Efficacy and Safety of a Novel Anhydrous 0.1% Retinal-Based Concentrate with Hydrophilic Actives for Photoaged Skin: A Six-Week Prospective Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study presents a novel formulation concept with potential value for cosmetic dermatology. However, several issues need to be addressed before the manuscript can be considered for publication.

1] The absence of a control or comparator group (e.g., vehicle, placebo, or active reference such as retinoic acid) is a significant limitation. This makes it difficult to distinguish the product effect from natural variation or placebo responses. Please acknowledge this more explicitly and discuss how it limits interpretation.

2] The study includes 27 participants without a power calculation. Please justify the sample size and discuss its impact on generalizability. Also, details about skin types or Fitzpatrick classification are missing and should be provided.

3] The test product, moisturizers, and cleanser were all manufactured by the authors’ company (Skinome). This potential conflict of interest should be transparently disclosed in the manuscript.

4] The figures are descriptive but resemble promotional material more than scientific results. Please consider adding statistical plots (boxplots, bar graphs with error bars, confidence intervals) to better illustrate group-level changes.

Author Response

Response to Reviewers

We thank the reviewer for your constructive and detailed feedback. Below we respond point by point.

 

 

1] The absence of a control or comparator group (e.g., vehicle, placebo, or active reference such as retinoic acid) is a significant limitation. This makes it difficult to distinguish the product effect from natural variation or placebo responses. Please acknowledge this more explicitly and discuss how it limits interpretation.

This topic has been more adequately addressed in the discussion section.

Manuscript excerpt (p. 28, Discussion):

“A key limitation of this study is the absence of a vehicle, placebo, or active comparator group. While the significant improvements observed across multiple objective parameters strongly suggest product-related effects, natural variation, placebo responses, the effect of moisturizer use or the combined use of the products cannot be excluded. This limits the ability to establish definitive causal inference. Future studies should therefore adopt randomized, placebo- or vehicle-controlled designs, and ideally include direct comparisons with reference compounds such as all-trans retinoic acid, to more precisely determine the relative efficacy and tolerability of the formulation.”

 

2] The study includes 27 participants without a power calculation. Please justify the sample size and discuss its impact on generalizability. Also, details about skin types or Fitzpatrick classification are missing and should be provided.

The number of participants was pragmatically based on previous experience from similar studies, and the predicted effect has now been added and discussed in the article. The Fitzpatrick photype of the subjects are added.

Manuscript excerpts:

• (p. 14, Statistical Analysis):

“The number of subjects needed were determined pragmatically based on previous experience from similar studies and the number of subjects needed to show a difference.”

• (p. 12, Participants):

“The majority of participants were Fitzpatrick phototype III (21 subjects) and the rest phototype II (6 subjects), with a mix of oily/combination (n=4), normal/dry (n=16) and dry/very dry (n=7) skin types.”

• (p. 29, Discussion):

“Another limitation relates to the relatively small sample size (n = 27), which was determined based on previous experience and feasibility rather than a priori power calculations. Although significant improvements were consistently observed, the limited sample restricts generalizability and increases the risk of type II error for outcomes with smaller effect sizes.”

 

3] The test product, moisturizers, and cleanser were all manufactured by the authors’ company (Skinome). This potential conflict of interest should be transparently disclosed in the manuscript.

Response: We agree that this potential conflict of interest should be fully transparent. In the revised manuscript, we have added an explicit Conflict of Interest statement:
“All study products were manufactured by Skinome Research AB, which employs three of the authors.”

Manuscript excerpt (p. 31, Funding):

“The authors received no funding for this work.”

4] The figures are descriptive but resemble promotional material more than scientific results. Please consider adding statistical plots (boxplots, bar graphs with error bars, confidence intervals) to better illustrate group-level changes.

Our response:
We thank the reviewer for this constructive suggestion. We have revised the figures and added difference plots including confidence intervals and p-values, to present the results in a more rigorous scientific manner and to better reflect group-level changes.

