A Study of the Potential for a Herbicide Formulation Containing 2,4-D and Picloram to Cause Male-Mediated Developmental Toxicity in Rats

D. J. Oakes*, W. S. Webster*,1, P. D. C. Brown-Woodman{dagger} and H. E. Ritchie{dagger}

* Department of Anatomy and Histology, University of Sydney, Sydney, NSW 2006, Australia; and {dagger} School of Biomedical Sciences, Faculty of Health Sciences, University of Sydney, Lidcombe, NSW 2141, Australia

Received November 15, 2001; accepted March 6, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Male Vietnam veterans have repeatedly expressed concern that exposure to herbicides in Vietnam may have caused birth defects in their offspring. The second most used herbicide was a mixture of 2,4-D and picloram called Agent White. This study is an investigation into the possible male-mediated reproductive toxicology of this herbicide. Male rats were gavaged for 5 days per week for 9 weeks with a mixture of 2,4-D and picloram called Tordon 75D® (the Australian derivative of Agent White). Three doses were tested; the high dose was considered the maximum tolerated dose. Each male was mated with two untreated females during weeks 2 and 3, 4 and 5, and 8 and 9 of treatment, and with four untreated females after an 11-week recovery period. Negative controls were males dosed with distilled water, and positive controls were males dosed with cyclophosphamide at 5.1 mg/kg/day. All mated females were killed on day 20 of gestation, and the fetuses were weighed and examined for either structural malformations or skeletal development. Litter size, fetal weight, and malformation rate were all unaffected by treatment. The cyclophosphamide positive controls showed the expected large increase in postimplantation loss. In general, within the limitations of the power of the study, the results did not show any evidence that exposure to a herbicide formulation containing 2,4-D and picloram is likely to cause male-mediated birth defects or other adverse reproductive outcomes.

Key Words: 2,4-D; picloram; Agent White; reproductive effects; rats.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There have been repeated claims that the men who served in the Vietnam War were exposed to a wide range of toxic chemicals and that as a consequence they now have an increased incidence of adverse health effects, including more birth defects in their offspring. Four major epidemiological studies have been undertaken to investigate this latter claim, but they did not find a positive relationship (CDC, 1988Go; Donovan et al., 1984Go; Erickson et al., 1984Go; Wolfe et al., 1995Go). The issue was revisited in 1996 when the U.S. government accepted evidence that Vietnam veterans had an increased risk of fathering children with spina bifida (U.S. Govt., 1996; Wolfe et al., 1995Go). More recently, an Australian investigation into the health of male Vietnam veterans and their children has also reported a significant excess of birth defects (AIHW, 1999; Australian Government, 1998).

These reports of increased numbers of birth defects in veterans' children would gain credibility if it were possible to demonstrate that one or more of the chemicals used in the Vietnam War could cause male-mediated developmental toxicity. For a number of years, the most suspect chemical was dioxin (TCDD), a contaminant of Agent Orange, the most commonly used herbicide in the Vietnam War. However, despite extensive studies, there is no evidence that dioxin can cause mutagenic changes to the testicular germ cells that would result in an increased incidence of birth defects in the offspring (Chahoud et al., 1989Go; Khera and Ruddick, 1973Go). Similarly, the two active herbicides in Agent Orange, 2,4-D and 2,4,5-T, do not show mutagenic activity (IOM, 1996Go; Lamb et al., 1981Go).

In 1996, the Department of Veterans' Affairs in Australia became aware of a report that a mixture of two herbicides, picloram and 2,4-D, when fed to male mice resulted in an increased incidence of birth defects and fetal growth retardation in their offspring (Blakley et al., 1989Go). The study had its limitations because the doses used were very high and the number of animals used was low. However, the study was of interest, as the two herbicides were also used in Vietnam, in a mixture code-named Agent White. Agent White was the second most used herbicide in Vietnam, with over 20 million liters sprayed (IOM, 1996Go). The Department of Veterans' Affairs in Australia subsequently commissioned a further animal study to determine whether Agent White fed to male rats would result in an increased incidence of birth defects in the offspring.

