Appendix B

Short-Term Adverse Health Responses

The committee responsible for Update 2008 advised the removal of chloracne, porphyria cutanea tarda (PCT), and early-onset peripheral neuropathy from the body of the Veterans and Agent Orange (VAO) reports, and the committee for Update 2010 removed them. The three conditions that occur in temporal proximity to exposure have little relevance to new claims from Vietnam veterans, and there has been minimal new evidence since they were classified as having evidence of an association with herbicide exposure

The three conditions have long been recognized by the Department of Veterans Affairs as presumptively related to service in Vietnam. This appendix has been retained in subsequent volumes in the VAO series to provide easy access to the distilled body of biomedical evidence upon which these decisions were made.

CHLORACNE

Chloracne is a skin disease that is characteristic of exposure to 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) and other diaromatic organochlorine chemicals. It shares some pathologic processes (such as the occlusion of the orifice of the sebaceous follicle) with more common forms of acne (such as acne vulgaris), but it can be differentiated by the presence of epidermoid inclusion cysts, which are caused by proliferation and hyperkeratinization (horn-like cornification) of the epidermis and sebaceous gland epithelium. Although chloracne is typically distributed over the eyes, ears, and neck, it can also occur on the trunk, genitalia, and buttocks of chemical-industry workers exposed to TCDD (Neuberger et al., 1998). It is resistant to acne treatments, but it usually regresses.

Chloracne has been used as a marker of exposure in epidemiologic studies of populations exposed to TCDD and related chemicals. It is one of the few findings in humans that are consistently associated with such exposure, and it is a well-validated indicator of high-dose exposure to TCDD and related chemicals (Sweeney et al., 1997/98). If chloracne occurs, then it appears shortly after the chemical exposure, not after a long latent period; therefore, new cases of chloracne among Vietnam veterans would not be the result of exposure during the Vietnam War. It should be noted that the absence of chloracne does not necessarily indicate an absence of substantial exposure to TCDD, as is apparent from studies of people who had documented exposure to TCDD after the Seveso incident in 1976 in Italy (Baccarelli et al., 2005a), nor is there necessarily a correlation between serum TCDD concentration and the occurrence or severity of chloracne. Susceptibility to the development of chloracne varies among individuals.

Recent data released following the poisoning of Victor Yushchenko have provided evidence suggesting that chloracne may be a detoxification mechanism. Close examination of the lesions over time demonstrate that the lesions are hamartomas, characterized by the loss of the sebaceous glands. These lesions concentrate the level of TCDD 10-fold, and show increased expression of enzymes involved in toxicant metabolism (Saurat et al., 2012). This suggests the hypothesis that chloracne lesions are an adaptive skin response to the exposure to dioxin and related compounds and that they play a functional role in the detoxification pathway for these compounds.

Conclusions from VAO and Previous Updates

The committee responsible for Veterans and Agent Orange: Health Effects of Herbicides Used in Vietnam (referred to as VAO; IOM, 1994) determined that there was sufficient evidence of an association between exposure to at least one chemical of interest (TCDD) and chloracne. Additional information available to the committees responsible for Veterans and Agent Orange: Update 1996 (IOM, 1996), Update 1998 (IOM, 1999), Update 2000 (IOM, 2001), Update 2002 (IOM, 2003a), Update 2004 (IOM, 2005), Update 2006 (IOM, 2007), Update 2008 (IOM, 2009), Update 2010 (IOM, 2011a), and Update 2012 (IOM, 2014) has not modified that conclusion.

Even in the absence of a full understanding of the cellular and molecular mechanisms that lead to the disease, several notable reviews (Panteleyev and Bickers, 2006; Sweeney and Mocarelli, 2000) have deemed the clinical and epidemiologic evidence of dioxin-induced chloracne to be strong. The occupational epidemiologic literature has many examples of chloracne in workers after reported industrial exposures (Beck et al., 1989; Bond et al., 1987, 1989a,b; Cook et al., 1980; Goldman, 1972; May, 1973, 1982; Oliver, 1975; Pazderova-Vejlupkova et al., 1981; Poland et al., 1971; Suskind and Hertzberg, 1984; Suskind et al., 1953; Zober et al., 1990). With relative-risk estimates as high as 5.5 in exposed

workers compared with referent non-exposed workers, Bond et al. (1989a) identified a dose–response relationship between probable exposure to TCDD and chloracne. Not everyone exposed to relatively high doses develops chloracne, and some with lower exposure may acquire it (Beck et al., 1989).

