LETTER TO THE EDITOR 297

Copyright © 2004 John Wiley & Sons, Ltd. J. Appl. Toxicol. 24, 297–306 (2004)

JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 24, 297–306 (2004)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jat.985

Copyright © 2004 John Wiley & Sons, Ltd.

Letters to the Editor

Dear Sir,We would like to comment on the publication by Darbre

et al. (2004), Concentrations of Parabens in Human BreastTumours, and the accompanying Editorial by Harvey andEverett (2004), the Significance of the Detection of Estersof p-Hydroxybenzoic Acid (Parabens) in Human BreastTumours. Darbre et al. (2004) reported the presence ofparabens (a family of the alkyl esters of p-hydroxybenzoicacid used as antimicrobial preservatives in some consumerproducts) in breast tumor tissue. Although this study didnot conclude that parabens cause breast cancer, the impli-cation was evident because it was noted that some parabenshave been shown to have very weak estrogenic activity insome in vitro and in vivo test systems. Furthermore, thestudy was published subsequent to a hypothesis publishedby the same authors that the presence of parabens indeodorants and antiperspirants might be a risk factor forbreast cancer (Darbre, 2003; Harvey, 2003). It is not theintent of this letter to debate this issue, but rather to pointout potential deficiencies in the design and interpretationof the study by Darbre et al. (2004).

The most noticeable deficiency in the study by Darbreet al. (2004) is the absence of control tissues. Althoughcause and effect is still impossible to infer from such studies,a finding of equal paraben concentrations in normal (i.e.tumor-free) breast tissue would have essentially negatedthe key finding from this study. Harvey and Everett (2004)acknowledged in their Editorial that normal breast tissuewas not analyzed, but they appeared to dismiss this byconcluding that apparently normal tissue at the time ofbiopsy may later develop a tumor. The comparison ofbreast adipose tissue concentrations of organochlorinecompounds in controls and breast cancer patients intable 4 of Darbre et al. (2004) highlights the potentialdangers of omitting a control group in such studies. Thisconcern is highlighted by contamination of some of theblanks employed, where in some instances higher parabenconcentrations were recorded than found in breast tumortissues. Although the authors acknowledged this, andsubtracted these values from tissue concentrations, thisraises the issue of whether paraben laboratory contamina-tion was prevalent. Using the data shown in table 2 ofDarbre et al. (2004), when mean concentrations of indi-vidual parabens in breast tumor tissue are compared tomean paraben concentrations in blanks, there is no statis-tical difference based on a t-test. We therefore concludethat the authors have not eliminated paraben contamina-tion within the laboratory as the source of the parabensreported to be in the breast tissue.

A related concern is the possible use of drugs con-taining parabens. One example is the aromatase inhibitorexemestane, which is used for the treatment of breast can-cer. This drug is reported to be part of the drug treatmentregimen for breast cancer at the Edinburgh Breast Unit,

and the exemestane formulation used at this treatmentcenter is preserved with methylparaben, which accountedfor 62% of the parabens detected in the breast tumortissue sampled by Darbre et al. Apart from the fact thatmethylparaben is devoid of in vivo estrogenic activity(Hossaini et al., 2000), its possible use in such studies shouldbe controlled by reference to breast tumor tissue frompatients who were not treated with a paraben-containingdrug. Among the parabens detected in breast tumor tissue,only isobutylparaben and n-butylparaben have been demon-strated to have weak in vivo estrogenic activity (Routledgeet al., 1998; Hossaini et al., 2000; Darbre et al., 2002).

Darbre et al. (2004) noted that because parabens arelipophilic compounds with increasing octanol/water parti-tion coefficients (Kow) with increasing molecular weightand length of the alkyl side-chain, it is possible that if theyenter the body intact they may accumulate in lipid-richtissues. However, table 2 of Darbre et al. (2004) does notreveal a pattern of greater paraben tissue levels consistentwith partition coefficients. For example, methylparaben,the least lipophilic of the parabens, was detected inthe highest concentrations in breast tumor tissue and itappears that tissue concentrations are almost in reverseorder to their partition coefficients.

Harvey and Everett (2004) allude in their Editorial tothe study by Mirick et al. (2002) reporting the absence of anassociation between breast cancer and the use of underarmcosmetic products. However, some additional informationabout this study is relevant to this discussion. The NationalCancer Institute (NCI) conducted a population-based case–control study involving 813 cases of breast cancer to invest-igate possible associations between the use of underarmdeodorants and increased risk of breast cancer in womenaged 20–74 years. Product use, including antiperspirantsand deodorants, and the use of a blade for underarm hairremoval were confirmed by personal interview with 810cases and 793 controls (Mirick et al., 2002). The absence ofreported associations between cosmetic use and breastcancer reduces the likelihood that parabens might be asso-ciated etiologically with the development of breast cancer.Further, the majority of underarm products sold in theUSA and the UK do not contain parabens (Dr G. McEwen,Vice-President-Science for the Cosmetic, Toiletry, andFragrance Association, personal communication).

