Clinical and Instrumental Evaluation of the Face Biorevitalization Effect With the Medical Device Foliage Hydrofil
Phase: NA
Conditions: Skin Ageing
Sponsor: Phitogen Beauty Labs SRL

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CLINICAL AND INSTRUMENTAL EVALUATION OF THE FACE 
BIOREVITALIZATION EFFECT WITH THE MEDICAL DEVICE 
FOLIAGE HYDROFIL 
 
 
Clinical Investigation Plan (CIP) code: FOLIAGE - 2022 
Version 1, 02/02/2022 
 
 
 
Sponsor 
Phitogen Holding S.p.A. 
 
Via Valtellina, 19/21/23 
63074 San Benedetto del Tronto (AP) 
Italy 
Phone: +39 0735-762020 
Fax +39 0735-758350  
 
 
Principal Investigator 
Dr. Tiziana Lazzari, MD 
Poliambulatorio di Medicina Estetica  
Casa della Salute  
Largo XII Ottobre 62 - Genova 
Phone: +39 010-8078631 
 
 
 
 
 
CONFIDENTIALITY STATEMENT 
The information in this document contains trade secrets and commercial information that are privileged 
or confidential and may not be disclosed unless such disclosure is required by law. In any event, persons 
to whom the information is disclosed must be informed that the information is privileged or confidential 
and may not be further disclosed by them. These restrictions on disclosure will apply equally to all future 
information supplied to you which is indicated as privileged or confidential. 

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Sponsor’s Approval Page 
 
 
 
Technical Director:  
Dott. Stefano Chahine 
 
 
 
Signature & Date: _________________________________________________________ 
 
 
 
 
 
Quality Assurance:          Dott.sa Giada Iachini 
 
 
 
 
Signature & Date: _________________________________________________________ 
 
 
 
 

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Principal Investigator’s Approval Page 
 
I understand that all information concerning the medical device Foliage Hydrofil supplied by 
Phitogen Holding SpA and/or Latis Srl in connection with this study protocol are confidential 
information. This information include: CIP, Case Report Form, product documents. 
 
I understand that any change in this study protocol must be approved in writing by Phitogen 
Holding SpA the Co-ordinating Investigator and the Ethics Committee before implementation, 
except where necessary to eliminate apparent immediate hazard to subjects. 
 
I confirm that I will conduct the study according to this protocol (except when mutually agreed to 
in writing with Phitogen Holding SpA or its delegate Latis Srl), the Good Clinical Practice (GCP), 
the UNI EN ISO 14155, the Declaration of Helsinki current edition and laws and regulations in 
the Country where the study is to be conducted. 
 
I confirm that I will record and report all adverse events occurring during the study, according to 
this protocol. 
I confirm that I am informed about the need of data records retention, according to current 
regulations and that no data can be destroyed without the written consent of Phitogen Holding 
SpA. 
I confirm that I will transfer adequate ownership of my responsibilities for the clinical investigation 
and will inform the Sponsor, in case I retire from my PI role. 
I confirm that in case the Trial Center File is stolen or anyhow damaged, I will promptly inform 
the Sponsor and declare it to the Competent Authorities. 
 
 
Principal Investigator:  …………………………………………………… 
 
 
 
 
 
 
Signature & Date: _________________________________________________________ 
 
 
 
 
 

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General information 
 
Sponsor Representative 
Contact 
Technical Director – Dr. Stefano Chahine 
Phone. +39 0735/762020 
E-mail. farmacovigilanza@phitogenspa.it  
 
Quality Assurance – Dr.ssa Giada Iachini  
Phone. +39 0735/762020 
E-mail. quality@phitogenspa.it  
 
  
Clinical CRO 
Latis Srl – Viale Sauli 39/5 – 16121 Genova 
Phone. +39 010 562234 
Fax. +39 010 540699 
E-mail. info@latiscro.it 
 
Contacts for safety reporting 
CRO Contact for Serious 
Adverse Events (SAEs) 
and Reportable Events 
Latis Srl – Viale Sauli 39/5 – 16121 Genova 
Phone. +39 010 562234 
Fax. +39 010 540699 
Laura Michellini Scientific Director 
Mobile. +39 347 4785898 
E-mail. michellini@latiscro.it 
 
Phitogen Holding S.p.A. - Via Valtellina, 19/21/23 
63074 San Benedetto del Tronto (AP) 
Dr Stefano Chahine  
Phone. +39 0735 762020 
E-mail. farmacovigilanza@phitogenspa.it 
 
 
 
 
 

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TABLE OF CONTENTS 
1. 
SYNOPSIS OF THE CLINICAL INVESTIGATION PLAN ................................................................ 7 
3. 
JUSTIFICATION FOR THE DESIGN OF THE CLINICAL INVESTIGATION ..................................................... 16 
4. 
BENEFITS AND RISKS OF THE INVESTIGATIONAL DEVICE, CLINICAL PROCEDURES AND CLINICAL 
INVESTIGATION ................................................................................................................................................ 17 
5. 
OBJECTIVES AND HYPOTHESES OF THE CLINICAL INVESTIGATION ........................................................ 18 
5.1  PRIMARY OBJECTIVE AND ENDPOINT ................................................................................................................... 18 
5.2  SECONDARY OBJECTIVES AND ENDPOINTS ............................................................................................................ 18 
5.3  TOLERABILITY AND SAFETY OBJECTIVES AND ENDPOINTS ......................................................................................... 19 
6. 
DESIGN OF THE CLINICAL INVESTIGATION ............................................................................................ 19 
6.1 GENERAL ........................................................................................................................................................ 19 
6.2 INVESTIGATIONAL DEVICE .................................................................................................................................. 20 
6.2.1 
Foliage Hydrofil ................................................................................................................................... 20 
6.2.1.1  
Packaging and labelling .............................................................................................................................. 20 
6.2.1.2 
Study device instructions for use ............................................................................................................... 21 
6.2.1.3 
Study product accountability and compliance ........................................................................................... 21 
6.2.2 
Concomitant Medications/Treatments ............................................................................................... 22 
6.3 SUBJECTS ........................................................................................................................................................ 23 
6.3.1 
Inclusion criteria .................................................................................................................................. 23 
6.3.2 
Exclusion criteria ................................................................................................................................. 23 
6.4 PROCEDURES ................................................................................................................................................... 25 
6.4.1 
Study Visits and Assessments .............................................................................................................. 25 
6.4.2 
Corneometer assessment .................................................................................................................... 27 
6.4.3 
Skin texture, skin tone, fine lines and wrinkles assessment ................................................................ 27 
The Investigator will evaluate at each visit the skin texture, skin tone, smoothing of fine lines and 
wrinkles modification in the treated area and will score each item as follows: .................................... 27 
Wrinkles will be assessed through the severity rating scale: .................................................................. 28 
6.4.4 
Global Aesthetic Improvement Scale (GAIS) by the Investigator ........................................................ 28 
6.4.5 
Patient’s Aesthetic Improvement Scale (PAIS) .................................................................................... 28 
6.4.5 
Subject Overall Satisfaction ................................................................................................................ 28 
6.5 MONITORING PLAN .......................................................................................................................................... 29 
7. 
STATISTICAL DESIGN AND ANALYSIS .................................................................................................... 30 
7.1 SAMPLE SIZE DETERMINATION ........................................................................................................................... 30 
7.2 DEFINITION OF STUDY POPULATIONS FOR ANALYSIS ............................................................................................... 30 
7.3 STATISTICAL ANALYSIS ...................................................................................................................................... 31 
7.3.1 
Missing Data ....................................................................................................................................... 31 
7.3.2 
Multiplicity .......................................................................................................................................... 31 
7.3.3 
Covariates, Interactions and Subgroups ............................................................................................. 31 
7.3.4 
Analysis of Demographics and Baseline Variables .............................................................................. 31 
7.3.5 
Efficacy analysis .................................................................................................................................. 32 
7.3.5.1 
Primary endpoint ....................................................................................................................................... 32 
7.3.5.2 
Secondary endpoints ................................................................................................................................. 32 
7.3.6 
Safety Analysis .................................................................................................................................... 33 
7.4 PLANNED INTERIM ANALYSIS.............................................................................................................................. 33 
8.        DATA MANAGEMENT ............................................................................................................................. 33 
9.        AMENDMENTS TO THE CIP ..................................................................................................................... 35 

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10. 
DEVIATIONS FROM CLINICAL INVESTIGATION PLAN............................................................................. 35 
11. 
DEVICE ACCOUNTABILITY ..................................................................................................................... 36 
12. 
STATEMENT OF COMPLIANCE .............................................................................................................. 36 
13. 
INFORMED CONSENT PROCESS ............................................................................................................ 36 
14. 
ADVERSE EVENTS, ADVERSE DEVICE EFFECTS AND DEVICES DEFICIENCIES ........................................... 37 
14.1 DEFINITIONS .................................................................................................................................................... 37 
14.2 ADVERSE EVENT INTENSITY/CAUSALITY ............................................................................................................... 38 
14.3 ADVERSE EVENT, DEVICE DEFICIENCY, ADVERSE DEVICE EFFECT, REPORTING ............................................................... 39 
15. 
VULNERABLE POPULATION .................................................................................................................. 40 
16. 
SUSPENSION OR PREMATURE TERMINATION OF THE CLINICAL INVESTIGATION ................................. 40 
16.1  STUDY DISCONTINUATION ................................................................................................................................. 40 
16.2  STUDY INTERRUPTION ....................................................................................................................................... 41 
17. 
PUBLICATION POLICY ........................................................................................................................... 41 
18. 
BIBLIOGRAPHY ..................................................................................................................................... 43 
Appendix 1 ................................................................................................................................................................ 44 
Appendix 2 ................................................................................................................................................................ 45 
 
 
 
 

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1. 
SYNOPSIS OF THE CLINICAL INVESTIGATION PLAN 
 
CIP Title 
CLINICAL AND INSTRUMENTAL EVALUATION OF THE FACE 
BIOREVITALIZATION EFFECT WITH THE MEDICAL DEVICE 
FOLIAGE HYDROFIL 
CIP Code 
FOLIAGE - 2022 
Sponsor 
Phitogen Holding S.p.A. 
Countries and 
Sites 
Italy – 1 Clinical Site 
Study Design 
Post market, prospective, single arm, clinical investigation. 
Objectives  
Primary objective 
To evaluate the clinical performance of Foliage Hydrofil in the 
improvement of the skin hydration in the treated area (face) at Day 98, 
from baseline. 
 
