Damage Reduction via Scanning Electron Microscopy · 2018-03-15 · Damage Reduction via Scanning...

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TR-B&R007-18-A-R2, Page 1 of 42, March 13, 2018. Confidential Document.

TEST REPORT B&R007-18-A, Review 2

Test Proposal B&R007-18, Review 1

Damage Reduction via Scanning Electron Microscopy

Products Evaluated

Monat Renew Shampoo

Monat Restore Leave-in Conditioner

Monat Volume Revive Shampoo

Monat Volume Revitalize Conditioner

Monat Black Shampoo + Conditioner

Sponsor: MONAT GLOBAL

Thomas J. Hoolihan

Senior Vice President and Chief Legal Officer

3470 N.W. 82nd Avenue, Suite 910. Miami, FL – USA 33122


SYNOPSIS OF TEST REPORT TR-B&R007-18-A-R2

Study Code: B&R007-18

Title of Study: Damage Reduction via Scanning Electron Microscopy.

Product Evaluated: Monat Renew Shampoo; Monat Restore Leave-in Conditioner; Monat Volume Revive Shampoo;

Monat Volume Revitalize Conditioner; Monat Black Shampoo + Conditioner.

Study plan code: PE-B&R007-18-R0

Purpose of study: The purpose of this study was to evaluate levels of damage to the surface of hair fibers submitted

to treatment with cosmetic products via scanning electron microscopy (SEM).

Study Director: Viviane C. Albarici

Chronology of study: The study was carried out from February 20th to March 2nd, 2018.

Experimental Design: In the present study, tresses of natural and bleached/dyed Caucasian hair were submitted 12

applications of the treatments as described below:

Virgin Caucasian Hair - group Virgin;

Bleached and Dyed Hair – group Bleached/Dyed;

Virgin Hair + Monat Renew Shampoo (Normal Condition) – group T01;

Virgin Hair + Monat Renew Shampoo (Modified Condition) – group T02;

Virgin Hair + Monat Renew Shampoo (Normal Condition) + Flat Iron – group T03;

Bleached and Dyed Hair + Monat Renew Shampoo (Normal Condition) – group T04;

Bleached and Dyed Hair + Monat Renew Shampoo (Modified Condition) – group T05;

Bleached and Dyed Hair + Monat Renew Shampoo (Normal Condition) + Flat Iron – group

T06;

Virgin Hair + Monat Volume Revive Shampoo (Normal Condition) – group T07;

Virgin Hair + Monat Volume Revive Shampoo (Modified Condition) – group T08;

Virgin Hair + Monat Volume Revive Shampoo (Normal Condition) + Flat Iron – group T09;

Bleached and Dyed Hair + Monat Volume Revive Shampoo (Normal Condition) – group T10;

Bleached and Dyed Hair + Monat Volume Revive Shampoo (Modified Condition) – group

T11;

Bleached and Dyed Hair + Monat Volume Revive Shampoo (Normal Condition) + Flat Iron

– group T12;

Virgin Hair + Monat Black Shampoo + Conditioner (Normal Condition) – group T13;

Virgin Hair + Monat Black Shampoo + Conditioner (Modified Condition) – group T14;

Virgin Hair + Monat Black Shampoo + Conditioner (Normal Condition) + Flat Iron – group

T15;

Bleached and Dyed Hair + Monat Black Shampoo + Conditioner (Normal Condition) –

group T16;

Bleached and Dyed Hair + Monat Black Shampoo + Conditioner (Modified Condition) –

group T17;

Bleached and Dyed Hair + Monat Black Shampoo + Conditioner (Normal Condition) + Flat

Iron – group T18;


Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Normal

Condition) – group T19;

Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Modified


Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Normal

Condition) + Flat Iron – group T21;

Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Modified


Bleached and Dyed Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner

(Normal Condition) – group T23;


(Modified Condition) – group T24;


(Normal Condition) + Flat Iron – group T25;


(Modified Condition) + Flat Iron – group T26;

Virgin Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner

(Normal Condition) – group T27;


(Modified Condition) – group T28;


(Normal Condition) + Flat Iron – group T29;


(Modified Condition) + Flat Iron – group T30;

Bleached and Dyed Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize

Conditioner (Normal Condition) – group T31;


Conditioner (Modified Condition) – group T32.


Conditioner (Normal Condition) + Flat Iron – group T33;


Conditioner (Modified Condition) + Flat Iron – group T34.

From the hair fibers collected, 5-mm long segments were taken from the center area. These

segments were fastened to the microscope sample holder using the conductive adhesive tape to

the carbon base. The samples were coated with a conductive layer of 90 Å Gold using the Spulter

Balzers SCD-050 Coater. For each treatment group, 05 micrographs were randomly obtained,

using the ZEISS™ 940-A scanning electron microscope at 15 kV.

Directions: Shampoo (Normal Condition): Apply 0,6 mL of Shampoo and rub the tresses for 30 seconds.

Leave it on hair for 3 minutes. Rinse for 30 seconds and remove excess water.


Shampoo (Modified Condition): Apply 0,3 mL of Shampoo and rub the tresses for 30 seconds.

Rinse for 30 seconds and remove excess water. Repeat the wash.

