The Effect on Sperm Production in Adult Sprague-Dawley Rats Exposed by Gavage to Bisphenol A between Postnatal Days 91–97

J. Ashby*,1, H. Tinwell*, P. A. Lefevre*, R. Joiner{dagger} and J. Haseman{ddagger}

* Syngenta Central Toxicology Laboratory, Alderley Park, Cheshire, SK10 4TJ, United Kingdom; {dagger} General Electric Company, Pittsfield, Massachusetts 01201; and {ddagger} National Institute of Environmental Health Sciences, Alexander Drive, Research Triangle Park, North Carolina 27709

Received October 30, 2002; accepted March 21, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
M. Sakaue et al. (2001,J. Occup. Health vol. 43, pp. 185–190) have described how oral exposure of sexually mature male rats to bisphenol A (BPA) between postnatal days (PND) 91–97 led to a reduction in daily sperm production (DSP) 5 weeks later (18 weeks of age). Activity was observed over the dose range 20 µg/kg–200 mg/kg BPA, with an absence of activity over the dose range 2 ng/kg–2 µg/kg BPA. There was no evidence of a dose response relationship over the active dose range (five orders of magnitude range). The observation of endocrine disruption (ED) effects for BPA at such low doses, and in sexually mature animals, was unexpected. It was therefore decided to mount an independent repeat of their study. A total of four independent studies were conducted according to the protocol used by Sakaue et al. Doses of 20 µg/kg, 2 mg/kg, or 200 mg/kg BPA were administered to adult Sprague-Dawley (SD) rats over PND 91–97, and the studies were terminated when the rats reached the age of 18 weeks. Three different rodent diets were employed (RM3, Purina 5002, and CE2), the last of which had been used by Sakaue et al. BPA failed to give any evidence of ED activities, including the changes in DSP reported by Sakaue et al. 2001Go. During the course of these studies, the test protocol was adapted to coincide more precisely with that used by Sakaue et al.; this included restricting the number of animals per cage, removing bedding from the cages, and changing to the use of glass water bottles in the cages. The only thing of interest to emerge from our studies was the observation of a significant difference in DSP between the control groups of our first and second study. As the change in diet from RM3 to Purina 5002 was the major difference between those two studies, we conducted a repeat of the second study, but we were unable to confirm the differences seen between the first and second study. The probability that those differences arose either by chance, or as the result of intrinsic study-to-study variability, was strengthened by the absence of significant differences in the sperm parameters in a final (fifth) study where the sperm parameters for control animals maintained on the three different diets were compared under the conditions of the main experiments. No explanation for our failure to replicate the effects reported by Sakaue et al. is evident. A review of DSP values reported in the recent literature is provided and discussed, and it is concluded that use of the term DSP/g testis rather than DSP/testis could increase the sensitivity of DSP assessments.

Key Words: bisphenol A; sperm production; testis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of two independent Sprague-Dawley (SD) rat multigeneration assays of bisphenol A (BPA) have recently been reported. The first was a two-generation assay conducted using the oral route over the dose range 0.2 µg/kg–200 µg/kg BPA (Ema et al., 2001Go). The second was a three-generation dietary assay conducted over the dose range 1 µg–500 mg/kg BPA (Tyl et al., 2002Go). Both assays were reported as negative with toxicity defining a no adverse effect dose level (NOAEL) of 5 mg/kg BPA. Two other evaluations of the endocrine toxicity of BPA were reported in the same period. In the first of these, Talsness et al.(2000)Go reported that oral exposure of SD rats to doses as low as 20 µg/kg BPA during gestation days 6–21 led to a wide variety of endocrine toxicities, including effects on testes weight and sperm production. We recently reported our inability to confirm those effects, and in the same article, listed all of the other published endocrine disruption (ED) data reported for BPA in the rat (Tinwell et al., 2002Go). Ten of those studies were reported as negative, and one of the remaining five contained reports of activity for BPA in the µg/kg dose range; that being a study reported by Sakaue et al.(2001)Go. Sakaue et al. described how oral exposure of sexually mature male SD rats to BPA between postnatal days (PND) 91–97 led to reduced daily sperm production 5 weeks later. Activity was observed over the dose range 20 µg/kg–200 mg/kg BPA, with an absence of activity over the dose range 2 ng/kg–2 µg/kg BPA. Sakaue et al.(2001)Go confirmed their initial observations in an independent experiment.

