Herbicide Exposure: Link to Disease

Share

Chairman Simpson and Members of the Committee, thank you forthis opportunity to discuss the mistaken assignment of a suggestivelink between Agent Orange exposure of fathers and the appearance ofspina bifida in their children. I will discuss three points: firstwill be a few words about science, second is a discussion of theAir Force's Ranch Hand Study [1] and theprocess by which the Institute of Medicine [2] reached its conclusion about Agent Orange andspina bifida, and third is an analysis that demonstrates that theIOM conclusion has no predictive value.

The late Karl Popper, the premier philosopher of science,[3] said that science consists of twoactivities. The first is the formulation of a hypothesis that is atentative description of the way a part of the universe operates.The second activity is testing the hypothesis. Without testing, wewill have no idea of whether the hypothesis is or is not a gooddescription of reality. Importantly, hypothesis testing cannotdepend on picking and choosing data because picking and choosingleads to ignoring information that might favor the null hypothesis.Popper and other scientists have emphasized the importance ofdisproving the null hypothesis, which is a shorthand way to referto the idea that the hypothesis that is being considered is wrongand evidence must be produced to support it. [4]

IOM considered a possible link between Agent Orange and spinabifida in both its 1993 [5] and its 1996report. In 1993, it suggested no link, and, as we all know, in1996, it concluded that there was "limited/suggestive evidence foran association between exposure to herbicides used in Vietnam andspina bifida in offspring" (IOM 1996 at p. 1-8). The reason for thechanged conclusion was information in the Ranch Hand study (IOM1996 at p. 9-8).

The Ranch Hand study compared birth outcomes of the childrenborn to Air Force personnel with known exposures to herbicides withthe outcomes for children born to a Comparison group that had noherbicide exposure. What would we expect if exposures to herbicideshad absolutely no impact on birth defects and we compared the kindsand numbers of birth defects in the children of a group ofherbicide-exposed men to the birth defects in a group ofnon-herbicide-exposed men? Would we expect exactly the same kindsof birth defects? Or exactly the same numbers? Of course not, basedon chance, we would expect that some birth defects would be morecommon in the exposed group and that some would be more common inthe non-exposed group.

That is exactly what we find when we look at the results of theRanch Hand study. There is no statistically significant differencebetween the frequency of birth defects in the Comparison and RanchHand populations. The ratio of birth defects/child was 0.21 in theComparisons and 0.22 in the Ranch Hands. When comparisons were madeof the frequency of birth defects in different organsystems--nervous system, genital system, digestive system,etc.--birth defects in four organ systems were more common in thechildren of the Comparisons and birth defects in the other sixorgan systems were more common in the children of the RanchHands.

Two specific anomalies and two developmental disabilities werecommon enough in the children of Ranch Hands for statisticalanalysis on the bases of the fathers' herbicide exposures. Thefrequencies of the two specific anomalies--major birth defects andmultiple birth defects--were comparable in the children of theComparisons and Ranch Hands. Six percent, of the Comparisons'children and eight percent of the Ranch Hands' children had majorbirth defects, and 0.4 percent of the Comparisons' children and 0.6percent of the Ranch Hands' children had multiple birth defects.[6] The small numbers of these defectsmakes the differences in percentage not statistically differentfrom each other.

To examine the possible effect of increasing herbicideexposures, the Ranch Hands were divided into three groups--(1)those with no evidence of exposure above background levels, (2)those with low exposures, and (3) those with high exposures. Ifherbicides cause birth defects, we would expect the frequency ofthe birth defects to increase along with exposures. That is notwhat was found. Instead, major birth defects and multiple birthdefects were most common in the children of Ranch Hands with lowexposures. There is no way to explain those results as beingrelated to herbicide exposures. If increasing exposures causedthose birth defects, the defects would be more common in thechildren of the "high" exposure group of Ranch Hands. The mostreasonable explanation is that herbicides had no effect on thesebirth defects and that the distribution of birth defects inchildren of men with background, low, and high exposures was amatter of chance.