Conditioner: Apply 0.45 mL of Conditioner and rub it for 60 seconds. Rinse the tress for 30

seconds and remove excess water.

Conclusion: Virgin Hair

According to the results, the tresses submitted to 12 cycles of treatments using “Monat Renew

Shampoo”, “Monat Volume Revive Shampoo”, “Monat Black Shampoo + Conditioner”, “Monat

Renew Shampoo + Monat Restore Leave-in Conditioner” and “Monat Volume Revive Shampoo

+ Monat Volume Revitalize Conditioner”, on Normal or Modified conditions showed no effect on

surface damage of natural hair.

Moreover, the tresses submitted to these treatments following flat iron application showed no

effect on surface damage of natural hair, either.

Bleached/Dyed Hair

According to the results, the tresses submitted to 12 cycles of treatments using “Monat Renew

Shampoo”, “Monat Volume Revive Shampoo”, “Monat Black Shampoo + Conditioner”, “Monat

Renew Shampoo + Monat Restore Leave-in Conditioner” and “Monat Volume Revive Shampoo

+ Monat Volume Revitalize Conditioner”, on Normal or Modified conditions showed no effect on

surface damage of bleached and dyed hair.

Furthermore, the tresses submitted to these treatments following flat iron application showed no

effect on surface damage of bleached and dyed hair ether.

Finaly, the the products “Monat Renew Shampoo + Monat Restore Leave-in Conditioner” and

“Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner” slightly contribuite to

improve smoothing of the cuticule when compared to the bleached and dyed hair.

The table below shows the Reduction of Damage, RD, calculated to the treatments T23, T24, T31

and T32:

“Reduction of Damage, RD”of the treatment group (in percentage and number of times) compared to the Bleached/Dyed group.

Study group % Number of

times

Bleached and Dyed Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Normal Condition)

17 1.2

Bleached and Dyed Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Modified Condition)

15 1.2

Bleached and Dyed Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Normal Condition)

15 1.2

Bleached and Dyed Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Modified Condition)

14 1.2


COPYRIGHT NOTICE

Copyright © 2018 Kosmoscience. All rights reserved.

This Study Report is a copyright material owned by the sponsor of the study and, therefore, all the rights herein are

reserved and protected by Law 9.610 dated 02/19/1998. This document will not be reproduced, whether totally or

partially, by any processes or means, and it will not be disclosed by any electronic means, such as the Internet, without

the prior written approval from Kosmoscience.

This document is authorized for internal use by the sponsor, and by government supervision agencies, including

ANVISA/Health Ministry for the purposes of enrolling the tested product.

The study Sponsor may mention the original document and make use of the reference results achieved in its own

published materials.

The test report may only be completely reproduced if an approval in writing by the lab is requested. In the same

way, the approval in writing should be requested when there be need the reproduction of the document parts, especially

including images or pictures.

The results and discussion described in this report refer only to tested items.


AMENDMENTS TO THE TEST REPORT

Test Report B&R007-18-A, Review “1”, issued on 03/13/2018, becomes obsolete after the issuance of

this report named: Test Report B&R007-18-A, Review “2” issued on 03/13/2018.

Any reproduction of data from the formerly issued Test Report B&R007-18-A, Review “1” becomes impractical and

invalid starting on the date of this new issuance.


CONTENTS

I – INTRODUCTION ........................................................................................................................ 8

II – OBJECTIVE ............................................................................................................................... 9

III – EXPERIMENTAL PROCEDURE ................................................................................................. 10

IV – RESULTS AND DISCUSSION ................................................................................................... 12

V – CONCLUSION ......................................................................................................................... 27

VI – REFERENCES ........................................................................................................................ 31

VII – HISTORY OF REVIEWS .......................................................................................................... 32

APPENDIX I – Formulation of the samples forwarded by the Sponsor ................................................... 33

APPENDIX II – Experimental Procedure ............................................................................................ 34

APPENDIX III – Experimental Data Obtained ..................................................................................... 36

APPENDIX IV – Statistical Analysis .................................................................................................. 37


I – INTRODUCTION

The scanning electron microscopy (SEM) is a technique that uses a beam of electrons providing images with

high resolution and high depth of field. This has been used, to characterize pathologies, to classify hair according to its

origin or age and for forensic studies. Besides, with this technique is possible to see and quantify changes on the surface

of hair fibers. Thus, the scanning electron microscopy technique allows the evaluation of the surface of the hair fibers about

to prevent and repair damage caused by straightening procedures, dyeing, bleaching, successive combability, exposure to

sun light and temperature (such as the use of dryers and flat iron).

The scanning electron microscopy (SEM) technique was used in this study to assess the surface of hair fibers

submitted to several hair care products. The image is the result of secondary electrons formed while the sample surface is

scanned by a high-intensity beam of electrons.

By means of image analysis software programs, it is possible to standardize images and quantify the

respective surface damage to enable a more precise comparison of the action of different hair care products.


II – OBJECTIVE

The purpose of this study is to evaluate levels of damage to the surface of hair fibers submitted to treatment

with cosmetic products via scanning electron microscopy (SEM).


III – EXPERIMENTAL PROCEDURE

1. Generalities

1.1 Study Plan

The study was conducted per guidelines of study plan PE-B&R007-18-R0.