Manuscript excerpt (pp. 19–24, Results):

“The differences plots are presented in Figures 1 to 8 and visualize the absolute calculated difference before versus after for each analyzed parameter and for each individual subject … The 98% confidence intervals are indicated for each parameter.”

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Major Comments

 

Study Design and Subject Selection

The description of the study design is somewhat limited and would benefit from further detail. In particular, the criteria for subject inclusion and exclusion, skin type classification, and Fitzpatrick phototype should be clearly reported. This information is crucial to assess the relevance of the findings and their generalizability. Moreover, it should be clarified whether the product was applied to the entire face or only to selected regions, and whether a split-face or comparator design was considered.

Product Application Protocol

While the manuscript specifies evening application following cleansing, it would be important to indicate whether a wash-out period was implemented prior to baseline to exclude potential confounding from previous skincare routines. The gradual introduction schedule is noted, but it is unclear if this was standardized across all participants or adjusted individually based on tolerability.

Moisturizer Selection

The protocol indicates that the retinal concentrate was mixed in a 1:2 ratio with a moisturizer chosen according to skin type. It should be clarified whether all participants used the same moisturizer or if different formulations were provided depending on skin characteristics. If multiple moisturizers were employed, the key ingredients, emulsion type (O/W or W/O), and potential impact on outcomes should be described in detail. Given the importance of the vehicle in cosmetic studies, the choice of moisturizer requires more justification.

Mixing Procedure and Ratio Control

The methodology regarding the 1:2 mixing ratio raises concerns about reproducibility. It seems unlikely that participants measured this precisely without standardized dispensing mechanisms. The authors should clarify how this ratio was ensured in practice, for example, whether predefined pump dispensers or visual dosing instructions were provided. Without such information, adherence to the intended ratio remains uncertain.

Sample Size and Demographics

The study reports 27 participants, but it is unclear whether this number was based on a power calculation or determined pragmatically. Further information on the distribution of age, gender, and skin type/phototype is necessary to contextualize the findings and evaluate the representativeness of the sample.

 

Method

 

 

 

The description of the equipment employed in this study requires further detail. Specifically, the manuscript should clarify by which optical and computational methods the Antera 3D system evaluates the reported skin parameters. It is important to note that this device does not directly measure skin hydration, as would be done with a corneometer, but rather approximates hydration indirectly through external features such as wrinkle depth and surface appearance. While such imaging systems are valuable for visualization and relative assessments, they may not provide sufficiently rigorous clinical data for certain biophysical endpoints.

I would recommend either

• complementing the current analysis with standardized biophysical instruments- g., a corneometer for hydration, a mexameter for melanin/erythema, and a cutometer for elasticity

 

or

 

• reframing the study as a preclinical or exploratory screening test rather than a full clinical trial. This distinction is important to ensure that the terminology and conclusions accurately reflect the strength of the evidence provided.

 

 

Subjects

 

The description of the study population raises several concerns. The panel includes both men and women; however, male and female skin differs substantially in terms of structure, thickness, sebum production, and aging patterns. This heterogeneity complicates interpretation, and in a small sample size such as this, it may have introduced confounding effects. For clarity and stronger scientific validity, I would recommend either excluding men from the study or analyzing them separately.

In addition, the age range (40–69 years) is very broad. Photoaging severity, skin physiology, and responsiveness to topical actives differ considerably between individuals in their early forties compared to those in their late sixties. A narrower age range, or at least stratified subgroup analyses, would provide more meaningful and interpretable results.

 

 

Statistical

 

While the statistical analysis is reported as being conducted with paired Wilcoxon signed-rank tests, the robustness of the analysis is limited by substantial heterogeneity within the study population and methodology. Several concerns arise:

Inclusion criteria: These are not clearly defined. Without strict criteria, variability in baseline characteristics may undermine the validity of the results.

