Tracking Down of a Selected Panel of Parabens: A Validated Method to Evaluate Their Occurrence in Skin Layers
, Ilaria Neri 1,†, Giacomo Russo 2, Sonia Laneri 1,* and Lucia Grumetto 1,*
Luisa Coderch
Round 1
Reviewer 1 Report
This is an interesting work to facilitate the analyses of a group of parabens in the skin. The Liquid Chromatography Method is quite well developed and validated, however, in my opinion it can not be considered a new method to evaluate bioaccumulation in skin. Both the Title and the Introduction seems to be focused on a new methodology to determine the penetration of parabens on skin but in stead it is only a new analytical method to quantify parabens. Please consider the following comments:
1. The title does not reflect the work since there is not “a new methodology to Evaluate their Potential Bioaccumulation in Skin” I would said “a new methodology to analyze their presence in the skin”
2. Introduction is vey difficult to follow, please separate it in paragraphs for subjects.
3. “Skin Sample Preparation and Extraction” In the methodology presented, the three skin layers SC, epidermis and dermis are separated first and the parabens are applied later followed by N2 evaporation and extraction. By this methodology you can study the matrix effect but not bioaccumulation. Parabens penetration was not performed in real conditions which have to be done in the total skin, followed by skin layer separation.
4. Both standards and blanc extraction were solubilized EtOH, while SC, Ep and De were extracted in EtOH:H2O 50%. What is the reason? It wouldn’t have been better to apply the same extracting solution?
5. In sentence “LOD and LOQ parameters, with values ranging from 0.026 to 0.419 and from 0.087 to 1.395”, these two high values 0.419 and 1.395 do not correspond to the values of Table 1. There is also an error in Matrix effect of BuP 0.81%, please delete %.
6. Perhaps it would be good to have in Table 3 also values of LOQ, which indicates the linearity range.
7. In general, there is a lack of discussion on the results obtained. Recovery of the different parabens in the different layers.
Author Response
Reviewer 1
This is an interesting work to facilitate the analyses of a group of parabens in the skin. The Liquid Chromatography Method is quite well developed and validated, however, in my opinion it can not be considered a new method to evaluate bioaccumulation in skin. Both the Title and the Introduction seems to be focused on a new methodology to determine the penetration of parabens on skin but in stead it is only a new analytical method to quantify parabens. Please consider the following comments:
- The title does not reflect the work since there is not “a new methodology to Evaluate their Potential Bioaccumulation in Skin” I would said “a new methodology to analyze their presence in the skin”
We agree with referee that this study is focused on a new analytical method to quantify parabens. The title of the submitted manuscript was “Tracking Down of a Selected Panel of Parabens: A Validated Method to Evaluate Their Potential Bioaccumulation in Skin Layers”, as you can see in the downloaded manuscript in Cosmetics web site. Indeed, we specified at the end of introduction section (pag. 2 lines 78-80) that “……we propose and validate an analytical method to simultaneously determine up to seven PBs in each skin layer, Stratum Corneum, Epidermis, and Dermis,…”
- Introduction is very difficult to follow, please separate it in paragraphs for subjects.
According to referee suggestion, to make the reading easier, we separated the paragraphs.
- “Skin Sample Preparation and Extraction” In the methodology presented, the three skin layers SC, epidermis and dermis are separated first and the parabens are applied later followed by N2 evaporation and extraction. By this methodology you can study the matrix effect but not bioaccumulation. Parabens penetration was not performed in real conditions which have to be done in the total skin, followed by skin layer separation.
We did not perform our research as a transdermal passage through the skin, already evaluated in another our published study (Neri et al., 2022), but we evaluated the occurrence of Parabens in each skin layers and quantified them as in other recent studies such as (Demurtas et al., 2021). In light of referee consideration, we changed the title as follow: “Tracking Down of a Selected Panel of Parabens: A Validated Method to Evaluate their occurrence in Skin Layers”.
- Both standards and blanc extraction were solubilized EtOH, while SC, Ep and De were extracted in EtOH: H2O 50%. What is the reason? It wouldn’t have been better to apply the same extracting solution.
We used standards solubilized in ethanol to enhance PBs penetration, while the choose of a mixture of organic solvent/water, as reported also in other studies (Pedersen et al., 2007; Seo et al., 2017), is a “green” solution that achieved the same recoveries of 100 % ethanol.
- In sentence “LOD and LOQ parameters, with values ranging from 0.026 to 0.419 and from 0.087 to 1.395”, these two high values 0.419 and 1.395 do not correspond to the values of Table 1. There is also an error in Matrix effect of BuP 0.81%, please delete %.
According to referee suggestion we amended in the main manuscript.
- Perhaps it would be good to have in Table 3 also values of LOQ, which indicates the linearity range.
We added new column in Table 3 reporting LOQ values.
- In general, there is a lack of discussion on the results obtained. Recovery of the different parabens in the different layers
We added some considerations about the recoveries in the amended manuscript.
REFERENCES
Demurtas, A., Pescina, S., Nicoli, S., Santi, P., Ribeiro de Araujo, D., Padula, C., 2021. Validation of a HPLC-UV method for the quantification of budesonide in skin layers. Journal of Chromatography B 1164, 122512.
