104CYc Liv M 2 19 18 9 104 91 64 36

F 5 20 20 7 104 86 68 33LAS Liv M 2 18 24 19 104 104 95 77

F 5 24 23 18 104 102 78 48DPNb Liv M 2 23 24 24 104 104 79 53

F 5 22 24 24 104 104 100 78CYc Kid M 0 5 12 6 104 91 64 36

F 0 6 16 3 104 86 68 33

Group 3AFb Liv M 2 6 14 21 104 104 104 104

F 5 6 19 20 104 104 104 104NBBNb Bla M 0 24 24 21 104 104 101 86

F 0 20 21 22 104 103 98 78LA Kid M 0 1 8 21 104 104 104 104

F 0 0 2 11 104 104 104 104TH Thy M 0 8 20 9 104 104 102 72

F 1 4 17 23 104 104 104 104

Group 4AFb Liv M 2 4 19 21 104 104 104 104

F 5 10 12 22 104 104 104 104D Liv M 2 1 0 4 104 104 104 104

F 5 2 1 3 104 104 104 104HEX Liv M 2 5 2 5 104 104 104 104

F 5 4 9 9 104 104 104 104AR Liv M 2 20 17 18 104 104 104 104

F 5 18 21 23 104 104 104 104

Source: SRI International (1978), Tables 4, 9, 15, 20; pp. 24, 29, 35, 40.a Bla, 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.

synergy, which will be discussed below. In addition theauthors dismissed three other possible synergisticeffects and did not mention a fifth example in whichpairwise exposure resulted in a lengthened MST and ahigher tumor number.

The SRI test results also present opportunities to lookfor antagonism in which simultaneous exposures to twocarcinogens result in a reduction of tumor numbers.The SRI report notes several examples of antagonism,many of which were accompanied or perhaps causedby life span shortening. Examples of antagonisms thatoccurred in the absence of life span shortening will be
discussed below.
Synergy

No statistical analysis of the SRI data has been un-dertaken (or, at least, it has not been made available; seeDiscussion). In this paper, both “synergy” and “antago-nism” are judgment calls, and an increase of more than50% in tumor number was necessary to invoke a sug-gestion of synergy. Five possible examples of synergy(identified by bold numbers in Tables 3–5) are discussedbelow.

Possible synergistic effect between NTA and DPN. Asshown in Table 3, the high dose of NTA caused 11 kid-ney or ureter tumors in male rats. The simultaneous

386 MICHAEL GOUGH

TABLE 3Number of Kidney or Ureter Tumors in Rats Fed NTP and Combinations of NTP and DPN

Number of rats with tumors MST (weeks)

Sex DPN dose NTA dose: 0 Low Mid High NTA dose: 0 Low Mid High

M 0 0 0 0 11 104 104 104 92Low 0 2 0 17 104 104 104 79Mid 0 1 0 6 77 102 84 70High 0 0 0 0 54 54 56 51

F 0 0 0 1 6 104 104 104 103Low 1 0 0 6 104 104 104 104Mid 0 1 1 3 94 104 98 94High 0 0 0 1 76 83 79 63

Source: SRI International (1978), Table 7, p. 27.

posed only to MNNG as a possible “underestimate, since

feeding of the low dose of DPN along with the high-dose NTA increased the number of tumors to 17.The increase is consistent with a synergistic interac-tion, and the SRI scientists cautiously interpreted theincrease:

Although low-level DPN appear to increase the inci-dence of target (kidney and ureter) tumors somewhatin high-level (NTA) males, there was no correspondingincrease in incidence in females even though they sur-vived longer and examination of food consumption datashowed that they accumulated nearly as much NTA(SRI International, 1978, pp. 13–24).