Secondary objectives 
▪ To evaluate the clinical performance of Foliage Hydrofil in the 
improvement of the skin hydration in the treated area at Day 21 
and 42, from baseline; 
▪ To evaluate the improvement of the skin texture, skin tone, 
smoothing of fine lines and wrinkles in the treated area at Day 21, 
42 and 98, from baseline;  
▪ To evaluate the aesthetic change from baseline as judged by the 
Investigator at Day 21, 42 and 98;  
▪ To evaluate the aesthetic change from baseline as judged by the 
subject at Day 21, 42 and 98;  
▪ To evaluate the subject’s satisfaction at Day 21, 42 and 98. 
 
Safety objectives 
▪ To assess the local safety at injection site (pain, erythema, 
edema, bruising) of the treatment with Foliage Hydrofil; 
▪ To assess the systemic safety of the treatment with Foliage 
Hydrofil. 

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Primary study 
endpoint of 
clinical 
performance 
Evaluation of the skin hydration modification from baseline to Day 98.  
Changes will be evaluated through corneometer skin examination. 
Secondary 
study 
endpoints of 
clinical 
perfomance 
- 
Evaluation of the skin hydration modification from baseline to Day 21 
and 42, through corneometer examination 
- 
To evaluate the number of responders to Foliage Hydrofil treatment 
at Day 98; 
- 
Evaluation of the of the skin texture, skin tone, smoothing of fine lines 
and wrinkles modification from baseline in the treated area at Day 21, 
42 and 98;  
- 
Investigator’s aesthetic change assessment using the Global 
Aesthetic Improvement Scale (GAIS) on Day 21, 42 and 98. GAIS is 
a categorical scale with 5-points, from worse than before treatment to 
“optimal improvement”; 
- 
Subject’s aesthetic change assessment using the Patient’s Aesthetic 
Improvement Scale (PAIS) on Day 21, 42 and 98. PAIS is a 
categorical scale with 5-points, from “worse” to “very much improved”; 
- 
The overall subject’s satisfaction at on the treatment at Day 21, 42 
and 98, through a 5-points scale (very much satisfied, satisfied, not 
satisfied nor unsatisfied, unsatisfied, very unsatisfied). 
Safety 
endpoints 
Number, typology, severity of local and systemic adverse events. 
Particular attention will be paid to local adverse events like pain, 
erythema, edema, bruising. 
Study 
Treatment 
Foliage Hydrofil is a CE marked, class III, resorbable medical device 
(sterile, non-pyrogenic and physiological gel) to be used for the hydration 
of the skin and for the correction of superficial skin imperfections of the 
face and body and in the process of repairing the dermal tissue. The 
main component of Foliage Hydrofil is HA sodium salt of non-animal 
origin, produced by bacterial fermentation. 
Each subject will receive three treatments, one vial 2mL (1.6% HA 
formulation) each, with Foliage Hydrofil, 3 weeks apart. Foliage Hydrofil 
will be injected with needles 30G 4mm only. The area treated with the 

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study treatment will be the face. The device administration could be done 
after an anesthetic cream application, according to Investigator’s 
judgement. 
Study Duration  For each subject the clinical investigation will be of about 14 weeks (3,5 
months). 
The enrolment period will be of 4-6 months; therefore, the overall project 
duration will be of maximum 10 months. 
Number of 
Subjects 
24 females and males planned. 
Target Study 
Population 
Caucasian women and man of 30-55 years, with signs of skin aging in 
the face. 
Selection 
Criteria 
Inclusion Criteria 
Subjects presenting all the following inclusion criteria will be eligible to 
enter the study: 
1. Generally healthy man and women, aged between 30 and 55 years 
(inclusive), looking for aesthetic procedures to improve the aspect 
of their face. 
2. Caucasian race (skin type from I to IV as per Fitzpatrick Skin Type 
scale) 
3. Visible signs of skin aging in the face (e.g. reduced skin elasticity, 
reduced turgor, actinic elastosis, smaller and larger wrinkles) 
according to the Investigators opinion; 
4. Corneometer Capacitance of the area treated < 80 
5. Subjects wishing not to modify their lifestyle during the study 
period; 
6. Subjects with cooperative attitude, able to comprehend the full 
nature and the purpose of the investigation, including possible 
risks, side effects and discomforts and willing to adhere to the 
study schedule (including attendance to the planned visits) and 
study prescriptions; 
7. Written informed consent release prior to any study-related 
procedures. 

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Exclusion criteria 
Subjects presenting one or more of the following exclusion criteria will 
not be eligible to enter the study: 
1. Pregnancy or breastfeeding woman or woman of childbearing 
potential not practicing adequate contraception. 
2. Subjects with Fitzpatrick skin type from V to VI  
3. Subjects 
with 
known 
allergy/hypersensitivity 
to 
product 
components. 
4. Extensively photo damaged and aged skin according to the 
Investigators opinion. 
5. Active skin disease, inflammation or related conditions, such as 
infection, perioral dermatitis, seborrheic eczema and rosacea in 
the face, hands or décolletage. 
6. Immune system disorders 
7. History of 
or active 
collagenosis (e.g. 
systemic lupus, 
erythematosis, rheumatic arthritis, skin or systemic sclerosis). 
8. Concomitant therapy with thrombolytics or anticoagulants, or have 
taken inhibitors of platelet aggregation, within 2 weeks before the 
first product administration. 
9. History of cancerous or pre-cancerous lesions in the face, neck 
and décolletage. 
10. Tattoo/s on the skin around the treatment site 
11. Previous tissue augmenting therapy with non-permanent filler or 
treatment with botulinum toxin, aesthetic surgical therapy, laser 
treatment, mesotherapy, or any form of peeling in the face, hands 
or décolletage within 12 months prior to the baseline visit. 
12. Use of injectable revitalization preparations within 12 months prior 
to the baseline visit. 
13. Use of retinoic acid within 6 months prior to the baseline visit. 
14. Previous 
or 
concomitant 
treatment 
with 
chemotherapy, 
immunosuppressive agents or corticoids. 
15. History of treatment with permanent filling materials. 

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16. Participation in any other clinical study within 30 days prior to the 
first visit or plan to participate in another clinical study during this 
study period. 
17. Planned or ongoing weight reduction program during the study. 
18. Known history of drug or alcohol abuse within 6 months prior to the 
baseline visit. 
19. Known hypersensitivity to one or more components of anesthetic 
creams 
20. Nicotine use during the study or stopped within 12 months before 
the first visit. 
21. Any medical condition that in the opinion of the Investigator makes 
the subject unsuitable for inclusion (e.g. severe chronic disease, 
malignancy, bleeding disorder, skin diseases etc). 
22. Other condition preventing the subject to entering the study in the 
Investigator's opinion e.g. subjects anticipated to be unreliable, 
unable to return for the follow-up visits, not likely to avoid other 
prohibited treatments or procedures or incapable of understanding 
the information or instructions. 
Previous and 
Concomitant 
Treatments 
Non permitted pharmacologic and non-pharmacologic treatments: 
▪ any tissue augmenting therapy with non-permanent filler or 
treatment with botulinum toxin, aesthetic surgical therapy, laser 
treatment, mesotherapy, or any form of peeling in the face, or 
décolléte; 
▪ use of injectable revitalization preparations; 
▪ use of retinoic acid; 
▪ treatment with permanent filling materials 
▪ chemotherapy, immunosuppressive agents or corticoids; 
▪ thrombolytics, anticoagulants, inhibitors of platelet aggregation 
▪ sun exposure or tanning booths or UV sources (SPF 50+ should 
applied in case sun exposure cannot be avoided). 

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To be avoided close to the treatment, the intake of any treatment 
(aspirin, N.S.A.I.D., Vitamin E) acting on the fluidity of the blood, to 
minimize the possibility of bruising or bleeding of the injected area. 
Sample size 
determination 
24 males and females will be enrolled.  Assuming an average difference 
in the skin hydration evaluated through corneometer skin examination 
between baseline and Day 98 of 7.5, with a standard deviation of 8, 20 
subjects will allow to appreciate this difference as statistically significant 
at an alpha = 0.05 and a 95% power. Considering the above and a 
possible drop-out rate of 20%, 24 subjects will be recruited. 
Statistical 
Analysis  
Descriptive statistical analysis of all relevant variables will be performed. 
Continuous variables will be summarized by the number of subjects (N), 
mean, standard deviation, median, minimum, maximum. Categorical 
variables will be summarized by the number (N) and the proportion of 
subjects (%). Where appropriate, 95% confidence intervals for the target 
variables will be estimated. 
The significance level of statistical tests will be set at 0.05. Parametric 
tests (e.g., paired t-test) will be used to analyze continuous variables; 
when continuous variables are not normally distributed, the 
corresponding non-parametric tests (e.g. Wilcoxon signed-sum rank 
test) will also be performed. Details of statistical analysis are provided in 
the following paragraphs. 
The statistical analysis will be performed using SAS 9.4 for Windows 
(SAS Institute Inc., Cary, NC, USA). 
Primary 
performance 
endpoint 
Analysis 
Paired t-test/Wilcoxon signed rank test will be used to assess a 
significant change in skin hydration, evaluated through corneometer skin 
examination, from baseline to Day 98. 
Statistical 
Analysis – 
Secondary 
performance 
endpoints 
Paired t-test/Wilcoxon signed rank test will be used to assess a 
significant change in skin hydration, evaluated through corneometer skin 
examination, from baseline to Day 21 and Day 42. 
Subjects will be considered responders when skin hydration increase is 
at least 10% between baseline and Day 98 and non-responders when 

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there is not any increase in skin hydration. The number (N) and 
proportion of subjects (%) for each category will be reported. 
The skin texture, skin tone, smoothing of fine lines and wrinkles will be 
summarized at all time-points in terms of number (N) and percentage 
(%). Subjects will be considered responders when Investigator’s 
evaluation of skin texture, skin tone and smoothing of fine lines 
increases at least one category on the assessment scale; otherwise, 
subjects will be considered non-responders. Subjects will be considered 
responders when Investigator’s evaluation of wrinkles decreases at least 
two grades on the severity rating scale; otherwise, subjects will be 
considered non-responders. Subjects whose grade on the severity rating 
scale of Investigator’s evaluation of wrinkles decreases from 2 “Slight” 
to 1 “Absent” will also be considered responders. 
The Global Aesthetic Improvement Scale (GAIS) filled-in by the 
investigator and the Patient’s Aesthetic Improvement Scale (PAIS) filled-
in by the subject will be summarized at all time-points in terms of number 
(N) and percentage (%). Subjects will be considered responders when 
the evaluation increases at least one level on the categorical scale; 
otherwise, subjects will be considered non-responders 
The overall subject’s evaluation of satisfaction with the treatment at Day 
98, performed by means of the 5-item scale, will be summarized through 
number (N) and proportion of subjects (%) for each item.  
Statistical 
Analysis – 
Safety 
endpoints 
The number of subjects who experienced at least one adverse event 
(AE) or adverse device event (ADE), study product-related AE or ADE, 
serious AE or ADE, severe AE or ADE and the number of subjects 
withdrawn due to AE will be summarized. 
For each system/organ class term (SOC) and preferred term (PT), 
summaries will be made with respect to the proportion of subjects having 
at least one occurrence of that event during the study and the total 
number of events.  