1.2 Dates

The study was approved to start on February 20rd, 2018 and it was carried out from February 20th to March

2nd, 2018.

2. Identifying samples supplied by the Sponsor for the Study

A27-156 Monat Renew Shampoo – 18J0115107;

A27-044 Monat Volume Revive Shampoo – 18J0115104;

A27-040 Monat Volume Revitalize Conditioner – 17J1227153;

A17-062D Monat Black Shampoo + Conditioner – 18J0117146;

A27-040 Monat Volume Revitalize Conditioner – 17J1227153;

A27-042 Monat Restore Leave-In Conditioner – 18J0111087.

The formulations of the samples, forwarded by the Sponsor, are listed in Appendix I. After the study, all

residues of samples were filed per Kosmoscience® Quality Procedures under unique study identification code B&R007-

18 for a six-month period.

3. Procedure for conducting evaluations

Fifty four tresses were prepared from Natural Caucasian hair and Fifty four tresses were prepared from

bleached/dyed Caucasian hair, weighing 3.0 g each and 25 cm long. All tresses underwent a standard pre-cleaning

process with 10% Sodium lauryl ether sulfate (SLES) solution for 1 minute then rinsed with running water.

The tresses were split in 36 groups and submitted to 12 applications of the treatments, as described below:

Treatment Study Code

Virgin Caucasian Hair Virgin

Bleached and Dyed Hair Bleached/Dyed

Virgin Hair + Monat Renew Shampoo (Normal Condition) T01

Virgin Hair + Monat Renew Shampoo (Modified Condition) T02

Virgin Hair + Monat Renew Shampoo (Normal Condition) + Flat Iron T03

Bleached and Dyed Hair + Monat Renew Shampoo (Normal Condition) T04

Bleached and Dyed Hair + Monat Renew Shampoo (Modified Condition) T05

Bleached and Dyed Hair + Monat Renew Shampoo (Normal Condition) + Flat Iron T06

Virgin Hair + Monat Volume Revive Shampoo (Normal Condition) T07

Virgin Hair + Monat Volume Revive Shampoo (Modified Condition) T08


Virgin Hair + Monat Volume Revive Shampoo (Normal Condition) + Flat Iron T09

Bleached and Dyed Hair + Monat Volume Revive Shampoo (Normal Condition) T10

Bleached and Dyed Hair + Monat Volume Revive Shampoo (Modified Condition) T11

Bleached and Dyed Hair + Monat Volume Revive Shampoo (Normal Condition) + Flat Iron T12

Virgin Hair + Monat Black Shampoo + Conditioner (Normal Condition) T13

Virgin Hair + Monat Black Shampoo + Conditioner (Modified Condition) T14

Virgin Hair + Monat Black Shampoo + Conditioner (Normal Condition)+ Flat Iron T15

Bleached and Dyed Hair + Monat Black Shampoo + Conditioner (Normal Condition) T16

Bleached and Dyed Hair + Monat Black Shampoo + Conditioner (Modified Condition) T17

Bleached and Dyed Hair + Monat Black Shampoo + Conditioner (Normal Condition) + Flat Iron T18

Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Normal Condition) T19

Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Modified Condition) T20

Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Normal Condition) + Flat Iron

T21

Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Modified Condition) + Flat Iron

T22


T23


T24

Bleached and Dyed Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Normal Condition) + Flat Iron

T25

Bleached and Dyed Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Modified Condition) + Flat Iron

T26

Virgin Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Normal Condition)

T27

Virgin Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Modified Condition)

T28

Virgin Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Normal Condition) + Flat Iron

T29

Virgin Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Modified Condition) + Flat Iron

T30


T31


T32

Bleached and Dyed Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Normal Condition) + Flat Iron

T33

Bleached and Dyed Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Modified Condition) + Flat Iron

T34

After the treatment, the hair fibers were collected for SEM analysis, 24 hours of drying in a controlled environment

at 55 ± 5% relative humidity and 22 ± 2 °C. The procedure to apply the products via SEM analysis is described in

Appendix II.


IV – RESULTS AND DISCUSSION

From the photomicrographs obtained, image analysis was made with the Scion® for Windows software

program to detect damage to the hair fiber surface (raised cuticles, fragments) characterized by lighter colored areas.

In order to quantify the level of damage to the hair fiber surface, the “Percentage of Damage, D parameter

was determined. This parameter represents the percentage of white pixels in relation to the total number of pixels

(kept constant) of images converted into a binary white: black system – Figure 1.

Figure 1. Illustration of detection of fragments and edges via image analysis.

Virgin Hair

Appendix III summarizes the results from image analysis for each sample, out of which Graph 1 was

obtained.

Graph 1: Results of percentage damage detected on hair surface from SEM image analysis. Mean ± SD.


The results obtained for the study groups were statistically compared to Virgin group using the method

one-way ANOVA, followed by a Dunnett’s post-test, both considering a 95% confidence interval. Table 1

summarizes the results obtained. The complete data are listed in Appendix IV.

Table 1. Statistical analysis. 95% confidence interval.

Comparison Test Are there statistical difference?