TABLE 4Number of Stomach and/or Small Intestine Tumors in Rats Fed MNNG and MNNG in Combination

with NBBN, NTA, and DPN

Number of rats with tumors MST (weeks)Second chemical

Sex dose MNNG dose: 0 Low Mid High MNNG dose: 0 Low Mid High

M 0 0 19 19 23 104 104 97 84Low NBBN 0 19 22 20 104 104 97 82Mid NBBN 1 21 20 20 102 98 94 76High NBBN 0 5 14 17 83 77 75 78Low NTA 0 19 23 24 104 104 100 80Mid NTA 0 18 22 22 104 104 104 86High NTA 1 1 1 14 92 88 84 81Low DPN 1 20 22 22 104 104 95 75Mid DPN 0 2 18 19 77 84 82 74High DPN 0 0 4 4 54 50 54 50

F 0 0 8 19 20 104 104 103 95Low NBBN 0 14 19 19 101 104 104 88Mid NBBN 0 6 16 20 92 92 88 80High NBBN 0 1 2 13 75 78 73 72Low NTA 0 12 19 23 104 104 102 97Mid NTA 0 9 18 20 104 104 104 90High NTA 1 0 3 8 104 104 102 102Low DPN 0 13 19 21 104 104 95 86Mid DPN 0 10 19 23 94 98 97 88High DPN 0 0 18 18 76 80 80 77


Possible synergy between MNNG and the other threeGroup 1 chemicals—NBBN, NTA, and DPN. The lowdose of MNNG caused 8 stomach or small intestine tu-mors in female rats. The addition of the low dose of anyof the three other Group 1 chemicals, NBBN, NTA, orDPN, increased the tumor numbers to 14, 12, and 13,respectively (Table 4). The number of tumors in malerats exposed to the low dose of MNNG was higher, 19,and exposures to the other three Group 1 chemicalshad no effect on that number. The SRI authors dis-miss the number of tumors in the female animals ex-


TABLE 5Number of Thyroid Tumors in Rats Fed TH and Combinations of TH and LA


Sex LA dose TH dose: 0 Low Mid High TH dose: 0 Low Mid High

M 0 0 8 20 9 104 104 104 72Low 0 6 23 16 104 104 104 84Mid 0 14 19 19 104 104 104 93High 0 2 15 18 104 99 101 97

F 0 1 4 17 23 104 104 104 104Low 0 3 20 21 104 104 104 104Mid 0 3 10 23 104 104 104 104High 1 3 13 22 104 104 104 104


TABLE 6NTA-Caused Reductions in the Number of Tumors Induced by MNNG, NBBN, or DPN

MNNG-caused stomach and small intestine tumors


Sex NTA dose MNNG dose: 0 Low Mid High MNNG dose: 0 Low Mid High

M 0 0 19 19 23 104 104 97 84Low 0 19 23 24 104 104 100 80Mid 0 18 22 22 104 104 104 86High 1 1 1 14 104 88 84 81

F 0 0 8 19 20 104 104 103 95Low 0 12 19 23 104 104 102 97Mid 0 6 16 20 104 104 104 90High 0 1 2 13 104 104 102 102


NBBN-caused bladder tumors


Sex NTA dose NBBN dose: 0 Low Mid High NBBN dose: 0 Low Mid High

M 0 0 19 23 22 104 104 102 83Low 0 24 23 24 104 104 104 84Mid 0 23 24 24 104 104 104 88High 2 8 14 19 92 100 96 86

F 0 0 23 23 24 104 101 92 75Low 0 21 21 21 104 104 99 74Mid 0 19 23 24 104 104 97 88High 4 13 12 19 104 104 104 104


DPN-caused liver tumors


Sex NTA dose DPN dose: 0 Low Mid High DPN dose: 0 Low Mid High

M 0 2 21 23 24 104 104 77 54Low 4 22 23 24 104 104 102 56Mid 1 24 24 23 104 104 84 56High 4 9 23 22 92 79 70 51

F 0 5 16 24 23 104 104 96 76Low 6 9 24 24 104 104 102 83Mid 7 13 24 24 104 104 98 79High 10 18 24 24 104 104 94 63


388 MICHAEL GOUGH

administration of the low MNNG with low NBBN, lowNTA and low DPN resulted in higher incidences (12–14animals)” (SRI International, 1978, p. 13).

While the explanation may be correct, there is noth-ing to corroborate it. Equally, it is possible that all threeof the other Group 1 chemicals acted synergisticallywith NBBN.

