Bullfrogs:
Almost all amphibians (toads and frogs) metamorphose prior to winter in their first year of life, and thus move the process along somewhat quicker than the bullfrog used in this study, which hibernates as a tadpole in the sediment and undergoes metamorphosis in the spring. Thus, instead of using a naturally metamorphed animal, the researchers induced the pre-metamorphic tadpoles (animals that were not yet ready to undergo this process naturally) by injecting them with the thyroid hormone, triodothyronine (T3) and then exposed them to water containing triclosan. Exposure to a toxin should demonstrate adverse effects following a dose-response curve, with adverse effects occurring at a greater number at higher doses. A dose-response curve was lacking in this study. Thus it is unclear how such exaggerated conclusions have been drawn with regard to this work. In fact, in this same publication, in animals that were not injected with T3, but exposed to T3 and triclosan in their water, no effect was seen on the tadpoles. This leads to the question, if the bullfrog tadpole is not in at least a pro-metamorphic state, how do we know a response is representative of what might be observed under true metamorphic conditions in nature?
Veldhoen, N; R. Skirrow, H. Osachoff, H. Wigmore, D. Clapson, M. Gunderson, G. Van Aggelen, C. Helbing. (2006). The bactericidal agent triclosan modulates thyroid hormone-associated gene expression and disrupts postembryonic anuran development. Aquatic Toxicology. Vol. 80, Issue 3, pages 217-227.
Rats:
In the study in rats, animals were dosed orally for 4 days with triclosan at concentrations of 0, 10, 30, 100, 300, and 1000 mg/kg/day. No clinical signs of toxicity were observed in any of the animals. The no observed adverse effect level (NOAEL) for circulating T4 was reported to be 30 mg/kg/day. It has been demonstrated that triclosan has a long-term (lifetime) oral NOAEL in rats of 48 mg/kg/day and the NOAEL for fetal development is 50 mg/kg/day. The current study data, presented as a dose-response curve, clearly supports the previously identified NOAEL value of 48 mg/kg/day as there were no significant changes seen at ~65 mg/kg/day. This leads to the question, if the findings of a study are at dose levels that exceed a biologically safe dose, what is their relevance for human health risk assessment? Additionally, the dose level identified by the authors as a safe dose in rats, is more than 120 times greater than the highest expected exposure level in humans using all product types in their routine daily personal hygiene (eg toothpaste, hand soap, shower gel, deodorant, mouthwash, etc.).
Crofton, K.; K. Paul, M. DeVito and J. Hedge. (2007). Short-term in vivo exposure to the water contaminant triclosan: Evidence for disruption of thyroxine. Env. Tox. Pharm 24: 194-197.
Cell Culture Experiments:
In the male body, testosterone (T) and dihydrotestosterone (DHT) bind to the androgen receptors (AR) in the prostate and Leydig cells of the testes to initiate responses such as the growth of the male reproductive tract, anabolic effects on body skeletal muscle mass and secondary sex characteristics. In the cell culture study, kidney cells were genetically altered to be unable to metabolize steroids, but to instead emit a flash of light in the presence of endogenous steroids as well as in the presence of synthetic compounds. The cells were then treated with the test compound alone, testosterone alone or a combination of testosterone and the test compound. The authors state that these engineered cells are “highly responsive to endogenous steroids as well as synthetic compounds.” However, “none of the compounds appeared to be androgenic when tested individually without testosterone. It is clear that the relative binding efficiencies, if any, of the test compounds for the AR are orders of magnitude below that of the natural ligands.” In the combination studies, the highest concentration of triclosan tested was approximately 100,000-fold higher than the T concentration used. The result was no measurable “flash of light” from the cells in the high dose concentrations. The design of the study leaves serious doubts as to the relevance of the experiment. It represents an artificial set-up of cell cultures, hormones and the tested chemical substances which is totally unrealistic and does not resemble the mechanisms in a live organism. Therefore, it would be frivolous to draw conclusions from this study about the effect of triclosan on animals or on the human body.
Chen J., K.C. Ahn, N.A. Gee, S.H. Gee, B.D. Hammock, and, B.L Lasley. (2007). Antiandrogenic properties of parabens and other phenolic containing small molecules in personal care products. Tox. Appl. Pharm. 221 (278-284).
