FINAL REPORT GLP STUDY Sponsor - ECOSYSTEM …...FINAL REPORT GLP STUDY N 2017/2391 of 25/08/2017 This document cannot be reproduced even in part without written authorization by Complife

MODULO

M POS 04-04A

rev 00 del 02/01/2017

COMPLIFE ITALIA Srl CENTRO di SAGGIO BPL Sede di PAVIA pag. 1 di 12

FINAL REPORT

FINAL REPORT GLP STUDY

Sponsor

Laboratoire ECOSYSTEM

Study Title

In vitro evaluation of the Ocular Irritation Potency - Neutral Red Uptake Assay –

Living Hair Batch: LH237

Study Identification

2017/2391

COMPLIFE ITALIA S.r.l.- CENTRO DI SAGGIO BPL - SEDE DI PAVIA FINAL REPORT GLP STUDY N° 2017/2391 of 25/08/2017

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This document cannot be reproduced even in part without written authorization by Complife Italia S.r.l.

M POS 04-04A Relazione Finale_rev00 pag 2 di 12

INDEX

1 STUDY INFORMATION pag. 3

2 STATEMENT OF COMPLIANCE TO GOOD LABORATORY PRACTICE PRINCIPLES pag. 4

3 RQAU STATEMENT pag. 5

4 IDENTIFICATION OF THE STUDY, TEST ITEM AND REFERENCE ITEM pag. 6

5 INFORMATION ABOUT SPONSOR AND TEST FACILITY pag. 6

6 EXPERIMENTAL STARTING AND COMPLETION DATES pag. 7

7 DESCRIPTION OF MATERIALS pag. 7

8 TEST METHOD pag. 8

9 RESULTS pag. 10

10 CONCLUSIONS pag. 10

11 AMENDMENTS AND DEVIATIONS TO THE STUDY PLAN pag. 11

12 ARCHIVES pag. 11

13 REFERENCES pag. 12

14 RAW DATA EXTRACT pag. 12


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1. STUDY INFORMATION

SPONSOR Laboratoire ECOSYSTEM ZI-Rue René Pinsard 45310 Patay – France Sponsor representative: Frédéric Pottecher

TEST FACILITY COMPLIFE ITALIA Srl – Sede di PAVIA Address: Via Angelini 21, 27100 Pavia

Study Director: Dr. Silvana Giardina Test Facility Manager: Dr. Angela Michelotti Quality Assurance Responsible: Dr. Francesco Tursi

STUDY PROGRAM Starting study date: 27/07/2017 Proposed experimental starting date: 02/08/2017 Proposed experimental ending date: 04/08/2017 Proposed Final Report editing date: 25/08/2017

STUDY PROGRAM APPROVAL / VERIFICATION / ACKNOWLEDGEMENT Study Director: Dr. Silvana Giardina, 27/07/2017

Responsabile dell’Assicurazione Qualità: Dr. Francesco Tursi, 27/07/2017

Direttore del Centro di Saggio: Dr. Angela Michelotti, 27/07/2017

Rappresentante del Committente: Frédéric Pottecher 02/08/2017


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2. STATEMENT OF COMPLIANCE TO GOOD LABORATORY PRACTICE

The undersigned Silvana Giardina, as a Study Direct of the Test Facility COMPLIFE ITALIA Srl - Sede di PAVIA, declares that the study described in the present Report has been carried out by myself and/or under my direct supervision in compliance with the requirements of Decreto Legislativo n.50 del 2 Marzo 2007 (recepting the Directive 2004/09/CE and 2004/10/CE concerning the inspection and verification of Good Laboratory Practice and harmonization of laws, regulations and administrative provisions relating to the application of the principles of good laboratory practice and the verification of their applications for tests on chemical substances) The study has been carried out at the Test Facility COMPLIFE ITALIA Srl - Sede di PAVIA, recognized by the Certificate of Compliance with the Principles of Good Laboratory Practice n. 2017/7 dated 20/02/2017.

San Martino Siccomario, 25/08/2017

Study Director Dr. Silvana Giardina


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3. RQAU STATEMENT The undersigned Francesco Tursi, responsible of the Quality Assurance Unit (RQAU) of the Test Facility COMPLIFE ITALIA Srl - Sede di PAVIA, declares that the present study has been carried out according the procedure described in the GLP Study Plan N° ……….…/…………. and in compliance with to the principles of Good Laboratory Practices (GLP). Such principles are in agreement with the Italian legislation according to the Legislative Decree n.50 of 2nd March 2007,(enforcement of Directive 2004/9/CE and 2004/10/CE on the on the inspection and verification of Good Laboratory Practice and the harmonization of laws, regulations and administrative provisions relating to the application of the principles of Good Laboratory Practice and the verification of their applications for tests on chemical substances) and with OCSE Principles concerning GLP, and fulfills the requirements of all OECD member states for a mutual acceptance. All phases of the study not described in the present Study Plan have been carried out according to Standard Operating Procedures (SOP) of the Test Facility. RQAU as responsible of the study monitoring and in order to assure the conformity to the GLP principles declares that: - he has conducted inspections on the present study at the data indicated below and he has verified:

- the compliance to BPL of the Study Plan …....../…....../…......,

- the availability of Study Plan …....../…....../…......, - the compliance of raw data recording ......./…...../…….., - the compliance of to the study plan …...../…...../….....,

- he has reported inspections results in writing to the Director of Test Facility and to the Study Director on ……. /…...…/….....,

- he has examined on …….../….…../…..… the present Final Report ;

- he has reported on ……../.…..../..….. the results of inspection of the Final Report to the Director of Test Facility and to the Study Director.