The inclusion of organochlorine data in table 4 ofDarbre et al. (2004) appears to confirm their concernsbut in fact does not. Over the past two decades almost30 studies have been conducted to address the concernthat weakly estrogenic organochlorine compounds, suchas polychlorinated biphenyls (PCBs) and DDT, might beassociated with an increased risk of breast cancer. Thesestudies fall into two distinct categories. Like Darbre et al.(2004), the small early studies (e.g. Wassermann et al., 1976;Unger et al., 1984; Falck et al., 1992; Dewailly et al., 1994)

298 R. GOLDEN AND J. GANDY

Copyright © 2004 John Wiley & Sons, Ltd. J. Appl. Toxicol. 24, 297–306 (2004)

were derived from active cases of breast cancer andappeared to indicate increased risk of disease. Virtuallynone of the numerous large studies (e.g. Hunter et al.,1997; Hoyer et al., 1998; Dorgan et al., 1999; Helzlsoueret al., 1999; Demers et al., 2000; Millikan et al., 2000;Stellman et al., 2000; Wolff et al., 2000; Zheng et al., 2000;Demers et al., 2002; Moysich et al., 2002). have detecteda statistically significant increased risk of breast cancerassociated with organochlorine exposure. Many of thesesubsequent studies were prospective in design, therebyeliminating the possible confounding effects of disease-related alterations in organochlorine serum levels. Demerset al. (2000) have concluded that ‘taken together, resultsfrom six large epidemiological studies reported during thelast 2 years, including our own, provide little indication thatorganochlorine exposure is a risk factor for [breast cancer]’.Specifically with respect to DDE, a recent extensivemeta-analysis by Lopez-Cervantes et al. (2004) concluded:‘Overall, these results should be regarded as strong evid-ence to discard the putative relationship between p,p′-DDE and breast cancer risk.’ In assessing the likelihoodthat breast cancer might be associated with exposure toendocrine-active compounds, the IPCS (2002) concluded:‘Overall, the current scientific evidence (from humanand animal studies) does not support a direct associationbetween exposure to environmental EDCs [endocrine-disrupting compounds] and increased risk of breastcancer.’ Consequently, it appears unwarranted to assume

that weakly estrogenic chemicals (including parabens)found in breast tumor tissue are etiologically associatedwith the disease.

Preliminary findings that have potential implications forpublic health should be published, but it should be done ina manner that reflects sound science; we suggest that thestudy by Darbre et al. (2004) does not meet this criterion.Appropriate tissue controls were not used, laboratory con-tamination with parabens was not eliminated, exposure toparabens via drug treatment was possible and was noteliminated, the implication that parabens bioaccumulatein breast tissue is inconsistent with both the literatureand the pattern of parabens found in the tissues, existingepidemiological data indicating the absence of an associa-tion between underarm deodorants and breast cancer wasnot fully discussed and, finally, the study findings were notconfirmed before publication — a necessary precaution insuch an emotive area.

ROBERT GOLDENToxLogic, LLC, Potomac, MD 20854, USA

JAY GANDYCTEH, LLC, Little Rock, AR 72201, USA

(Both R. G. and J. G. have served as consultants to theCosmetic, Toiletry, and Fragrance Association (CTFA) inreviewing the toxicity data on parabens.)

REFERENCES

Darbre PD. 2003. Underarm cosmetics and breast cancer. J. Appl.Toxicol. 23: 89–95.

Darbre PD, Byford JR, Shaw LE, Horton RA, Pope GS, Sauer MJ.2002. Oestrogenic activity of isobutylparaben in vitro and invivo. J. Appl. Toxicol. 22: 219–226.

Darbre, PD, Aljarrah A, Miller WR, Coldham NG, Sauer MJ,Pope GS. 2004. Concentrations of parabens in human breasttumours. J. Appl. Toxicol. 24: 5–13.

Demers A, Ayotte P, Brisson J, Dodin S, Robert J, Dewailly E.2000. Risk and aggressiveness of breast cancer in relation toplasma organochlorine concentrations. Cancer Epidemiol.Biomarkers Prev. 9: 161–166.

Demers A, Ayotte P, Brisson J, Dodin S, Robert J, Dewailly E.2002. Plasma concentrations of polychlorinated biphenylsand the risk of breast cancer: a congener-specific analysis.Am. J. Epidemiol. 155: 629–635.