Almost 200 cases of chloracne were recorded in those residing in the vicinity of the accidental industrial release of dioxin in Seveso. Most cases occurred in children, particularly those who lived in the highest-exposure zone, and most cases resolved within 7 years (Assennato et al., 1989a,b; Caramaschi et al., 1981; Mocarelli et al., 1991). No cases of chloracne were identified in conjunction with the non-extreme environmental dioxin contamination at Times Beach, Missouri (Webb et al., 1987).

Exposures of Vietnam veterans were substantially lower than those observed in occupational studies and in environmental disasters, such as the one in Seveso. The long period since the putative exposure has imposed methodologic limitations on studies of Vietnam cohorts for chloracne. Nonetheless, the Vietnam Experience Study (CDC, 1988) found that chloracne was self-reported more often by Vietnam veterans than by Vietnam-era veterans (odds ratio [OR] = 3.9). An excess incidence was also found in Vietnam versus era veterans among subjects who were physically examined (OR = 7.3). In comparison with a non-exposed group, Air Force Ranch Hand personnel potentially exposed to Agent Orange reported a significant excess of acne (OR = 1.6) (Wolfe et al., 1990), but no cases of chloracne or postinflammatory scars were found on physical examination 20 years after possible herbicide exposure (AFHS, 1991b).

Biologic Plausibility

Previous updates have reported that chloracne-like skin lesions have been observed in several animal species in response to exposure to TCDD but not to purified phenoxy herbicides. Data accruing over the past several decades demonstrated that TCDD alters the differentiation of human keratinocytes, and more recent studies have illuminated how. Geusau et al. (2005) found that TCDD accelerates the events associated with early differentiation but also obstructs the completion of differentiation. Panteleyev and Bickers (2006) proposed that the major mechanism of TCDD induction of chloracne is activation of the stem cells in the basal layer of the skin to differentiate and inhibition of their ability to commit fully to a differentiated status. Ikuta et al. (2010) have investigated the expression of B-lymphocyte maturation protein 1 in epidermal keratinocytes and sebocytes in mice after induction of the aryl hydrocarbon receptor (AHR). Using a constitutively activated form of the AHR, Tauchi et al. (2005) demonstrated that TCDD-mediated chloracne involves inflammatory processes, which was confirmed by a recent finding that reactive oxygen species (ROS) can accelerate keratinocyte differentiation (Kennedy et al., 2012). Recent studies using an in vitro model of normal human epidermal keratinocytes confirmed the involvement

of the AHR in chloracne development (Forrester et al., 2014). The data support a biologically plausible mechanism for the induction of chloracne by TCDD.

Synthesis

No epidemiologic data in the past decade have refuted the conclusion of prior VAO committees that the evidence of an association between exposure to dioxin and chloracne is sufficient. The 2004 poisoning case of Ukrainian politician Victor Yushchenko has provided a high-profile instance that supports the idea that this condition can be a response to high-level exposure to TCDD, and the careful monitoring of his case has demonstrated the course of chloracne’s resolution in conjunction with subsiding serum concentrations (Sorg et al., 2009). The formation of chloracne lesions after the administration of TCDD has been observed in some species of laboratory animals.

Conclusion

On the basis of numerous epidemiologic studies of occupationally and environmentally exposed populations and supportive toxicologic information, the committee for Update 2014 concurs with all previous VAO committees that there is sufficient evidence of an association between exposure to at least one chemical of interest and chloracne. Because TCDD-associated chloracne becomes evident shortly after exposure, there is no risk of new cases long after service in Vietnam.

PORPHYRIA CUTANEA TARDA (PCT)

Porphyrias are uncommon disorders caused by deficiencies of enzymes involved in the pathway of biosynthesis of heme, the iron-containing nonprotein portion of the hemoglobin molecule. PCT, the most common of the porphyrias, is a heterogeneous group of disorders caused by a deficiency of a specific enzyme, uroporphyrinogen decarboxylase. It can be inherited but usually is acquired. Type I PCT, which accounts for 80 to 90 percent of all cases, is an acquired disease that typically becomes evident in adulthood. It can occur spontaneously but usually occurs in conjunction with environmental factors, such as alcohol consumption, exposure to estrogens, or use of some medications.

The most important clinical finding in PCT is cutaneous photosensitivity. Sensitivity to sunlight is thought to result from the excitation of excess porphyrins in the skin by long-wave ultraviolet radiation, which leads to cell damage. Fluid-filled vesicles and bullae develop on sun-exposed areas of the face and on the dorsal surfaces of the hands, feet, forearms, and legs. Other features include hypertrichosis (excess hair) and hyperpigmentation (increased pigment), especially on the face. People with PCT have increased porphyrins in the liver, plasma, urine, and stools. Iron, estrogens, alcohol, viral hepatitis, and chlorinated

hydrocarbons can aggravate the disorder. Iron overload is almost always present in people who have PCT.

