Multicentric Clinical Investigation on the Use of Single-dose Ophthalmic Solution Based on Hyaluronate Sodium Phosphate in the Treatment of Eye Discomfort in Particular in Case of Ocular Dryness
Conditions: Ocular Dryness
Sponsor: SIFI SpA

Page 1 of 64 
Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
 
 
 
 
 
Clinical Investigation Plan 
 
 
 
 
“MULTICENTRIC CLINICAL INVESTIGATION ON THE USE OF 
SINGLE-DOSE OPHTHALMIC SOLUTION BASED ON SODIUM 
HYALURONATE IN THE TREATMENT OF EYE DISCOMFORT 
IN PARTICULAR IN CASE OF OCULAR DRYNESS” 
 
Version 2 March 14th, 2023 
 
 
STUDY CODE: 052/SI Hyalistil Bio PF Mono 
 
Post Market Clinical Follow up (PMCF)  
(Observational Study-Profit) 
 
 
 
 
 
 
 
 
Confidentiality statement 
 
This document contains information that is confidential and proprietary to SIFI SpA. This information is being provided to you solely for the 
purpose of evaluation and/or conducting a clinical trial for SIFI SpA. You may disclose the contents of this document only to study personnel 
under your supervision or to your Ethics Committee who need to know the contents for this purpose: The contents of this document may not be 
used in any other clinical trial and may not be disclosed to any other person or entity without the prior written permission of SIFI SpA. The 
foregoing shall not apply to disclosure required by governmental regulations or laws. 

Page 2 of 64 
Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
Clinical Investigation Plan Status: FINAL 
Clinical Investigation Plan No.: 
052/SI Hyalistil Bio PF Mono 
Study Type: Post Market Clinical Follow up (PMCF) (Prospective, observational, non-
interventional clinical investigation) 
Products Name: 
Hyalistil Bio PF 
Date of Issue: 
March 14th, 2023 
Amendment: 052/SI Hyalistil Bio PF Mono/EM version 1 of March 14th,2023 
Study Title 
Multicentric clinical investigation on the use of single-dose ophthalmic solution based on 
sodium hyaluronate in the treatment of eye discomfort in particular in case of ocular dryness 
Sponsor 
SIFI SpA  
Via Ercole Patti 36 
95025 Aci S. Antonio (CT) – (Italy) 
Dr. Fabrizio Chines 
Tel: +39 0957922136 
Mob: +39 3389472159 
Email: fabrizio.chines@sifigroup.com 
Contract Research Organization  
UNIFARM Research Centre 
Viale Andrea Doria 21  
95125 Catania (Italy) 
Prof. Gian Marco Leggio 
Tel: +39 095 7167514 
Fax: +39 095 7167514 
Email: direzione@unifarm.org 
Principal Investigators 
 
Dr. Davide Scollo 
Oculistica, P.O. San Marco, Azienda Ospedaliero Universitaria Policlinico  
"G.Rodolico - San Marco"  
Viale Carlo Azeglio Ciampi, 95121 Catania (Italy) 
Tel: +39 095378121 
Email: davidescollo@hotmail.com 
 
 Prof. Vincenza Maria Elena Bonfiglio 
U.O. di Oculistica, Presidio Belmonte, Azienda Ospedaliero Universitaria Policlinico  
“Paolo Giaccone”  
Via Rampolla Mariano Cardinale, 10B, 90142 Palermo (Italy) 
Tel: +39 091 6553903  
Email: enzabonfiglio@gmail.com 
 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
Study Sites:  
1) P.O. San Marco, Azienda Ospedaliero Universitaria Policlinico "G. Rodolico - San 
Marco" Oculistica,  
Viale Carlo Azeglio Ciampi, 95121 Catania (Italy) 
Tel: +39 0953781213 
Email:davidescollo@hotmail.com 
 
2) U.O. di Oculistica, Presidio Belmonte, Azienda Ospedaliero Universitaria Policlinico  
“Paolo Giaccone”  
Via Rampolla Mariano Cardinale, 10B, 90142 Palermo (Italy) 
Email: enzabonfiglio@gmail.com 
 
 
 
 
 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
APPROVAL SHEET   
I, here undersigned, approve the content of the Clinical Investigational Plan. 
 
SPONSOR REPRESENTATIVE 
Dr. Fabrizio Chines 
SIFI S.p.A. 
95025 Aci S. Antonio (CT) – (Italy) 
Tel: +39 0957922136 
Mob: +39 3389472159 
Email: fabrizio.chines@sifigroup.com 
                          
_____________________________________________  
Signature Date 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
APPROVAL SHEET   
I, here undersigned, approve the content of the Clinical Investigational Plan. 
 
CRO REPRESENTATIVE 
Prof. Gian Marco Leggio 
UNIFARM Research Centre 
Viale Andrea Doria 21 
95125 Catania (Italy) 
Tel: +39 095 7167514 
Fax: +39 095 7167514 
Email: direzione@unifarm.org  
______________________________________ 
         Signature Date 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
APPROVAL SHEET   
I, here undersigned, approve the content of the Clinical Investigational Plan. 
 
Dr. Davide Scollo 
Oculistica, P.O. San Marco,  
Azienda Ospedaliero Universitaria Policlinico  
"G.Rodolico - San Marco"  
Viale Carlo Azeglio Ciampi,  
95121 Catania (Italy) 
Tel: +39 0953781213 
       Email: davidescollo@hotmail.com 
 
                          
_________________________________________  
Signature Date 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
 
APPROVAL SHEET   
I, here undersigned, approve the content of the Clinical Investigational Plan. 
 
Prof. Vincenza Maria Elena Bonfiglio 
U.O. di Oculistica, 
Presidio Belmonte,  
Azienda Ospedaliero Universitaria Policlinico  
“Paolo Giaccone”  
Via Rampolla Mariano Cardinale,  
10B, 90142 Palermo (Italy) 
Tel: +39 091 6553903 
Email: enzabonfiglio@gmail.com 
 
                                                                              
__________________________________________  
  Signature Date 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
 
APPROVAL SHEET   
I, here undersigned, approve the content of the Clinical Investigational Plan. 
 
 
RESPONSIBLE FOR STATISTICS  
Dr. Filippo Palermo 
UNIFARM Research Centre 
Viale Andrea Doria 21 
95125 Catania (Italy) 
Tel: +39 095 7167514 
Fax: +39 095 7167514 
Email: filippo.palermo@unifarm.org 
                          
____________________________________________  
Signature Date 
 
 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
 
INVESTIGATOR SIGNATURE 
 
I have read and understood this Clinical Investigation Plan. I will conduct the study in 
accordance with all applicable national and regional laws and regulations and will strictly 
follow the study procedures.  
I agree to provide all the information requested in the electronic Case Report Forms. 
I also agree that all information provided to me by the Sponsor (or Sponsor’s designees), 
including clinical data, clinical investigation plans, Case Report Forms, and any verbal and 
written information, will be kept strictly confidential. It is recognized that this information 
may be relayed in confidence to the Ethics Committee or regulatory authorities. 
In addition, no reports or information about this observational study will be provided to anyone 
not involved in the study other than the Sponsor (or Sponsor’s designees), the Ethics 
Committee(s) or regulatory authorities. Any such submission will indicate that the material is 
confidential. 
 
Dr. Davide Scollo 
Oculistica, P.O. San Marco,  
Azienda Ospedaliero Universitaria Policlinico  
"G. Rodolico - San Marco"  
Viale Carlo Azeglio Ciampi,  
95121 Catania (Italy) 
Tel: +39 0953781213 
       Email: davidescollo@hotmail.com 
 
 
_________________________________________  
Signature Date

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
 
INVESTIGATOR SIGNATURE 
 
I have read and understood this Clinical Investigation Plan. I will conduct the study in 
accordance with all applicable national and regional laws and regulations and will strictly 
follow the study procedures.  
I agree to provide all the information requested in the electronic Case Report Forms. 
I also agree that all information provided to me by the Sponsor (or Sponsor’s designees), 
including clinical data, clinical investigation plans, Case Report Forms, and any verbal and 
written information, will be kept strictly confidential. It is recognized that this information 
may be relayed in confidence to the Ethics Committee or regulatory authorities. 
In addition, no reports or information about this observational study will be provided to anyone 
not involved in the study other than the Sponsor (or Sponsor’s designees), the Ethics 
Committee(s) or regulatory authorities. Any such submission will indicate that the material is 
confidential. 
 
Prof. Vincenza Maria Elena Bonfiglio 
U.O. di Oculistica, 
Presidio Belmonte,  
Azienda Ospedaliero Universitaria Policlinico  
“Paolo Giaccone”  
Via Rampolla Mariano Cardinale,  
10B, 90142 Palermo (Italy) 
Tel: +39 091 6553903 
Email: enzabonfiglio@gmail.com 
 
              __________________________________________  
  Signature Date 
 
 
 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
 
CHANGE HISTORY RECORD 
 
Clinical 
Investigation 
Plan 
version 
Clinical 
Investigation 
Plan 
date 
Description of 
changes 
 
Amendment 
version 
Amendment 
date 
1 
October 10th 2022 
First release  
- 
- 
2 
March 14th, 2023 
Change on: 
Primary objective 
Statistical Analysis 
Number of Box to delivery 
Procedures 
 
      1 
March 14th, 2023 
  
 
 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
 
CLINICAL INVESTIGATION PLAN SYNOPSIS 
 
TITLE 
Multicentric clinical investigation on the use of single-dose ophthalmic 
solution based on sodium hyaluronate in the treatment of eye discomfort 
in particular in case of ocular dryness 
CLINICAL 
INVESTIGATION 
CODES 
052/SI Hyalistil Bio PF Mono 
AMENDMENT 
052/SI Hyalistil Bio PF Mono/EM version 1 of March 14th,2023 
AIM OF THE 
CLINICAL 
INVESTIGATION   
To assess the performance and safety resulting from the treatment of 
an ophthalmic solution for the ocular discomfort and in particular in 
presence of ocular dryness. 
CLINICAL 
INVESTIGATION 
SITES 
1) P.O. San Marco, Azienda Ospedaliero Universitaria Policlinico  
  "G. Rodolico - San Marco" Oculistica,  
Viale Carlo Azeglio Ciampi, 95121 Catania (Italy) 
Dr. Davide Scollo 
Tel: +39 0953781213 
Email: davidescollo@hotmail.com  
2) U.O. di Oculistica, Presidio Belmonte, Azienda Ospedaliero 
Universitaria Policlinico “Paolo Giaccone”  
Via Rampolla Mariano Cardinale,  
10B, 90142 Palermo (Italy) 
Prof. Vincenza Bonfiglio  
Tel: +39 091 6553903 
Email: enzabonfiglio@gmail.com 
 
CLINICAL 
INVESTIGATION 
PHASE 
Post Market Clinical Follow up (PMCF) (Investigator sponsored per 
profit, prospective, observational, non-interventional clinical 
investigation) 
 
CLINICAL 
INVESTIGATION   
PERIOD 
Approximately 4 months from first patient first visit (FPFV) to last 
patient last visit (LPLV)  
Each patient enrolled will be treated for 35± 4 days. 
Enrollment period: 3 months. 
 
CLINICAL 
INVESTIGATION 
DESIGN 
Multicenter, prospective, observational, open-label, non- interventional 
clinical investigation evaluating the performance and safety of 4 daily 
instillations of the ophthalmic solution in the treatment of mild irritation 
of the ocular surface. 
 
CLINICAL 
28 male and female subjects with signs and symptoms of ocular 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
INVESTIGATION 
POPULATION 
discomfort and ocular irritation resulting from ocular dryness who 
qualifies for Hyalistil Bio PF treatment. 
NUMBER OF 
SUBJECTS 
28 subjects recruited with competitive enrolment at the involved study 
sites. 
SUBJECT 
SELECTION 
CRITERIA 
 
 
 
 
 
 
 
 
 
Inclusion criteria 
1. Subjects (male or female) must be ≥ 18 years of age; 
2. Subject able to provide Informed Consent, in compliance with the 
good clinical practice and local laws (thus, subject able to 
comprehend the full nature and purpose of the study, including 
possible risks and side events); 
3. Subjects with ocular discomfort resulting from mild to moderate dry 
eye in one or both eyes determined by: 
- Scoring of ocular surface staining with fluorescein using the 
National Eye Institute (NEI) scale. Total score per single eye 
range 6-33 summing the score of cornea and conjunctiva.  
(Considering a normal score of 0-33); 
- Tear film break-up time with fluorescein (TFBUT) ≤ 10 seconds; 
The TFBUT value will be recorded as the average of 3 
measurements; 
- Symptom Assessment in Dry Eye (SANDE) questionnaire ≥ 35. 
4. Subject able to be compliant with the requirements of the clinical 
investigation plan, according to the Investigator; 
5. Subject who qualifies for Hyalistil Bio PF treatment according to the 
approved indication; 
6. Subject who in physician's opinion will benefit from this treatment. 
 