San Martino Siccomario, …………………….

Quality Assurance Responsible

Dr. Francesco Tursi


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4. IDENTIFICATION OF THE STUDY, TEST ITEM AND REFERENCE ITEM

4.1 TITLE OF STUDY In vitro evaluation of the ocular irritation potency – Neutral Red Uptake Assay


This study has been carried out for regulatory purposes according to Regulation (EC) n° 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products (Text with EEA relevance) and to its annexes.

4.2 IDENTIFICAZION OF THE TEST ITEM Living Hair Batch: LH237

Arrived on 14/07/2017 Storage condition RT

Tested product is a shampoo, with the following INCI: PFAFFIA PANICULATA EXTRACT, AVENA SATIVA, COCOS NUCIFERA OIL, PERSEA GRATISSIMA OIL, ILLITE, SODIUM COCOYL ISETHIONATE, SODIUM COCO SULFATE, HYDROLYZED RICE PROTEIN

4.3 IDENTIFICAZION OF THE REFERENCE ITEM Sodium Dodecyl Sulfate (SDS) in culture medium

5. INFORMATION ABOUT SPONSOR AND TEST FACILITY

5.1 SPONSOR IDENTIFICATION

Laboratoire ECOSYSTEM ZI-Rue René Pinsard 45310 Patay – France Sponsor representative: Frédéric Pottecher

5.2 IDENTIFICATION OF ALL TEST FACILITIES INVOLVED IN THE STUDY

COMPLIFE ITALIA Srl – Sede di PAVIA Address: Via Angelini 21, 27100 Pavia

5.3 NAME AND ADDRESS OF STUDY DIRECTOR

Dr. Silvana Giardina, c/o COMPLIFE ITALIA – Sede di PAVIA


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6. EXPERIMENTAL STARTING AND COMPLETION DATE

6.1 EXPERIMENTAL STARTING DATE

02/08/2017

6.2 EXPERIMENTAL COMPLETION DATE

04/08/2017

7. DESCRIPTION OF MATERIALS

Reference items Sodium Dodecyl Sulfate (SDS) Test system BALB/c 3T3 cells

Reagents NR (Neutral Red) SDS (Sodium Dodecyl Sulphate) Ethanol Acetic acid Deionized water DMEM high glucose Foetal bovine serum Penicillin-Streptomycin L-glutamine

Trypsin-edta solution PBS

Instruments Personal protective equipment Cell incubator 37°C, 95% RH, 5% CO2 RH BIOTECH Laminar flow cabin with HEPA filters TUV ASALAIR 1200 Optical microscope OPTIKA Well plates Steril tubes (0.5, 2, 15 e 50 ml) Sierologial pipettes (5 e 10 ml) Automatic pipettor HAMILTON Mechanical micropipettes Tweezers Scissors Analytical balance KERN ALJI60-4NM Magnetic mixer GDV Plate reader GDV DV990 BV6

Preparation of test item The product was tested by dilution (8 concentrations were tested) in culture medium.


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8. TEST METHOD

TESTING PROCEDURE

BALB/c 3T3 cells were seeded and exposed to tested product in a concentration range. After 24h incubation, Neutral Red Uptake in the lysosomes/endosomes and in vacuoles of living cells was measured and used as quantitative indicator of cell number and viability.

1st DAY: BALB/c 3T3, clone 31 (ATCC CCL-163) was seeded in 96-well plate at 1 x 104 cells/well and maintained for 24h (37°C, 95% RH, 5% CO2).

2nd DAY: after 24 h incubation, medium was removed and new medium containing tested product, positive controls was added to the wells. Plates were incubated for 24h (37°C, 95% RH, 5% CO2). Cells treated with culture medium were used as negative control.

3rd DAY: cells were observed at microscope for a visual evaluation, then, medium, sample, positive and negative control were removed. Cells were washed with PBS and new medium containing Neutral Red (NR) was added to the wells. Cells were incubated for 3h (37°C, 95% RH, 5% CO2). After incubation, NR-medium was removed and cells washed with PBS and solubilising solution (ETOH/acetic acid/deionized water) was added to the wells. Plate was shaked for 10 minutes and then red at 540 nm.

QUALITY ASSURANCE OF THE RESULTS

Positive control Sodium Dodecyl Sulfate is the most frequently used positive control, with an historical NR50 value of 0.093 mg/ml. The test complies the quality criteria if NR50 value of the positive control is between 0.070 – 0.116 mg/ml (confidence interval of 95%).

