Introduction
Metals, ceramics, and resin are used for dental materials, but only after fulfilling strict conditions. They must
demonstrate stability in the demanding intraoral environment, safety in their contact with the human body, andfunctionality in use. As technological innovation continues apace, the advanced development of new materials foruse with the human body offers medical professionals and patients many new advantages. However, when advancing
product development with new chemical substances and new technology, safety obviously is and must remain the
single most important concern. In fact, the revised Japanese Pharmaceutical Affairs Law of April 2005 places
stronger emphasis than ever before on the evaluation of medical devices, including dental materials, in terms of theirsafety and effectiveness.

Table of Contents
Introduction ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･2Outline ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･3

Methods

8

Results･･････････････････････････････････ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ 14

Discussion ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ 20
Conclusion ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ 20
References ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ 21

At Yamamoto Precious Metals Co., Ltd. (hereafter Yamakin), we have performed extensive fundamental research onindirect composite resin for the purpose of new materials development. We have also conducted a wealth of researchon the physical and chemical properties of monomers and inorganic fillers, their mechanical properties and otherrelated topics. Moreover, in order to progress to product development in a way that will guarantee product safety
along with excellent mechanical and physical properties, since 2003, we have conducted joint research with the Oral
Tumor Control Science Tumor Pathology Course at Kochi Medical School, Kochi University. In 2005, The Biological
Science Safety Laboratory was established in the Department of Oral and Maxillofacial Surgery, Kochi University.
Yamakin has undertaken these initiatives because we see ourselves as a manufacturer which provides safety to bothpatients and the medical professionals who serve them. For evaluation of biological safety, The Biological Science
Safety Laboratory carries out the “Biological Evaluation of Medical Devices” conforming to ISO 10993, theinternational standard, and also our own examinations based on cell, cell tissue, and genetic engineering.
Even though cytotoxicity has been reported for the main components of indirect composite resin – monomers,
urethane dimethacrylate and triethylene glycol dimethacrylate – they are still used as main components in mostproducts on the market because of their longstanding results in clinical practice. In addition, endocrine disruptor
action has been reported in some of the polymers used in indirect composite resin. Therefore, it is necessary to fully
consider potential impacts on the human body when developing new products.
The indirect composite resin which Yamakin has developed based on the results of our fundamental research is a
nano-composite resin that takes the safety concerns mentioned above into full consideration. At the same time,
nano-technology is exploited to the utmost, and our product has the highest filling rate among the composite resinscovered by the Japanese national health insurance system. This composite resin is compounded of 20-nm sphericalfillers in the resin matrix and is improved greatly in its strength and abrasion resistance by being compounded with
100-nm spherical fillers; as a result, it can achieve the requirements demanded of a basic indirect composite resin.
With “Indirect Composite Resin for Biological Safety”as its overriding development concept, a wide range of tests on
the safety of basic indirect composite resin, has been conducted based on the stipulations of ISO 10993. These tests
include: 1) Acute Toxicity Test on mice 2) Colony-Forming Assay with Cultured Cells (In Vitro Cytotoxicity Test) 3)
Reverse Mutation Test on bacteria (Mutagenicity Test) 4) Skin Sensitization Test on guinea pigs, and 5) Oral Mucosa
Irritation Test on hamsters. Through this testing process, safety for human usage is confirmed.
Luna-Wing, designed as an indirect composite resin, has become the final product in this process of development,
adding to the basic indirect composite resin a small amount of color pigment along with a fluorescent pigmentcompound for color adjustment with natural teeth. There are various kinds of additives, depending on theirproperties, but it remains a fact that there are no reported cases of their having undergone biological evaluation.
Even if basic indirect composite resin is considered to be safe for the human body, if the relevant additive displays
any physical impact, however slight, the resin itself is not safe.
To purse safety even further, Yamakin has carried out tests conforming to ISO 10993, along with its own testing,based on cells, cell tissue and genetic engineering. Testing was undertaken in collaboration with Kochi Medical
School, Kochi University, centering on color pigments, fluorescent pigments, and primers. We now bring together the
results of this collaborative research testing, and report the data as Indirect Composite Resin Safety Evaluation
Report: Biological Evaluation of “Luna-Wing” (Vol. 4 in a Series on Safety Reports). We are confident that the safety
information provided here can help to ensure security for medical professionals and patients.
June 2006
Yamamoto Precious Metal Co., Ltd.
Biological Science Safety Laboratory

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《 Outline 》

2. Colony-Forming Assay with Cultured Cells (In Vitro Cytotoxicity Test)

1. Acute Toxicity Test
Purpose of the Test
The Systemic Toxicity Test is carried out in order to predict the safety of a sample in an environment that closely
resembles that of the human body. There are different appearances for toxicity: Acute (toxicity shows quickly) and
Chronic (toxicity shows over a long time); this test is for Acute.