 Exclusion criteria 
1. Corneal injuries or abrasions of traumatic origin in the eye of study; 
2. Ocular infection or clinically significant inflammation (such as 
Herpes Simplex infection, corneal virus infection, bacterial, viral or 
fungal conjunctivitis, tuberculosis and mycosis of the eye, purulent 
and herpetic blepharitis, stye); 
3.  Sjögren's syndrome; 
4. Stevens-Johnson syndrome;  
5. Systemic lupus erythematosus; 
6. Pathologies associated with corneal thinning; 
7. Taking drugs that may interfere with tear gland secretion (beta -
blockers); 
8. Patients using any topical therapies such as non-steroidal anti-
inflammatory drugs, cortisone, cyclosporine, vasoconstrictor, 
artificial tears (different than the investigated product) the eye of 
study;  

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
9. Treatment with topical or systemic corticosteroids in the 4 weeks 
preceding the study; 
10. 
Known or potential hypersensitivity and/or history of allergic 
reactions to one of the components of the topical medical device. 
11. Participation in another clinical trial within the previous 30 days; 
12. Evidence of severe or uncontrolled systemic disease or any other 
significant disorders, which in the opinion of the Investigator does 
not allow the participation in the study or could compromise the 
results. 
CLINICAL 
INVESTIGATION 
OBJECTIVES 
  PRIMARY OBJECTIVE 
• Evaluation of Tear film break-up time with fluorescein (TFBUT) 
at Study Termination Visit (Day 35 ± 4 of treatment) compared to 
Visit 1 (Day 0 of treatment - baseline).  
 SECONDARY OBJECTIVE 
• Evaluation of the change in total score (score from 6 to 33, 
considering a normal score 0-33) resulting from the sum of the 
corneal staining score (score from 0 to 15) and conjunctival 
staining score (score from 0 to 18) with fluorescein using the 
National Eye Institute Scale (NEI) at Visit 2 (Day 14 ± 2 of 
treatment) and at the Study Termination Visit (Day 35 ± 4 of 
treatment) compared to Visit 1 (Day 0 of treatment - baseline); 
• To evaluate the tear film stability per group as objectified by the 
tear break up time with fluorescein (TFBUT) at Study 
Termination Visit (Day 35 ± 4 of treatment) compared to Visit 2 
(Day 14 ± 2 of treatment). 
• Changes about Best Corrected Visual Acuity (BCVA) measured 
by the “Early Treatment Diabetic Retinopathy Study” (ETDRS) 
at Visit 2 (Day 14 ± 2 of treatment) and Study Termination Visit 
(Day 35 ± 4 of treatment) compared to Visit 1(Day 0 of treatment – 
baseline). 
• Patient reported outcomes:  
To compare patients reported outcomes (PRO) measures per 
group, including:  
- Patient’s reported symptoms (SANDE) at Visit 2 (Day 14 ± 2 of 
treatment) and Study Termination Visit (Day 35 ± 4 of treatment) 
compared to Visit 1(Day 0 of treatment – baseline). 
- Assessment of the quality of life (QOL) by “Questionnaire 
about Eye Symptoms and Daily Life” (DEQS) at Study 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
Termination Visit (Day 35 ± 4 of treatment) compared to Visit 1 
(Day 0 of treatment - baseline).  
 - Evaluation of the degree of satisfaction to the treatment 
reported by patients through the use of the visual analogue scale 
(VAS) , at the Study Termination Visit (Day 35 ± 4 of treatment). 
In the event that, for both trial centers, the DEQS questionnaire is not 
available, the assessment of the quality of the life will be performed by the 
DEQ-5 questionnaire. 
SAFETY EVALUATIONS 
• Evaluation of the safety and the compliance of medical device.  
 
ENDPOINTS^ 
PRIMARY ENDPOINTS  
• Clinical performance: 
- 
25 30% increase from Visit 1 (Day 0 of treatment - baseline) to 
Study Termination Visit (Day 35 ± 4 of treatment) of tear film 
break-up time with fluorescein (TFBUT). [Time frame: 
Visit 1 and Study Termination Visit]. 
 
SECONDARY ENDPOINTS 
• Reduction from baseline (Visit 1- Day 0 of treatment) in the corneal and 
conjunctival fluorescein staining score using the NEI scale.  
Fluorescein staining of the ocular surface is assessed as the total sum 
of the corneal and conjunctival subregions.  
- Corneal Fluorescein Staining (NEI score, sum of 5 subregions, 
maximum score 15. Each subregion will have a maximum score of 3.  
(0=no staining, 3 = maximum staining).  
- Conjunctival Fluorescein Staining (NEI score, sum of 6 subregions, 
maximum score 18. Each subregion will have a maximum score of 3.  
(0=no staining, 3 = maximum staining).  
    [Time frame: Visit 1, Visit 2 and Study Termination Visit].  
• Tear film break-up time with fluorescein (TFBUT)
ϕ. [Time frame: 
Visit 2 and Study Termination Visit].  
•  BCVA is measured using the standard Early Treatment Diabetic 
Retinopathy Study (ETDRS) acuity chart and reported in ETDRS 
letters. [Time frame: Visit 1, Visit 2 and Study Termination Visit]; 
• Patient reported outcomes: 
- Change in intensity and frequency of dry eye symptoms assessed by 
completing the questionnaire SANDE. [Time frame: Visit 1, Visit 2 
and Study Termination Visit].  
- Evaluation of the degree of satisfaction to the treatment reported by 
patients through the use of the visual analogue scale (VAS) , 
ranging from 0 to 100 mm, at Study Termination Visit (Day 35 ± 4 of 

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Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
treatment). [Time frame: Study Termination Visit]. 
Patients will be asked to rate their degree of satisfaction to the 
treatment and answer the following questions: 
• “I feel satisfied using this treatment? '',  
• “With this treatment, I have a feeling of freshness?”, 
• “With this treatment, I have a feeling of relief?”, 
• “This treatment contributed to reduce my pain due to eye 
dryness?'', 
• “This treatment is comfortable? '', 
 Patients will be asked to place a vertical mark on a horizontal line, 
from 0 to100 mm, to indicate the degree of satisfaction to the 
treatment, where: 
✓ 0-10 mm=none  
✓ 11-30 mm=very mild 
✓ 31-50 mm=mild 
✓ 51-70 mm=moderate 
✓ 71-90 mm=strong 
✓ 91-100 mm=very strong. 
-Assessment of the quality of life (QOL) by “Questionnaire about 
Eye Symptoms and Daily Life” (DEQS) at Study Termination Visit 
(Day 35 ± 4 of treatment) compared to Visit 1 (Day 0 of treatment - 
baseline). [Time frame: Visit 1 and Study Termination Visit]. 
In the event that, for both trial centers, the DEQS questionnaire is not 
available, the assessment of the quality of the life will be performed by the 
DEQ-5 questionnaire. 
 
EVALUATION OF SAFETY PARAMETERS 
✓ Evaluation of safety of daily instillation of ophthalmic solution during all the 
study period through:  
• Investigator Global Assessment of Safety (IGAS): using the 4-
point scale:1= very good safety, 2 =good safety, 3 = moderate 
safety and 4 = poor safety. IGAS will be evaluated at the Study 
Termination Visit. [ Time Frame: Study Termination Visit];  
• Evaluation of reported adverse events/incidents. [Time Frame: 
During the treatment period].  
• Evaluation of intraocular pressure (IOP). [Time frame: Visit 1, 
Visit 2 and Study Termination Visit];  
• Evaluation of compliance through verification of correct 
instillation of the medical device, counting of single dose 
containers and boxes. [Time Frame: Visit 2 and Study 
Termination Visit].  
^According to clinical practice, the Investigator will perform all clinical 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
evaluations on both eyes of the individual patient. 
In the case that both eyes are affected by ocular dry eye, the statistical 
analysis will be performed considering the eye with the lowest level of 
TFBUT score “worse eye”. If the level of TFBUT score at baseline is the 
same in both eyes, then the eye with the highest level of NEI score will 
be designated as the “worse eye.” 
If the level of NEI score at baseline is the same in both eyes, then the right 
eye will be designated as the “worse eye.” 
 
The primary end point will be evaluated on the PP population and also 
in ITT population. 
STATISTICS 
Sample size estimate 
A previous study report baseline level of tear film break-up time with 
fluorescein (TFBUT) of 6.23±1.75 at baseline, to detect a 25  increase 
of TFBUT at the Study Termination Visit compared to baseline (Visit 
1) (mean difference 1,56, sd 1.91), with an of alpha=0.01(two-tailed) 
and a power (1 – ß) of 0.80, 23 subjects are requires (Wilcoxon signed-
rank test). Considering a dropout of 20%, it is expected to enroll a total 
of 28 subjects. 
 
Statistical analysis 
Descriptive analysis will be performed by using absolute rate, 
percentage and frequency tables for qualitative variables. 
Normally distributed data will be reported as minimum, maximum, 
means and standard deviations, whereas non- normally distributed data 
as medians and interquartile range (IQR). 
To compare qualitative variables for not-paired data will be used the 
Chi-square test with Yates corrections or Fisher's exact test when 
appropriate and the Mc Nemar's test for paired data. 
Differences in endpoints between baseline and different timepoints will 
be assessed using paired t-test for normally distributed data, whereas the 
Wilcoxon signed-rank test will be used for non- normally distributed 
data. 
Data distribution will be tested by using the Kolmogorov-Smirnov test. 
A p-value <0.05 will be considered statistically significant. 
All the Analysis will be performed using SAS version 9.4 for Windows 
(SAS Institute Inc., Cary, NC, USA). 
Analysis set 
All patients enrolled will be included in the safety population. 
All patients enrolled having a primary post-baseline -performance 
assessment will be included in the ITT population. 
If the primary performance measurement at Visit 2 is missing, it will be 
imputed with the score at baseline. If the primary performance 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
measurement at Study Termination Visit is missing, it will be imputed 
with the score at Visit 2, if it is not missing, or otherwise with the score 
at baseline.  
All patients of the ITT population with any major clinical investigation 
plan deviation will be included in the PP population. 
The analysis of safety endpoints will be performed in the safety 
population.  
Analysis of performance endpoints will be performed on the ITT 
population. The analysis of the primary endpoint will be performed on 
the PP population but will be also repeated in the ITT population. 
MEDICAL DEVICE 
Hyalistil Bio PF  
ROUTE OF 
ADMINISTRATION 
AND DOSAGE  
 
1 drop of Hyalistil Bio PF in the conjunctival fornix, 4 times a day. 
Hyalistil Bio PF can also be used by contact lens wearers. 
 It is not necessary to remove contact lenses before administering 
Hyalistil Bio PF. 
Moreover, advised not to administer other drugs or medical devices for 
ocular use within 10 minutes from the instillation of Hyalistil Bio PF 
INVASIVE 
PROCEDURES 
 
There are no invasive techniques outside of normal clinical practice for 
the purposes of the study. Regarding Tear Film Break-Up Time test with 
fluorescein (TFBUT), fluorescein staining is an invasive procedure and 
is normally used in clinical practice in the assessment of ocular 
discomfort due to dry eye. 
CHRONOGRAM OF 
VISITS AND 
EVALUATIONS  
 
Visit 1 (screening-enrolment)  
The following procedures will be performed:  
- 
 Explanation to the patient of study aims, procedures and possible 
risks of the observational study; 
- 
 Informed consent signature; 
- 
 Allocation screening number; 
 Demographic data collection 
- 
In case of female patients (collection of information on pregnancy 
or breastfeeding self-reported by the patient); 
- 
 Medical and surgical history/current medical conditions; 
- 
 Prior/concomitant ocular and systemic medications; 
- 
 Ocular examination of both eyes: 
o External Ocular Examination; 
o BCVA test through the ETDRS at 4 m distance; 
o Symptom Assessment in Dry Eye by SANDE questionnaire; 
o Evaluation of Intraocular pressure (IOP); 
o Tear film break-up time with fluorescein (TFBUT);  
o Ocular surface staining (NEI score) with fluorescein; 

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Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
- DEQS questionnaire administration; 
- 
Patient eligibility: inclusion/exclusion criteria evaluation; 
- 
Assignment of the study and allocation subject clinical 
investigational number;  
- 
Generation of unique subject identifiers code*; 
- 
Medical device dispensation; 
- 
Beginning of the treatment*. 
 
The Investigator will dispense to the patients 5 boxes of medical device 
each containing 30 single-dose containers of medical device each 
containing 30 single-dose containers for the following 35±4 days. After 
completing the baseline evaluation, patients will start the study 
treatment as instructed through self-instillation of medical device under 
investigation at home*.  
Patients will return to the clinical site on day 14±2 from the first 
instillation of medical device under investigation (Visit 2). 
Visit 2 (on the 14th day from the first instillation of medical device 
under investigation (MD)) (window ± 2 days)*** 
The following procedures will be performed:  
- 
Assessment of treatment compliance; 
- 
Current medical conditions; 
- 
Concomitant medications; 
- 
Evaluation of any reported adverse events /incidents**; 
- 
Ocular examination of both eyes: 
o External Ocular Examination; 
o BCVA test through the ETDRS at 4 m distance; 
o Symptom Assessment in Dry Eye by SANDE questionnaire; 
o Evaluation of Intraocular pressure (IOP); 
o Tear film break-up time with fluorescein (TFBUT);  
o Ocular surface staining (NEI score) with fluorescein; 
Patients will return to the clinical site on day 35±4 days from the first 
instillation of medical device under investigation (Study Termination 
Visit). 
Study Termination Visit (on the 35th day from the first instillation 
of medical device under investigation) (window ± 4 days) 
The final visit is defined as the visit performed on 35 ± 4 days after the 
first instillation of medical device under investigation°. 
In case of premature study discontinuation, patients will undergo 
an Early Termination Visit (ETV)'. 
The following procedures will be performed: 
- 
Assessment of treatment compliance (reconciliation of the 
amount of used single dose containers and empty/unused medical 

Page 20 of 64 
Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
device boxes returned by patients to the centre)§; 
- 
Current medical conditions; 
- 
Concomitant medications; 
- 
Evaluation of any reported adverse events/incidents**; 
- 
Ocular examination of both eyes: 
o External Ocular Examination; 
o BCVA test through the ETDRS at 4 m distance; 
o Symptom 
Assessment 
in 
Dry 
Eye 
by 
SANDE 
questionnaire; 
o Evaluation of Intraocular pressure (IOP); 
o Tear film break-up time with fluorescein (TFBUT); 
o Ocular surface staining (NEI score) with fluorescein; 
- Administration of the VAS scale on the degree of satisfaction to 
the treatment; 
- DEQS questionnaire administration; 
- Evaluation of the safety of the experimental product through the 
compilation of the IGAS scale; 
- Restitution of remaining medical devices§. 
* Start of the treatment (window + 1 day). 
** In case of adverse events/incidents all assessments considered necessary by the 
Investigator will be performed, including the possible suspension of treatment. 
*** Variable because it is based on the planned visit schedule according to clinical 
practice for the individual patient Visit 2 (windows of ± 2 days). 
° All patients enrolled will be treated for 35± 4 days.  The Study Termination Visit is 
variable because it is based on the planned visit schedule according to clinical practice 
for the individual patient (window ± 4 days).  
§ Restitution of the amount of used single dose containers and empty/unused medical 
device boxes returned by patients to the centre. 
'In case of premature study discontinuation, patients will undergo an Early 
Termination Visit (ETV). During the ETV the patients will return the amount of used 
single dose containers and empty/unused medical device boxes returned by patients to 
the centre. 
* The “unique subject identifiers code” consists of the 5-digit screening number (e.g. 
S1001, S1002,….S1028 etc.), and, if applicable, the 4-digit subject clinical 
investigational number (e.g. 1001, 1002,…1028 etc.). Specifically, the first subject 
clinical investigational number was referred to the study site. 
Allocation screening number and subject clinical investigational number are separated 
by slashes (e.g. “S1001/1001”). (e.g. “S1001/1001” for site number 1, “S2001/2001” 
for site number 2). 
 The TFBUT value will be recorded as the average of 3 measurements. 

