GLP-compliant Studies Research Articles

Ethyl t‐butyl ether: review of reproductive and developmental toxicity

Ethyl t-butyl ether (ETBE) is a motor fuel oxygenate used in reformulated gasoline. Knowledge of developmental and reproductive toxicity potential of ETBE is critical for making informed decisions about acceptance and regulations. This review discusses toxicology studies providing information about effects on reproduction and the conceptus. Seven GLP-compliant studies following widely accepted protocols have focused specifically on developmental and reproductive toxicity (DART) in rats and rabbits exposed to ETBE by gavage with doses up to 1,000 mg/kg body weight/day, the limit specified in standardized test guidelines. Other repeat-dose general toxicology studies have administered ETBE to rodents for up to 180 days, and included reproductive organ weights, histology, or other indications of reproductive system structure or function. DART potential of the main ETBE metabolite t-butyl alcohol and class-related MTBE has also been studied. More GLP-compliant studies exist for evaluating ETBE using well-established, currently recommended protocols than are available for many other chemicals used today. The database for determining ETBE DART potential is adequate, although not all study details are currently easily accessible for peer-review. ETBE does not appear to be selectively toxic to reproduction or embryofetal development in the absence of other manifestations of general toxicity. Studies using recommended methods for sample preservation and analysis have shown no targeted effect on the reproductive system. No embryofetal effects were observed in rabbits. Early postnatal rat pup deaths show no clear dose-response and have largely been attributed to total litter losses with accompanying evidence of maternal neglect or frank maternal morbidity.

Good Laboratory Practices and Safety Assessments: Another View

In a letter responding to an article by Myers et al. (2009), Becker et al. (2009) claimed that industry’s Good Laboratory Practices (GLP)-compliant studies are superior to traditional academic peer-review in predicting the risk of toxic agents. I have read almost 30,000 experimental, etiologic, and epidemiologic papers (most in part), and it is evident that industry GLP studies do not report the same risks of a chemical when published in peer-reviewed studies from academia. This may be explained by biases in industry experiments and epidemiology, especially in design, due to the financial interests of industry sponsors—some receiving billions of dollars in revenue per chemical each year. For pharmaceuticals, dozens of published reviews show a strong correlation between industry sponsorship and findings of safety; I know of four such strong correlations in studies of industrial chemical risks (Bekelman et al. 2003; Fagin and Lavelle 1999; Swaen and Meijers 1988; vom Saal and Hughes 2005). Becker et al. (2009) relied on a commentary by a former editor at the Nature research journals (Jennings 2006) to claim that peer-review gives inferior data compared with GLP studies. Actually, Jennings (2006) wrote about improving, not abandoning, peer review. He presented data showing that the long-term value of scientific papers in neuroscience (judged by experts) correlates with the quality of the journals in which they were published (based on impact factor). That is a cardinal finding because industry supports various journals and their scientific associations, but their GLP studies are rarely published in high-quality journals (again, based on my readings). Evidently, industry’s GLP data are not reliable enough to publish, while financial independence of authors and editors, as well as peer review, are markers of good quality data. Since the widespread experimental testing frauds at Industrial Bio-Test Laboratories (Schneider 1983) and Craven Laboratories (U.S. Environmental Protection Agency 1994), which generated the GLP reforms, industry has issued oceans of GLP-compliant studies for submission to regulatory agencies. Few are submitted for publication, but almost all (in my experience) are submitted to journals that publish many industry-sponsored studies. Critically, industry and their regulatory agencies took the opportunity proferred by the requirement to comply with GLP to exclude almost all academic high-quality, non-GLP studies from risk assessments of existing chemicals (and the toxicity of new agents are primarily evaluated by the parties who want to sell it). For existing chemicals, I have always found that the effective toxicity doses in regulatory (GLP) studies are higher than those in the peer-reviewed literature, for several end points. It is important for individuals who value the contributions that science makes to society (reliable data)—or those who are cautious about toxicity of low-dose and cocktail agents that may affect biochemical signals, especially during development—to continue lobbying public agencies to incorporate academia’s peer-reviewed studies and to use disclosure of financial interests to give appropriate credence to industry’s data in chemical risk assessments. I also call on independent academics to be less competitive and make their methods and data more freely available.