Increasing MST and increasing tumor number inmale rats exposed to high-dose 2-thiouracil (TH) andlead (II) acetate trihydrate (LA). The dose responsefor TH-caused thyroid tumors in male rats indicatesthat the high dose is so toxic that it decreases the MST(Table 5). The reduced life span in male rats is accompa-nied by a reduction in tumor number perhaps becausethe shorter life spans are not sufficient to allow the de-velopment of tumors.

All three doses of LA increase the MST in male ratsexposed to the high dose of TH, and the number of tu-mors seen in the TH + LA-exposed males exceed thenumber following exposure only to TH. The increasein MST coupled with the increase in tumor numbercan be interpreted as opposite in effect from the morefrequently observed phenomenon in which decreasedMSTs are accompanied by reduced tumor numbers.

Increasing doses of TH caused increasing numbersof tumors in female rats, and there was no possibilityto observe synergism in female rats, if it occurs. Thehigh dose of TH did not shorten life spans in femalerats, making it impossible to see if the addition of LAincreased the life span of TH-exposed females.

Antagonism

In contrast to the absence of the word “increase”from the SRI “Summary and Conclusions” (SRI In-ternational, 1978, pp. 19–20), the words “reduce” and“reductions” are used several times to describe the num-ber of tumors observed in animals fed two carcinogens.In some cases, the tumor reduction went hand in hand

TABLE 7NBBN-Caused Reductions in the Number of Tumors Induced by NTA

NTA-caused kidney or ureter tumors


Sex NBBN dose NTA dose: 0 Low Mid High NTA dose: 0 Low Mid High

M 0 0 0 0 11 104 104 104 92Low 0 1 1 6 104 104 100 100Mid 0 0 1 9 104 104 104 96High 0 0 0 11 83 84 88 86

F 0 0 0 1 6 104 104 103 95Low 0 0 0 8 104 104 104 104Mid 0 0 0 3 92 99 97 104High 0 0 1 0 75 74 88 104


with shortened MST and is of little interest. In 20 cases,however, the feeding of two carcinogens halved or nearlyhalved the number of tumors observed when either car-cinogen was fed singly and had no effect on MST. Thosecases are identified by bold numbers in Tables 6–11 andare discussed below.

NTA reduces tumor rates from exposures to MNNG,NBBN, and DPN. High doses of NTA reduced thenumber of stomach and small intestine tumors causedby MNNG, the number of bladder cancers caused byNBBN, and the number of kidney or ureter cancerscaused by DPN. The antagonistic effect of NTA onMNNG-, NBBN-, and DPN-caused tumors, in theabsence of any effect on the MST, was limited to femalerats.

In female rats, the high dose of NTA reduced tumornumbers at all levels of MNNG exposure and had noeffect on MST (Table 6). In male rats, the addition of thehigh dose to NTA to the food reduced tumor number atall doses of MNNG but decreased the MST seen at lowand mid doses of MNNG.

High doses of NTA also reduced the number of blad-der cancers caused by NBBN in both male and fe-male rats. The reduction in tumor number in maleswas accompanied by reductions in MST. In females, thecombination of high-dose NTA and low or middle dosesof NBBN reduced the tumor number and, at the sametime, lengthened MST (Table 6).

NTA had a similar effect on DPN-caused liver tumorsin rats. As shown in Table 6, the high dose of NTA re-duced the number of liver tumors in male rats exposedto low-dose DPN, but that reduction was accompaniedby a shortened MST. In female rats, the low dose of NTAreduced the number of tumors caused by the low levelof 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 kid-ney and/or ureter tumors in female rats simultaneously


TABLE 8AF-Caused Reductions in the Number of Tumors Induced by LA

LA-caused kidney tumors


Sex AF LA dose: 0 Low Mid High LA dose: 0 Low Mid High

M 0 0 1 8 21 104 104 104 104Low 0 0 9 18 104 104 104 104Mid 1 1 12 19 104 104 104 104High 0 0 11 19 104 104 104 104

F 0 0 0 2 11 104 104 104 104Low 0 0 2 11 104 104 104 104Mid 0 0 3 14 104 104 104 104High 0 0 1 5 104 104 104 104


exposed to the high dose of NTA. Remarkably, the low,middle, and high dose of NBBN increased MST in allfemale rats exposed to the high dose of NTA. In malerats, NBBN did not affect NTA-caused tumor inductionand high-dose NBBN-shortened MSTs (Table 7).