Page 21 of 64 
Hyalistil Bio PF Mono 
Medical device 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n. 2 dated March 14th, 2023 
 
 
ORGANIZATION 
DELEGATED TO 
CONDUCTION THE 
CLINICAL 
INVESTIGATION   
CRO (Contract 
Research 
Organization) 
Unifarm Research Centre 
Viale Andrea Doria 21, 
95125 Catania (Italy) 
Tel: +39 095 7167514 
Fax: +39 095 7167514 
Email:  direzione@unifarm.org 
Prof. Gian Marco Leggio 
ETHICAL 
COMMITTEE  
 
Comitato Etico “Catania 1” 
Azienda Ospedaliero Universitaria Policlinico "G. Rodolico - San 
Marco", Via S. Sofia, 78 - 95123 Catania 
Comitato Etico Palermo 1 
Azienda Ospedaliero-Universitaria Policlinico “Paolo Giaccone” 
Via del Vespro, 129 
90127 Palermo 

Page 22 of 64 
Version n. 2 March 14th, 2023 
Hyalistil Bio PF  
Medical device 
   Study Code: 052/SI Hyalistil Bio PF Mono 
 
 
 
 
FLOW CHART 
 
Study Procedure 
Visit 1 
(screening-
enrolment- start of 
treatment)* 
Visit 2*** 
(on the 14th day from the 
first instillation of 
medical device) 
(window ± 2 days) 
Study termination 
Visit° 
(on the 35th day from 
the first instillation of 
medical device) 
(window ± 4 days) 
Informed Consent 
√ 
 
 
Allocation screening number   
√ 
 
 
Demographic data collection 
√ 
 
 
Medical and surgical history 
√ 
 
 
Current medical conditions 
√ 
√ 
√ 
Prior ocular local and systemic medication 
√ 
 
 
Concomitant ocular local and systemic 
medication 
 
√ 
√ 
External Ocular Examination 
√ 
√ 
√ 
BCVA evaluation through ETDRS grade 
√ 
√ 
√ 
SANDE questionnaire 
√ 
√ 
√ 
Evaluation of Intraocular pressure (IOP) 
√ 
√ 
√ 
Ocular surface staining 
(Fluorescein- NEI score) 
√ 
√ 
√ 
Tear Film Break-up Time 
(TFBUT- Fluorescein) 
√ 
√ 
√ 
Administration of DEQS questionnaire 
√ 
 
√ 
Inclusion/Exclusion Criteria 
 
√ 
 
 

Page 23 of 64 
Version n. 2 March 14th, 2023 
Hyalistil Bio PF  
Medical device 
   Study Code: 052/SI Hyalistil Bio PF Mono 
 
 
 
Enrolment and allocation subject clinical 
investigational number 
√ 
 
 
 
Generation of unique subject identifiers 
code* 
√ 
 
 
 
 
Medical device dispensing* 
√ 
 
 
Check Compliance (check proper 
treatment with medical device, use of 
other medication and medical devices) 
 
√ 
√ 
Medical device restitution§ ' 
 
 
√ 
Evaluation of satisfaction to the treatment 
by VAS scale 
 
 
√ 
Evaluation of IGAS 
 
 
√ 
Adverse event /Incidents** 
 
√ 
√ 
* Start of the treatment (window + 1 day). 
** In case of adverse events incidents all assessments considered necessary by the Investigator will be performed, including the possible suspension of treatment. 
*** Variable because it is based on the planned visit schedule according to clinical practice for the individual patient Visit 2 (windows of ± 2 days). 
° All patients enrolled will be treated for 35 ± 4 days.  The Study Termination Visit is variable because it is based on the planned visit schedule according to 
clinical practice for the individual patient (window ± 4 days).  
§ Restitution of the amount of used single dose containers and empty/unused medical device boxes returned by patients to the centre. 
'In case of premature study discontinuation, patients will undergo an Early Termination Visit (ETV). During the ETV the patients will return the amount of used 
single dose containers and empty/unused medical device boxes returned by patients to the centre. 
*The “unique subject identifiers code” consists of the 5-digit screening number (e.g. S1001, S1002,….S1028 etc.), and, if applicable, the 4-digit subject clinical 
investigational number (e.g. 1001, 1002,…1028 etc.). Specifically, the first subject clinical investigational number was referred to the study site. 
Allocation screening number and subject clinical investigational number are separated by slashes (e.g. “S1001/1001”). (e.g. “S1001/1001” for site number 1, 
“S2001/2001” for site number 2). 
 The TFBUT value will be recorded as the average of 3 measurements. 
In the event that, for both trial centers, the DEQS questionnaire is not available, the assessment of the quality of the life will be performed by the DEQ-5 questionnaire.

Hyalistil Bio PF  
Medical device 
 
 
Study code: 052/SI Hyalistil Bio PF Mono 
 
 
Page 24 of 64 
Version n. 2 dated March 14th, 2023 
 
 
 
LIST OF ABBREVIATION 
BVCA= Changes about Best Corrected Visual Acuity  
CA = Competent Authority 
CIP = Clinical Intestigational Plan  
CRF = Case Report Forms 
CRO = Contract Research Organization 
EC= Ethic Committee 
eCRF = electronic Case Report Form  
ETDRS = Early Treatment Diabetic Retinopathy Study 
ETV = Early Termination Visit  
FPFV = First Patient First Visit  
GCP = Good Clinical Practice 
HCE = Human Corneal Epithelial  
ICF = Informed Consent Form 
ICH = International Conference on Harmonization 
IMP =Investigational Medicinal Product  
IOP= Intraocular Pressure  
IS = Investigational Site 
LPLV = Last Patient Last Visit  
MD = Medical Device 
MedDRA= Medical Dictionary for Regulatory Activities  
NA = Not Applicable 
NEI = National Eye Institute  
PI = Principal Investigator 
PP = Per Clinical investigation plan Set  
PT = Preferred Term  
RA = Regulatory Authorities 
SAF = Safety Analysis Set  
SANDE = Symptom Assessment in Dry Eye  
SAP = Statistical Analysis Plan 
SOC= System / Organ class 
SOPs = Standard Operating Procedures 
TFBUT = Tear Film Break-Up Time  
TSP = Tamarind seed polysaccharide 
VAS = Visual Analog Scale 
 
 

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Page 25 of 64 
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TABLE OF CONTENTS 
APPROVAL SHEET ....................................................................................................................... 4 
APPROVAL SHEET ....................................................................................................................... 5 
APPROVAL SHEET ....................................................................................................................... 6 
APPROVAL SHEET ....................................................................................................................... 7 
APPROVAL SHEET ....................................................................................................................... 8 
INVESTIGATOR SIGNATURE ..................................................................................................... 9 
INVESTIGATOR SIGNATURE ................................................................................................... 10 
CHANGE HISTORY RECORD .................................................................................................... 11 
CLINICAL INVESTIGATION PLAN SYNOPSIS ...................................................................... 12 
FLOW CHART .............................................................................................................................. 22 
LIST OF ABBREVIATION .......................................................................................................... 24 
1. 
Investigator and Facility ......................................................................................................... 27 
1.1 
Principal Investigator and Clinical Research Facility .................................................... 27 
1.2 
Statistics ......................................................................................................................... 27 
1.3 
Quality Assurance Unit .................................................................................................. 28 
2. 
Rational and background ........................................................................................................ 29 
3. 
Aim of the Clinical Investigation ........................................................................................... 31 
4. 
Clinical Investigation Design ................................................................................................. 32 
5. 
Patient risk and benefit ........................................................................................................... 33 
6. 
Inclusion criteria ..................................................................................................................... 33 
7. 
Exclusion criteria .................................................................................................................... 33 
8. 
Subject withdrawal criteria ..................................................................................................... 34 
9. 
Screening Failure .................................................................................................................... 34 
10. Primary objective ................................................................................................................... 35 
11. Secondary objective ............................................................................................................... 35 
12. Clinical Investigation Scheme ................................................................................................ 36 
13. Ethical considerations and Informed Consent ........................................................................ 38 
14. Investigational medical device ............................................................................................... 38 
14.1 Description of product ........................................................................................................ 39 
14.2 Description as to how traceability shall be achieved during and after the clinical 
investigation ................................................................................................................... 41 
14.3 Details concerning the manufacturer of Medical Device ................................................... 41 
14.4 Name or number of the model/type to permit full identification ....................................... 41 
15. Investigational medical device storage and handling ............................................................. 41 
16. Investigational medical device accountability ....................................................................... 42 
17. Other allowed therapies .......................................................................................................... 42 
18. Procedures .............................................................................................................................. 43 
19. Primary and secondary outcomes^ ......................................................................................... 46 
20. Equipment to assess clinical investigation variables and primary and secondary outcomes . 47 
21. Statements of compliance ....................................................................................................... 53 
22.1 Declaration of Helsinki ...................................................................................................... 53 
22.2  Ethic Committee and Regulatory Authority Approvals and Any Additional 
Requirements .................................................................................................................. 53 
22.3 
Informed Consent ........................................................................................................... 53 
22.4 
 Amendments and EC Additional Requirements ........................................................... 54 
22.5 
Insurance Policy ............................................................................................................. 55 
22. Subject Compliance ................................................................................................................ 55 
23. Statistic ................................................................................................................................... 55 

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24. Data collection – eCRFs ......................................................................................................... 57 
25.1 Unique subject identifier .................................................................................................... 58 
25.2 Coding dictionaries ............................................................................................................ 59 
26 Monitoring, Data and Quality Management .......................................................................... 59 
26.1 
Clinical monitoring and identification of source data .................................................... 59 
26.2 Source Documents .............................................................................................................. 60 
26.3 Audit ................................................................................................................................... 61 
26.4 
 Inspections ..................................................................................................................... 61 
25. Safety Data Evaluation ........................................................................................................... 61 
26. Adherence to Clinical Investigation Plan ............................................................................... 61 
27. Clinical Investigation Termination ......................................................................................... 61 
28. Financing of the Clinical Investigation .................................................................................. 62 
29. Clinical Investigation Report ................................................................................................. 62 
30. Publication policy ................................................................................................................... 62 
REFERENCES ............................................................................................................................... 63 
 
 
 
 
 
 
 
 

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Page 27 of 64 
Version n. 2 dated March 14th, 2023 
 
 
 
1. Investigator and Facility 
 
1.1 Principal Investigator and Clinical Research Facility 
       SITE Number 1(Coordinator experimental site) 
 Dr. Davide Scollo 
Oculistica, P.O. San Marco, Azienda Ospedaliero Universitaria Policlinico  
"G.Rodolico - San Marco"  
Viale Carlo Azeglio Ciampi, 95121 Catania (Italy) 
Tel: +39 095378121 
Email: davidescollo@hotmail.com 
 
SITE Number 2 
Prof. Vincenza Bonfiglio 
U.O. di Oculistica, 
Presidio Belmonte,  
Azienda Ospedaliero Universitaria Policlinico  
“Paolo Giaccone”  
Via Rampolla Mariano Cardinale,  
10B, 90142 Palermo (Italy) 
Tel: +39 091 6553903 
Email: enzabonfiglio@gmail.com 
 
 
1.2 Statistics 
All data collected will be analysed by a statistical expert, who will produce a report. 
 
Dr. Filippo Palermo 
UNIFARM Research Centre 
Viale Andrea Doria 21 
95125 Catania (Italy) 
Tel: +39 095 7167514 
Fax: +39 095 7167514 
Email: filippo.palermo@unifarm.org 
 
 
 
 
 
 
 
 
 

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Page 28 of 64 
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1.3 Quality Assurance Unit 
The study clinical investigation plan review, inspection of the clinical facilities and methods 
used as well as verification of data records and eCRFs for accuracy and compliance will be 
performed by the UNIFARM Research Centre Quality Assurance personnel. 
 
UNIFARM Research Centre  
Viale Andrea Doria 21  
95125 Catania (Italy) 
Tel: +39 095 7167514 
Fax: +39 095 7167514 
Email: direzione@unifarm.org 

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Version n.2 dated March 14th, 2023 
Page 29 of 64 
 
 
2. Rational and background 
 
Dry eye disease (DED) is a common ocular condition of the ocular surface and tear film caused 
by tear deficiency or excessive evaporation, leading to damage to the interpalpebral ocular surface 
[1] and is associated with subjective symptoms, including ocular discomfort, visual disturbance, 
dryness, and soreness [1, 2]. 
The disease has a multifactorial origin and linked to pathological conditions of one of the 
functional unit portions that includes the tear film, and the ocular surface (cornea, conjunctiva, 
accessory lacrimal glands, the meibomian glands, the junction epidermal mucus, a master gland 
and nervous connection systems, secretory ducts and lacrimal sac nose). 
The prevalence of DED is high, affecting up to 34% of certain populations, particularly female 
and elderly (> 65 years old) patients with a significant impact patients’ quality of life and a 
significant burden on society [3].  
From a pathophysiological point of view, ocular surface inflammation, hyperosmolarity, and tear 
film instability have been identified as the main triggers for the development and progression of 
the disease [1].  
The stability of the tear film is influenced by its individual components as lipids, water and mucins 
[4,5]. The open eye is constantly subjected to desiccating stress through evaporation of the tears; 
however, it is protected from damage by homeostatic mechanisms that regulate tear secretion and 
distribution in response to signals from the ocular surface. A failure of these mechanisms leads 
to a quantitative or qualitative deficiency of tears that typically induces tear film instability, 
wetting defects and hyperosmolar stress, increased friction and chronic mechanical irritation at 
the ocular surface and an increased risk of infections.  
Moreover, the alteration of the precorneal tear film may rapidly result in gland dysfunctions of 
the eyelids and conjunctiva and ultimately affect the visual acuity (VA) [6].  
The classic tear film model is composed of 3 major components: an outer lipid layer, an aqueous 
middle layer, and the inner mucus layer. Intrinsic or extrinsic changes to any of these three 
components of the tear film can affect ocular surface homeostasis and encourage tear film 
instability and tear hyperosmolarity [3]. 
As extrinsic changes, one major causative risk factor of semi-acute eye complaints is air pollutants 
causing chemesthesis (sensory irritation) by trigeminal stimulation of the ocular surface [7].  
In general hormonal or autoimmune factors aggravated by pollution, local infection, ultraviolet 
irradiation, tobacco smoke or allergic or iatrogenic phenomena results in a cascade of cellular 
reactions, in which apoptosis may be closely linked. 
Patients affected by ocular dryness are usually treated with topical lubricants with the aim to 
replace the tear volume, stabilize the tear film, protect the ocular surface and reduce friction of 
the eyelids on the cornea while blinking. Commercially available tear lubricants differ in their 
composition and include cellulose derivatives, lipids, polyvinyl alcohol, hydroxypropyl guar and 
hyaluronic acid [8]. 
Sodium hyaluronate is a high-molecular-weight anionic polysaccharide with several mechanical 
properties useful for lubricating the ocular surface. It is a natural polymer whose physical 
characteristics confer to solutions important viscoelastic properties. It is high hydrophilic and 
therefore it can bind high quantities of water in a reversible manner, to increase the tear volume 
and to hydrate the ocular surface. In addition, sodium hyaluronate has an excellent 