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Local tolerability at the site of administration will be reported and 
summarized (with particular attention to pain, erythema, edema, 
bruising). 
Population 
Safety analysis set (SAF): all subjects enrolled who sign informed 
consent and receive the treatment. 
Full analysis set (FAS): all subjects of the SAF who have performed the 
baseline assessments and have at least one post-baseline assessment 
of any performance endpoint (primary or secondary).  
Per-Protocol analysis set (PPAS): all subjects of the FAS who also meet 
all inclusion/exclusion criteria and who do not have any major protocol 
deviation. 
 
 
Figure 1: Study design 
 
 
 
 
 
 
Visit 1 – treatment 1 
 
  Visit 2 – treatment 2 
 
 Visit 3 – treatment 3 
 
            Visit 4 – end of study 
 
 
 
 
Day 1 
 
 
 
Day 21 (±4) 
 
 
Day 42 (±4) 
 
                           Day 98 (±7) 
 
 

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Table 1: Study Flow-Chart  
 
 
 
 
 
Visit number  
V1 
V2 
V3 
V4 
Time 
Day 1 
 
Day 21 
(allowed window: 
±4 days) 
Day 42 
(allowed window: 
±4 days) 
Day 98  
(allowed window:  
±7 days) 
Phase 
Screening, baseline, 
Treatment 1 
Treatment 2 
Treatment 3 
Follow-up / End of 
study 
Informed consent 
X 
 
 
 
Inclusion/exclusion criteria 
X 
 
 
 
Demography 
X 
 
 
 
Medical and surgical history 
X 
 
 
 
Previous/Concomitant treatments 
X 
X 
X 
X 
Physical examination  
X 
X 
X 
X 
Pregnancy test (urine dipstick, 
where applicable)  
X 
 
 
 
FOLIAGE HYDROFIL 
ADMINISTRATION  
X 
X 
X 
 
Photos of the treated areas  
X* 
X* 
X* 
X 
Corneometer analysis of treated 
areas 
X* 
X* 
X* 
X 
Skin surface characteristics 
(texture, tone etc.) 
X* 
X* 
X* 
X 
PAIS (Subject) 
 
X 
X 
X 
GAIS (Investigator) 
 
X 
X 
X 
Overall satisfaction by subject 
 
 
 
X 
AEs collection 
X 
X 
X 
X 
  
*before the administration of the study product. 
 
 
 
 
 
 

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2. 
Identification and Description of the Investigational Device 
Foliage Hydrofil is a CE marked, class III, resorbable medical device (sterile, non-
pyrogenic and physiological gel) to be used for the hydration of the skin and for the 
correction of superficial skin imperfections of the face and the body and in the process 
of repairing the dermal tissue. The main component is HA sodium salt of non-animal 
origin, produced by bacterial fermentation, with a molecular weight similar to the human 
HA. Foliage Hydrofil does not contain human tissues or their derivatives.  
Foliage Hydrofil is indicated for superficial cutaneous blemishes of the face or other body 
areas. 
The manufacturer, Phitogen Holding S.p.A. is also the Sponsor of this post-market 
clinical follow-up study. 
 
3. 
Justification for the Design of the Clinical Investigation 
The slowing of aging process is interested by numerous researches; more figures 
(plastic surgeon, cosmetic surgeon etc) pay attention to aging of face in modern society 
[1]. Skin aging process could be viewed as an alteration occurring in collagen, elastin 
and, mainly, in HA content [2, 3]. HA degradation, together with the bone resorption, 
plays a major role in the formation of wrinkles and other signs of aging, and structural 
proteins are essential to skin health and youthfulness [4, 5]. The increasing concern to 
maintain a youthful appearance has driven the growth of new dermatological procedures 
for treatment of skin aging. So, in recent years there has been an increasing emphasis 
on minimally invasive treatments and techniques designed to treat volume loss, 
wrinkles, and skin photodamage [6].  
Among treatments today available, bio-revitalization is a process which triggers the 
skin’s own, natural collagen production and cell rejuvenation through the injection of 
hyaluronic acid serums. Hyaluronic acid, (HA) is a naturally occurring molecule in the 
body which is found in the skin, joints, cartilage and other areas, providing natural 
lubrication. This substance is hydrophilic, drawing many times its weight in water. As we 
age, we have less naturally produced HA in our tissues and skin dehydration becomes 
a telltale sign of aging. The HA owns a great hydrating power, due to this property is 
widely used in the biorevitalization treatments. Its use results in supple, younger-looking 
skin with reduced wrinkles and improved elasticity [7, 8].  

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In bio-revitalization procedures that involve intradermal micro-injections of natural HA, 
with a molecular weight close to the endogenous one, carried out in correspondence 
with the imperfections, the viscoelastic properties of HA allow to rehydrate the tissues, 
allowing to prevent and contrast the aging process skin and promote their remodeling. 
Although the procedure can be performed on almost any area of the skin, the most 
popular treatment areas are the face, neck and décolleté area and the back of the hands. 
These areas of the body tend to be more susceptible to sun damage and as a result 
people often notice aging in these areas first. 
Today, intradermal injections of biological substances able to induce a revitalization of 
the dermis can stimulate qualitative and quantitative improvements in aging skin 
alterations. The most frequently used substance is natural noncross-linked hyaluronic 
acid (HA), which is the main ingredient of the medical device Foliage Hydrofil. 
The aim of this study is to confirm the clinical performance and safety of the medical 
device Foliage Hydrofil on the face bio-revitalizing treatment. To this purpose no. 24 
adults (males and females) searching for bio-revitalization treatment will be enrolled and 
treated with three injective sessions apart 3 weeks each other. All the subjects will 
receive the same treatment in an open label fashioned study. 
 
 
4. 
Benefits and Risks of the Investigational Device, Clinical Procedures 
and Clinical Investigation 
Foliage Hydrofil belongs to a class of minimally invasive treatments used to maintain a 
youthful appearance. All the subjects will be treated in the face sites agreed with the 
physician. 
The product will be injected through micro-injections in the derma. Injections, even if 
done with micro needles could generate some risks and a certain level of discomfort to 
the subject. In order to avoid risks, the Investigator will duly disinfect the injection sites 
before treatments. If necessary, an anesthetic cream could be used before injections. 
Potential side effects due to the treatment are mainly pain, erythema, edema, bruising, 
but they are usually of mild intensity and transient. In such cases the Investigator will 
suggest the appropriate treatment to manage any side effect. 

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No tests or invasive examinations are foreseen in this study that can increase the risk 
for participants.  
In the light of the above, no additional risk arising from the participation to this clinical 
investigation can be envisioned. 
 
 
5. 
Objectives and Hypotheses of the Clinical Investigation 
5.1  
Primary Objective and Endpoint 
The primary objective of this clinical investigation is to evaluate and confirm the clinical 
performance of Foliage Hydrofil in the improvement of the skin hydration in the treated 
area at Day 98, from baseline. 
 
The primary endpoint of the study is the change in skin hydration from baseline to Day 
98, evaluated through corneometer skin examination. 
 
5.2  
Secondary Objectives and Endpoints 
The secondary objectives of this clinical investigation are: 
- 
to evaluate the clinical performance of Foliage Hydrofil la in the improvement of 
the skin hydration in the treated area at Day 21 and 42, from baseline; 
- 
to evaluate the improvement of the skin texture, skin tone, smoothing of fine lines 
and wrinkles in the treated area at Day 21, 42 and 98, from baseline;  
- 
to evaluate the aesthetic change from baseline as judged by the Investigator at 
Day 21, 42 and 98;  
- 
to evaluate the aesthetic change from baseline as judged by the subject at Day 
21, 42 and 98;  
- 
to evaluate the subject’s satisfaction at Day 21, 42 and 98. 
 
Performance secondary endpoints of this clinical investigation are: 
- 
the evaluation of the skin hydration modification from baseline to Day 21 and 42, 
through corneometer examination.  
- 
the evaluation of the of the skin texture, skin tone, smoothing of fine lines and 
wrinkles modification from baseline in the treated area at Day 21, 42 and 98;  

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- 
the Investigator’s aesthetic change assessment using the Global Aesthetic 
Improvement Scale (GAIS) on Day 21, 42 and 98. GAIS is a categorical scale 
with 5-points: worse than before treatment, no change, minimal improvement, 
good improvement, optimal improvement; 
- 
the subject’s aesthetic change assessment using the Patient’s Aesthetic 
Improvement Scale (PAIS) on Day 21, 42 and 98. PAIS is a categorical scale 
with 5-points: worse, no change, somewhat improved, moderately improved  
improved, very much improved; 
- 
the evaluation of the overall subject’s satisfaction at on the treatment at Day 21, 
42 and 98, through a 5-points scale (very much satisfied, satisfied, not satisfied 
nor unsatisfied, unsatisfied, very unsatisfied). 
 
5.3  
Tolerability and Safety Objectives and Endpoints 
To evaluate the local safety at injection site (pain, erythema, edema, bruising) and the 
systemic safety of the treatment with Foliage Hydrofil. 
 
Safety endpoints are: 
- 
the number, typology, severity of local and systemic adverse events. Particular 
attention will be paid to local adverse events like pain, erythema, edema, bruising. 
 