Purpose of the Test
The test is performed to evaluate toxicity at the cell level. For this test, a Colony-Forming Assay with Cultured Cells
is selected from among possible evaluation methods using cells. One of the characteristics of this test is that it canshow severe reactivity to toxicity. It is also recommended in ISO 10933-5.
Summary of the Test
Ⅰ. V79 (Chinese Hamster Lung) cells are incubated and grown.

Summary of the Test
Ⅰ. Four-week-old mice are prepared and kept for 1 week.

Incubation of Cells
Mice in a Cage

Cages

Ⅱ. Approximately 100 cells are placed in each hole on culture plates.

Ⅱ. Normally, a single suspension of a sample is forcibly administered (when necessary, multiple doses) to miceusing a feeding tube.
FeedingTube
胃ゾンテ

Seeding Cell Suspension on Culture Plates
Forced Oral Dosage Using a Feeding Tube

Ⅲ. Fourteen days after dosing, weight and other health conditions are observed. If toxicity is shown, weight
reduction or weakening will be observed. Death may be observed when toxicity is strong.

Observing Health Condition

Ⅳ. After observation, all of the mice used in the examination are vivisected and shape change to the organs is
ascertained along with mass change.

Ⅲ. Solution composition is extracted for 24 hours from a sample using culture fluid. The extract is added on thecells and incubated for 6 days.

Extraction from Samples

6-day Incubation

Ⅳ. The number of colonies is counted. If the number of colonies formed is close to the original number of cells, there
is no toxicity.
Cell

Cultured Cells Attaching

Colonies

Culture Fluid①Seeding Cells
Observation of Shape Change to the Organs after Vivisection―3―②Culturing of Cells
Fig.1: Flow from Addition of Cells to Colony Formation―4―③Colonies

3. Reverse Mutation Test (Mutagenicity Test)

Ⅱ. Test samples are attached to the flank of the guinea pig as in a skin patch test and left for 48 hours.

Purpose of the Test
If a component or components of products have an influence on DNA, it will cause various problems. It may cause
cancer in cases where DNA is impacted after birth, and in cases where DNA in general somatic cells is impacted, it
will cause deformation in descendant cells or even in further generations. Therefore, it is absolutely essential toascertain the influence on DNA of products component(s). This test is carried out in order to predict such risks.
Summary of the Test
Ⅰ. Bacteria (salmonella and colon bacteria) are incubated and soaked for 8 hours at 37°C.
Application of Samples

Ⅲ. The attached samples are removed after 48 hours, and the presence or absence of an allergic reaction is
confirmed. If there is no allergic reaction, the skin remains the same as shown in the picture on the bottom left (thereddened area is caused by surgical tape), and if there is an allergic reaction, it will cause inflammation and form ascab as shown in the picture on the bottom right.

Culture Fluid with Bacteria

Automatic Thermostat Shaker

Ⅱ. The incubated bacteria and materials are compounded and added into an agar medium which has almost no
nutritional ingredients. It is then left for 48 hours.

No Allergic Reaction

Allergic Reaction

5. Oral Mucosa Irritation Test

Compound of Bacteria and Materials

Seeding Bacteria into Agar Medium

Ⅲ. After 48 hours, the number of bacterial colonies is counted. If the sample produces abnormality in DNA,
several hundred to more than 1,000 colonies will have formed.

Purpose of the Test
A wide range of toxic substances adversely impact the human body by being
absorbed into it. However, other toxic substances cause abnormality on thesurface texture, as with cigarette tar. This test is to analyze impact on surface
texture in the mouth.
Summary of the Test
Ⅰ. Samples are placed into the cheek pouches of a hamster and left for 2 weeks.
They are then removed.

Anesthetization

Ⅱ. A permanent preparation of the cheekpouches is made.

Seeding Cell Suspension on Culture Plates

4. Skin Sensitization Test

Ⅲ. The permanent preparation is dyed.

