Natural variability and the influence of concurrent control values on the detection and interpretation of low-dose or weak endocrine toxicities.

While defining the no effect level for the 5[alpha]-reductase inhibitor finasteride in the Hershberger assay, we encountered an inverted-U low-dose trophic effect on the prostate gland of the rat. Two attempts to confirm this observation were unsuccessful, and we concluded that the positive effect initially observed was associated with normal biologic variability. During the same period we attempted, unsuccessfully, to repeat our own observation of weak uterotrophic activity in the rat for the sunscreen 3-(4-methylbenzylidene)camphor (4MBC). Further evaluation led us to conclude that 4MBC is uterotrophic only when the control uterine weights are at the low end of their normally encountered range. This led us to reevaluate our earlier mouse uterotrophic assay data for bisphenol A (BPA). Originally we had concluded that BPA gave irreproducible evidence of weak uterotrophic activity, but upon ordering the eight experiments we had conducted, according to decreasing control uterine weight, we confirmed reproducible weak uterotrophic activity for BPA when the control uteri were at the low end of their normal range. In this article, we describe these observations, together with a reanalysis of the data associated with several reported instances of weak or low-dose endocrine effects that have proven difficult to confirm in independent laboratories. These include the activity of BPA on the CFI mouse prostate; the activities of BPA, octylphenol, and nonylphenol on the rat testis; and the effect of polycarbonate caging on control mouse uterine weight. In all of these cases, variability among controls provides a major obstacle to data interpretation and confirmation. Our recommendations on experimental design are also presented, with a view to ending the current impasse on the reality, or otherwise, of low-dose or weak endocrine toxicities. Key words: 3-(4-methylbenzylidine)camphor, bisphenol A, endocrine disruption, finasteride, low dose, nonylphenol. Environ Health Perspect 112:847-853 (2004). doi:10.1289/ehp.6862 available via http://dx.doi.org/[Online 18 February 2004]

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Two issues in the field of chemically mediated endocrine disruption (ED) are the possible occurrence of ED effects at dose levels lower than the no effect level defined by classical toxicity evaluations, and how to interpret weak chemically induced ED effects that lie within the range of normally encountered control variability (Ashby 2003). We have studied several reports of low-dose ED effects over the past 6 years, so far without being able to confirm any of them; these chemicals include butylbenzylphthalate (BBP) (Ashby et al. 1997; Sharpe et al. 1995, 1998); bisphenol A (BPA) (Ashby et al. 1999, 2003; Nagel et al. 1997; Sakaue et al. 2001; Talsness et al. 2000; Tinwell et al. 2002a); and nonylphenol (NP) (Colerangle and Roy 1996; Lee 1998; Odum and Ashby 2000; Odum et al. 1999). In contrast, we were able to confirm (Tinwell et al. 2002b) the weak uterotrophic activity of 3-(4-methylbenzylidene)camphor (4MBC) reported by Schlumpf et al. (2001a, 2001b), despite the inability of Bolt et al. (2001) to confirm this activity.

Recently, we encountered two instances that may throw light on the origins of these reproducibility problems. The first of these was the observation of an apparent inverted-U low-dose effect for the 5[alpha]-testosterone reductase inhibitor finasteride (FIN) in the Hershberger antiandrogen assay, an effect we were unable to reproduce. The second was our inability (after publication of Tinwell et al. 2002b) to reconfirm our own observation of uterotrophic activity for 4MBC. In each case, further studies revealed that control variability between experiments was probably at the root of these reproducibility problems. This conclusion led us to reevaluate earlier findings for BPA in the mouse uterotrophic assay (Tinwell et al. 2000) and to analyze earlier reports of low-dose effects for BPA (Howdeshell et al. 2003; Nagel et al. 1997; Nonneman et al. 1992; Sakaue et al. 2001) and NP (Lee 1998).

Materials and Methods

Chemicals. We obtained testosterone propionate (TP) and FIN from Sigma Chemical Company (Poole, Dorset, UK). Tocopherol-stripped corn oil (ICN Biomedicals Inc., Basingstoke, Hampshire, UK) was used to suspend FIN and TP by homogenization (Ashby 1987). We obtained 4MBC (CAS Registry No. 38102-62-4; Merck batch TT805785 029) from ChemQuest (Wilmslow, Cheshire, UK) and diethylstilbestrol (DES) from Sigma Chemical Company. Antarelix (ANT) was a gift from Europeptides (Argenteuil, France). We used arachis oil (AO; Sigma Chemical Company) as the vehicle for 4MBC (homogenized as described previously; Tinwell et al. 2002b), DES, and ANT. All chemicals were stored at room temperature except ANT, which was stored at 4[degrees]C. All preparations were shaken vigorously just before and during dosing. All experiments were conducted according to U.K. Home Office guidelines as described in the appropriate Home Office license for animal use.

Hershberger assays. Animals. Male Alpk:APfSD rats (animal breeding unit at AstraZeneca Pharmaceuticals, Macclesfield, Cheshire, UK) were castrated at 6 weeks of age via midline incision followed by 8 days of recovery. In each study, the animals were randomized into groups of six rats of approximately the same initial mean body weight and range. They were group housed at a maximum of six per cage in solid-bottomed polypropylene cages containing sawdust (Wood Treatments Ltd., Macclesfield, Cheshire, UK) and shredded paper as bedding. Fun tubes and houses were provided as environmental enrichment. All animals were given RM1 diet (Special Diet Services Ltd., Witham, Essex, UK) and water ad libitum for the duration of the experiment.

Study design. The experimental method has been described previously (Ashby and Lefevre 2000). Rats were given 10 consecutive daily treatments consisting of a subcutaneous (sc) injection of either the corn oil vehicle or TP (0.4 mg/kg) and an oral dose of either vehicle or the relevant dose of FIN. We used 5 mL/kg and 2 mL/kg dosing volumes for oral and subcutaneous administrations, respectively.

The animals were killed 24 hr after the last treatment by an overdose of halothane (Concord Pharmaceuticals Ltd., Essex, UK). The following tissues were removed and weighed at necropsy: liver and combined kidneys (to 1 mg), Cowper's glands, levator ani/bulbo cavernosus muscles (LA/BC), seminal vesicles (with coagulating gland), ventral prostate, glans penis, and combined adrenal glands (all to 0.1 mg). Changes in tissue weights were evaluated for statistical significance using analysis of variance (ANOVA) and analyses of covariance (ANCOVA) with final body weight.

