Phthalate Esters Research Summary

American Chemistry Council
Phthalate Esters Panel

Dr. Tim Zacharewski while at the University of Western Ontario (UWO) conducted several studies on behalf of ACC's Phthalate Esters Panel to investigate the potential of phthalate esters (PAEs) to act as an estrogen. The commercial phthalates that were tested include: butylbenzl phthalate (BBP), dihexyl phthalate (DHP), diiso-heptyl phthalate (DiHP), diisononyl phthalate (DINP), dibutyl phthalate (DBP), di-2-ethyl hexyl phthalate (DEHP), di-n-octyl phthalate (DnOP), and diiso-decyl phthalate (DIDP). Specifically, research was designed to focus on the ability of PAEs and the hydrolysis products - monoester and alcohol - to interact with the estrogen receptor.

In Vitro Screening Tests

The first phase of studies used cell cultures as a way of determining if these substances act directly on the estrogen receptor. Since binding of a substance to a receptor does not mean that the substance can produce an estrogenic response, the laboratory developed methods to measure whether the receptor was "activated." Investigators at UWO used several activation/detection schemes rather than one method to determine if activation occurred.

The studies were used to determine estrogen-receptor activation (not just binding) in:

• MCF-7 cells (a human breast cancer cell line),
• HeLa cells (a human cervical cancer cell line),
• Yeast cells (an estrogen-dependent growth assay), and
• The MCF-7 Bus cells (the "E-screen" cell proliferation assay).

All are quantitative assessments (assays) except for the yeast cell growth assay which is difficult to quantify. In addition, the ability of PAEs to competitively bind to the estrogen receptor was used to evaluate if the PAE inhibits the activity of natural hormones.

Estradiol, or natural estrogen, was used to compare the levels at which an estrogenic effect was observed. The research results show that at dose levels 10,000 times higher than natural estrogen, three phthalate esters (DBP, BBP, DHP) showed a weak estrogenic response (less than 50% activity). To ensure a thorough investigation, however, the Panel decided not to rely simply on the screening mechanism, but chose to conduct further in vivo (live animal) studies for estrogenic activity on all PAEs.

In Vivo (Laboratory Animals) Tests

Research on PAEs, monoesters, and alcohols continued using intact-animal methods that are standard in the pharmaceutical industry for evaluating estrogen effects. These methods include two assays using female rats to determine:

• the response of the uterus to an estrogen (the uterotrophic assay), which measures the thickness or weight of the uterus following treatment with a potential estrogen-like substance, and
• the response of the vagina to an estrogen (the vaginal epithelial cell cornification assay), which determines if the vaginal epithelial cells change their appearance as they do during the estrous cycle.

In each case, the activities of PAEs were compared to the activity of estradiol (the natural estrogen). Dr. Zacharewski concluded that none of the phthalate esters elicited in vivo effects as determined by the monitoring of uterine wet weight and vaginal cell cornification. Under a long-standing ACC policy, the Phthalate Esters Panel will make publicly available copies of all final reports.