A 90-Day Toxicological Evaluation of Compound 1080 (Sodium Monofluoroacetate) in Sprague-Dawley Rats

Charles T. Eason*,1 and Patricia Turck{dagger}

* Landcare Research New Zealand Ltd., Canterbury Agriculture & Science Center, Gerald St., Lincoln 8152, New Zealand; and {dagger} MPI Research Inc., 54943 North Main Street, Mattawan, Michigan 49071-9399

Received January 10, 2002; accepted July 9, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Groups of Sprague-Dawley rats received sodium monofluoroacetate (Compound 1080) at 0.025, 0.075, and 0.25 mg/kg by oral gavage once daily for 90 days and were then euthanized. The control and 0.25 mg/kg/day groups included additional rats of each sex that were treated for 90 days, then maintained without treatment for a further 56-day recovery period. Microscopic changes were restricted to the testes and the heart, and were seen only in males dosed with 1080 at 0.25 mg/kg/day and included severe hypospermia in the epididymides, severe degeneration of the seminiferous tubules of the testes, and cardiomyopathy. Sperm evaluation indicated severe decreases in all three sperm parameters evaluated (concentration, % motile, and % abnormal) at 0.25 mg/kg/day. There were no microscopic changes or 1080-related effects on sperm parameters at 0.025 and 0.075 mg/kg/day. The no observable effects level (NOEL) for rats administered 1080 via oral gavage for 90 days was 0.075 mg/kg/day. The lowest observable effects level (LOEL) dose was 0.25 mg/kg/day. After dosing with the LOEL dose of 0.25 mg/kg/day, mean concentrations of 1080 in rat plasma were 0.26 µg/ml at 1 h and 0.076 µg/ml at 12 h. Rat urine collected from the same animals contained 0.059 µg/ml.

Key Words: sodium monofluoroacetate (1080); 90-day exposure; no observable effect level (NOEL); cardiomyopathy; testicular degeneration.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulatory toxicology studies are usually conducted before the launch of new drugs or pesticides. Alternatively, they may be conducted on established products, such as compound 1080 (sodium monofluoroacetate), to update toxicology data generated before new standards and data requirements became commonplace. Compound 1080 is widely used in New Zealand and Australia as a vertebrate pesticide. Early investigations and toxicology studies on 1080 and its mode of action as an inhibitor of the Kreb’s cycle have been extensively reviewed elsewhere (Eisler, 1995Go; Seawright and Eason, 1994Go).

Before the suite of regulatory toxicology studies was commissioned by New Zealand agencies, including the study in rats reported in this document, a battery of regulatory studies had been completed in the USA because 1080 is used there in livestock protection collars. This included 15 studies on product chemistry, five studies on wildlife hazards, and three studies relating to human health. The results from these studies, which related to skin and eye irritation and transdermal absorption and toxicity of 1080, have been summarized elsewhere (Fagerstone et al., 1994Go).

In response to demand for clarification of the potential risk from chronic, low-level exposure to 1080 and to support regulatory assessments of risk in the use of 1080 as a vertebrate pesticide, the first phase of new (New Zealand-directed) regulatory toxicology studies was completed in 1998. Results of three different complementary toxicity studies indicate that 1080 is not mutagenic, and therefore, is not anticipated to be a carcinogen (Eason et al., 1999Go). Results of developmental toxicology studies indicate that 1080 causes developmental defects in rats when pregnant females are exposed to relatively high doses (0.33 and 0.75 mg/kg) on a daily basis during the period of organogenesis from Day 6 through to Day 17 of gestation. The NOEL for developmental effects was 0.1 mg/kg/day, based on observations of bent ribs at 0.33 mg/kg/day (Eason et al., 1999Go).

After completion of this study we identified a similar unpublished 90-day study in rats (Wolfe, 1988Go) that we had not been aware of. In this article, we compare our findings with those of Wolfe (1988), with earlier findings relating to the sublethal effects of 1080 in animals and with results of the teratogenicity study by Eason et al.(1999). The current study differs from all previous studies of the sublethal effects of 1080 in animals, as blood and urine analyses for 1080 concentrations have been undertaken so that LOELs and NOELs can be related to 1080 levels in blood and urine as well as the amount of 1080 ingested in mg/kg/day.