Uterotrophic assays. Animals and dosing. The experimental methods were as described previously (Odum et al. 2001). Female Alpk:APfSD (Wistar derived) rats were obtained from the barriered animal breeding unit (AstraZeneca). These were 19-20, 20-21, 21-22, or 22-23 days of age at the beginning of the study. The animals were housed up to a maximum of six per cage as described for the Hershberger assay. Each female received an sc injection of the appropriate compound on 3 consecutive days and was killed 24 hr after the last dose. On the basis of previously published data, 4MBC was given at 1 g/kg (Tinwell et al. 2002b) and ANT was given at 300 [micro]g/kg (Odum et al. (2001); 5 [micro]g/kg DES was used as a positive control. We used a dosing volume of 5 mL/kg body weight when animals were exposed to a single compound. However, in studies in which the animals were exposed to test compound and ANT, we used a dosing volume of 2.5 mL/kg body weight. Females were exposed to ANT 15 min before receiving the appropriate test compound (AO, 4MBC, or DES).

Study design. 4MBC had previously been shown to induce a significant increase in uterine weight in immature rats (Tinwell et al. 2002b). Thus, the initial experiment in the present study was designed to repeat those original observations. We also included additional groups of animals exposed to 4MBC together with the gonadotropin-releasing hormone (GnRH) antagonist ANT. In this experiment, 4MBC failed to significantly increase the uterine weight; however, the control uterine weights were greater than usual. Therefore, we repeated the experiment. In the final two experiments, we used animals of different ages at the start of the uterotrophic assays. All animals were killed 24 hr after the last dose by an overdose of fluothane followed by cervical dislocation. The uterus was removed, trimmed free of fat, gently blotted, and weighed. It was then placed in a preweighed vial, dried overnight (24 hr) at 70[degrees]C, and reweighed. All data were assessed for statistical significance using a two-sided Student's t-test.

Results

Hershberger assays. The assays for FIN described in this article represent a continuation of previous experiments (Ashby and Lefevre 2000; Blohm et al. 1986) and studies conducted as part of the current Organisation for Economic Co-operation and Development (OECD) validation of the Hershberger assay. The OECD studies demonstrated that a dose of FIN as low as 200 [micro]g/kg/day inhibited the growth of the seminal vesicles, ventral prostate, and glans penis of TP-treated rats (J. Ashby, unpublished data). Subsequently, we have shown that doses of FIN as low as 8 [micro]g/kg/day are active in the assay (Ashby et al. 2004), with the prostate gland being the most sensitive tissue (Figure 1). The present experiments were designed to determine the no effect level for the inhibitory effect on the growth of the prostate in TP-treated animals. In all of the experiments, a dose of 5,000 [micro]g/kg/day FIN significantly inhibited the growth of the seminal vesicles, ventral prostate, Cowper's glands, and glans penis compared with animals administered only TP (Table 1). In one study (experiment 1, Table 1) growth of the LA/BC was also significantly inhibited.

[FIGURE 1 OMITTED]

In the first low-dose experiment (experiment 1, Table 1), doses ranging from 5,000 [micro]g/kg/day to 8 [micro]g/kg/day FIN were all clearly active in the assay. The lowest dose tested (8 [micro]g/kg/day FIN) affected all tissues except the LA/BC and the Cowper's glands. In experiment 2 (Table 1), the only tissues significantly inhibited at 8 [micro]g/kg/day FIN were the seminal vesicles and glans penis. The glans penis was reduced in weight at all dose levels of FIN down to 0.1 [micro]g/kg/day (Figure 2). However, the ventral prostate weight was significantly increased (p < 0.01) for the 2 [micro]g/kg/day dose of FIN. In attempts to repeat this inverse prostate weight dose response (Figure 1), we conducted two further experiments (experiments 3 and 4, Table 1) using a tighter dose range of FIN around the dose that had increased prostate weight in experiment 2. In the first of these repeat studies (experiment 3, Table 1), the only significant weight change observed was an increase in the weight of the Cowper's glands at 5 [micro]g/kg/day FIN. This increase is probably associated with biologic variation and is probably not biologically significant (compare values for Cowper's glands weights in experiments 1-4, Table 1). However, seemingly in support of the previous experiment, we observed an inverted-U shaped response for increases in prostate weight around the 2 [micro]g/kg/day dose of FIN, but none of the effects were statistically significant (Figure 1). In experiment 4, we did not observe the same pattern in the increase of the Cowper's glands weight seen in the experiment 3 (Table 1), and no change in prostate weight was observed below the 5,000 [micro]g/kg/day positive control dose of FIN (Figure 1). In that final experiment, we observed a statistically significant reduction in the weight of the seminal vesicles at 5 [micro]g/kg/day FIN. Inhibition of the growth of the glans penis was not consistently observed in these experiments (Figure 2).

[FIGURE 2 OMITTED]

The data sets recorded for the prostate gland during the course of these experiments are shown in Figure 1, both in terms of the weight of the prostate gland relative to the concurrent control value (taken as 100%) and in terms of absolute prostate weights.

Variability in control tissue and body weights. We calculated a mean of each control tissue weight and of control body weights from the four experiments, yielding a new mean weight value for all controls (corn oil + TP) combined. These means, multiplied by the SD of the mean and divided by 100, yield a percentage of the variation in control weights between experiments. The control prostate weights showed the highest variability between experiments, as follows: prostate, 11%; Cowper's glands, 6%; seminal vesicles, 5%; glans penis, 4%; LMBC, 4%; liver, 1.8%; kidneys, 1.5%; body weight, 1.1%. The relatively high variability of all of the sex accessory tissues, compared with the liver and kidney weights and body weight, probably relates to the fact that the stimulatory dose of TP used in these Hershberger assays causes submaximal growth of the tissues, being just below the plateau-inducing dose of TP (Ashby and Lefevre 2000). Thus, small pharmacodynamic differences between experiments may yield small differences in the stimulatory dose of TP experienced by the tissues between experiments.

Uterotrophic assays. The data for the uterotrophic studies of 4MBC are presented in Table 2. We have previously shown 4MBC to be reproducibly active in the immature rat uterotrophic assay in this laboratory (Tinwell et al. 2002b) (experiment 1, Table 2). However, in the initial repeat study (experiment 2, Table 2), 4MBC failed to induce a significant increase in uterine weight. In this experiment the control uterine weights were heavier than usual: 35.1 [+ or -] 5.9 compared with 23.3 [+ or -] 4.3 (experiment 1, Table 2; Tinwell et al. 2002b); thus, a repeat of this study was undertaken (experiment 3, Table 2). In experiment 3, control uterine weights were within our normal range, and 4MBC induced a weak, although significant (p < 0.01), increase in uterine weight. This activity was abolished by the GnRH inhibitor ANT, suggesting that the uterotrophic effects were mediated centrally via a temporal advance in puberty rather than via a direct effect of 4MBC on the uterus. Two further experiments were therefore performed in which we investigated the effect of the initial age of the rat on the activity of 4MBC (experiments 4 and 5, Table 2). Although 4MBC led to an increase in uterine weight in experiments 4 and 5, the significance of the response diminished with the older animals. Thus, the response to 4MBC was highly significant (p < 0.01) when 19-20-day-old animals were used; this was reduced to p < 0.05 with 20-21-day-old animals, and the response was not significant when the older animals (21-22 and 22-23 days old) were used. These effects were reproducible between experiments 4 and 5 (Table 2).