 
6. 
Design of the Clinical Investigation 
6.1 
General 
This is a monocenter, open label post-market clinical follow-up investigation. 
Each subject will be treated with the study device. 
The subject will start treatment on the day (Day 1) after the first visit and two further 
treatments at 3 weeks distance. Two months after the last treatment, a final visit will be 
planned to evaluate the final aesthetic result. 
Each subject for whom written consent is obtained will be identified during the study by 
a “Subject Code”, automatically generated and assigned by the e-CRF.   

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All screened subjects will receive the Subject Code regardless of whether they receive 
the treatment or not. If a subject discontinues from the study at any time, the Subject 
Code will not be re-used. 
All subjects who will sign the informed consent and receive the Subject Code will be 
entered into a Subject’s Register, containing the name and surname of the subjects and 
their Subject Codes. The Subject’s Register will be accessible for source data 
verification but will be preserved only at clinical site. 
The Investigator will keep record of all enrolled subjects in the Subject’s 
Screening/Enrolment Log: Subject Code, the date of consent, or eventually the reason 
for not actively entering the study or interrupting the study will be recorded. 
 
6.2 
Investigational device  
6.2.1 Foliage Hydrofil 
Foliage Hydrofil is a CE marked, class III, resorbable medical device (sterile, non-
pyrogenic and physiological gel) to be used for the hydration of the skin and for the 
correction of superficial skin imperfections of the face and body and in the process of 
repairing the dermal tissue. The main component is HA sodium salt of non-animal origin, 
produced by bacterial fermentation, with a molecular weight similar to the human HA. 
Foliage Hydrofil does not contain human tissues or their derivatives.  
Foliage Hydrofil is indicated for superficial cutaneous blemishes of the face or other body 
areas. 
The medical device is formulated as injectable vials of 2 mL (1.6% HA formulation). In 
this clinical investigation each subject will receive three treatments, one vial 2mL each, 
with Foliage Hydrofil, 3 weeks apart. Foliage Hydrofil will be injected with 30G 4mm only. 
The area treated will be the face. The device administration could be done after an 
anesthetic cream application, according to Investigator’s judgement. 
 
6.2.1.1  Packaging and labelling 
The Foliage Hydrofil will be provided for the study by the Sponsor of this clinical 
investigation.  
The device shipment to the study site will be done only after the completion of Ethics 
and administrative procedures.  

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The market packaging and labelling will be used. Only and additional label will be added 
to the outer box, specific for the clinical investigation.  
Each package of treatment will contain: 
• Instruction for use (IFU) 
• 1 sealed blister containing 1 single-dose / single-use sterile pre-filled syringe (1x2 ml) 
• adhesive labels to be applied on the clinical chart/patient card for product tracking 
• 1 needle 
 
6.2.1.2 Study device instructions for use 
Foliage Hydrofil is intended for injection in the derma, for the hydration of the skin and 
the correction of skin blemishes, thanks to its HA content.  
The device will be administered at clinical site by a qualified physician, already 
experienced with the use of similar devices. In any case specific instructions for the 
correct management of the study product will be provided by the Sponsor before the 
enrolment start. Product instructions for use (IFU) shall be fully complied.  
Each treatment session will involve multiple microinjections, with needles 30G 4mm, in 
the face (external corner of the eye and cheek). The amount of product injected will be 
up to 2 mL, according to Investigator’s judgement; the microinjections will be performed 
at a distance of 1–2 cm from each other. 
An anesthetic cream could be used before injections, at Investigator’s discretion. 
 
The study product shall be carefully stored at the study site, in a safe area and 
separately from other devices/drugs/products. It shall be stored at room temperature 
(Min 2°C; Max 25°C), possibly in a dry and cool place, away from direct sunlight or heat. 
 
6.2.1.3 Study product accountability and compliance 
The Investigator is responsible for ensuring the accountability of the study product. 
Accountability records will include: 
- confirmation of product delivery and receipt to/at the clinical site 
- records concerning the product administration to each subject  
- the return to the Sponsor or alternative disposition of unused products. 
 
The Investigator should maintain records that adequately document: 

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- that the subjects were treated with the quantities specified by the clinical 
investigation plan/amendment(s) 
- that all study products provided by the Sponsor were fully reconciled. 
Unused products must not be discarded or used for any purpose other than the present 
clinical investigation.  
Subjects will be reminded of the importance of strictly complying with the instructions 
received from the Investigator. 
Compliance to the treatment will be done through Investigator’s records and vials 
accountability. 
 
6.2.2 Concomitant Medications/Treatments 
Any medication/treatment (other than those excluded by the clinical investigation plan) 
that is considered necessary for the subjects’ well-being and does not interfere with the 
study product can be given/administered/prescribed at the Investigator’s discretion. 
The following prior and concomitant medications shall be avoided, as far as possible, 
during the study participation: 
- 
any tissue augmenting therapy with non-permanent filler or treatment with 
botulinum toxin, aesthetic surgical therapy, laser treatment, mesotherapy, or 
any form of peeling in the face, or décolléte; 
- 
use of injectable revitalization preparations; 
- 
use of retinoic acid; 
- 
treatment with permanent filling materials 
- 
chemotherapy, immunosuppressive agents or corticoids; 
- 
thrombolytics, anticoagulants, inhibitors of platelet aggregation 
- 
sun exposure or tanning booths or UV sources (SPF 50+ should 
applied in case sun exposure cannot be avoided). 
 
To be avoided close to the treatment, the intake of any treatment (aspirin, N.S.A.I.D., 
Vitamin E) acting on the fluidity of the blood, to minimize the possibility of bruising or 
bleeding of the injected area. 
 
Any concomitant medication/treatment shall be recorded in the appropriate section of 
the eCRF. 

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6.3 
Subjects 
In this clinical investigation 24 caucasian man and women of 30-55 years, with signs of 
skin aging in the face and fulfilling the following eligibility criteria will be enrolled.  
Some eligibility criteria reflect the device contraindications/precautions and other criteria 
have the purpose of avoiding confounding factors that could interfere with study results.  
 
6.3.1 Inclusion criteria 
Subjects presenting all the following inclusion criteria will be eligible to enter the study: 
1. Generally healthy man and women, aged between 30 and 55 years (inclusive), 
looking for aesthetic procedures to improve the aspect of their face. 
2. Caucasian race (skin type from I to IV as per Fitzpatrick Skin Type scale); 
3. Visible signs of skin aging in the face (e.g. reduced skin elasticity, reduced turgor, 
actinic elastosis, smaller and larger wrinkles) according to the Investigators 
opinion; 
4. Corneometer Capacitance of the area treated < 80 
5. Subjects wishing not to modify their lifestyle during the study period; 
6. Subjects with cooperative attitude, able to comprehend the full nature and the 
purpose of the investigation, including possible risks, side effects and discomforts 
and willing to adhere to the study schedule (including attendance to the planned 
visits) and study prescriptions; 
7. Written informed consent release prior to any study-related procedures. 
 
6.3.2 Exclusion criteria 
Subjects presenting one or more of the following exclusion criteria will not be eligible to 
enter the study: 
 
1. Pregnancy or breastfeeding woman or woman of childbearing potential not 
practicing adequate contraception. 
2. Subjects with Fitzpatrick skin type from V to VI  
3. Subjects with known allergy/hypersensitivity to product components. 

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4. Extensively photo damaged and aged skin according to the Investigators 
opinion. 
5. Active skin disease, inflammation or related conditions, such as infection, 
perioral dermatitis, seborrheic eczema and rosacea in the face, hands or 
décolletage. 
6. Immune system disorders 
7. History of or active collagenosis (e.g. systemic lupus, erythematosis, rheumatic 
arthritis, skin or systemic sclerosis). 
8. Concomitant therapy with thrombolytics or anticoagulants, or have taken 
inhibitors of platelet aggregation, within 2 weeks before the first product 
administration. 
9. Tattoo/s on the skin around the treatment site 
10. History of cancerous or pre-cancerous lesions in the face, neck and 
décolletage. 
11. Previous tissue augmenting therapy with non-permanent filler or treatment with 
botulinum toxin, aesthetic surgical therapy, laser treatment, mesotherapy, or 
any form of peeling in the face, hands or décolletage within 12 months prior to 
the baseline visit. 
12. Use of injectable revitalization preparations within 12 months prior to the 
baseline visit. 
13. Use of retinoic acid within 6 months prior to the baseline visit. 
14. Previous or concomitant treatment with chemotherapy, immunosuppressive 
agents or corticoids. 
15. History of treatment with permanent filling materials. 
16. Participation in any other clinical study within 30 days prior to the first visit or 
plan to participate in another clinical study during this study period. 
17. Planned or ongoing weight reduction program during the study. 
18. Known history of drug or alcohol abuse within 6 months prior to the baseline 
visit. 
19. Known hypersensitivity to one or more components of anesthetic creams 
20. Nicotine use during the study or stopped within 12 months before the first visit. 

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21. Any medical condition that in the opinion of the Investigator makes the subject 
unsuitable for inclusion (e.g. severe chronic disease, malignancy, bleeding 
disorder, skin diseases etc). 
22. Other condition preventing the subject to entering the study in the Investigator's 
opinion e.g. subjects anticipated to be unreliable, unable to return for the follow-
up visits, not likely to avoid other prohibited treatments or procedures or 
incapable of understanding the information or instructions. 
 
6.4 
Procedures 
6.4.1 Study Visits and Assessments 
Visit 1 screening – baseline- treatment start (day 1):  
Subjects will be evaluated with regard to the inclusion and exclusion criteria that will 
allow their participation into the study. 
Before any study specific evaluation is carried out, subjects will receive all the 
information about the study by the Investigator and will sign an informed consent form. 
The following activities will be performed at first visit:  
▪ Collection of demographic information  
▪ Medical and surgical history  
▪ Physical examination (focused on the skin) 
▪ Previous (in the 30 days before) and concomitant medications / 
treatments recording  
▪ Urine pregnancy test (where applicable) 
▪ Inclusion/exclusion criteria assessment 
▪ Photos of the treated area (before injections) 
▪ Corneometer examination of treated area (before injections) 
▪ Skin surface characteristics, texture, tone, fine lines and wrinkles 
(before injections) 
▪ First treatment 
▪ Local and systemic adverse events after the first treatment. 
 