Previous studies have focused on either the metabolism and fate of 1080 in animals, or separate experiments on the toxic effects of 1080 (Eason et al., 1994aGo,bGo). This study further differs from the study by Wolfe (1988) in that a substantial recovery period was included. Estrous cyclicity and additional assessment on the testes were included as there have been concerns regarding the effects of 1080 on reproductive systems in male and female animals (Eason et al., 1999Go; Shinoda et al., 2000Go; Sullivan et al., 1979Go; Wolfe, 1988Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was run in accordance with the United States Environmental Protection Agency Guidelines (U.S. EPA, 1998Go), which define the number of treatment groups, the number of animals per group, and provide guidelines for the parameters assessed. In addition to the standard procedures outlined in these guidelines, special tests were included to assess the effect of 1080 on known target organs (e.g., testes), recovery groups were included, and 1080 concentration was assessed in the blood and urine.

Animals and treatment.
Sixty male and 60 female Sprague-Dawley rats (about six weeks of age) were obtained from Charles River Laboratories, USA, and acclimatized for three weeks at MPI Research, Michigan. Prior to assignment to study groups, the rats were weighed, sexed, and examined for evidence of disease and other physical abnormalities. All rats available for the selection process had body weights that were within ± 20% of the mean body weight for each sex. A randomization procedure was used to assign the rats to control and treatment groups, so that there were 20 of each sex in the control group (0 mg/kg/day), 10 of each sex in the 0.025 mg/kg/day dose group, 10 of each sex in the 0.075 mg/kg/day dose group, and 20 of each sex in the 0.25 mg/kg/day dose group The standard group size (rats/sex/group) recommended by the U.S. EPA (1988) for a 90-day study is 10. An extra 10 rats of each sex in the control and highest 1080 dose group were utilized as recovery subgroups.

The dose levels were selected on the basis of results from the preceding 28-day pilot study. In this pilot study, a NOEL was not determined when doses ranging from 0.2 to 0.8 mg/kg/day were administered, because significant effects on the testes were noted at all dose levels. Hence, for the 90-day study, one dose level was chosen (0.25 mg/kg/day) in which these toxic effects would be manifest. Because there was no indication of a NOEL between 0.2 and 0.8 mg/kg/day, substantially lower doses were selected for the mid- and low-dose groups. The two lower-dose groups were designed to span different degrees of exposure so that a NOEL could be identified in one or both groups. The oral route was chosen as it is one of the potential routes of human exposure to 1080.

Compound 1080 obtained from Tull Chemical Company, USA, was dissolved in HPLC Grade water (Aldrich Chemical Company) and administered by gastric intubation once a day for 90 consecutive days at a dose volume of 10 ml/kg/day. The control group was given water at the same dose volume.

To ensure the correct dose was administered, test article analyses for concentration and stability were undertaken at Landcare Research’s toxicology laboratory. Dosing solutions were freshly prepared within each 21-day interval and periodically analyzed for 1080 concentration and stability using a validated gas-chromatography technique.

Following the 90-day exposure period, treatment was discontinued for half the animals in Groups 1 and 4 for a further period of 56 days.

The rats were individually identified with implanted transponders (Biomedic Data Systems, Inc.) and housed individually in suspended, stainless steel wire-mesh cages. Fluorescent lighting was provided for approximately 12 h per day. Temperature and humidity were monitored and maintained at 18–24°C and 36–75%, respectively.

Rodent Diet (certified Rodent Chow® #5002, PMI Nutrition International, St. Louis, MO) and water were freely available to all rats.

General examinations.
Standard OECD and U.S. EPA (U.S. EPA, 1998) guidelines were followed in terms of the selection of tissues and procedures, which included comprehensive general observations, blood analyses for hematology and clinical chemistry parameters, and tissue pathology, including gross and microscopic examination, to determine whether 1080 adversely affected any particular target organ. All rats were observed for morbidity, mortality, and evidence of toxic effects. Individual food consumption of each rat was measured and recorded during the study, including the recovery period for Groups 1 and 4. Detailed clinical examinations were conducted weekly during the study and recovery periods.

Individual body weights were measured and recorded for all rats two days after receipt, weekly during the study, and recovery periods, and before postmortem examination at the end of the treatment or recovery periods. Food consumption was not measured. Ophthalmoscopic examinations were conducted before dosing and after 90 days of treatment to determine whether or not 1080 caused ocular toxicity.