Discussion

FIN inhibits the production of dihydrotestosterone from testosterone and consequently shows activity as a male rat developmental toxin with a lowest active dose of 10 [micro]g/kg/day (Bowman et al. 2003). FIN also shows reproducible antiandrogenic activity in the Hershberger assay (Hershberger et al. 1953) at doses of [greater than or equal to] 8 [micro]g/kg (Figure 1A; Ashby et al. 2004). The present studies initially demonstrated a statistically significant low-dose trophic effect on the prostate gland at 2 [micro]g/kg (experiment 2, Figure 1A), suggesting an androgenic, as opposed to antiandrogenic, effect. In the first attempted repeat of this trophic effect (experiment 3), we observed a statistically nonsignificant increase in prostate weight at the 2-[micro]g/kg dose, and in experiment 4 no effects were seen (Figure 1A). Because reproducibility of an effect is the primary criterion of its validity, we conclude that FIN does not show low-dose endocrine effects. In seeking an explanation for the initially observed low-dose trophic effect of FIN, we noticed that control prostate weights were the most variable of all of the tissue weights recorded in the Hershberger assay: 11% variance for prostate compared with values as low as 1.1% for body weights (see "Variability in control tissue and body weights" in "Results"). This variability is evident in Figure 1B, which shows absolute prostate weights for the series of experiments with FIN. Thus, the control prostate weight in experiment 1 is essentially the same as the statistically increased prostate weight recorded for the FIN group in experiment 2. This variability in control prostate weights raises the strong possibility that the significant effect seen for the low dose of FIN was a chance observation occurring within the normal interexperimental control range. Similarly, although the variability in control glans penis weights (4%) was lower than that for the prostate (11%), it is probable that the nonreproducible decreases in glans penis weight also induced by low doses of FIN in experiment 2 were associated with normal biologic variability (Figure 2). The key points from these data for FIN are that the increase in prostate weight and the decreases in glans penis weights induced by FIN were all within the control range encountered for these tissues in the present experiments, and the effects induced were not reproducible.

The uterotrophic activity of 4MBC (Table 2, Figure 3) was dependent upon the weight of the control uteri in the relevant experiment. There is no way to select for low control uterine weights because there is not a predictive correlation between initial body weight and uterine weights observed 4 days later (Table 2). Thus, it is a matter of chance whether an experiment has control uteri in the low weight range thereby giving rise to a positive response induced by 4MBC or whether the control uteri will be in the high range in which case 4MBC will be negative. The data shown in Figure 3 confirm, as expected, that the uterus grows marginally during postnatal days (PND) 19-25, before the major growth phase starting at puberty (around PND 29-30). By the time the maximum uterine weight for this prepubertal growth is reached (~40 mg), the assay is no longer able to detect 4MBC as uterotrophic. The fact that the prepubertal growth of the uterus, and the uterotrophic activity of 4MBC, is abolished by GnRH inhibition indicates that both effects are centrally mediated, perhaps via modulation of centrally controlled release of low levels of estradiol from the ovaries or the adrenal glands. These data therefore indicate that there are two mechanisms by which an agent can induce an uterotrophic response. The first mechanism, as illustrated by the reference estrogen DES, involves direct action of the estrogen on the uterus leading to maximal growth of the tissue (from 20 to > 100 mg; Table 2). This mechanism, as expected, is not influenced by coadministration of the GnRH inhibitor ANT. The second mechanism, as illustrated by 4MBC, involves the test agent influencing the centrally mediated stimulation of prepubertal growth of the uterus. This activity is blocked by GnRH inhibition, and the magnitude of the uterotrophic effect is limited to the maximum control uterine weight that can be encountered in prepubertal animals (~40 mg).

[FIGURE 3 OMITTED]

The fact that the activity of 4MBC in the uterotrophic assays was dependent upon the concurrent control uterine weight led us to reevaluate our earlier data for BPA in the mouse uterotrophic assay (Tinwell et al. 2000) in which eight uterotrophic assays were described for the BPA dose range of 0.02 [micro]g/kg to 300 mg/kg. Although we found some evidence of weak uterotrophic activity at some doses, none of the effects was consistently reproducible. The clearest example of this was for the 200-mg/kg dose of BPA, which gave four weak positive responses and four negative responses (Figure 4A). When those data are reordered from chronological order into the order of descending control uterine weights (Figure 4B), the results become consistent with the effects seen for 4MBC. Thus, the positive BPA results were weak and only became significant when the control uterine weights were low. Further, the magnitude of the effects induced by BPA remained constant: the decreasing control values created the statistically significant effects for BPA.

[FIGURE 4 OMITTED]

These data for FIN, 4MBC, and BPA indicate that concurrent control values and interexperimental variation in control levels can influence the interpretation of weak endocrine toxicities and thereby affect the qualitative outcome of bioassays. This conclusion is possibly relevant to several other instances of low-dose or weak endocrine effects that have proven resistant to independent confirmation. For example, Sharpe et al. (1995) reported that octylphenol (OP) and BBP were able to reduce rat testes weights. We were unable to confirm those effects for BBP (Ashby et al. 1997), and this led Sharpe et al. to repeat their evaluation of OP (Sharpe et al. 1998). In that repeat study, the control testes weights had fallen and OP was no longer active (Sharpe et al. 1998; Figure 5). Uncertain of the origins of these effects, these researchers ran a further control group and recorded control testes weights similar to their original 1995 study (Figure 5; Sharpe et al. 1998). They were unable to account for this variability in control testes weights, and the activity of BBP and OP in the assay remains unconfirmed. A similar situation exists for the effects of NP on the rat prostate gland (Lee 1998). Lee described a study in which NP reduced prostate weight and in which access to five identical control groups was possible. In the first four of these, control prostate weights were approximately 60 mg/100 g body weight, but in the fifth study this value was significantly lower (~40 mg/100 g body weight; Figure 6). Lee (1998) did not comment on this unexpected and statistically significant change in control prostate weight, which itself led to the conclusion that NP was uniquely inactive in that fifth experiment (Figure 6). We were subsequently unable to show any effects for NP (Odum and Ashby 2000) when tested under the conditions described by Lee (1998), and the control prostate weights were similar to the first four studies reported by Lee (1998).