Virgin vs. T01 No

Virgin vs. T02 No

Virgin vs. T03 No

Virgin vs. T07 No

Virgin vs. T08 No

Virgin vs. T09 No

Virgin vs. T13 No

Virgin vs. T14 No

Virgin vs. T15 No

Virgin vs. T19 No

Virgin vs. T20 No

Virgin vs. T21 No

Virgin vs. T22 No

Virgin vs. T27 No

Virgin vs. T28 No

Virgin vs. T29 No

Virgin vs. T30 No

According to the results obtained, the tresses submitted to the treatments T01, T02, T03, T07, T08, T09,

T13, T14, T15, T19, T20, T21, T22, T27, T28, T29 and T30 showed no statistically significant differences in values

of damage in comparison to the to the Virgin tresses (Virgin group).

The comparison between the conditions of application of the treatments was performed using the method

one-way ANOVA for repeated measures, followed by a Tukey’s post-test, considering a 95% confidence interval.

The statistical analysis is described in Appendix IV. Table 2 shows the results obtained.

Table 2. Statistical analysis. 95% of confidence interval. Comparison Test Are there statistical difference?

T01 vs. T02 No

T01 vs. T03 No

T02 vs. T03 No

T07 vs. T08 No

T07 vs. T09 No

T08 vs. T09 No

T13 vs. T14 No

T13 vs. T15 No

T14 vs. T15 No

T19 vs. T20 No

T19 vs. T21 No

T19 vs. T22 No

T20 vs. T21 No

T20 vs. T22 No

T21 vs. T22 No


T27 vs. T28 No

T27 vs. T29 No

T27 vs. T30 No

T28 vs. T29 No

T28 vs. T30 No

T29 vs. T30 No

According to the results obtained, the tresses submitted to the treatments T01, T02 and T03 showed no

statistically significant differences in values of damage when compared to each other.

The tresses submitted to the treatments T07, T08 and T09 showed no statistically significant differences in

values of damage when compared to each other.

The tresses submitted to the treatments T19, T20, T21 and T22 showed no statistically significant

differences in values of damage when compared to each other.



Figure 2 illustrates images of the hair fibers for the treatment groups.

Virgin

T01


T02

T03

T07

T08


T09

T13

T14

T15


T19

T20

T21

T22


T27

T28

T29

T30

Figure 2. Examples of images for treatment groups.


Bleached/Dyed Hair

Appendix III summarizes the results from image analysis for each sample, out of which Graph 2 was

obtained.

Graph 2: Results of percentage damage detected on hair surface from SEM image analysis. Mean ± SD.

The results obtained for the study groups were statistically compared to Bleached/Dyed group using the

method one-way ANOVA, followed by a Dunnett’s post-test, both considering a 95% confidence interval. Table 3

summarizes the results obtained. The complete data are listed in Appendix IV.

Table 3. Statistical analysis. 95% confidence interval.


Bleached/Dyed vs. T04 No









Bleached/Dyed vs. T23 Yes









According to the results obtained, the tresses submitted to the treatments T04, T05, T06, T10, T11, T12,

T16, T17, T18, T25, T26, T33 and T34 showed no statistically significant differences in values of damage in

comparison to the to the Bleached/Dyed group.

The tresses submitted the treatments T23, T24, T31 and T32 showed significantly lower results of damage

values in comparison to the to the Bleached/Dyed group.

The comparison between the conditions of application of the treatments was performed using the method

one-way ANOVA for repeated measures, followed by a Tukey’s post-test, considering a 95% confidence interval.

The statistical analysis is described in Appendix IV. Table 4 shows the results obtained.

Table 4. Statistical analysis. 95% of confidence interval.


T04 vs. T05 No

T04 vs. T06 Yes

T05 vs. T06 No

T10 vs. T11 No

T10 vs. T12 No

T11 vs. T12 No

T16 vs. T17 No

T16 vs. T18 No

T17 vs. T18 No

T23 vs. T24 No

T23 vs. T25 Yes

T23 vs. T26 Yes

T24 vs. T25 No

T24 vs. T26 Yes

T25 vs. T26 No

T31 vs. T32 No

T31 vs. T33 No

T31 vs. T34 No

T32 vs. T33 No

T32 vs. T34 No

T33 vs. T34 No

According to the results obtained, the tresses submitted to the treatments T04 and T06 showed no

statistically significant differences in values of damage when compared to T05 group.

The tresses submitted to the treatment T04 showed significant lower values of damage when compared to

the treatment T06.

The tresses submitted to the treatments T10, T11 and T12 showed no statistically significant differences in

values of damage when compared to each other.

The tresses submitted to the treatments T23 and T24 showed no statistically significant differences in values

of damage when compared to each other.


the treatments T25 and T26.

The tresses submitted to the treatments T24and T26 showed no statistically significant differences in values

of damage when compared to the treatment T25.



the treatment T26.



Table 5 shows the “Reduction of Damage, RD”, in percentage and in number of times, calculated for T23,

T24, T31 and T32 treatments in comparison to Bleached/Dyed group, according to Equations 1 and 2.