The first obstacle in planning the study was that the precise formula for Agent White is unknown. Agent White was the military code name for Tordon 101®, a commercial herbicide used in the United States in the 1960s. The active herbicides in the mixture were picloram and 2,4-D (Table 1Go). What remains unknown is what other chemicals were included. Typically, herbicides contain surfactants to aid penetration of foliage, antifoaming agents, and solvents, but these details are not available for Agent White. As we were unable to duplicate the exact formula for Agent White because of the unavailability of published details, it was decided instead to use a herbicide mixture known as Tordon 75D®. This herbicide mixture contains the same two active ingredients as Agent White. The manufacturer, Dow Agrosciences, Australia, revealed the other so-called inert components on a confidential basis. The use of Tordon 75D® made the experiments relevant to both the Vietnam veterans and the contemporary Australian farmer. As can be seen from Table 1Go, the composition of Agent White, Tordon 101®, and Tordon 75D® is very similar.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Details of Agent White and Related Herbicide Formulations Containing 2,4-D and Picloram
 
In the original study by Blakley and colleagues (1989), male mice were treated with Tordon 202c®, another 2,4-D/picloram mixture (Table 1Go). The male mice were treated for 60 days (the entire period of spermatogenesis) and then mated to untreated females. This pattern of treating and mating meant that it was not possible to determine if any adverse effects were due to damage to the spermatogonial cells (which is of great importance because it represents permanent damage to the male genome) or due to damage to sperm undergoing maturation (which is much less significant as it does not represent permanent damage; all abnormal sperm should disappear about 60 days after cessation of treatment). In the present study, male rats were treated with Tordon 75D® for a period of 9 weeks, during which time they were mated at 2- to 3-week intervals with untreated females. This permitted the effects of Tordon 75D® on the various stages of sperm development to be examined. At the end of the 9-week treatment period, the male rats were untreated for a further 11 weeks, after which they were again mated with untreated females. This final mating should detect any adverse effects associated with damage to the testicular stem cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Source and age of animals.
Twelve-week-old male Sprague-Dawley rats were purchased from Perth Animal Resource Centre (ARC), Perth, Western Australia. The animals were allowed 1 week to acclimatize in the new animal facility before beginning experimental procedures. Female rats were delivered as required so that whenever females were needed for mating they were 12 weeks of age and had been acclimatized in the animal house for 2 weeks. Because of the large number of animals involved, the entire experiment was performed over 2 years. The animals were treated and mated as described below during year 1, and the complete experiment was replicated during the following year (year 2).

Determination of male fertility.
Before starting treatment of male rats with Tordon 75D®, the fertility of the males was determined by mating each male with two untreated females over a 2-week period. The males were placed overnight with two females, which were checked the next morning for the presence of sperm in vaginal smears. The pregnant females were killed on day 20 of gestation, and the fetuses were examined as described below.

Chemicals used in the study.
Commercial-grade Tordon 75D® was purchased from Dow Agrosciences, Sydney, Australia. According to the material safety data sheet, it contained 2,4-D (300 g acid equivalents/l) and picloram (75 g acid equivalents/l), both present as their triisopropanolamine salts, as well as 300–600 g/l of surfactants, water, and inerts. Cyclophosphamide (CP) was purchased from Baxter Pharmaceuticals, Sydney, Australia in 100-ml plastic sealed syringes containing 60 ml of a 1.0-mg CP/ml distilled water solution.

Dosing with Tordon 75D®.
Extensive preliminary studies were performed with male rats to determine the maximum tolerated dose of Tordon 75D® that could be administered for 5 days per week for 9 weeks. The constraints imposed upon the study by the Animal Ethics Committee at the University of Sydney was that a 10% loss of body weight relative to controls during the treatment period was the maximum permitted.