Cheek Pouches are Wrapped tomake Paraffin Blocks

A Permanent Preparation is Made

Purpose of the Test
The test is performed to measure the level of allergenic risk asample exhibits.
Summary of the Test
Ⅰ. A guinea pig is subcutaneously injected with a reagent in orderto make it physically sensitive to allergy.
After Subcutaneous Injection―5―

A Glass Cover is Installed to Complete The Permanent Preparation is Dyed
a Permanent Preparation―6―

Ⅳ. The structure is observed. If the sample is not a substance that exhibits adverse impact in the mouth, thetissue sequence will be in order; however if adverse impact has occurred, the structure is thickened and a mass ofneutrophil, eosinophile, basophile leucocyte and macrophage will appear.

Normal Tissue

9. DNA Fragmentation Test
Purpose of the Test
The test is performed in order to ascertain whether or not apoptosis (programmed cell death) has occurred, by
analyzing DNA fragmentation.
Summary of the Test
Ⅰ. Cells are placed on a culture plate. Incubation is for 6 hours.
Ⅱ. Sample solution is added to the incubated cells. Incubation takes place for 48 hours.
Ⅲ. The DNA is removed.
Ⅳ. The DNA is dyed.
Ⅴ. The DNA is electronically isolated.
Ⅵ. The presence or absence of apoptosis is confirmed by analyzing the DNA in its isolated condition.

6. Cell Growth Inhibition Test
Purpose of the Test
As with the Colony-Forming Assay with Cultured Cells, this test is performed to evaluate toxicity at the cell level.
Among the characteristics of the test is that it is the most popular of in vitro Cytotoxicity tests and that the evaluationis conducted based on the cell reproduction rate.
Summary of the Test
Ⅰ. Cells are placed on a culture plate.
Ⅱ. The extract is placed on the cells from a sample. Incubation is for 48 hours.
Ⅲ. The cell reproduction rate is measured.
The evaluation method is comparing the number of cells reproducing before and after adding the extract.

7. DNA Synthesis Test
Purpose of the Test
The cells composing the human body perform cytokinesis repeatedly every day, and the old cells are discharged
outside the body. The test is performed to ascertain whether there is toxicity in the sample or not by observing if thereaction is performed normally, paying attention to reproduction and composition of DNA, which always performs
the cytokinesis.
Summary of the Test
Ⅰ. Cells are placed on a culture plate.
Ⅱ. The extract is placed on the cells from a sample. Incubation is for 42 hours.
Ⅲ. After 42 hours, a reagent that reacts with the site of synthesis when DNA is compounding is added.
Ⅳ. Incubation takes place for another 6 hours.
Ⅴ. The quantity of the reacted reagent is measured. With the quantity of compounded DNA without adding the extract
serving as a base, evaluation is conducted by observing how much the quantity of compounded DNA changes after adding
the extract.

8. Cytotoxicity Test
Purpose of the Test
Cell death takes place in two major ways. One is apoptosis, and the other is necrosis. Apoptosis is DNA-programmed
cell death, for example, the disappearance of cells between the fingers and toes in a developing human embryo.
Necrosis is cell death caused by external factors, such as burns. This test is performed in order to ascertain whether
or not both forms of cell death have occurred.
Summary of the Test
Ⅰ. Cells are placed onto a culture plate. Incubation is for 6 hours.
Ⅱ. Culture fluid is removed after incubation.
Ⅲ. The extract is placed on the cells from a sample. Incubation is for 48 hours.
Ⅳ. Coloring liquid is added and left for 10 minutes in order to confirm apoptosis and necrosis.
Ⅴ. Appropriate equipment is used to count the number of normal cells and dead cells (flow cytometry).

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10. Protein Synthesis Test
Purpose of the Test
The test is carried out in order to evaluate toxicity by observing increase or decrease of the synthesis quantity afterRNA-synthesized cells proteins have been dyed.
Summary of the Test
Ⅰ. Cells are placed onto a culture plate. Incubation is for 6 hours.
Ⅱ. Culture fluid is removed after incubation.
Ⅲ. The extract is added to the cells from a sample. Incubation takes place for 48 hours.
Ⅳ. Protein is extracted.
Ⅴ. The protein is dyed.
Ⅵ. The absorptivity is measured.