The following treatment session will be scheduled three weeks later, with a window of ± 
4 days. The subject will be solicited to contact the Investigator for any problem between 
visits.  

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Visit 2 (Day 21 ± 4) and Visit 3 (Day 42 ± 4):  
The following activities will be done at Visit 2, and 3: 
▪ Physical examination (focused on the skin) 
▪ Change in concomitant medications / treatments recording  
▪ Photos of the treated area (before second/third injection session) 
▪ Corneometer examination of treated area (before second/third injection 
session) 
▪ Skin surface characteristics, texture, tone, fine lines and wrinkles 
(before second/third injection session) 
▪ PAIS Subject 
▪ GAIS Investigator 
▪ Second/third treatment 
▪ Adverse events (occurred since previous visit) recording 
▪ Local and systemic adverse events after the second/third treatment. 
The second and third treatment sessions will be scheduled three weeks apart from each 
other, with a window of ± 4 days. The subject will be solicited to contact the Investigator 
for any problem between visits.  
The final visit will be scheduled about 2 months after last treatment session.  
 
Visit 4 – End of study (Day 98 ±7):  
The following activities will be done at Visit 4: 
▪ Physical examination (focused on the skin) 
▪ Change in concomitant medications / treatments recording  
▪ Photos of the treated area  
▪ Corneometer examination of treated area  
▪ Skin surface characteristics, texture, tone, fine lines and wrinkles 
▪ PAIS Subject 
▪ GAIS Investigator 
▪ Overall satisfaction by subject  
▪ Adverse events (occurred since previous visit) recording. 
Should the subject interrupt or conclude in advance the clinical study, for any reason, a 
Visit 4 will be completed.  

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6.4.2 Corneometer assessment  
Corneometer is one of the most popular measurement instrument to reproducibly and 
accurately determine the hydration level of the skin surface (stratum corneum).  
The corneometer indicates the hydration level of the superficial layers of the skin via 
measurement of skin dielectric properties. 
The measurements are performed by the application of a probe to the skin surface. 
Upon contact, an electric field passes through the stratum corneum and the dielectric 
constant is obtained. 
The value of the dielectric constant (in arbitrary units) is directly proportional to the level 
of skin hydration. 
On the probe head there is a fine piece of glass to ensure that only the capacitance 
changes due to water content are identified. Even small changes in water can be 
detected. The measurement time is short at only 1 second minimising occlusion effects. 
The depth of the measurement is 10-20µm, therefore limited to the stratum corneum, 
ensuring that deeper skin layers do not influence the measurement. 
The measuring surface is around 49mm2, so even small areas can be accurately 
measured. 
  
6.4.3 Skin texture, skin tone, fine lines and wrinkles assessment 
The Investigator will evaluate at each visit the skin texture, skin tone, smoothing of fine 
lines and wrinkles modification in the treated area and will score each item as follows: 
 
 
Skin texture 
Poor 
Good 
Very good 
□ 
□ 
□ 
 
Fine lines 
Poor 
Good 
Very good 
□ 
□ 
□ 
 
 
Skin tone 
Poor 
Sufficient 
Middle 
Good 
□ 
□ 
□ 
□ 
 

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Wrinkles will be assessed through the severity rating scale: 
Grade Description 
1 
Absent 
2 
Slight 
3 
Moderate 
4 
Severe 
5 
Extreme 
 
6.4.4 Global Aesthetic Improvement Scale (GAIS) by the Investigator  
GAIS is a categorical scale with five levels:  
Grade Description 
1 
worse than before treatment 
2 
no change 
3 
minimal improvement 
4 
good improvement 
5 
optimal improvement 
 
The Investigator will be requested to judge the aesthetic improvement of the treated 
area at all visits following the first one.  
 
6.4.5 Patient’s Aesthetic Improvement Scale (PAIS)  
PAIS is a categorical scale with five levels: 
 
Grade Description 
1 
worse  
2 
no change 
3 
somewhat improved 
4 
moderately improved 
5 
very much improved 
 
The subject will be requested to judge the aesthetic improvement of the treated area at 
all visits following the first one.  
 
6.4.5 Subject Overall Satisfaction 
Overall subject’s satisfaction on the treatment received will be defined by means of a 5-
items scale at the end of the study (Visit 4). 

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The subject will be asked “Please express your degree of satisfaction with the treatment 
received” 
 
Grade Description 
1 
very much satisfied 
2 
satisfied 
3 
not satisfied nor unsatisfied 
4 
unsatisfied 
5 
very unsatisfied 
 
 
6.5 
Monitoring Plan 
The study will be monitored on a regular basis by the CRO’s adequately qualified and 
trained clinical Monitors throughout the study period to ensure the proper conduct of the 
clinical Investigation.  
The purposes of study monitoring are to verify that the rights and well-being of study 
subjects are protected, that the reported study data are accurate, complete and 
verifiable against the source documents, and that the study is conducted in accordance 
with the current clinical investigation plan, Good Clinical Practice guideline (UNI EN ISO 
14155) and applicable regulatory requirements. 
During the monitoring visits, Monitors will verify the following, including but not limited 
to: subject informed consent, subject’s eligibility, safety data and reporting, quality of 
source documents and CRF data against subject’s medical records. If inconsistencies 
are found, the corresponding corrections to the CRF data will have to be made by the 
Investigator or designated person. Monitors will also check subject compliance, accrual, 
delegation of responsibilities within the Investigator’s team, relevant communications 
with family doctors, if any, ancillary equipment and facilities, etc. The Investigator and 
other site staff involved in the study must allocate enough time to the Monitor at these 
visits. 
Given the current COVID-19 pandemic situation also proper approaches will be 
implemented remotely to allow the performance of the monitoring activities, in line with 
applicable guidelines of competent authorities and best practices.  
Upon request by the Sponsor, on-site study audits may be conducted in order to ensure 
the study is in compliance with GCP, applicable regulatory requirements, and the clinical 
investigation plan. The auditing activities may also be conducted after study completion. 

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The Investigator agrees to allow Sponsor/auditors/CRO monitors to have direct access 
to his/her study records for review, being understood that they are bound by professional 
secrecy, and as such will not disclose any personal identity or personal medical 
information. 
The Investigator will take all appropriate measures required by the Sponsor to 
implement corrective actions for all problems found during audits. 
 
 
7. 
Statistical Design and Analysis 
This section summarizes the statistical principles and methods planned to analyze the 
data for this clinical investigation. 
 
7.1 
Sample Size Determination 
Assuming an average difference in the skin hydration evaluated through corneometer 
skin examination (capacitance) between baseline and Day 98 of 7.5, with a standard 
deviation of 8, 20 subjects will allow to appreciate this difference as statistically 
significant at an alpha = 0.05 and a 95% power. Considering the above and a possible 
drop-out rate of 20%, 24 subjects will be recruited. 
 
7.2 
Definition of Study Populations for Analysis 
The following populations are defined for this investigation: 
• Safety analysis set (SAF): all subjects enrolled who sign informed consent and 
receive at least one administration of the investigational device. 
• Full analysis set (FAS): all subjects of the SAF who have performed the baseline 
assessments and have at least one post-baseline assessment of any 
performance endpoint (primary or secondary).   
• Per-Protocol analysis set (PPAS): all subjects of the FAS who also meet all 
inclusion/exclusion criteria and who do not have any major protocol deviation. 
 
The analysis of safety endpoints will be performed in the SAF population. Analysis of 
performance endpoints will be performed on the FAS population. Additionally, analysis 
of primary endpoint will also be performed on the PPAS population. 

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7.3 
Statistical Analysis 
Descriptive statistical analysis of all relevant variables will be performed. Continuous 
variables will be summarized by the number of subjects (N), mean, standard deviation, 
median, minimum, maximum. Categorical variables will be summarized by the number 
(N) and the proportion of subjects (%). Where appropriate, 95% confidence intervals for 
the target variables will be estimated. 
The significance level of statistical tests will be set at 0.05. Parametric tests (e.g., paired 
t-test) will be used to analyze continuous variables; when continuous variables are not 
normally distributed, the corresponding non-parametric tests (e.g., Wilcoxon signed rank 
test) will also be performed. Details of statistical analysis are provided in the following 
paragraphs. 
The statistical analysis will be performed using SAS 9.4 for Windows (SAS Institute Inc., 
Cary, NC, USA). 
 
7.3.1 Missing Data 
Missing data will not be replaced in any statistical analysis. 
 
7.3.2 Multiplicity 
No adjustment is needed. 
 
7.3.3 Covariates, Interactions and Subgroups 
There are no planned subgroup analyses. 
 
7.3.4 Analysis of Demographics and Baseline Variables 
Demographic (gender, age) and baseline characteristics will be summarized using 
mean, median, standard deviation, minimum and maximum for continuous variables and 
frequencies and percentages for categorical variables.   
 

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7.3.5 Efficacy analysis 
7.3.5.1 Primary endpoint 
Paired t-test, when the skin hydration is normally distributed, or the corresponding non-
parametric Wilcoxon signed rank test will be used to assess a significant change in skin 
hydration, evaluated through corneometer skin examination (capacitance), from 
baseline to Day 98.  
 