Estrous cyclicity.
To evaluate potential effects of 1080 on the estrous cycle, all females were evaluated during the last three weeks of the 90-day exposure period. Each female received a vaginal lavage daily and the stage of estrus was determined by microscopic examination of the cell types present.

Sperm evaluation.
The testes are known to be a target organ for 1080-induced toxicity (Sullivan et al., 1979Go). Because of this, the testes were the focus for observations. A section of the right vas deferens was used for videotaping a prepared sperm sample for automated evaluation of sperm motility utilizing the Hamilton-Thorne Computer-Assisted Semen Analysis (CASA) System. The right cauda epididymis was separated, weighed, and used for manual (visual) assessment of sperm concentration. Slides were prepared for assessment of sperm morphology from sperm concentration preparations.

Clinical pathology: Hematology and clinical chemistry studies.
Blood samples (3–4 ml) were collected by cardiac puncture after carbon dioxide anesthesia at termination of study and at the end of the 56-day recovery period. Whole blood was used for hematological examination and separate subsamples were centrifuged and plasma retained for clinical chemistry analyses.

Hematological parameters determined included leukocyte count, erythrocyte count, hemoglobin, hematocrit, MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration), and platelet numbers assessed on whole blood samples using a Baker 9000 hematological counter. Differential leukocyte counts were assessed by light microscopy and prothrombin time (PT) using an MLA Electra 900.

Clinical chemistry parameters determined included blood levels of sodium, potassium, chloride, calcium, phosphorus, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen, creatinine, total protein, albumin, globulin, and glucose determined using an automated Olympus AU600.

Urine and plasma for 1080 concentration.
On the day before euthanasia (at least 16 h before), 5 rats of each sex from each group were placed in stainless steel metabolism cages to collect urine samples. The animals had free access to water, but were fasted overnight. The urine samples were frozen at –20°C after collection pending 1080 analyses.

Blood samples (approximately 1.5–2.0 ml) were collected from the orbital plexus for determination of plasma concentration of 1080. Blood samples were collected at 1 and 12 h after dosing on Days 10 and 77, from three male rats per group at each time point for each interval. Samples were placed in tubes containing EDTA. Plasma samples were frozen at approximately –20°C pending analysis. The selection of Day 10 as a sampling point, rather than Day 1, was to allow 1080 concentration to reach steady state equilibrium. Day 77 was selected as a second assessment to evaluate whether a difference in the rate of excretion and metabolism occurred during the course of the study. The 1 h time point was chosen to approximate the likely t-max for 1080 in rats (Eason et al., 1994bGo).

Frozen samples of urine and plasma were analyzed for 1080 concentration using gas chromatography with ECD detection, following derivatization to form a dichloroanide (Eason et al., 1994aGo).

Macroscopic examination.
All rats were given a complete postmortem examination after euthanasia by carbon dioxide inhalation. Each rat was examined carefully for external abnormalities. The abdominal, thoracic, and cranial cavities were examined for abnormalities, and the organs were removed, examined, and fixed. Organ weights (brain, adrenal, heart, kidney, liver, ovary, or testis) were recorded for all rats at postmortem. Appropriate organ weight ratios were calculated (relative to body and brain weights).

Microscopic examination.
A full complement of organs and tissues was prepared and microscopically examined by a veterinary pathologist for all rats in the control and 0.25 mg/kg/day dose groups. The heart, kidneys, liver, lungs, epididymides (male), and testes (male) were determined to be potential target organs and were microscopically examined in the 0.025 and 0.075 mg/kg/day dose groups. In addition, all gross lesions were microscopically examined in all groups. A 4-step grading system of trace, mild, moderate, and severe was used to define gradable lesions for comparison between treatment groups.

Statistics
Statistical comparisons were made between the control group and each treatment group. If more than one set of comparisons was required, all analyses were conducted separately on each set unless stated otherwise. The sexes were analyzed separately.

By convention in toxicology, an observable effect is one for which there is positive evidence, i.e., one that is statistically significant. Following this convention, significance tests were used to screen results as described below. Where a statistically significant effect was found, typically between the high rate and control, means for lower rates and for associated variables were examined for any trends suggesting physiologically significant effects masked by experimental variability.

