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RAPPORTO DI PROVA 14/000031110
Data di emissione
data di emissione 31/01/2014
Codice intestatario
Spett.le
WORLD WORK SRL
Via del Progresso, 47
36054 MONTEBELLO
VICENTINO (VI)
IT
0069864
Dati campione
Numero di accettazione
13.037479.0001
Consegnato da
The Courier il 29/10/2013
Data ricevimento
29/10/2013
Proveniente da WORLD WORK SRL Via del Progresso, 47 36054 MONTEBELLO VICENTINO (VI) IT
Descrizione campione CAMPIONE CANNULE ASPIRASALIVA MONOUSO TRASPARENTI LOTTO573/13
Dati campionamento
Campionato da
Cliente
RISULTATI ANALITICI
Valore
U.M.
LoQ
LoD
Data inizio
fine analisi
Unità Riga
op.
SUL CAMPIONE TAL QUALE
TEST DI CITOTOSSICITA' IN VITRO
Met.: UNI EN ISO 10993­5:2009
1
vedasi relazione
12/11/2013­
09
2
­28/01/2014
Unità Operative
Unità 09 : Via Fratta Resana PHARMA (TV)
Responsabile prove biologiche
Direttore laboratorio
Dott.ssa Federica Cattapan
Dott. Tiziano Conte
Ordine nazionale dei biologi
Albo professionale n.045961 sez.A
Chimico
Ordine dei chimici ­ Provincia di treviso
Iscrizione n. 148
­ LoD: limite di rilevabilità, individua un intervallo di confidenza dello zero ad un livello di probabilità del 99%. ­ LoQ: limite di quantificazione; "n.r.": non rilevato, indica un valore inferiore
a LoD; "tracce (x)": indica un valore compreso tra LoD e LoQ, tale valore è puramente indicativo; "<x" o ">x" indicano rispettivamente un valore inferiore o superiore al campo di misura
della prova. ­ Se non diversamente specificato, le sommatorie sono calcolate mediante il criterio del lower bound (L.B.). ­ Iscrizione al numero 7 dell'elenco regionale della Regione
Veneto dei laboratori che effettuano analisi nell’ambito delle procedure di autocontrollo delle industrie alimentari, come da Allegato A del DDR n. 73 del 16 gennaio 2008. ­ Se non
diversamente specificato le prove microbiologiche quantitative (esclusi MPN) sono eseguite su singola replica e due diluizioni consecutive conforme alla ISO 7218:2007.
Modello 756/SQ rev. 3
Pagina 1
di 1
Documento con firma digitale avanzata ai sensi della normativa vigente.
I risultati contenuti nel presente Rapporto di prova si riferiscono esclusivamente al campione oggetto di analisi. Il presente Rapporto di prova non può essere riprodotto
parzialmente, salvo autorizzazione scritta di Chelab.
Chelab S.r.l, a Mérieux NutriSciences company
Head office: Via Fratta 25 31023 Resana, Italy Phone. + 39 0423.7177 / Fax + 39 0423.715058 www.chelab.it
VAT nr. 01500900269, R.E.A Treviso n. 156079 Fully paid up € 103.480,00.
