* Syngenta Central Toxicology Laboratory, Alderley Park, Cheshire, SK10 4TJ, United Kingdom; and
National Institute of Environmental Health Sciences, Alexander Drive, Research Triangle Park, North Carolina 27709
Received February 8, 2002; accepted April 2, 2002
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ABSTRACT |
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Key Words: bisphenol A; Sprague-Dawley rats; Alderley Park rats; sexual development; in utero exposure.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Animals and housing.
Young (8 weeks old) pregnant female rats were obtained either from Harlan Olac UK (SD rats; 185 ± 20 g) or from the Barriered Animal Breeding Unit, AstraZeneca, Alderley Park, Macclesfield UK (AP rats; 230 ± 20 g) and allowed 5 days acclimatization prior to dosing. All animals were individually housed in plastic solid bottomed cages with sawdust (Wood Treatments Ltd.; Macclesfield, Cheshire, UK) and shredded paper as bedding (SI Supplies, Poynton, Cheshire, UK). All animals were subjected to a 12-h light/dark schedule and controlled humidity and temperature. The females were allowed Rat and Mouse No. 3 (RM3) breeding diet (Special Diet Services Ltd.; Witham, Essex, UK; 18.5% soya content) and water ad libitum. The females were maintained on RM3, which is specifically designed for breeding, lactation, and growth of young stock, until weaning was complete. Pups were maintained on Rat and Mouse No 1 (RM1) diet (Special Diet Services Ltd.; Witham, Essex, UK; 6.5% soya content) following weaning.
Group allocation and dosing.
Animals were weighed upon arrival in the laboratory (before acclimatization) and again on GD 5. Females (n = 7/group) were placed into 1 of 5 groups based primarily on the rank ordering of the body weight gains between GD 0 and GD 5, while also ensuring that there were no significant differences in group mean body weight at GD 5.
The pregnant females were dosed by gavage from GD 6 through GD 21 with daily doses of either AO (Group 1), 20 µg/kg BPA in AO (Group 2), 100 µg/kg BPA in AO (Group 3), 50 mg/kg BPA in AO (Group 4), or EE in AO (Group 5) using a dosing volume of 10 ml/kg body weight. The doses selected for evaluation were as described by Chahoud and colleagues (Table 1). Analyses of the BPA dosing solutions as described above indicated that the actual concentrations were 24 µg/kg; 109 µg/kg, and 50.65 mg/kg for those used to dose the AP rats and 23 µg/kg, 108 µg/kg, and 49.15 mg/kg for those used to dose the SD rats. The dose of EE (200 µg/kg body weight) caused vaginal bleeding coupled with mild signs of toxicity (e.g., piloerection, hunched stance) and body weight loss (
3% for AP females and
1.4% for SD rats) during the first 9 days of dosing, a time when the pregnant control animals were gaining weight. The daily dose level was therefore reduced to 100 µg/kg on GD 11 for AP rats and on GD 14 for SD females, dependent on when toxicity was first observed.
Fresh dosing solutions were prepared on GD 6, 7, 9, 13, 16, and 19 for the AP rats and on GD 6, 8, 11, 14, 17, and 19 for the SD rats. Samples of all dosing solutions were stored at -70°C for further analysis.
Littering and weaning.
Parturition occurred between GD 21 and GD 23, with the majority occurring on GD 22. Those females that started to litter on GD 21 did not receive a final dose of compound. The pups in each litter were counted, sexed, weighed, and the anogenital distance (AGD) measured 24 h after birth (defined as postnatal day [PND] 1). Five days after birth (defined as PND 5) all pups were weighed again and each litter was culled to give a combination, where possible, of 4 males and 4 females, and where not possible, a maximum of 8 pups/litter. Culling took no account of pup size, although one AP male pup was removed from the study, as it was significantly smaller than its littermates (body weight of 3.7 g compared to a mean bodyweight of 9.7 ± 1.6); this rat would probably not have survived for the duration of the study. All litters and dams were maintained on RM3 and were not handled again until weaning (PND 23). At weaning, littermates were identified by a unique number, weighed, and then group-housed according to sex. All pups were placed on RM1 diet and water ad libitum at this time.
Weighing of pups, monitoring of puberty, and termination.
Pups were weighed at weaning (PND 23) and then every third day until termination (PND 9091 for males and PND 98 for females). The onset of vaginal opening (VO) was monitored daily from weaning and the body weight for each female was recorded on the day that this was observed. As soon as VO was observed vaginal smears were taken from each female and assessed for first estrus (defined as the first day on which only cornified epithelial cells were observed on a vaginal smear). Preputial separation (PPS) was monitored daily in all males from PND 35 and body weights were recorded on the day that this occurred.
All females were terminated on PND 98 by an overdose of fluothane followed by cervical dislocation; each animal being weighed just prior to termination. The liver and organs of the reproductive tract (vagina, cervix, uterus, and ovaries) were removed and weighed. The vagina and the uterus were fixed in Bouins for histopathological examination as described by Talsness et al. (2000a,b). All males were terminated at PND 90 as described above. Body weights were recorded just prior to termination. Liver, testes, epididymides, seminal vesicles, and ventral prostate were removed and weighed. The left testis and, in the case of the SD rats, both epididymides, were fixed in Bouins for histopathological examination as described by Fialkowski et al.(2000). The prostate, as well as the decapsulated right testis, was flash frozen in liquid nitrogen and stored at -80°C for further investigation.