[FIGURES 5-6 OMITTED]

A further example of how the variability of control prostate weights can influence the interpretation and confirmation of chemically induced effects is illustrated by the activity of BPA on the CF1 mouse prostate gland (Figure 7). Nonneman et al. (1992) described the effect of intrauterine position on the subsequent weight of the prostate gland of CF1 mice. Subsequently, Nagel et al. (1997), from the same laboratory, reported the ability of BPA to increase the weight of the mouse prostate gland, with the implication that the estrogenic activity of BPA had simulated the presence of female fetuses being next to male fetuses in utero. Cagen et al. (1999) and Ashby et al. (1999) were unable to confirm those effects of BPA, despite the fact that Cagen et al. (1999) recorded control prostate weights as low as those of Nagel et al. (1997). Within the present context, the most interesting aspect of the combined data shown in Figure 7 is the variability of the control prostate weights between laboratories and, most particularly, within the laboratory of Nonneman and Nagel. Again, the most interesting questions are what factors influence the unexplained variations in control prostate weights, within and between laboratories, and how these variations influence interpretation of the effects seen for BPA.

[FIGURE 7 OMITTED]

A further example of the importance of control variability is illustrated by the reports of weak effects induced by BPA on rat testes (Sakaue et al. 2001; Talsness et al. 2000). We were unable to confirm the studies of either Sakaue et al. (Tinwell et al. 2002) or Talsness et al. (Ashby et al. 2003) after extensive studies. The results reported by Sakaue et al. (2001) and related results from the same laboratory (Ohsako et al. 2001; Sakaue et al. 1999) enable reference to four sets of control observations for rat daily sperm production (DSP) (Figure 8). The most marked reduction in DSP induced by BPA in the two experiments was reported by Sakaue et al. (2001) (Figure 8). However, neither of these effects was significantly different from the control DSP value reported in the same year by Ohsako et al. (2001; bar 2, Figure 8). This therefore provides a further instance of unaccounted control variability enveloping chemically induced effects from another study conducted in the same laboratory. In four independent repeat studies (Ashby et al. 2003), we found that test and control DSP values were not significantly different from each other and were similar to the BPA values reported by Sakaue et al. (2001) (Figure 8). We also have established that diet was not the cause of the different assay outcomes (Ashby et al. 2003; Odum et al. 2001).

[FIGURE 8 OMITTED]

The remaining question posed by the low-dose studies on FIN is how to interpret effects that show a nonsignificant change, as illustrated by the nonsignificant increase in prostate weight seen for FIN (experiment 2, Figure 1). If the statistical methods used are appropriate, the absence of significance should indicate the absence of a chemically induced effect. An example of this problem is provided by Howdeshell et al. (2003), who discussed in detail a nonsignificant increase (p = 0.31) in uterine weight for CF1 mice housed in worn polycarbonate cages equipped with worn polycarbonate drinking bottles (Figure 9). Howdeshell et al. (2003) associated this effect with the BPA (~300 ng;mL) that was reported to leach from the cages during the course of the experiment, but it may equally have been related to the heavier body weights of the test animals at the start of the experiment (p = 0.17). The group sizes in that study were large (57/group), but this represented the pooling of three replicate experiments. It would have been useful to know the results of those three individual experiments to evaluate whether the nonsignificant increase in uterine weight was reproducible between replicates, especially given the problems we encountered (Tinwell et al. 2000) when evaluating the uterotrophic activity of BPA in mice.

[FIGURE 9 OMITTED]

Some endocrine-mediated toxicities are highly specific to the conditions of the test, as exemplified by the specificity of the effects induced by BPA in the vagina of F344 rats but not Sprague-Dawley rats (Long et al. 2000) and by the resistance of C57 mice, as opposed to CD1 mice, to the reproductive tract developmental effects of neonatal DES exposure (Couse et al. 2001). Such clear-cut specificities, usually defined in the same laboratory, are distinct from the several recent failures to confirm endocrine toxicities between laboratories when using ostensibly the same test protocol. We suggest that the latter instances can only be resolved, and avoided in future, if the following actions are taken:

* For the end points being studied, it is useful to have a historical database with which to compare current assay performance and test results. Inclusion of this database into publications would aid resolution of the problems described herein.

* Most laboratories experience variability in the end points they measure. Understanding the origin of these variations is a necessary precursor to concluding weak endocrine activity for test agents.

* When low-dose or weak endocrine effects are indicated, it is important to confirm the observations before publication.

* Unadjusted data from separate experiments should be shown, as opposed to pooled and adjusted data. Adjusted data are subsidiary to the original data.

* A distinction should be drawn between effects that lie within recent historical control levels and those that exceed those levels. Different mechanisms may operate in these two situations, leading to different approaches to data extrapolation.

* Use of appropriate statistical methods enables objective qualitative judgments to be made. Subjective discussion of statistically nonsignificant effects should be avoided.

Received 14 November 2003; accepted 18 February 2004.

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Talsness C, Fialkowski O, Gericke C, Merker H-J, Chahoud I. 2000. The effects of low and high doses of bisphenol A on the reproductive system of female and male rat offspring. Congenital Anom 40:S94-S107.

Tinwell H, Haseman J, Lefevre PA, Wallis N, Ashby J. 2002a. Normal sexual development of two strains of rat exposed in utero to low doses of bisphenol A. Toxicol Sci 68:339-348.

Tinwell H, Joiner R, Pate I, Soames A, Foster J, Ashby J. 2000. Uterotrophic activity of bisphenol A in the immature mouse. Regul Toxicol Pharmaco 132:118-126.

Tinwell H, Lefevre PA, Moffat G J, Burns A, Odum J, Spurway TD, et al. 2002b. Confirmation of uterotrophic activity of 3-(4-methylbenzylidine)camphor in the immature rat. Environ Health Perspect 110:533-536.

John Ashby, Helen Tinwell, Jenny Odum, and Paul Lefevre Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, United Kingdom

Address correspondence to J. Ashby, Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

The authors declare they have no competing financial interests.

Table 1. Tissue weights from castrated rats administered
corn oil (oral), corn oil + TP (0.4 mg/kg sc), or corn
oil +TP +FIN (oral), each for 10 consecutive days.