Enclosed to the test reports number: 14/000031110
FINAL REPORT 13.037479.01
TEST FOR IN VITRO CYTOTOXICITY,
ACCORDING TO ISO 10993-5:2009
Pag. 1 di 12
Chelab Silliker
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parzialmente, salvo autorizzazione scritta di Chelab
Enclosed to the test reports number: 14/000031110
Sponsor:
Test Facility:
WORLD WORK SRL
CHELAB SILLIKER
Via del Progresso, 47
Via Fratta, 25
36054 MONTEBELLO VICENTINO (VI)
31023 Resana (TV)
IT
ITALY
SUMMARY
1. STUDY PURPOSE .................................................................................................................................. 3 2. REFERENCE ITEM .................................................................................................................................. 3 3. TEST SYSTEM ....................................................................................................................................... 3 4. REAGENTS AND REFERENCE SOLUTIONS ................................................................................................ 4 5. MATERIALS AND APPARATUS ................................................................................................................ 4 6. CULTURING OF CELL LINE ...................................................................................................................... 5 6.1 THAWING .......................................................................................................................................... 5 6.2 MAINTENANCE OF CELL CULTURES .................................................................................................... 5 PREPARATION OF TEST SOLUTIONS ....................................................................................................... 5 7 7.1 TEST SUBSTANCE .............................................................................................................................. 5 7.2 POSITIVE CONTROL ........................................................................................................................... 6 7.3 NEGATIVE CONTROL (VEHICLE CONTROL VC) .................................................................................... 6 7.4 REAGENT CONTROL (OR SUPPORT CONTROL) ..................................................................................... 6 PROCEDURE ......................................................................................................................................... 6 8 8.1 NRU TEST ......................................................................................................................................... 6 8.2 MICROSCOPE OBSERVATION .............................................................................................................. 8 9 DATA ELABORATION .............................................................................................................................. 8 10 VALIDATION OF THE ANALYTICAL METHOD .......................................................................................... 9 11 RESULTS ......................................................................................................................................... 10 11.1 ACCEPTANCE CRITERIA ................................................................................................................... 10 Pag. 2 di 12
Chelab Silliker
I risultati contenuti nel presente documento si riferiscono esclusivamente al campione oggetto di analisi. Il presente documento non può essere riprodotto
parzialmente, salvo autorizzazione scritta di Chelab
Enclosed to the test reports number: 14/000031110
11.2 RESULTS OF THE TEST ..................................................................................................................... 10 12 REFERENCE AND GUIDELINES .......................................................................................................... 12 13 CONCLUSIONS ................................................................................................................................. 12 1.
STUDY PURPOSE
The purpose of the analysis is to assess the in vitro cytotoxicity of:
CAMPIONE CANNULE ASPIRASALIVA MONOUSO TRASPARENTI LOTTO573/13
2.
REFERENCE ITEM
Name: Sodium dodecyl sulfate (SDS)
Ref: 71729
Supplier: Sigma
3.
TEST SYSTEM
Cell line: BALB\3T3 Clone A31 (recommended by Annex A of ISO 10993-5:2009)
ATCC CCL-163
NIP 46
Embryo Fibroblast
Mouse
Doubling time: 24 hours
Culture conditions: 37 °C  1 °C, 5 % CO2, 90 %  10% relative humidity
Growth medium: D-MEM with 10 % Newborn Calf Serum (NCS) and 4 mM L-Glutamine
Treatment medium: D-MEM with 5 % Newborn Calf Serum (NCS) and 4 mM L-Glutamine
Pag. 3 di 12
Chelab Silliker
I risultati contenuti nel presente documento si riferiscono esclusivamente al campione oggetto di analisi. Il presente documento non può essere riprodotto
parzialmente, salvo autorizzazione scritta di Chelab
Enclosed to the test reports number: 14/000031110
4.
REAGENTS AND REFERENCE SOLUTIONS
-
Acetic Acid, Glacial. VWR 1.018.302.500
-
Purified Agar. Oxoid LP0028
-
ddH2O. Autoclaved at 121 °C for 15 minutes
-
Dulbecco's Modified Eagle's Medium (D-MEM) High Glucose. Sigma D6546
-
Dulbecco's Phosphate Buffered Saline (D-PBS) without Ca2+/Mg2+. Sigma D8537
-
Dulbecco's Phosphate Buffered Saline (D-PBS) with Ca2+/Mg2+. Sigma D8662
-
Ethanol, absolute. VWR 20821321
-
L-Glutamine. Sigma G7513
-
Neutral Red Solution. Sigma N2889
-
Newborn Calf Serum. Sigma N4637
-
Penicillin-Streptomycin BioReagent. Sigma P0781
-
Trypsin-EDTA Solution (1X). Sigma T3924
All reagents employed directly with the cells were bought sterile and kept sterile during the test.
5.