CTRL

TRTCTRL

D

DDRD *100(%)

Equation 1. Reduction of Damage (%) of treatments groups relative to the CTRL group. CTRL= Bleached/Dyed

TRT

CTRL

D

DRD

Equation 2. Reduction of Damage (number of times) of treatments groups relative to the CTRL group. CTRL= Bleached/Dyed

Table 5. “Reduction of Damage, RD”of the treatment group (in percentage and number of times) compared to the

Bleached/Dyed group.

Study group % Number of times

T23 17 1.2

T24 15 1.2

T31 15 1.2

T32 14 1.2

Figure 3 illustrates images of the hair fibers for the treatment groups.

Bleached


T04

T05

T06

T10


T11

T12

T16

T17


T18

T23

T24

T25


T26

T31

T32

T33


T34

Figure 3. Examples of images for treatment groups.


V – CONCLUSION

In the present study, tresses of natural and bleached/dyed Caucasian hair were submitted 12 applications

of the treatments as described below:

Virgin Caucasian Hair - group Virgin;

Bleached and Dyed Hair – group Bleached/Dyed;

Virgin Hair + Monat Renew Shampoo (Normal Condition) – group T01;

Virgin Hair + Monat Renew Shampoo (Modified Condition) – group T02;

Virgin Hair + Monat Renew Shampoo (Normal Condition) + Flat Iron – group T03;

Bleached and Dyed Hair + Monat Renew Shampoo (Normal Condition) – group T04;

Bleached and Dyed Hair + Monat Renew Shampoo (Modified Condition) – group T05;

Bleached and Dyed Hair + Monat Renew Shampoo (Normal Condition) + Flat Iron – group T06;

Virgin Hair + Monat Volume Revive Shampoo (Normal Condition) – group T07;

Virgin Hair + Monat Volume Revive Shampoo (Modified Condition) – group T08;

Virgin Hair + Monat Volume Revive Shampoo (Normal Condition) + Flat Iron – group T09;

Bleached and Dyed Hair + Monat Volume Revive Shampoo (Normal Condition) – group T10;

Bleached and Dyed Hair + Monat Volume Revive Shampoo (Modified Condition) – group T11;

Bleached and Dyed Hair + Monat Volume Revive Shampoo (Normal Condition) + Flat Iron – group T12;

Virgin Hair + Monat Black Shampoo + Conditioner (Normal Condition) – group T13;

Virgin Hair + Monat Black Shampoo + Conditioner (Modified Condition) – group T14;

Virgin Hair + Monat Black Shampoo + Conditioner (Normal Condition) + Flat Iron – group T15;

Bleached and Dyed Hair + Monat Black Shampoo + Conditioner (Normal Condition) – group T16;

Bleached and Dyed Hair + Monat Black Shampoo + Conditioner (Modified Condition) – group T17;

Bleached and Dyed Hair + Monat Black Shampoo + Conditioner (Normal Condition) + Flat Iron – group

T18;

Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Normal Condition) – group

T19;

Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Modified Condition) – group

T20;

Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Normal Condition) + Flat Iron

– group T21;

Virgin Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Modified Condition) + Flat

Iron – group T22;


– group T23;


Bleached and Dyed Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Modified



+ Flat Iron – group T25;

Bleached and Dyed Hair + Monat Renew Shampoo + Monat Restore Leave-in Conditioner (Modified


Virgin Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Normal Condition) –

group T27;


– group T28;

Virgin Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Normal Condition) +

Flat Iron – group T29;


+ Flat Iron – group T30;

Bleached and Dyed Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Normal


Bleached and Dyed Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Modified

Condition) – group T32.

Bleached and Dyed Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Normal


Bleached and Dyed Hair + Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner (Modified

Condition) + Flat Iron – group T34.

Virgin Hair

According to the results, the tresses submitted to 12 cycles of treatments using “Monat Renew Shampoo”,

“Monat Volume Revive Shampoo”, “Monat Black Shampoo + Conditioner”, “Monat Renew Shampoo + Monat

Restore Leave-in Conditioner” and “Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner”, on

Normal or Modified conditions showed no effect on surface damage of natural hair.

Moreover, the tresses submitted to these treatments following flat iron application showed no effect on

surface damage of natural hair, either.

Bleached/Dyed Hair

According to the results, the tresses submitted to 12 cycles of treatments using “Monat Renew Shampoo”,

“Monat Volume Revive Shampoo”, “Monat Black Shampoo + Conditioner”, “Monat Renew Shampoo + Monat


Restore Leave-in Conditioner” and “Monat Volume Revive Shampoo + Monat Volume Revitalize Conditioner”, on

Normal or Modified conditions showed no effect on surface damage of bleached and dyed hair.

Furthermore, the tresses submitted to these treatments following flat iron application showed no effect on

surface damage of bleached and dyed hair ether.

Finaly, the the products “Monat Renew Shampoo + Monat Restore Leave-in Conditioner” and “Monat

Volume Revive Shampoo + Monat Volume Revitalize Conditioner” slightly contribuite to improve smoothing of the

cuticule when compared to the bleached and dyed hair.

The table below shows the Reduction of Damage, RD, calculated to the treatments T23, T24, T31 and T32:

“Reduction of Damage, RD”of the treatment group (in percentage and number of times) compared to the Bleached/Dyed

group.

