IBAN: DE 50 3006 0601 0005 5786 98BIG: DAAEDEDD
HygCenCentrum fur Hygiene undmedizinische Produktsicherheit GmbH
Centrum fur Hygiene,und medizinische Produktsicherheit
HygCen GmbH • Postfach 11 01 35 • D-19001 SchwerinKerox Kft.H-2049 DiosdHomokbanya ut 77
(( DAkkS*^ n,,...
Cytotoxicity Test to DIN EN ISO 10993-5SOP 09-001
DeutscheAkkreditierungsstelleD-PL-18818-02-01D-PL-18818-02-02
Anertiannt durch/Hecognized by*, Zemralstel «der Lander £* fur Gcsundhcitsschutz £* bei Arzneimittalr und t
* Mcdizinproduk-j-n >**++* ZLG-AP-314.10.23
2014-06-23
Test Protocol
Identification of thetest laboratory:
Delivery date:
Product:
Customer:
SN 17120 a
2014-06-05
Kerox ZircoStar®N1 Zirconia Blanks and Blocks
Kerox Kft.
Test method:
Test time period:
Test conditions:
Cytotoxicity of eluates according to theDIN EN ISO 10993-5:2009-10 *Biological evaluation of medical devicesPart 5: tests for cytotoxicity: in vitroSOP 09-001
2014-06-11 until 2014-06-13
Examining climate: 25°C / 47% rel. humidity
Incubation: 24 hours
The samples were checked in the delivery state.
SN 17120 a Page 1 of 4
HyrjC«n Centrum fur Hygiene und medi/mischB Prncuktsirherhwt GmbHBornhov»dstra(Je 78 • D-10055 SchwerinTelefon: +49 (0) 385 56 82 65Telefax: +49 (0) 385 56 82 67E-Maii: infc3KPhyycen.deInternet: www.hygcen.de
Deutsche Apotheker-u Arzl^hank BLZ 300 606 01 Konto 0 005 5786fl8Deutsche Bank AG Schv. s BLZ 130 700 24 Kont
Deutsche Apotheksr- u. Ar/tebark IBAN DE 50 3006 0601 0005 5786 5)8BIC DAAEDEDD
Description of the method
Extraction conditions:
Cell culture
Exposition
Measuring principle
Measurement
Controls
2.3 g material into 11.5 ml MEM + 9 % serum +1 % antibioticsolution at 37°C for 24 h = extraction medium
Fl-cells are derived from the human amnion. The stockcultures were carried out into 250 ml culture flasks (GreinerGmbH). The cells were trypsinised all 4 days. Only cells up to100 passages were used.Trypsinised cells were seeded in tissue culture plates.The culture medium consists of MEM (Minimum EssentialMedium) supplemented with 9 % calf serum, 1 % antibioticsolution (Penicilline G, Streptomycin sulfate, Neomycin) andL-glutamine.
After 24hours of cultivation the cells were available asmonolayer. A medium change with extraction medium wasaccomplished. Therefore the culture medium was decantedand the extraction medium carefully pipetted into the wells(0.1 ml per well).An incubation for 24h is following.
Vital cells incorporate the dye neutral red. Destroyed cellscannot incorporate the dye and remain unstained. Theintensity of colour of the elution solution can be measured witha photometer.
At the end of the incubation time the microtiterplate will bewashed with PBS (Phosphate Buffered Saline). Culturemedium containing the dye neutral red (50ug/ml) was given tothe cells. After an incubation time of 3 hours themicrotiterplate was washed again to remove the spare dye.With a special elution solution (1% acetic acid in 50% ethylealcohol) the dye was solved out of the cells. After 1 hour ofelution the photometric measurement was conducted.
As a negative control culture medium without a test solutionwas established.To verify the sensitivity of the test system a positive control(1.5mg/ml Sodiumdodecylsulfate) in culture medium wasexposed in the cell culture system.
Evaluation The optical density of 12 parallel tests was determined andused for statistical evaluation.