Examination of the two developmental disabilities that weresufficiently common for statistical analysis supports theconclusion that there is no relationship between herbicideexposures and frequency of birth defects. [7] Delayed development was more common in thechildren of the "low" exposure group than in the children offathers in either the "background" or "high" exposure group.Moreover, delayed development was more common in the children ofthe "background" exposure group than in those of the "high"exposure group. Hyperkinetic syndrome was most common in thechildren of the background exposure group.

Rather than dwelling on the differences in the frequency ofthese anomalies and developmental disabilities in the children ofmen with different exposure histories, it makes more sense to saythat there is no relationship between exposure and these anomaliesand disabilities. Indeed, that is how IOM interpreted thosedata.

IOM interpreted the spina bifida data differently. The RanchHand study reported the numbers of 14 specific anomalies ordevelopmental disabilities identified in the children ofComparisons or Ranch Hands or both for which "Counts andrates...[were] too sparse to analyze..." (Wolfe et al.1995, p. 20).

Two neural tube anomalies--one case of anencephaly and threecases of spina bifida--occurred only in the children of RanchHands. Those two conditions account for four of the five cases ofnervous system anomalies reported in Ranch Hand children. Therewere three nervous system anomaly cases in the Comparisons'children. In addition, two other birth defects--one case ofpolydactyly and one case of reduced limb deformity--occurred onlyin the Ranch Hands' children.

In contrast, cleft palate and cleft lip/palate occurred only inthe Comparison children. In the past, these conditions had oftenbeen suggested as possibly associated with dioxin because exposureof pregnant mice to dioxin increased the frequency of cleft palatein their offspring. [8] There is,however, no evidence for exposure of male breeding mice to AgentOrange having any effect on their offspring. [9] The Ranch Hand study is consistent with theconclusion that parental exposure does not cause cleft palate. Inaddition to cleft palates that occurred only in Comparisons'children, the single case of hydrocephalus that was seen in thestudy occurred in the child of a Comparisons.

Given the distribution of birth defects between Comparisons andRanch Hands, there is as much logic in suggesting that exposures toherbicides protects against cleft palate and hydrocephalus as therewas in IOM's concluding that the results about spina bifida aresuggestive of an association.

Neither the authors of the Ranch Hand study, the Department ofHealth and Human Services committee that reviewed the study beforeits publication, [10] the reviewers andeditors of the journal Epidemiology that published the AirForce study, or a scientist who commented on the Air Force studyfor Epidemiology [11] concludedthat there was "limited/suggestive" evidence for a connectionbetween herbicide exposure and spina bifida. Indeed, none of themmentioned any evidence about that possible association at all.

Why did IOM reach its conclusion that the evidence was"limited/suggestive"? In my opinion, the reason is obvious. IOMassigns that classification when

Evidence is suggestive of an association between herbicides andthe outcome but is limited because chance, bias, and confoundingcould not be ruled out with confidence. For example, at least onehigh-quality study shows a positive association, but the results ofother studies are inconsistent. [12]

That criterion flies in the face of Popper's and otherscientists' insistence that all the data be considered and weighedtogether. In fact, it literally throws out any consideration of thenull hypothesis because it lets the analysts focus on a single,isolated finding as the proof of their case. This is bad science.It was not, as is sometimes suggested, forced on IOM by Congress.IOM set its criterion of "one good study" for itself.

There is no known biological mechanism by which parentalexposure to herbicides can cause spina bifida in the veterans'children. [13] Neither 2,4-D nor2,4,5-T, the principle components of Agent Orange, [14] nor dioxin [15]is a mutagen, and none can affect the DNA in a sperm cell. Both2,4-D and 2,4,5-T are eliminated from the body in a period ofweeks, so that those chemicals would not have been present in thefathers' bodies when children were conceived after Vietnam service.Dioxin, on the other hand, is very persistent in the body. Sincedioxin cannot affect DNA, the only method by which it could causebirth defects is by being transferred from the father to the motherthrough semen. The few molecules of dioxin that would betransferred would be added to the trillions more molecules in themother's body. How could that tiny addition have an effect? Itcould not.