Group pair-wise comparisons.
For each specified end point (body weight, body weight change, hematology except leukocyte counts, clinical chemistry, absolute and relative to body and brain organ weights, mean estrous cycle time, number of estrous cycles/period, and epididymal sperm concentration) and for all collection intervals, Levene’s test (Milliken and Johnson, 1992Go) was used to assess homogeneity of group variances. If Levene’s test was not significant (p > 0.01), Dunnett’s test (Dunnett, 1955Go) was used to compare each treatment group with the control group. If Levene’s test was significant (p < 0.01), comparison with the control group was made using Welch’s t-test (Welch, 1937Go) with a Bonferroni correction. Results of all pair-wise comparisons were reported at the 0.05 and 0.01 significance levels.

Log transformation.
For data not distributed normally (total and differential leukocyte counts), a log transformation was performed and then analyzed as described above.

Arcsin-square-root transformation.
Data comprising percentage values (spermatogenesis parameters; % abnormal and % motility) were transformed using the arcsin of the square root (Steel and Torrie, 1980Go). Group pair-wise comparisons were used to analyze the transformed percentage values.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of 1080 Solutions
The 0.025 mg/kg/day dosing solution was found to be stable when stored under refrigeration for 21 days. Dosing solutions used for each group at Weeks 1, 2, 3, 4, 8, and 12, upon analysis, had a mean 1080 concentration nearly identical to the nominal concentrations (recovery of 1080 in the dosing solution was greater than 93% for up to 21 days), indicating that the dosing solutions were accurately prepared.

General Examinations
All animals survived to the end of treatment or recovery, and no general clinical signs relating to 1080 toxicity (e.g., behavior changes and mortality) were noted in the treated groups when compared with controls during the treatment or the recovery periods.

No 1080-related changes in body weight occurred in the treatment groups when compared with controls during the treatment or recovery periods.

Statistically significant decreases in body weight change were noted in males treated with 0.25 mg/kg/day when compared with controls during Days 77–79 (treatment period) and Days 126–133 (recovery period). However, these changes did not form a consistent pattern and are, therefore, not considered 1080-related. Increase in body weight in females at 0.25 mg/kg/day was statistically different from controls at the Week 17–18 (recovery) interval, but this was not considered to be an adverse or 1080-related effect. Body weight and body weight increase at 0.25 mg/kg/day for all other intervals, including the recovery period, were similar to controls. No statistically significant changes were noted at 0.025 or 0.075 mg/kg/day when compared with controls during the treatment period.

No 1080-related changes in food consumption or food efficiency were noted during the treatment or recovery periods. No 1080-related ophthalmoscopic findings were noted in the 1080-treated groups when compared with controls.

Clinical Pathology
At Day 90, there was a small decrease (approximately 6%) in erythrocyte counts in males at 0.25 mg/kg/day when compared with controls. An association with 1080 administration was not clear, and no differences were noted following the recovery period. Occasionally, other values were statistically different from controls, but these were considered incidental and not related to 1080 administration.

There were no apparent 1080-related alterations in the clinical chemistry parameters evaluated. Occasional values were statistically different from controls, but these were considered incidental and not associated with 1080 administration.

Urine and Plasma 1080 Concentration Studies
The dose levels of 0.025, 0.075, and 0.25 mg/kg/day showed a dose-related, but not a dose-proportional trend, with mean concentrations of 0.006, 0.032, and 0.059 mg/ml in rat urine at higher dose levels (Table 1Go). However, urine levels of 1080 were variable (noting that these data were not corrected for urine volume or creatinine content, which were not measured). The plasma concentrations 1 and 12 h after dosing on Days 10 and 77 also appeared to increase in a dose-related manner (Table 2Go). There was no evidence of any difference between the concentrations on Day 10 and Day 77.


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TABLE 1 Mean 1080 Concentration (Day 90) in Rat Urine following Oral Dosing with 1080 solution
 

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TABLE 2 1080 Concentration in Rat Plasma at Day 10 and Day 77 following Oral Dosing with 1080 solution
 
Estrous Cyclicity
No 1080-related changes were observed in the treatment groups when compared with controls. The mean cycle length and number of cycles were similar in all groups including the controls.

Sperm Evaluation
Statistically significant changes in sperm parameters were noted at 0.25 mg/kg/day when compared with controls at the end of the treatment period (Table 3Go). The 56-day recovery period appeared to have no effect on improving sperm parameters seen after 90 days exposure at 0.25 mg/kg/day. The values in male rats in the 0.25 mg/kg/day group after the recovery were, if anything, lower than the values seen at the conclusion of the treatment period (Table 6Go).