MATERIALS AND APPARATUS
-
Autoclave system 121 °C ± 1 °C, 15 min, SRA 12
-
Analytical balance (± 0.0001 g), SRA 48
-
Technical balance (± 0.01 g), SRA 46
-
Stirrer, SRA 39
-
Incubator: 37 °C ± 1 °C, 90 % ± 10 % RH and 5 % ± 1 % CO2/air, SRA 29
-
Centrifuge, SRA 79
-
Microplate reader (Beckmann), SRA 37
-
25 mL, 10 ml, 5 ml, 2 ml, 1 ml (nominal value) graduate pipette
-
0-20 µl (SRA 88), 20-200 µl (SRA 232) and 200-1000 µl (SRA 99) automatic pipette
-
Thermostatic bath 37 °C ± 1 °C, SRA 96
-
Sterile disposable filters 0.22 µm and 0.45 µm porosity
-
Vortex mixer
Pag. 4 di 12
Chelab Silliker
I risultati contenuti nel presente documento si riferiscono esclusivamente al campione oggetto di analisi. Il presente documento non può essere riprodotto
parzialmente, salvo autorizzazione scritta di Chelab
Enclosed to the test reports number: 14/000031110
-
Sterile tubes
-
Laminar flux hood, SRA 21
-
Burker counting chamber
-
Various laboratory glassworks and tools
-
Phase contrast microscope (Zeiss), SRA 185
All glassware employed directly with the culture media, the reagents or with the sample were sterilized in
autoclave at 121 °C for 15 min.
6.
CULTURING OF CELL LINE
6.1 THAWING
The cryovials containing the cell lines were removed from the liquid nitrogen tank. The entire volume was
transferred into the appropriate culture medium and spinned for 5 minutes at 200 g. Surnatant was
discarded and cell pellet was resuspended in fresh culture medium. Finally, cells were transferred into
flasks containing growth medium and incubated at 37 °C ± 1 °C, 5 % CO2, 90 ± 10 % RH.
6.2 MAINTENANCE OF CELL CULTURES
The cell cultures were incubated at 37 °C ± 1 °C in humidified atmosphere with 5 % CO2. At confluence
the cells were trypsinized, counted on a Burker chamber and passaged into new flasks at the desired
concentration.
7
PREPARATION OF TEST SOLUTIONS
7.1 TEST SUBSTANCE
An extract was obtained by adding 25.5 ml of treatment medium to 5.10 g of sample, to reach the
extraction ratio of 0.2 g/ml (w/V) (see UNI EN ISO 10993-12:2009, Table 1, par. 10.3.3).
The sample was incubated at 37 °C ± 1 °C for 72 hours under constant agitation (according to UNI EN
ISO 10993-12:2009, Par. 10.3.3). Afterwards, 1: 6√10 serial dilutions of the eluate were made in
treatment medium. Note that the obtained eluate was not filtered (see UNI EN ISO 10993-12:2009, par.
10.3.9).
The following dilutions were selected for analysis: 100 % (neat extract) , 68 %, 46.3 %, 31.5 %, 21.4 %,
14.6 %, 9.9 % and 6.7 %.
Pag. 5 di 12
Chelab Silliker
I risultati contenuti nel presente documento si riferiscono esclusivamente al campione oggetto di analisi. Il presente documento non può essere riprodotto
parzialmente, salvo autorizzazione scritta di Chelab
Enclosed to the test reports number: 14/000031110
7.2 POSITIVE CONTROL
An SDS solution 0,5 mg/ml will be obtained in treatment medium. Dilutions will be made in treatment
medium, with a dilution factor of 1,47 (6√10).
The following dilutions were selected for analysis: 0,5 mg/ml, 0,34 mg/ml, 0,23 mg/ml, 0,16 mg/ml, 0,11
mg/ml, 0,07 mg/ml, 0,05 mg/ml and 0,03 mg/ml.
7.3 NEGATIVE CONTROL (VEHICLE CONTROL VC)
12 wells were used to control the treatment medium.
7.4 REAGENT CONTROL (OR SUPPORT CONTROL)
To the same support employed to produce the eluate of the sample, 10 ml of treatment medium were
added. The support was then incubated at 37 °C for 72 hours under constant agitation (according to ISO
10993-12:2009, Par. 10.3.3).
8
PROCEDURE
8.1 NRU TEST
Cells were trypsinized and counted on a Burker chamber.