1. Heterogeneity of the sample: The age range is very broad (40–69 years), encompassing distinct stages of photoaging. Furthermore, men and women exhibit significant physiological differences in skin structure and aging patterns. Pooling them together without stratified analyses reduces interpretability.
2. Moisturizer variability: The test product was mixed with moisturizers adapted to skin type, but it is not specified how many participants used each formulation. Since vehicle composition (ingredients, emulsion type) can strongly influence skin outcomes, this introduces a confounding factor that is not controlled for.
3. Multiplicity of parameters: Numerous endpoints were assessed (pigmentation, wrinkles, texture, pores, redness, etc.), yet no correction for multiple comparisons is mentioned. This increases the likelihood of Type I errors.

Taken together, the number of uncontrolled variables makes it difficult to draw statistically robust and clinically meaningful conclusions from the reported analysis. I would strongly recommend clarifying subject selection, moisturizer allocation, and considering subgroup analyses or adjustments for multiple testing.

 

 

Ethics

 

The ethical approval statement in the manuscript is incomplete. It is necessary to clearly indicate:

• the name of the ethics committee that granted approval, (ii) the official approval/registration number,
• and (iii) the validity period of the approval. These elements are essential for transparency and to ensure compliance with international ethical standards.

 

 

Results

 

The results are currently expressed only as percentage changes with p-values. For greater clarity and scientific rigor, it would be preferable to present both baseline and post-treatment absolute values (mean ± SD or 95% CI), alongside the calculated percentage change. This would allow readers to better interpret the magnitude and clinical relevance of the improvements, rather than relying solely on relative differences.

 

 

The demographic description of the study population lacks sufficient detail. The manuscript does not specify how many of the female participants were post-menopausal, perimenopausal, or premenopausal. This information is critical, as hormonal status strongly influences skin physiology, collagen synthesis, and pigmentation patterns. In addition, the inclusion of both male and female subjects introduces further heterogeneity, as their skin characteristics differ significantly.

Given this variability, the presentation of results appears somewhat overstated and less plausible without acknowledging these confounding factors. The authors should provide a clear breakdown of the participants by menopausal status and sex, and explicitly discuss the limitations arising from the heterogeneity of the study population. Acknowledging these factors would improve the transparency and scientific credibility of the manuscript.

Comments for author File: Comments.pdf

Author Response

Response to Reviewers

We thank the reviewer for your constructive and detailed feedback. Below we respond point by point.

Kindest Regards

Major Comments

 

Study Design and Subject Selection

The description of the study design is somewhat limited and would benefit from further detail. In particular, the criteria for subject inclusion and exclusion, skin type classification, and Fitzpatrick phototype should be clearly reported. This information is crucial to assess the relevance of the findings and their generalizability. Moreover, it should be clarified whether the product was applied to the entire face or only to selected regions, and whether a split-face or comparator design was considered.

Our response:
We thank the reviewer for this important comment. We have expanded the Methods section to include explicit inclusion and exclusion criteria, as well as detailed reporting of Fitzpatrick phototype and skin type classification. It is also clarified that the mixture was applied to the entire face and that no split-face or comparator design was used in this exploratory trial.

Manuscript excerpts:

• (p. 11, Study Design):

“…the mixture was then applied to the entire face … The study included no wash out period prior to using the study products.”

• (p. 12, Participants):

“Exclusion criteria included: Diagnosed skin disorder (rosacea, eczema, acne, psoriasis); Pregnancy and breastfeeding; Exposure to any cosmetic treatment (microneedling, strong chemical peels) in the last 6 months … Any use of botox or fillers prior to the study.”

• (p. 12, Participants):

“The majority of participants were Fitzpatrick phototype III (21 subjects) and the rest phototype II (6 subjects), with a mix of oily/combination (n=4), normal/dry (n=16) and dry/very dry (n=7) skin types.”

 

Product Application Protocol

While the manuscript specifies evening application following cleansing, it would be important to indicate whether a wash-out period was implemented prior to baseline to exclude potential confounding from previous skincare routines. The gradual introduction schedule is noted, but it is unclear if this was standardized across all participants or adjusted individually based on tolerability.