AF reduces tumor rate from exposure to LA. High-dose AF reduced the number of tumors associated with
the high dose of LA and did not affect MST in female
TABLE 9TH-Caused Reductions in the Number of Tumors Induced by AF and LA

AF-caused liver tumors


Sex TH dose AF dose: 0 Low Mid High AF dose: 0 Low Mid High

M 0 2 6 14 21 104 104 104 104Low 1 5 19 21 102 104 100 100Mid 2 3 11 23 104 104 104 104High 0 0 0 0 72 48 46 47

F 0 5 6 19 20 104 104 104 104Low 3 3 14 15 104 104 104 104Mid 1 3 5 3 104 104 104 104High 0 0 2 3 104 87 85 98


LA-caused kidney tumors


Sex TH dose LA dose: 0 Low Mid High LA dose: 0 Low Mid High

M 0 0 1 8 21 104 104 104 104Low 0 1 8 18 102 104 104 99Mid 0 0 5 22 104 104 104 101High 0 0 0 14 72 84 93 97

F 0 0 0 2 11 104 104 104 104Low 0 1 2 4 104 104 104 104Mid 0 0 1 10 104 104 104 104High 0 0 0 5 104 104 104 104


rats (Table 8). The addition of AF to the diet of malerats fed LA had no effect.

TH reduces tumor rates from exposure to AF and LA.TH antagonized tumor induction by AF in both maleand female rats (Table 9). In males, the reduction in tu-mors associated with high-dose TH was accompanied bysevere reductions in MST. In females, the reduction intumors associated with the middle dose of TH occurred

390 MICHAEL GOUGH

TABLE 10D-Caused Reductions in the Number of Tumors Induced by AF



Sex D dose AF dose: 0 Low Mid High AF dose: 0 Low Mid High

M 0 2 4 19 21 104 104 104 104Low 1 2 19 21 104 104 104 104Mid 0 5 16 24 104 104 104 104High 4 8 17 21 104 104 104 104

F 0 5 10 12 22 104 104 104 104Low 2 13 11 21 104 104 104 104Mid 1 3 7 21 104 104 104 104High 3 5 10 19 104 104 104 104


in the absence of any effect on MST. At the high dose ofTH, both tumor number and MST were reduced.

Low- and high-dose TH were associated with lowertumor numbers in female rats fed high-dose LA, withno effect on MST (Table 9), but the dose response toTH in rats fed high-level LA is not simple. The numberof tumors at middle-dose TH and high-dose LA (10) ishigher than the numbers seen at low and high-dose THand high-dose LA (4 and 5). The simpler explanationfor the observed numbers is that the result at middle-dose LA is higher than would be expected on repeatedtesting than that the observations at both low- andhigh-level LA are lower than would be found on re-peated testing.

The text of the SRI report that discusses the resultsof feeding LA and TH to rats states, “Pairing of LA withTH tended to reduce kidney tumor incidences some-what, and there was some associated reduction in lifes-pan” (SRI International, 1978, p. 17). That statementdoes not, however, describe the results for female rats,
in which there was no shortening of MST (Table 9). (Table 11).
TABLE 11HEX-Caused Reductions in the Number of Tumors Induced by AF



Sex HEX dose AF dose: 0 Low Mid High AF dose: 0 Low Mid High

M 0 2 4 19 21 104 104 104 104Low 2 4 20 23 104 104 104 104Mid 2 3 19 19 104 104 104 104High 5 3 18 20 104 104 104 104

F 0 5 10 12 22 104 104 104 104Low 4 4 10 22 104 104 104 104Mid 9 3 14 24 104 104 104 104High 9 2 8 19 104 104 104 104


D reduces tumor rate from exposure to AF. D, in theSRI tests, “did not induce liver tumors to any discernibleextent in either sex” (SRI International, 1978, p. 17).The addition of the middle dose of D to female rats ex-posed to the low and middle dose of AF reduced thenumber of tumors with no effect on MST (Table 10),and the addition of the high dose of D reduced the num-ber of tumors caused by the low dose. D did not affecttumor number or MST in male rats.