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TABLE 3 Summary of Sperm Parameters After 56 days recovery period without08.76108.65907.89502.218*
 

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TABLE 6 Organ Weight Values—90-Day Male Rats
 
All three sperm parameters were statistically lower than controls in the 0.25 mg/kg/day male rats after both the treatment and recovery periods. Sperm motility was reduced to 0%, more than 99% of all sperm was abnormal, and a reduction of 75% in the concentration of sperm was observed in the 0.25 mg/kg/day dose group at the end of the treatment period. At the end of the 56-day recovery period no improvement occurred in all three parameters. The majority of sperm seen in these male rats appeared to be fragmented, with heads and tails separated. Although sperm were present in samples used to analyze motility, all appeared dead, and no movement was evident. No changes were observed in the male rats dosed with 0.025 or 0.075 mg/kg/day when compared with the control group.

Macroscopic Examination
1080-related macroscopic effects were observed in the epididymides and the testes (Tables 4 and 5GoGo). The epididymides were notably small in 3 of 10 males receiving 1080 at 0.25 mg/kg/day, but normal in all other treated groups and the control. The testes were small in all males at 0.25 mg/kg/day, but normal in all other treated groups and the control group. Treatment-related effects were still present in the testes after 56 days of recovery, where the testes were small in 9 of 10 males at 0.25 mg/kg/day, but normal in the control animals.


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TABLE 4 Incidence of Macroscopic Observations Relating to Effects on the Testes—Terminal Sacrifice: Male Rats   Moderatemg/kg/daymg/kg/daymg/kg/daymg/kg/day
 

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TABLE 5 Incidence of Macroscopic Observations Relating to Effects on the Testes—Recovery Sacrifice: Male Rats Number of animals examinedDOSSACDOSSAC
 
These macroscopic observations were correlated with 1080-related organ weight changes in the testes and epididymides of male rats following exposure to 0.25 mg/kg/day 1080 for 90 days (Tables 6, 7, 8, and 9GoGoGoGo). There were also 1080-related changes in heart weight in both males and females at 0.25 mg/kg/day after 90 days.


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TABLE 7 Organ Weight Values—Terminal Sacrifice: Female Rats
 

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TABLE 8 Organ Weight Values—Recovery Sacrifice: Male Rats Adrenal/body weight (% x 103)000.0000.30810003.43**00.38310
 

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TABLE 9 Organ Weight Values—Recovery Sacrifice: Female Rats Adrenal/body weight (% x 103)000.0000.1049004.3400.07110
 
In male rats dosed at 0.25 mg/kg/day, the heart to body weight-ratio was significantly increased when compared to controls, while the testes weight, testes to body weight-ratio, and testes to brain weight-ratio were all significantly decreased at 0.25 mg/kg/day when compared with controls. In female rats dosed at 0.25 mg/kg/day, the heart weight, heart to body weight-ratio, and heart to brain weight-ratio were all significantly increased when compared with controls.

Treatment-related organ weight changes in the heart and testes of rats were still evident after 56 days of recovery without 1080 exposure. In male rats, the heart to body weight-ratio was statistically significantly increased when compared to controls at 0.25 mg/kg/day, while the testes weight, testes to body weight-ratio, and testes to brain weight-ratio were all statistically significantly decreased at 0.25 mg/kg/day when compared with the controls. There were no significant organ weight changes in the female rats at 0.25 mg/kg/day after 56 days of recovery when compared with the control group.

Microscopic Examination
1080-related microscopic changes were present in the epididymides and testes in male rats exposed to 0.25 mg/kg/day (Table 10Go). There was severe hypospermia of the epididymis and severe degeneration of the seminiferous tubules of the testes in all males after exposure to 1080 at 0.25 mg/kg/day for 90 days. There was also an increase in the incidence of cardiomyopathy in males exposed to 0.25 mg/kg/day when compared with the control group. Incidence was 3 out of 10 at the end of the recovery period versus 5 out of 10 after 90 days of 1080 exposure.


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TABLE 10 Incidence of Microscopic Observations—Terminal Sacrifice: Male Rats  Infiltration, mononuclearmg/kg/daymg/kg/daymg/kg/daymg/kg/day
 
1080-related microscopic changes were still present in the heart, epididymides, and testes in male rats in the 0.25 mg/kg/day group following the 56 days of recovery. There was hypospermia of the epididymis in 9 of 10 males, and degeneration of the seminiferous tubules of the testes in 10 of 10 males. Both changes were less severe at the end of recovery than at the end of treatment, suggesting a partial reversal for both of these lesions following 56 days of recovery (Table 11Go).