 

Our response:
No wash-out period was implemented, and this is now explicitly stated in the Methods. The gradual introduction schedule was standardized across all participants and is described in detail.

Manuscript excerpt (p. 11, Study Design):

“All subjects were instructed to phase in the test product by using it every third night the first week, every other night the second week, and then every night for the last four remaining weeks. The study included no wash out period prior to using the study products.”

 

Moisturizer Selection

The protocol indicates that the retinal concentrate was mixed in a 1:2 ratio with a moisturizer chosen according to skin type. It should be clarified whether all participants used the same moisturizer or if different formulations were provided depending on skin characteristics. If multiple moisturizers were employed, the key ingredients, emulsion type (O/W or W/O), and potential impact on outcomes should be described in detail. Given the importance of the vehicle in cosmetic studies, the choice of moisturizer requires more justification.

Our response: Different moisturizers was assigned depending on the skin type, this has been clarified in the article. The formulation type (o/w) for the moisturisers has also been specified. All ingredient list are included since before.

The moisturisers were assigned based on skin type, and the products used are existing product in Skinome’s portfolio and is recommended for the skin type respectively. As the skin type varied between the subjects it was decided that one moisturizer will not be suitable for all. It is specified how many participants that got which moisturizer.

• (p. 11, Study Design):

“The moisturizer was chosen based on the participant’s skin type at the start of the study.”

• (pp. 13–14, Test Products):

“The moisturisers used in the study were all o/w emulsions and selected based on the different skin types included in the study … The different moisturisers ingredient lists in Table III–V.”

 

Mixing Procedure and Ratio Control

The methodology regarding the 1:2 mixing ratio raises concerns about reproducibility. It seems unlikely that participants measured this precisely without standardized dispensing mechanisms. The authors should clarify how this ratio was ensured in practice, for example, whether predefined pump dispensers or visual dosing instructions were provided. Without such information, adherence to the intended ratio remains uncertain.

Our response: The packaging used has a dosage of 0,2 ml per push, and it was specified that 1 push of test products and 2 pushes of moisturizer should be used and the amount recommended is therefore standardized. This is more clearly written in the article.

Manuscript excerpt (p. 11, Study Design):

“Subjects were instructed to self-dose by taking 1 pump of the test product and 2 pumps of the moisturizer from packages that both dispensed 0.2 ml/pump.”

 

Sample Size and Demographics

The study reports 27 participants, but it is unclear whether this number was based on a power calculation or determined pragmatically. Further information on the distribution of age, gender, and skin type/phototype is necessary to contextualize the findings and evaluate the representativeness of the sample.

Our response: The number of subjects was determined based on previous experience from similar trials, so more a pragmatical approach then a power calculation. The requested Information about the study participants has also been added more clearly in the article.

Manuscript excerpts:

• (p. 14, Statistical Analysis):

“The number of subjects needed were determined pragmatically based on previous experience from similar studies…”

• (p. 12, Participants):

“A total of twenty-seven caucasian healthy adults (4 men and 23 women) aged 40–69 years … The majority of participants were Fitzpatrick phototype III … with a mix of oily/combination (n=4), normal/dry (n=16) and dry/very dry (n=7) skin types.”

• (p. 18, Results):

“The participants ranged in age from 40–69 (mean 49.1 ± 6.3 years, median 47 years).”

 

 

Method

 The description of the equipment employed in this study requires further detail. Specifically, the manuscript should clarify by which optical and computational methods the Antera 3D system evaluates the reported skin parameters. It is important to note that this device does not directly measure skin hydration, as would be done with a corneometer, but rather approximates hydration indirectly through external features such as wrinkle depth and surface appearance. While such imaging systems are valuable for visualization and relative assessments, they may not provide sufficiently rigorous clinical data for certain biophysical endpoints.

Our response: More information about the Antera camera has been added. The camera is an instrument that has been used in numerous studies and is considered a reliable instrument for this type of measurement.