SN 17120 a Page 2 of 4
Results
Figure 1: box plot of the cellvitality
*̂(0c
re
a0
1 ,._UU
•i nnn1 ,UUU •
O Qf\f\U H
,OUU •
U,HUU
0,200 •Onnn,UUU •
^—^5— i— — 5£— ii=̂ r̂ ~~^~^~' LJJ — • •*=
;neg. control pos. control SN 17120
N = 12
Table 1: Descriptive statistics (cellvitality)
NegativecontrolPositivecontrol
SN 17120 a
N
9
9
12
Mean
0,942
0,058
0,965
cell vitality (%)
100,00
6,20
102,49
minimum
0,862
0,053
0,864
maximum
1,052
0,064
1,070
Std. Deviation
0,070
0,004
0,062
P*
-
-
0,9973
*U test (Man Whitney) vs. Control
Archiving:
Information:
The raw data with respect to this test and a copy of the report willbe stored in the archive of HygCen.
The test results exclusively refer to the samples described above. Accountof extracts of this test report is only possible by written approval fromHvaCen.
Prof. Dr. med. H.-P. WernerManager of scientific-technical affairs
*Dipl. Llmweltwiss. J. KohnleinVice department manager
SN 17120 a Page 3 of 4
Annex of testreport SN 17120a of 2014-06-17
Figure 2: Kerox ZircoStar N1 Zirconia Blanks and Blocks
SN 17120 a Page 4 of 4
Centrum fur Hygieneund medizinische Produktsicherheit
HygCen GmbH • Postfach 11 01 35 • D-19001 Schwerin
Kerox Kft.H-2049 DiosdHomokbanya ut 77
DAkkS
*****
DeutscheAkkreditierungsstelleD-PL-18818-02-01D-PL-18818-02-02
Anerkannt durch/Ftecognizad byZemralstel e der Lander £
T fur Gcsundhoitsschutz S** be Arzneimittelr und ^«• Medizinprodukten I
2LG-AP-314.10.23
2014-06-23
Cytotoxicity Test to DIN EN ISO 10993-5SOP 09-001
T E S T R E P O R T
Identification of thetest laboratory:
Delivery date:
Product:
Customer:
Test method:
Test time period:
Test conditions:
SN 17120 b
2014-06-05
Kerox ZircoStar® N1 Zirconia Blanks and Blocks
Kerox Kft.
Biological evaluation of medical devicesCytotoxicity of eluates according to theDIN EN ISO 10993-5:2009-10 *Part 5: tests for Cytotoxicity: in vitroTests for irritation and sensitization accordingDIN EN ISO 10993-10:2010Part 10: Tests for membrane integritySOP 09-001
2014-06-11 until 2014-06-13
Examining climate: 25 °C / 47 % rel. humidity
Incubation: 24 hours
The samples were checked in the delivery state.SN17120b page 1 of 3
PruiinstitutHygCen Centrum fur Hygiene und medi.'inischfi Produktsicherheit GmbH
vBdstrafte 78 - D-19055 SchwerinTelefon: +49 (0) 385 56 82 6i>Telefax: +49 (0) 385 56 82 67E-Mail: info^hyycen.deInternet: www.hygcen.de
Deutsche Apotheker- u. ArztefiankDeutsche Bank AG Schwerin
Deutsche Apotheker- .1. Arztcbank
Geschaftsfufirenn Dipl.-lng, (FH) Margiit Wernet Amtsgerichl Schwerin HRB 4792 UST-Nr: DE178599849
BLZ30060601 Kent o 0005 57S 6988LZ 130 700 24 Konto 316
IBAN DE bU 3U06 0601 0005 5786 98BIG DAAEDEDD
Steuet-Nr. 090
uy/^iI ^^1if^E"A/l\sCl\l
Description of the method
Extractionconditions:
Cell culture
Exposition
Measuringprinciple
Control
Evaluation
2.3 g material into 11.5 ml MEM + 9 % serum +1 % antibioticsolution at 37°C for 24 h = extraction medium
Fl-cells are derived from the human amnion. The stock cultureswere carried out into 250 ml culture flasks (Greiner GmbH). Thecells were trypsinised all 4 days. Only cells up to 100 passageswere used.Trypsinised cells were seeded in tissue culture plates.The culture medium consists of MEM (Minimum EssentialMedium) supplemented with 9 % calf serum, 1 % antibioticsolution (Penicilline G, Streptomycin sulfate, Neomycin) and L-glutamine.
