Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work I was asked to review deals with precisely analyzing, as indicated by the title, the design and development of a shampoo with dark semi-permanent dyes for the gradual coverage of gray hair. The article stands out for its excellent methodology; in fact, there are already constant references to well-known guidelines such as ANVISA on line 138. There is a precise description of the process leading to the experimentation, both in terms of pigment selection and in terms of stability and performance tests, all assessable through analyses of both organoleptic and physicochemical parameters (lines 205-241). The results are encouraging, considering that even after stress conditions and washing cycles (lines 329-335), it is shown that the formulations were the ones maintaining capacity and stability (lines 329-335). Despite this, I believe that a more critical discussion is necessary on the limitations of the study, such as the lack of analysis between natural and bleached hair, which is (to be honest) only briefly mentioned in lines 525-528, or the complete absence of in vivo testing. Therefore, I believe that the article is acceptable but that a revision is necessary based on the suggestions I have given.

Author Response

Comments: Despite this, I believe that a more critical discussion is necessary on the limitations of the study, such as the lack of analysis between natural and bleached hair, which is (to be honest) only briefly mentioned in lines 525-528, or the complete absence of in vivo testing.

Response:  

The authors agree with the reviewer and have expanded the Discussion section to more critically address the study limitations. The correction is in the manuscript, highlighted in red on line 564-584.

The main limitations include:
a) Bleached hair vs gray hair
Bleached hair was initially used as an experimental model due to its reproducibility and sensitivity for detecting color deposition. However:
Bleached hair presents higher porosity and structural damage, which may overestimate dye uptake.
This difference was partially addressed by including hair tresses with 90% gray hair in later stages of the study, where different deposition behavior was observed.
b) in vitro evaluations
The study was limited to in vitro evaluations, which:
Allow controlled and reproducible conditions
But do not fully replicate real-use scenarios (e.g., scalp interaction, user variability)
c)  instrumental evaluations
Color evaluation was based on visual analysis and digital tools, which, although useful, could be complemented by:
CIELab measurements
Spectrophotometric analysis

Despite these limitations, the study provides:
a robust proof of concept for the development of a shampoo capable of gradual gray hair coverage using semi-permanent dyes, supported by stability and functional performance data.
Future studies should include in vivo evaluations, long-term use analysis, and instrumental color measurements to further validate these findings.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors describe a formulation of a shampoo for gradual hair color correction. The study is of interest, since consumers are seeking these types of products that they can use on a regular basis without causing damage to the hair, in contrast to dyes for example. The work addressed a knowledge gap of finding new marketed products for hair coloring. References are adequate and mostly up to date. After addressing few minor comments, the manuscript can be accepted for publication:

1- The authors need to highlight the novelty of their shampoo in the introduction section, in contrast to the already existing shampoos in the market.

2- Could repeated pigment deposition lead to buildup or uneven coloration over time? This needs to be addressed in the discussion.

3- Are there regulatory concerns with using these specific dyes in rinse-off vs leave-on products?

4- Were skin irritation or safety considerations evaluated, especially given the use of synthetic dyes?

Author Response

Comments 1: The authors need to highlight the novelty of their shampoo in the introduction section, in contrast to the already existing shampoos in the market.

Response 1: We agree with the reviewer and have revised the Introduction section to clearly highlight the novelty of the proposed shampoo, emphasizing its differences from existing products on the market. The correction is in the manuscript, highlighted in red on line 78-83.

Unlike conventional color-depositing shampoos available on the market, which often produce immediate and sometimes non-natural tonal effects (e.g., bluish or violet hues), the formulation developed in this study was specifically designed to achieve a progressive and controlled gray-to-black tonal transition.

In addition, the novelty of this work lies in:

The rational selection and combination of cationic semi-permanent dyes (Basic Blue 124, Basic Yellow 87, Basic Orange 31, and Basic Red 51) to obtain a natural black-gray shade.
The incorporation of these dyes into a rinse-off shampoo system, optimized to allow gradual pigment deposition through repeated use, rather than immediate coloration.
The demonstration of functional stability, including color retention after repeated washing cycles, which is not always systematically evaluated in similar formulations.

These aspects differentiate the developed formulation from existing commercial products and provide a more controlled and natural alternative for gray hair coverage.

Comments 2: Could repeated pigment deposition lead to buildup or uneven coloration over time? This needs to be addressed in the discussion.

Response 2: We agree with the reviewer and have expanded the Discussion section to more critically address the study limitations. The correction is in the manuscript, highlighted in red on line 544-550.

The possibility of pigment accumulation or uneven coloration with repeated use is an important consideration in formulations based on semi-permanent dyes. In the present study, the gradual color deposition observed suggests that the dyes interact primarily with the outer layers of the hair fiber, leading to a progressive tonal change rather than abrupt color buildup.

Furthermore, the in vitro wash-resistance (color retention) results showed that:

On bleached hair, color intensity decreased after repeated washing, indicating limited accumulation and higher pigment release due to increased porosity.
On gray hair (90%), the color remained stable after eight wash cycles, suggesting controlled deposition without excessive buildup.

These findings indicate that the formulation tends to reach a color equilibrium rather than continuous accumulation.

However, the authors acknowledge that long-term use could potentially lead to variability in color uniformity depending on hair condition (porosity, prior treatments). Therefore, this aspect has been added to the Discussion as a limitation and as a topic for future in vivo studies.

Comments 3: Are there regulatory concerns with using these specific dyes in rinse-off vs leave-on products?

Response 3: We agree with the reviewer and have expanded the Discussion section to address the area of regulatory standards more critically. The correction is in the manuscript, highlighted in red on line 355-364.

The dyes used in this study (Basic Blue 124, Basic Yellow 87, Basic Orange 31, and Basic Red 51) belong to the class of direct (semi-permanent) dyes commonly used in hair coloring products.

From a regulatory perspective:

In regions such as the European Union (Regulation (EC) No 1223/2009), hair dyes are subject to specific restrictions regarding concentration limits and permitted use conditions.
The classification of the product as a rinse-off formulation is relevant, as exposure time and systemic absorption are generally lower compared to leave-on products.
Nevertheless, compliance depends on the maximum allowed concentrations and safety assessments for each specific dye, which must be verified according to the regulatory framework of the target market.

In this study, the dyes were used at low concentrations (0.3–0.45%), consistent with typical cosmetic applications. However, the authors recognize that a complete regulatory assessment, including toxicological evaluation and compliance verification, is required prior to commercialization.

Comments 4: Were skin irritation or safety considerations evaluated, especially given the use of synthetic dyes?

Response 4: We agree with the reviewer and have expanded the Discussion section to more critically address the study limitations. The correction is in the manuscript, highlighted in red on line 571-576.

No in vivo skin irritation or sensitization tests were performed in this study, as the primary objective was focused on formulation development, physicochemical stability, and in vitro performance evaluation.

However:
The formulation was designed using cosmetic-grade ingredients commonly used in shampoo formulations.
The slightly acidic pH (5.7–5.9) is within the range considered compatible with scalp physiology, which may help minimize irritation.
Semi-permanent dyes are generally associated with a lower risk of adverse reactions compared to oxidative dyes, as they do not require hydrogen peroxide or ammonia.

Nevertheless, the authors acknowledge that:
Safety evaluation, including dermatological testing, is essential prior to product use in humans.
This limitation and the need for future safety studies have been added to the manuscript.