The fact that Sakaue et al. had observed ED effects for BPA at such a low dose, and in sexually mature animals, led us to mount an independent repeat of their study. As in all previous cases of us repeating reports of low dose effects, the objective was to establish a model of such effects in order to study their mechanism at the molecular level. Pursuant to the negative outcome of our first experiment we discussed with Sakaue et al. the precise details of their test protocol, and our subsequent experiments were designed to simulate more closely the experimental conditions that they had employed.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
BPA was purchased from Aldrich (Gillingham, Dorset, UK) as a solid (99+% pure; mp 158–159°C). Ethanol (99.86% v/v), purchased from Hayman Ltd. (Witham, Essex, U.K.) and corn oil, obtained from Mazola, were used to prepare a solution of 6.5% ethanol in corn oil (CO/EtOH). BPA was homogenized in this test vehicle to give a final concentration of 40 mg/ml. Additional dose preparations (400 µg/ml and 4 µg/ml) were prepared by serial dilutions with vehicle of this top dose. All dose preparations, as well as the test vehicle, were stored at room temperature for the duration of the study. Samples of each preparation were taken for analyses and stability measurements.

Analysis of dosing solutions and stability measurements.
Each dosing solution was analyzed using reverse-phase high-pressure liquid chromatography (HPLC). Stability measurements were performed by analyzing samples of the dosing solutions on the first day of dosing, storing these samples at room temperature, and then reanalyzing them 24 h after the end of dosing. Samples were diluted with solvent [methanol/tetrahydrofuran (1:2; v/v)] to give concentrations within the range of the calibration standards selected. Samples were analyzed using a 2487 Series UV detector (Waters) and a flow rate of 1 ml/min. The limit of detection was determined to be 0.2 µg/ml BPA (equivalent to a 1 µg/kg dosing solution).

Dietary analysis.
The phytoestrogen aglycone content of the diets was determined as described in detail by Wiseman et al.(2002)Go. Portions of the diets (200 mg) were extracted by shaking with aqueous methanol at 60°C for 1 h. The extracts were defatted with hexane and hydrolyzed to the aglycones with dilute HCl. The aglycones were then extracted with ether. Daidzein, genistein, and coumestrol were detected and quantified against reference samples by liquid chromatography–mass spectometry (LC-MS). Data were corrected for extraction efficiency. Quality control was determined by the concurrent analysis of a soya flour of known daidzein and genistein content, results were <9% different from those expected. The limits of detection for daidzein and genistein were 0.05 µg/g diet and for coumestrol were 0.1 µg/g diet.

