Report on the Assessment of Contact Hypersensitivity to 2-Ethylhexyl p-methoxycinnamate in Female BALB/c Mice (CAS No. 5466-77-3)
Report Date: August 2012
The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of the NTP or the U.S. Government.
2-Ethylhexyl p-methoxycinnamate is a common active ingredient in sunscreens and sunscreen-containing products that absorbs in the ultraviolet B (UVB, 290-320 nm) range. In 2006, the National Cancer Institute nominated EHMC for complete toxicological characterization and evaluation of carcinogenic potential based on high production volume, widespread use, and reported estrogenic and reproductive effects. The NTP is currently evaluating the potential reproductive effects of EHMC using a modified one-generation design and is also investigating absorption, distribution, metabolism, and excretion and toxicokinetics. Hayden et al. (2005) reported that 2% EHMC in mineral oil applied to human epidermal membranes in vitro penetrated the membranes and that measurable amounts of EHMC were detected in the stratum corneum and epidermis. EHMC has been detected in both the plasma and urine of humans 3 to 4 hours after topical application of 10% EHMC in lotion (Janjua et al., 2004). Several reports indicate that EHMC may be a contact sensitizer in humans. Four people have tested positive to EHMC in patch skin tests, seven people in photopatch skin tests, and one person tested positive in both tests (Collaris et al., 2008; Ferriols and Boniche, 2000; Kimura and Katoh, 1995; Ricci et al., 1997; Schauder and Ippen, 1997; Trevisi et al. 1994). The doses applied varied between the reported studies and ranged from 1% to 10%, although the current regulatory limit for EHMC in consumer products in the United States is 7.5% (Personal care 2008). Several studies have evaluated the potential for EHMC lotions, both alone and in conjunction with UVB radiation, to modulate the hypersensitivity response to known sensitizers. Specifically, EHMC effectively protected against UVB-induced suppression of the hypersensitivity response to both 2,4-dinitrofluorobenzene (Reeve et al., 1991; Wolf et al., 1993) and oxazalone (Reeve et al., 1994), but it had no effect when 2,4,6 trinitrochlorobenzene was used as the sensitizing agent (Ho et al., 1992). Furthermore, EHMC lotion (5-10%) did not alter the hypersensitivity response to any of those sensitizers in the absence of UVB. However, there are no published studies that have evaluated the direct contact hypersensitivity potential of EHMC. Therefore, EHMC was selected by the NTP to be tested for its potential to induce contact allergy following dermal application in a rodent model.
The objective of this study was to determine the sensitizing potential of EHMC when applied dermally to female BALB/c mice. Measurement of the contact hypersensitivity response was initially accomplished using the combined local lymph node assay and irritancy assay. Two LLNA/IRR studies were conducted. In the first LLNA study, the exposure levels of EHMC were 50%, 25%, 10%, 5% and 0.25% (v/v) in an acetone:olive oil vehicle (4:1 v:v; AOO). In the second LLNA study, the exposure levels of EHMC were 5%, 2.5%, 1%, 0.5% and 0.25% (v/v). In both LLNA studies, there were no statistically significant changes in lymph node cell proliferation in any of the EHMC-treated groups when compared to the vehicle control. Additionally, lymph node cell proliferation in each of the EHMC treatment groups was below the three-fold level of the vehicle response. In the IRR assays, there were statistically significant increases in percent ear swelling following exposure to EHMC starting at 1.0%, with the greatest increase being observed at 25%, when compared to the vehicle control.
Two Mouse Ear Swelling Tests were conducted to further investigate the potential contact hypersensitivity effects of EHMC. In the first study, the sensitization levels of EHMC were 1.0%, 2.5% and 5.0%, and the challenge level was 5% EHMC. There were no significant changes in percent ear swelling in any of the EHMC-sensitized groups when compared to the vehicle irritancy control group at 24 hr post-challenge. At 48 hr post-challenge, a significant increase in percent ear swelling was observed in mice that had been both sensitized and challenged with 5% EHMC. However, the increase in percent ear swelling in the positive control group was not significantly different from the positive control challenge only group at either 24 hr or 48 hr post-challenge, necessitating a repeat of the study. In the second MEST study, the EHMC sensitization levels utilized were 0.25%, 0.5% and 1.0%, and the challenge level was 0.5%. In this study, there were no significant changes in percent ear swelling in any of the EHMC-sensitized groups when compared to the VHIC group at either 24 hr post-challenge or 48 hr post-challenge. In the repeat MEST study, the positive control, 2,4-Dinitrofluorobenzene, significantly increased the percent ear swelling when compared to the PCCO group, as expected.
In conclusion, under the experimental conditions utilized in this study of female BALB/c mice, EHMC at 0.25% – 50% produced no changes in lymph node cell proliferation in the LLNA. Furthermore, the results of the MEST were negative when mice were sensitized with 0.25% – 1.0% EHMC and challenged with 0.5% EHMC. A questionable increase in the MEST was observed when mice were sensitized with 5% EHMC and challenged with 5% EHMC. Finally, EHMC was identified as an irritant at concentrations as low as 1% in the IRR assay.
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