Assessment of Pristine Carbon Nanotubes Toxicity in Rodent Models

Round 1

Reviewer 1 Report

 This manuscript reviews literature focusing on in vivo toxicity of carbon nanotubes in, primarily rodent model, which reveals toxicities including inflammation, fibrosis and pathology at organ levels. The authors provide current guidelines for testing nanomaterials, and summarized in vivo studies primarily from 2009 to 2022.

Major concerns:

1)      Criteria for inclusion in the review should be clarified. Many nanotoxicity-related animal studies in the literature are invalid and misleading. Did the included literature follow IACUC guideline for animal study? Did the included studies have enough numbers of the animals in each group? It is required to have at least five animals in each group. Did the included studies use appropriate methods for statistical analysis? The authors should not include the literature that does not follow general requirement for a valid in vivo study.

2)      The authors indicate that the size of CNTs is closely related to the toxicity, without providing comment on the dose. Is there a range of threshold dose generally considered safe in application of CNTs as nanomedicine in these animal model?

3)      The authors should provide specific features of CNTs that cannot be replaced with other nanomaterials that is more safe, in spite of all different toxicities observed and described in the literature.

Minor concerns:

1)      The writing and wording requires editing. For instance, Line 154- Recent studies have recently shown that…..; line 437 etc.

2)      Inconsistent format problems, including references from #99-110 are listed with two numbering system, double bracket for reference numbers in Tables etc.

Author Response

Firstly, we would like to express our deepest thanks to the Reviewer for devoting time to reviewing our manuscript, the corrections and suggestions. We have carried out a revision of the manuscript and we believe the paper has been improved.

The Reviewer's comment: Criteria for inclusion in the review should be clarified. Many nanotoxicity-related animal studies in the literature are invalid and misleading. Did the included literature follow IACUC guideline for animal study? Did the included studies have enough numbers of the animals in each group? It is required to have at least five animals in each group. Did the included studies use appropriate methods for statistical analysis? The authors should not include the literature that does not follow general requirement for a valid in vivo study.

The Authors’ answer: Due to the fact that all publications have been published, we would not like to undermine the reliability of the work of other authors. It is true, however, that many studies follow different guidelines in the case of animals or statistical evaluation, but we believe that all these results can be used in assessing the toxicity of carbon nanotubes. As we mentioned in the review of the in vivo study after exposure to carbon nanotubes, they differ very significantly in various respects and it is necessary to develop specific rules for conducting these studies in order to make a reliable assessment.

The Reviewer's comment:. The authors indicate that the size of CNTs is closely related to the toxicity, without providing comment on the dose. Is there a range of threshold dose generally considered safe in application of CNTs as nanomedicine in these animal model?

The Authors’ answer: As correctly noticed, the dosage information was not written. According to the Reviewer's suggestion, we added a fragment about aspect of dose in toxicity. However, the literature lacks the exact doses that affect toxicity in an animal model, which may be due to the fact that the toxicity of carbon nanotubes is difficult to define unambiguously. Comparing the studies individually, however, it can be seen that some toxic effects occurred at higher doses, but this is a topic that is not yet fully understood.

The Reviewer's comment: The authors should provide specific features of CNTs that cannot be replaced with other nanomaterials that is more safe, in spite of all different toxicities observed and described in the literature.

The Authors’ answer: We would like to thank the Reviewer for this comment and careful review of our manuscript. The concept of our manuscript was to show pristine carbon nanotubes used in in other research and their toxic effects in different organs, because their properties described in the manuscript still make them interesting material to use in biomedical applications, so there is a need to understand their toxic effects thoroughly. In order to enrich manuscript we added some more information’, following suggestion.

The Reviewer's comment: The writing and wording requires editing. For instance, Line 154- Recent studies have recently shown that…..; line 437 etc. Inconsistent format problems, including references from #99-110 are listed with two numbering system, double bracket for reference numbers in Tables etc.

The Authors’ answer: According to the Reviewer's suggestion, the changes have been marked up using the “Track Changes” function.

Reviewer 2 Report

The present manuscript proposes to carry out a review regarding carbon nanotubes in vivo toxicity. Several other recent publications present well-updated and important reviews on the topic, so the content of this manuscript does not make a significant contribution to the topic. In addition, when the references cited by the authors are observed, important references involving carbon nanotubes and their toxicity published by important research groups around the world are not mentioned. Another factor that must be taken into account is the little critical and in-depth text on this subject. Therefore, more figures and discussion should be added.

Author Response

Firstly, we would like to express our deepest thanks to the Reviewer for devoting time to reviewing our manuscript, the corrections and suggestions. We have carried out a revision of the manuscript and we believe the paper has been significantly improved.

The Reviewer's comment: The present manuscript proposes to carry out a review regarding carbon nanotubes in vivo toxicity. Several other recent publications present well-updated and important reviews on the topic, so the content of this manuscript does not make a significant contribution to the topic.

The Authors’ answer: We would like to thank the Reviewer for this comment and careful review of our manuscript. Our manuscript differs from such publications, as its focuses on toxic effects of pristine carbon nanotubes in many organs. Our manuscript also concentrate on conveying how important it is do develop standardized procedures for the toxicological assessment of carbon nanotubes, because when evaluating publications, it can be noticed that compering them with each other is very difficult. This suggest that it is difficult to make a reliable toxicological assessment.

The Reviewer's comment: In addition, when the references cited by the authors are observed, important references involving carbon nanotubes and their toxicity published by important research groups around the world are not mentioned.

The Authors’ answer: The concept of the manuscript was to focus on pristine carbon nanotubes, which were only administrated to mice and rats, with their toxicological evaluations. There is indeed a lot of publications focusing on functionalized MWCNTs and SWCNTs, however functionalization has a huge impact on carbon nanotubes properties and toxic effects, and compering them with pristine carbon nanotubes would not be reliable.

           The Reviewer's comment: Another factor that must be taken into account is the little critical and in-depth text on this subject. Therefore, more figures and discussion should be added.

The Authors’ answer: According to the Reviewer's suggestion, we reviewed the text and the changes have been made in some places of the manuscript.