Study group % Number of times Bleached and Dyed Hair + Monat Renew Shampoo + Monat Restore Leave-in

Conditioner (Normal Condition) 17 1.2


15 1.2


15 1.2


14 1.2


This document is authorized for internal use by the sponsor, and by government supervision agencies, including ANVISA/Health Ministry for the purposes of enrolling the tested product.

Copyright © 2018 Kosmoscience. All rights reserved. No part of this publication may be reproduced, distributed, or transmitted in any means, including photocopying,

recording, or other electronic or mechanical method, without the prior written permission of the Publisher.

Administrative Head: Technical Head: Quality Head:

Adriano S. Pinheiro Chemist CRQ-IV 04245232

Viviane Albarici Chemist, PhD

Jefferson R. Bongarthner Chemist CRQ-IV 04261924


VI – REFERENCES

1. GAMEZ-GARCIA, M. Effects of some oils, emulsions, and other aqueous systems on the mechanical

properties of hair at small deformations. J. Soc. Cosmet. Chem. 44, p. 69-87 (1993).

2. CHAHAL, S. P.; JONES, R. T. Moisture regulation of hair by cosmetic proteins as demonstrated by dynamic

vapour sorption. A novel efficacy testing technique, Proc. 14th Latin American and Therlan Cong. Cosmet.

Chem. Santiago Chile. p. 45-56 (1999).

3. WILSON, T. Ed., Confocal Microscopy (Academic Press, San Diego, 1990).

4. SWIFT, J. A. “The Histology of Keratin Fibers” in the Chemistry of Natural Proteins Fibers, R. S. Asquith

(Plenum Press, New York, 1977), p. 81-146.

5. PINHEIRO, S. A. et al. Identificação e Prevenção dos Danos Oxidativos em Fibras Capilares por Marcadores

Fluorescentes. XIX Congresso Brasileiro de Cosmetologia, 2005, São Paulo. Revista Cosmetics & Toiletries.

6. KASPRZYK, E.; DELDOTTO, L. Hydrolyzed Quinoa for Hair Repair and Gloss, Cosmetics & Toiletries,

Formulation/Hair, 2013.

7. MICHAELIS: moderno dicionário da língua portuguesa. São Paulo: Companhia Melhoramentos, 1998-

(Dicionários Michaelis). 2259p.

8. JONES, R. T.; CHAHAL, S. P. The use of radiolabelling techniques to measure substantivity and penetration

into hair of protein hydrolysated, Internat. J. Cosmet. Sci. 19, p. 215-226 (1997).

9. GRUBER, J. V.; WINNIK, F. M. Cationic Polymer Deposition, J. Cosmet. Sci. 2001.

10. WEIGMANN H-D., KAMATH Y.K., REUTSCH S. B., BUSCH P. , TESMANN H. “Characterization of surface

deposits on human hair fibers”. J Soc Cosmet Chem, 41:379 (1990).

11. ANA LUÍSA SILVA SANT´ANNA, “Estudo da deposição de ceramidas sobre a fibra capilar para o combate

de danos cuticulares”. Dissertação de Mestrado. Universidade Estadual Paulista, 2000.

12. JOHNSON, D .H. “Hair and Hair Care”. Marcel Dekker, Inc. New York, (1997).


VII – HISTORY OF REVIEWS

Review Description Date

0 First issuance March 12th, 2018

1 The Sponsor identification was replaced, the code number of samples

was added on page 9 and the conclusions were re-written. March 13th, 2018

2 The identification of samples was updated on page 9 March 13th, 2018


APPENDIX I – Formulation of the samples forwarded by the Sponsor

Unreported.


APPENDIX II – Experimental Procedure

1. Preparing hair samples

Fifty four tresses were prepared from Natural Caucasian hair and Fifty four tresses were prepared from

bleached/dyed Caucasian hair, weighing 3.0 g each and 25 cm long. All tresses underwent a standard pre-cleaning

process with 10% Sodium lauryl ether sulfate (SLES) solution for 1 minute then rinsed with running water.

2. Treatments of the Tresses

2.1. Groups T01, T04, T07, T10, T13 e T16

a) Wet the tress for 20s and remove excess water.

b) Apply 0,6 mL of Shampoo and rub the tresses for 30 seconds. Leave it on hair for 3 minutes. Rinse for 30


c) Repeat the steps a and b until 12 cycles.

2.2. Groups T02, T05, T08, T11, T14 e T17


b) Apply 0,3 mL of Shampoo and rub the tresses for 30 seconds. Rinse for 30 seconds and remove excess water.

Repeat the wash.

c) Repeat the steps a and b until 12 cycles.

d) Comb the tresses for 5 times to remove the knots.

2.3. Groups T03, T06, T09, T12, T15 e T18




c) Apply a cycle of 10 strokes with the flat iron at approximately 230°C. Each stroke was made starting from the

hair roots to the tips for 10 seconds.

d) Repeat the steps a and c until 12 cycles.

2.4. Groups T19, T23, T27 e T31




c) Apply 0.45 mL of Conditioner and rub it for 60 seconds. Rinse the tress for 30 seconds and remove excess

water.

d) Repeat the steps a, b and c until 12 cycles.