Initially the Tordon 75D® was administered in drinking water, as this was the method of administration used by Blakley and colleagues in their mouse study. However, as the concentration of Tordon 75D® was increased in the drinking water, the amount of water consumed by the rats decreased, and this was associated with dramatic weight loss, perhaps due to dehydration. This form of dosing was eventually abandoned in favor of gavage, which allowed higher daily doses of Tordon to be administered. The maximum tolerated dose administered by gavage 5 days per week for 9 weeks was eventually determined to be 5 ml of 10% Tordon 75D®/kg body weight. This represented a dose of 150 mg/kg/day 2,4-D and 37.5 mg/kg/day picloram. As part of the experimental design, the male rats were dosed with this high dose (HD) or with a middle dose (MD = 50% of the HD) or a low dose (LD = 25% of the HD). The treated male rats were weighed on each treatment day and monitored for signs of toxicity on a daily basis.

Dosing with cyclophosphamide.
As a positive control, CP was administered to male rats 5 days per week for 9 weeks by gavage at a dose of 5.1 mg/kg body weight (5.1 ml of 1.0 mg/ml solution/kg body weight). This dose has previously been shown to have a mutagenic effect in male rats leading to an increased incidence of resorption and birth defects in mated untreated female rats (Jenkinson and Anderson, 1990Go; Trasler et al., 1985Go, 1986Go, 1987Go).

Treatment of males.
Males of proven fertility were acclimatized for 1 week to daily gavage with distilled water. At the end of this week, males (17 weeks old) were placed at random into one of five groups, five males per group. Each male was dosed daily by gavage five times each week (Monday to Friday) for a total of 9 weeks.

Group 1 was a negative control group (5 ml of distilled water/kg body weight).

Group 2 was LD Tordon group (5 ml of 2.5% Tordon 75D®/kg body weight).

Group 3 was MD Tordon group (5 ml of 5% Tordon 75D®/kg body weight).

Group 4 was HD Tordon group (5 ml of 10% Tordon 75D®/kg body weight).

Group 5 was positive control group treated with CP (5.1 mg/kg).

Mating regimen.
Each treated male was given the opportunity to mate with two untreated females as described above during weeks 2 and 3, weeks 4 and 5, and weeks 8 and 9 of treatment. The males were then allowed an 11-week recovery period, during which they were untreated. At the end of this recovery period, the males were given the opportunity to mate with four untreated females over a 2-week period.

F2 generation.
A small additional experiment was performed. Two control males, two high-dose males, and two CP-treated males were each mated with two untreated females, and the females were allowed to litter to give an F1 generation. The litters were allowed to grow to maturity, and the males from each litter were mated with the females from the other (same treatment) litter. These pregnant females were killed on day 20 of gestation, and the F2 generation was examined as described below.

Examination of the pregnant females and fetuses.
All mated females were killed on day 20 of gestation. The ovaries were removed and preserved in Bouins fluid for subsequent counting of corpora lutea. All live and dead fetuses and visible resorptions were recorded. The fetuses were weighed; fetuses with a weight that was two standard deviations less than the mean litter weight were designated as runts. After weighing, fetuses were placed in either Bouins fluid for subsequent examination by razor sectioning and dissection (as described by Wilson, 1965Go) or in 95% alcohol for subsequent staining with alizarin red and skeletal examination (Taylor, 1986Go).

Statistical analysis.
For the reproductive studies, the litter was considered the experimental unit. Data were analyzed by one-way analysis of variance with the Fisher exact test using Minitab Statistical Analyses software. Binomial distribution test was used to determine significance of sex ratio. The significance level used throughout was p < 0.05.

Serum levels of 2,4-D and picloram after single and repeated treatment with Tordon 75D®.
Twenty-one male rats (17 weeks old) were gavaged with 5 ml of 10% Tordon 75D®/kg. At various times posttreatment (7, 15, 30 min; 1, 2, 6, and 24 h), three rats per time interval were anesthetized with isoflurane and 10 ml of blood was collected from the abdominal aorta. The blood was immediately centrifuged at 3000 rpm, and the serum was collected and stored in the dark at –20°C. The rats were killed by anesthetic overdose and cervical dislocation. A further 21 male rats were dosed with the same high dose of Tordon 75D® for 5 days per week for 9 weeks and then bled after the final dose as described for the single-dose animals.