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Medical device 
 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n.2 dated March 14th, 2023 
Page 30 of 64 
 
 
pseudoplasticity (i.e. non-newtonian behaviour). This means that an ophthalmic topical device 
containing sodium hyaluronate is more viscous at rest (i.e. when eyes are open) and more liquid-
like when a stress is applied (i.e. during blinking), thus minimizing friction between lids and 
ocular surface acid [9, 10]. This effect is reversible, so that such solution is able to recover its 
viscosity. 
Solutions containing sodium hyaluronate are able to prolong their ocular surface contact time in 
respect to other ophthalmic products. 
A growing number of studies have been published demonstrating good tolerability of sodium 
hyaluronate and its ability to improve symptoms of dry eye [11] A recent systematic review [12] 
examined 18 clinical trials comparing the efficacy of sodium hyaluronate against alternative 
lubricant preparations in the treatment of dry eye syndrome. Majority of the 18 studies selected 
for review showed superiority of sodium hyaluronate in improving ocular staining and symptoms. 
Sodium hyaluronate demonstrated greater improvement of Schirmer’s test compared to other 
preparations (p < 0.001). 
It is generally recognized that elimination of preservatives such as benzalkonium chloride is 
important in the long-term safety and tolerability of ocular preparations, especially for individuals 
with dry eye [13]. 
In this field, the hydrating and viscoelastic properties of sodium hyaluronate make him the ideal 
candidate in all conditions of ocular redness and/or ocular dryness due to alterations of the 
lachrymal film. In addition, sodium hyaluronate retains water and therefore, it is proficient to 
maintain the ocular surface moisturized and protect it from external agents. 
Hyalistil BIO PF (listed in the Medical Device Technical File as HA MONO 0.2% Phosp. Free-) 
is sterile medical devices (eye drops) for ophthalmic use containing 0.2% sodium hyaluronate. 
The device is presented in unidose containers phosphate-free. Therefore, HA MONO 0.2% Phosp. 
Free- is indicated to lubricate and hydrate the ocular surface able to provide a stable coating on 
the surface of the eye performing a moisturizing and lubricating action on the ocular surface, thus 
allowing a temporary relief to burning, irritation and all dry-eye related discomfort. 
In addition, the device assures the protection of the ocular surface during the healing processes.  
Moreover, since the presence of phosphates in ophthalmic formulations may rarely cause, in 
patients with compromised cornea, corneal deposits or corneal opacities [14, 15], HA MONO 
0.2% Phosp. Free- is a specific formulation without phosphates (and alternative buffer systems) 
mainly indicated in all cases of ocular discomfort and ocular dryness due to (as example) dry eye 
syndrome, ocular surgery, allergy, 
environmental factors (sun exposure, wind, smoke, pollution, conditioned air), excessive use of 
computer monitors, contact lenses use, aging, Moreover, they protect the ocular surface during 
the process of wound healing after corneal abrasions. 
One-two drops of HA MONO 0.2% Phosp. Free- should be instilled in the conjunctival fornix 
three to four times daily, unless otherwise indicated. 
No safety concerns have been identified and reported on the leaflet with the exception of 
occasional burning and local irritation upon instillation. 
Rolando (2007) [16], published a paper describing the activity of HA MONO 0.2% or Tamarind 
seed polysaccharide (TSP) in the treatment of dry eye syndrome. This was an open-label, 
randomized, single-centre clinical study. Thirty patients were randomized to receive three or more 
applications per day of either TSP 0.5%, TSP 1% or HA MONO 0.2%. over a period of 90 days. 

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Version n.2 dated March 14th, 2023 
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The primary objective of tolerability was assessed by visual analogue scale, scoring of specific 
symptoms and the incidence of adverse events. Secondary objectives included improvement in 
stability of the precorneal tear film, subjective symptoms and corneal and conjunctival staining. 
All 3 treatments had a comparable performance. In this study, higher TSP concentration gave 
higher benefit for the subjective symptoms. 
Monaco (2011) [17], analyzed the effects of HA MONO 0.2% or 0.5% carboxymethylcellulose 
(Optive) on the ocular surface in 20 patients with glaucoma using antiglaucoma drugs. This was 
a prospective, comparative, randomized, double-blind study with a crossover design. Each group 
was administered a 4-week course administered quarter in a day. After a 2-week washout period, 
the course of treatment was reversed. The primary efficacy criteria consisted of assessing 
symptoms according to the OSDI; 
The secondary efficacy criteria consisted of evaluating tear film confocal microscopy, central 
corneal thickness, corneal and conjunctival staining. At the end of the study only 
carboxymethylcellulose induced a significant improvement in OSDI and staining compared to 
baseline values. No significant differences were observed for other parameters evaluated. Both 
solutions were well tolerated. No adverse events were reported. 
Cagini (2017) [18], compared the stability of the tear film after a single instillation of eye drops 
containing HA MONO 0.2% or 0.2% sodium hyaluronate crosslinked (ICROSS). Forty subjects 
were included in this study: 20 healthy volunteers and 20 patients suffering from Sjogren 
syndrome. The surface regularity index and surface asymmetry index were registered before and 
5, 30, and 60 min after instillation of eye drops. In healthy subjects the two compounds had no 
effect, whereas in patients with Sjogren syndrome topographic indexes improved with both 
compounds; crosslinked 0.2% sodium hyaluronate had a better performance (P < 0.05) for both 
surface regularity index and surface asymmetry index at 60 min after instillation. 
Under these premises, considering that HA-MONO-0.2%-Phosp.free was marketed in Italy with 
the name “Hyalistil Bio PF”, the objective of the present study is to assess the clinical 
performance, tolerance, and safety of Hyalistil Bio PF device after 35 ± 4 days of treatment in 
patients affected by eye discomfort in particular in case of ocular dryness. 
 
3. Aim of the Clinical Investigation 
The purpose of this prospective observational study is to assess, during the conventional clinical 
practice, the performance and safety of the daily instillation of Hyalistil Bio PF device in the 
treatment of patients affected by eye discomfort in particular in case of ocular dryness. 
A subject will be included in the study if:  
• 
has been fully informed by the Investigator about the details of the observational study; 
• 
has signed the Informed Consent Form; 
• 
fulfils all the inclusion criteria and if he has not any exclusion criteria. 
 
The assignment of a subject to medical device treatment is not decided in advance by CIP but is 
part of current clinical practice. The use of the medical device is separated from the decision to 
include the subject in the clinical investigation. No additional invasive or burdensome diagnostic 
or monitoring procedures outside at the normal clinical practice are applied to the subjects. 
Regarding Tear Film Break-Up Time test with fluorescein (TFBUT), fluorescein staining is an 

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Version n.2 dated March 14th, 2023 
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invasive procedure and is normally used in clinical practice in the assessment of ocular 
discomfort due to dry eye. 
Once a subject's eligibility has been established, the Investigator will provide all the relevant 
information about this clinical investigation. 
The Informed Consent will be reviewed, discussed, dated and signed by both the subject and the 
Investigator before the beginning of screening procedures. Each subject will be interviewed by 
the Investigator who will record medical history, previous/concomitant pharmacological 
treatments, and will conduct a routine medical examination. 
No laboratory tests are planned. 
The data subject (including personal data and special categories of personal data) will be 
processed only by the authorised scientific staff, as reported in the staff signature and the 
delegation log.  
The Investigator will assign two progressive numerical codes (screening number) to the screened 
patient and (subject clinical investigation number) to the enrolled patient.   
The Investigator will not assign subjects into intervention groups and will not be made any 
attempts to collect data beyond those available throughout the course of normal clinical practice. 
Only the authorised scientific staff will know any additional information regarding the 
identifiability of the patient. The Sponsor and/or the delegated staff of CRO will receive and/or 
known anonymized patient data through the numerical code (unique subject identifiers code) 
assigned by the Investigator. 
The unique subject identifiers code consists of the 5-digit screening number (e.g. S1001, 
S1002,….S1028 etc.), and, if applicable, the 4-digit subject clinical investigational number (e.g. 
1001, 1002,   1028, etc.). Specifically, the first subject clinical investigational number was 
referred to the study site. 
Allocation screening number and subject clinical investigational number are separated by 
slashes (e.g. “S1001/1001”). (e.g. “S1001/1001” for site number 1, “S2001/2001” for site 
number 2). 
The Investigator will only report the information and data in coded form in the clinical study 
record, referring exclusively to the unique subject identifiers code assigned to the patient at the 
time of enrolment. 
In the unlikely event that authorized staff of CRO becoming aware of the patient's identity or 
parts of their identity, the CRO can neither transcribe/ report/ copy nor save this information or 
even communicate or transmit to the Sponsor. 
The Investigator and the Sponsor are autonomous data controllers. 
 
4. Clinical Investigation Design 
This clinical investigation is a multicentric prospective observational study investigating the 
effect of daily instillation in patients suffering from eye discomfort in particular in case of ocular 
dryness under normal clinical practice. Withdrawn subjects will be replaced until reaching a total 
number of 28 subjects. 
The study will run for 4 months from first patient’s enrolment until data evaluation. All patients 
will be followed from enrolment until study end date, which will occur when, in compliance 
with the normal clinical practice, the last patient will undergo the Study Termination Visit (35± 
4 days from the first instillation of study product). 

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The duration of the enrolment period is expected to be 3 months. 
 
5. Patient risk and benefit 
This is a non-interventional clinical investigation and therefore all medical procedures will be 
performed per standard of care. The clinical investigation plan itself does not therefore introduce 
any additional risk for the patient. 
This clinical investigation plan will collect routine data at specified time points and will not 
require additional patient visits or investigations. 
 
6. Inclusion criteria 
1. Subjects (male or female) must be ≥ 18 years of age; 
2. Subject able to provide Informed Consent, in compliance with the good clinical practice 
and local laws (thus, subject able to comprehend the full nature and purpose of the study, 
including possible risks and side events); 
3. Subjects with ocular discomfort resulting from mild to moderate dry eye in one or both 
eyes determined by: 
- Scoring of ocular surface staining with fluorescein using the National Eye Institute 
(NEI) scale. Total score per single eye range 6-33 summing the score of cornea and 
conjunctiva.  (Considering a normal score of 0-33); 
- Tear film break-up time with fluorescein (TFBUT) ≤ 10 seconds; The TFBUT value 
will be recorded as the average of 3 measurements; 
- Symptom Assessment in Dry Eye (SANDE) questionnaire ≥ 35. 
4. Subject able to be compliant with the requirements of the clinical investigation plan, 
according to the Investigator; 
5. Subject who qualifies for Hyalistil Bio PF treatment according to the approved 
indication; 
6. Subject who in physician's opinion will benefit from this treatment. 
 
7. Exclusion criteria 
1. Corneal injuries or abrasions of traumatic origin in the eye of study; 
2. Ocular infection or clinically significant inflammation (such as Herpes Simplex infection, 
corneal virus infection, bacterial, viral or fungal conjunctivitis, tuberculosis and mycosis of 
the eye, purulent and herpetic blepharitis, stye); 
3.  Sjögren's syndrome; 
4. Stevens-Johnson syndrome;  
5. Systemic lupus erythematosus; 
6. Pathologies associated with corneal thinning; 
7. Taking drugs that may interfere with tear gland secretion (beta -blockers); 
8. Patients using any topical therapies such as non-steroidal anti-inflammatory drugs, 
cortisone, cyclosporine, vasoconstrictor, artificial tears (different than the investigated 
product) the eye of study;  
9. Treatment with topical or systemic corticosteroids in the 4 weeks preceding the study; 
10. Known or potential hypersensitivity and/or history of allergic reactions to one of the 

Hyalistil Bio PF  
Medical device 
 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n.2 dated March 14th, 2023 
Page 34 of 64 
 
 
components of the topical medical device. 
11. Participation in another clinical trial within the previous 30 days; 
12. Evidence of severe or uncontrolled systemic disease or any other significant disorders, 
which in the opinion of the Investigator does not allow the participation in the study or could 
compromise the results. 
 
8. Subject withdrawal criteria 
It will be documented whether or not each subject completed the observational study. If, for a 
subject, observations are discontinued, the reason will be reported. 
• 
Patients can be withdrawn for the following reasons: 
✓ Worsening condition despite treatment; 
✓ Voluntary subject withdrawal for any reason and at any time without prejudice;  
✓ At the discretion of the Investigator, (e.g. lack of patient compliance, etc.); 
✓ If an adverse reaction (including a concomitant illness) develops which in the view of the 
Investigator is incompatible with the continuation of the study; 
✓ Failure to comply with requirements of the clinical investigation plan; 
✓ Onset of conditions requiring additional treatments or surgery that could influence the 
results of the observational study; 
✓ Any treatment discontinuation must be recorded on the electronic case report form (eCRF) 
by the Investigator, who will indicate date and reason(s) for treatment withdrawal. 
 
8.1 Discontinuation procedures 
For any patient discontinuing the observational study, the Investigator will: 
- Ask the patient to undergo, as far as possible, an Early Termination Visit (ETV) to examine 
the patient's health conditions. This examination will verify that all values tested at screening 
have remained within a clinically acceptable range (i.e. not clinically significant changes 
compared to screening); 
- Arrange for alternative medical care of the withdrawn patient, if necessary; 
- Report in the eCRF the date and time of administration of the last dose and the date and primary 
reason of study discontinuation.  
 
9. 
Screening Failure 
Screening Failures are considered “Potential subjects who were screened for the participation in 
the clinical investigation, but were not enrolled”.  
Primary reason for screening failure could be:  
• Adverse Event; 
• Patient Non-compliance; 
• Withdrawn of Consent;  
• Inclusion / exclusion criteria not met;  
• Other. 
 