We have also included 2 more references to the reference list including the method and the use of Antera 3D:

Matias, A. R., M. Ferreira, P. Costa and P. Neto (2015). "Skin colour, skin redness and melanin biometric measurements: comparison study between Antera((R)) 3D, Mexameter((R)) and Colorimeter((R))." Skin Res Technol 21(3): 346-362.

 

Messaraa, C., A. Metois, M. Walsh, S. Hurley, L. Doyle, A. Mansfield, C. O'Connor and A. Mavon (2018). "Wrinkle and roughness measurement by the Antera 3D and its application for evaluation of cosmetic products." Skin Res Technol 24(3): 359-366.

 

 

I would recommend either

• complementing the current analysis with standardized biophysical instruments- g., a corneometer for hydration, a mexameter for melanin/erythema, and a cutometer for elasticity  

or

 

• reframing the study as a preclinical or exploratory screening test rather than a full clinical trial. This distinction is important to ensure that the terminology and conclusions accurately reflect the strength of the evidence provided.

Our response: The study has been reframed and it’s limitations are more clearly discussed.

 

Subjects

 

The description of the study population raises several concerns. The panel includes both men and women; however, male and female skin differs substantially in terms of structure, thickness, sebum production, and aging patterns. This heterogeneity complicates interpretation, and in a small sample size such as this, it may have introduced confounding effects. For clarity and stronger scientific validity, I would recommend either excluding men from the study or analyzing them separately.

In addition, the age range (40–69 years) is very broad. Photoaging severity, skin physiology, and responsiveness to topical actives differ considerably between individuals in their early forties compared to those in their late sixties. A narrower age range, or at least stratified subgroup analyses, would provide more meaningful and interpretable results.

Our response:  Although the skin differs somewhat between men and women and in between different age groups the effect provided by the product should be the same. We agree that we have to be careful in the conclusions drawn, but the overall results is very clear and significant, in the complete study population. The limitations are discussed and it’s stated that it would be interesting to conduct future studies on narrower or even more  defined subgroups.

 

Statistical

 

While the statistical analysis is reported as being conducted with paired Wilcoxon signed-rank tests, the robustness of the analysis is limited by substantial heterogeneity within the study population and methodology. Several concerns arise:

Inclusion criteria: These are not clearly defined. Without strict criteria, variability in baseline characteristics may undermine the validity of the results.

Our response: Inclusion criterias are more defined.

1. Heterogeneity of the sample: The age range is very broad (40–69 years), encompassing distinct stages of photoaging. Furthermore, men and women exhibit significant physiological differences in skin structure and aging patterns. Pooling them together without stratified analyses reduces interpretability.

Our response: We are aware of this and the limitations it brings to the conclusions, and it is more clearly discussed in the article.

1. Moisturizer variability: The test product was mixed with moisturizers adapted to skin type, but it is not specified how many participants used each formulation. Since vehicle composition (ingredients, emulsion type) can strongly influence skin outcomes, this introduces a confounding factor that is not controlled for.

Our response: It is stated how many subjects that got each moisturizer. Due to that the group had different skin types it was decided that they should get a moisturizer that was based on their skin type. The product used are regular products in Skinome’s portfolio and are adapted to and recommended for the same skin types as used in the study. The limitation that the moisturizer can bring an added affect, and thereby influence the result, is now discussed in the article.

1. Multiplicity of parameters: Numerous endpoints were assessed (pigmentation, wrinkles, texture, pores, redness, etc.), yet no correction for multiple comparisons is mentioned. This increases the likelihood of Type I errors.

Our response: A conservative Bonferroni correction (for 8 parameters) has been added in the analysis and the results are still significant.

Taken together, the number of uncontrolled variables makes it difficult to draw statistically robust and clinically meaningful conclusions from the reported analysis. I would strongly recommend clarifying subject selection, moisturizer allocation, and considering subgroup analyses or adjustments for multiple testing.

 Our response: This has been addressed more clearly in the article.