2.5. Groups T20, T24, T28 and T32



Repeat the wash.


water.

d) Repeat the steps a, b and c until 12 cycles.

2.6. Groups T21, T25, T29 and T33





water.

d) Apply a cycle of 10 strokes with the flat iron at approximately 230°C. Each stroke was made starting from the


e) Repeat the steps a, b, c and d until 12 cycles.

2.7. Groups T22, T26, T30 and T34



Repeat the wash.


water.

d) Apply a cycle of 10 strokes with the flat iron at approximately 230°C. Each stroke was made starting from the


e) Repeat the steps a, b, c and d until 12 cycles.


APPENDIX III – Experimental Data Obtained

Image Analysis: Software Program: Scion® for Windows.

Values of percentage of damage obtained via Scion® for Windows software program.

Virgin T01 T02 T03 T07 T08 T09 T13 T14

20.55 21.41 20.39 22.82 20.94 19.65 22.31 20.40 21.29

18.80 20.65 21.06 19.97 17.14 20.82 19.44 19.84 16.16 18.98 17.60 19.84 21.58 20.83 19.47 19.96 18.89 18.00

20.72 21.41 20.85 21.45 20.04 20.59 20.12 18.48 20.52

18.26 20.69 20.59 21.29 19.91 18.91 19.82 17.89 20.69

T15 T19 T20 T21 T22 T27 T28 T29 T30

21.65 18.45 19.12 21.81 24.58 18.06 18.90 17.92 20.09

18.38 18.31 17.18 18.94 19.72 15.94 18.32 19.25 21.19 17.71 20.37 20.56 19.78 18.47 21.46 19.15 22.47 18.64

18.41 17.25 19.64 19.34 18.87 15.66 19.88 17.60 20.39

23.47 18.88 22.04 20.81 21.52 19.78 16.96 21.45 21.72

Bleached/Dyed T04 T05 T06 T10 T11 T12 T16 T17

22.28 23.07 22.80 24.65 24.78 21.38 21.45 21.89 21.38

24.64 20.97 22.40 23.52 20.11 20.90 23.80 23.03 24.53

25.42 21.33 23.13 24.48 22.22 23.14 22.72 22.73 21.54

23.91 23.00 21.65 24.17 20.72 23.45 24.34 22.79 23.25 26.82 23.49 23.13 23.11 23.45 23.33 22.14 21.83 23.59

T18 T23 T24 T25 T26 T31 T32 T33 T34

22.21 20.94 21.25 22.67 25.66 18.25 19.95 24.53 24.51

21.75 20.22 21.66 23.28 22.60 22.17 18.90 22.24 23.76

24.61 19.04 20.79 22.04 26.14 20.60 20.36 25.64 23.13 22.70 20.09 20.37 24.96 23.17 21.82 23.35 21.85 20.27

24.54 21.39 20.69 21.20 21.88 21.93 23.07 22.28 22.66


APPENDIX IV – Statistical Analysis

Method: One-way ANOVA with Dunnett’s post-test. Confidence Interval: 95%.

Data: Damage %, listed in Appendix III. Virgin vs Treatments

Software: GraphPad™ Prism® 6.0.

One-way analysis of variance Virgin vs Treatments

F 1.253

P value 0.2481

P value summary ns Are differences among means statistically significant? (P < 0.05) No

R square 0.2284

ANOVA table SS DF MS Treatment (between columns) 55.43 17 3.261

Residual (within columns) 187.3 72 2.601

Total 242.7 89

Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant?

Virgin vs. T01 -0.8900 -3.891 to 2.111 No Virgin vs. T02 -1.084 -4.085 to 1.917 No

Virgin vs. T03 -1.960 -4.961 to 1.041 No

Virgin vs. T07 -0.3100 -3.311 to 2.691 No

Virgin vs. T08 -0.4260 -3.427 to 2.575 No Virgin vs. T09 -0.8680 -3.869 to 2.133 No

Virgin vs. T13 0.3620 -2.639 to 3.363 No

Virgin vs. T14 0.1300 -2.871 to 3.131 No

Virgin vs. T15 -0.4620 -3.463 to 2.539 No

Virgin vs. T19 0.8100 -2.191 to 3.811 No Virgin vs. T20 -0.2460 -3.247 to 2.755 No

Virgin vs. T21 -0.6740 -3.675 to 2.327 No

Virgin vs. T22 -1.170 -4.171 to 1.831 No

Virgin vs. T27 1.282 -1.719 to 4.283 No Virgin vs. T28 0.8200 -2.181 to 3.821 No

Virgin vs. T29 -0.2760 -3.277 to 2.725 No

Virgin vs. T30 -0.9440 -3.945 to 2.057 No


Method: One-way ANOVA with Tukey’s post-test. Confidence Interval: 95%.

Data: Damage %, listed in Appendix III. Comparison Between condition of application on Virgin hair.


One-way analysis of variance

F 1.300

P value 0.3084

P value summary ns

Are differences among means statistically significant? (P < 0.05)

No

R square 0.1781



Total 18.25 14

Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant?