Animals and housing.
Young adult (12.5 weeks old) SD (International Genetic Standard) male rats were obtained from Charles River U.K. (350–450 g) and allowed 4 or 5 days for acclimatization prior to dosing. All animals were housed in stainless steel cages and were subjected to a 12-h light/dark schedule. Five independent experiments were performed, with Experiments 1–4 involving the exposure of animals to either vehicle or BPA (20 µg/kg; 2 mg/kg; 200 mg/kg). In the final study animals were exposed to vehicle only. Slight modifications to animal husbandry and/or diet occurred with each subsequent study. Thus, in the first experiment, animals were housed three to a cage, which was reduced to two per cage as the experiment progressed. The animals were given bedding and received Rat and Mouse No. 3 (RM3) breeding diet (pelleted; Special Diet Services Ltd., Witham, Essex, U.K.) and water (via an automatic feeding system) ad libitum. In the other four experiments, the animals were housed two/cage in stainless steel cages without any bedding and received water via glass water bottles. These experimental conditions were based on those used by Sakaue et al. (2001 and personal communication)Go. In Experiments 2 and 3 the animals received 5002 diet (powdered; Purina Mills, Inc., Nottingham, U.K.); in Experiment 4 they were given the same diet as was used in the original Japanese study reported by Sakaue et al., 2001Go (CE2 diet, pelleted; CLEA, Tokyo, Japan). In the final experiment, the animals received RM3, 5002, or CE2 diet. Group sizes of 10 were used in all experiments with the exception of the control groups in Experiments 3 and 4. In these two experiments the control group size was increased to n = 20. These larger control groups were randomly split into two identical subgroups of n = 10 prior to the start of dosing to determine any interanimal variability. The data generated from each subgroup were pooled prior to the analysis of the test data.

Dosing.
Animals (age ~13 weeks) were exposed to the appropriate compound by oral gavage using a dose volume of 5 ml/kg body weight. In Experiments 1–4 each animal received a single dose of either CO/EtOH or BPA at the appropriate concentration in CO/EtOH (200 mg/kg, 2 mg/kg, or 20 µg/kg) on each of 6 days. In the final experiment, animals were exposed to CO/EtOH only for 6 days.

Body weight measurements.
All animals were weighed on a daily basis during dosing and then weighed on a weekly basis until termination.

Termination.
Animals were terminated 5 weeks after the start of dosing (i.e., 18 weeks of age) by an overdose of Halothane followed by cervical dislocation. Each rat was weighed just prior to termination. Liver, kidney, testes, epididymides, seminal vesicles, and ventral prostate were removed and weighed. Both testes as well as the prostate were flash frozen in liquid nitrogen and stored at -80°C for further investigation.

Estimation of daily sperm production.
Homogenization resistant sperm counts were determined as described previously (Ashby et al., 1997Go) using the method of Blazak et al.(1993)Go. Each frozen right testis (decapsulated) was homogenized in 50 ml 0.9% (w/v) NaCl containing merthiolate (0.01% w/v) and Triton X-100 (0.05%, v/v) using a Waring blender. The number of released sperm heads was determined in duplicate using an improved Neubauer haemocytometer. The number of sperm present in the area of the counting chamber used was equivalent, when multiplied by 104, to the number of sperm/ml homogenate. The variation between duplicate readings was less than 10%. Daily sperm production (DSP) values were obtained using the transit time factor of 6.1 (Blazak et al., 1993Go).

Statistical analyses.
For each of the four experiments in which animals were exposed to BPA, organ weights and sperm data were evaluated by ANOVA procedures to determine the significance of differences among the four treatment (three BPA and one control) groups. For organ weights, body weight was also included as a covariable (ANCOVA). Pairwise comparisons of BPA groups vs. controls were made by Dunnett’s test. In addition, in order to increase the power of the study for detecting any subtle BPA effects, an ANOVA was carried out using the data from all four experiments together in a single analysis to determine the significance of any BPA effects, experiment-to-experiment variability, and the interaction between these two factors. In the final study, (different diets) Fisher’s least significant difference test (LSD test) was used to make all possible pairwise comparisons among the different diet groups.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of Dosing Solutions and Stability Measurements
Analysis of the dosing solutions indicated that the top two dosing preparations (i.e., 200 mg/kg and 2 mg/kg) were within 7% of their nominal concentrations. However, analysis of the lowest dose preparation (20 µg/kg) suggested that this solution was, on average, 146% of the nominal concentration. All dosing solutions were stable for at least 7 days.