 

Ethics

 

The ethical approval statement in the manuscript is incomplete. It is necessary to clearly indicate:

• the name of the ethics committee that granted approval, (ii) the official approval/registration number,

Our response: Added to the article more clearly.

• and (iii) the validity period of the approval. These elements are essential for transparency and to ensure compliance with international ethical standards.

Our response: The approval date is specified, no other information available.

 

 

Results

 

The results are currently expressed only as percentage changes with p-values. For greater clarity and scientific rigor, it would be preferable to present both baseline and post-treatment absolute values (mean ± SD or 95% CI), alongside the calculated percentage change. This would allow readers to better interpret the magnitude and clinical relevance of the improvements, rather than relying solely on relative differences.

Our response: Difference plots where absolute values are compared have been added, to show the variation in between different subjects and also confidence intervals. The values used in the statistical analysis are combined values from many areas and repetitive measurements in the same area, for each parameter, to reduce variation and to get as reliable values as possible to compare. The absolute values are therefore not more easy to interpret, rather the opposite. It is more clearly written and discussed in the article.

 

 

The demographic description of the study population lacks sufficient detail. The manuscript does not specify how many of the female participants were post-menopausal, perimenopausal, or premenopausal. This information is critical, as hormonal status strongly influences skin physiology, collagen synthesis, and pigmentation patterns. In addition, the inclusion of both male and female subjects introduces further heterogeneity, as their skin characteristics differ significantly.

Our response: We don’t have information about the subjects hormonal status and it is also other factors influencing the skin status. The intention was to make the study on a relevant target group of users, and to identify the difference that could be seen. It is more clearly discussed in the article about the limitations and future studies on different subgroups, to identify differences between gender, age and/or hormonal status.

Given this variability, the presentation of results appears somewhat overstated and less plausible without acknowledging these confounding factors. The authors should provide a clear breakdown of the participants by menopausal status and sex, and explicitly discuss the limitations arising from the heterogeneity of the study population. Acknowledging these factors would improve the transparency and scientific credibility of the manuscript.

Our response: The limitations of the study are discussed and acknowledged, and the interpretation and conclusions are toned down.

Reviewer 3 Report

Comments and Suggestions for Authors

This single-arm, six-week, prospective cosmetic study (n=27; 40–69 y) evaluates an anhydrous 0.1% retinal concentrate co-formulated with hydrophilic actives (N-acetyl-glucosamine, niacinamide, ascorbic acid, alpha-glucan oligosaccharide). Subjects mixed the concentrate 1:2 with a Skinome moisturizer before nightly application (after a two-week ramp-up). Antera 3D imaging reported statistically significant median improvements in pigmentation (−12%), fine lines (−14%), wrinkle depth (−5%), texture (+12%), volume irregularities (−15%), pore visibility (−24%), and brightness (+4% ITA°), with no change in redness; self-assessments were favorable. The study argues that the mix-activated anhydrous format enhances stability and efficacy with good tolerability. The manuscript is promising, especially the practical “mix-activated anhydrous” concept and multi-parameter imaging outcomes, but milder issues limit interpretability and external validity at this stage.

Queries:

1. The concentrate contains 0.1% retinal but is mixed 1:2 with moisturizer before use; doesn’t that reduce the applied dose to ~0.033%? How do you justify labeling the study as “0.1% retinal”?

2. What are the exact percentages (w/w) of N-acetyl glucosamine, niacinamide, ascorbic acid, and alpha-glucan oligosaccharide in the concentrate, and what are their post-mix applied concentrations?

3. What is the post-mix pH with each moisturizer type, and is there evidence that all hydrophilic actives and retinal remain solubilized and stable immediately before skin contact?

4. Can you provide validated stability data for retinal and ascorbic acid (under ICH conditions, and in-use after mixing) to support the claim that the anhydrous system preserves potency?

5. How was reproducibility of Antera 3D ensured (ROI alignment, intra-/inter-session repeatability, operator blinding)?

6. Did you apply any correction for multiple comparisons across the many endpoints tested, and if not, can you provide adjusted p-values?