HEX reduces tumor rate from exposure to AF. HEX,like D in the SRI tests, was not carcinogenic; “Livertumor incidences in females receiving mid and highlevels of HEX were slightly higher than the median con-trol value, but not appreciably so; consequently HEXdoes not appear to be a liver carcinogen” (SRI In-ternational, 1978, pp. 17–18). All doses of HEX re-duced the number of tumors caused by low-dose AFin female rats with no effect on MST, and no doseof HEX affected tumor number or MST in males


Low doses of AF produced different numbers oftumors in male rats (4) and female rats (10). Thedisparity makes it reasonable to question the apparentreductions in tumor numbers in female rats followingexposures to D and HEX. For instance, if the number oftumors at the low dose of AF in females was spuriouslyhigh, the apparent reductions could represent a regres-sion to the means. That explanation seems less likelybecause the number of tumors reported in Tables 10and 11 in female rats exposed to the low dose of AF byitself is consistent with the numbers of tumors (13, 6,and 10) determined in three different tests of low doseAF (Table 2). The number of tumors associated with thelow dose of AF in male rats (4) reported in Tables 10 and11 is consistent with the results (5, 6, and 4) reportedfor three tests of the low dose in male rats (Table 2). If,however, the lowest number of tumors reported in fe-male rats exposed to the low dose of AF (6) is more rep-resentative of the effect of that dose level, the apparentantagonism between D and AF and between HEX andAF would disappear.

DISCUSSION

The authors of the SRI report note increases and de-creases in tumor number and state that a more quan-titative analysis of the results was being prepared by agroup at the University of California at Los Angeles. Ithas not been possible to locate that analysis, if it wasprepared. In the absence of such an analysis or repeatedtesting, any discussion of the results is limited neces-sarily to “eye-balling” the reported numbers of tumorsand lengths of MSTs.

The first comment about the results of the SRI re-port is that it contains nothing to suggest that syner-gism, even between known carcinogens, is common orstriking. Indeed, the SRI scientists dismissed four ofthe five examples of possible synergism. In the case oflow-level DPN appearing to cause an increase in malerats exposed to high-level NTA (Table 3), the SRI scien-tists pointed to the absence of any such effect in femalesfed high-level NTA and do not discuss the increase be-yond that. NBBN, NTA, and DPN increased the numberof tumors in female rates exposed to low-dose MNNG(Table 4). The SRI scientists did not interpret the resultas suggesting synergy. Quite the contrary, they suggestthat the increase seen in all three cases where a secondchemical was fed indicates that the number of tumorsseen in the females exposed only to MNNG was an “un-derestimate.”

Give the limited information available, the four caseswhere tumor numbers increased with the addition of asecond chemical could be interpreted as either synergyor the result of fluctuations in the numbers of tumorsproduced in the tests. Depending on which interpreta-

tion is correct, there is either no or very scant evidencefor synergy.

The fifth possible example of synergy is more perplex-ing. The highest tested dose of TH is toxic to both maleand female rats (Table 5). The addition of LA to the dietof male rats fed the highest TH dose increased theirMST and the number of thyroid tumors. In this case,increasing the MST—a good thing—led to an increasein cancer number—a bad thing, as could be expectedregardless of synergism. The result is a parallel to thechanges in human life expectancy and cancer risk inthe past century in this country, in which increasinglife spans have resulted in populations with a largerproportion of older individuals who are more likely todevelop cancer.

The interpretation of the possible cases of antagonismis subject to the same limitations as for possible casesof synergism. In particular, tumor number fluctuationscould result in spurious conclusions. Any reported testresult of pairwise testing might represent a “low” num-ber or an “underestimate” of the number of tumors thatwould be seen upon repeated tests and produce an ap-parent antagonism, when none was present. Still, theevidence that NTA reduces tumor numbers resultingfrom feeding MNNG and NBBN is strengthened be-cause it does not depend on a difference seen at a singledose level of MNNG or NBBN.