Dietary Analysis
The phytoestrogen content of the batches of diet used in the present studies is shown in Table 1Go. CE2 diet had the highest phytoestrogen content when the daidzein, genistein, and coumestrol contents were combined and expressed as total genistein equivalents. The results of these analyses were compared to those obtained for diets in the OECD program to validate the rat uterotrophic bioassay (Phase Two; Owens et al., 2003Go) where RM3, 5002, and CE2 were also used (Table 2Go). The phytoestrogen contents of RM3 and CE2 were similar between the two studies whereas 5002 had a much lower phytoestrogen content in the present study. The diet analysis for the two studies was carried out by different laboratories but a previous comparison of the analysis methods using a standard soya flour gave comparable results (Dr. D. Clarke, personal communication). We conclude therefore that the difference for 5002 is due to batch variability in manufacture.


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TABLE 1 Phytoestrogen Content of the Diet Batches Used in the Present Studies
 

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TABLE 2 The Phytoestrogen Content of the Diets Used in the OECD Phase 2 Validation Studies of the Uterotrophic Assay
 
It has been suggested that the responsiveness of the immature rat uterotrophic bioassay may be compromised when using diets with a total genistein equivalent level above ~350 µg/g diet (Owens et al., 2003Go). The total genistein equivalent levels in all the diets used in the present study were below this, although the level at which dietary phytoestrogens may impact on male sexual organ growth is unknown.

Exposure to BPA
The group mean (± SD) data for the terminal body and organ weights generated in Experiments 1–4 are presented in Table 3Go. These data indicated that liver and kidney weights were significantly correlated (p < 0.01) with body weight, but that other organ weights were not. More importantly, there were no significant effects on either body or organ weights induced by BPA at any of the dose levels tested in any of the four studies. Analysis of the sperm data (Table 4Go) gave a similar picture in that BPA did not significantly affect sperm counts or DSP at any of the doses tested in any of the studies conducted. Experiments 3 and 4 had 20 control animals divided at random into two groups of 10. These groups were pooled after preliminary analyses revealed no biologically important differences between them.


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TABLE 3 Individual Terminal Body Weight and Organ Weight Data following Exposure to BPA
 

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TABLE 4 Individual Study Data for Sperm Counts and Daily Sperm Production (DSP) as Performed According to Blazak et al. (1993)Go Using the Right Testis
 
Evaluating the data from all four studies together in a single analysis (summarized in Table 5Go) lends further support to the observation that BPA did not adversely affect any of the parameters measured. The only variable showing evidence of being marginally affected by BPA was body weight, inasmuch as the overall ANOVA showed a significant (p < 0.05) difference among the test groups. However, after adjusting for experiment-to-experiment variability, no pairwise comparisons with the controls were significant, the largest difference being a 5% body weight reduction in the 200 mg/kg BPA group relative to the controls.


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TABLE 5 Group Mean ± SD for All Parameters Measured after Combining Data from Experiments 1–4
 
It should, however, be borne in mind that several parameters showed significant (p < 0.01 or greater) study-to-study variability, which was apparent in both control and BPA-exposed groups. For example, prostate weights of animals in Experiment 1 (Table 3Go) were ~10% lighter than those in Experiments 2–4. Similarly, animals in Experiment 4 were ~7% heavier at termination than those in Experiments 1–3. Nonetheless, BPA had no effect on any of the parameters measured in any of the four experiments. Moreover, these negative findings were consistent from experiment to experiment (i.e., there were no significant experiment by treatment group interactions). The interexperiment variation is discussed below, with particular reference to the control data.

Interexperiment Variability
Although it is clear that BPA did not affect any of the parameters measured in any of the four studies (Tables 3Go and 4Go), it was apparent that there was significant experiment-to-experiment variability as described above. For example, consideration of the control data highlights significant variation for particular parameters. Specifically, a comparison of the control sperm count and DSP data from Experiments 1 and 2 (carried out before completion of Experiments 3 and 4) showed that the two experiments were significantly (p < 0.01) different. As it was thought that such differences were unlikely to be due to random variability, attempts to address these differences were made in subsequent studies.

