Results

We identified 1567 people who were potentially exposed to 2,4-D between 1945 and 1994, inclusive (table 1). One subject died outside the United States. Death certificates were obtained for all remaining deaths. Most of the cohort was first exposed before the age of 35. Only 50 women were in the cohort. All women were living at the end of 1994, except for one who died from external causes. Due to the few women, the limited number of person-years at risk, and the single death due to causes unrelated to exposure to 2,4-D, detailed statistical analyses were confined to men.

Included were 863 male subjects from the original 2,4-D cohort, although previous publications of the cohort have reported on 878 subjects. Excluded were 14 women and three part time workers (two of whom were also women). Part time workers do not meet the criteria for entry into the current epidemiology database. An additional 405 employees were identified who would have been eligible for the original cohort, if the work history records had been computerised at the time. Most of these subjects were potentially exposed for less than a year with resulting very low and low cumulative dose indices (table 2). Also new to the updated cohort were 249 men who began working in 2,4-D production areas since 1983. These jobs were classified as low or very low exposure.

The resulting cohort under study comprised 1517 male employees who contributed 39 799 person-years at risk for an average follow up of 26.2 years. There were 330 deaths, representing an additional 198 deaths since the previous update. The results of the life table analyses are presented in table 3. Fewer deaths than expected were found due to all causes and due to all malignant neoplasms. A significantly decreased SMR was found for non-malignant respiratory diseases for zero years latency (SMR 0.35, 95% CI 0.15 to 0.68) and for 20 years latency (SMR 0.38, 95% CI 0.15 to 0.77). No significantly increased SMR was found for any of the underlying causes of death categories analysed. No cases of soft tissue sarcoma were found upon a review of the death certificates.

The three deaths due to NHL in this cohort were equivalent to the number of expected NHL deaths in the United States (SMR 1.00, 95% CI 0.21 to 2.92). All three subjects were first employed before 1965. However, one was not identified on the employee rosters used to create the original cohort. This employee was employed in the 2,4-D plant for less than 5 months during 1955 in a low exposure job and died 23 years later. The other two deaths occurred a relatively short time after first exposure (within 3 and 11 years). Due to their short time between date of recruitment in a 2,4-D job and date of death, they were excluded from the 20 year latency analyses.

The results of the internal analyses are presented in table 4. There were 40 600 unexposed manufacturing workers in the comparison group with 14 516 deaths and more than one million person-years. These analyses have the advantage of a comparison population similar to the exposed cohort as well as separating the RR by dose level. The RR was slightly higher than the corresponding SMR for all categories. The overall RRs for deaths due to all causes and all cancer were 1.10 (95% CI 0.98 to 1.22) and 1.05 (95% CI 0.85 to 1.30), respectively.

Expected rates for NHL (ICD 200 and 202) were computed with the internal Dow Chemical population. The resulting pooled RR for NHL was 2.63 (95% CI 0.85 to 8.33). Two of the deaths due to NHL occurred in men with moderate cumulative exposure, suggestive of a dose response. However, due to the short period between first exposure and death, all deaths were classified with very low cumulative exposure after lagging the exposure by 20 years.

Closer inspection of individual death certificates showed that three of the six deaths from diseases of nervous and sensory organs were attributed to amyotrophic lateral sclerosis (ALS). The RR was significantly increased when we limited the analyses to deaths due to ALS (RR 3.45, 95% CI 1.10 to 11.11). Lagged analyses were similar as all three employees died more than 20 years after their first exposure. The cases were employed in the manufacture or formulation of 2,4-D at different periods (1947–9, 1950–1, and 1968–86), and for varying durations of time (1.3, 1.8, and 12.5 years).

Discussion

The previous Dow Chemical 2,4-D cohort studies have been criticised for their small sample size. Indeed, for rare diseases such as NHL, it is difficult to identify any cohort sufficiently large to attain a great deal of statistical power, especially one that would have the nearly complete follow up of this research. This update of the Dow Chemical 2,4-D cohort provided 8 additional years of follow up data for the original cohort and nearly 20 000 person-years. The cohort was expanded by more than 600 employees and 198 additional deaths were observed. We found no increased risk of NHL compared with the United States population. When compared to the other Midland Dow Chemical employees, the relative risk was increased, but with a wide confidence interval (95% CI 0.85 to 8.33). We cannot explain why the expected number of deaths attributed to NHL is higher in the United States than in our local comparison population. The three employees who died of NHL were occupationally exposed to 2,4-D for only 4.1, 4.7, and 31.2 months. None of the workers held the same type of job, and the last death due to NHL occurred in 1979. These exposure characteristics are not indicative of a causal association of 2,4-D and NHL. This is consistent with the lifetime studies in the mouse, rat, and dog that have found no oncogenic response to 2,4-D.29 30