For the simultaneous HPLC determination of 2,4-D and picloram in the serum samples, the serum was acidified with methanol-HCl, and an internal standard of 2,4,5-T (20 µg/100 µl) was added. A supernatant was collected after two centrifugations at 11,000 x g for 5 min. The supernatant was injected onto a Hypersil ODS C-18 steel column with a mobile phase of methanol:distilled water (55:45) containing the ion-pairing reagent 0.005 M tetrabutylammoniumhydrogen sulfate. The column effluent was monitored by UV absorption at 231 nm. The retention times for picloram, 2,4-D, and 2,4,5-T were 4.6, 14.7, and 27.9 min, respectively. The peak area ratios of picloram or 2,4-D, relative to internal standard, were calculated for each standard extract and plotted against the concentration of picloram or 2,4-D to give a calibration curve that was linear over the range studied. After linear regression, the concentration of picloram and 2,4-D in each extract of rat serum was determined from the standard curve. The extraction recovery from serum was 83% and 77% for 2,4-D and picloram, respectively. The limit of detection was 1 µg/ml serum. Regression analysis was performed using Microsoft Excel. Pharmacokinetic parameters were determined using NCSS 2000 statistical software.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of the Tordon 75D® Dosing on the Male Rats
Generally, all the rats appeared healthy during the study. Immediately postdosing, some rats appeared lethargic, but recovered in about 1 h. The effect of 9 weeks chronic treatment with Tordon 75D® on the body weight of male rats is shown in Figure 1Go. The control males steadily gained weight during the 9-week treatment and the 12-week recovery period. The LD and MD animals showed a similar pattern of weight gain. In contrast, the HD animals had decreased weight gain during the 9-week treatment period compared with the controls (Fig. 1Go). The animals lost about 3% of their weight during the first 4 weeks of treatment, whereas the controls gained about 5%. In the last 5 weeks of the treatment period, weight gain for the HD animals was similar to that of the controls. In the 11-week posttreatment period, the HD males showed some catch-up, with their weight increasing 11% in the 6 weeks immediately after treatment, whereas the controls gained only 5%. At the end of the posttreatment recovery period, there were no significant weight differences between the groups.



View larger version (26K):
[in this window]
[in a new window]
 
FIG. 1. Effect of Tordon 75D® treatment on the body weight of male rats. Each point on the graph represents the mean of 10 rats.

 
Breeding Results
This main reproduction experiment was performed in two parts over 2 years. It was expected that the results of each part of the study would be approximately the same and that the data could be combined into a single group. A few differences did emerge; specifically, mean litter weight was reduced in 1999 by about 4% compared with 1998. This was seen in both control and treated groups and was therefore not considered an obstacle to combining the data (Table 2Go).


View this table:
[in this window]
[in a new window]
 
TABLE 2 Reproductive Outcome (F1 Generation) of Male Rats Treated by Gavage for 9 weeks (5 days per week) with Either Low-, Mid-, or High-Dose Tordon 75D® or 5.1 mg/kg Cyclophosphamide
 
Pretreatment matings.
All the male rats were tested for fertility before allocation to groups for dosing. This data set of 104 litters from untreated males and females provides baseline figures for subsequent comparison. There were only six major malformations, at an incidence of 0.52%.

Matings 2–3 weeks after commencement of dosing.
The three Tordon 75D® treatments did not have any effect on fertility, postimplantation loss, mean litter size or weight, number of runts, malformations, or skeletal variations. Preimplantation loss was increased to 15.2% in the HD group, but it was not significant compared with the control group. There was a significant increase (p < 0.05) in the percentage of hydronephrosis in the HD group (5.8%) compared with the control group (0.4%). For the CP group, there were significantly increased pre- and postimplantation losses, decreased mean litter size, and more female fetuses (Table 2Go).

Matings 4–5 weeks after commencement of dosing.
There were no significant differences between the control litters and Tordon 75D® groups. For the CP group, there was a marked increase in postimplantation loss (46.3%) compared with the controls (2.7%). This was accompanied by a marked reduction in litter size. There were some minor differences in skeletal parameters, with the number of forelimb metacarpal calcification centers increased compared with controls; this may reflect advanced development associated with small litter size.

