* Department of Anatomy and Histology, University of Sydney, Sydney, NSW 2006, Australia; and
School of Biomedical Sciences, Faculty of Health Sciences, University of Sydney, Lidcombe, NSW 2141, Australia
Received November 15, 2001; accepted March 6, 2002
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ABSTRACT |
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Key Words: 2,4-D; picloram; Agent White; reproductive effects; rats.
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INTRODUCTION |
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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., 1989; Khera and Ruddick, 1973
). Similarly, the two active herbicides in Agent Orange, 2,4-D and 2,4,5-T, do not show mutagenic activity (IOM, 1996
; Lamb et al., 1981
).
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., 1989). 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, 1996
). 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 1). 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 1
, the composition of Agent White, Tordon 101®, and Tordon 75D® is very similar.
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MATERIALS AND METHODS |
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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 300600 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, 1990; Trasler et al., 1985
, 1986
, 1987
).
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, 1965) or in 95% alcohol for subsequent staining with alizarin red and skeletal examination (Taylor, 1986
).
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.
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RESULTS |
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Matings 23 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 2).
Matings 45 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 89 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 3).
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DISCUSSION |
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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 23 and 45 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 249284 µg 2,4-D/ml serum and 1720 µg picloram/ml serum. The serum half-lives were about 6 h for 2,4-D and 24 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, 1982; Kolmodin-Hedman and Erne, 1980
; Lavy et al., 1987
; Nash et al., 1982
). For a 70-kg man, this would represent exposure to 0.111 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., 1974
). 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., 1984
). 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, 1966; Grunow and Bohme, 1974
), and picloram is thought to be excreted unchanged (Nolan et al., 1984
).
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, 1990; Trasler et al., 1985
, 1986
, 1987
). 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., 1989) 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 1). 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., 1992). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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