-
1x104 cells/well were seeded in 60 wells of 96-well plates (i.e. 100 µl of a 1x105 cell/ml cell
suspension); in the peripheral wells only 100 µl of Growth medium (=blanks) were dispensed (see
Table 1).
Table 1: Seed plan
Plate
1
2
3
4
5
6
7
8
9
10
11
12
A
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank
B
Blank
cells
cells
cells
cells
cells
cells
cells
cells
cells
cells
Blank
C
Blank
cells
cells
cells
cells
cells
cells
cells
cells
cells
cells
Blank
D
Blank
cells
cells
cells
cells
cells
cells
cells
cells
cells
cells
Blank
E
Blank
cells
cells
cells
cells
cells
cells
cells
cells
cells
cells
Blank
F
Blank
cells
cells
cells
cells
cells
cells
cells
cells
cells
cells
Blank
G
Blank
cells
cells
cells
cells
cells
cells
cells
cells
cells
cells
Blank
Pag. 6 di 12
Chelab Silliker
I risultati contenuti nel presente documento si riferiscono esclusivamente al campione oggetto di analisi. Il presente documento non può essere riprodotto
parzialmente, salvo autorizzazione scritta di Chelab
Enclosed to the test reports number: 14/000031110
H
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank
-
Plates were incubated for 24 h (37 °C; 5 % CO2), to 50 % confluence of the cells.
-
Cells were treated with 100 µl of the test substance (see 7.1), according to the following plate design
(see Table 2) and incubated for 24 h (37 °C; 5 % CO2). In column 1, 2, 11 and 12 only Treatment
medium was placed. In columns from 3 to 10, serial dilutions of the sample were placed.
Table 2: Treatment plate
Plate
A
1
Blank
2
Blank
VC1
3
Blank
C1
4
Blank
C2
5
Blank
C3
6
Blank
C4
7
Blank
C5
8
Blank
C6
9
Blank
C7
10
Blank
C8
11
Blank
VC2
12
Blank
B
C
D
E
F
G
Blank
Blank
Blank
Blank
Blank
Blank
VC1
VC1
VC1
VC1
VC1
VC1
C1
C1
C1
C1
C1
C1
C2
C2
C2
C2
C2
C2
C3
C3
C3
C3
C3
C3
C4
C4
C4
C4
C4
C4
C5
C5
C5
C5
C5
C5
C6
C6
C6
C6
C6
C6
C7
C7
C7
C7
C7
C7
C8
C8
C8
C8
C8
C8
VC2
VC2
VC2
VC2
VC2
VC2
Blank
Blank
Blank
Blank
Blank
Blank
H
Blank
Blank
VC1
Blank
C1
Blank
C2
Blank
C3
Blank
C4
Blank
C5
Blank
C6
Blank
C7
Blank
C8
Blank
VC2
Blank
-
Wells were washed with 150 µl of D-PBS with Ca2+/Mg2+.The washing solution was removed by
gentle tapping.
-
A 50 μg/ml Neutral Red solution (NR) (3-amino-7-dimethylamino-2-methylphenazine hydrochloride,
CAS number 553-24-2) was prepared in medium without serum.
-
100 µl of the 50 μg/ml NR were added in each well, and the plate was incubated for 3 hours (37 °C;
5 % CO2).
-
After incubation, the NR medium was removed, and wells were washed with 150 µl of D-PBS with
Ca2+/Mg2+. Excess buffer was decanted and removed by gentle tapping.
-
150 µl of NR desorb solution (freshly prepared as 49 parts water + 50 parts ethanol + 1 part acetic
acid) were added.
-
Plates were gently shaken for 10 min in darkness.
-
Optical density of the NR extract at 540 nm was measured in a spectrophotometer.
