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• W4225420147 abstract "Vol. 130, No. 5 Letter to the EditorOpen AccessComment on “Application of an in Vitro Assay to Identify Chemicals That Increase Estradiol and Progesterone Synthesis and Are Potential Breast Cancer Risk Factors”is accompanied byApplication of an in Vitro Assay to Identify Chemicals That Increase Estradiol and Progesterone Synthesis and Are Potential Breast Cancer Risk Factorsis a letter which has replyResponse to “Comment on ‘Application of an in Vitro Assay to Identify Chemicals That Increase Estradiol and Progesterone Synthesis and Are Potential Breast Cancer Risk Factors’” Lyle D. Burgoon and Christopher J. Borgert Lyle D. Burgoon Address correspondence to Lyle D. Burgoon. Email: E-mail Address: [email protected] https://orcid.org/0000-0003-4977-5352 Raptor Pharm & Tox, Ltd., Apex, North Carolina, USA Search for more papers by this author and Christopher J. Borgert Applied Pharmacology and Toxicology, Inc., Gainesville, Florida, USA Search for more papers by this author Published:4 May 2022CID: 058002https://doi.org/10.1289/EHP11083AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit The article by Cardona and Rudel (2021) analyzed ToxCast results for the H295R steroidogenesis assays and reported that “296 chemicals increased [estradiol] (182) or [progesterone] (185), with 71 chemicals increasing both.” This result is both fascinating and rather striking in suggesting that in vitro 32% of chemicals increase estrogen production, 32% increase progesterone production, and 12% increase both. Based on our reanalysis of the data (available on Github: https://github.com/DataSciBurgoon/toxcast_steroidogenesis/blob/main/Raptor_SSI_Steroidogenesis.ipynb) and based on the fact that most ToxCast chemicals have small sample sizes (generally n=2, atypically n=8) and therefore likely suffer from sampling bias, most if not all of the chemicals identified by Cardona and Rudel (2021) are false positives (Christley 2010; Gelman and Carlin 2014; Lin 2018).Peculiarly, Cardona and Rudel (2021) did not base their analysis directly on ToxCast data from the U.S. Environmental Protection Agency’s invitrodb database. Rather, they used data from Haggard et al. (2018, 2019); using data from only a single plate for each chemical, ignoring replicate information from other plates. Ignoring available replicates is a violation of common statistical standards and will result in flawed and biased results, mostly due to sampling bias and failure to account for plate effects.The authors also overlooked data quality issues reported for several of the chemicals in the ToxCast dataset in the National Toxicology Program’s Integrated Chemical Environment (ICE) database (Abedini et al. 2021). Thirty-seven and 42 of the chemicals that Cardona and Rudel (2021) identified as increasing estrogen or progesterone production, respectively, have quality flags that indicate that the chemical used may not be what was intended, or that the concentration may be 30% or less than the intended concentration.Furthermore, the progesterone data exhibit a major day effect, indicating they are not of suitable quality for analysis. Without additional information, the progesterone data should be removed entirely.Our reanalysis identified either 17 or 3 chemicals (depending upon stringency) that we allege may be stimulating the production of estrogen in vitro; however, all of those are likely false positives due to small sample sizes and sampling bias. This is far less than the number identified by Cardona and Rudel (2021), suggesting most of the chemicals they identified are false positives. Therefore, we suggest that these 17 or 3 chemicals would be good starting points for further research at human-relevant concentrations, with the understanding that they very well could be false positives due to the limited number of replicates.ReferencesAbedini J, Cook B, Bell S, Chang X, Choksi N, Daniel AB, et al.2021. Application of new approach methodologies: ICE tools to support chemical evaluations. Comput Toxicol 20:100184, 10.1016/j.comtox.2021.100184. Crossref, Google ScholarCardona B, Rudel RA. 2021. Application of an in vitro assay to identify chemicals that increase estradiol and progesterone synthesis and are potential breast cancer risk factors. Environ Health Perspect 129(7):077003, PMID: 64287026, 10.1289/EHP8608. Link, Google ScholarChristley RM. 2010. Power and error: increased risk of false positive results in underpowered studies. Open Epidemiol J 3(1):16–19, 10.2174/1874297101003010016. Crossref, Google ScholarGelman A, Carlin J. 2014. Beyond power calculations: assessing Type S (sign) and Type M (magnitude) errors. Perspect Psychol Sci 9(6):641–51, PMID: 26186114, 10.1177/1745691614551642. Crossref, Medline, Google ScholarHaggard DE, Karmaus AL, Martin MT, Judson RS, Setzer RW, Paul Friedman K. 2018. High-throughput H295R steroidogenesis assay: utility as an alternative and a statistical approach to characterize effects on steroidogenesis. Toxicol Sci 162(2):509–534, 10.1093/toxsci/kfx274. Crossref, Medline, Google ScholarHaggard DE, Setzer RW, Judson RS, Paul Friedman K. 2019. Development of a prioritization method for chemical-mediated effects on steroidogenesis using an integrated statistical analysis of high-throughput H295R data. Regul Toxicol Pharmacol 109:104510, PMID: 31676319, 10.1016/j.yrtph.2019.104510. Crossref, Medline, Google ScholarLin L. 2018. Bias caused by sampling error in meta-analysis with small sample sizes. PLoS One 13(9):e0204056, PMID: 30212588, 10.1371/journal.pone.0204056. Crossref, Medline, Google ScholarL.D.B. has no conflicts to declare and received no funding for this work. C.J.B. has received funding from the Endocrine Policy Forum for evaluation of issues relevant to endocrine disruption.FiguresReferencesRelatedDetailsRelated articlesApplication of an in Vitro Assay to Identify Chemicals That Increase Estradiol and Progesterone Synthesis and Are Potential Breast Cancer Risk FactorsJul 21, 2021, 12:00:00 AMEnvironmental Health PerspectivesResponse to “Comment on ‘Application of an in Vitro Assay to Identify Chemicals That Increase Estradiol and Progesterone Synthesis and Are Potential Breast Cancer Risk Factors’”May 4, 2022, 12:00:00 AMEnvironmental Health Perspectives Vol. 130, No. 5 May 2022Metrics Downloaded 494 times About Article Metrics Publication History Manuscript received9 February 2022Manuscript accepted12 April 2022Originally published4 May 2022 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days." @default.
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