                                 Body weight
                              (mean [+ or -] SD)

Experiment   Treatment
no.          ([micro]g/kg)    Initial weight (g)

1            Corn oil alone   244.5 [+ or -] 13.0
             TP + corn oil    247.2 [+ or -] 8.6
             TP + FIN 8       242.8 [+ or -] 13.9
             TP + FIN 40      243.7 [+ or -] 13.4
             TP + FIN 200     246.5 [+ or -] 10.0
             TP + FIN 5,000   248.8 [+ or -] 10.2

2            TP + Corn oil    264.0 [+ or -] 8.6
             TP + FIN 0.1     258.7 [+ or -] 7.4
             TP + FIN 0.5     267.2 [+ or -] 9.2
             TP + FIN 2.0     261.5 [+ or -] 13.0
             TP + FIN 8.0     263.0 [+ or -] 4.6
             TP + FIN 5,000   264.0 [+ or -] 8.8

3            TP + Corn oil    255.7 [+ or -] 10.6
             TP + FIN 0.5     256.3 [+ or -] 12.6
             TP + FIN 1.0     252.3 [+ or -] 8.6
             TP + FIN 2.0     255.2 [+ or -] 5.9
             TP + FIN 5.0     256.8 [+ or -] 9.1
             TP + FIN 5,000   251.2 [+ or -] 8.1

4            TP + Corn oil    252.5 [+ or -] 10.5
             TP + FIN 0.5     252.7 [+ or -] 7.1
             TP + FIN 1.0     252.8 [+ or -] 7.2
             TP + FIN 2.0     250.3 [+ or -] 9.3
             TP + FIN 5.0     251.0 [+ or -] 10.6
             TP + FIN 5,000   254.8 [+ or -] 8.9

                                 Body weight
                              (mean [+ or -] SD)

Experiment   Treatment
no.          ([micro]g/kg)      Final weight (g)

1            Corn oil alone   305.5 [+ or -] 23.2
             TP + corn oil    342.5 [+ or -] 17.1
             TP + FIN 8       335.2 [+ or -] 18.9
             TP + FIN 40      335.8 [+ or -] 20.4
             TP + FIN 200     333.8 [+ or -] 18.8
             TP + FIN 5,000   338.2 [+ or -] 11.9

2            TP + Corn oil    349.2 [+ or -] 11.1
             TP + FIN 0.1     350.2 [+ or -] 13.8
             TP + FIN 0.5     354.0 [+ or -] 14.3
             TP + FIN 2.0     348.0 [+ or -] 24.9
             TP + FIN 8.0     351.2 [+ or -] 8.7
             TP + FIN 5,000   359.2 [+ or -] 14.2

3            TP + Corn oil    340.5 [+ or -] 18.5
             TP + FIN 0.5     339.2 [+ or -] 12.8
             TP + FIN 1.0     334.3 [+ or -] 14.2
             TP + FIN 2.0     342.8 [+ or -] 18.8
             TP + FIN 5.0     341.0 [+ or -] 22.4
             TP + FIN 5,000   332.8 [+ or -] 11.7

4            TP + Corn oil    343.3 [+ or -] 14.5
             TP + FIN 0.5     344.7 [+ or -] 6.2
             TP + FIN 1.0     341.5 [+ or -] 7.1
             TP + FIN 2.0     330.0 [+ or -] 18.9
             TP + FIN 5.0     336.5 [+ or -] 14.3
             TP + FIN 5,000   341.7 [+ or -] 14.3

                                Tissue weight
                              (mean [+ or -] SD)

Experiment   Treatment
no.          ([micro]g/kg)         Liver (g)

1            Corn oil alone   13.69 [+ or -] 1.53
             TP + corn oil    16.37 [+ or -] 1.28
             TP + FIN 8       15.88 [+ or -] 1.32
             TP + FIN 40      15.71 [+ or -] 1.22
             TP + FIN 200     15.34 [+ or -] 0.50
             TP + FIN 5,000   15.73 [+ or -] 1.13

2            TP + Corn oil    16.63 [+ or -] 0.82
             TP + FIN 0.1     16.98 [+ or -] 1.12
             TP + FIN 0.5     17.28 [+ or -] 0.98
             TP + FIN 2.0     16.82 [+ or -] 1.31
             TP + FIN 8.0     17.75 [+ or -] 1.03*
             TP + FIN 5,000   17.49 [+ or -] 0.64

3            TP + Corn oil    16.90 [+ or -] 1.41
             TP + FIN 0.5     16.44 [+ or -] 0.87
             TP + FIN 1.0     16.35 [+ or -] 1.35
             TP + FIN 2.0     17.20 [+ or -] 2.00
             TP + FIN 5.0     16.63 [+ or -] 1.83
             TP + FIN 5,000   16.10 [+ or -] 0.74

4            TP + Corn oil    17.05 [+ or -] 1.06
             TP + FIN 0.5     17.43 [+ or -] 0.70
             TP + FIN 1.0     17.23 [+ or -] 0.98
             TP + FIN 2.0     16.27 [+ or -] 1.51
             TP + FIN 5.0     16.99 [+ or -] 1.16
             TP + FIN 5,000   17.52 [+ or -] 0.86

                                 Tissue weight
                              (mean [+ or -] SD)

Experiment   Treatment
no.          ([micro]g/kg)        Kidneys (g)

1            Corn oil alone   2.22 [+ or -] 0.11
             TP + corn oil    2.45 [+ or -] 0.21
             TP + FIN 8       2.47 [+ or -] 0.17
             TP + FIN 40      2.34 [+ or -] 0.26
             TP + FIN 200     2.36 [+ or -] 0.14
             TP + FIN 5,000   2.38 [+ or -] 0.20

2            TP + Corn oil    2.47 [+ or -] 0.22
             TP + FIN 0.1     2.43 [+ or -] 0.11
             TP + FIN 0.5     2.51 [+ or -] 0.12
             TP + FIN 2.0     2.42 [+ or -] 0.10
             TP + FIN 8.0     2.53 [+ or -] 0.14
             TP + FIN 5,000   2.44 [+ or -] 0.08

3            TP + Corn oil    2.40 [+ or -] 0.22
             TP + FIN 0.5     2.30 [+ or -] 0.06
             TP + FIN 1.0     2.44 [+ or -] 0.13
             TP + FIN 2.0     2.49 [+ or -] 0.09
             TP + FIN 5.0     2.49 [+ or -] 0.21
             TP + FIN 5,000   2.34 [+ or -] 0.12

4            TP + Corn oil    2.48 [+ or -] 0.08
             TP + FIN 0.5     2.54 [+ or -] 0.11
             TP + FIN 1.0     2.44 [+ or -] 0.12
             TP + FIN 2.0     2.38 [+ or -] 0.09
             TP + FIN 5.0     2.40 [+ or -] 0.10
             TP + FIN 5,000   2.46 [+ or -] 0.22

                                Tissue weight
                              (mean [+ or -] SD)

Experiment   Treatment
no.          ([micro]g/kg)       Adrenals (mg)

1            Corn oil alone   64.2 [+ or -] 4.4
             TP + corn oil    52.0 [+ or -] 7.4
             TP + FIN 8       49.3 [+ or -] 4.2
             TP + FIN 40      53.7 [+ or -] 9.7
             TP + FIN 200     54.8 [+ or -] 4.8
             TP + FIN 5,000   53.5 [+ or -] 3.2

2            TP + Corn oil    53.4 [+ or -] 8.7
             TP + FIN 0.1     58.1 [+ or -] 8.8
             TP + FIN 0.5     61.4 [+ or -] 7.9
             TP + FIN 2.0     55.6 [+ or -] 7.2
             TP + FIN 8.0     50.0 [+ or -] 8.3
             TP + FIN 5,000   49.9 [+ or -] 8.6