IOM states (IOM 1996 p. 9-18) that "Laboratory studies of thepotential developmental toxicity...of TCDD as a result of exposureto adult male animals are too limited to permit conclusions." Thismay be a correct summation, but it ignores the study by Lamb etal. that showed that feeding Agent Orange to male mice causedno birth defects even when it caused toxic effects in the malemice.

The IOM also brushed aside the study of 15,291 births toresidents of Seveso, Italy. Seveso was the scene of a chemicalplant accident in 1976 that spread dioxin and other chemicals overan area with a population of 37,000. In 1988, a group of Italianphysicians published a paper that compared the frequency and kindsof birth defects in children born in the Seveso area in the sixyears after the accident to those in children born in thesurrounding, uncontaminated area during the same period. [16]

The contaminated area of Seveso is divided into three zones, Abeing most contaminated, B less contaminated, and R still lesscontaminated. The noncontaminated comparison area is callednonABR.

I am going to use the IOM interpretation of the Air Force studyas the basis of a hypothesis and use the Seveso results to test it.This exercise fits into Popper's description of how science isdone, and it is nothing that IOM could not have done. It issomething that IOM should have done.

The Air Force reported 5 nervous system anomalies in the RanchHands and 3 in the Comparisons. If there is a relationship betweendioxin exposure and nervous system anomalies, those birth defectswould be expected to be higher in the children born to the exposedparents at Seveso. There were 2 such defects among the 2900children born to the exposed parents and 22 in the 12391 childrenborn to the unexposed parents. The frequency of such birth defectsis 0.07 percent in the exposed group and 0.17 percent in theunexposed group, [17] or 2.5-timeshigher in the unexposed group. The Seveso data provide no supportfor the idea that dioxin exposure causes central nervous systemdefects.

Some dioxin exposures were higher at Seveso than thoseexperienced by the Ranch Hands (see table 1), more people wereexposed at Seveso, and, very importantly, both men and women wereexposed. Table 2 presents the results of calculating the expectednumber of cases of spina bifida in the Seveso population if IOM'sconclusion about the relationship between dioxin and spina bifidais correct. (Blood samples were obtained from many Sevesoresidents, and as they are assayed for dioxin, information aboutexposures there will become more certain.)

The absence of spina bifida from the residents of Seveso Zones Aand B and its absence or near absence from Zone R is a strongargument against the conclusion reached by IOM. In contrast to themale-only exposure of the Ranch Hands, both sexes were exposed atSeveso, and couples composed of highly exposed men and women havehad children. [18] The animalexperiments indicate that sufficiently high exposures of pregnantmice and rats can cause birth defects. If that is the case inhumans, even the exposures of the women at Seveso were notsufficiently high to increase the rates of neural tube defects oroverall birth defects (Mastroiacovo et al. 1988). Theanimal experiments provide no evidence for dioxin causing birthdefects when administered to the male parent, and there is nobiological theory to explain how dioxin could have that effect. Theabsence of excess birth defects in the Seveso population conformswith predictions from the animal experiments and with theory. TheSeveso study contradicts the IOM's conclusion.

Conclusion

No biological mechanism is known that would explain how dioxinexposure of men could cause birth defects in their children; awell-done animal experiment demonstrated that exposing male mice toherbicides did not increase birth defects among their offspring;none of the other experiments that IOM cites as supporting itsconclusions about a "limited/suggestive" association between dioxinand spina bifida has any verifiable information about exposure andthere is no evidence that men classified on the basis of records aslikely exposed were actually exposed, [19] the Ranch Hand study is best interpreted asshowing no connection between paternal dioxin exposure and birthdefects, and the absence of spina bifida from the Seveso populationwith higher exposures and many more births than in the Ranch Handstudy directly contradicts IOM's conclusion. The IOM committeemisled itself by deciding to use the criterion that one goodpositive study would provide sufficient evidence for alimited/suggestive association. That decision when combined withpicking through the available data and selecting one finding thatsupports their conclusion is not scientific. It led IOM to adecision that has no support.