Pag. 7 di 12
Chelab Silliker
I risultati contenuti nel presente documento si riferiscono esclusivamente al campione oggetto di analisi. Il presente documento non può essere riprodotto
parzialmente, salvo autorizzazione scritta di Chelab
Enclosed to the test reports number: 14/000031110
8.2 MICROSCOPE OBSERVATION
After 24 hours of cell treatment with the test substances and prior to proceed with cellular staining with
Neutral Red, 96-well plates were observed under a phase contrast microscope (Zeiss). Morphological
abnormalities shown by the cells, caused by the toxicity of the test substance, were summarized and
described using the following numerical code (see ISO 10993-5:2009, Par. 8.5.1, Table 2):
Table 3: Qualitative morphological grading of cytotoxicity
GRADE
REACTIVITY
0
None
CONDITIONS OF ALL CULTURES
Discrete intracytoplasmatic granules, no cell lysis, no reduction of cell
growth.
Not more than 20 % of the cells are round, loosely attached and without
1
Slight
intracytoplasmatic granules, or show changes in morphology; occasional
lysed cells are present; only slight growth inhibition observable.
Not more than 50 % of the cells are round, devoid of intracytoplasmatic
2
Mild
granules, no extensive cell lysis; not more than 50 % growth inhibition
observable.
Not more than 70 % of the cell layers contain rounded cells or are lysed; cell
3
Moderate
layers not completely destroyed, but more than 50 % growth inhibition
observable.
4
9
Severe
Nearly complete or complete destruction of the cell layers.
DATA ELABORATION
Data elaboration was performed using the validate data form (Mod. 102/GxP rev.2 Test for in vitro
cytotoxicity according to ISO 10993-5:2009).
Absorbance at 540 nm was measured for every 96-well plate (OD) (i.e. test sample and positive control),
and rounded to the third decimal digit (OD rounded).
Pag. 8 di 12
Chelab Silliker
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parzialmente, salvo autorizzazione scritta di Chelab
Enclosed to the test reports number: 14/000031110
The mean of blanks (Mean Blank) (values in column 1 and 12 in Table 2) was calculated. For each
column from 3 to 11, the mean of the highest and lowest cells was calculated (see Table 2) to verify that
the residual sample does not interfere with the absorbance reading.
A correction was made, subtracting the mean of blanks to each absorbance value (OD corrected).
The mean of the absorbances of Vehicle control (VC1 and VC2), after the correction, was calculated
(Mean Vehicle Control).
Standard deviation (SD) was also calculated for each parameter.
Relative cell viability (RV) was calculated as follows:
(OD corrected / Mean Vehicle Control) · 100
Cellular viability was expressed in terms of percentage relative to the viability of the Vehicle Control (cell
viability in the Vehicle control = 100 %). Variation coefficient (% CV) was calculated as follows: SD of the
Relative Viability / Mean Relative Viability · 100.
Average values of the VC on the left (VC1) and on the right (VC2) was subtracted from Mean Vehicle
Control, to verify that the acceptance criterion is met.
Viability data for the test sample and the controls were then analyzed using the statistical analysis
software Phototox 2.0 in order to obtain the IC50 value. This software is validated by OECD for the
phototoxicity analysis, which includes an IC50 calculation; this parameter was also calculated in this
cytotoxicity test, but it was not relevant to define whether the test substance is cytotoxic or not.
All data were expressed with 3 decimal digits, except the Relative viability values, which shows 1
decimal digit. All data were rounded as follows: if the last digit is < 5, data were rounded down; if the last
digit is ≥ 5, data were rounded up.
Data elaboration will be performed using the validate data form.
10 VALIDATION OF THE ANALYTICAL METHOD
Negative control was used to verify the absence of cytotoxicity of the reagents used in the test, as well
as cell viability.
Positive control was employed to verify cell sensitivity to cytotoxic substances.
Pag. 9 di 12
Chelab Silliker
I risultati contenuti nel presente documento si riferiscono esclusivamente al campione oggetto di analisi. Il presente documento non può essere riprodotto
parzialmente, salvo autorizzazione scritta di Chelab
Enclosed to the test reports number: 14/000031110
11 RESULTS
11.1 ACCEPTANCE CRITERIA
-
Mean OD of VC > 0.3
-
IC50 for positive control (SDS) is within the 95 % confidence interval (0.070 mg/ml – 0.116 mg/ml,
see Annex A, ISO 10993-5:2009, Par. A.2.3.2),
-
The average value of the VC on the left and on the right do not differ by more than 15 % from the
mean of all VC (Mean RV VC – Mean RV VC1 and Mean RV VC – Mean RV VC2 < 15 %).