Matings 8–9 weeks after commencement of dosing.
Again, there were mostly no differences between the control and Tordon 75D® groups. The MD group showed a small but significant reduction in litter size compared with the controls, and there was a reduced number of runts in the HD group. For the CP group, preimplantation loss was moderately elevated to 19.8% compared with 5.1% in the controls. Postimplantation loss was markedly increased to 60.7% compared with 2.5% in the controls, and the percentage of runts was significantly decreased compared with the controls.

Matings after the 11-week recovery period.
The three Tordon 75D® groups were not significantly different from the controls or from each other. The CP group had also returned to normal values.

F2 generation fetuses.
There were no significant differences between the control, CP, and HD Tordon 75D® groups (Table 3Go).


View this table:
[in this window]
[in a new window]
 
TABLE 3 Reproductive Outcome (F2 Generation) of the F1 Generation from F0 Male Rats Treated by Gavage for 9 Weeks (5 days per week) with Tordon 75D® (5 ml of 10% Tordon 75D®/kg weight) or Cyclophosphamide (5.1 mg/kg body weight)
 
Pharmacokinetic Results
Pharmacokinetic parameters were obtained for 2,4-D and picloram after single and multiple 9-week oral dosing with Tordon 75D® at HD. For 2,4-D, the single dose profile differed from that taken at the end of the chronic dosing (Fig. 2aGo). The maximum serum concentration (Cmax) was 249 ± 39 µg/ml at 2 h after the single dose and 284 ± 59 µg/ml at 6 h after chronic dosing. The areas under the curve (AUC0-24h) for the single and chronic doses were 2974 µg.h/ml and 4857 µg.h/ml, respectively. Plasma half-life was 6.1 and 5.7 h, respectively.



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 2. Concentration of (a) 2,4-D and (b) picloram in rat serum following oral administration by gavage (5 ml of 10% Tordon 75D®/kg body weight). Each value is the mean of three rats.

 
For picloram, the graphs looked more similar in shape (Fig. 2bGo). The Cmax was 17 ± 4.2 µg/ml at 0.5 h after a single dose and 20 ± 6.3 µg/ml at 1 h after chronic dosing. The AUC0-24h was 97 µg.h/ml for the single dose and 224 µg.h/ml for the chronic dosing.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Within the statistical limitations of this study, there is no indication that Tordon 75D® caused male-mediated developmental toxicity such as birth defects, resorptions, or fetal growth retardation. The incidence of birth defects in the control rats was only about 0.5%; this influenced the power of the study to detect increases in this particular parameter. With approximately 10 litters in each group and 120–140 fetuses, the power of the study (0.8) would only be sufficient to detect an 8-fold or greater increase in birth defects at p < 0.05.

The relatively low power of the study is partly counteracted by the high dose of Tordon 75D® administered. This dose was associated with weight loss during the first 4 weeks of dosing and was considered to be the maximum tolerated dose. As such, this dose should maximize any toxic effects on the testes. Despite the high dose, when these males were mated to untreated females at various times during and after dosing, the major parameters of reproductive outcome were almost all within the control range. This included postimplantation resorptions, litter size, mean litter weight, percentage of runts, malformations, and parameters of skeletal development.

A possible treatment-related effect was the increased preimplantation loss for HD Tordon 75D® after 2–3 and 4–5 weeks treatment. Rates of preimplantation loss for controls ranged from 7.9 to 11%, whereas for these two Tordon 75D® groups the rate was 15.2% and 16.2%, respectively. For all HD Tordon 75D® groups, mean litter size was less than controls, but these differences were not significant. There was no dose effect for these differences.