After 24hours of cultivation the cells were available asmonolayer. A medium change with extraction medium wasaccomplished. Therefore the culture medium was decanted andthe extraction medium carefully pipetted into the wells (0.1 ml perwell).An incubation for 24h is following.
Lactatdehydrogenase (LDH), a stable cytoplasmatic enzyme,exists in all cells and will be released into the culture medium, ifthe cell membrane is damaged or in case of cell lysis. LDHreduces pyruvate to lactate, by oxidation of NADH to NAD"1". Theuptake rate of NADH was determined photometric with a cineticabout 25 min.
As negative control culture medium without extraktion mediumwas applied. To prove the maximum LDH-release Triton X wasused as positive control.
The LDH-release of 12 parallel tests was determined and usedfor statistical evaluation.
SN 17120b page 2 of 3
Fig. 1: Release of LDH
inn
Ofi _
EV DU(0«£5>- /in
a_ionZ\J
Triton X Negative control SN 17120
Table 1: Descriptive statistics
Positive-control
Medium-control
SN 17120 b
N
6
6
6
LDH-release (%)
100.0
16.0
15.8
Archiving: The raw data with respect to this test and a copy of the report willbe stored in the archive of HygCen.
Information: he/dThe/test results exclusively refer to the samples described above.Account of extracts of this test report is only possible by written
roval from HygCen.
Prof. Dr. med. H.-P. WernerManager of scientific-technical affairs
Dipy. Umweltwiss. J. KohnleinVice department manager
SN 17120 b page 3 of 3
Centrum fur Hygieneund medizinische Produktsicherheit
HygCen GmbH • Postfach 11 01 35 • D-19001 Schwerin
Kerox Kft.H-2049 DiosdHomokbanya ut 77
((DAkkS
•at,
DeutscheAkkreditierungsstelleD-PL-18818-02-01D-PL-18818-02-02
Anertoinnt tJurch/RffcaqniziKl byw Zentralstel e der Lander 3
§^ fur Gosundhoitsschutz ^^ be. Arcneimittelr und g
'fr Medizinprodukten $> ZLG-AP-314.10.23
2014-06-23
Identification of the testlaboratory:
Delivery date:
Product:
Manufacturer:
Test method:
Period of analysis:
Test conditions:
T E S T R E P O R T
SN 17120 c
2014-06-05
Kerox ZircoStar® N1 Zirconia Blanks and Blocks
Kerox Kft.
Epikutan testTests for irritation and delayed-type hypersensitivityaccording to the DIN EN ISO 10993-10, 2010-12Biological evaluation of medical devicesSOP 09-013
2014-06-17 until 2014-06-20
Conditioning: 24 h
SN 17120 c Page 1 of 5
PrutinstitutHyn>'-«n Centrum fur Hygiene und medumischfi Prndtiktsicherheit GmbHBomhovadstrsHe 78 • D-19055 ScliwennTelefon: + 49 (0) 385568265Telefax: +49 (0) 385 56 82 67E-Mail: infoOhygcen.de
www.hygcen.de
Deutsche Apotheker- u. Arztebank BLZ 300 606 (J1 Konfo 0 005 578 698Deutsche Bank AG Schwerin BIZ 13070024 Konto.318.i645
Deutsche Apotheker- u. Aritcbank IBAN DE 50 3006 0601 0005 5786 98BIC DAAEDEDD
Geschattsfuhrenn Dipi.-lnq. (FH| Margnt Werner Amtsgericht Schwerin HRB UST.-Nr: DE178599849 r-lr: 090/110/03882
ISj Centrum nir Hygiene und
Test method
The patch test is a model designed to obtain proof of a primary irritative effect orcontact allergy (by provocation of allergic skin reactions in not sensitized testpersons) due to epicutaneous, local and lasting contact with the preparation underinvestigation.