Cancers such as NHL are not uncommonly found among patients with immunodeficiencies, particularly in patients infected with human immunodeficiency virus (HIV). Some have suggested that immunotoxicity may be the mechanism by which some pesticides may be carcinogenic.31 32 The supporting evidence is inconclusive. Immunosuppressive effects in mice were found after acute high doses of n-butylester of 2,4-D, although the authors, Blakley and Schiefer, concluded that “it is unlikely that 2,4-D esters will have any major immunotoxicological significance in agricultural communities.”33 Two other mouse studies found immunostimulatory effects and no effects.34 35 The reason for the disparate findings of the three studies has not been clarified. Results of subchronic and chronic toxicology studies in the dog have found that 2,4-D is not immunotoxic.30

Contributing to the debate is a human study of 10 farmers for which blood samples were collected before and after application of 2,4-D and another phenoxy herbicide.36 Based upon concentrations of lymphocyte subsets—for example, circulating T helper cells, natural killer cells, etc—Faustini et al concluded that the participants experienced immunosuppressive effects due to exposure to phenoxy herbicides. No external control group was investigated. A control group—such as farmers who were merely cultivating—is important as exposure to sunlight may37 or may not38 39 be immunosuppressive.

If 2,4-D were immunosuppressive, not only an excess of lymphatic and haematopoietic deaths, but also an excess of infectious and opportunistic diseases would be expected. In the current study, we found no significant excess of deaths due to infectious and parasitic diseases (SMR 1.19, 95% CI 0.38 to 2.77), pneumonia (SMR 0.56, 95% CI 0.15 to 1.43), or lymphatic and haematopoietic cancer (SMR 1.06, 95% CI 0.48 to 2.01).

A non-significant increased SMR was found for ill defined cancers. This increase was similarly found in the previous Dow Chemical studies.21 22 All other and ill defined cancers had unspecified sites. By definition, these cancers are difficult to interpret as a group because the site of origin is unknown.

An unexpected significant excess of deaths attributed to ALS was found. Increased mortality from ALS was not an initial hypothesis in this study, as extensive state of the art animal toxicology studies have shown no evidence of 2,4-D toxicity to the nervous system,40 nor have other cohort studies of people with exposure to 2,4-D reported increased rates of ALS.11 19The disease ALS is a largely fatal neurological disease with an unknown aetiology. A plethora of risk factors from heavy metals, trauma, and occupation, to physical activity have been investigated, but only sex (male) and genetic predisposition seem to be consistently found.41 42 In the context of 2,4-D, exposure to pesticides and agricultural chemicals have shown no significant association in several studies.43-45 A significant association of exposure to pesticides was found in a recent case-control study of ALS.46 However, the investigators did not find a significant association of exposure to herbicides.

The human health significance of the current ALS finding is unknown. Whether the significant RR of ALS found is a true or false positive must be questioned. The molecule 2,4-D does not bioaccumulate, is rapidly cleared from the body, and is excreted unchanged within 48 hours. The paucity of confirming animal or human data on neurotoxicity of 2,4-D and the lack of any association of herbicides with ALS suggests that the three cases of ALS in this cohort are unrelated to exposure to 2,4-D.

Overall this update was limited by the unavailability of potentially confounding lifestyle factors such as diet and smoking. The study is also limited by the end point of mortality. Mortality is greatly influenced by the stage of disease at diagnosis and the quality of treatment, which may in turn be influenced by social factors such as education, access to health care, and membership to a health insurance plan. Incidence studies, however, are also problematic because there is not a national tumour registry system in the United States. The major limitation of these studies, as noted by other Dow Chemical investigators, is finding the residential data for employees that have left the company.47 48

Conversely, the current study had several strengths. Follow up of the cohort was nearly 100% complete. We were also able to use an internal comparison population. National mortalities, although more stable, may introduce bias because workers must initially be healthy to gain employment and having access to health insurance may reduce early mortality. Another strength of the current cohort is that potential exposure to 2,4-D has been estimated from industrial hygiene monitoring data. As well as objective exposure data, the cohort members were potentially exposed to 2,4-D on a daily basis. This contrasts with farmers who typically only apply the herbicide a few times each year and then may apply a myriad of other herbicides, insecticides, and fungicides over their lifetime. We were also not reliant upon the memory of the cohort members to estimate exposure.

Conclusion

In summary, this study represents an updated cohort of workers potentially exposed to 2,4-D. In general, we found no evidence of an increased risk of death due to all causes or total malignant neoplasms. No significant risk of NHL was found. A significantly reduced SMR was found for non-malignant respiratory diseases. A higher than expected number of cases of ALS was found. This finding is not consistent with previous human or animal studies and may be unrelated to exposure to 2,4-D .