 

Hyalistil Bio PF  
Medical device 
 
 
Study code: 052/SI Hyalistil Bio PF Mono 
Version n.2 dated March 14th, 2023 
Page 35 of 64 
 
 
10. Primary objective 
 
• 25%increase of Tear film break-up time with fluorescein (TFBUT) at Study Termination 
Visit (Day 35 ± 4 of treatment) compared to Visit 1 (Day 0 of treatment - baseline).  
 
11. 
Secondary objective 
• Evaluation of the change in total score (score from 6 to 33, considering a normal score 0-
33) resulting from the sum of the corneal staining score (score from 0 to 15) and 
conjunctival staining score (score from 0 to 18) with fluorescein using the National Eye 
Institute Scale (NEI) at Visit 2 (Day 14 ± 2 of treatment) and at the Study Termination Visit 
(Day 35 ± 4 of treatment) compared to Visit 1 (Day 0 of treatment - baseline); 
• To evaluate the tear film stability per group as objectified by the tear break up time with 
fluorescein (TFBUT) at Study Termination Visit (Day 35 ± 4 of treatment) compared to Visit 
2 (Day 14 ± 2 of treatment). 
• Changes about Best Corrected Visual Acuity (BCVA) measured by the “Early Treatment 
Diabetic Retinopathy Study” (ETDRS) at Visit 2 (Day 14 ± 2 of treatment) and Study 
Termination Visit (Day 35 ± 4 of treatment) compared to Visit 1(Day 0 of treatment – baseline). 
• Patient reported outcomes:  
To compare patients reported outcomes (PRO) measures per group, including:  
- Patient’s reported symptoms (SANDE) at Visit 2 (Day 14 ± 2 of treatment) and Study 
Termination Visit (Day 35 ± 4 of treatment) compared to Visit 1(Day 0 of treatment – baseline). 
 - Evaluation of the degree of satisfaction to the treatment reported by patients through the 
use of the visual analogue scale (VAS) , at the Study Termination Visit (Day 35 ± 4 of 
treatment). 
- Assessment of the quality of life (QOL)  by “Questionnaire about Eye Symptoms and 
Daily Life” (DEQS)at Study Termination Visit (Day 35 ± 4 of treatment) compared to Visit 
1 (Day 0 of treatment - baseline). 
In the event that, for both trial centers, the DEQS questionnaire is not available, the assessment 
of the quality of the life will be performed by the DEQ-5 questionnaire. 
 
  Safety Evaluations 
• Evaluation of the safety and the compliance of medical device.  
 
 

Page 36 of 64 
Version n. 2 dated March 14th, 2023 
 
Study code: 052/SI Hyalistil Bio PF mono 
Hyalistil Bio PF  
Medical device  
 
 
 
 
 
 
12. Clinical Investigation Scheme 
 
Study Procedure 
Visit 1 
(screening-
enrolment- start of 
treatment)* 
Visit 2*** 
(on the 14th day from the 
first instillation of 
medical device) 
(window ± 2 days) 
Study termination 
Visit° 
(on the 35th day from 
the first instillation of 
medical device) 
(window ± 4 days) 
Informed Consent 
√ 
 
 
Allocation screening number   
√ 
 
 
Demographic data collection 
√ 
 
 
Medical and surgical history 
√ 
 
 
Current medical conditions 
√ 
√ 
√ 
Prior ocular local and systemic medication 
√ 
 
 
Concomitant ocular local and systemic 
medication 
 
√ 
√ 
External Ocular Examination 
√ 
√ 
√ 
BCVA evaluation through ETDRS grade 
√ 
√ 
√ 
SANDE questionnaire 
√ 
√ 
√ 
Evaluation of Intraocular pressure (IOP) 
√ 
√ 
√ 
Ocular surface staining 
(Fluorescein- NEI score) 
√ 
√ 
√ 
Tear Film Break-up Time 
(TFBUT- Fluorescein) 
√ 
√ 
√ 
Administration of DEQS questionnaire 
√ 
 
√ 
Inclusion/Exclusion Criteria 
√ 
 
 

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Version n. 2 dated March 14th, 2023 
 
Study code: 052/SI Hyalistil Bio PF mono 
Hyalistil Bio PF  
Medical device  
 
 
 
 
 
Enrolment and allocation subject clinical 
investigational number 
√ 
 
 
 
Generation of unique subject identifiers 
code* 
√ 
 
 
 
 
Medical device dispensing* 
√ 
 
 
Check Compliance (check proper 
treatment with medical device, use of 
other medication and medical devices) 
 
√ 
√ 
Medical device restitution§ ' 
 
 
√ 
Evaluation of satisfaction to the treatment 
by VAS scale 
 
 
√ 
Evaluation of IGAS 
 
 
√ 
Adverse events /Incidents** 
 
√ 
√ 
 
* Start of the treatment (window + 1 day). 
** In case of adverse events/incidents all assessments considered necessary by the Investigator will be performed, including the possible suspension of treatment. 
*** Variable because it is based on the planned visit schedule according to clinical practice for the individual patient Visit 2 (windows of ± 2 days). 
° All patients enrolled will be treated for 35± 4 days.  The Study Termination Visit is variable because it is based on the planned visit schedule according to clinical 
practice for the individual patient (window ± 4 days).  
§ Restitution of the amount of used single dose containers and empty/unused medical device boxes returned by patients to the centre. 
'In case of premature study discontinuation, patients will undergo an Early Termination Visit (ETV). During the ETV the patients will return the amount of used single 
dose containers and empty/unused medical device boxes returned by patients to the centre. 
* The “unique subject identifiers code” consists of the 5-digit screening number (e.g. S1001, S1002,….S1028 etc.), and, if applicable, the 4-digit subject clinical 
investigational number (e.g. 1001, 1002,…1028 etc.). Specifically, the first subject clinical investigational number was referred to the study site. 
Allocation screening number and subject clinical investigational number are separated by slashes (e.g. “S1001/1001”). (e.g. “S1001/1001” for site number 1, 
“S2001/2001” for site number 2). 
 The TFBUT value will be recorded as the average of 3 measurements. 
In the event that, for both trial centers, the DEQS questionnaire is not available, the assessment of the quality of the life will be performed by the DEQ-5 questionnaire. 

 
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Page 38 of 64 
 
 
 
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13. Ethical considerations and Informed Consent 
 
This prospective observational clinical investigation will be performed in compliance with 
current GCP, including the archiving of essential documents, in compliance with CIP, 
Declaration of Helsinki, ISO14155:2020, Regulation (EU) 2016/679 concerning the protection 
of individuals with regard to the processing of personal data (also GDPR), Regulation (EU) 
2017/745 (article 63) and other applicable international and national regulatory requirements. 
Before starting the observational study, it is necessary that an appropriate Ethics Committee 
(EC) approve the CIP and Informed Consent Form (ICF). 
This is a non-interventional clinical investigation plan and standards of care have to be followed 
at all times. 
Prior to enrolment, at all patients will be provided the ICF. It is the responsibility of the 
Investigator to ensure that all potential patients are fully informed about the requirements of the 
study and that they are made aware of the potential risks and benefits of participating in the 
study. Written confirmation of informed consent has to be obtained from each patient prior to 
the performance of any study procedure in the trail. Written informed consent must be 
documented using the study specific ICF. The Investigator (or study personnel) will retain a 
copy of the signed ICF document and a copy have to be provided to the patient. The date that 
the ICF is signed must be recorded in the patient’s medical notes. 
Once approved by the EC, no changes should be made to these documents without first seeking 
approval from the relevant EC and the Sponsor. 
 
 
14. Investigational medical device 
 
The medical devices Hyalistil Bio PF is moisturizing and lubricating ophthalmic solution 
helpful in relieving ocular discomfort, such as dry eye syndrome, eye surgery, allergy, and 
irritation due to environmental conditions (such as pollution, air conditioning, smoke, wind, 
chlorine in swimming pools, etc.), prolonged use of electronic devices, use of contact lenses, 
menopause, aging, chronic use of topical drugs (eg. Ocular hypotonizing agents, eye drops 
containing preservatives, etc.), taking systemic drugs (eg antihypertensives, antidepressants, 
antihistamines, hormone replacement therapy, etc.). 
Hyalistil Bio PF contains sodium hyaluronate (0.2%), a natural polymer presents in various 
parts of the human body, whose chemical-physical characteristics give the solution viscoelastic, 
mucoadhesive and moisturizing properties. 
Hyalistil Bio PF coats the surface of the eye ensuring adequate lubrication and reducing the 
friction caused by eyelid movements, hydrates the ocular surface and ensures protection during 
the repair processes of the corneal epithelium. 
Therefore, Hyalistil Bio PF guarantees a feeling of lasting well-being and maximum comfort. 
The product does not contain preservatives and phosphates, therefore can also be used by 
contact lens wearers. 

 
Hyalistil Bio PF Mono  
Medical device  
Study code: 052/SI Hyalistil Bio PF Mono 
Page 39 of 64 
 
 
 
Version n. 2 dated March 14th, 2023 
 
 
     14.1 Description of product 
 
Composition 
 
Components 
Amount 
(100 ml) 
Sodium hyaluronate 
g 0.200 
Trometamol (TRIS) 
g 0.242 
Sodium chloride 
g. 0.600 
Hydrochloric acid q.s. to  
pH 7.2 
Potassium chloride 
g 0.250 
Purified water q.s. to 
ml 100 
 
 
Indications 
Hyalistil Bio PF contains sodium hyaluronate, a natural polymer also presents in structures of the 
human eye, whose chemical physical characteristics gives the solution hydrating, muco-mimetic 
and viscoelastic properties. 
Hyalistil Bio PF coats the eye surface, guaranteeing its adequate lubrification and reducing the 
friction caused eyelid movements. It also hydrates the ocular surface and assures protection during 
the healing processes of corneal epithelium. 
Therefore, Hyalistil Bio PF combines lasting relief with maximum comfort Characteristics and 
mechanism of action 
 
Presentation 
30  single-dose containers of 0.25 ml 
 
Dosage and administration  
The patient will instill 1 drop of Hyalistil Bio PF in the conjunctival fornix, 4 times a day, in the 
affected eye. 
 
 
     Instructions for use 
Wash / sanitize your hands. Before use make sure that the single-dose container is intact. Detach 
the single-dose from the strip. Open by turning the top without pulling. Instill avoiding that the tip 
of the container comes into contact with the eye or any other surface. 
 
 

 
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Medical device  
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Contraindications 
Hypersensitivity to one of the ingredients. 
 
Warnings 
There are no known interactions of the product with the administration of drugs for ophthalmic 
use. However, it is recommended not to administer other drugs or medical devices for ophthalmic 
use within 10 minutes after administration of Hyalistil Bio PF. 
It is not necessary to remove contact lenses before administering Hyalistil Bio PF 
as the product does not alter the physical characteristics, including transparency, of the most 
common soft and rigid contact lenses. 
The use of the product may occasionally cause intolerance (mild burning or irritation). 
 
Undesirable effect 
The use of the product may occasionally cause intolerance (mild burning or irritation). 
 
Interactions with other drugs or medical device for ocular use: 
There are no known interactions of the product with the administration of drugs for ophthalmic 
use. However, it is recommended not to administer other drugs or medical devices for ophthalmic 
use within 10 minutes after administration of Hyalistil Bio PF It is not necessary to remove the 
contact lenses before the administration of Hyalistil Bio PF since the product does not alter the 
physical characteristics, including the transparency, of the most common soft and rigid contact 
lenses. 
 
Precautions for use and storage 
       For topical ocular use only. 
Keep out of reach of children. 
Do not use the device if the single-dose is opened or damaged; 
Avoid touching the eye or any surface with the tip of the opened container; 
Each single-dose container should be used immediately after opening; 
Any residual product should be not used. since all devices do not contain preservative and 
they may become contaminated, exposing the eye to the risk of infection; 
Do not use the devices after the expiry date; 
Do not dispose the devices in the environment after use; 
The administration to children or people with limited ability must be carried out by a 
responsible adult; 
It is advised not to use other drugs or medical device for ocular use within 10 minutes from 
the administration of the device 
The use of the product may occasionally cause intolerance (mild burning or irritation). 
Store at a temperature not exceeding 25 ° C. 
Validity for intact packaging: 24 months. 
 

 
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Medical device  
Study code: 052/SI Hyalistil Bio PF Mono 
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14.2 Description as to how traceability shall be achieved during and after the 
clinical investigation 
The traceability will be achieved during and after observational period by the lot number 
“CC141” assigned to medical device Hyalistil Bio PF will be used in the observational study. 
 
14.3 Details concerning the manufacturer of Medical Device 
SIFI S.p.A. – Via Ercole Patti, 36 
95025 Aci S. Antonio (CT) – ITALY 
 
14.4 Name or number of the model/type to permit full identification 
The name of the model to permit full identification of the medical devices is: Hyalistil Bio PF. 
 
15. Investigational medical device storage and handling 
The Sponsor will offer, like free samples, individual boxes containing an adequate supply of the 
investigational product to be instilled during the study to each subject. 
The Pharmacist and/or Investigator will be responsible for receipt, proper storage, and usage of 
experimental product, as well as for the investigational product distribution, collection of used 
and unused vials and final disposal of the remaining investigational product. 
The investigational product must be stored not exceeding 25 °C at the investigational sites, in an 
appropriate locked room accessible only to the pharmacist, the Investigator, or a duly designated 
person. 
A temperature probe and data logger will accompany the investigational product on shipment. It 
is essential that the investigational sites verify the temperature excursion during shipment against 
acceptable storage conditions in order to identify potential stability concerns during shipment. 
These must be immediately communicated to the Sponsor that will decide upon appropriate 
actions to be taken. The investigational product will be stored in a locked place, sheltered from 
light. 
PI, or a delegate, will maintain an inventory record of the investigational product received. 
At the conclusion of the study, a final medical device accountability will be performed. If any 
supplies are missing, this will be indicated together with an explanation for the discrepancy. 
During the Visit 1 the study personnel will give the patient boxes containing the investigational 
product. 
Patient should bring the investigational product, boxes containing for 35±4 days of treatment, at 
home and store not exceeding 25 °C. 
As previously described, each single-dose container must be used immediately after opening. 
Do not touch the end of the open container and do not put it in direct contact with the eye. 
Each single-dose container has the amount needed to treat both eyes. 
Any residue must not be reused, as Hyalistil Bio PF does not contain preservatives and the 
remaining product could become contaminated, exposing to the risk of eye infections. 
Any deviations from the recommended storage conditions should be reported by Pharmacist 
and/or Investigator to the Sponsor, and the use of the investigational product should be suspended 

 
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until they have given authorization for its continued use. The investigational product supplies are 
to be used only in accordance with this clinical investigation plan. 
 