Negative control Negative control is represented by the culture medium without tested product and that is subjected to the same treatment of tested product. The use of a negative control in a toxicity test allows to evaluate the possible aspecific effects of reagents and dye. The absolute Optical density value (OD540 of NR) obtained in the negative control reading shows that the cells seeded in the wells grow exponentially with a normal division time. The test complies the quality criteria if OD540 value of the negative control reading is ≥ 0.3.

RESULTS PRESENTATION AND CALCULATION

Evaluation of cytotoxicity has been carried out by cell viability determination. It is assumed that Negative control represents the 100% viability. For each sample and/or dilution, for the positive control cell viability was calculated according to the following formula:

% Cell viability = 100 * OD540 treated cells/OD540 negative control

Lower is the % cell viability, higher is the product cytotoxic potential.


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The product classification is performed according to COLIPA model derived by a validation study:

Endpoint value Modified Maximum Average Score (MMAS)

Classification

NR50 > 1000 µg/ml < 25 Not/mild irritant

15 µg/ml < NR50 < 1000 µg/ml 80.46/log10 NR50 Moderate irritant

NR50 < 15 µg/ml > 60 Strong irritant


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9. RESULTS

NEGATIVE CONTROL OD540= 1.288 ± 0.035

POSITIVE CONTROL (SDS)

NR50= 0.100 ± 0.0020 mg/ml


NR50 = 214.97 ug/ml MMAS = 34.50

10. CONCLUSIONS

According to obtained results, the product


is MODERATE IRRITANT


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11. AMENDMENTS AND DEVIATION TO THE STUDY PLAN

No amendment or deviation.

12. ARCHIVES

Documents and materials Archives Responsable Retention period

Study plan (signed copy) BPL Archive Study director 10 years

Raw data BPL Archive RQAU 10 years

Final Report BPL Archive Study director 10 years

Test item and reference item BPL Archive Study director 5 years

Records of all inspections performed by the quality assurance program

BPL Archive RQAU 10 years

Records of qualifications, training, experience and job descriptions of personnel

BPL Archive Test Facility Manager

10 years

Records and reports of the maintenance and calibration of apparatus

BPL Archive RQAU 10 years


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13. REFERENCES

- Invittox BALB/c 3T3 Neutral Red Uptake (NRU) Cytotoxicity Test - Protocol n°: 46. - Botham P.A., Earl L.K., Fentem J.H., Roguet R., van deSandt J.J.M. (1998) Alternative methods for skin irritation testing: the current status. ATLA 26: 195-211. - Ohno T., Futamura Y., Harihara A., Hatao M., Hayasaka A. (1998) Validation study on five cytotoxicity assays by JSAAE- VIII. Details of the neutral red uptake assay. Alternatives to animal testing and experimentation, 5:131-145. - Balls M. Botham P.A., Bruner L.H., spielmann H. (1995) The EC/HO international validation study on alternatives to the Draize eye irritation test. Toxicol. In vitro, 9: 871-929. - Borenfreund, E. and Puerner, J. (1985). Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24:119-124. - Mossman, T. (1993). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63. - Seibert, H., Balls, M., Fentem, J.H., Bianchi, V., Clothier, R.H., Dierickx, P.J., Ekwall, B., Garle, M.J., Gòmez-Lechòn, M.J., Gribaldo, L., Gülden, M., Liebsch, M., Rasmussen, E., Roguet, R., Shivrastava, R. and Walum, E. (1996). Acute toxicity testing in vitro and the classification and labelling of chemicals. ATLA 24:499-510.

14. RAW DATA EXTRACT

abs abs media dev.st. vitalità %

CTR- 1,276 1,278 1,288 0,037 100,00%

1,306 1,256 3,65%

1,333 1,254

1,347 1,255

mg/ml abs vitalità % NR50 NR50 medio= 0,100

CTR+ 0,25 0,1 0,095 7,76% 7,38% 0,099 dev.st.= 0,0020

SDS 0,125 0,246 0,286 19,10% 22,20% 0,102

0,0625 1,197 1,242 92,93% 96,42%

0,03125 1,254 1,296 97,35% 100,61%

ug/ml abs vitalità % MEDIA

BPL/2017/2391 2000,0 0,126 0,11 9,78% 8,54% 9,16% NR50 NR50 medio= 214,97

1000,0 0,109 0,113 8,46% 8,77% 8,62% 212,00 dev.st.= 4,20

500,0 0,189 0,153 14,67% 11,88% 13,28% 217,94

250,0 0,368 0,408 28,57% 31,67% 30,12%

125,0 1,257 1,284 97,58% 99,68% 98,63% MMAS MMAS medio= 34,50

62,5 1,268 1,314 98,44% 102,01% 100,22% 34,59 dev.st.= 0,13

31,3 1,28 1,275 99,37% 98,98% 99,18% 34,41

15,6 1,26 1,299 97,82% 100,84% 99,33%