7. Different participants used different moisturizers; how was this heterogeneity controlled or accounted for statistically?

8. How were adverse events captured and graded, and can you provide a table of expected retinoid-related side effects (peeling, dryness, stinging) even if absent?

9. Figures show improvements up to 62–67% for single areas while median global results are smaller; are these figures best-responder exemplars, and how do they relate to group medians?

10. Was the study preregistered and was a sample size or power calculation performed to justify the cohort of 27 participants?

Suggestions:

1. Report effect sizes with confidence intervals and standardized effect sizes, not only p-values.

2. Provide full quantitative composition of the test product and in vitro permeation or tape-strip data confirming active delivery post-mix.

3. Add detailed stability and handling data including forced degradation and consumer-relevant storage guidance.

4. Perform sensitivity analysis or subgroup analysis according to moisturizer type to check for confounding.

Author Response

Response to Reviewer

We thank the reviewer for your constructive and detailed feedback. Below we respond point by point.

Kindest Regards

Queries:

1. The concentrate contains 0.1% retinal but is mixed 1:2 with moisturizer before use; doesn’t that reduce the applied dose to ~0.033%? How do you justify labeling the study as “0.1% retinal”?

Our response: The product used in the study is a 0,1% retinal concentrate and the pump dosage  (0,2ml) and the application is now clearly specified . This will standardize the dose given in each application.

1. What are the exact percentages (w/w) of N-acetyl glucosamine, niacinamide, ascorbic acid, and alpha-glucan oligosaccharide in the concentrate, and what are their post-mix applied concentrations?

Our response: The concentrations of the mentioned ingredients are specified in the article. Regarding the applied dose this is relating to the pump dosage which is 0,2ml.

1. What is the post-mix pH with each moisturizer type, and is there evidence that all hydrophilic actives and retinal remain solubilized and stable immediately before skin contact?

Our response: We have not measured the post mix pH of the product combination for each moisturiser but given the pH of the products and buffer capacity of the ingredients used in the formulations it will be between 5 to 6.

We have not measured the stability or solubility during the application procedure, but as this is a very short period of time (seconds rather than minutes) there is no reason to expect a significant degradation.

1. Can you provide validated stability data for retinal and ascorbic acid (under ICH conditions, and in-use after mixing) to support the claim that the anhydrous system preserves potency?

Our response: We have done internal studies during development and shown the stability of both retinal and vitamin C in anhydrous system. A part of the development work has also been performed as two separate Master Thesis projects done at KTH Royal Institute of Technology, Stockholm in collaboration with Skinome: Ref: 1

https://kth.diva-portal.org/smash/record.jsf?dswid=6974&pid=diva2%3A1598594&c=1&searchType=SIMPLE&language=sv&query=skinome&af=%5B%5D&aq=%5B%5B%5D%5D&aq2=%5B%5B%5D%5D&aqe=%5B%5D&noOfRows=50&sortOrder=author_sort_asc&sortOrder2=title_sort_asc&onlyFullText=false&sf=all

ref2: https://kth.diva-portal.org/smash/record.jsf?pid=diva2%3A1722942&dswid=6974

1. How was reproducibility of Antera 3D ensured (ROI alignment, intra-/inter-session repeatability, operator blinding)?

Our response: The Antera camera has been used and validated in several studies and is acknowledged to be a reliable instrument. More information about the camera is added in the article. Also two references regarding the use of Antera 3D is added to the article:

Matias, A. R., M. Ferreira, P. Costa and P. Neto (2015). "Skin colour, skin redness and melanin biometric measurements: comparison study between Antera((R)) 3D, Mexameter((R)) and Colorimeter((R))." Skin Res Technol 21(3): 346-362.

Messaraa, C., A. Metois, M. Walsh, S. Hurley, L. Doyle, A. Mansfield, C. O'Connor and A. Mavon (2018). "Wrinkle and roughness measurement by the Antera 3D and its application for evaluation of cosmetic products." Skin Res Technol 24(3): 359-366.