To enhance absorption of the test substances, they are applied under occlusiveconditions during the patch test. The sensitivity threshold must be crossed in order toelicit a positive reaction.
An eluate of the product is applied to clinically healthy skin on the outside of thelower arm of test volunteers and fixed there (Leukotest, Hartmann).
The volunteers were clarified about the risks of the patch test and gave their writtenagreement.
The test patch is removed after an exposure duration of 24 hours, 48 hours and 72hours at which time an initial evaluation is done.
SN 17120 c page 2 of 5
HVCIl I ***! nntrum fur Hygiene und
f*C A /Vxt/V
Results of Epikutan test. Tests for irritation and delaved-type hypersensitivitvaccording to the DIN EN ISO 10993-10. 2010-12
Identification of the testlaboratory:
Description of test sample:
Size of the test sample:
Size of the test patch area:
Application of the test sample:
Volunteers:
Period of analysis:
Test date :
Results of 10 test person:
SN 17120 c
Kerox ZircoStar® N1 Zirconia Blanks andBlocks
100|jl oftheeluate
2.5 cm x2.5 cm
100|jl oftheeluate
7 women, 3 men; age 23-52 years
24 h, 48 h, 72 h
Begin: 2014-06-17 until 2014-06-20
Test person1.2.3.4.5.6.7.8.9.10.
after 24 h0000000000
after 48 h0000000000
after 72 h0000000000
Legend: 0 = no reaction1 = light erythem or oedema2 = pronounced erythem or oedema3 = massive erythem or oedema
Result
In all 10 test volunteers, no erythem or oedema were recorded in the test patch areaafter 24, 48 and 72 hours.
SN17120c page 3 of 5
Archiving: The raw data with respect to this test and a copy of the report willbe stored in the archive of HygCen.
Information: The test results exclusively refer to the samples describedabove. Account of extracts of this test report is only possible bywritten approval from HygCen.
Prof. Dr. med. H.-P. WernerManager of scientific-technical affairs
Dipl.(Umweltwiss. J. KbhnleinVice department manager
SN17120C page 4 of 5
Centra-• i und
Annex of Testreport SN 17120 c of 2014-06-20
Fig. 1: Kerox ZircoStar® N1 Zirconia Blanks and Blocks
SN17120c page 5 of 5
Centrum fur Hygieneund medizinische Produktsicherheit
HygCen GmbH • Postfach 11 01 35 • D-19001 Schwerin
Kerox Kft.H-2049 DiosdHomokbanya ut 77
((DAkkSDeutscheAkkreditierungsstelleD-PL-18818-02-01D-PL-18818-02-02
•if if -fa Arwrtuinnt duneh/Ritcognized by.* * Zemralstel e der Lander f
fur Gosundhoitsschutz ̂
•*• Medizinprodukten IZLG-AP-31 4.1 0.23
Genotoxicity (SOP 09-003)
2014-06-23
P R U F B E R I C H T
Identification of thetest laboratory:
Delivery date:
SN 17120d
2014-06-05
Product:
Customer:
Test method:
Test time period:
Test conditions:
Kerox ZircoStar® N1 Zirconia Blanks and Blocks
Kerox Kft.
Bacterial reverse mutation test (OECD 471) accordingISO 10993-3:2009Biological evaluation of medical devicesPart 3: Tests for genotoxicity, carcinogenity andreproductive toxicity (SOP 09-003)
2014-06-20 until 2014-06-23
Examining climate: 22°C, 46 %, rel. FeuchteConditioning: 24 hThe samples were checked in the delivery state.
