-->PONE-D-25-65913-->-->Excessive manganese content in children’s multivitamin supplements: potential for neurodevelopmental harm and other adverse health outcomes-->-->PLOS One

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The authors have declared that no competing interests exist. EJM’s affiliation is with Better Life Laboratories, Inc., a nonprofit organization that conducts scientific research and provides technical expertise, equipment, and training to help needy people around the world. Better Life Laboratories received no specific funding for this project from any donors. Donors to Better Life Laboratories provided no input in choosing the subject matter of this project, the hypotheses that were tested, the method of analysis, the research findings, or the manner of disseminating the results. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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Additional Editor Comments:

Dear Authors,

The reviewers have recommended reconsideration of the manuscript following major revisions. Please consider the following points during revision:

1. The use of complementary analytical techniques is generally desirable. However, for trace metal determination, ICP-OES provides greater precision, making the additional use of spectrometric methods, as presented in the Results section, less justifiable. Unless scientifically necessary, ICP-OES should be used as the sole analytical technique in the present study, consistent with what is summarized in the Abstract.
2. Reference articles suggested by reviewers should be cited only if they add value to or improve the manuscript. Editorial decisions are not based on whether such articles are cited.
3. Additional comments and suggestions are provided in the attached MS Word file.

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Partly

Reviewer #2: No

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: No

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-->3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: Dear editor and authors,

The manuscript entitled "Excessive manganese content in children’s multivitamin supplements: potential for neurodevelopmental harm and other adverse health outcomes" evaluated Mn contents in multivitamin and mineral supplements using ICP OES. It presents scientific relevance for Chemistry, Health, Food, Medicine, Biological and others area. The language (English) is satisfactory (but, I suggest the final revision)! However, you need to change some details/information in the Title, Abstract, Introduction, Material and Methods, results and discussion, and conclusions.

1. Title: Adequate!

2. Abstract:

- The abstract is well-written but lacks the main objective of the study. Additionally, it lacks details on the methods and parameters used. What are the optimal conditions for acid digestion? I suggest including information about analytical validation. Has the method been validated? What were the validated parameters? I suggest inserting numerical data related to analytical validation and analyte concentration (range, content in mg/kg).

- Please replace "ICP-OES" with "ICP OES" (without the hyphen) here and throughout the manuscript, as it is not a hyphenated analytical technique.

- Lastly, I suggest highlighting the advantages, innovations, and disadvantages of the proposed method.

- Graphical Abstract/Highlights: ??

3. Introduction section:

- Since an article proposal is also the development/validation of analytical methods, I suggest including more discussions on sample preparation and information/references on analytical techniques (ICP OES) and/or other techniques based on ICP. I suggest consulting and inserting these manuscripts that discuss acid digestion, bioacessibility and ICP technique:

- Total and Bioaccessible contents of Microelements in Multivitamin Formulations Exposed to Consumption in Brazil using in vitro Gastrointestinal Digestion (INFOGEST Protocol). Journal of Trace Elements and Minerals (2025). https://doi.org/10.1016/j.jtemin.2025.100224

- In vitro Monitoring of Macro and Microelements in Multimineral Preparations across Dissolution Profiles by Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). Journal of the Brazilian Chemical Society (2017). (http://dx.doi.org/10.21577/0103-5053.20170065).

- Analytical approach of elemental impurities in pharmaceutical products: A worldwide review. Spectrochimica Acta Part B-Atomic Spectroscopy (2023). (https://doi.org/10.1016/j.sab.2023.106689)

- Multielement Determination of Macro and Micro Contents in Medicinal Plants and Phytomedicines from Brazil by ICP OES. Journal of the Brazilian Chemical Society (2017). (http://dx.doi.org/10.5935/0103-5053.20160187).

- At the end of the introduction, I suggest at the end of the introduction, to highlight the "innovative" proposal of the method, as well as the advantages/innovation/disadvantages.

4. Material and methods

- In section “Sample Selection”: What are the conditions for collection/acquisition and storage of samples? What is the time/period (from acquisition to analysis)? The text “To assess the accuracy of our results, we also purchased Multielement Tablets Standard Reference Material® (SRM 3294) from the National Institute of Standards and Technology (NIST)” should be moved to section “Quality Assurance and Quality Control”.