3            TP + Corn oil    56.6 [+ or -] 11.1
             TP + FIN 0.5     56.5 [+ or -] 4.1
             TP + FIN 1.0     57.8 [+ or -] 2.8
             TP + FIN 2.0     54.4 [+ or -] 7.0
             TP + FIN 5.0     54.1 [+ or -] 5.4
             TP + FIN 5,000   53.3 [+ or -] 2.9

4            TP + Corn oil    61.5 [+ or -] 7.7
             TP + FIN 0.5     60.5 [+ or -] 10.3
             TP + FIN 1.0     60.1 [+ or -] 10.9
             TP + FIN 2.0     58.8 [+ or -] 6.4
             TP + FIN 5.0     56.5 [+ or -] 9.7
             TP + FIN 5,000   51.4 [+ or -] 8.2

                                 Tissue weight
                              (mean [+ or -] SD)

Experiment   Treatment
no.          ([micro]g/kg)          LA/BC (mg)

1            Corn oil alone   148.8 [+ or -] 18.2
             TP + corn oil    381.2 [+ or -] 35.6
             TP + FIN 8       353.1 [+ or -] 37.7
             TP + FIN 40      348.0 [+ or -] 20.7
             TP + FIN 200     317.2 [+ or -] 30.9 **
             TP + FIN 5,000   314.3 [+ or -] 51.5 **

2            TP + Corn oil    359.4 [+ or -] 76.5
             TP + FIN 0.1     366.8 [+ or -] 25.0
             TP + FIN 0.5     358.7 [+ or -] 41.7
             TP + FIN 2.0     367.1 [+ or -] 35.9
             TP + FIN 8.0     354.9 [+ or -] 45.5
             TP + FIN 5,000   333.7 [+ or -] 12.6

3            TP + Corn oil    345.3 [+ or -] 41.1
             TP + FIN 0.5     373.3 [+ or -] 30.6
             TP + FIN 1.0     333.8 [+ or -] 35.9
             TP + FIN 2.0     362.6 [+ or -] 29.2
             TP + FIN 5.0     365.0 [+ or -] 21.3
             TP + FIN 5,000   319.6 [+ or -] 32.9

4            TP + Corn oil    365.3 [+ or -] 31.5
             TP + FIN 0.5     360.4 [+ or -] 48.1
             TP + FIN 1.0     361.9 [+ or -] 34.5
             TP + FIN 2.0     360.5 [+ or -] 30.4
             TP + FIN 5.0     376.3 [+ or -] 18.7
             TP + FIN 5,000   340.0 [+ or -] 21.9

                                Tissue weight
                              (mean [+ or -] SD)

Experiment   Treatment
no.          ([micro]g/kg)          CG (mg)

1            Corn oil alone    5.6 [+ or -] 0.4
             TP + corn oil    37.7 [+ or -] 2.1
             TP + FIN 8       36.9 [+ or -] 3.2
             TP + FIN 40      34.0 [+ or -] 4.6 *
             TP + FIN 200     32.1 [+ or -] 1.3 **
             TP + FIN 5,000   22.0 [+ or -] 3.7 **

2            TP + Corn oil    39.4 [+ or -] 2.4
             TP + FIN 0.1     38.9 [+ or -] 2.7
             TP + FIN 0.5     35.9 [+ or -] 3.2
             TP + FIN 2.0     38.6 [+ or -] 3.0
             TP + FIN 8.0     39.6 [+ or -] 4.6
             TP + FIN 5,000   23.9 [+ or -] 3.7 **

3            TP + Corn oil    34.3 [+ or -] 3.6
             TP + FIN 0.5     36.8 [+ or -] 4.5
             TP + FIN 1.0     37.3 [+ or -] 1.2
             TP + FIN 2.0     36.6 [+ or -] 3.7
             TP + FIN 5.0     38.7 [+ or -] 3.7 * [up arrow]
             TP + FIN 5,000   24.6 [+ or -] 2.2 **

4            TP + Corn oil    37.9 [+ or -] 3.4
             TP + FIN 0.5     41.8 [+ or -] 2.9
             TP + FIN 1.0     39.2 [+ or -] 6.1
             TP + FIN 2.0     37.8 [+ or -] 4.3
             TP + FIN 5.0     35.5 [+ or -] 4.0
             TP + FIN 5,000   21.1 [+ or -] 4.9 **

                                 Tissue weight
                               (mean [+ or -] SD)

Experiment   Treatment
no.          ([micro]g/kg)          SV (mg)

1            Corn oil alone    64.8 [+ or -] 10.9
             TP + corn oil    506.8 [+ or -] 35.1
             TP + FIN 8       405.0 [+ or -] 42.4 **
             TP + FIN 40      335.5 [+ or -] 37.0 **
             TP + FIN 200     295.0 [+ or -] 27.8 **
             TP + FIN 5,000   188.9 [+ or -] 25.2 **

2            TP + Corn oil    508.1 [+ or -] 71.5
             TP + FIN 0.1     516.7 [+ or -] 89.4
             TP + FIN 0.5     479.5 [+ or -] 32.6
             TP + FIN 2.0     528.6 [+ or -] 66.3
             TP + FIN 8.0     423.6 [+ or -] 52.5 *
             TP + FIN 5,000   215.7 [+ or -] 42.4 **

3            TP + Corn oil    466.5 [+ or -] 35.6
             TP + FIN 0.5     474.8 [+ or -] 36.0
             TP + FIN 1.0     443.9 [+ or -] 49.0
             TP + FIN 2.0     474.9 [+ or -] 60.6
             TP + FIN 5.0     457.9 [+ or -] 38.1
             TP + FIN 5,000   182.2 [+ or -] 24.3 **

4            TP + Corn oil    458.1 [+ or -] 36.9
             TP + FIN 0.5     482.8 [+ or -] 44.9
             TP + FIN 1.0     432.0 [+ or -] 49.2
             TP + FIN 2.0     463.1 [+ or -] 45.6
             TP + FIN 5.0     380.7 [+ or -] 71.7 *
             TP + FIN 5,000   160.8 [+ or -] 23.5 **

                                 Tissue weight
                               (mean [+ or -] SD)

Experiment   Treatment
no.          ([micro]g/kg)        Prostate (mg)

1            Corn oil alone    24.8 [+ or -] 14.0
             TP + corn oil    168.6 [+ or -] 13.1
             TP + FIN 8       114.9 [+ or -] 12.4 **
             TP + FIN 40       97.6 [+ or -] 23.2 **
             TP + FIN 200      76.9 [+ or -] 10.1 **
             TP + FIN 5,000    58.0 [+ or -] 8.5 **