Investigational Medical Devices Delivery Plan 
Box delivery  
 
Boxes to be provider 
 Visit 1 (0 days) 
5 Boxes of 30 single dose containers 
 
16. Investigational medical device accountability 
The Pharmacist and/or Investigator will confirm the receipt of the investigational product supply 
in writing by signing and dating standard of investigational product accountability forms. 
At the Study Termination Visit the patients will return the used or unused study boxes to the 
Investigator. 
In case of premature study discontinuation, patients will undergo an Early Termination Visit 
(ETV). During the ETV the patients will return the amount of used single dose containers and 
empty/unused medical device boxes returned by patients to the centre. The Pharmacist and/or 
Investigator will keep a cumulative inventory and dispensing records and will maintain all 
supplies under adequate security. 
An accurate product disposition record will be kept, specifying the date and amount dispensed to 
each patient. 
Inventory record of receipt, use or loss of investigational product will be retained. This inventory 
record must be available for inspection by the Sponsor and regulatory inspection at any time. 
Copies of this record will be provided to the Sponsor by the CRO throughout the duration of the 
study. 
Partially used or unused study investigational product boxes will be verified and returned by the 
Investigator/ Institution to the investigational product manufacturer, at the end of the study. 
During the observational study, the Investigator will complete the "investigational product 
accountability forms". After completion of the clinical investigation, the unused study 
investigational product will be destroyed after authorization by the Sponsor by an authorized 
company according to GCP regulations. 
 
17. Other allowed therapies  
All medications (including over-the-counter drugs, herbal products, vitamins, and antacids) taken 
prior to start the study or taken for clinical reason during the observational period, will be 
recorded in the appropriate Case Report Form reporting generic name, date(s) and time(s) of 
intake, reason(s) for use, and dosage information. 
Medication entries should be specific to product name (if a combination drug product) and spelled 
correctly. The dose, unit, frequency, route of administration, start date, discontinuation date, and 
indication should also be recorded. For medications administered only one time, the frequency 
column may reflect "once." 
Concomitant treatments should be taken according to current medical practice. All therapies, 
topic or systemic, that might interfere with the evaluation of the treatment under investigation are 

 
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forbidden. This decision depends on the type of illness and the kind of drug to be used.  If the 
subject must take drugs, the PI will decide whether the subject can continue to participate in the 
study. 
Patients that: 
- 
using any topical therapies such as non-steroidal anti-inflammatory drugs, cortisone, 
cyclosporine, vasoconstrictor, artificial tears (different than the investigated product) the eye 
of study;  
- 
must be treated with therapies forbidden (treatment with topical or systemic corticosteroids 
in the 4 weeks preceding the study); 
- 
treatment with drugs that may interfere with tear gland secretion (beta -blockers); 
- 
must be treated with therapies, that in the opinion of the Investigator does not allow the 
participation in the study or could compromise the results; 
- 
will be excluded from the prosecution of the study, but their data until that moment will be 
used in the statistical analysis. 
 
18. Procedures 
Study visits and procedures 
In this observational study each enrolled patient will be evaluated during 3 scheduled visits.  
Maximum observation period will be 35 ± 4 days. A written informed consent will be obtained 
before any study assessment or procedure. 
The first patient first visit (FPFV) is defined as the 1st visit to the clinical site by the 1st screened 
patient. The “Last Patient Last Visit” (LPLV) is defined as the last visit to the clinical site by the 
last patient (i.e., the last visit foreseen by the study clinical investigation plan), independently of 
whether the patient completed or withdrew from the study. 
All enrolled subjects will be treated for 35 ± 4 days and followed for a maximum of 39 days. 
They will be evaluated during 3 scheduled visits according to clinical practice for the individual 
patient: at baseline (Visit 1), at 14 ± 2 days from the first instillation of medical device under 
investigation (Visit 2) and at 35 days from the first instillation of medical device under 
investigation (Study Termination Visit) with window of ± 4 days. 
 
Visit 1 (screening-enrolment)  
The following procedures will be performed:  
-  Explanation to the patient of study aims, procedures and possible risks of the observational 
study; 
-  Informed consent signature; 
-  Allocation screening number; 
-  Demographic data collection; 
- In case of female patients (collection of information on pregnancy or breastfeeding self-
reported by the patient); 
-  Medical and surgical history/current medical conditions; 
-  Prior/concomitant ocular and systemic medications; 

 
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-  Ocular examination of both eyes: 
o External Ocular Examination; 
o BCVA test through the ETDRS at 4 m distance; 
o Symptom Assessment in Dry Eye by SANDE questionnaire; 
o Evaluation of Intraocular pressure (IOP); 
o Tear film break-up time with fluorescein (TFBUT);  
o Ocular surface staining (NEI score) with fluorescein; 
- DEQS questionnaire administration; 
- Patient eligibility: inclusion/exclusion criteria evaluation; 
- Assignment of the study and allocation subject clinical investigational number;  
- Generation of unique subject identifiers code*; 
- Medical device dispensation; 
- Beginning of the treatment*. 
 
The Investigator will dispense to the patients 5 boxes of medical device each containing 30 single-
dose containers of medical device each containing 30 single-dose containers for the following 
35±4 days. After completing the baseline evaluation, patients will start the study treatment as 
instructed through self-instillation of medical device under investigation at home*.  
Patients will return to the clinical site on day 14±2 from the first instillation of medical device 
under investigation (Visit 2). 
 
Visit 2 (on the 14th day from the first instillation of medical device under investigation (MD)) 
(window ± 2 days)*** 
The following procedures will be performed:  
- Assessment of treatment compliance; 
- Current medical conditions; 
- Concomitant medications; 
- Evaluation of any reported adverse events/incidents**; 
- Ocular examination of both eyes: 
o 
External Ocular Examination; 
o 
BCVA test through the ETDRS at 4 m distance; 
o 
Symptom Assessment in Dry Eye by SANDE questionnaire; 
o 
Evaluation of Intraocular pressure (IOP); 
o 
Tear film break-up time with fluorescein (TFBUT);  
o 
Ocular surface staining (NEI score) with fluorescein; 
Patients will return to the clinical site on day 35±4 days from the first instillation of medical 
device under investigation (Study Termination Visit). 
 
Study Termination Visit (on the 35th day from the first instillation of medical device under 
investigation) (window ± 4 days) 
The final visit is defined as the visit performed on 35 ± 4 days after the first instillation of medical 

 
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device under investigation°. 
In case of premature study discontinuation, patients will undergo an Early Termination 
Visit (ETV)'. 
The following procedures will be performed: 
- Assessment of treatment compliance (reconciliation of the amount of used single dose 
containers and empty/unused medical device boxes returned by patients to the centre)§; 
- Current medical conditions; 
- Concomitant medications; 
- Evaluation of any reported adverse events/incidents**; 
- Ocular examination of both eyes: 
o External Ocular Examination; 
o BCVA test through the ETDRS at 4 m distance; 
o Symptom Assessment in Dry Eye by SANDE questionnaire; 
o Evaluation of Intraocular pressure (IOP); 
o Tear film break-up time with fluorescein (TFBUT); 
o Ocular surface staining (NEI score) with fluorescein; 
- Administration of the VAS scale on the degree of satisfaction to the treatment; 
- DEQS questionnaire administration; 
- Evaluation of the safety of the experimental product through the compilation of the IGAS 
scale; 
- Restitution of remaining medical devices§. 
 
* Start of the treatment (window + 1 day). 
** In case of adverse events/incidents all assessments considered necessary by the Investigator will be performed, 
including the possible suspension of treatment. 
*** Variable because it is based on the planned visit schedule according to clinical practice for the individual 
patient Visit 2 (windows of ± 2 days). 
° All patients enrolled will be treated for 35 days.  The Study Termination Visit is variable because it is based on 
the planned visit schedule according to clinical practice for the individual patient (window ± 4 days).  
§ Restitution of the amount of used single dose containers and empty/unused medical device boxes returned by 
patients to the centre. 
'In case of premature study discontinuation, patients will undergo an Early Termination Visit (ETV). During the 
ETV the patients will return the amount of used single dose containers and empty/unused medical device boxes 
returned by patients to the centre. 
* The “unique subject identifiers code” consists of the 5-digit screening number (e.g. S1001, S1002,….S1028 etc.), 
and, if applicable, the 4-digit subject clinical investigational number (e.g. 1001, 1002,…1028 etc.). Specifically, 
the first subject clinical investigational number was referred to the study site. 
Allocation screening number and subject clinical investigational number are separated by slashes (e.g. 
“S1001/1001”). (e.g. “S1001/1001” for site number 1, “S2001/2001” for site number 2). 
 The TFBUT value will be recorded as the average of 3 measurements. 
 
 
 
 

 
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19. Primary and secondary outcomes^ 
Primary Endpoints  
• Clinical performance: 
-25 % increase from Visit 1 (Day 0 of treatment - baseline) to Study Termination Visit (Day 
35 ± 4 of treatment) of tear film break-up time with fluorescein (TFBUT)f. [Time frame: Visit 
1 and Study Termination Visit]. 
 
 Secondary Endpoints 
• Reduction from baseline (Visit 1- Day 0 of treatment) in the corneal and conjunctival fluorescein 
staining score using the NEI scale.  
Fluorescein staining of the ocular surface is assessed as the total sum of the corneal and 
conjunctival subregions.  
- Corneal Fluorescein Staining (NEI score, sum of 5 subregions, maximum score 15. Each 
subregion will have a maximum score of 3.  
(0=no staining, 3 = maximum staining).  
- Conjunctival Fluorescein Staining (NEI score, sum of 6 subregions, maximum score 18. 
Each subregion will have a maximum score of 3.  
(0=no staining, 3 = maximum staining).  
     [Time frame: Visit 1, Visit 2 and Study Termination Visit].  
• Tear film break-up time with fluorescein (TFBUT)
ϕ. [Time frame: Visit 2 and Study 
Termination Visit].  
•  BCVA is measured using the standard Early Treatment Diabetic Retinopathy Study (ETDRS) 
acuity chart and reported in ETDRS letters. [Time frame: Visit 1, Visit 2 and Study Termination 
Visit];  
• Patient reported outcomes: 
- Change in intensity and frequency of dry eye symptoms assessed by completing the 
questionnaire SANDE. [Time frame: Visit 1, Visit 2 and Study Termination Visit].  
- 
Evaluation of the degree of satisfaction to the treatment reported by patients through the use 
of the visual analogue scale (VAS) , ranging from 0 to 100 mm, at Study Termination Visit (Day 
35 ± 4 of treatment). [Time frame: Study Termination Visit]. 
Patients will be asked to rate their degree of satisfaction to the treatment and answer the following 
questions: 
• “I feel satisfied using this treatment? '',  
• “With this treatment, I have a feeling of freshness?”, 
• “With this treatment, I have a feeling of relief?”, 
• “This treatment contributed to reduce my pain due to eye dryness?'', 
• “This treatment is comfortable? '', 
 Patients will be asked to place a vertical mark on a horizontal line, from 0 to100 mm, to indicate 
the degree of satisfaction to the treatment, where: 
✓ 0-10 mm=none 

 
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✓ 11-30 mm=very mild 
✓ 31-50 mm=mild 
✓ 51-70 mm=moderate 
✓ 71-90 mm=strong 
✓ 91-100 mm=very strong. 
 
- 
Assessment of the quality of life (QOL) by “Questionnaire about Eye Symptoms and 
Daily Life” (DEQS) at Study Termination Visit (Day 35 ± 4 of treatment) compared to 
Visit 1 (Day 0 of treatment - baseline). [Time frame: Visit 1 and Study Termination Visit]. 
 
In the event that, for both trial centers, the DEQS questionnaire is not available, the assessment of the quality 
of the life will be performed by the DEQ-5 questionnaire. 
 
Evaluation of safety parameters 
      Evaluation of safety of daily instillation of ophthalmic solution during all the study period 
through: 
•  Evaluation of safety of daily instillation of ophthalmic solution during all the study period 
through:  
- Investigator Global Assessment of Safety (IGAS): using the 4-point scale:1= very good 
safety, 2 =good safety, 3 = moderate safety and 4 = poor safety. IGAS will be evaluated at 
the Study Termination Visit. [ Time Frame: Study Termination Visit];  
- Evaluation of reported adverse events/incidents. [Time Frame: During the treatment period].  
• Evaluation of intraocular pressure (IOP) [Time frame: Visit 1, Visit 2 and Study Termination 
Visit];  
• Evaluation of compliance through verification of correct instillation of the medical device, 
counting of single dose containers and boxes. [Time Frame: Visit 2 and Study Termination 
Visit].  
 
^According to clinical practice, the Investigator will perform all clinical evaluations on both 
eyes of the individual patient. 
The statistical analysis will be performed considering right eye as the “study eye.” 
The primary end point will be evaluated on the ITT population and in PP population. 
 
20. Equipment to assess clinical investigation variables and 
primary and secondary outcomes 
Ophthalmological Evaluations  
Ocular evaluations will be performed on both eyes. The assessment will be performed at 
Baseline (Visit 1), Visit 2, and at the Study Termination Visit. The ophthalmological 
assessment will include:  
 
 

 
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External Ocular Examination  
External Ocular Examination assesses the motility of the extraocular muscles and the 
appearance and function of the eyelids before instillation of any dilating or anesthetic eye 
drops.  
 
Best Corrected Visual Acuity 
Best Corrected Visual Acuity BCVA was measured using the standard Early Treatment 
Diabetic Retinopathy Study (ETDRS) acuity chart lighting, and procedures and reported in  
Best-corrected visual acuity is measured at all trial visits using standard charts, (chart 1 is used 
for testing the visual acuity of the RIGHT eye; Chart 2 for testing the LEFT eye).  
Patients should not be allowed to see any of the charts before the examination. 
A distance of 4 meters is required between the patient’s eyes and the visual acuity chart. With 
the box light off, not more than 15 foot-candles of light (161.4 Lux) should fall on the center of 
the chart. To measure the amount of light, the room is set up for visual acuity testing, but with 
the box light off. The light meter is placed at the fourth line from the top of the chart, with its 
back against the chart and the reading is taken. If more than one line available for testing visual 
acuity, the visual acuity of an individual patient should be measured in the same line at each 
visit, if possible. If different lines are used to test visual acuity, they must each meet the same 
standards. 
 