- In section “Sample Preparation”: For pretreatment, did the authors follow their own protocol? Or did they follow a reference? If so, please indicate the reference! What is the residual acidity of the samples? Was the dissolved carbon content measured in the samples?

- In section “Sample Analysis by Spectrophotometry”: This information is not included in the abstract! What is the purpose of using molecular absorption spectrophotometry if the proposal is ICP OES? They are different techniques! Was there analytical validation? Did the authors follow their own protocol? Or did they follow a reference?

- In section “Sample Analysis by Inductively Coupled Plasma-Optical Emission Spectroscopy”: How were the ICP OES conditions optimized? Spectral lines? Flux? Did you follow the manufacturers' recommendations or study (uni/multivariately) until the optimal conditions were defined? Has any spectral interference or matrix effect study been carried out?

- In section “Quality Assurance and Quality Control”: And the aspects associated with analytical validation? Have the methods been validated? What protocol was followed? How were the parameters evaluated: LOD, LOQ, precision, accuracy, robustness and linearity? Which concentration ranges were studied? What concentration levels are used to assess accuracy? I suggest detailing the proposed method in more detail... Information on “potential interferents” should be described here!

5. Results and Discussion

The results are interesting, but I suggest expanding the discussions and compare them with other studies published in the literature.

- In section “Accuracy and Precision of the Determination of Manganese Results by Spectrophotometry”: There is no discussion here! I suggest combining it with section “Hypothesis About the Bioavailability of Manganese in Some of the Children’s Supplement Products in our Study” and expanding the discussions and compare them with other studies published in the literature.

- In section “The Detection Limit, Accuracy, and Precision of the Determination of Manganese by Inductively Coupled Plasma-Optical Emission Spectroscopy”: I suggest naming this section: “analytical validation” or “Quality Assurance and Quality Control”. How was the matrix effect evaluated? I suggest expanding the discussions. Please replace "ICP-OES" with "ICP OES" (without the hyphen) here and throughout the manuscript, as it is not a hyphenated analytical technique.

- In section “The Manganese Concentrations Reported on Labels Compared to the Concentrations Measured in Samples”: Table 2 should be better presented, with upper and lower lines. Standard deviation values should be added. There is no discussion here! I suggest incorporating toxicological aspects of Mn into the mansucript for humans. I suggest expanding the discussions and compare them with other studies published in the literature (using the same matrix or others). A good suggestion would be to create a table with data on Mn determination in this matrix in different countries, comparing the sample digestion preparation and analytical technique used.

- In section “Regulations Applicable to Manganese in Children’s Supplements in the United States”: This section should be combined with the previous one “The Manganese Concentrations Reported on Labels Compared to the Concentrations Measured in Samples”; “The Generally Recognized as Safe List and the Labeled Supplemental Manganese Ingredients” and “Governmental Recommendations Applicable to the Manganese Content in Children’s Supplements”. Please, to expand the discussions.

- The sections “Daily Exposure Estimates Based on Labeled Values”, “Daily Exposure Estimates Based on Measured Values” and “Daily Exposure Estimates Based on Ordinary Dietary Intakes in Addition to Measured Values in Supplements” should be combined.

- I suggest, at the end of the "results and discussion", to write a paragraph summarizing the findings and their impacts on the research proposal.

6. Conclusion: I suggest inserting the main findings (numerical data), and to indicate disadvantages/limitations of the method and the study!

7. Table and Figures: Adequate! See comments above.

8. References: I suggest inserting the indicated references into the manuscript. Please, check if the references are in accordance with the journal's rules.

