The association between the exposure and bioaccumulation of endocrine disruptor chemicals (EDCs) and their adverse effects on human and wildlife populations has raised concern worldwide. Regulatory agencies such as the Environmental Protection Agency (EPA) and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) in the United States; the European Commission for the Validation of Alternative Methods (ECVAM) in the European Union; and the Japanese Commission for the Validation of Alternative Methods (JaCVAM) in Japan have conducted studies to validate new test methods for EDCs.1, 2 This concern has also reached producers such as the Campbell Soup Company, which is phasing bisphenol A (BPA) out of its product containers.3
Estrogenic EDCs can alter the normal levels of the hormone estrogen in humans and wildlife populations. Altering hormonal levels is particularly problematic in developing fetuses and young children, as altering their hormonal levels can alter their life course.4–10 The greatest concern is the early onset of cancers, e.g., breast and prostate.6–8 Other issues of concern are precocious puberty and childhood obesity. According to one article published in 2010,9 puberty starts as much as two years earlier now than it did for children born 10 to 30 years ago, which may lead to the early onset of several forms of cancer.9 There have also been recent studies linking EDCs to adult and childhood obesity.10
Several chemicals used in the personal care industry are potential endocrine disruptors. One area of concern is sunscreens, as their use has increased during the past decades due to growing concern over skin damage such as sunburn, photoaging and skin cancer. Several in vitro, internationally validated bioassays evaluating the active components of sunscreens, i.e., UV filters such as 3-(4-methylbenzylidene) camphor (4-MBC), octyl methoxycinnimate and benzophenone-3, have shown them to be estrogenic.4, 11–14
More clinical studies are required to determine their effects via topical application, although studies by Janjua et al. have shown these compounds to be present in urine and blood plasma after topical application.15 The same researchers also found changes in levels of the hormones estradiol and testosterone in participants after topical application.15 In these studies, 32 healthy volunteers, 15 young males and 17 postmenopausal females, were assigned to apply daily, over their entire body, 2 mg/cm2 of basic cream formulation without (week 1) and with (week 2) the three sunscreens at 10% w/w each. Such UV filters are commonly used in children’s sunscreen products.
Two other synthetic chemicals considered by regulatory agencies to be estrogenic endocrine disruptors are BPA and diethylstilbestrol (DES). BPA is a plasticizer found in plastic bottles, the plastic lining of drinking bottles and canned goods including baby food. DES was originally designed as a drug to prevent miscarriages, which failed in its intended use and caused side effects such as increased risk of breast cancer, clear cell adenocarcinoma (CCAC) of the vagina and cervix, and reproductive anomalies.16 There also are naturally occurring estrogenic compounds, called phytoestrogens, such as genistein in soy-based infant formulas and vegetarian diets, which have been associated with hypospadias in male offspring and an increase in autoimmune diseases, giving rise to considerable concern among health care practitioners.16–18
Taken together, the data shows a pattern of the detrimental effects of EDCs, and while unsafe levels of the compounds have not yet been specified, understanding the estrogenic potency of these and other chemicals is important, considering the potential for exposure from personal care products as well as concern over these ingredients. Further, since these chemicals are ubiquitous, highly lipophilic and often chlorinated, they maintain a persistent presence in the environment, resulting in their bioaccumulation in the food chain.19–23
In 1996, two congressional acts brought to light the need to develop an EDC testing program: the Food Quality Act and the Safe Water Reauthorization Act and Amendments.24 These Acts prompted governmental agencies including the EPA, ICCVAM and the National Institute of Environmental Health Sciences (NIEHS) to develop bioassays for the detection of endocrine disruptors. One assay developed was the BG1Luc4E2 or BG1-ER assay, a transcriptional activation bioassay created by stably transfecting a DNA plasmid, pGudLuc7.0, into BG1 human ovarian carcinoma cells. The plasmid contained a luciferase gene under the transactivational control of four estrogen-responsive DNA elements. Luciferase is the protein that makes fireflies glow, thus the BG1-ER cells would glow upon contact with estrogenic compounds. The resulting cell line, BG14LucE2, therefore responds in a chemical-specific and dose-dependent manner and is capable of detecting as little as 0.1 pmol 17 β-estradiol.
In initial studies by Rogers and Denison, treatment of BG1-ER cells resulted in the significant induction of luciferase activity for estrogen when dosed with: DES, 17 β-estradiol, o,p′-DTT, BPA, nonylphenol, genistein and daidzein. Luciferase activity was not increased by any other steroid tested, which included progesterone, testosterone, all-trans retinoic acid, thyroid hormone, dihydrotestosterone and dexamethasone. This demonstrated the specificity of the assay for estrogenic compounds.19
The BG1-ER assay has been through several US and international validation studies with governmental agencies. It was nominated by the Scientific Advisory Committee for the evaluation of Alternative Toxicological Methods (SACATM) in March 2004 for a high priority for validation, which put into motion a US Protocol Standardization Study as well as the International Validation Study. In July of 2006, ICCVAM completed the US protocol standardization study, consisting of: standardization of the high-throughput 96-well plate layout; standards and quality control (QC) testing; quality assurance procedures; and the testing of eight blind coded agonist and antagonist chemicals.