Conclusions from VAO and Previous Updates

On the basis of strong animal studies and case reports demonstrating TCDD-induced PCT and resolution after cessation of exposure, the committee responsible for VAO determined that there was sufficient evidence of an association between exposure to TCDD and PCT in genetically susceptible people.

Epidemiologic studies of occupational populations have indicated inconsistent associations between the chemicals of interest and increased urinary uroporphyrin. Bleiberg et al. (1964) reported increased urinary uroporphyrin in 11 of 29 workers in a factory that manufactured 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), with the manifestation of some clinical evidence of PCT in three of them. In a follow-up study of the same facility 6 years later, no abnormalities in urinary porphyrins were observed (Poland et al., 1971). Calvert et al. (1992) reported no difference in porphyrinuria or the occurrence of PCT between 281 workers in the National Institute for Occupational Safety and Health (NIOSH) cohort who were involved in the production of trichlorophenol and were exposed to TCDD and 260 non-exposed workers. Serum TCDD concentration was not associated with uroporphyrin or coproporphyrin concentrations.

Among people who were exposed to TCDD as a result of the 1976 chemical-plant explosion in Seveso, Italy, clinical PCT was observed only in a brother and a sister who had a mutant enzyme that confers susceptibility in the heterozygous state. In 1977, 60 Seveso residents were tested for increased porphyrins, and 13 had secondary coproporphyrinuria; increased concentrations persisted in only three cases, and this was thought to be due to liver damage and alcohol consumption (Doss et al., 1984). In the Quail Run mobile-home park in Missouri, residents exposed to dioxin as a result of the spraying of waste oil contaminated with TCDD were found to have higher urinary uroporphyrins than controls, but no cases of clinical PCT were diagnosed (Hoffman et al., 1986; Stehr-Green et al., 1987).

The baseline study of the US Air Force Ranch Hands (AFHS, 1984) showed no difference in uroporphyrin or coproporphyrin concentrations in urine between Ranch Hands and controls. There were no indications of the clinical appearance of PCT in Ranch Hands. Follow-up studies of the Ranch Hand cohort revealed that mean uroporphyrin was greater in the comparison group than in the Ranch Hands, whereas mean coproporphyrin was higher in Ranch Hands. The clinical significance of the small differences between the Ranch Hands and the comparison groups was uncertain.

The committee responsible for Update 1996 considered three additional non-positive citations of populations that had substantial exposure to TCDD. Jung et

al. (1994) presented porphyrin data on former workers in a German pesticide plant that had manufactured 2,4-D and 2,4,5-T. Of 170 men tested, 27 had present or past chloracne. The study found no difference in porphyrin concentrations between subjects with and without chloracne. There was also no relationship between abnormal results of liver-function tests or porphyrin concentrations and the presence of chloracne. In addition, there was no relationship between porphyrin concentrations in urine, red blood cells, or plasma and TCDD concentrations in adipose tissue. Three cases of chronic hepatic porphyria (none with overt PCT and none with chloracne) were identified—a number that did not exceed the expected prevalence in this population. Von Benner et al. (1994) found no indication of clinical porphyria in self-referred workers in six other German chemical plants. Another report on the NIOSH cohort (Calvert et al., 1994) was negative. On the basis of the cumulative findings, the committee responsible for Update 1996 concluded that there was only limited or suggestive evidence of an association. The committees for later updates have not changed the revised conclusion.

Because PCT is manifested shortly after exposure to TCDD, new cases of PCT attributable to exposure during the Vietnam War are not expected to occur.

Biologic Plausibility

PCT has not been exactly replicated in animal studies of the effects of TCDD although other porphyrin abnormalities have been reported. Administration of TCDD to mice results in an accumulation of uroporphyrin that occurs in a manner that requires the AHR, cytochrome P450 1A1 (CYP1A1), and CYP1A2 (Robinson et al., 2002; Smith et al., 2001; Uno et al., 2004), but the underlying mechanism of action has not been fully illuminated (Smith and Chernova, 2009; Smith and Elder, 2010). In a recent study, Chavan and Krishnamurthy (2012) treated human liver–derived cell lines with TCDD, showing that this activated ABCB6 expression, an important requirement to support the increased heme demand that occurs when animals are exposed to xenobiotics.