Reviewer #2: Introduction

The Introduction addresses an important and timely issue of potential manganese exposure in children from dietary supplements; however, the argumentation relies primarily on cross-sectional studies and biomarker data whose methodological limitations are not sufficiently discussed. The authors present associations between manganese levels and health effects as largely established, despite the fact that the available evidence is observational in nature and does not demonstrate causality. There are no direct data linking manganese supplementation to actual health disorders in children. The narrative is distinctly alarmist and does not precisely refer to doses that cause documented clinical effects. The bioavailability of manganese from supplements and its interactions with other dietary components are also not discussed, which limits a reliable risk assessment. The formulated research hypotheses are weakly connected to the presented risk narrative, as they are limited to assumptions regarding the agreement between measured and labeled manganese content and non-exceedance of tolerable levels, without directly addressing children’s health or the toxicological mechanisms that are strongly emphasized earlier.

Materials and Methods

The Materials and Methods section describes the analytical procedures in relatively sufficient detail to allow reproduction of the laboratory work; however, it contains significant limitations. A major concern is the sampling strategy. The authors limited product selection exclusively to the Amazon platform, which does not constitute a random or representative sample of the U.S. children’s supplement market. There is no justification as to why Amazon would reflect the actual market structure, nor is there any information on market share of individual brands or brick-and-mortar retail sales (pharmacies, retail chains). The statement that the selected 26 products constitute a “reasonable representation” of the market is not supported by any data and is arbitrary. Consequently, generalization of the results to the entire U.S. market is methodologically unjustified.

There is also a problem with the comparison group. Products without declared manganese were randomly selected from Amazon brands, but their pharmaceutical forms (mainly gummies) differ substantially from those of manganese-containing supplements. There is no justification as to whether and how this control group constitutes an appropriate reference.

An important methodological flaw appears in the statistical section. The calculation of “percent differences” is unusual and non-intuitive (division by the mean of the differences rather than by the labeled value), which may lead to under- or overestimation of relative deviations and hinders interpretation in the context of label compliance. In addition, there is no justification for the selection of statistical tests with respect to specific research hypotheses, and the description of the analyses is very general.

Results and Discussion

This section provides valuable analytical data on manganese content in children’s supplements; however, conclusions regarding bioavailability, health risk, and “exceedances” of ULs are largely over-interpreted, based on simplified assumptions, and lead to a narrative of potential hazard that is not fully justified by the presented data.

In the initial part, the authors demonstrate that the spectrophotometric PAN method is unreliable in the studied matrix, which should be acknowledged as appropriate. However, the subsequent explanation for the method’s failure is problematic. The authors suggest that poor recoveries may indicate strong binding of manganese in supplements and potential “removal” of manganese from the child’s body, which is a far-reaching and unjustified speculation. The digestion conditions (nitric acid + hydrogen peroxide, 120°C) are far more aggressive than physiological conditions, and lack of recovery in such a matrix is a strictly analytical phenomenon and cannot be used to infer bioavailability or possible “sequestration” of minerals in the gastrointestinal tract.

The most serious concern relates to the estimation of exposure and UL exceedances. The authors treat Adequate Intake as equivalent to median intake and use it as an “average dietary background,” which is a simplification and may lead to overestimation of total exposures. Moreover, ULs for manganese in children are based on very limited data, as the authors themselves acknowledge, yet they are nevertheless used as rigid toxicity thresholds, creating an alarmist narrative of “exceedances” without accounting for the uncertainty of these values or individual variability in dietary and water-borne manganese intake. The regulatory discussion and references to the GRAS list also contain significant inaccuracies. The absence of a particular manganese form from the GRAS list does not automatically imply illegality or unsafety in dietary supplements, which is indirectly suggested and may mislead readers.

Conclusions

The conclusions presented in the Conclusions section largely go beyond what can be directly inferred from the empirical data obtained in the study, and the narrative has a distinctly normative and alarmist character that is not fully justified by the scope and quality of the collected data.

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: mn draft 14 AE COMMENTS.docx

Excessive manganese content in children’s multivitamin supplements: potential for neurodevelopmental harm and other adverse health outcomes

PONE-D-25-65913R1

Dear Dr. Frisbie,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Timothy Omara

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: N/A

Reviewer #2: Yes

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-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: Although they did not agree with some of my suggestions, the authors improved the manuscript. Accept.

Reviewer #2: Thank you to the authors for their appropriate adjustments to the sections of concern and their astute observations and comments back.

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Reviewer #1: No

Reviewer #2: No

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