Ocular surface staining (NEI score - fluorescein)  
 
Figure from: NEI Industry grading system, American Academy of Ophthalmology: https://www.aao.org/ 

 
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As a grading scale for the corneal and conjunctiva damage, the NEI/Industry Workshop 
guidelines will be used. The cornea is divided into five sectors (central, superior, inferior, nasal 
and temporal), each of which is scored on a scale of 0–3, with a maximal score of 15. Both 
nasally and temporally, the conjunctiva is divided into a superior paralimbal area, an inferior 
paralimbal area and a peripheral area with a grading scale of 0–3 and with a maximal total score 
of 18 for the nasal and temporal conjunctiva.  
For a better reading it is also essential not to use an intense illumination beam, which may 
reduce the contrast and lead to an underestimation of grading. 
 
Intraocular pressure (IOP) assessment 
Intraocular pressure (IOP) measurement was performed using a Goldmann applanation 
tonometer after topical anesthesia with unpreserved 0.4% oxybuprocaine hydrochloride. 
 
       Symptom Assessment in Dry Eye (SANDE):  
The Symptom Assessment in Dry Eye (SANDE) questionnaire is a short questionnaire to 
evaluate both the intensity and frequency of dry eye using a 100 mm VAS [19]. The patient’s 
symptoms of ocular dryness and/or irritation will be quantified on the scale based on two 
questions assessing both severity and frequency of symptoms. A VAS is a measurement 
instrument that tries to measure a characteristic or attitude that is believed to range across a 
continuum of values and cannot easily be directly measured. For example, the amount of 
irritation that a patient feels ranges across a continuum from none to an extreme amount of 
irritation. From the patient's perspective this spectrum appears continuous (i.e. their irritation 
does not take discrete jumps, as a categorization of none, mild, moderate and severe would 
suggest). It was to capture this idea of an underlying continuum that the VAS was devised.  
For the assessment, the patients mark on the 100 mm VAS line the point that they feel represents 
their perception of their current state. The VAS score is determined by measuring in millimeters 
from the left-hand end of the line to the point that the patient marks. The SANDE scores will 
be then evaluated for the 2 questions severity (dryness and irritation with a range of 0-100 for 
each question) and frequency (dry and irritated with a range of 0-100 for each question). To 
simplify the procedure the patient will be informed that to provided anchors for the question 
measuring the frequency of symptoms, the extreme left of the 100 mm line indicated “rarely” 
and the extreme right indicated “all of the time”. Similarly, for the question that measured the 
severity of symptoms, the words “very mild” and “very severe” were placed at the left and right 
ends of the 100 mm line, respectively. 
 

 
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Tear Film Break-up Time (TFBUT)  
Patients with a Tear Film Break-up Time TFBUT test ≤ 10 in the study eye (study eye) at the 
screening visit are eligible for enrollment.  
TFBUT will be measured by determining the time to tear break-up. The TFBUT will be 
performed after instillation of 5 l of 2% preservative-free sodium fluorescein solution into the 
inferior conjunctival cul-de-sac of each eye. The patient will be instructed to blink several times 
to thoroughly mix the fluorescein with the tear film. To achieve maximum fluorescence, the 
examiner should wait approximately 30 seconds after instillation before evaluating TFBUT. 
With the aid of a slit lamp at 10X magnification using cobalt blue illumination, the examiner 
will monitor the integrity of the tear film, noting the time it takes to form lacunae (clear spaces 
in the tear film) from the time that the eye is opened after the last blink. This measurement will 
be performed within 10 seconds maximum. The TFBUT will be measured twice during the first 
minute after the instillation of the fluorescein. The TFBUT value will be the average of 3 
measurements.  
Relevant TFBUT findings will be entered in the eCRF. 
 
Safety of Medical Devices 
Considering the observational nature of the study, a surveillance program to collect adverse 
events /incidents/potential incidents was not planned. 
Adverse events /incidents/potentially incidents should be reported in the same way as 
spontaneous post-marketing reports. 
The safety will be evaluated by the collection of eventual presence of adverse events 
/incidents/potentially incidents that happened from the ICF signature until the end of the 35 ± 
4 days study. All adverse effect/incidents/potentially incidents (independently from the 
seriousness) experienced during the study, spontaneously reported by the patient, or elicited as 

 
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a result of general questioning by the site staff, will be recorded in the source documents 
(Clinical Record) and they will be reported on the eCRF. 
Furthermore, the Safety of medical device will be assessed using the Investigator Global 
Assessment of Safety (IGAS): using the 4-point scale:1= very good safety, 2 =good safety, 3 = 
moderate safety and 4 = poor safety. IGAS will be evaluated during the Study Termination 
Visit. [ Time Frame: Study Termination Visit]. 
 
Visual Analogue Scales 
Patients will be asked to rate their degree of satisfaction to the treatment and answer the following 
questions: 
• “I feel satisfied using this treatment? '',  
• “With this treatment, I have a feeling of freshness?”, 
• “With this treatment, I have a feeling of relief?”, 
• “This treatment contributed to reduce my pain due to eye dryness?'', 
• “This treatment is comfortable? '', 
 Patients will be asked to place a vertical mark on a horizontal line, from 0 to100 mm, to indicate 
the degree of satisfaction to the treatment, where: 
✓ 0-10 mm=no  
✓ 11-30 mm=very mild 
✓ 31-50 mm=mild 
✓ 51-70 mm=moderate 
✓ 71-90 mm=strong 
✓ 91-100 mm=very strong. 
Evaluation of the degree of satisfaction to the treatment reported by patients through the use 
of the visual analogue scale (VAS) ranging from 0 to 100 mm, at Study Termination Visit 
[Time frame: Study Termination Visit].  

 
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 Dry Eye-Related Quality-of-Life Score (DEQS) 
The DEQS questionnaire consists of 15 items to assess symptoms and their effect on daily living 
throughout the previous week.  
During the study will be asks to patients to rate the frequency and severity of various ocular 
symptoms on a scale of 0 to 4 from “not at all” to “always” and “not at all” to “very much.” 
The first six questions focus on ocular symptoms, while the other nine focus on how the patient's 
daily life has been affected. It questions patients on things like light sensitivity and difficulty 
using screens, whether their work is being impacted and whether they are feeling depressed as a 
result of their symptoms. A quality-of-life score ranging from 0 to 100 will be then calculated 
with the cutoff value for DED being 15 points.  
 
In the event that, for both trial centers, the DEQS questionnaire is not available, the assessment 
of the quality of the life will be performed by the DEQ-5 questionnaire. 
     Dry Eye Questionnaire (DEQ-5) measures symptom severity over the last month. The test 
contains only five questions, making it one of the quickest to complete and grade. During the 
study will be asks patients to rate the severity of eye discomfort, dryness and wateriness each 
from 0 to 4, with 0 indicating "never" and 4 indicating "constantly." The patient will be asked 
about the intensity of the symptom, with a score of 0 meaning it is not intense at all, and a score 
of 5 meaning it is very intense. The total score is a number between 0 and 22. 
All measurements will be taken by a trained optometrist and all instruments will be calibrated 
immediately prior to the study. 
 
 

 
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21. 
Statements of compliance  
 
22.1 Declaration of Helsinki 
The CRO/Sponsor and the Investigator ensure that all procedures described in this clinical 
investigation plan, data evaluation and documentation will be produced strictly according to 
normal clinical practice and the current version of Declaration of Helsinki.  
 
22.2  Ethic Committee and Regulatory Authority Approvals and Any 
Additional Requirements   
The clinical investigation plan and essential documents (ICF, etc..) will be approved by the Ethics 
Committees (ECs) and notified to the Ministry of Health. The approval of the clinical 
investigation will be obtained before the beginning of the study. 
The study can begin only after full approval by the EC. Medical device will not be sent to 
Investigator/s before the EC. 
 
 22.3 
Informed Consent 
Before enrollment in the study, the Investigator will: 
1. Explain, in a clear and detailed manner, the scope, the procedures and the possible 
consequences of the clinical investigation. The information will be given in both oral and 
written form.  
2. Obtain signed informed consent from the potential subject before performing any of the 
procedures included in the observational study. 
3. Assign screening number; 
4. Determine subject eligibility; 
5. Assignment of the study and allocation of subject clinical investigational number; 
6. Generation of unique subject identifiers code that consist of the 5-digit screening number (e.g. 
S1001, S1002,…S10028 etc.), and, if applicable, the 4-digit subject clinical investigational 
number (e.g. 1001, 1002,…1028 etc.). Specifically, the first subject study number was referred 
to the study site. 
Allocation screening number and subject clinical investigational number are separated by 
slashes (e.g. “S101/1001”). 
If a subject withdraws from participation in the observational study, his/her unique subject 
identifiers code can’t be reused. 
Before being enrolled in the clinical investigation, the patients must have expressed their consent 
to participate by provide a written consent form. The information sheet and informed consent 
form will be prepared in the local language by the CRO and must be approved by the EC. It will 
include all the elements required by law according to the ICH-GCP recommendations. 
In addition to the standard requirements that Investigators are obliged to observe in providing 
information, the following points must be covered: 
• a description of the aims of the observational study and how it will be organised, in 

 
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accordance with the normal clinical practices; 
• the type of treatment; 
• any potential adverse events attributable to the clinical investigation treatment; 
• the freedom to ask for further information at any time; 
• the right of subjects to withdraw from the clinical investigation at any time without giving 
reasons and without compromising their further course of medical treatment; 
• adequate time to answer possible questions. 
Adequate time and opportunity to satisfy questions will be given to the patients and the time will 
be recorded. 
The Investigator will be supplied with an adequate number of blank informed consent forms to 
be used. The forms will be signed and dated by both the Investigator and the subjects. 
A copy of the signed form will be given to the patient. 
To ensure medical confidentiality and data protection, the signed informed consent forms will be 
stored in the Investigator’s study file according to the regulatory requirements. 
The Investigator will allow inspection/audit of the forms by authorized representatives of the 
Sponsor, EC members and Competent Authorities (CA). He will confirm, by signing and dating 
the forms, that informed consent has been obtained. In case any new information regarding the 
IDs will be available during the study the subject will be contacted by the Investigator and in the 
case a new version of informed consent, will be prepared, submitted to the EC and after approval 
proposed to the subject for signature. 
The Informed Consent will incorporate (or, in some cases, be accompanied by a separate 
document incorporating) the wording that complies with relevant data protection and privacy 
legislation (Regulation (EU) 2016/679 concerning the protection of individuals with regard to the 
processing of personal data, also GDPR). 
 
22.4  Amendments and EC Additional Requirements 
 
In order to obtain interpretable results, neither the Investigator nor the Sponsor will alter the 
clinical investigation conditions agreed upon and set out in this clinical investigation plan. 
Amendments should be made by mutual agreement between the Investigator and the Sponsor. 
Any amendment to this study clinical investigation plan must be set out in writing, giving the 
reasons, being signed by all concerned parties and will be sent to the EC.  All amendments will 
be sent to the ECs. 
Any amendments that may lead to increased risks and/or inconvenience to the subject must first 
be approved by the EC, if appropriate, and such approval in writing addressed to the Sponsor. 
The amendment becomes then part of the clinical investigation plan. 
Copies of all documents must be stored also by the Investigator/s. Any additional EC 
requirements will be followed as appropriate. 
 
 
 

 
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22.5 Insurance Policy 
 
As the study is non-interventional, there is no need for insurance policies in addition to those 
already provided for normal clinical practice. 
 
22. 
Subject Compliance 
The Subjects’ compliance will be monitored by study staff during visits and evaluated by the 
Investigator. Any subject found not to follow pre-study directions or to be non-compliant, will be 
withdrawn from the study. Details of the reason for removal of subjects will be recorded and 
reported. 
 
23. 
Statistic 
Sample size estimate 
A previous study report baseline level of TFBUT of 6.23±1.75 in patients with moderate dry eye 
disease. After 30 days of treatment with tear eyedrops containing hyaluronic acid 0.3%, TFBUT 
was 8.10 ± 2.06 s [20]. To detect a 25% of increase of TFBUT at Study Termination Visit 
compared to the baseline (Visit 1) (mean difference 1.56, s.d. 1,91), considering an alpha = 0.01 
(two-tailed) and a power of 1 – ß = 0.80, 23 subjects are required (Wilcoxon signed-rank test). 
Taking into account an expected dropout of 20%, it is expected to enroll a total of 28 subjects. 
 
Analysis set 
All patients enrolled will be included in the safety population. 
All patients enrolled having a primary post-baseline performance assessment will be included in 
the PP population and ITT population. 
If the primary performance measurement at Visit 2 is missing, it will be imputed with the score at 
baseline. If the primary performance measurement at Study Termination Visit is missing, it will 
be imputed with the score at Visit 2, if it is not missing, or otherwise with the score at baseline. 
All patients of the ITT population who did not have any major clinical investigation plan deviation 
will be included in the PP population. 
The analysis of safety endpoints will be performed in the safety population. Analysis of 
performance endpoints will be performed on the ITT population. The analysis of primary endpoint 
will be performed on the PP population but this later will be repeated in the ITT population. 
 
Statistical Methods 
Descriptive analysis will be performed by using absolute rate, percentage and frequency tables 
for qualitative variables. 
Normally distributed continuous data will be reported as minimum, maximum, mean and standard 
deviation, whereas non- normally distributed data as medians and IQR. 
Categorical variables will be analyzed by using Yates-corrected chi-square test or Fisher's exact 
test when appropriate. The McNemar's test will be used to analyze paired nominal data. 
Differences in endpoints between baseline and different timepoints will be assessed using paired 

 
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t-test for normally distributed data, whereas the Wilcoxon signed-rank test will be used for non- 
normally distributed data. 
Data distribution will be evaluated through the Kolmogorov-Smirnov test. 
A p-value <0.05 will be considered statistically significant. 
Analysis will be performed using SAS version 9.4 for Windows (SAS Institute Inc., Cary, NC, 
USA). 
 