Serum 2,4-D and Picloram Levels in Humans
The HD of Tordon 75D® used in this study resulted in the male rats being exposed to daily Cmax values of 249–284 µg 2,4-D/ml serum and 17–20 µg picloram/ml serum. The serum half-lives were about 6 h for 2,4-D and 2–4 h for picloram. The serum levels of 2,4-D and picloram of men who were exposed to Agent White in Vietnam are unknown, of course. Commercial applicators and forestry workers using 2,4-D without protective precautions had estimated exposures ranging from 0.9 to 160 µg/kg/day (Draper and Street, 1982Go; Kolmodin-Hedman and Erne, 1980Go; Lavy et al., 1987Go; Nash et al., 1982Go). For a 70-kg man, this would represent exposure to 0.1–11 mg. In terms of estimated serum levels, men given an oral dose of 5 mg 2,4-D/kg (= 350 mg for a 70-kg man) had a Cmax of 40 µg/ml (Kohli et al., 1974Go). Hence, an 11-mg intake would give an estimated plasma level of 1 µg/ml. This level can be compared to the level of 284 µg/ml seen in the HD Tordon 75D® rats. Occupational exposure data is not available for picloram, but a 5-mg/kg oral dose in volunteer men produced plasma levels of 3.6 µg/ml (Nolan et al., 1984Go). This can be compared to picloram levels of 20 µg/ml in the HD rats. Hence, based on the 2,4-D data, it would appear that exposure in the HD rats was at least 200 times greater than the likely human exposure.

Metabolites of 2,4-D and picloram are generally not considered important toxicologically. For 2,4-D, metabolites other than conjugates have not been reported (Erne, 1966Go; Grunow and Bohme, 1974Go), and picloram is thought to be excreted unchanged (Nolan et al., 1984Go).

Cyclophosphamide was used in this study as a positive control. In general, the results obtained in the present study were comparable to other studies in which male rats were dosed at 5.1 mg/kg/day and then mated to untreated females (Jenkinson and Anderson, 1990Go; Trasler et al., 1985Go, 1986Go, 1987Go). In particular, the pattern of postimplantation loss was consistent with a dominant lethal effect. The small increase in malformations was also consistent with other studies. Increased numbers of small fetuses (runts) have also been associated with paternal treatment with CP, but there was no evidence of an increase in the present study.

The major aim of this study was to test the hypothesis that a herbicide mixture of 2,4-D and picloram, chronically administered to male rats, would lead to increased adverse reproductive effects when the males were mated with untreated females. Similar experiments in mice (Blakley et al., 1989Go) had shown decreased fetal weight and increased fetal abnormalities. The failure to confirm these results in rats may be due to a number of factors.

The main difference in the two studies was that mice were used in one study and rats in the other. There is no obvious reason why there should be a major species difference in the mutagenic response. The mice were exposed to Tordon 202C®, which is similar but not identical to Tordon 75D® (Table 1Go). The total daily intake of 2,4-D and picloram in the HD mice was 336 and 20 mg/kg, respectively, administered for 60 days, whereas in the rats the HD dose was 150 and 37.5 mg/kg, respectively, administered for 9 weeks.

Importantly, in the mouse study, the HD (0.84% Tordon 202C®) was greater than the LD50 for the male mice under the study conditions. Hence, this dose reduced the number of HD male mice available for mating from 10 to 4. Similarly the MD was approximately LD10. In comparison, the HD used in the rat study did not result in any deaths. A consequence of the HD used in the mouse study was that there were only four males to mate to untreated females, resulting in five litters. In the comparable part of the rat study, there were 16 litters. The small numbers in the mouse study make the results uncertain.

Two major adverse effects were seen in the mouse study. First, fetal weight in the HD group (1.08 ± 0.02 g) was significantly reduced compared with the controls (1.40 ± 0.03 g). Second, total abnormal fetuses were increased in a dose-dependent manner (controls, 0.6%; LD, 8.3%; MD, 10.8%; and HD, 19.2%). Neither of these effects was seen in the comparable parts of the rat study, which had much larger numbers of fetuses. The definition of total abnormal fetuses in the mouse study was unusual, as it included malformed fetuses and variants. There were very few malformed fetuses: none in the controls, 1 (0.6%) (ablepharon) in the LD group, 4 (2.4%) (one cleft palate, three agenesis of the testis) in the MD group, and 1 (1.7%) (agenesis of the testis) in the HD group. Fetuses with variants were more common: 1 (0.6%) in the controls, 12 (7.7%) in the LD group, 5 (6.1%) in the MD group, and 8 (17.6%) in the HD group. Variants were mainly extra ribs and decreased skeletal ossification.