Table 1
Dioxin Levels in the Ranch Hands and the Seveso Population RanchHands, calculated initial dioxinconcentrations.
b

Parental Concentrations (ppt)______________

Classification Na mean 75%tile maximum

background 283 --- --- <10

low exposure 241 60 --- 109

high exposure 268 294 --- 2020

Seveso population, measured concentrations in samples taken soonafter exposure

Parental Concentrations (ppt)_______________

Classification Na mean 75%tile maximum

Zone A 198c ca. 500d ca. 2000d 56,000e

Zone B 435f ca. 125d --- ---

Zone R 2439f ca. 60g --- ---

__________

a. Number of children born to parents in the indicatedclassification.

b. Joel Michalek, principal investigator, Air Force Ranch Handstudy, email Aug. 28, 1996.

c. Number of births through December 1994 reported in Mocarelli,P., P. Brambilla, P.M. Gerthoux, et al. 1996. TheLancet 348: No birth defects have been reported among thechildren born to parents who lived in Zone A, although there was anexcess of female births during the first seven years afterexposure. In contrast, less than 50 percent of the children born toRanch Hands were female (Joel Michalek, email, September 10,1996.)

d. Approximations supplied by Larry Needham, Centers for DiseaseControl, telephone conversation, Sept. 9, 1996.

e. Mocarelli, P., D.G. Patterson, A. Marocchi, and L.L. Needham.1990. Chemosphere 20:967-974.

f. Mastroiacovo, P., A. Spagnolo, M. Ernesto, et al.1988. Journal of the American Medical Association259:1668-1672.

g. There are no published values for dioxin concentrations inresidents of Zone R. I have approximated the concentration at 1/2the Zone B concentration because: The dioxin level in the soil ofZone R is 1/3 the level in Zone B and animal mortality and theprevalence of chloracne (a skin disease that is indicative ofdioxin exposure) are essentially the same in Zones B and R(Mastroiacovo et al. 1988. Journal of the AmericanMedical Association 259:1668-1672.)

Table 2
Expected Numbers of Cases of Spina Bifida at Seveso If the IOM'sConclusion is Correct Compared to the Reported Numbers

Area at Seveso Calculated Number Observed Number

of spina bifida casesa of spina bifida casesb

Zone A 2.4 to 6.5 0

Zone A (75%tile)c >2.4 to >6.5 0

Zone B 1.3 to 3.6 0

Zone R 3.5 to 9.7 0

___________

a. 1 of 241 children fathered by Ranch Hands with low exposures(60 ppt dioxin) had spina bifida, and 2 of 268 children fathered byRanch Hands with high exposures (294 ppt dioxin) had spina bifida.The expected frequency of cases of spina bifida in the Sevesochildren based on those relationships are calculated as:

frequency spina bifidarh = predicted frequency spina bifidas

dioxin concentrationrh dioxin concentrations.

For instance, the frequency of spina bifida in the low exposureRanch Hands is 1/241=0.4%, and their dioxin concentration is 60ppt. The dioxin concentration of residents of Zone A is 500 ppt,and the relationship becomes

0.4% spina bifida = X% spina bifida, and X = 3.3%.

60 ppt 500 ppt

For the high exposure Ranch Hands the relationship is

0.7% spina bifida = X% spina bifida, and X = 1.2%.

294 ppt 500 ppt

Multiplying a frequency times the number of births yields anestimate of the expected number of births with spina bifida. Forinstance 198 births in Zone A multiplied times the expectedfrequency of 3.3% is 6.5, and multiplied by the expected frequencyof 1.2% is 2.4.

b. No birth defect was reported in Zone A. One neural tubedefect was reported in Zone B; from other information inMastroiacovo et al. (1988), it appears that that birthdefect was a brain tumor and not spina bifida. One neural tubedefect was reported in Zone R, and it is impossible to tell fromthe paper if that defect was a spina bifida. At my request, ascientist at the Centers for Disease Control contacted Dr.Mastroiacovo and asked him if any of the neural tube defects atSeveso was spina bifida. The scientist emailed on September 16, "Ie-mailed Dr. Mastroiacovo but unfortunately all the records are instorage and he is leaving today for a meeting. I'm sure he would behappy to try to get the information for you at a later time, but hewon't be able to access them before the hearing." (I'm surprisedthat this important information is not readily available.) However,an Italian scientist visiting Dr. L. Needham at CDC stated thatthere was no spina bifida in zones A, B, or R (telephoneconversation, September 16, 1996).