One obvious variable between the first two studies was diet (RM3 was used in Experiment 1 and 5002 was used in Experiment 2). Thus, in the third experiment animals were again fed 5002 diet and the control group was increased to n = 20, which was randomly split (prior to dosing) into two subgroups of n = 10. All other experimental conditions were identical to those of the second study. Thus, if diet was the causal agent for the significant variation, then it was expected that the control response in this third study would be similar to that observed in Experiment 2 and would be significantly different from that of Experiment 1. Comparison of the two control subgroups of 10 animals showed that they were not significantly different, so they were pooled prior to evaluating the data from all three studies. Comparison of the sperm count and DSP data measured for the three control groups showed that the data from Experiment 3 were, in fact, intermediate between the controls of Experiments 1 and 2 (Table 4Go), a response that, despite increased sample size, did not differ significantly from either of the other control groups.

A fourth experiment was conducted, which was identical to the third experiment with the exception that the animals received CE2 diet (as used by Sakaue et al., 2001Go) rather than 5002 or RM3 diet. In this study the two control subgroups did differ significantly (p < 0.05) with regard to sperm/testis and DSP/testis (Table 4Go). However, this "stray" significance is consistent with chance expectations given the number of variables assessed and could, therefore, be attributed to no more than interanimal variation and/or random variability, as there were no differences between the two subgroups in terms of animal husbandry and compound exposure. Thus, for the purpose of comparing the control and test data, the two subsets were pooled (as was done in Experiment 3).

When all four control groups were considered, the experiment-to-experiment variability for body weight, all sperm and DSP parameters, and epididymis weight were significant (p < 0.05). However, since Experiments 1–4 were carried out over a nine month period, this interexperiment variation may have been due to some other confounding factors (e.g., different animal shipments, subtle differences in animal husbandry) of which diet could have been one. Thus, one final experiment was conducted to evaluate any possible diet effect (Table 6Go), in which the only difference between the groups was the diet (RM3, 5002, CE2) the animals received. Comparison of these data showed that there were no significant differences in all parameters measured, with the exception of the right epididymis weight in the CE2 diet group, which was significantly (p < 0.05) reduced (by ~9%) relative to the other two diet groups. However, such an observation is more than likely to be spurious for three reasons. First, given the number of variables evaluated, one spurious significant finding is consistent with chance expectation. Second neither the left epididymis nor total epididymides weight was affected. Finally, this finding is not supported by the results in Experiments 1–4 in which (for example) the RM3 diet was associated with right epididymis weights that were lower, not higher, than that seen for the CE2 diet (see Table 3Go).


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TABLE 6 Group Mean ± SD Data for All Parameters Measured in Experiment 5 (Assessment of Diets)
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present studies BPA failed to give any evidence of changes in reproductive tissue weights or DSP, the latter having been reported by Sakaue et al.(2001)Go. This conclusion is based on the results of four independent repeat experiments using three different diets (RM3, 5002, and CE2), the final one of which (CE2) was imported to the U.K. from Japan and was the same as that used by Sakaue et al. During the course of these experiments our test protocol was sequentially adapted in order for it to coincide more exactly with that used by Sakaue et al. These adaptations included restricting the number of animals per cage, removing bedding from the cages and changing to the use of glass water bottles in the cages. The only thing of interest to emerge from our studies was the observation of a significant difference in the sperm parameters between the control groups of the first and second study. As the change in diet from RM3 to Purina 5002 was the major difference between those two studies we conducted a repeat of the second study, but we were unable to confirm the differences seen between the first and second study. The probability that those differences arose either by chance, or as the result of intrinsic study-to-study variability, was strengthened by the absence of significant differences in the sperm parameters in a final study where control groups maintained on the three different diets were compared under the conditions of the main experiments.