The results in Table 6 show that high-dose NTAreduced the number of tumors caused by high-doseMNNG and had little effect on MST. This is the onlyunequivocal example of antagonism of carcinogenic ef-fects reported in male rats in the SRI results.

High-dose NTA also caused reductions in the num-ber of tumors in female rats exposed to both low- andmiddle-dose MNNG (Table 6). The NTA had no effecton MST in the low-dose MNNG group or in the middle-dose MNNG group (103 to 102 weeks), and lengthenedthe MST in the high-dose MNNG from 95 to 102.

High-dose NTA reduced the number of tumors in fe-male rats exposed to low- and middle-dose NBBN. Inthose cases, the addition of NTA to the diets of the an-imals increased their MSTs, while reducing the tumornumbers.

More than one combination of D and AF and of HEXand AF resulted in numbers of tumors less than thosecaused by AF alone. Both middle- and high-dose Dcaused a reduction in tumors in female rats exposed tolow-dose AF, and all doses of HEX caused a reduction inthe number of tumors from low-dose AF. Neither AF, D,nor HEX nor any paired combination of those chemicalsaffects MSTs in males or females (Tables 10 and 11).

An example of the more typical result that indicatesantagonism is shown on Table 6. The low-dose DPN–low-dose NTA combination caused 9 tumors comparedto 16 tumors observed in the female rats exposed tolow-dose DPN only. The evidence, here, is a difference in

392 MICHAEL GOUGH

the two numbers of a pair of numbers, and fluctuation ineither or both might account for an apparent differencewhen no actual difference exists.

Although only the high-dose NBBN reduced the tu-mor number in female rats exposed to high-dose NTA,all NBBN doses extended the MST of female rats ex-posed to high-dose NTA (Table 7). This effect on life spansupports the idea of antagonistic interactions betweenthe two chemicals that reduce the toxic effects of NTA.

There are three other examples of antagonism be-tween pairs of chemicals: High-dose AF reduced the tu-mors observed in female rats exposed to high-dose LA(Table 8) and did not affect MST. High-dose TH reducedthe number of tumors seen in female rats exposed tohigh-dose AF without affecting MST, and both low- andhigh-dose TH reduced the number of tumors resultingfrom exposure to high-dose LA (Table 8).

The SRI report noted many of these reductions inthe “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. Thereport did not, however, use the words “antagonism” or“antagonistic.” In contrast to the discussion of reduc-tions in the “Summary and Conclusions” section, thereis no mention of increases.

The SRI tests are one example of how testing of pairsof chemicals can be done, and examination of the me-chanics of those tests would provide guidance for ad-ditional or better tests. Such tests, costly and time-consuming as they could be, could inform the manydiscussions that are concerned about synergism.

Needless to say, additional tests would be usefulonly to the extent that the results are published. TheSRI results, inexplicably buried for almost a quartercentury, provide experimental evidence that synergism
may be less likely than antagonism in mixed exposures
to known carcinogenic substances, but they have notbeen published or publicized.

CONCLUSIONS

Testing of a dozen chemicals in pairwise combina-tions produced no convincing evidence for synergisticcarcinogen interactions. Four of the five possible syner-gistic interactions that were observed may be explainedby fluctuations in the numbers of tumors observed inthe tests, and one could be the effect of lengthened lifespan.

Also observed were 20 possible cases of antagonismin which combined exposures to two chemicals resultedin fewer tumors than exposure to either chemical alone.Antagonism to generalized toxic effects were also appar-ent in several tests when the addition of a second chem-ical increased the life span of the test animals withouta concomitant increase in tumors.

Most significantly, perhaps, given public concernabout synergies, the tests described here, done undercontract to the NCI in the mid-1970s, produced no goodevidence of synergy. By contrast, the same tests pro-duced several examples of antagonism, in which expo-sure to two chemicals resulted in fewer cancers thanexposure 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 CaseNo. 27066, November 6, 2001.

SRI International (1978). Combined Effects of Chemical Carcinogensand 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

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