2            TP + Corn oil    143.3 [+ or -] 26.3
             TP + FIN 0.1     141.2 [+ or -] 24.3
             TP + FIN 0.5     140.9 [+ or -] 16.2
             TP + FIN 2.0     178.6 [+ or -] 8.9 ** [up arrow]
             TP + FIN 8.0     129.6 [+ or -] 13.3
             TP + FIN 5,000    60.2 [+ or -] 13.5 **

3            TP + Corn oil    132.5 [+ or -] 32.9
             TP + FIN 0.5     153.3 [+ or -] 27.9
             TP + FIN 1.0     159.9 [+ or -] 31.7
             TP + FIN 2.0     151.6 [+ or -] 29.3
             TP + FIN 5.0     140.2 [+ or -] 14.0
             TP + FIN 5,000    60.8 [+ or -] 6.6 **

4            TP + Corn oil    142.0 [+ or -] 21.1
             TP + FIN 0.5     134.1 [+ or -] 27.8
             TP + FIN 1.0     130.4 [+ or -] 17.7
             TP + FIN 2.0     128.6 [+ or -] 10.0
             TP + FIN 5.0     125.5 [+ or -] 17.3
             TP + FIN 5,000    50.5 [+ or -] 9.7 **

                                 Tissue weight
                               (mean [+ or -] SD)

Experiment   Treatment
no.          ([micro]g/kg)      Glans penis (mg)

1            Corn oil alone    70.7 [+ or -] 6.1
             TP + corn oil    122.4 [+ or -] 5.7
             TP + FIN 8       110.1 [+ or -] 8.2 **
             TP + FIN 40      103.4 [+ or -] 7.6 **
             TP + FIN 200     101.6 [+ or -] 7.2 **
             TP + FIN 5,000    98.7 [+ or -] 8.4 **

2            TP + Corn oil    121.9 [+ or -] 8.9
             TP + FIN 0.1     112.9 [+ or -] 9.3 *
             TP + FIN 0.5     110.1 [+ or -] 6.5 **
             TP + FIN 2.0     112.5 [+ or -] 11.5 *
             TP + FIN 8.0     111.3 [+ or -] 4.6 *
             TP + FIN 5,000    99.2 [+ or -] 4.4 **

3            TP + Corn oil    120.8 [+ or -] 8.0
             TP + FIN 0.5     112.5 [+ or -] 7.1
             TP + FIN 1.0     110.9 [+ or -] 11.0
             TP + FIN 2.0     115.9 [+ or -] 8.4
             TP + FIN 5.0     114.4 [+ or -] 12.3
             TP + FIN 5,000    96.6 [+ or -] 7.6 **

4            TP + Corn oil    112.5 [+ or -] 5.3
             TP + FIN 0.5     108.9 [+ or -] 5.7
             TP + FIN 1.0     110.2 [+ or -] 9.9
             TP + FIN 2.0     113.5 [+ or -] 9.2
             TP + FIN 5.0     107.4 [+ or -] 10.9
             TP + FIN 5,000    93.2 [+ or -] 6.4 **

Abbreviations: CG, Cowper's glands; SV, seminal vesicles;
[up arrow], elevated tissue weight.

* p < 0.05 and ** p < 0.01, FIN + TP versus corn oil + TP
by ANOVA and ANCOVA.

Table 2. Activity of 4MBC in the immature rat uterotrophic assay.

                  Age at
                 start of
                  dosing
Experiment no.    (days)    Compound        Dose (per kg)

1 (Tinwell                  AO         5 mL
  et al.                    4MBC       500 mg
  2002b)                    4MBC       1,000 mg
                            DES        5 [micro]g

2                 19-20     AO         5 mL
                            4MBC       1,000 mg
                            DES        5 [micro]g
                            ANT        300 [micro]g
                            4MBC/ANT   1 g/300 [micro]g
                            DES/ANT    5 [micro]g/300 [micro]g

3                 19-20     AO         5 mL
                            4MBC       1,000 mg
                            DES        5 [micro]g
                            ANT        300 [micro]g
                            4MBC/ANT   1 g/300 [micro]g
                            DES/ANT    5 [micro]g/300 [micro]g

4                 19-20     AO         5 mL
                            4MBC       1,000 mg
                            DES        5 [micro]g
                  20-21     AO         5 mL
                            4MBC       1,000 mg
                  21-22     AO         5 mL
                            4MBC       1,000 mg

5                 19-20     AO         5 mL
                            4MBC       1,000 mg
                            DES        5 [micro]g
                  20-21     AO         5 mL
                            4MBC       1,000 mg
                            DES        5 [micro]g
                  21-22     AO         5 mL
                            4MBC       1,000 mg
                            DES        5 [micro]g
                  22-23     AO         5 mL
                            4MBC       1,000 mg
                            DES        5 [micro]g

                                          Uterine weight
                                        (mean [+ or -] SD)

                  Age at
                 start of
                  dosing
Experiment no.    (days)    Compound       Blotted (mg)

1 (Tinwell                  AO          23.3 [+ or -] 4.3
  et al.                    4MBC        31.1 [+ or -] 4.6 **
  2002b)                    4MBC        29.4 [+ or -] 3.5 **
                            DES        123.6 [+ or -] 8.2 **

2                 19-20     AO          35.1 [+ or -] 5.9
                            4MBC        35.1 [+ or -] 4.4
                            DES        115.5 [+ or -] 3.3 **
                            ANT         21.9 [+ or -] 4.0
                            4MBC/ANT    20.0 [+ or -] 2.2
                            DES/ANT    125.1 [+ or -] 5.1 **

3                 19-20     AO          20.0 [+ or -] 1.9
                            4MBC        26.7 [+ or -] 2.8 **
                            DES         99.2 [+ or -] 6.2 **
                            ANT         16.2 [+ or -] 0.9
                            4MBC/ANT    16.8 [+ or -] 0.8
                            DES/ANT    113.7 [+ or -] 10.9 **

4                 19-20     AO          23.7 [+ or -] 3.8
                            4MBC        32.1 [+ or -] 2.7 **
                            DES        108.9 [+ or -] 15.6 **
                  20-21     AO          28.0 [+ or -] 4.1
                            4MBC        33.1 [+ or -] 3.8 *
                  21-22     AO          33.5 [+ or -] 9.8
                            4MBC        38.2 [+ or -] 6.0

5                 19-20     AO          23.0 [+ or -] 1.5
                            4MBC        29.1 [+ or -] 4.8 **
                            DES        111.8 [+ or -] 11.5 **
                  20-21     AO          22.5 [+ or -] 3.2
                            4MBC        26.8 [+ or -] 3.5 *
                            DES        109.9 [+ or -] 4.3 **
                  21-22     AO          26.5 [+ or -] 3.2
                            4MBC        28.9 [+ or -] 4.2
                            DES        121.9 [+ or -] 28.5 **
                  22-23     AO          29.8 [+ or -] 4.1
                            4MBC        33.4 [+ or -] 6.3
                            DES        108.6 [+ or -] 14.4 **