11.2 RESULTS OF THE TEST
Table 4: Data collected for the NRU test on BALB cells treated with the test sample 13.037479.01
CONCENTRAZIONE
OSSERVAZIONE AL
OD CORR
%
MEDIA VC
MICROSCOPIO
Media SD
0,705 0,023
Media
0
100,0
0
0,692 0,045
98,2
6,4
68,0
0
0,748 0,059
106,1
8,4
46,3
0
0,753 0,072
106,8
10,2
31,5
0
0,733 0,044
103,9
6,2
21,4
0
0,667 0,041
94,6
5,9
14,6
0
0,682 0,029
96,7
4,1
9,9
0
0,692 0,029
98,2
4,1
6,7
0
0,693 0,025
98,2
3,5
VITALITA' RELATIVA
SD
100
Mean OD corrected of VC= 0.705 > 0.3
Mean RV VC – Mean RV VC1 = -1.1% < 15 %
Mean RV VC – Mean RV VC2 = 1.2% < 15 %
Relative viability of the highest concentration >70 % of the control group.
Pag. 10 di 12
Chelab Silliker
I risultati contenuti nel presente documento si riferiscono esclusivamente al campione oggetto di analisi. Il presente documento non può essere riprodotto
parzialmente, salvo autorizzazione scritta di Chelab
Enclosed to the test reports number: 14/000031110
Table 5: Data collected for the NRU test on BALB cells treated with the SDS
CONCENTRAZIONE
OSSERVAZIONE AL
MICROSCOPIO
mg/ml
MEDIA VC
0,5
0,34
0,23
0,16
0,11
0,07
0,05
0,03
0
4
4
4
4
2
1
0
0
VITALITA'
RELATIVA
OD CORR
Media
0,442
0,001
0,001
-0,003
-0,002
0,138
0,317
0,400
0,454
SD
0,020
0,001
0,003
0,001
0,001
0,011
0,015
0,029
0,014
Media
SD
100
0,1
0,2
0,2
0,6
-0,7
0,2
-0,4
0,3
31,3
2,5
71,8
3,4
90,4
6,6
102,7
3,2
Mean OD corrected of VC 0.442 > 0.3
Mean RV VC – Mean RV VC1 = -1.4 % < 15 %
Mean RV VC – Mean RV VC2 = 1.2 % < 15 %
IC50 = 0.097mg/ml (between 0.070 mg/ml – 0.116 mg/ml)
Table 6: Data collected for the NRU test on BALB cells treated with negative and reagent controls
OD CORR
OSSERVAZIONE AL
MICROSCOPIO
Media
SD
VITALITA' RELATIVA
Media
SD
MEDIA VC
0
0,677 0,048
C REAGENT
0
0,676 0,058
99,8
8,6
C NEGATIVE
0
0,725 0,050
107,1
7,4
100
Mean OD corrected of VC =0.677 > 0.3
Mean RV VC – Mean RV VC1 = 4.1% < 15 %
Mean RV VC – Mean RV VC2 = - 4.2% < 15 %
Pag. 11 di 12
Chelab Silliker
I risultati contenuti nel presente documento si riferiscono esclusivamente al campione oggetto di analisi. Il presente documento non può essere riprodotto
parzialmente, salvo autorizzazione scritta di Chelab
Enclosed to the test reports number: 14/000031110
12 REFERENCE AND GUIDELINES
ISO 10993-5:2009
UNI-EN-ISO 10993-12:2012
13 CONCLUSIONS
According to ISO 10993-5:2009, if the relative cell viability for the highest concentration of the sample
(100 %) is < 70 % of the control group, then the material shall be considered cytotoxic, if ≥ 70 % of the
control group, then the material shall be considered non-cytotoxic.
According to ISO 10993-5:2009, under the test conditions applied, the following sample is considered to
be NON cytotoxic: 13.037479.01
Pag. 12 di 12
Chelab Silliker
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