 

1. Did you apply any correction for multiple comparisons across the many endpoints tested, and if not, can you provide adjusted p-values?

Our response: Bonferroni correction has been used and provide adjusted p-values, and it did not change the outcome.

1. Different participants used different moisturizers; how was this heterogeneity controlled or accounted for statistically?

Our response: No correction has been made based on that the subjects used different moisturisers, this has been discussed in the article as a limitation.

 

1. How were adverse events captured and graded, and can you provide a table of expected retinoid-related side effects (peeling, dryness, stinging) even if absent?

Our response: All side effects reported by the subjects are included in the manuscript.

1. Figures show improvements up to 62–67% for single areas while median global results are smaller; are these figures best-responder exemplars, and how do they relate to group medians?

Our response: The pictures are selected because they provide a visually clear response, and should be considered as single points more than the median difference.

1. Was the study preregistered and was a sample size or power calculation performed to justify the cohort of 27 participants?

Our response: The study was a cosmetic study and no preregistration was therefore required. No power calculation was performed to justify the study cohort, it’s based on pragmatical experience from previous studies.

Suggestions:

1. Report effect sizes with confidence intervals and standardized effect sizes, not only p-values. Our response: More data including confidence intervals has been added.
2. Provide full quantitative composition of the test product and in vitro permeation or tape-strip data confirming active delivery post-mix.

Our response: The quantitative compositions of the actives have been added. We have only literature penetration data for the actives and have no reason to believe that it is different from the already available information. Data is not provided in the manuscript.

1. Add detailed stability and handling data including forced degradation and consumer-relevant storage guidance.

Our response: Stability studies were not part of the clinical test but has been part of the development. Recommended storage conditions and guidance have been added to the manuscript.

1. Perform sensitivity analysis or subgroup analysis according to moisturizer type to check for confounding.

Our response: The limitations with the study are more clearly discussed in the manuscript, both when it comes to the moisturizers used and the relatively heterogeneous study group. Future studies on more narrow and specific groups of subjects are of interest and an opportunity for future research. 

 

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All four comments have been acknowledged and acted upon in the revised manuscript. 

Please correct "photoype".

Author Response

Dear Reviewer

Many thanks for your prompt feedback. We have now corrected the word: phototype. Many thanks again for your invaluable review and feedback on our paper. Kindest Regards Johanna Gillbro

Reviewer 2 Report

Comments and Suggestions for Authors

The protocol indicates that the retinal concentrate was mixed in a 1:2 ratio with a moisturizer chosen according to skin type. It should be clarified whether all participants used the same moisturizer or if different formulations were provided depending on skin characteristics. If multiple moisturizers were employed, the key ingredients, emulsion type (O/W or W/O), and potential impact on outcomes should be described in detail. Given the importance of the vehicle in cosmetic studies, the choice of moisturizer requires more justification.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 

We thank you for your invaluable feedback and comments. Your comment should already have been addressed in the revised submission. We hope that this is sufficient. 

Kindest Regards Johanna Gillbro 

 The study design section states that “The moisturizer was chosen based on the participant’s skin type at the start of the study.”

 

In the participant section, the number of subjects with the different skin types has been clarified “The majority of participants were Fitzpatrick phototype III (21 subjects) and the rest phototype II (6 subjects), with a mix of oily/combination (n=4),  normal/dry (n = 16) and dry/very dry (n = 7) skin types.”

 

In the Test products section the formulation types of the moisturisers and for what skin type they were used has been stated “the moisturisers used in the study were all o/w emulsions and selected based on the different skin types included in the study”. The complete ingredient lists of all moisturisers including for what skin types they were used are also included in table III to V.  

Reviewer 3 Report

Comments and Suggestions for Authors

All comments are addressed.

Author Response

Dear Reviewer 

Many thanks for your invaluable comments, feedback and suggestions during this review process. Kindest Regards Johanna Gillbro