                                          Uterine weight
                                        (mean [+ or -] SD)

                  Age at
                 start of
                  dosing
Experiment no.    (days)    Compound         Dry (mg)

1 (Tinwell                  AO          4.9 [+ or -] 0.8
  et al.                    4MBC        6.0 [+ or -] 0.7 **
  2002b)                    4MBC        5.6 [+ or -] 0.7 **
                            DES        20.9 [+ or -] 1.7 **

2                 19-20     AO          6.6 [+ or -] 0.8
                            4MBC        6.3 [+ or -] 0.5
                            DES        20.0 [+ or -] 0.3 *
                            ANT         4.4 [+ or -] 0.7
                            4MBC/ANT    4.3 [+ or -] 0.5
                            DES/ANT    19.8 [+ or -] 2.5 **

3                 19-20     AO          3.8 [+ or -] 0.6
                            4MBC        4.2 [+ or -] 1.3
                            DES        16.9 [+ or -] 1.3 **
                            ANT         3.4 [+ or -] 0.3
                            4MBC/ANT    3.3 [+ or -] 0.4
                            DES/ANT    19.0 [+ or -] 2.4 **

4                 19-20     AO          4.8 [+ or -] 0.8
                            4MBC        5.8 [+ or -] 0.5 **
                            DES        19.0 [+ or -] 2.5 **
                  20-21     AO          5.3 [+ or -] 0.6
                            4MBC        6.3 [+ or -] 0.6
                  21-22     AO          6.3 [+ or -] 1.6
                            4MBC        7.1 [+ or -] 1.0

5                 19-20     AO          4.6 [+ or -] 0.4
                            4MBC        5.4 [+ or -] 0.8 **
                            DES        19.1 [+ or -] 1.7 **
                  20-21     AO          4.5 [+ or -] 0.7
                            4MBC        5.0 [+ or -] 0.6
                            DES        18.1 [+ or -] 1.0 **
                  21-22     AO          5.2 [+ or -] 0.5
                            4MBC        6.8 [+ or -] 3.5
                            DES        20.1 [+ or -] 2.3 **
                  22-23     AO          5.7 [+ or -] 0.8
                            4MBC        6.5 [+ or -] 1.0
                            DES        19.4 [+ or -] 1.9 **

                                          Body weight
                                       (mean [+ or -] SD)

                  Age at
                 start of
                  dosing
Experiment no.    (days)    Compound      Initial (g)

1 (Tinwell                  AO         46.9 [+ or -] 2.6
  et al.                    4MBC       47.3 [+ or -] 3.6
  2002b)                    4MBC       46.4 [+ or -] 2.7
                            DES        46.1 [+ or -] 5.1

2                 19-20     AO         46.0 [+ or -] 1.7
                            4MBC       46.3 [+ or -] 2.4
                            DES        46.2 [+ or -] 2.6
                            ANT        45.1 [+ or -] 3.2
                            4MBC/ANT   43.8 [+ or -] 3.0
                            DES/ANT    46.6 [+ or -] 1.7

3                 19-20     AO         38.5 [+ or -] 2.6
                            4MBC       39.4 [+ or -] 2.1
                            DES        39.5 [+ or -] 1.5
                            ANT        40.0 [+ or -] 2.6
                            4MBC/ANT   40.7 [+ or -] 2.6
                            DES/ANT    40.7 [+ or -] 2.6

4                 19-20     AO         43.8 [+ or -] 4.3
                            4MBC       43.2 [+ or -] 3.3
                            DES        42.7 [+ or -] 5.2
                  20-21     AO         49.2 [+ or -] 3.1
                            4MBC       48.6 [+ or -] 2.6
                  21-22     AO         54.9 [+ or -] 5.2
                            4MBC       53.3 [+ or -] 4.3

5                 19-20     AO         41.8 [+ or -] 1.4
                            4MBC       41.3 [+ or -] 2.4
                            DES        40.8 [+ or -] 2.4
                  20-21     AO         42.0 [+ or -] 3.2
                            4MBC       42.3 [+ or -] 3.6
                            DES        42.7 [+ or -] 5.5
                  21-22     AO         49.6 [+ or -] 2.5
                            4MBC       47.9 [+ or -] 2.6
                            DES        50.1 [+ or -] 1.4
                  22-23     AO         49.1 [+ or -] 2.8
                            4MBC       49.6 [+ or -] 2.9
                            DES        52.1 [+ or -] 1.9

                                          Body weight
                                       (mean [+ or -] SD)

                  Age at
                 start of
                  dosing
Experiment no.    (days)    Compound     Terminal (g)

1 (Tinwell                  AO         56.4 [+ or -] 3.2
  et al.                    4MBC       56.9 [+ or -] 5.2
  2002b)                    4MBC       56.5 [+ or -] 4.1
                            DES        56.4 [+ or -] 5.3

2                 19-20     AO         56.4 [+ or -] 2.3
                            4MBC       57.6 [+ or -] 4.4
                            DES        56.2 [+ or -] 2.9
                            ANT        57.2 [+ or -] 4.2
                            4MBC/ANT   57.4 [+ or -] 3.7
                            DES/ANT    57.7 [+ or -] 2.2

3                 19-20     AO         48.1 [+ or -] 2.7
                            4MBC       49.3 [+ or -] 2.0
                            DES        51.2 [+ or -] 3.7
                            ANT        48.8 [+ or -] 3.5
                            4MBC/ANT   48.9 [+ or -] 4.1
                            DES/ANT    50.8 [+ or -] 3.2

4                 19-20     AO         53.9 [+ or -] 5.1
                            4MBC       53.1 [+ or -] 4.5
                            DES        51.4 [+ or -] 6.1
                  20-21     AO         60.9 [+ or -] 4.0
                            4MBC       60.6 [+ or -] 5.0
                  21-22     AO         65.5 [+ or -] 4.7
                            4MBC       66.0 [+ or -] 4.9

5                 19-20     AO         52.1 [+ or -] 2.0
                            4MBC       50.6 [+ or -] 3.6
                            DES        51.7 [+ or -] 3.9
                  20-21     AO         51.1 [+ or -] 4.7
                            4MBC       51.9 [+ or -] 4.0
                            DES        53.6 [+ or -] 6.9
                  21-22     AO         62.4 [+ or -] 3.5
                            4MBC       60.5 [+ or -] 3.6
                            DES        62.7 [+ or -] 3.4
                  22-23     AO         62.2 [+ or -] 3.1
                            4MBC       61.4 [+ or -] 4.0
                            DES        64.3 [+ or -] 3.8

All compounds were given by sc injection.

* p < 0.05, and ** p < 0.01 by two-sided Student's t-test.