Dewailly E, Dodin S, Verreault R, Ayotte P, Sauve L, Morin J,Brisson J. 1994. High organochlorine body burden in womenwith estrogen receptor-possitive breast cancer. J. Natl. Can-cer Inst. 86: 232–234.

Dorgan JF, Brock JW, Rothman N, Needham LL, Miller R,Stephenson Jr HE, Schussler N, Taylor PR. 1999. Serumorganochlorine pesticides and PCBs and breast cancer risk:results from a prospective analysis (USA). Cancer CausesControl 10: 1–11.

Falck Jr F, Ricci Jr A, Wolff MS, Godbold J, Deckers P. 1992.Pesticides and polychlorinated biphenyl residues in humanbreast lipids and their relation to breast cancer. Artch.Environ. Health 47: 143–146.

Harvey PW. 2003. Parabens, oestrogenicity, underarm cosmeticsand breast cancer: a perspective on a hypothesis. J. Appl.Toxicol. 23: 285–288.

Harvey PW, Everett DJ. 2004. Significance of the detection ofesters of p-hydroxyvenzoic acid (parabens) in human breasttumours. J. Appl. Toxicol. 24: 1–4.

Helzlsouer KJ, Alberg AJ, Huang HY, Hoffman SC, Strickland PT,Brock JW, Burse VW, Needham LL, Bell DA, Lavigne JA,Yager JD, Comstock GW. 1999. Serum concentrations oforganochlorine compounds and the subsequent develop-

ment of breast cancer. Cancer Epidemiol. Biomarkers Prev.8: 525–532.

Hossaini A, Larsen JJ, Larsen JC. 2000. Lack of oestrogenic ef-fects of food preservatives (parabens) in uterotropic assays.Food Chem. Toxicol. 38: 319–323.

Hoyer AP, Grandjean P, Jorgensen T, Brock JW, Hartvig HG.1998. Organochlorine exposure and risk of breast cancer.Lancet 352: 1816–1820.

Hunter DJ, Hankinson SE, Laden F, Colditz GA, Manson JE,Willett WC, Speizer FE, Wolff MS. 1997. Plasma organo-chlorine levels and the risk of breast cancer. New Eng. J.Med. 337: 1253–1258.

International Programme on Chemical Safety (IPCS). 2002.Global Assessment of the State-of-the-Science of EndocrineDisruptors, WHO/PCS, EDC/02.2. World Health Organization:Geneva.

Lopez-Cervantes M, Torres-Sanchez L, Tobias A, Lopez-Carrillo L.2004. Dichlorodiphenyldichloroethane burden and breastcancer risk: a meta-analysis of the epidemiologic evidence.Environ. Health Perspect. 112: 207–214.

Millikan R, DeVoto E, Duell EJ, Tse CK, Savitz DA, Beach J,Edmiston S, Jackson S, Newman B. 2000. Dichloro-diphenyldichloroethene, polychlorinated biphenyls, andbreast cancer among African–American and White womenin North Carolina. Cancer Epidemiol. Biomarkers Prev. 9:1233–1240.

Mirick DK, Davis S, Thomas DB. 2002. Antiperspirant use and therisk of breast cancer. J. Natl. Cancer Inst. 94: 1578 –1580.

Moysich KB, Ambrosone CB, Mendola P, Kostyniak PJ,Greizerstein HB, Vena JE, Menezes RJ, Swede H, Shields PG,Freudenheim JL. 2002. Exposures associated with serumorganochlorine levels among postmenopausal womenfrom western New York State. Am. J. Ind. Med. 41: 102–110.

Routledge EJ, Parker J, Odum J, Ashby J, Sumpter JP. 1998.Some alkyl hydroxy benzoate preservatives (parabens) areestrogenic. Toxicol. Appl. Pharmacol. 153: 12–19.

Stellman SD, Djordjevic MV, Britton JA, Muscat JE, Citron ML,Kemeny M, Busch E, Gong L. 2000. Breast cancer risk in

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relation to adipose concentrations of organochlorinepesticides and polychlorinated biphenyls in Long Island,New York. Cancer Epidemiol. Biomarkers Prev. 9: 1241–1249.

Unger M, Kiaer H, Blichert-Toft M, Olsen J, Clausen J. 1984.Organochlorine compounds in human breast fat from de-ceased with and without breast cancer and in a biopsy ma-terial from newly diagnosed patients undergoing breastsurgery. Environ. Res. 34: 24–28.

Wassermann M, Nogueira DP, Tomatis L, Mirra AP, Shibata H,Arie G, Cucos S, Wassermann D. 1976. Organochlorine com-

pounds in neoplastic and adjacent apparently normal breasttissue. Bull. Environ. Contam. Toxicol. 15: 478–484.