《 Methods 》
1. Materials
The materials used in the tests are shown in the table below.
Materials

Main Components or Characteristics

Fluorescent Pigment

Europium Alkaline-Earth Phosphate

Color Pigment

Inorganic Pigment (TiO2, Fe2O3, ZrO2, etc.)
Organic Pigment

PrimerThiol Primer

Resin (Test Model)

Dentine (A3 Shade) Hardened Material

UDMA

Urethane Dimethacrylate

TEGDMA

Triethylene Glycol Dimethacrylate

Note: The basic indirect composite resin (Test Model) has already been confirmed as ISO10993-compliant by
testing; it was therefore evaluated under company-internal criteria. UDMA and TEGDMA are added for
purposes of comparison.

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2. Acute Toxicity Test
2-1 Test Animals
Four-week-old male and female ICR-type mice (10 males and females for both test and control groups) are prepared-and kept for approximately one week. They are used for testing after the absence of abnormalities is confirmed. Fiveof the test animals are kept in once polycarbonate cage, with room temperature set at 23°C±2°C with the lighting
time at 14 hours per day.
2-2 Preparation of Sample Solution
Fluorescent pigment, color pigment, and primer are suspended in pure water and sample solution is prepared at
100mg/ml.
2-3 Evaluation Method
Test animals fast for approximately 4 hours before administering. After measuring their weight, 2g/kg of a single
dose of sample solution is forcibly administered to both male and female test groups using a feeding tube. For thecontrol group, 0.6ml of pure water for female and 0.7ml for male is also administered. The observation term is 14
days; administration is performed frequently and observed once a day from the day following administration.
Weights are measured from 7 to 14 days after administration, and t-testing is used to compare groups with a
5%-significant level of difference. The test animals are dissected after the observation term.

4-3 Method
Preincubation assay (with and without metabolic activation for the sample) is performed for the test. 0.1mL of thesample solution diluted at a set density, S9Mix or 0.5mL of 0.1mol/L of sodium phosphate buffer solution (pH704),
and 0.1mL of bacteria suspension respectively is added into a sterilized tube. After 20 minutes of pre-incubation in athermostat inspection chamber at 37°C, 2mL of top agar is added and mixed in, spread evenly and solidified inminimal glucose agar plate culture medium. Incubation takes place for 48 hours at 37°C and the number of colonies
which have appeared through reverse mutation is counted.

5. Skin Sensitization Test
5-1 Test Animals
The test group is composed of nine guinea pigs; six more are used for each negative control group and positive
control group (prompt sanitization material treatment group).

3. Colony-Forming Assay (In Vitro Cytotoxicity Test)

5-2 Method
On 2cm × 2cm filter paper at 0.1m/L each, a 10%, 1%, and 0.1% Vaseline mixture of fluorescent pigment, color
pigment, and primer for the test group and a 10%, 1%, and 0.1% Vaseline mixture of Vaseline and a sample for thecontrol group is applied. (The same test under the same testing conditions as the sample is performed for thenegative control group in order to confirm that the result of the sample is not an error.). The preparations are thenapplied on the sheared and shaved flank of the guinea pig. For the positive control group, apply 0.01% (w/v) of
acetone solution of DNCB (2,4-dinitrochlorobenzen) at 0.01m/L each.

3-1 Performing Cells
V79 Cells (Chinese Hamster Lung)

6. Oral Mucosa Irritation Test

3-2 Preparation of Sample Solution
Fluorescent Pigment, Color Pigment
The sample solution (100%) is made by adding 1mL of MO5 culture medium to 0.1g of a sample and extracting for
24 hours at 37°C. It is then sterilized with a 0.22µm filter and diluted with MO5 culture medium.
Fluorescent Pigment : 3.13, 6.25, 12.5, 25, 50 and 100%
Color Pigment : 3.13, 6.25, 12.5, 25, 50 and 100%
Primer
For primer, the sample solution（1000µg/mL-100%）is made by diluting with MO5 culture medium. It is thensterilized with a 0.22µm filter and diluted with MO5 culture medium.
Primer : 3.13, 6.25, 12.5, 25, 50 and 100%
3-3 Method
V79 cells (Chinese Hamster Lung) propagated in a single layer by trypsinization are exfoliated and 200 pieces/mL ofcell suspensions is prepared using MO5 culture medium. The cell suspensions are scattered with 0.5m/L over eachwell on a tissue culture plate and incubated for approximately 6 hours at 5% CO2 at 37°C. After incubation,
attachment of the cells to the bottom of the wells is confirmed and the culture medium is removed. 0.5m/L each ofsample solution at each respective density, negative control sample solution, and the extract is placed in each of four
wells , and incubation takes place for 6 days at 5% CO2 at 37°C. After the incubation, each well is dipped into a 10％
neutral phosphoric acid buffer formalin solution for 30 minutes and the wells are dyed with 0.1% of methylene blue
solution for 15 minutes. The number of colonies is then counted. For the samples, the test is repeated twice.