T01 vs. T02 -0.1940 -2.081 to 1.693 No T01 vs. T03 -1.070 -2.957 to 0.8166 No

T02 vs. T03 -0.8760 -2.763 to 1.011 No


F 17.19

P value < 0.0001 P value summary ****

Are differences among means statistically significant? (P < 0.05) Yes

R square 0.1895

ANOVA table SS DF MS

Treatment (between columns) 439.2 2 219.6 Residual (within columns) 1878 147 12.78

Total 2317 149


T07 vs. T08 -0.8204 -2.513 to 0.8722 No T07 vs. T09 3.149 1.457 to 4.842 Yes

T08 vs. T09 3.97 2.277 to 5.662 Yes


F 0.2248

P value 0.8020

P value summary ns Are differences among means statistically significant? (P < 0.05)

No

R square 0.03611



Total 49.99 14

Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? T13 vs. T14 -0.2320 -3.613 to 3.149 No

T13 vs. T15 -0.8240 -4.205 to 2.557 No

T14 vs. T15 -0.5920 -3.973 to 2.789 No



F 1.172 P value 0.3513

P value summary ns


No

R square 0.1802


Treatment (between columns) 10.65 3 3.550


Total 59.12 19 Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant?

T19 vs. T20 -1.056 -4.205 to 2.093 No

T19 vs. T21 -1.484 -4.633 to 1.665 No


T20 vs. T22 -0.9240 -4.073 to 2.225 No

T21 vs. T22 -0.4960 -3.645 to 2.653 No


F 1.538

P value 0.2432 P value summary ns

Are differences among means statistically significant? (P < 0.05) No R square 0.2238





T27 vs. T28 -0.4620 -3.773 to 2.849 No

T27 vs. T29 -1.558 -4.869 to 1.753 No

T27 vs. T30 -2.226 -5.537 to 1.085 No


T29 vs. T30 -0.6680 -3.979 to 2.643 No


Method: One-way ANOVA with Dunnett’s post-test. Confidence Interval: 95%.

Data: Damage %, listed in Appendix III. Bleached/Dyed vs. treatments


One-way analysis of variance Virgin vs Treatments

F 3.359

P value 0.0002

P value summary ***

Are differences among means statistically significant? (P < 0.05) Yes

R square 0.4423


Treatment (between columns) 108.2 17 6.362 Residual (within columns) 136.4 72 1.894

Total 244.5 89

Dunnett's multiple comparisons test Mean Diff. 95% CI of diff. Significant?

Bleached/Dyed vs. T04 -0.8900 -3.891 to 2.111 No

Bleached/Dyed vs. T05 -1.084 -4.085 to 1.917 No Bleached/Dyed vs. T06 -1.960 -4.961 to 1.041 No



Bleached/Dyed vs. T12 -0.8680 -3.869 to 2.133 No Bleached/Dyed vs. T16 0.3620 -2.639 to 3.363 No

Bleached/Dyed vs. T17 0.1300 -2.871 to 3.131 No



Bleached/Dyed vs. T24 -0.2460 -3.247 to 2.755 No Bleached/Dyed vs. T25 -0.6740 -3.675 to 2.327 No



Bleached/Dyed vs. T32 0.8200 -2.181 to 3.821 No Bleached/Dyed vs. T33 -0.2760 -3.277 to 2.725 No


Method: One-way ANOVA with Tukey’s post-test. Confidence Interval: 95%.

Data: Damage %, listed in Appendix III. Comparison Between condition of application on Bleached/Dyed vs. hair.



F 5.373

P value 0.0216

P value summary *


Yes

R square 0.4724



Total 15.97 14


T04 vs. T05 -0.2500 -1.664 to 1.164 No T04 vs. T06 -1.614 -3.028 to -0.1999 Yes

T05 vs. T06 -1.364 -2.778 to 0.05006 No



F 0.2440 P value 0.7872

P value summary ns

Are differences among means statistically significant? (P < 0.05) No R square 0.03908





T10 vs. T11 -0.1840 -2.675 to 2.307 No

T10 vs. T12 -0.6340 -3.125 to 1.857 No

T11 vs. T12 -0.4500 -2.941 to 2.041 No


F 0.4807

P value 0.6297

P value summary ns


No

R square 0.07418



Total 17.01 14


T16 vs. T17 -0.4040 -2.337 to 1.529 No



F 8.113

P value 0.0016

P value summary ** Are differences among means statistically significant? (P < 0.05)

Yes

R square 0.6034



Total 67.36 19


T23 vs. T24 -0.6160 -2.954 to 1.722 No T23 vs. T25 -2.494 -4.832 to -0.1558 Yes

T23 vs. T26 -3.554 -5.892 to -1.216 Yes

T24 vs. T25 -1.878 -4.216 to 0.4602 No

T24 vs. T26 -2.938 -5.276 to -0.5998 Yes T25 vs. T26 -1.060 -3.398 to 1.278 No



F 2.397

P value 0.1062

P value summary ns Are differences among means statistically significant? (P < 0.05) No

R square 0.3100



Total 69.39 19



T31 vs. T34 -1.912 -5.042 to 1.218 No

T32 vs. T33 -2.182 -5.312 to 0.9480 No

T32 vs. T34 -1.740 -4.870 to 1.390 No T33 vs. T34 0.4420 -2.688 to 3.572 No

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