However, the only really significant difference between the two studies was the use of a greater than LD50 dose in the mouse study. It is possible that if a male mouse is very sick (but still able to mate), the quality of the sperm/semen may be changed in some way to cause reduced fetal weight or abnormality. Reduced fetal weight is the most robust finding of Blakley and colleagues (1989), but because of the small numbers of animals, it may be a chance finding. However, if it is a real effect, it would not necessarily be unique to 2,4-D and picloram, but could presumably result from any substance that makes a male animal very sick.

This study has extended beyond a simple repeat of the mouse study by Blakley and colleagues (1989). The mating protocol allowed potential separation of the effects of Tordon 75D® on various stages of sperm development and the stem cell population of the testes. Breeding of the F2 generation potentially allowed detection of balanced translocations or abnormal genes of low penetrance (Hales et al., 1992Go). All of these studies with Tordon 75D® were negative and indicate, within the limitations of the power of the study, that herbicide mixtures of 2,4-D and picloram are unlikely to be male reproductive toxicants.


    ACKNOWLEDGMENTS
 
This project was supported by a grant from the Commonwealth Department for Veterans' Affairs. The authors gratefully acknowledge the support and cooperation of Dr. Keith Horsley (Department of Veterans' Affairs) and Dow Agrosciences, Australia. The authors also thank Ann Korabelnikoff for her excellent animal handling skills.


    NOTES
 
1 To whom correspondence should be addressed. Fax: 61 2 9351 6556. E-mail: billweb{at}anatomy.usyd.edu.au. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Australian Government. (1998). Morbidity of Vietnam Veterans—A Study of the Health of Australia's Vietnam Veterans Community—Volume 1. Male Vietnam Veterans Survey and Community Comparison Outcomes. Government Printing Services, Canberra, Australia.

Australian Institute of Health and Welfare (AIHW). (1999). A Study of the Health of Australia's Vietnam Veterans Community—Volume 3. Validation study. Government Printing Office, Canberra, Australia.

Blakley, P. M., Kim, J. S., and Firneisz, G. D. (1989). Effects of paternal subacute exposure to Tordon 202c on fetal growth and development in CD-1 mice. Teratology 39, 237–241.[ISI][Medline]

CDC (1988). Health status of Vietnam Veterans. III. Reproductive outcomes and child health. The Centers for Disease Control Vietnam Experience Study. JAMA 259, 2715–2719.[Abstract]

Chahoud, I., Krowke, R., Schimmel, A., Merker, H. J., and Neubert, D. (1989). Reproductive toxicology and pharmacokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin. 1. Effects of high doses on the fertility of male rats. Arch. Toxicol. 63, 432–439.[ISI][Medline]

Donovan, J. W., MacLennan, R., and Adena, M. (1984). Vietnam service and the risk of congenital anomalies. A case-control study. Med. J. Aust. 140, 394–397.[ISI][Medline]

Draper, W. M., and Street, J. C. (1982). Applicator exposure to 2,4-D, dicamba, and a dicamba isomer. J. Environ. Sci. Health B. 17, 321–339.[ISI][Medline]

Erickson, J. D., Mulinare, J., McClain, P. W., Fitch, T. G., James, L. M., McClearn, A. B., and Adams, M. J., Jr. (1984). Vietnam veterans' risks for fathering babies with birth defects. JAMA 252, 903–912.[Abstract]

Erne, K. (1966). Studies on the animal metabolism of phenoxyacetic herbicides. Acta Vet. Scand. 7, 264–271.[ISI][Medline]

Grunow, W., and Bohme, C. (1974). [Metabolism of 2.4.5-T and 2.4-D in rats and mice (author's transl.)]. [German]. Arch. Toxicol 32, 217–225.[ISI][Medline]

Hales, B. F., Crosman, K., and Robaire, B. (1992). Increased postimplantation loss and malformations among the F2 progeny of male rats chronically treated with cyclophosphamide. Teratology 45, 671–678.[ISI][Medline]

Institute of Medicine (IOM). (1996). Committee to Review the Health Effects on Vietnam Veterans of Exposure to Herbicides. Veterans and Agent Orange Update 1996. National Academy Press, Washington, DC.