Termination of dams.
All dams that had littered were terminated as described above following weaning. Any dams that had not littered were terminated on either GD 24 or GD 25 and pregnancy confirmed by staining the uterus with 10% ammonium polysulphide to determine the presence of implantation scars (Salewski, 1964). Two SD dams exposed to EE were terminated before the end of dosing because of severe vaginal bleeding. At parturition, one AP rat and one SD rat were terminated because of difficulties encountered during littering. In these cases the females were terminated as described above and the contents of the uterus examined for the presence of fetuses and the number of macroscopically identifiable implantation sites.
Calculation of daily sperm production.
Counts of homogenization resistant sperm were determined as described previously (Ashby et al., 1997) using the method ofBlazak et al.(1993). 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) with a Waring blender. The number of released sperm heads was determined in duplicate with an improved Neubauer haemocytometer. The number of sperm present in the area of the counting chamber 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 derived with the transit time factor of 6.1 (Blazak et al., 1993
).
Statistical analyses.
ANOVA procedures were used to assess the impact of BPA and litter (dam) effects on the variables of interest. In some cases, the variance-stabilizing logarithmic transformation was used in these analyses. Analysis of covariance (ANCOVA) procedures (Snedecor and Cochran, 1980) were used for organ weights to adjust for differences in body weight. Pairwise comparisons were made by Dunnetts test (Miller, 1966
). The statistical analyses shown for the data are using the litter as the statistical unit. However, analyses were performed using the grouped individuals as the unit, and that revealed no additional statistically significant changes from those found using the litter as the statistical unit.
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RESULTS |
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DISCUSSION |
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The repeat study reported here deviated from that reported by Chahoud and colleagues in two ways. First, AO was employed as a common vehicle while Chahoud used corn oil for the EE group and culinary cornstarch for the control and BPA groups. Second, an intermediate termination time of PND 9091 (males) and PND 98 (females) was chosen based on the repeat observations made by Chahoud at PND 70 and 170. These intermediate termination times were fully compatible with the endpoints being assessed and the reports being evaluated, and the use of a single sampling time enabled larger group sizes to be used while reducing animal usage. We also paralleled the study in SD rats with our own strain of Wistar-derived AP rats to enable us to relate any positive findings in SD rats to our own strain of animals (Wistar and SD rats are primarily used internationally in regulatory reproductive toxicity studies).
The dose of EE used by Chahoud and colleagues (200 µg/kg) was very high given that the EC10 value for EE in oral rat uterotrophic assays is 0.5 µg/kg (Kanno et al., 2001
). In the present experiments, the 200 µg/kg dose of EE gave gross evidence of reproductive impairment in the form of a high incidence of fetal resorptions. There were also effects at the level of the litter on prostate and right epididymis weights and an advance in the time of VO. However, among all of the endpoints evaluated for the 3 doses of BPA, the only statistically significant effects observed were a reduction in sperm count and DSP and a delay in VO for the 50 mg/kg dose of BPA in AP rats. This dose of BPA is within the active uterotrophic assay dose range for BPA (Ashby, 2001
) and represents an unexceptional observation beyond providing another example of a strain difference in the response of rats to BPA (Long et al., 2000
).
The general absence of effects for BPA in the present study is in clear contrast to the results reported by Chahoud and his colleagues (Table 1). Those investigators observed positive results for most of the endpoints determined and for all of the doses of BPA and EE evaluated. While some of these changes were consistent across the 5 test groups, some were not. Instances of changes that were not dose-related across the 3 doses of BPA were the day of PPS, male organ weights at PND 70, and progesterone levels in diestrus (Table 1
). Interpretation of the often subtle and sometimes nondose-related changes shown in Table 1
must be influenced by the values for individual parameters recorded for concurrent and historical control animals. However, no concurrent control animals were available in the studies by Chahoud and colleagues (NTP, 2001
).
The present results, within the context of those shown in Table 2, add to earlier examples of endocrine toxicity data for some other chemicals not being capable of independent confirmation (e.g., Ashby, 2000
; NTP, 2001
; Sharpe et al., 1998
). Such conflicts of findings may eventually influence the interpretation of results observed for chemicals after their routine assessment for endocrine toxicity. Two separate influences may contribute to such conflicts of findings, and these are worthy of study. First, some manifestations of endocrine toxicity may be intrinsically dependent on subtle and unspecified conditions of the bioassay (for example, diet, housing conditions, noise levels, degree of animal handling, strain of animal, and changes in body weight). In these situations genuine positive responses will exist that are difficult to reproduce in apparently similar, but critically different, experiments. Second, the multiplicity of biological endpoints typically evaluated and the intrinsic variability among animals for these endpoints may result in some instances of statistical false positives. Pending the resolution of this problem of data reproducibility, adequate intra- and interlaboratory confirmation of endocrine toxicities is indicated, especially in cases of new and subtle effects being observed. Further, attention should be given to the use of adequate concurrent control groups and the compilation of historical control data against which to assess the biological significance of new findings.
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NOTES |
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