Neri, I., Laneri, S., Di Lorenzo, R., Dini, I., Russo, G., Grumetto, L., 2022. Parabens Permeation through Biological Membranes: A Comparative Study Using Franz Cell Diffusion System and Biomimetic Liquid Chromatography. Molecules.
Pedersen, S., Marra, F., Nicoli, S., Santi, P., 2007. In vitro skin permeation and retention of parabens from cosmetic formulations. International Journal of Cosmetic Science 29, 361-367.
Seo, J.E., Kim, S., Kim, B.H., 2017. In vitro skin absorption tests of three types of parabens using a Franz diffusion cell. J Expo Sci Environ Epidemiol 27, 320-325.
Reviewer 2 Report
The Authors presented a study entitled: “Tracking Down of a Selected Panel of Parabens: A Validated Method to Evaluate Their Potential Bioaccumulation in Skin Layers”.
The work is very interesting and could be helpful for determine the toxicological risk related to the attentioned categories ingredients in cosmetic products, as the preservatives.
However, as a suggestion, regarding the allowed concentrations for parabens in cosmetic products in accordance with the SCCS opinions about their toxicological profile, and the EU Regulation 1223/2009, the difference between the leave-on and rinse-off products should be considerd.
Moreover, the Authors should consider just some advice before the publication for improving the manuscript, as follows:
In the “Materials and Methods” section, at paragraph “2.8. Selectivity”, please, explain better the expression “after the verify the occurrence” reported at line 161.
Again, in the “Materials and Methods” section, at paragraph “2.10. Matrix Effect”, please, explain better the sentence “The matrix effect was investigated by calculating the ratio of the peak area in the presence of matrix (matrix spiked with PBs mixed solution) to the peak area in the absence of matrix (PBs mixed pure solution)”, reported at lines 168-170.
In the “Results and Discussion” section, at paragraph “3.2. Method Validation”, please check the correspondence between the LOD and LOQ values 0.419 μg mL-1, and 1.395 μg mL-1, reported in table 1, and those reported at lines 209, 210, respectively.
The same check should be accomplished for the recovery values 62.26 and 63.68 attributed to SC and Epidermis, respectively, both reported at line 214, and those reported in table 2. Furthermore, the standard deviation should be reported for the recovery values.
In my opinion, the peaks reported in all the panels of figure 1 should be labelled along with their relative retention time.
Again, in the “Results and Discussion” section, the Authors, reported the following sentence (“Furthermore, these studies assess PBs in other matrices, such as serum, cosmetic and pharmaceutical samples”) at lines 259-260; could they explain better the sentence if they are referring to a cosmetic serum formulation?
Author Response
Reviewer 2
The Authors presented a study entitled: “Tracking Down of a Selected Panel of Parabens: A Validated Method to Evaluate Their Potential Bioaccumulation in Skin Layers”.
The work is very interesting and could be helpful for determine the toxicological risk related to the attentioned categories ingredients in cosmetic products, as the preservatives.
However, as a suggestion, regarding the allowed concentrations for parabens in cosmetic products in accordance with the SCCS opinions about their toxicological profile, and the EU Regulation 1223/2009, the difference between the leave-on and rinse-off products should be considerd.
We thank the reviewer for the advice. The information about PB concentration in leave-on and rinse-off products is reported in the cited references. We aim at keeping the discussion brief and concise and believe this suffice.
Moreover, the Authors should consider just some advice before the publication for improving the manuscript, as follows:
-In the “Materials and Methods” section, at paragraph “2.8. Selectivity”, please, explain better the expression “after the verify the occurrence” reported at line 161.
We apologize, but it is a typing error that now we amended
-Again, in the “Materials and Methods” section, at paragraph “2.10. Matrix Effect”, please, explain better the sentence “The matrix effect was investigated by calculating the ratio of the peak area in the presence of matrix (matrix spiked with PBs mixed solution) to the peak area in the absence of matrix (PBs mixed pure solution)”, reported at lines 168-170.
To better clarify we added a sentence in the section 2.10. Matrix effect.
-In the “Results and Discussion” section, at paragraph “3.2. Method Validation”, please check the correspondence between the LOD and LOQ values 0.419 μg mL-1, and 1.395 μg mL-1, reported in table 1, and those reported at lines 209, 210, respectively.
According to referee suggestion we amended in the main manuscript.
-The same check should be accomplished for the recovery values 62.26 and 63.68 attributed to SC and Epidermis, respectively, both reported at line 214, and those reported in table 2. Furthermore, the standard deviation should be reported for the recovery values.
We amended the values in the manuscript and added the standard deviation in Table 2
-In my opinion, the peaks reported in all the panels of figure 1 should be labelled along with their relative retention time.
We added the acronyms and retention time for each PB in Figure 1, as suggested by referee.
-Again, in the “Results and Discussion” section, the Authors, reported the following sentence (“Furthermore, these studies assess PBs in other matrices, such as serum, cosmetic and pharmaceutical samples”) at lines 259-260; could they explain better the sentence if they are referring to a cosmetic serum formulation?
According to the referee suggestion, we clarified in the main text that “serum” is intended as blood serum and not as cosmetic formulation.
