Huff et al. carcinogenicity results. Interestingly, Löser (1980) exposed animals to cadmium for 24 months, about 6 months longer than did Takenaka et al. (1983), who exposed animals for 18 months but observed them 13 months longer than did Löser. Toluene. Soffritti et al. (2004) orally treated Sprague-Dawley rats with toluene for 2 years and observed them for an additional 6 months. At the end of this observation period, signiﬁcant increases occurred in several tumors, including total malignant tumors (Figure 1); tumors of subcutaneous tissue, mammary glands, and oral cavity, lips, and tongue; and lymphomas/leukemias. In con- trast, the National Toxicology Program’s (NTP) 2-year inhalation bioassays in Fischer rats and B6C3F1 mice did not detect any chemically related tumors (Huff 2003). Thus, the 26 extra weeks of observation without additional exposure in the Sprague-Dawley study (Soffritti et al. 2004) allowed time for tumors to develop, progress, and become detectable. An animal strain–response differ- ence between laboratories is possible, but this was not the case in all other comparisons of carcinogenicity studies performed by the Ramazzini Foundation and the NTP, nor have such differences been shown generically (Huff 2002). Discussion Advantages of beginning exposure during intrauterine development. Over the years, the NTP has tested chemicals for possible car- cinogenicity in about 600 2-year bioassays, most often using both sexes of two strains of rodents, rats and mice. Yet only 7 (~ 1%) of those involved in utero/perinatal exposures: • Azidothymidine transplacental carcinogenesis study (NTP 2006) • t-Butylhydroquinone (TBHQ) (NTP 1997a) • D&C yellow no. 11 (NTP 1997b) • 5,5-Diphenylhydantoin (phenytoin) (NTP 1993a) • Ethylene thiourea (NTP 1992) • Genistein (NTP 2007) • Polybrominated biphenyl mixture (Firemaster FF-1) (NTP 1993b). The developing fetus is particularly sensi- tive to effects of hormones and estrogenic (and other) chemicals. For instance, prenatal expo- sure of humans to diethylstilbestrol (DES) provides a classic example of transplacental carcinogenesis predicted in animals (Newbold and McLachlan 1982) and confirmed in humans (Herbst et al. 1971; Palmlund 1996; Palmer et al. 2006). Because exposures to phytoestrogens and synthetic hormonally active compounds, such as bisphenol A (BPA), are increasing in diets of infants and children, these materials need to be investi- gated in prenatal and perinatal bioassays. Newbold et al. (2001) evaluated the endocrine disruptor genistein in a 5-day neonatal expo- sure in CD-1 mice and after 18 months found increases in adenocarcinomas of the uterus. They then designed expanded bioassay with three exposure regimens: a) continuous expo- sure from conception through 2 years of age; b) exposure from conception through 20 weeks (140 days) followed by a control diet to 2 years of age; and c) dosing from concep- tion through weaning (21 days) followed by the control diet to 2 years. Increases in tumors (mammary gland and pituitary gland tumors) were observed only in the ﬁrst experiment. An in utero exposure protocol used to study effects of TBHQ (Abdo and Kari 1996; NTP 1997a) 2.5 years after exposure began should be considered by the NTP and regula- tory agencies as a model for cancer bioassays. Female F0 rats were fed diets containing vary- ing amounts of TBHQ, beginning 2 weeks before cohabitation with males and continu- ing through gestation and lactation until F1 pups were weaned. F1 rats continued to receive postweaning diets containing TBHQ for 123 weeks for males and 129 weeks for females. Interestingly, despite this inclusive exposure protocol from conception up to nearly 30 months of age, there was no evi- dence of carcinogenicity. In fact, decreases in mammary gland tumors occurred in both )100 (%90 ec 80 len 70 va 60 rep 50 e 40 v tia 30 lu 20 um10 C 0 1440 sexes. These ﬁndings certainly contradict the notion that studies using in utero exposure with follow-up for about 2.5 years invariably result in carcinogenic activity. In contrast, recent studies of the widely used plasticizer additive BPA indicate that pre- natal exposures can induce obesity in offspring (Dolinoy et al. 2007), a phenomenon that has not been observed with postnatal exposures alone. Prenatal exposure of pregnant Agouti Avy mice to BPA, which is a major component of polycarbonate plastics and epoxy resins, sig- niﬁcantly reduced DNA methylation (Dolinoy et al. 2007). Others have reported that prenatal BPA also increases mammary tumor develop- ment and other developmental abnormalities (Soto et al. 2008), and may increase recurrent miscarriages (Sugiura-Ogasawara et al. 2005). Thus, experiments of prenatal exposure to BPA find that such exposures imprint mammary cells, leaving them especially sensitized to later cell growth and hormonal stimulation— characteristics also found in tumors. It may also be relevant that low levels of BPA have been found to activate genes in noncancerous breast cells in a way that mimics that seen in highly aggressive breast cancer (Dairkee et al. 2008), indicating that BPA plays a critical role both prenatally and postnatally. Advantages of studies with longer observa- tion periods versus 2-year studies. Experimental studies should be designed with optimum sensi- tivity to identify likely adverse health problems throughout humans’ increasing life span. Humans, of course, consume or are exposed to countless natural and synthetic substances dur- ing gestation, nursing, and the rest of their lives. In modern societies, proportionally more peo- ple are living until their 70s, 80s, and 90s, long after prenatal and childhood exposures and retirement from workplace exposures. Because most long-term rodent carcinogenesis studies do not involve in utero exposure and are inten- tionally terminated after 2 years (104 weeks) of exposure, they cannot shed light on the effects of chemicals on embryos/fetuses/neonates or “elderly” animals. Likewise, studies truncated after 2 years of exposure do not allow sufﬁcient latency periods for late-developing tumors, such as the 80% of all human cancers that occur after 60 years of age. Because a 2-year-old rat is roughly equivalent to a 60- to 65-year-old per- son, conventional 2-year-long bioassays cannot detect tumors that will develop later in life. Although some researchers have suggested reducing the duration of rodent studies to 18 months (e.g., Davies et al. 2000), scientists at the National Institute of Environmental Health Sciences reject that recommendation, noting that animals in such studies would be the equivalent of humans only 30–50 years of age and would reduce statistical power (Bucher 2002; Haseman et al. 2001; Kodell et al. 2000). Instead, a number of researchers VOLUME 116 | NUMBER 11 | November 2008 • Environmental Health Perspectives Total malignant tumors in females Total malignant tumors in males Toluene 500 mg/kg bw Control (olive oil alone) )100 (%90 ec 80 len 70 va 60 re 50 ep 40 tiva 30 lu 20 um10 C 0 16 32 48 64 80 96 112 128 144 160 Age at death (weeks) 16 32 48 64 80 96 112 128 144 160 Age at death (weeks) Figure 1. Cumulative prevalence of total malignant tumors in females and males histopathologically observed, by age at death, among Sprague-Dawley rats treated with 500 mg/kg body weight/day of toluene for 104 weeks and then kept under control conditions until death or 130 weeks (experiment BT 903). Dashed lines indicate 112 weeks, the typical termination of NTP studies. Arrows indicate beginning of exposure. Reproduced from Soffritti et al. (2004) with permission from the European Ramazzini Foundation.