Synthesis

No epidemiologic data have emerged in the past decade that refute the conclusion of previous VAO committees that there is limited or suggestive evidence of an association between the chemicals of interest and PCT.

Conclusion

On the basis of the evidence reviewed here and in previous VAO reports, the committee for Update 2014 concurs with prior VAO committees that there is limited or suggestive evidence of an association between exposure to at least one chemical of interest and PCT. PCT is rare, and its occurrence may be influenced

by a genetic predisposition in people who have low concentrations of protoporphyrinogen decarboxylase.

EARLY-ONSET PERIPHERAL NEUROPATHY

Since Update 1996, VAO committees have partitioned their consideration of peripheral neuropathy into early-onset (implicitly transient) peripheral neuropathy and chronic peripheral neuropathy. Primarily on the basis of reports of short-term health effects after industrial accidents, the committee responsible for Update 1996 concluded that there was limited or suggestive evidence of an association between exposure to the chemicals of interest and “acute and subacute” neuropathy, which was redesignated early-onset transient peripheral neuropathy by the committee responsible for Update 2004. The committee for Update 2010 recognized the imprecision in the nomenclature that had been used to characterize the type of peripheral neuropathy that is regarded as service-connected. The diagnosis in question is, in fact, contingent on the proximity of its occurrence to the time of exposure rather than on the transitory nature of the adverse outcome. Clinically, in cases of an immediate response of peripheral neuropathy after toxicant exposure, stabilization or improvement is the rule after the exposure ends. However, the recovery may not be complete; the degree of recovery can depend on the severity of the initial deficits and the particular exposure. Furthermore, there is a possibility of “subclinical” effects, and a person might be unaware of symptoms, although evidence of nerve dysfunction can be found through a detailed neurologic examination or electrodiagnostic testing. Thus, the committee chose to delete the word transient to recognize that symptoms of early-onset peripheral neuropathy may be protracted and that recovery from them may be incomplete.

The information about peripheral neuropathy presented in this appendix demonstrates that it may occur soon after an exposure to extremely high concentrations of dioxin. In addition, this appendix addresses the evidence that some people who experience early-onset peripheral neuropathy (that is, during or shortly after dioxin exposure) may continue to manifest the problem long after the exposure has ceased; this would show that early-onset peripheral neuropathy is not necessarily transient.

Conclusions from VAO and Previous Updates

Several occupational studies have evaluated whether herbicide exposure or production may lead to early-onset neuropathy. In March 1949, an explosion occurred in a reactor vessel in a chemical plant in Nitro, West Virginia, where 2,4,5-T was being produced. Many workers reported health effects (toxic hepatitis, increased serum lipids, and central nervous system involvement), including a severe acute neuropathy in four workers who had chloracne (Ashe and Suskind,

1949, 1950). Thirty years later, an attempt was made to identify workers who had been exposed during that accident and workers who may have been chronically exposed from 1948 through 1969 (Suskind and Hertzberg, 1984). Neurologic examination and nerve-conduction studies did not demonstrate abnormalities compared with a cohort of non-exposed controls; however, the procedure for obtaining controls did not ensure equivalence. It is unclear whether the four subjects who had acute neuropathy were included in the effort.

In April 1979, chlorinated dibenzo-p-dioxin contamination was found in a community in Arkansas that was close to a plant where 2,4,5-T and 2,4-D had been produced since 1957. Fifty-five workers in that plant who had no history of diabetes or alcohol abuse were identified from the total workforce; they underwent neurologic examination and nerve-conduction studies (Singer et al., 1982). Both median motor and calf sensory nerve-conduction studies showed significantly lower conduction velocity in the plant workers than in control subjects.

Other industrial accidents have also suggested a link between the chemicals of interest and early-onset neuropathic symptoms, which persisted in some people. Jirasek et al. (1974) studied 55 of 80 workers who complained of a variety of symptoms after chronic exposure to 2,4,5-T in a manufacturing facility in the Czech Republic; of the 55, the results of physical examinations of 17 suggested neuropathy that was said to have been confirmed with electromyographic abnormalities. A follow-up of 44 of the 55 was conducted 10 years after the exposure had ceased; about 30 percent of them were reported to have continued neuropathic symptoms (Pazderova-Vejlupkova et al., 1981). More recently, Urban et al. (2007) evaluated long-term sequelae in subjects who developed neuropathy after the original exposure. Subjects had increased serum TCDD concentrations more than 30 years after exposure, and evidence of continued neuropathy was noted in 9 of 15 subjects who were available for study.

