IBAN: DE 50 3006 0601 0005 5786 98 BIG: …...DIN EN ISO 10993-5:2009-10 * Part 5: tests for Cytotoxicity: in vitro Tests for irritation and sensitization according DIN EN ISO 10993-10:2010

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.


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


Annex of testreport SN 17120a of 2014-06-17

Figure 2: Kerox ZircoStar N1 Zirconia Blanks and Blocks


Centrum fur Hygieneund medizinische Produktsicherheit

HygCen GmbH • Postfach 11 01 35 • D-19001 Schwerin

Kerox Kft.H-2049 DiosdHomokbanya ut 77

DAkkS

*****


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


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


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.


Dipy. Umweltwiss. J. KohnleinVice department manager

SN 17120 b page 3 of 3




((DAkkS

•at,


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 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


Information: The test results exclusively refer to the samples describedabove. Account of extracts of this test report is only possible bywritten approval from HygCen.


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




((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


Delivery date:

SN 17120d

2014-06-05

Product:

Customer:

Test method:

Test time period:

Test conditions:


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!.;


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.


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


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

Documents

IBAN: DE 50 3006 0601 0005 5786 98 BIG: …...DIN EN ISO 10993-5:2009-10 * Part 5: tests for Cytotoxicity: in vitro Tests for irritation and sensitization according DIN EN ISO 10993-10:2010