Good Laboratory Practices: Tyl Responds

I am responding to the comments by vom Saal and Myers on my commentary (Tyl 2009), which was in response to their commentary (Myers et al. 2009). Our dietary BPA mouse study was performed under Organisation for Economic Co-operation and Development (OECD) Test Guideline 416 (OECD 2001) and Good Laboratory Practices (GLPs) (OECD 1998), with in-house quality control, and formal RTI quality assurance, and expert Developmental & Reproductive Toxicology (DART) panel oversight. We submitted all of our summary data online with our published mouse study (Tyl et al. 2008). I also discussed our BPA data, especially prostate weights (ventral and dorsolateral lobes separately) and ages of animals at scheduled necropsy, two areas of concern to vom Saal and Myers, in my commentary (Tyl 2009). We found no BPA effects on mouse prostate weights at any dietary dose, from 3 μg/kg/day to 600 mg/kg/day, whereas vom Saal and colleagues reported increased prostate weights at 2 and 20 μg/kg/day BPA, administered on gestational days 11–17 (Nagel et al. 1997). Vom Saal and Myers suggested that prostate weights were very large in our control mice (Tyl et al 2008), in his view, likely due to prostatitis and/or poor dissection techniques resulting in extraneous tissue left on the prostates. We provided histopathologic confirmation of low (normal) rates of prostatitis, no increased incidences or severities from BPA, and no evidence of extraneous tissue from examination of the prostate paraffin block faces and histology slides. For animal ages at termination, we initially presented approximate ages of our animals at demise because we were not aware of their concerns at that time. In a letter to the editor of Toxicological Sciences, where the multigenerational BPA rat and mouse studies (Tyl et al. 2002, 2008) were published, I (Tyl 2009) explained in great detail the ages of our F0, F1, and F2 animals at scheduled necropsy; the ages of the F1 animals varied at most by 3 weeks in all groups, based on when the F0 animals mated during the 2-week mating period, the need to have all F1 offspring exposed for at least 8 weeks during the prebreeding period, and the need for all F1s to be available for pairing in all groups at the same time. FDA auditors recently spent 11 days at RTI (30 March to 9 April 2009) inspecting our BPA rat (Tyl et al. 2002) and mouse (Tyl et al. 2008) multigenerational reproductive toxicity study data and records, with no study findings. It is clear that vom Saal and Myers apparently still do not understand or appreciate the discipline, power, importance, and usefulness of GLPs on study design, performance, documentation, and interpretation (which is why GLP-compliant studies are preferentially used in formal hazard identification and risk assessment). The effects of dose levels, route, timing (life stages), and duration of BPA exposures on reported early and late effects, and whether there is a linkage between the low-dose early end points from short-term, small, basic exploratory studies and the outcomes from long-term guideline studies, need to be evaluated. Long-term, robust oral studies (Ashby et al. 1999; Cagen et al. 1999) and guideline-compliant oral multigenerational studies (Ema et al. 2001; Tyl et al. 2002, 2008), regardless of sponsorship, have not confirmed the low-dose effects reported in the basic studies, nor any long-term consequences anticipated from these reported early effects. Determination of whether there is hazard or risk of BPA to humans and wildlife from low, environmentally relevant doses by relevant exposure routes is based on available appropriate data. To date, governmental and other organizational hazard, risk, and weight-of-evidence assessments have concluded, based on the data, that there is no evidence of any adverse effects from oral BPA at low doses.

Cancer of the lungs and other diseases after exposure to asbestos dust

A range of fibrous materials, including several types of asbestos and carbon fibres with nano scale diameters that had reported positive genotoxicity data (predominantly clastogenicity), were tested in the in vitro micronucleus test (OECD 487) in GLP-compliant studies in Chinese Hamster Ovary cells. Out of eight materials tested, only one (crocidolite, an asbestos fibre) gave a positive response either in the presence or absence of metabolic activation (S9) and at short (3 h) or extended (24 h) exposure times (p ≤ 0.001). Our data suggest that the commonly used tests for clastogenicity in mammalian cells require extensive modification before fibrous materials are detected as positive, raising questions about the validity of these tests for detecting clastogenic and aneugenic fibrous materials.