Acute neuropathic symptoms were reported after the Seveso accident, and persistent signs were noted. Gilioli et al. (1979) evaluated 35 subjects who had been exposed during the accident and noted abnormalities in a variety of neurophysiologic measures compared with age-matched controls 2 years after the exposure. However, it is unclear how the exposed subjects were selected for study. In a more complete survey, Boeri et al. (1978) studied 470 subjects from two exposure zones about a year after the accident and found a higher incidence of neurophysiologic abnormalities in the exposed subjects than in non-exposed controls; the residents of the zone of greater exposure were more severely affected than those of the less exposed zone. The same group (Filippini et al., 1981) found an increased prevalence of peripheral neuropathy in residents who had indicators of exposure compared with those who did not (relative risk [RR] = 2.6, 95% confidence interval [CI] 1.0–7.2 for those with choracne; RR = 3.6, 95% CI 1.3–10.2, for those with increased hepatic enzymes) when they were evaluated 21 months after the accident. Improvement may have occurred in the years following the accident. For example, Assennato et al. (1989a,b) studied 193 exposed

residents of the area 9 years after the accident and did not find neurophysiologic abnormalities; however, the number of residents in the group who originally complained of neuropathic symptoms was not discussed. Similarly, 6 years after the accident, Barbieri et al. (1988) examined 153 residents who had developed chloracne. World Health Organization criteria for neuropathy were not fulfilled for any subjects, but there was a statistically significant increase in neurophysiologic abnormalities compared with those in non-exposed age-matched controls.

There have been a number of case reports of exposure-associated early-onset neuropathy. Goldstein et al. (1959) reported the cases of three patients seen at the Mayo Clinic who had acute weakness and sensory loss demonstrated to be due to peripheral neuropathy; symptoms occurred within hours of an exposure to 2,4-D sufficient to wet both clothes and skin. The three patients recovered incompletely; in one, a cerebrospinal fluid (CSF) examination was normal except for minimally increased protein. Todd (1962) reported another case of neuropathy that occurred about 4 days after 2,4-D exposure, again in sufficient quantities to cause large areas of skin to be wet. Clinical examination demonstrated a sensory motor poly-neuropathy; CSF examination showed slightly increased protein but was otherwise normal. The patient recovered substantially but not completely over 2 years. Finally, Berkley and Magee (1963) reported a case of a 39-year-old man who had symptoms of acute neuropathy that progressed to an inability to walk starting 4 days after 2,4-D exposure; CSF analysis was normal, including normal protein concentrations, and he recovered nearly completely over the course of 8 months.

Case reports do not provide conclusive evidence of causal relationships, but the cases discussed above showed a close temporal relationship between a high exposure to 2,4-D and neuropathy. The most likely non-toxicant-related acute neuropathy is Guillain-Barré syndrome; however, this syndrome is associated with characteristic findings on clinical neurophysiologic examination and highly increased protein in CSF. In the three cases above that had CSF evaluation, protein concentrations were either normal or increased to a minimal extent not consistent with Guillain-Barré syndrome. In addition, patients who had clinical neurophysiologic studies also showed abnormalities not consistent with Guillain-Barré. Thus, it seems likely that the cases represent the results of 2,4-D exposure.

Biologic Plausibility

Neuronal cell cultures treated with 2,4-D showed a decreased neurite extension associated with intracellular changes, including a decrease in microtubules, inhibition of the polymerization of tubulin, disorganization of the Golgi apparatus, and inhibition of ganglioside synthesis (Rosso et al., 2000a,b). Those mechanisms are important for maintaining synaptic connections between nerve cells and supporting the mechanisms involved in axon regeneration during recovery from peripheral neuropathy. Grahmann et al. (1993) and Grehl et al. (1993) reported observing, respectively, electrophysiologic and pathologic abnormalities in the

peripheral nerves of rats treated with TCDD. When the animals were sacrificed 8 months after exposure, there was pathologic evidence of axonal nerve damage and histologic findings typical of toxicant-induced injury. Those results constitute evidence of the biologic plausibility of an association between exposure to the chemicals of interest and peripheral neuropathy.

Conclusions

On the basis of studies of health effects after industrial accidents and the well-documented cases reported above, the current committee reaffirms the conclusion of VAO committees since Update 1996 that there is limited or suggestive evidence of an association between exposure to the chemicals of interest and early-onset peripheral neuropathy. Inasmuch as new data on this subject, especially with regard to Vietnam veterans, are unlikely to emerge, the committee for Update 2014 reaffirms that finding.

REFERENCES

The Appendix B references can be found in the References chapter.