PriifinstrtutHygCen Centrum fur Hygiene und medi7inische Prnduktsicnerhert GmbHB;)mhovn(lstra.'i« 78 • D-19055 SchwennTelefon: +49 (0) 385 56 82 6i>Telefax: +49 (0) 385 56 82 67E-Mail: nifoS8riygcen.deInternet: www.hygcen.de
SN 17120d page 1 of 4
Deutsche Apotheker- u. Arztebank BLZ 300 606 U1 Konto 0 .Deutsche Bank AG Schwenr BLZ 13070024 <cmo3^
Deutsche Apothekef- u, Ai%;k:r;ar,k
Geschaftsfuhrenn D!pl.-lnq. (FH) Marqnt Werner Amtsqericht Scdwerin HRB 47Q2 UST-Nr: DE178599849
IBAN DE 50 3006 0601 0005 5786 98BIC DAAEDEDD
Stcu-ji-Nr 09!.;
Description of the method
Test principle:
Testgerms:
The bacterial reverse mutation test (Ames-Test) serves for theverification of the mutagenic effect of test substances.The mutagenic effect can be identified by the induction of areversion of His" to His+. Applied are different strains of thetestgerm Salmonella typhimurium, which are not able tosynthezise histidine. After a remutation the testgerm is able tosythezise histidine and grow on histidine free nutrient medium.For the simulation of the Zur Simulation des mammalianmetabolism the testprocedure is carried out stoffwechsels erfolgtdie Testdurchfuhrung additionally in presence of rat liverenzymes (S9-Mix).
Salmonella typhimurium (TA 98) fort he detection of frameshift-mutationen.Mixed strains (TA 7001, TA 7002, TA 7003, TA 7004, TA 7005,TA 7006) for the detection of base pair substitutions.
Cultivation of thetestgerms: 12h at 36 ± 1 °C, at 150 U/min
Negative control: Dilution solution (tryptone-sodium chloride - solution)
Positive control : 2-Nitrofluorene, 20ug/ml (without S9),2-Aminoanthracene 100ug/ml (with S9)
S9-Mix: 10% in phosphate buffer with 1.2% NADPH
Incubation: 48h at 36 ± 1 °C
Evaluation Calculation of the mean of the 3-fold determination andcalculation of the induction factor with regard to the spontaneousmutation of the negative control.
SN17120d page 2 of 4
j l I *^Y Centrum fur Hygiene u.
If*Phi
Results of the Bacterial reverse mutation test (OECD 471) accordingISO 10993-3. SOP 09-003
Testproduct:
Extraction:
Testgerms
Test time period:
cfu of the testsuspension:
Control values
SN 17120d
2.3g material were extracted about 24h at37°C in 11.5 ml 0.85% NaCI-solution.
Salmonella typhimurium (TA 98)Mixed strains (TA 7001, TA 7002, TA 7003,TA 7004, TA 7005, TA 7006)
2014-06-20 until 2014-06-23
9.23 Ig/ml TA 989.27 Ig/ml TA Mix
control
Negative controlwithout S9 Mix
Negative controlwith S9 Mix
2-Nitrofluorenewithout S9 Mix2-Aminoanthracenewith S9 Mix
Test-germ
TA98
TAMix
TA98
TAMix
TA98
TAMix
plate 1cfu/plate
94
19
59
16
800
370
plate 2cfu/plate
68
25
69
12
710
380
plate 3cfu/plate
81
15
68
16
530
400
mean
81
20
65
15
680
383
Legend: cfu= colony forming units
Evaluation: The cfu of the positive control should be 2.5 fold higher than the cfu of the negativecontrol
SN17120d page 3 of 4
Results of the Bacterial reverse mutation test (OECD 471) accordingISO 10993-3. SOP 09-003
Test without S-9 Mix
SN No.
17120d
Test-germ
TA98
TAMix
plate 1cfu/plate
87
20
plate 2cfu/plate
79
14
plate 3cfu/plate
62
16
mean
76
17
IF
0.94
0.85
Test with S-9 Mix
SN No.
17120d
Test-germ
TA98
TAMix
plate 1cfu/plate
77
20
plate 2cfu/plate
64
21
plate 3cfu/plate
53
12
mean
65
18
IF
1.00
1.20
Legend: cfu = colony forming units
IF = Induction faktor
Induction factor =Induced number of reversed bacteria (product)
Spontaneous number of reversed bacteria (Negativkontrolle)
An induction faktor > 2,0 is classified as genotoxic.