Cell cultures are not normal tissues, but are engineered pieces of organisms often operating in the absence of normal feedback systems, metabolic processes and general physiological dynamics that are found in the whole organism. As such, it is generally agreed that the information derived from these types of in vitro systems are not relevant for human health risk assessments.
Recently, UC Davis researchers evaluated the in vitro biological activity of triclosan in several in vitro cellular system screening tools. According to the authors, the purpose of these studies was to evaluate the mechanistically-derived cell-based assay systems readily available at UC Davis to further their development as potential probes for identifying pharmaceutical candidates. The studies were not designed to make human health related risk evaluations on triclosan.
These studies used 3 different cellular in vitro systems including recombinant rat hepatoma (liver cancer) cells, human ovarian cancer cells, and skeletal myotube primary cultures from mice. The cell cultures were exposed to triclosan concentrations ranging from 1 to 10 µM; while in vitro exposures are difficult to extrapolate directly to whole body dosages, by rough estimation the cell cultures received an approximate dose of 100-1000 µg/kg-body weight/day. The amount of triclosan that people use in their personal care products could result in an estimated systemic (circulating in blood) average dose on the order of 4.5 µg/kg-body weight/day . Thus the cell culture experiments are not representative of human exposure levels.
The focus of this work is on developing new potential therapeutic or diagnostic tools from these cell systems. Nevertheless, based on the level of activity found in the different cell culture systems, there is little likelihood of human risk based on the concentrations of triclosan tested. Instead, triclosan is helping the UC Davis research team to elucidate new detection systems and pathways that could lead to better targeted pharmaceuticals.
Ahn KC, Zhao B, Chen J, Cherednichenko G, Sanmarti E., Denison M., Lasley B., Pessah I., Kultz D., Chang D., Gee S., and Hammock B. (2008). In vitro biological activities of the antimicrobials triclocarban, its analogues, and triclosan in bioassay screens: receptor-based bioassay screens. Env. Health. Perspec. 16 May 2008
Earthworms:
Kinney et al. (2008) collected worms and soil samples from three sites several times during a growing season: a soybean field not amended with biosolids or manure (Site 1), a soybean field amended with wastewater treatment plant (WWTP) biosolids (which were not tilled into the soil) (Site 2), and a corn field treated with swine manure (which was tilled into the soil) (Site 3). Site 1 and 3 can be considered control sites for triclosan since Site 1 had not received any amendments in at least 7 years and Site 3 received amendments which should not contain consumer product ingredients. Prior to the analysis of the worms, they were fasted and allowed to empty the contents of their guts so that whole-body analyses would provide an idea of what was carried in their tissues. These values were then reported side by side with measured soil concentrations of the constituent under evaluation in order to estimate bioaccumulation potential.
There are some significant inconsistencies with this study. Soil triclosan concentrations in WWTP biosolids treated soil were reported to range from <50 µg/kg up to 160 µg/kg, but control soils also contained TCS. In fact, the highest soil triclosan (TCS) concentration, 833 µg/kg, was in the ‘control’ soil, expected to have no consumer product chemicals. Since other consumer product chemicals were also detected in this control soil, we suspect the samples or the instruments used were contaminated. This is supported by the fact that worms collected with the soil, contained no measurable triclosan. Further, Nuria Lozano, a visiting scientist at the U.S. Department of Agriculture (USDA), comments in ES&T Science News that triclosan levels in particular were an order of magnitude higher than those she has found in her own work. Looking at other compounds measured in the Kinney et al. study, we see similar issues. Given the likelihood of laboratory contamination of control soil, it is difficult to support the finding that TCS bioconcentration factors in earthworms can be as high as reported. Clearly, additional work is needed to fully understand soil and earthworm concentrations of triclosan.
Kinney C, Furlong E, Kolpin D, Burkhardt M, Zaugg S, Werner S, Bossio J and Benotti J. (2008). Bioaccumulation of Pharmaceuticals and Other Anthropogenic Waste Indicators in Earthworms from Agricultural Soil Amended With Biosolid or Swine Manure. Environ. Sci. Technol. Published on Web 02/22/2008. DOI: 10.1021/es702304c
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