c. The 75th percentile measurement in Zone A is about 2000.These calculations were based on an assumed average exposure of2000 ppt, which is too low, and the assumption that the 25 percentof people with high exposures were parents of 25 percent of thechildren in Zone A.


Notes

[1] Wolfe, W.H., J.E.Michalek, J.C. Miner, et al. 1995. Epidemiology6:17-22.

[2] Institute ofMedicine. 1996. Veterans and Agent Orange: Update 1996.National Academy Press: Washington, DC.

[3] Bondi, H. 1992. Thephilosopher for science. Nature 358:363.

[4] Popper, K., inMiller, D. (Ed). 1985. Popper Selections. PrincetonUniversity Press: Princeton, NJ. pp. 133-151.

[5] Institute ofMedicine. 1993. Veterans and Agent Orange. NationalAcademy Press: Washington, DC.

[6] There were 56 majorbirth defects in the children of the Comparisons; 59 in the RanchHands and 4 cases of multiple birth defects in the children of boththe Comparisons and Ranch Hands.

[7] There were 71 casesof delayed development in the children of both the Comparisons andRanch Hands and 32 cases of hyperkinetic syndrome in the childrenof Comparisons and 30 cases in the children of Ranch Hands.

[8] Courtney, K.D. andJ.A. Moore. 1971. Toxicology and Applied Pharmacology20:396-403.

[9] Lamb, J.C., J.AMoore, and T.A. Marks. 1980. Evaluation of2,4-dichlorophenoxyacetic acid (2,4-D) and2,4,5-trichlorophenoxyacetic acid (2,4,5-T), and2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in C57BL-6mice: Reproduction and fertility in treated male mice andevaluation of congenital malformations in their offspring.National Toxicology Program: Research Triangle Park, NC.[NTP-80-44].

[10] I chaired thatcommittee when it reviewed the Air Force birth defects study.

[11] Lindbohm, M-L.1995. Epidemiology 6:4-6.

[12] Institute ofMedicine. 1993. Veterans and Agent Orange. NationalAcademy Press: Washington, DC. At p. 6.

[13] IOM soft-pedalsthis idea, but it is clear from its discussion in the 1993 reportat pp. 593-595 that any connection between paternal exposures tononmutagenic agents and effects in offspring is speculative.

[14] Mortelmans, K.,S. Haworth, W. Speck, et al. 1984. Toxicology andApplied Pharmacology 75:137-146.

[15] EnvironmentalProtection Agency. 1994. Health Assessment Document for2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) and RelatedCompounds. USEPA: Washington, DC. [EPA/600/BP-92-001a.] VolumeI of III at pp. 6-12 to 6-14.

[16] Mastroiacovo,P., A. Spagnolo, M. Ernesto, et al. 1988. Journal ofthe American Medical Association 259:1668-1672.

[17] I included"neural tube defect," "microencephaly," and "other CNS defects" inthis tabulation. Two other categories, "eye defects" and "eardefects" that involve the nervous system are also tabulated byMastroiacovo et al. Adding those in does not affect theconclusion about no relationship between dioxin exposure andnervous system effects, but it would increase the number of birthdefects from 2 to 5 among the exposed group and from 22 to 39 amongthe unexposed group. The corresponding frequencies are 0.017 amongthe exposed group and 0.031 among unexposed group.

[18] Mocarell, P., P.Brambilla, P.M. Gerthoux, et al. Change in sex ratio withexposure to dioxin. The Lancet 348: .

[19] Centers forDisease Control. 1988. Journal of the American MedicalAssociation 260:1249-1254.

Committee on Veterans' Affairs
United States Senate