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TABLE 11 Incidence of Microscopic Observations—Recovery Sacrifice: Male Rats  Within normal limitsDOSSACDOSSAC
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study has confirmed that the NOEL for repeat-dose exposure to 1080 is low. In a recently completed teratogenicity assessment in rats, a NOEL of 0.1 mg/kg was derived (Eason et al., 1999Go). In the present study, after 90 days exposure, a similar NOEL of 0.075 mg/kg was identified. We also conclude that during sublethal exposure to 1080, the most sensitive sites for toxicity are the heart, epididymides, testes, and fetus in rats. However, the absence of changes in body weight, food consumption, hematology, and clinical chemistry in this study indicate that the sublethal toxicity of 1080 is relatively specific to identified organs.

Similar histological changes after exposure to fluorocitrate (the toxic metabolite of 1080) or 1080 itself have been previously reported in rat and lizard testes (Smith et al., 1977Go; Sullivan et al., 1979Go; Twigg et al., 1988Go; Wolfe, 1988Go), cat brain (Koenig, 1969Go), rat kidney (McDowell, 1972aGo,bGo), and sheep and guinea pig hearts (Schultz et al., 1982Go; Whitten and Murray, 1963Go). Previous researchers have suggested that the testes may be the most sensitive organ as morphological damage to the testes occurred in rats exposed to 1080 in their drinking water at doses that caused no morphological changes in the liver and kidneys (Savarie, 1984Go). In contrast, Whitten and Murray (1963) working with sheep concluded that the heart is the most sensitive organ. Acute multifocal injury to the myocardium occurred after doses as low as 0.11 mg/kg/day for 3–7 days, and lesser effects were also noted at doses as low as 0.055 mg/kg/day and attributed to 1080-induced damage.

This 90-day study has confirmed that the epididymides, testes, and the heart are target organs for sublethal effects of 1080. Earlier reports suggest that seminiferous tubules degenerated after rats drank water containing 1080, but completely regenerated after treatment had been withdrawn (Sullivan et al., 1979Go). By contrast, recovery from testicular damage did not occur in our current 90-day study even after 56 days without treatment. Mazzanti (1965) described regressive modifications of the seminiferous tubules caused by sodium monofluoroacetate in albino rats, and described the action of sodium monofluoroacetate as similar to that of fluoroacetamide. Treatment of normal rats with fluoroacetamide also produced atrophic testes (Mazzanti et al., 1968Go), with observations of morphological changes showing that testicular germinal epithelium was fully regenerated 165 days after treatment. This suggests potential for the reversal of the type of testicular damage described in this study outside a 56-day recovery period.

In the study by Sullivan et al. (1979), Sprague-Dawley rats were dosed with 0, 0.07, 0.18, or 0.71 mg/kg/day for 7 days followed by 21 days without treatment. Testicular atrophy and nonreversible tubular degeneration were found in the mid- and high-dose groups. At the lowest dose (0.07 mg/kg/day), it is unclear whether or not there was an effect. This publication reports "the testes were histologically normal, but acute degenerative changes were evident, there being no evidence of spermatocyte depletion or of seminiferous tubular atrophy," which appears to be contradictory. Our interpretation is that this dose of 0.07 mg/kg/day could be considered close to the borderline of the LOEL and NOEL.

These earlier 1080 studies, particularly the drinking water study in rats (Sullivan et al., 1979Go) and the multidose study in sheep (Whitten and Murray, 1963Go), demonstrate that there could be deleterious effects at dose levels lower than the NOEL of 0.075 mg/kg/day determined by the current 90-day rat study. It is likely that the different results are due to different routes of exposure, and interspecies differences in susceptibility between sheep and rats. Other vertebrate pesticides have similar toxicity. For example, in a five-day study in rats, the NOEL for brodifacoum, a rodenticide used throughout the world, was 0.02 mg/kg/day (WHO, 1995Go).