7.3.5.2 Secondary endpoints 
Paired t-test, when the skin hydration is normally distributed, or the corresponding non-
parametric Wilcoxon signed rank test will be used to assess a significant change in skin 
hydration, evaluated through corneometer skin examination (capacitance), from 
baseline to Day 21 and Day 42. 
Subjects will be considered responders when skin hydration increase is at least 10% 
between baseline and Day 98 and non-responders when there is not any increase in 
skin hydration. The number (N) and proportion of subjects (%) for each category will be 
reported. 
The skin texture, skin tone, smoothing of fine lines and wrinkles will be summarized at 
all time-points in terms of number (N) and percentage (%). Subjects will be considered 
responders when Investigator’s evaluation of skin texture, skin tone and smoothing of 
fine lines increases at least one category on the assessment scale, from baseline; 
otherwise, subjects will be considered non-responders. Subjects will be considered 
responders when Investigator’s evaluation of wrinkles decreases at least two grades on 
the severity rating scale, from baseline; otherwise, subjects will be considered non-
responders. Subjects whose grade on the severity rating scale of Investigator’s 
evaluation of wrinkles decreases from 2 “Slight” to 1 “Absent” will also be considered 
responders. 
The Global Aesthetic Improvement Scale (GAIS) filled-in by the investigator and the 
Patient’s Aesthetic Improvement Scale (PAIS) filled-in by the subject will be summarized 
at all time-points in terms of number (N) and percentage (%). Subjects will be considered 
responders when the evaluation increases at least one level on the categorical scale, 
from baseline; otherwise, subjects will be considered non-responders 

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The overall subject’s evaluation of satisfaction with the treatment at Day 98, performed 
by means of the 5-item scale, will be summarized through number (N) and proportion of 
subjects (%) for each item. 
7.3.6 Safety Analysis 
All enrolled subjects receiving at least one treatment injection will be included in the 
safety analysis.  
Adverse events (AEs) and Adverse Device Events (ADEs) will be coded using the last 
updated version of the Medical Dictionary for Regulatory Activities (MedDRA) dictionary 
to give a preferred term (PT) and a system/organ class term (SOC) for each event. The 
number of subjects who experienced at least one AE or ADE, study product-related AE 
or ADE, serious AE or ADE, severe AE or ADE and the number of subjects withdrawn 
due to AE will be summarized. 
For each SOC and preferred term, summaries will be made with respect to the 
proportion of subjects having at least one occurrence of that event during the study and 
the total number of events. The incidence of AEs and ADEs will be presented overall, 
by SOC and preferred term, and additional grouping by severity and relationship to the 
study treatment.  
Local tolerability at the site of administration will be reported and summarized. 
 
7.4     Planned Interim Analysis 
No interim analysis is planned.  
 
8.        Data Management 
During each study visit, the study Investigator (or designee) will collect and report study 
data in the relevant subject’s chart, documenting all significant observations. 
Any contact with the subject via telephone or other means that provides significant 
clinical information shall be documented in the source data. 
An Electronic Case Report Form (e-CRF) will be used to record subject’s study data.  
All data relating to the study will be documented in the electronic CRF (e-CRF). This 
CRF is developed to record the data requested by the protocol.  
The Investigator will maintain a list of all persons authorized to make entries and/or 
corrections on the e-CRFs. Each authorized person will be provided with a user-specific 

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ID protected by a renewable password. Data entries and corrections will be made only 
by the authorized persons. The e-CRF system will record date and time of any entry and 
/or correction and the user ID of the person making the entry/correction. The system will 
keep track of all old and new values (audit trail). It is the responsibility of the Investigator 
to ensure that the CRFs are properly and completely filled in. The CRFs must be 
completed for all subjects who have been included in the study. The Investigator will 
review all CRFs and electronically sign and date them for each subject, verifying that 
the information is complete, true and correct. All fields on the CRF must be completed 
as applicable. 
Checks to assist during the data entry and to assess the appropriateness and 
consistency of data will be developed on the e-CRF system. E-CRF pages will be 
reviewed both on site by the monitor of the center and remotely, by the data 
management staff of the CRO. Data Clarification Sheets (DCS) will be generated 
through the e-CRF system, both automatically, through edit checks, and manually, by 
CRAs and/or data managers, and the Investigator will have to check and solve them. 
The Investigator is responsible for the review and approval of all query resolutions. 
Subjects will be provided with paper questionnaires/scales. Such documents will be 
filled by the subjects during the study visits, to record data concerning their opinion on 
the aesthetic result of the treatment and the consequent satisfaction with the treatment 
received.  
It is responsibility of the Investigators to instruct the study participants on how to fill in 
questionnaires in a clear way and preferably in black ball-point pen. The 
questionnaires/scales will be anonymous, each subject is identified through the Subject 
Code. Questionnaires/scales data will be reported on the e-CRF by the Investigator (or 
designee). Questionnaires will be considered source data. 
 
At the beginning of the study, a Study Master File will be established according to 
Sponsor or CRO SOPs, in order to maintain the study documents as specified in the 
UNI EN ISO 14155 Guideline and as required by the applicable regulatory requirements. 
The CRO will take measures to prevent accidental or premature destruction of these 
documents. 
Clinical sites will permit study-related monitoring, audits, and regulatory inspection 
providing direct access to source data / documents. 

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Essential documents of the clinical investigation, including the subject identification list, 
source data and case report forms will be retained for 10 years after the completion or 
discontinuation of the clinical investigation at clinical sites. The sponsor will retain the 
study master file for 10 years after the CE mark for the study product has been 
discontinued in all countries. 
 
 
9.        Amendments to the CIP 
Changes to the clinical investigation plan may only be made by means of a written 
amendment, which has to be approved and signed by the authorized representatives of 
the Sponsor, and by the Investigator. 
Exhaustive justifications that motivate the amendment to the clinical investigation plan 
should clearly be addressed in the document.  
All substantial Clinical Investigation Plan amendments must be submitted for approval 
to IEC and notified to the Regulatory Authority. In case of non-substantial amendment 
the IEC and the Regulatory Authority (when applicable) will be only notified of the 
amendment without the request to review and approve it. 
The Investigator, the Sponsor and IEC, separately or together, should decide whether 
the subject’s informed consent form needs to be changed. 
 
 
10. Deviations from Clinical Investigation Plan 
The Investigator is to conduct the study in accordance with the relevant, current clinical 
investigation plan and will only deviate when necessary to protect the safety, rights and 
welfare of the subjects. In the event that an isolated, unforeseen instance occurs 
resulting in a clinical investigation plan deviation, the Investigator is to document this 
deviation and notify the CRO or the Sponsor as soon as possible. In no instance should 
this increase the subject’s risk or affect the validity of the study. 
 
 

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11. Device Accountability 
The device will be administered only at clinical site under the responsibility of the 
Investigator. The Investigator is responsible of ensuring the correct device 
administration and for registering data of the treatment administered. 
The study product will be provided to the clinical site once ethical authorization is 
obtained. 
 
 
12. Statement of Compliance 
The study will be conducted in compliance with the current version of the Declaration of 
Helsinki (Appendix 2), with the clinical investigation plan, the MDR 745/2017, the UNI 
EN ISO 14155, the Italian laws in force and the principles of the Good Clinical Practice.  
The clinical investigation will start at clinical site only after obtaining the approval of the 
relevant Ethics Committee. 
  
13. Informed Consent Process 
The Investigator is responsible for and will obtain informed consent from each subject, 
in accordance with the UNI EN ISO 14155, and the current version of the Declaration of 
Helsinki.  
All subjects invited to participate in the study are entitled to make their voluntary decision 
based on all current available information provided to them by the Investigator/designee. 
In addition, they will be given a document in native language written in clear concise lay 
language for review and consideration. The document will previously have been 
approved by the relevant independent Ethics Committee (IEC) and may further be 
updated as new important information becomes available that may affect subject’s 
willingness to participate or continue in the study. 
The subject must be made aware that he/she may refuse to join the study or may 
withdraw his/her consent at any time without prejudicing further medical care and that 
he/she is covered by the Sponsor's indemnity insurance in the event of a study related 
injury. Subjects must also know that their personal medical records may be reviewed in 
confidence by the Sponsor's staff or representatives and by Regulatory Authority and 

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IEC and that personal information will be collected and retained in a confidential 
database. Consent will always be given in writing after the subject has had adequate 
time to review the information and ask questions, if need be.  
 
14. Adverse Events, Adverse Device Effects and Devices Deficiencies 
14.1 Definitions 
Adverse Event (AE) 
Any untoward medical occurrence, unintended disease or injury or any untoward clinical 
signs, including an abnormal laboratory finding, in subjects, users or other persons, in 
the context of a clinical investigation, whether or not related to the investigational device. 
 
Adverse Device Effect (ADE) 
Any untoward and unintended response to a medical device. This includes any event 
resulting from insufficiencies or inadequacies in the instructions for use or the 
deployment of the device and any event that is a result of a user error. 
Device Deficiency 
Any inadequacy in the identity, quality, durability, reliability, safety or performance of an 
investigational device, including malfunction, use errors or inadequacy in information 
supplied by the manufacturer. 
 
Serious Adverse Event (SAE) 
Any adverse event that led to any of the following: 
a) death, 
b) serious deterioration in the health of the subject, that resulted in any of the following: 
i. life-threatening illness or injury, 
ii. permanent impairment of a body structure or a body function, 
iii. hospitalisation or prolongation of patient hospitalisation, 
iv. medical or surgical intervention to prevent life-threatening illness or injury or 
permanent impairment to a body structure or a body function, 
v. chronic disease 
c) foetal distress, foetal death or a congenital physical or mental impairment or birth 
defect. 

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Serious Adverse Device Effect (SADE) 
Adverse device effect that has resulted in any of the consequences characteristic of a 
serious adverse event or that might have led to any of these consequences if suitable 
action had not been taken or intervention had not been made or if circumstances had 
been less opportune. 
 
Unanticipated Serious Adverse Device Effect (USADE) 
Serious adverse device effect that by its nature, incidence, severity or outcome has not 
been identified in the risk analysis report. 
 
Incident 
Any malfunction or deterioration in the characteristics and/or performance of a device, 
as well as any lack of labelling or instructions for use which, directly or indirectly, may 
cause or have caused death or serious deterioration in the health of the patient or of a 
user or of other persons. 
 
14.2 Adverse Event Intensity/Causality 
The Investigator, based on his direct observation or on subjects' report, will record the 
event according to the current version of CTCAE: 
Description 
Definition 
Grade 1 (Mild): 
asymptomatic 
or mild 
symptoms; 
clinical 
or 
diagnostic 
observations only; intervention not indicated. 
Grade 2 (Moderate): 
 
 
minimal, local or noninvasive intervention indicated; limiting age-
appropriate instrumental daily life activities. 
Grade 3 (Severe):  
 
 
medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; 
disabling; limiting self -care daily life activities. 
Grade 4 (Life-
threatening 
consequences):  
urgent intervention indicated. 
Grade 5: 
death related to AE 
 

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The relationship of any AE to the product will be classified by the Investigator as follows: 
Description 
Definition 
Not related 
Relationship to the device, comparator or procedures can be 
excluded. 
 
Probable 
The relationship with the use of the investigational device or 
comparator, or the relationship with procedures, seems relevant 
and/or the event cannot be reasonably explained by another 
cause. 
 
Possible 
The relationship with the use of the investigational device or 
comparator, or the relationship with procedures, is weak but 
cannot be ruled out completely. 
 