The results of the present studies, together with the original data reported by Sakaue et al.(2001)Go are shown in Figure 1Go. Three points of interest emerge. First, the DSP values for all of our test and control groups were in the same range as the significantly reduced DSP values reported by Sakaue et al.(2001)Go. Second, although the decline in DSP induced by BPA (Sakaue et al., 2001Go, Fig. 1Go) was dose-related over the range 20 ng/kg–20 µg/kg, there was a plateau for the depressed DSP values over the dose range 20 µg/kg–200 mg/kg BPA (five orders of magnitude). Third, there were higher SDs for the DSP values in the control and the low (unaffected) BPA dose groups in the studies reported by Sakaue et al. than for those groups where DSP was reduced by BPA. The lower SD values in the last groups were also similar in magnitude to all of the DSP SD values seen in the present study (Fig. 1Go). These observations led us to compare our DSP values, and those of Sakaue et al.(2001)Go, with rat DSP values reported by our two laboratories, and by other investigators, over the past seven years (Fig. 2Go and Table 7Go). Included in Table 7Go are the results from a preliminary evaluation of BPA using Holtzman rats (Sakaue et al., 1999Go). The report of that study was only available as an abstract in Japanese; however, the data were made available to us for use in Table 7Go and Figure 2Go by the authors.



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FIG. 1. Compilation of the DSP data generated in adult SD rats following exposure to BPA. Those columns entered for the two studies performed by Sakaue et al. (2001; black and gray columns)Go were based on their published figures as well the mean ± SD data, which were kindly provided by the authors. These data were recalculated using the same time transit factor as employed herein (i.e., 6.1 instead of 6.3 as used by Sakaue et al.). The four studies reported herein are shown as open columns. Group sizes shown above the control entries applied equally to the appropriate test groups except that test groups of 10 were used in the two studies with 20 control animals. The hatched bars show the data from the final experiment where the three diets alone were compared. {ddagger}p < 0.01 when 5002 control data of Experiment 2 were compared to the RM3 control data of Experiment 1. *Reported to be statistically significantly different from concomitant control values.

 


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FIG. 2. Comparison of control DSP data as described herein and previously for our laboratory, with those for the laboratory of Tohyama (Ohsako and Sakaue). Three strains of rat were used in the studies shown above; Alderley Park (black columns), Sprague-Dawley (dark gray columns), and Holtzman (white columns). The hatched column is the mean + SD for all historical data given in Table 7Go with the exception of the shaded entries in the table. All data are shown as the mean + SD. All DSP calculations were based on the transit factor of 6.1. Thus, the data of Sakaue et al.(1999, 2001)Go and Ohsako et al.(2001)Go, which were published using a transit factor of 6.3, have been recalculated using 6.1 for the purpose of this figure.

 

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TABLE 7 Comparison of Control DSP Values for Previously Published Data
 
The DSP values shown for the three studies abstracted at the top of Table 7Go (in italics) are dramatically different from the 34 other literature values shown. A possible explanation for these high values may be the unusual method of tissue homogenization used. Whatever, they are so different to the rest of the values that they were excluded from the mean values shown at the foot of Table 7Go. The mean values for DSP/testis among control animals from these 34 literature citations (31.8 ± 8.6 [mean ± SD]) is independent of the homogenization instrument employed (Waring 29.15 ± 2.8; Polytron 30.8 ± 12.4). Figure 2Go shows the control DSP/testis data of immediate relevance to interpretation of the present results. On the right side of Figure 2Go are shown the control DSP values reported by Sakaue et al. and the two other sets of DSP values reported by that laboratory over the past two years. The left side of Figure 2Go illustrates the control DSP values reported in the present studies, together with values we have reported over the past five years. On the extreme left is shown the mean of the 34 literature citations shown in Table 7Go. The data shown in Figure 2Go indicate that the DSP values reported by Sakaue et al. are higher and more variable than is general in the literature, and this may provide a lead to future understanding of the difference in test outcomes for BPA. Inspection of Table 7Go also reveals an interesting point relating to the manner in which DSP data are reported. The most commonly used unit is DSP/testis. However, changes may have occurred in testes weights during a study, and this leads most investigators also to express their data as DSP/g testis. Comparison of the overall mean DSP/testis ± SD (31.8 ± 8.6) for the entries in Table 7Go with the overall mean DSP/g testis ± SD (18.5 ± 3.5) demonstrates that the variability in DSP/g testis is considerably less than the corresponding variability in DSP/testis. In addition, the coefficient of variation is also reduced. This reduced variability suggests that DSP/g testis may be more sensitive than DSP/testis for detecting possible chemical effects on DSP.