Conclusion:With the extract of the product "Kerox ZircoStar® N1 Zirconia Blanks and Blocks"tested with the Ames-test, no genotoxic effects could be observed.
Archiving: The raw data with respect to this test and a copy of the report willbe stored in the archive of HygCen.
Information test results exclusively refer to the samples described above,count of extracts of this test report is only possible by written
pproval from HygCen.
Prof. Dr. meti. H.-P. WernerManager of scientific-technical affairs
Dipl./Umweltwiss. JTKb'hnleinVice department manager
SN 17120d page 4 of 4
PROF. DR. MED. H. P. WERNERFACHARZT FUR HYGIENE
of. Dr. H.-P. WeyiprKerox Kit:H-2049 DiosdHomokbanya ut 77
• c/o HygCen GmbH - Bornhovedstr. 78 - D -19055 Schwer i
c/o HygCen Centrum fur Hygiene undmedizinische Produktsicherheit GmbHBornhovedstraBe 78D-19055 Schwerin
Tel.: +49 (0)385 / 56 82 65
Fax: +49 (0)385 / 56 82 67
E-Mai l : [email protected]
2014-06-23
Kerox ZircoStar® N1 Zirconia Blanks and Blocks
Judgement
After testing the biocompatibility of the Material "Kerox ZircoStar® N1 Zirconia Blanks
and Blocks" according to DIN EN ISO 10993-5, ISO 10993-10 and ISO 10993-3 -
(testreports SN 17120 a-d of 2014-06-23) - I give the following statement:
An evaluation of the scope of biological testing was carried out as per EN ISO 10993-
1:2010-04 and ISO 7405:2009-6.
The intended use of the product, declared by the producer, involves contact with
dentin for more than 30 days. The product has no long term contact with bone, tissue
and blood and is therefore no implant device. To evaluate the biocompatibility of the
product, a cytotoxicity test as per EN ISO 10993-5:2009 with the neutral red method
to determine the metabolic activity of the cells and an in vitro test to determine the
membrane integrity, an epikutan test according ISO 10993-10:2010-12 and tests for
genotoxicity according ISO 10993-3:2009 were therefore considered sufficient.
Any knowledge to be gained from further biocompatibility testing (such as ISO
10993-11, point 5 and Annex A4 - acute systemic toxicity - application by inhalation;
page 1 of 2
ISO 10993-11, point 6 and Annex A9 - subchronic systemic toxicity - oral application
and ISO 10 993-11, point 6 and Annex A4 - subchronic systemic toxicity - application
by inhalation) with this product would not justify the unnecessarily high level of harm
to experimental animals involved. As per EN ISO 10993-1:2009, chapter 4.6 and
chapter 6.2.1 8), such tests will therefore not be performed in these cases. The type
and scope of the tests performed complies with the specifications as per EN ISO
10993-1:2010-04.
From the tested material no cytotoxic compounds were extracted at 37°C. The
extract of the test material did not reduce the cell growth in comparison to the control
(testreport SN 17120, Fig. 1 and Tab. 1).
The test of the membrane integrity on the basis of the measurement of the LDH-
release follows no increased LDH (Lactatdehydrogenasis) in comparison to the
negative control. Relating to the maximal LDH-release with the positive control a
value of 15.8% was calculated (testreport SN 17120b, Fig. 1 and
Tab. 1). The extract of the materials did not damage the cell membrane.
Using the test material as mentioned before described by the manufacturer no
cytotoxic effects should be expected.
The performance of the Ames test for testing the genotoxicity according to
ISO 10993-3:2009 did not result in genotoxic effects of the product (testreport
SN 17120d).
The tests for irritation and delayed-type hypersensitivity according to the DIN EN ISO
10993-10:2010-12 were performed with an epikutan test with 10 volunteers. No
negative effects wefe observed (testreport SN 17120c).
Prof. Dr. med. H.-P. Werner
page 2 of 2