Jenkinson, P. C., and Anderson, D. (1990). Malformed foetuses and karyotype abnormalities in the offspring of cyclophosphamide and allyl alcohol-treated male rats. Mutat. Res. 229, 173–184[ISI][Medline]

Khera, K. S., and Ruddick, J. A. (1973). Polychlorinated dibenzo-p-dioxins: Perinatal effects and the dominant lethal test in Wistar rats. Adv. Chem. 120, 70–84.

Kohli, J. D., Khanna, R. N., Gupta, B. N., Dhar, M. M., Tandon, J. S., and Sircar, K. P. (1974). Absorption and excretion of 2,4-dichlorophenoxyacetic acid in man. Xenobiotica 4, 97–100.[ISI][Medline]

Kolmodin-Hedman, B., and Erne, K. (1980). Estimation of occupational exposure to phenoxy acids (2,4-D and 2,4,5-T). Arch. Toxicol. Suppl. 4, 318–321.[Medline]

Lamb, J. C., IV, Moore, J. A., Marks, T. A., and Haseman, J. K. (1981). Development and viability of offspring of male mice treated with chlorinated phenoxy acids and 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Toxicol. Environ. Health 8, 835–844.[ISI][Medline]

Lavy, T. L., Norris, L. A., Mattice, J. D., and Marx, D. B. (1987). Exposure of forestry ground workers to 2,4-D, picloram and dichloroprop. Environ. Toxicol. 6, 209–224.

Nash, R. G., Kearney, C. R., Maitlen, J. C., Sel, C. R., and Fertig, S. N. (1982). Agricultural applicators exposure to 2,4-dichlorophenoxyacetic acid. In: Pesticide Residue and Exposure (J. R. Plimmer, Ed.). pp. 119–132. American Chemical Society, Washington, DC.

Nolan, R. J., Freshour, N. L., Kastl, P. E., and Saunders, J. H. (1984). Pharmacokinetics of picloram in male volunteers. Toxicol. Appl. Pharmacol. 76, 264–269.[ISI][Medline]

Taylor, P. (1986). Practical Teratology. Academic Press, London.

Trasler, J. M., Hales, B. F., and Robaire, B. (1985). Paternal cyclophosphamide treatment of rats causes fetal loss and malformations without affecting male fertility. Nature 316, 144–146.[ISI][Medline]

Trasler, J. M., Hales, B. F., and Robaire, B. (1986). Chronic low dose cyclophosphamide treatment of adult male rats: Effect on fertility, pregnancy outcome and progeny. Biol. Reprod. 34, 275–283.[Abstract]

Trasler, J. M., Hales, B. F., and Robaire, B. (1987). A time-course study of chronic paternal cyclophosphamide treatment in rats: Effects on pregnancy outcome and the male reproductive and hematologic systems. Biol. Reprod. 37, 317–326.[Abstract]

United States Government (U.S. Govt.). (1996). Agent Orange Benefits Act, 1996. Signed by U.S. President Clinton on 26 September, 1996.

Wilson, J. G. (1965). Embryological considerations in teratology. In: Teratology—Principles and Techniques. (J. G. Wilson and J. Warkany, Eds.), pp. 251–279. University of Chicago Press, Chicago.

Wolfe, W. H., Michalek, J. E., Miner, J. C., Rahe, A. J., Moore, C. A. Needham, L. L., and Patterson, D. G., Jr. (1995). Paternal serum dioxin and reproductive outcomes among veterans of Operation Ranch Hand. Epidemiology 6, 17–22.[ISI][Medline]

Young, A. L., and Reggiani, G. M. 1988. Agent Orange and Its Associated Dioxin—Assessment of a Controversy. p. 11. Elsevier, Amsterdam.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Oakes, D. J.
Articles by Ritchie, H. E.
PubMed
PubMed Citation
Articles by Oakes, D. J.
Articles by Ritchie, H. E.