Other instances of unexplained variability in control animal ED parameters possibly affecting the outcome of ED studies have been described (e.g., Ashby, 2001Go; Ashby et al., 1999Go; Odum and Ashby, 2000Go; Sharpe et al., 1998Go; Tinwell et al., 1999, 2002Go; Welsch et al., 2001Go). Probably the most relevant of these citations concerns the reported trophic effect of BPA on the mouse prostate gland. In that case the weight of the control prostate glands in the original study (Nagel et al., 1997Go) were lower than in our repeat study that failed to confirm this property of BPA (Ashby et al., 1999Go). However, in an earlier study from the laboratory of Nagel et al., control prostate weights higher than those reported by Ashby et al. had been described (Ashby, 2001Go; Nonneman et al., 1992Go,). Resolution of such inconsistencies among control groups should perhaps act as a prelude to resolution of different test outcomes from studies of low-dose chemical-induced ED effects.

We currently have no explanation for the different test outcomes between the present studies and those of Sakaue et al. (2001)Go. The only possibility that we have not explored (for practical reasons) is that the SD rats used in our two laboratories have subtly different genetic make-ups leading to different sensitivities to BPA. Charles River IGS SD rats were used in the present study. These IGS SD rats are available in Japan, but the studies by Sakaue et al. employed Jcl:SD rats obtained from Japan CLEA. These latter were derived over 30 years ago by Japan CLEA from the original Charles River Cr:CD(SD) colony. If such subtle intrastrain differences were shown to account for the different assay outcomes on BPA it would solve one problem, but create a more serious one for the conduct and international comparability of chemical toxicity evaluations.


    ACKNOWLEDGMENTS
 
We are grateful to Professor C. Tohyama and Dr. S. Ohsako for many helpful discussions of their data, and for provision of some of their primary test data.


    NOTES
 
1 To whom correspondence should be addressed. Fax (0) 44 1625 590996. E-mail: john.ashby{at}syngenta.com. Back

The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Cagen, S. Z., Waechter, J. M., Jr., Dimond, S. S., Breslin, W. J., Butala, J. H., Jekat, F. W., Joiner, R. L., Shiotsuka, R. N., Veenstra, G. E., and Harris, L. R. (1999). Normal reproductive organ development in wistar rats exposed to bisphenol A in the drinking water. Reg. Toxicol. Pharmacol. 30, 130–139.[CrossRef][ISI][Medline]

Elbetieha, A., Da’as, S. I., Khamas, W., and Darmani, H. (2001). Evaluation of the toxic potentials of cypermethrin pesticide on some reproductive and fertility parameters in the male rats. Arch. Environ. Contam. Toxicol. 41, 522–528.[CrossRef][ISI][Medline]

El-Sabeway, F., Wang, S., Overstreet, J., Miller, M., Lasley, B., and Enan, E. (1998). Treatment of rats during pubertal development with 2,3,7,8-tetrachlorodibenzo-p-dioxin alters both signalling kinase activities and epidermal growth factor receptor binding in the testis and the motility and acrosomal reaction of sperm. Toxicol. Appl. Pharmacol. 150, 427–442.[CrossRef][ISI][Medline]

Ema, M., Fujii, S., Furukawa, M., Kiguchi, M., Ikka, T., and Harazono, A. (2001). Rat two-generation reproductive toxicity study of Bisphenol A. Reprod. Toxicol. 15, 505–523.[CrossRef][ISI][Medline]

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