Causal relationship 
The adverse event is associated with the investigational device, 
comparator or with procedures beyond reasonable doubt. 
 
 
 
14.3 Adverse Event, Device Deficiency, Adverse Device Effect, reporting 
The Investigator will record any AEs, Device Deficiency and ADEs occurring during the 
study. He/She will also co-operate with the Sponsor in connection with the reporting of 
any “reportable event” to the Competent Authority and to the Independent EC, if 
applicable.  
All AEs regardless of severity occurring between recruitment and completion of the 
study by a subject must be recorded on the AE form provided with the CRF. 
If there is a significant worsening of a medical condition that was present before starting 
the study, this should be considered as a new AE and a complete evaluation recorded. 
Signs and symptoms considered as lack of efficacy and occurring during the study will 
not be recorded on the AEs Section of the CRF except on the condition that, in the 
Investigator’s opinion, these signs and symptoms are caused by any reason different 
from lack of efficacy of the study product or meet the definition of serious AE. 
 
In the event of a SAE (including a reportable event) the Investigator has to: 
▪ 
Complete the relevant CRF pages and a reporting form with the available initial 
information, 
▪ 
Immediately inform the Sponsor or the CRO by telephone,  

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▪ 
Fax the completed report form to the contact person at the Sponsor or the CRO as 
soon as possible but no later than 3 calendar days after the investigation site study 
personnel’s awareness of the event. 
Reporting contacts are on page 4 of this Clinical Investigation Plan. 
The Investigator is responsible for ensuring the follow-up of any subject who 
experiences a serious adverse event (including any reportable event) during the study. 
The investigator must re-examine the subject at regular intervals until the symptoms 
have completely disappeared or stabilized. 
The Sponsor or its delegate will be responsible for reporting the SAE/SADE/Serious 
Incident to the appropriate Authorities and EC, according to the local regulations. 
 
 
15. Vulnerable Population 
No vulnerable population will be included in this clinical investigation. 
 
 
16. Suspension or Premature Termination of the Clinical Investigation 
Unless premature interruption occurs, the end of the study will be the closure visit at 
clinical site. 
 
16.1  Study Discontinuation 
Subjects may be discontinued at any time from the study for any of the following 
reasons: 
➢ 
An AE occurs that, in the opinion of the Investigator, makes it unsafe for the subject 
to continue in the study 
➢ 
Lack of compliance of the subject to the study treatment or assessments 
➢ 
The subject needs to use a medication/treatment not allowed  
➢ 
The subject is lost to follow-up 
➢ 
The subject dies 
➢ 
The subject withdraws consent 

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➢ 
The Investigator, for any reason, terminates the entire study, or terminates the 
study for that subject or the attending physician requests that the subject be 
withdrawn for any medical reason 
➢ 
The Sponsor or the Regulatory Authority or the Ethics Committee, for any reason, 
terminates the entire study or terminates the study for this site or for a particular 
subject. 
If a subject is discontinued from the study, the Investigator will, as far as possible, 
complete the end of study visit (Visit 4) e-CRF fields. The Investigator should try to 
ascertain the reason(s) for withdrawal, while fully respecting the subject’s rights. 
 
16.2  Study Interruption 
The Sponsor may consider study closure at the clinical site if the following occurs: 
➢ serious and/or persistent non-compliance with the Clinical Investigation Plan 
➢ inadequate collaboration of site personnel with CRO/Sponsor 
➢ administrative reasons 
➢ non-compliance with GCP, SOPs or regulatory requirements 
➢ lack of confidentiality and/or non-compliance with the contract spread with the 
Sponsor. 
17. Publication Policy 
All information obtained as a result of the study will be regarded as confidential. 
The results of the clinical study will be documented in an integrated clinical study report 
according to UNI EN ISO 14155. 
The Sponsor and the Investigator agree that no publications presenting or discussing 
data and/or results from this clinical study sponsored by Phitogen Holding S.p.A. will 
take place until the participating center has completed the study, the data have been 
interpreted, and the final report has been issued. 
As a rule, the Sponsor is free to use the data collected in the sponsored study for world-
wide scientific product documentation, and for publication. 
In general, the Sponsor has no objections if the Investigators publish the results of the 
study. However, the Investigator is requested to provide the Sponsor with a copy of the 
manuscript for review before submitting it to the publisher with a cover letter informing 
the Sponsor about the intention to publish the study results. When permission for 

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presentation or for publication is granted, Investigator, prior to submission of a 
manuscript or abstract to the publisher, shall forward a copy of said manuscript or 
abstract to the Sponsor who shall have 90 days to request any reasonable amendment 
thereto, which shall be taken into due account and consideration by the Investigators. 
The Sponsor is entitled to include as authors of the publication all Sponsor’s personnel 
who contributed substantially to the theoretical or experimental work and also to take 
part in the decision that establishes the order in which the authors’ names will be given. 
Costs for publication must be regulated by written agreement between the parties.  
If publication of the results of the study, either in part or in full, is prepared by the 
Sponsor, the Investigator will be provided with a copy of the manuscript before the 
submission to the publisher and asked to give approval of the document. The 
Investigator will be asked in writing if they accept to be included as author of the 
publication. Answers should be sent in writing to the Sponsor within a reasonable time 
limit (30 days). If no answer is received, it is assumed that the Investigator agrees to the 
Sponsor’s proposal. 
 
 

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18. Bibliography 
 
1. Brandi C, Cuomo R, Nisi G, Grimaldi L, D'Aniello C. Face Rejuvenation: a new 
combinated protocol for biorevitalization. Acta Biomed. 2018 Oct 8;89(3):400-
405. 
 
2. Donejko M, Przylipiak A, Rysiak E, Miltyk W, Galicka E, Przylipiak J, et al. 
Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis 
in cultured human fibroblasts. DrugDes Devel Ther. 2015;24;9:6225–33. 
 
3. Stellavato A, Corsuto L, D'Agostino A, La Gatta A, Diana P, Bernini P, De Rosa 
M, Schiraldi C. Hyaluronan Hybrid Cooperative Complexes as a Novel Frontier 
for 
Cellular 
Bioprocesses 
Re-Activation. 
PLoS 
One. 
2016 
Oct 
10;11(10):e0163510. doi: 10.1371/journal.pone.0163510. PMID: 27723763; 
PMCID: PMC5056743. 
 
4. Papakonstantinou E, Roth M, Karakiulakis G. Hyaluronic acid: A key molecule in 
skin aging.Dermato-endocrinol. 2012. 1;4(3):253–8. 10.4161  
 
5. Croce MA, Dyne K, Boraldi F, Quaglino D Jr, Cetta G, Tiozzo Ret al.Hyaluronan 
affects proteinand collagen synthesis by in vitro human skin fibroblasts. Tissue 
Cell. 2001;33(4):326 
 
6. Sparavigna A, Tenconi B, De Ponti I. Antiaging, photoprotective, and brightening 
activity in biorevitalization: a new solution for aging skin. Clin Cosmet Investig 
Dermatol. 2015 Feb 10;8:57-65. 
 
7. Tedeschi A, Lacarrubba F, Micali G. Mesotherapy with an Intradermal Hyaluronic 
Acid Formulation for Skin Rejuvenation: An Intrapatient, Placebo-Controlled, 
Long-Term Trial Using High-Frequency Ultrasound. Aesthetic plastic surgery. 
2015;39(1):129–33.  
 
8. Gao F, Liu Y, He Y, et al. Hyaluronan oligosaccharides promote excisional wound 
healing through enhanced angiogenesis. Matrix biology: journal of the 
International Society for Matrix Biology.2010;29(2):107–16.  
 
 

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Appendix 1  
 
List of Abbreviations  
 
ADE 
Adverse Device Effect 
AE 
Adverse Event 
AIDS 
Acquired Immune Deficiency Syndrome 
CIP 
Clinical Investigation Plan 
CRF 
Case Report Form 
CRO 
Contract Research Organization  
CTCAE 
Common Terminology Criteria for Adverse Events 
FAS 
Full Analysis Set 
GAIS 
Global Aesthetic Improvement Scale 
GCP 
Good Clinical Practice 
GMP 
Good Manufacturing Practice 
HA 
Hyaluronic Acid 
HIV 
Human Immunodeficiency Virus 
ICH  
International Conference on Harmonization 
IFU 
Instructions For Use 
IEC 
Indipendent Ethics Committee 
MedDRA 
Medical Dictionary for Regulatory Activities 
ml 
Milliliters 
N 
Number 
PAIS 
Patient’s Aesthetic Improvement Scale 
PI 
Principal Investigator 
PT 
Preferred Term 
QoL 
Quality of Life 
SADE 
Serious Adverse Device Effect 
SAF 
Safety Analysis Set 
SAE 
Serious Adverse Event 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
USADE 
Unanticipated Serious Adverse Device Effect 
 

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Appendix 2  
 
 
WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI 
Ethical Principles for Medical Research Involving Human Subjects 
 
Adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964 
and amended by the: 
29th WMA General Assembly, Tokyo, Japan, October 1975 
35th WMA General Assembly, Venice, Italy, October 1983 
41st WMA General Assembly, Hong Kong, September 1989 
48th WMA General Assembly, Somerset West, Republic of South Africa, October 1996 
52nd WMA General Assembly, Edinburgh, Scotland, October 2000  
53rd WMA General Assembly, Washington DC, USA, October 2002 (Note of Clarification added) 
55th WMA General Assembly, Tokyo, Japan, October 2004 (Note of Clarification added) 
59th WMA General Assembly, Seoul, Republic of Korea, October 2008 
64th WMA General Assembly, Fortaleza, Brazil, October 2013 
Preamble 
1.         The World Medical Association (WMA) has developed the Declaration of Helsinki as a statement 
of ethical principles for medical research involving human subjects, including research on identifiable 
human material and data. 
            The Declaration is intended to be read as a whole and each of its constituent paragraphs should 
be applied with consideration of all other relevant paragraphs. 
2.         Consistent with the mandate of the WMA, the Declaration is addressed primarily to physicians. 
The WMA encourages others who are involved in medical research involving human subjects to adopt 
these principles.  
 