Primary endpoint analysis 
• 25 % increase from Visit 1 (Day 0 of treatment - baseline) to Study Termination Visit (Day 35 ± 
4 of treatment) of tear film break-up time with fluorescein (TFBUT) will be reported as 
minimum, maximum, mean, standard deviation or median and IQR. According to the data 
distribution, paired t-test or Wilcoxon signed-rank test will be used to test the statistical 
significance of the change from baseline to each time points. [Time frame: Visit 1, and Study 
Termination Visit]. 
 
Secondary endpoint analysis 
 
• Changes in NEI score from baseline will be summarized as minimum, maximum, mean, 
standard deviation or median and IQR. Paired t-test or Wilcoxon signed-rank test will be used 
to test the statistical significance of the change from baseline to each timepoints. [Time frame: 
Visit 1, Visit 2 and Study Termination Visit].  
 
• Change in the SANDE scores will be summarized as minimum, maximum, mean, standard 
deviation or median and IQR. Paired t-test or Wilcoxon signed-rank test will be used to test 
the statistical significance. Details of changes (i.e., difference in SANDE scores between 
baseline and end of treatment) will be summarized as well. [Time frame: Visit 1, Visit 2 and 
Study Termination Visit]. 
 
• VAS will be summarized reporting the number of patients and related percentage in each 
category. [Time frame: Study Termination Visit]. 
 
• Changes in TFBUT from baseline will be reported as minimum, maximum, mean standard 
deviation or median and IQR. Paired t-test or Wilcoxon signed-rank will be used to test the 
statistical significance of the change from baseline to each timepoints. [Time frame: Visit 2, 
and Study Termination Visit]. 
 
• Change in the BVCA scores will be summarized as minimum, maximum, mean, standard 
deviation or median and IQR. Paired t-test or Wilcoxon signed-rank test will be used to test 
the statistical significance. Details of changes (i.e., difference in BVCA scores between 
baseline and end of treatment) will be summarized as well. [Time frame: Visit 1, Visit 2 and 
Study Termination Visit]. 
 

 
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• IGAS will be summarized reporting the number of patients and related percentage in each 
category. [Time frame: Study Termination Visit]. 
 
• Incidents will be coded using the last updated version of the MedDRA dictionary to give a 
preferred term (PT) and a system/organ class term (SOC) for each event. The number of 
patients who experienced at least one incident, study product-related incidents, serious 
incidents, and the number of patients withdrawn due to an incident will be summarized. The 
frequency of incidents will be presented overall, by SOC and PT, and additional grouping by 
severity and relationship to the observed treatment will be performed. [Time Frame: During 
the treatment period]. 
 
• Changes from baseline in IOP will be summarized as minimum, maximum, mean standard 
deviation or 18 median and IQR. Paired t-test or Wilcoxon signed-rank will be used to test 
the statistical significance of the change from baseline to each timepoints. [Time frame: Visit 
1, Visit 2 and Study Termination Visit]. 
 
• Changes from baseline in DEQS score will be summarized as minimum, maximum, mean, 
standard deviation or median and IQR. Paired t-test or Wilcoxon signed-rank test will be 
used to test the statistical significance of the change from baseline to the study termination. 
[Time frame: Visit 1 and Study Termination Visit]. 
 
 
24. 
Data collection – eCRFs 
 
An electronic data capture system will be used for this study. An electronic CRF (eCRF) is 
designed to collect all the data required by the clinical investigation plan and collected by the 
investigator for each patient. 
The Investigator must ensure that the clinical data required by the clinical investigation plan, 
collected during the study, are carefully reported, in the eCRFs. He must also check that the data 
reported in the eCRFs correspond to those in the patients’ source documents. 
Details of eCRF completion and correction will be explained to the Investigator. If the 
Investigator authorizes other persons to make entries in the eCRF, the names, positions, 
signatures, and initials of these persons must be supplied to the Sponsor. 
The Investigator or the designated personnel of their team agrees to complete all documents 
provided by the Sponsor at each patient’s visit and complete the eCRF within 2 days from the 
patient’s visit.  
Any outstanding entries must be completed immediately after the final examination. An 
explanation should be given for all missing data. 
The monitors must make certain that all data are completed on the eCRF and are according to 
source. 
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by entering his/her user’s name and password: 
• the authenticity of the data collected in the eCRF; 
• the coherence between the data in the eCRF and those in the source documents.  
 
The Investigator must keep source documents for each subject in the clinical investigation. All 
information on eCRFs must be traceable to these source documents, which are generally stored 
in the subject’s medical file. The source documents will contain all demographic and medical 
information, as well as the original signed informed consent form. 
The monitor, in accordance with the Sponsor's requirements, should ensure that the trial is 
properly conducted and documented.  
The monitor checks the accuracy and completeness of eCRF entries, source documents, and other 
records related to the trial and notifies (monitoring report) the Sponsor of deviations from the 
clinical investigation plan, SOPs, GCP, and applicable regulatory requirements, taking 
appropriate action to prevent recurrence of identified deviations. 
The monitor should submit a written report to the Sponsor after each trial- site visit or clinical 
investigation-related communication (monitoring report). 
Reports should include a summary of what the monitor reviewed and the monitor's statements 
concerning the significant findings/facts, deviations and deficiencies, conclusions, actions taken 
or to be taken and/or actions recommended to secure compliance. The review and follow-up of 
the monitoring report with the Sponsor should be documented by the Sponsor’s designated 
representative. 
Any change or correction to eCRF should be dated, initialed, and explained (if necessary) and 
should not obscure the original entry.  
The systems are designed to permit data changes in such a way that the data changes are 
documented and that there is no deletion of entered data (i.e. maintain an audit trail, data trail, 
edit trail). 
 
25.1 Unique subject identifier  
All the patients who sign the informed consent form for this study will be coded with “unique 
subject identifiers”. 
The unique subject identifier consists of the 5-digit screening number (e.g. S1001, 
S1002,…S10028 etc.), and, if applicable, the 4-digit subject clinical investigational number (e.g. 
1001, 1002, etc.). Specifically, the first subject study number was referred to the study site. 
Allocation screening number and subject clinical investigational number are separated by slashes 
(e.g. “S101/1001” for site number 1, “S201/2001” for site number 2, “S301/3001” for site number 
3). 
 
Study Sites 
Screening Number 
Subject Clinical 
Investigational 
Number 
SITE Number 1 
S1001, S1002, S1003, 
1001, 1002, 1003, 1004, 

 
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P.O. San Marco, Azienda Ospedaliero 
Universitaria Policlinico "G. Rodolico 
- San Marco" Oculistica,  
Viale Carlo Azeglio Ciampi, 95121 
Catania (Italy) 
Tel: +39 0953781213 
Email:davidescollo@hotmail.com 
 
 
S1004, S1010,  
S1028etc. 
etc. 
SITE Number 2 
U.O. di Oculistica, Presidio Belmonte, 
Azienda Ospedaliero Universitaria 
Policlinico  
“Paolo Giaccone”  
Via Rampolla Mariano Cardinale, 
10B, 90142 Palermo (Italy) 
Tel: +39 091 6553903 
Email: enzabonfiglio@gmail.com 
 
S2001, S2002, S2003, 
S2004, S2028 etc. 
2001, 2002, 2003, 2004, 
etc. 
 
25.2 Coding dictionaries  
Medical/surgical history and underlying diseases, physical examination abnormalities and AEs 
will be coded using the Medical Dictionary for Regulatory Activities (MedDRA™).  
Prior and concomitant medications will be coded using the WHO Drug Dictionary Enhanced 
(WHODDE). Version of coding dictionaries will be stated in the SAP and the study report. Study 
Monitoring, Quality Control and Quality Assurance. 
 
26  Monitoring, Data and Quality Management 
 
 
26.1 Clinical monitoring and identification of source data 
Monitoring is the act of overseeing the progress of a clinical trial and of ensuring that it is 
conducted, recorded and reported in accordance with the clinical investigation plan, SOPs, Good 
Clinical Practice (GCP), and the applicable regulatory requirement(s). 
UNIFARM will designate qualified person(s) to maintain a close liaison with the Investigators 
and study staff to ensure the study is being conducted with respect to GCP and according to the 
approved clinical investigation plan and subsequent amendment(s). 
This liaison will consist of documented visits and follow-up letters, and/or telephone call or Email 
communication, in accordance with monitoring plan, before the start of study and at regular 
intervals during the study to allow for the periodic reviews of the progress of the study.  
UNIFARM is responsible to verify that all incidents have been transmitted to the Sponsor and to 
the Competent Authority and the Ethic Committee if deemed necessary. 

 
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The CRA will explain the clinical investigation plan and study related procedures to all study 
staff, including the Investigator. If new collaborators are included during the clinical 
investigation, additional training sessions will be organized by the Investigator and/or the CRA.  
The main responsibilities of the monitor CRA are to verify Investigators’ adherence to the clinical 
investigation plan and that informed consent is obtained and recorded for all patients prior to 
implementation of any study procedure. 
The nature and location of all source documents will be identified to ensure that all sources of 
original data required to complete the eCRF are known to the Sponsor and participating study‐
site personnel and are accessible for verification by the CRA. 
The CRA will contact and visit the Investigator at regular intervals during the study. He will 
compare the eCRFs with medical records and other relevant documentation through direct access, 
during the on‐site monitoring visits. He verifies the completeness, consistency and accuracy of 
the data being recorded in the CRF by the Investigator. 
The Sponsor expects that, during any monitoring visits, the relevant participating study‐site 
personnel will be available, the source documentation will be accessible, and a suitable 
environment will be provided for review of observational study‐related documents. 
The confidentiality of the subjects' identities shall be well protected consistent with local and 
national regulations when the source documents are subject to direct access. 
The monitor will provide the Investigator with all the study materials before the study starts and, 
during the course of the study, will check at least the following: 
• Full compliance with ICH-GCP, ISO 14155:2020, EU Regulation 745/2017, GDPR, the clinical 
investigation plan and applicable regulatory requirements; 
• Subject recruitment; 
• Subject compliance; 
• Medical Device accountability; 
• Completeness and accuracy of the eCRF data and their consistency with the source documents 
(appropriate source data verification will be conducted on all eCRFs); 
• Verification of the facilities; 
• Investigator's Site File. 
The monitor will ensure completeness of eCRFs on an ongoing basis. 
Study monitor will have access to all records (including source documents when applicable) 
necessary to ensure the integrity of the recorded data. During monitoring visits, the Investigators 
and authorized staff will assist the monitor to check the eCRFs and other documents pertinent to 
the project. 
As part of the supervision of the study progress, other Sponsor personnel or the CRO may, on 
request, accompany the CRA on visits to the study site. The Investigator and his/her collaborators 
commit to cooperate with the CRA to resolve any problems, corrections or possible 
misunderstandings concerning the findings detected in the course of these monitoring visits. 
 
26.2 Source Documents 
Source documents are defined as original documents, data and records. This may include hospital 

 
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records, clinical and office charts, laboratory data/information, pharmacy dispensing and other 
records. Data collected during this study must be recorded on the appropriate source documents. 
 
 26.3 Audit  
The audit is an independent verification, separate from the monitoring activity, of the activities 
and the documents to ensure that the activities pertinent to the study were duly carried out and 
that they were recorded, analyzed and transferred in compliance with the clinical investigation 
plan, GCP, relevant SOPs and with applicable legislation. 
 
 26.4 Inspections 
Authorized representatives of national and foreign Competent Authorities may perform an 
official review of the study site, including, study site and source data verification. The purpose of 
inspection is to systematically and independently examine all study-related activities and 
documents, to determine whether these activities were conducted, and data were recorded, 
analysed, and accurately reported according to the clinical investigation plan, GCP, ICH 
guidelines, and any applicable regulatory requirements. The Investigator/Institution will contact 
immediately the Sponsor if contacted by a regulatory agency about an inspection at the study site. 
 
25. 
Safety Data Evaluation 
All safety data from medical examinations will be documented in the eCRFs, summarized by 
descriptive statistics and the results will be included in the clinical investigation Report. 
 
26. 
Adherence to Clinical Investigation Plan 
Except for an emergency situation in which proper care or medical treatment is required for 
protection, safety and well-being of the subjects, the clinical investigation will be conducted as 
described in the approved clinical investigation plan in accordance with the normal clinical 
practice. Any deviation from the normal clinical practice will be recorded and explained. 
 
27. 
 Clinical Investigation Termination 
• Regular Termination of the clinical investigation 
The observational study completion date is the date on which the last subject is examined or 
receives an intervention for the purpose of final data collection, if the observation period for this 
subject is completed according to the clinical investigation plan. 
• Premature Termination of the clinical investigation 
The Sponsor and Investigator reserve the right to termination the observational study at any time. 
Reasons for discontinuation must be documented appropriately. 
Upon completion of the clinical investigation, the patients will be ensured medical care at the 
same clinical site, according to the times and procedures required by normal clinical practice. 
 
 

 
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28. 
Financing of the Clinical Investigation  
 The financial aspects of this clinical investigation are described in detail in the contract between 
Sponsor and CRO and between CRO and clinical site involved in this study.  
 
29. 
Clinical Investigation Report 
The final Clinical Investigation Report will be written by the CRO and then approved by 
“Sponsor”, and the Principal Investigator. It will be written in compliance with the ISO: 
14155:2020, EU Regulation 745/2017 (Annex XV) and GDPR for both content and format. 
 
30. 
Publication policy  
The Sponsor agrees that the clinical results (including negative and inconclusive as well as positive 
results) can be made publicly available by the Investigator publishing in peer reviewed journals; 
presenting results at scientific congresses; and posting information and results on internet-based 
public registers and databases.  
In any case, study results will be communicated in full to the competent Authorities by the 
submission of a complete Clinical Study Report.  
As the Sponsor agrees that the study results can be published by the Investigator(s), the Investigator 
agrees to submit any manuscript (abstract, publication, paper etc.) to the Sponsor before any public 
disclosure.  
This will be done in order to ensure that clinical trial results are reported in an objective, accurate 
and balanced manner. The Sponsor reviews proposed manuscripts prior to submission within a 
reasonable period of time (30-90 business days in relation with the complexity of the work).  
The Investigator(s) will also be provided by the Sponsor with the clinical study report and the 
results of any additional analysis, tables, figures etc. undertaken for the purposes of the article, in 
order to take responsibility for the content of the publication(s).  
On an exceptional basis, the Sponsor may temporarily delay registration of certain data elements 
(e.g. compound, name, outcome, measures etc.) to seek necessary intellectual property protection. 
This is because early disclosure of such a data could, in some circumstances, prevent or negatively 
impact patentability. 

 
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