In January of 2010, the international validation study was completed by ICCVAM, ECVAM and JaCVAM. The international study included laboratories in these regions and focused on the consistency and robustness of the assay in all three labs, looking at standards and QC testing, as well as testing of 67 blind coded agonist and antagonist chemicals.25 The Federal Registry Notice accepting the method was published in February 2012, recommending the method for use in identifying substances with in vitro estrogen agonist and antagonist activity. In addition, ICCVAM noted the accuracy of the assay was at least equivalent to the current ER TA assay included in regulatory testing guidance.2 The Organization for Economic Cooperation and Development (OECD) voted to accept the method in April 2012.26
In relation, ICCVAM has proposed guidelines for interpreting the results of assays performed using the internationally accepted protocols.1 The protocol requires an 11-point standard curve of 17 β-estradiol, four negative controls, dimethyl sulfoxide (DMSO), and three positive controls, i.e., methoxychlor. The 11-point standard of 17 β-estradiol forms a standard sigmoidal or S-shaped curve.
ICCVAM’s proposed guidelines state that if a sample reading is above background but below 20% of the 17 β-estradiol curve, the sample is considered a “detect” but not an “active EDC.” If the sample is above the 20% of the 17 β-estradiol curve, the sample is considered a “detect” and an “active EDC” (see Figure 1). While these are proposed guidelines and not set rules, they are often included in the analysis of data to demonstrate the relative potency of a product.
Materials and Methods
To evaluate the content of personal care products for EDCs, the BG1-ER assay was used to test commercially available sunscreens, foundations and moisturizing lotions purchased in the United States. Also tested for their putative estrogenic EDC content were several nutritional supplements. The 2-g samples to be tested were first extracted using organic solvents, then evaporated and mixed with DMSO prior to incubation with the BG1-ER cells. After a 24-hr incubation period, the amount of estrogenic activity was determined using a luminometera.
The ICCVAM international agonist protocol for analysis of estrogenic endocrine disruptors was used for all analyses. Briefly, an 11-point 17 β-estradiol standard curve was used along with four DMSO negative controls and three methoxychlor positive controls. Solvent blanks were analyzed to determine any background contamination from the solvents used. Also, samples were spiked with a known quantity of 17 β-estradiol and extracted to determine percent recovery from the extraction procedure.
Each analysis was performed using a 96-well highthroughput method, which allows for complete analysis in less than 48 hr. All data from the 96-well plates was analyzed and evaluated using the luminometera. The four parameter Hill equation was used to convert the luminometer raw data into 17 β-estradiol equivalent values, which were then calculated per extracted weight and internal recoveries.
Figure 2 on shows the results from analysis of the sunscreens. Two products, sunscreens A and C, demonstrated a level of detection greater than the 20% threshold on the 17 β- estradiol standard curve. Using ICCVAM’s proposed guidelines, this would mean for both sunscreens A and C, estrogenic EDCs were detected and would be considered active endocrine disruptors at the levels found. Sunscreen B showed no detectable levels of estrogenic EDCs.
Besides sunscreens, of the several other cosmetic products examined (see Figure 3), one beige foundation and moisturizing lotion B had detectable activity greater than the 20% threshold on the 17 β-estradiol standard curve. Using ICCVAM’s proposed guidelines, this would mean for the foundation and moisturizing lotion B, estrogenic EDCs were detected and would be considered active endocrine disruptors at the levels found. Moisturizing lotion A showed no detectable levels of estrogenic EDCs.
The analysis of several nutritional supplements (see Figure 4) showed a mix of EDC detects and non-detects. A prenatal supplement, soy protein and fish oil supplements showed detectable levels of activity greater than the 20% threshold on the 17 β-estradiol standard curve. For the soy protein, this is not surprising since two of the main components of soy are genistein and daidzein. Both genistein and daidzein are considered estrogenic endocrine disruptors. Also, finding endocrine disruptors in fish oil is not surprising since most EDCs are lipophilic. Two other supplement products including whey protein and children’s gummy vitamins were below the limits of detection and thereby classified as non-detect and not active endocrine disruptors, per ICCVAM’s proposed guidelines.
These findings, taken together, demonstrate there is a potential health risk associated with currently marketed personal care products and dietary supplements. However, the presence of EDCs in a product does not necessarily mean the product is unsafe or safe, or that it should be pulled from shelves. Clinical studies must be conducted to determine safe levels for the individual EDCs for consumer products. Further, these clinical studies need to determine which formulations will result in greater dermal penetration by these chemicals of concern, allowing manufacturers to adjust their product formulations accordingly.
With the growing concern over EDCs in consumer products, there is a need for a reliable and fast assay with easily interpreted results that can detect estrogenic EDCs. The BG1-ER bioassay presented is capable of detecting low levels of estrogenic active compounds in consumer products. As an internationally accepted high-throughput method for the detection of estrogenic EDCs, the bioassay allows development chemists the ability to quickly access testing results and not interfere with the product development cycle.
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