General Principles 
3.         The Declaration of Geneva of the WMA binds the physician with the words, “The health of my 
patient will be my first consideration,” and the International Code of Medical Ethics declares that, “A 
physician shall act in the patient's best interest when providing medical care.” 
4.         It is the duty of the physician to promote and safeguard the health, well-being and rights of 
patients, including those who are involved in medical research. The physician's knowledge and 
conscience are dedicated to the fulfilment of this duty. 
5.         Medical progress is based on research that ultimately must include studies involving human 
subjects. 

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6.         The primary purpose of medical research involving human subjects is to understand the causes, 
development and effects of diseases and improve preventive, diagnostic and therapeutic interventions 
(methods, procedures and treatments). Even the best proven interventions must be evaluated 
continually through research for their safety, effectiveness, efficiency, accessibility and quality. 
7.         Medical research is subject to ethical standards that promote and ensure respect for all human 
subjects and protect their health and rights. 
8.         While the primary purpose of medical research is to generate new knowledge, this goal can never 
take precedence over the rights and interests of individual research subjects. 
9.         It is the duty of physicians who are involved in medical research to protect the life, health, dignity, 
integrity, right to self-determination, privacy, and confidentiality of personal information of research 
subjects. The responsibility for the protection of research subjects must always rest with the physician 
or other health care professionals and never with the research subjects, even though they have given 
consent. 
10.       Physicians must consider the ethical, legal and regulatory norms and standards for research 
involving human subjects in their own countries as well as applicable international norms and standards. 
No national or international ethical, legal or regulatory requirement should reduce or eliminate any of 
the protections for research subjects set forth in this Declaration. 
11.       Medical research should be conducted in a manner that minimises possible harm to the 
environment. 
12.       Medical research involving human subjects must be conducted only by individuals with the 
appropriate ethics and scientific education, training and qualifications. Research on patients or healthy 
volunteers requires the supervision of a competent and appropriately qualified physician or other health 
care professional. 
13.       Groups that are underrepresented in medical research should be provided appropriate access to 
participation in research. 
14.       Physicians who combine medical research with medical care should involve their patients in 
research only to the extent that this is justified by its potential preventive, diagnostic or therapeutic 
value and if the physician has good reason to believe that participation in the research study will not 
adversely affect the health of the patients who serve as research subjects. 
15.       Appropriate compensation and treatment for subjects who are harmed as a result of participating 
in research must be ensured. 
 
Risks, Burdens and Benefits  

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16.       In medical practice and in medical research, most interventions involve risks and burdens. 
           Medical research involving human subjects may only be conducted if the importance of the 
objective outweighs the risks and burdens to the research subjects. 
17.       All medical research involving human subjects must be preceded by careful assessment of 
predictable risks and burdens to the individuals and groups involved in the research in comparison with 
foreseeable benefits to them and to other individuals or groups affected by the condition under 
investigation. 
           Measures to minimise the risks must be implemented. The risks must be continuously monitored, 
assessed and documented by the researcher.  
18.       Physicians may not be involved in a research study involving human subjects unless they are 
confident that the risks have been adequately assessed and can be satisfactorily managed. 
           When the risks are found to outweigh the potential benefits or when there is conclusive proof of 
definitive outcomes, physicians must assess whether to continue, modify or immediately stop the 
study.            
 
Vulnerable Groups and Individuals 
19.       Some groups and individuals are particularly vulnerable and may have an increased likelihood of 
being wronged or of incurring additional harm. 
           All vulnerable groups and individuals should receive specifically considered protection. 
20.       Medical research with a vulnerable group is only justified if the research is responsive to the 
health needs or priorities of this group and the research cannot be carried out in a non-vulnerable group. 
In addition, this group should stand to benefit from the knowledge, practices or interventions that result 
from the research. 
 
Scientific Requirements and Research Protocols  
21.       Medical research involving human subjects must conform to generally accepted scientific 
principles, be based on a thorough knowledge of the scientific literature, other relevant sources of 
information, and adequate laboratory and, as appropriate, animal experimentation. The welfare of 
animals used for research must be respected. 
22.       The design and performance of each research study involving human subjects must be clearly 
described and justified in a research protocol. 
           The protocol should contain a statement of the ethical considerations involved and should indicate 
how the principles in this Declaration have been addressed. The protocol should include information 

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regarding funding, sponsors, institutional affiliations, potential conflicts of interest, incentives for 
subjects and information regarding provisions for treating and/or compensating subjects who are 
harmed as a consequence of participation in the research study. 
           In clinical trials, the protocol must also describe appropriate arrangements for post-trial provisions. 
 
Research Ethics Committees 
23.       The research protocol must be submitted for consideration, comment, guidance and approval to 
the concerned research ethics committee before the study begins. This committee must be transparent 
in its functioning, must be independent of the researcher, the sponsor and any other undue influence 
and must be duly qualified. It must take into consideration the laws and regulations of the country or 
countries in which the research is to be performed as well as applicable international norms and 
standards but these must not be allowed to reduce or eliminate any of the protections for research 
subjects set forth in this Declaration.  
           The committee must have the right to monitor ongoing studies. The researcher must provide 
monitoring information to the committee, especially information about any serious adverse events. No 
amendment to the protocol may be made without consideration and approval by the committee. After 
the end of the study, the researchers must submit a final report to the committee containing a summary 
of the study’s findings and conclusions.  
 
Privacy and Confidentiality  
24.       Every precaution must be taken to protect the privacy of research subjects and the confidentiality 
of their personal information. 
 
Informed Consent  
25.       Participation by individuals capable of giving informed consent as subjects in medical research 
must be voluntary. Although it may be appropriate to consult family members or community leaders, 
no individual capable of giving informed consent may be enrolled in a research study unless he or she 
freely agrees. 
26.       In medical research involving human subjects capable of giving informed consent, each potential 
subject must be adequately informed of the aims, methods, sources of funding, any possible conflicts of 
interest, institutional affiliations of the researcher, the anticipated benefits and potential risks of the 
study and the discomfort it may entail, post-study provisions and any other relevant aspects of the study. 
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consent to participate at any time without reprisal. Special attention should be given to the specific 
information needs of individual potential subjects as well as to the methods used to deliver the 
information. 
           After ensuring that the potential subject has understood the information, the physician or another 
appropriately qualified individual must then seek the potential subject’s freely-given informed consent, 
preferably in writing. If the consent cannot be expressed in writing, the non-written consent must be 
formally documented and witnessed.       
           All medical research subjects should be given the option of being informed about the general 
outcome and results of the study. 
27.       When seeking informed consent for participation in a research study the physician must be 
particularly cautious if the potential subject is in a dependent relationship with the physician or may 
consent under duress. In such situations the informed consent must be sought by an appropriately 
qualified individual who is completely independent of this relationship. 
28.       For a potential research subject who is incapable of giving informed consent, the physician must 
seek informed consent from the legally authorised representative. These individuals must not be 
included in a research study that has no likelihood of benefit for them unless it is intended to promote 
the health of the group represented by the potential subject, the research cannot instead be performed 
with persons capable of providing informed consent, and the research entails only minimal risk and 
minimal burden. 
29.       When a potential research subject who is deemed incapable of giving informed consent is able 
to give assent to decisions about participation in research, the physician must seek that assent in 
addition to the consent of the legally authorised representative. The potential subject’s dissent should 
be respected. 
30.       Research involving subjects who are physically or mentally incapable of giving consent, for 
example, unconscious patients, may be done only if the physical or mental condition that prevents giving 
informed consent is a necessary characteristic of the research  group. In such circumstances the 
physician must seek informed consent from the legally authorised representative. If no such 
representative is available and if the research cannot be delayed, the study may proceed without 
informed consent provided that the specific reasons for involving subjects with a condition that renders 
them unable to give informed consent have been stated in the research protocol and the study has been 
approved by a research ethics committee. Consent to remain in the research must be obtained as soon 
as possible from the subject or a legally authorised representative. 

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31.       The physician must fully inform the patient which aspects of their care are related to the research. 
The refusal of a patient to participate in a study or the patient’s decision to withdraw from the study 
must never adversely affect the patient-physician relationship. 
32.       For medical research using identifiable human material or data, such as research on material or 
data contained in biobanks or similar repositories, physicians must seek informed consent for its 
collection, storage and/or reuse. There may be exceptional situations where consent would be 
impossible or impracticable to obtain for such research. In such situations the research may be done 
only after consideration and approval of a research ethics committee. 
 
Use of Placebo 
33.       The benefits, risks, burdens and effectiveness of a new intervention must be tested against those 
of the best proven intervention(s), except in the following circumstances: 
           Where no proven intervention exists, the use of placebo, or no intervention, is acceptable; or 
           Where for compelling and scientifically sound methodological reasons the use of any intervention 
less effective than the best proven one, the use of placebo, or no intervention is necessary to determine 
the efficacy or safety of an intervention and the patients who receive any intervention less effective than 
the best proven one, placebo, or no intervention will not be subject to additional risks of serious or 
irreversible harm as a result of not receiving the best proven intervention.  
           Extreme care must be taken to avoid abuse of this option. 
 
Post-Trial Provisions 
34.       In advance of a clinical trial, sponsors, researchers and host country governments should make 
provisions for post-trial access for all participants who still need an intervention identified as beneficial 
in the trial. This information must also be disclosed to participants during the informed consent process. 
 
Research Registration and Publication and Dissemination of Results 
35.       Every research study involving human subjects must be registered in a publicly accessible 
database before recruitment of the first subject. 
36.       Researchers, authors, sponsors, editors and publishers all have ethical obligations with regard to 
the publication and dissemination of the results of research. Researchers have a duty to make publicly 
available the results of their research on human subjects and are accountable for the completeness and 
accuracy of their reports. All parties should adhere to accepted guidelines for ethical reporting. Negative 
and inconclusive as well as positive results must be published or otherwise made publicly available. 

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Sources of funding, institutional affiliations and conflicts of interest must be declared in the publication. 
Reports of research not in accordance with the principles of this Declaration should not be accepted for 
publication. 
 
Unproven Interventions in Clinical Practice 
37.       In the treatment of an individual patient, where proven interventions do not exist or other known 
interventions have been ineffective, the physician, after seeking expert advice, with informed consent 
from the patient or a legally authorised representative, may use an unproven intervention if in the 
physician's judgement it offers hope of saving life, re-establishing health or alleviating suffering. This 
intervention should subsequently be made the object of research, designed to evaluate its safety and 
efficacy. In all cases, new information must be recorded and, where appropriate, made publicly 
available.