In addition to the sublethal effect of 1080 previously reported in the literature, we have recently sourced a previously unknown and unpublished 90-day study (Wolfe, 1988Go) conducted by Hazleton Laboratories in 1988. In this study, Sprague-Dawley rats were dosed with 1080 at doses of 0, 0.05, 0.20, or 0.50 mg/kg/day. The NOEL for 1080 in rats defined by Wolfe was 0.05 mg/kg/day. The LOEL was 0.20 mg/kg/day based on dose-related effects similar to those reported for this study (e.g., absolute and/or relative weight of the heart and spleen, abnormal sperm and decreased size and weight of testes and epididymides in males). The study by Wolfe (1988) did not include 1080 analyses in blood and urine for correlation of toxic effects with exposure levels, and no recovery periods were included.

Our current study and Wolfe’s earlier study confirm the principal target organ of concern for sublethal effects from 1080 exposure in rats to be the testes. Oligospermia or aspermia have been associated with exposure of mink to sublethal doses of 0.08 mg/kg/day 1080 for 2 months (Hornshaw et al., 1986Go), providing further evidence that the sublethal effects of 1080 occur across a range of species. Although no lesions or microscopic effects on sperm abundance or development were observed in starlings fed sublethal doses of 1080 over a four-week period, a reduction in testis weight (although not at statistically significant levels) was reported (Balcomb et al., 1983Go). These authors suggested a difference in the sensitivity of birds and mammals to the effects of sublethal, chronic doses of 1080 on testes.

In a recent publication on 1080, a single dose at 1.0 mg/kg (4 times higher than the top dose of 0.25 mg/kg/day used in this 90-day study) administered to male Sprague-Dawley rats was associated with testicular toxicity (Shinoda et al., 2000Go). The study by Shinoda et al. highlights the "steep" dose-response curve associated with 1080 toxicity, and that even a single exposure could affect sperm production and viability (heart tissue was not examined).

The current 90-day study has established blood and urine concentrations in rats that can be correlated with LOEL and NOEL doses of 1080 on a mg/kg/day basis. After administration of 0.25 mg/kg/day 1080 for 10 or 77 days, the LOEL dose, correlated with mean urine concentrations of 0.059 µg/ml and plasma concentrations of 0.234–0.283 µg/ml, 1 h after exposure and 0.069–0.089 µg/ml, 12 h after exposure. The NOEL dose of 0.075 mg/kg/day correlated with a mean urine concentration of 0.032 µg/ml and a mean plasma concentration of 0.088–0.84 µg/ml, 1 h after exposure and 0.021–0.023 µg/ml, 12 h after exposure. However, previous studies have established a lower LOEL of 0.20 mg/kg/day (Wolfe, 1988Go) and 0.07 mg/kg/day (Sullivan et al., 1979Go). Hence caution is required with regard to emphatically stating that blood and urine concentrations below those detected in rats exposed in this study would not be associated with sublethal effects. Doses below 0.25 mg/kg/day (i.e., 0.20 and 0.07 mg/kg/day) have been associated with toxicity in rats, hence, concentrations of 1080 in blood and urine below those reported in rats receiving 0.25 mg/kg/day could be associated with damage to the testes and the heart.

We defined a higher NOEL than Wolfe (1988) as a result of using different doses. This study involved treatment of rats with 0.025, 0.075, and 0.25 mg/kg/day, whereas in the study by Wolfe (1988) there was a gap between the dose level of 0.05 mg/kg/day and 0.20 mg/kg/day. Based on the results of this study, the NOEL for rats administered 1080 via oral gavage for 90 days was 0.075 mg/kg/day and is similar to the NOEL of 0.1 mg/kg/day established in a teratogenicity study in rats (Eason et al., 1999Go). Target organs in rats include the testes and the heart. Severe hypospermia, severe degeneration of the seminiferous tubules of the testes, and cardiomyopathy were seen in males at 0.25 mg/kg/day. Sperm physiology was affected by 1080 exposure at 0.25 mg/kg/day and did not return to normal after 56 days cessation of exposure. The potential for reversibility of, or recovery from testicular damage resulting from sublethal exposure to 1080, especially from doses below the LOEL, could be addressed in future studies incorporating longer recovery periods and measurement of additional fertility parameters.


    NOTES
 
1 To whom correspondence should be addressed. Fax: + 64 3 325 6705. E-mail: easonc{at}landcareresearch.co.nz. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Balcomb, R., Bowen, C. A., 2nd, and Williamson, H. O. (1983). Acute and sublethal effects of 1080 on starlings. Bull. Environ. Contam. Toxicol. 31, 692–698.[ISI][Medline]

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