Wolff MS, Zeleniuch-Jacquotte A, Dubin N, Toniolo P. 2000.Risk of breast cancer and organochlorine exposure. CancerEpidemiol. Biomarkers Prev. 9: 271–277.

Zheng T, Holford TR, Mayne ST, Tessari J, Ward B, Carter D,Owens PH, Boyle P, Dubrow R, Archibeque-Engle S,Dawood O, Zahm SH. 2000. Risk of female breast cancerassociated with serum polychlorinated biphenyls and 1,1-dichloro-2,2′-bis(p-chlorophenyl)ethylene. Cancer Epidemiol.Biomarkers Prevent. 9: 167–174.

Reply to Robert Golden and Jay Gandy

Dear Sir,We thank you for the invitation to reply to this

letter that has been sent as a comment on our publicationentitled ‘Concentrations of Parabens in Human BreastTumours’ (Darbre et al., 2004). In reply to the suggestionof inappropriate interpretation of the results, we wouldwish to begin by reiterating that the purpose of this studywas to investigate ‘whether parabens can be detected inhuman breast tissue’ (introduction, p. 6). It was furtherstated in the paper that, from this study, it was not possibleto identify the sources of any measured parabens (dis-cussion, p. 9): ‘these studies cannot identify either thesource of the parabens or whether they entered thehuman body by an oral or by a topical route.’ Nowhere inthe manuscript was any claim made that the presenceof parabens had caused the breast cancer, indeed themeasurement of a compound in a tissue cannot provideevidence of causality.

Although inclusion in these studies of non-tumour breasttissue or tissue from other organs would have providedadditional information on the absorption and distributionof parabens within the body, it could not negate the nu-merical measurements made in the reported study. Theaim of this study was not to implicate causation of breastcancer from increased levels of parabens in tumour versusnormal tissue. The aim simply was to determine whetherparabens can be measured in human breast tissue at all.Indeed, comparison of increased levels in breast tumourversus non-tumour breast would not provide any evidenceof causality of the tumour unless at least longitudinal stud-ies were performed in those women who did and didnot subsequently develop breast cancer. Furthermore,even then, measurement of tissue chemical concentrationsalone cannot provide evidence of causality for a cancerthat can have a long latency period and for which issues ofindividual susceptibility are contributory (Brody and Rudel,2003).

The authors of the critique raise the issue of theblank values. These were fully tabulated and discussed.Owing to the paired nature of blanks and samples, statis-tical analysis was performed using the paired t-test andderived confidence limits (table 3) under statistical adviceacknowledged in the paper. In order to apply a Student’s

t-test across all the samples, the blank values would needto show a normal distribution around a mean. The blanksdid not show equal variation because some blanks werehigher than others and this influenced the tumour extractvalues. Under these circumstances, it is not a valid statis-tical test to compare the average blank value with each ofthe tumour extracts. The only valid statistical test is tocompare each extract with the corresponding blank valuecarried out at the same time. However, whether there is‘no statistical difference’ based on a Student’s t-test acrossall samples, as stated by the authors of the critique,depends upon qualification of the definition of significance.If such a comparison is made between the 6 blank valuesfor total paraben and the 20 tumour extract values fortotal paraben (table 2, lines 17 and 8, respectively) usinga Student’s t-test (two-tailed, two samples, unequal vari-ance), a P value of 0.035 is obtained. If similar t-testsare performed for measurement of individual parabensagainst their corresponding blank values, then P valuesare obtained as follows: P = 0.008 (methylparaben) P =0.005 (ethylparaben), P = 0.178 (n-propylparaben), P =0.474 (n-butylparaben), P = 0.077 (isobutylparaben).

With regard to the use of exemestane in breast cancertherapy, none of these patients were exposed to treatmentwith systemic anticancer drugs (including exemestane andadriamycin) prior to tumour removal.

The authors of this critique point out that 62% of theparaben detected in our study was methylparaben, andthat methylparaben has been reported as inactive in vivoin a rodent uterotrophic study reported by Hossaini et al.(2000). The authors question the relevance of oestrogenicactivity of parabens and cite three studies showinguterotrophic activity for only isobutylparaben and n-butylparaben. However, we would point out the although62% was methylparaben, the remaining 38% was com-prised of ethylparaben, n-propylparaben, n-butylparabenand isobutylparaben. Furthermore, in addition to the stud-ies cited showing in vivo uterotrophic activity for n-butylparaben and isobutylparaben, other parabens havebeen shown to have in vivo oestrogenic activity. Table 1summarizes the available published data on the oestrogenicactivity of parabens in in vivo and in vitro assays. In thestudy by Hossaini et al. (2000), no paraben tested gave a