SUMMARYThe major protocol features of the immature rat uterotrophic assay have been evaluated using a range of reference chemicals. The protocol variables considered include the selection of the test species and route of chemical administration, the age of the test animals, the maintenance diet used and the specificity of the assay for estrogens. It is concluded that three daily oral old rats, followed by determination of absolute uterus weights on the fourth day, provides a sensitive and toxicologically relevant in vivo estrogenicity assay. Rats are favoured over mice for reasons of toxicological practice, but the choice of test species is probably not a critical protocol variable, as evidenced by the similar sensitivity of rats and mice to the uterotrophic activity of methoxychlor. Vaginal opening is chemicals to reduce or abolish the uterotrophic response of estradiol is suggested to provide a useful extension of the uterotrophic assay foThe results of a series of studies on the environmental estrogen nonylphenol (NP), and its ng of the aliphatic sidechain is important for activity. 17 similar activity to estradiol in the uterotrophic assay, and is suggested to represent the 'parent' estrogen of NP. Benzoylation of r uterotrophic activity, in contrast to the enhancing effect of benzoylation on estradiol.Selected chemicals shown to be active in the immature rat uterotrophic assay were also evaluated in an in vitro yeast human estrogen receptor transactivation assay. Most of the chemicals gave similar qualitative responses to those seen in the uterotrophic assay, and the detection of the estrogen methoxychlor by the yeast assay evidenced a degree of intrinsic metabolic competence. However, the assay had a reduced ability (compared to rodents) to hydrolyse the benzoate ester of estradiol, and the estrogenic benzoate derivative of NP was not active in the yeast assay. These last results indicate that current metabolic deficiencies of in vitro estrogenicity assays will limit the value of negative data for the immediate future.The results described illustrate the intrinsic complexity of evaluating chemicals for estrogenic activities and confirm the need for rigorous attention to experimental design and criteria for assessing estrogenic activity.3

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Six short term tests for detecting carcinogenicity have been evaluated using 120 compounds, of which half were carcinogens and the rest non-carcinogens. The results obtained indicate that the Ames test and a "cell transformation" assay are both sufficiently sensitive to carcinogenicity, or the lack of it, in the compounds studied to enable them to be employed for detecting potential carcinogens. The consequences of using short term tests under various screening conditions have been explored. In order to have confidence in the results obtained for new or previously untested compounds it is important to use such tests in a carefully controlled manner.

Summary.-A number of tests have been described which are thought to be capable of identifying carcinogens without using the actual induction of cancer as an endpoint. This study compares the performance of 6 such tests on a selection of 120 organic chemicals. The tests studies were: (1) mutation of Salmonella typhimurium; (2) cell transformation; (3) degranulation of endoplasmic reticulum; (4) sebaceous gland suppression; (5) tetrazolium reduction and (6) subcutaneous implant. A further 4 tests were examined briefly, but were not included in the complete evaluation.The chemicals were classified into carcinogens (58) and non-carcinogens (62) on the basis of published experimental data, and into 1 of 4 broad chemical classes.There was considerable variation between tests in their ability to predict carcinogenicity, with the cell-transformation test and the bacterial-mutation test being the most accurate (94% and 93% accurate respectively). These 2 tests were considered to be of general use in screening, since they were clearly more accurate than the others. Statistical consideration of various combinations of these tests showed that the use of cell transformation and bacterial mutation together, provide an advantage over the use of either test alone. The inclusion of the other 4 tests in a screening battery predictably resulted in a great increase in overall inaccuracy and loss of discrimination, even though the detection of carcinogens is improved. All the tests were shown to generate both false positive and false negative results, a situation which may be controlled by the use, where possible, of appropriate chemical-class controls, to identify the test which is optimal for the class of chemical under test. Structural

Cultured hepatocytes have been treated with either DMSO or dimethylnitrosamine (NDMA) for either 1 or 2 h and the cells assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay). A strong positive test response was observed producing comet tails of a length and DNA content not observed in either viable or dead control cells. A stronger test response was observed after a 2 h, as opposed to a 1 h, incubation of hepatocytes with NDMA. The method of processing the image of the comet is discussed and it is proposed that measurement of the length of the comet tail should commence at the estimated trailing edge of the cell, rather than at the leading edge or the estimated centre of the cell. Using this criterion, many control cells have no comet trails thereby enabling chemically induced tails to be more readily assessed. A simplified version of defining the comet tail moment is proposed.

While defining the no effect level for the 5α-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 CF1 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.

ArticlesIn this study we found that the ultraviolet sunscreen component 3-(4-methylbenzylidine)camphor (4MBC) is uterotrophic in immature rats when administered by either subcutaneous injection or oral gavage. These data confirm earlier reports of uterotrophic activity for this agent when administered to immature rats in the diet or by whole-body immersion; however, they are in contrast to negative unpublished immature rat uterotrophic assay results. Data also indicate that 4MBC binds to isolated rat uterine estrogen receptors and shows activity in a human estrogen receptor yeast transactivation assay; however, we considered both of these effects equivocal. In this study, we confirmed the original observation that 4MBC was active as a mitogen to MCF-7 breast cancer cells. We evaluated and discounted the possibility that the estrogenic activity of 4MBC is related to its bulky camphor group, which is of similar molecular dimensions to that of the weak estrogen kepone. Uncertainty remains regarding the mechanism of the uterotrophic activity of 4MBC.

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