6-1 Test Animals
Five-week-old male Syrian hamsters (golden hamsters) are prepared and kept for approximately 2 weeks; 15
hamsters per sample are used for the tests after confirming the absence of abnormalities. Two to three of the test
animals are put into one polycarbonate cage and kept at a room temperature of 23°C±2°C with the lighting time set
for 14 hours per day.
6-2 Method
After measuring weights of the test animals, 40mg/kg of pentobarbital sodium is administered in the abdominal
cavity to produce general anesthesia. The mucous membrane of the cheek pouches is removed carefully so as to avoid
scratches and it is confirmed if there is any damage, such as inflammation, on the mucous membrane after thoroughcleaning with buffered saline solution. Excess water is removed with paper; the cheek pouches are restored to their
original condition. The samples are applied on one side of all of the check pouches and solvent control on the other.
After application, the cheek pouches are sewn together in order to maintain the sample and the solvent control.
Application is set for 2 weeks, and the test animals are anesthetized with ether and euthanized by bleeding. The
cheek pouches are removed after the application term. The cheek pouches are visually checked and solidified withformalin liquid. Following the usual directions, the tissue is dehydrated, penetrated, embedded in paraffin and dyedwith hematoxylin eosin (H.E.) after cutting thinly. The permanent preparation created here is observed
histopathologically with an optical microscope.

7. Cell Growth Inhibition Test
7-1 Performing Cells
3T3 Cells (mouse embryonic fibroblast cells)

4. Reverse Mutation Test (Mutagenicity Test)
4-1 Performing Bacterial Strains
The bacterial strains used for the test are mutated salmonella strains（TA100, TA98, TA1535, TA1537） and colon
bacterium (WP2uvrA).
4-2 Preparation of Sample Solution
Fluorescent pigment, color pigment, and primer are compounded with dimethyl sulfoxide (DMSO) in a centrifuge
tube at 50mg/mL and sample solution (100%) is created by mixing with a test-tube mixer. This sample solution isthen diluted using DMSO. Fluorescent pigment, color pigment, primer : 3130, 6250, 12500, 25000 and 50000µg/mL
DMSO is used as a negative control group.

―9―

7-2 Preparation of Sample Solution
Fluorescent Pigment, Color Pigment
The sample solution is made by adding 1mL of culture medium per 0.1g and extracting it at 37°C for24, 48, and 168
hours.

― 10 ―

Primer, UDMA, TEGDMA
Because it is difficult to dissolve them into culture medium for the primer, UDMA and TEGDMA are dissolved in
DMSO in advance and diluted step by step as shown below.
P r i m e r : 62.5, 125, 500, 1,000µg/mL
U D M A : 470 (1,000) , 235 (500) , 117.5 (250) , 58.8 (125) µg/mL (µM)
TEGDMA: 286 (1,000) , 143 (500) , 71.5 (250) , 35.75 (125) µg/mL (µM)
The density of DMSO in the sample solution here is prepared at less than 1% without toxicity for cultured cells.
For the sample solution, DMSO-diluted solution of the same density as diluted is used as a reference value.

9. Cytotoxicity Test

Resin (Test Model)
After washing resin (test model) with tap water and distilled water for injection of the Japanese pharmacopeia (fromHIKARI PHARMACEUTICAL CO., LTD.) and drying, ultraviolet sterilization is performed on both sides for 30
minutes on a clean bench. 1mL of culture medium is added to each 5 cm2 of surface area of the sample and extracted
to make a sample solution in incubation at 5% CO2 for 24, 48, and 168 hours at 37°C. All of the sample solution isthen sterilized with a 0.22µm filter and used for the test.

Primer, UDMA, TEGDMA
Because it is difficult to dissolve them into culture medium for the primer, UDMA and TEGDMA are dissolved in
DMSO in advance, with the density prepared as shown below.
P r i m e r : 500µg/mL
U D M A : 286 (1) µg/mL (mM)
TEGDMA: 470 (1) µg/mL (mM)
The density of DMSO in the sample solution here is prepared at less than 1% without toxicity for cultured cells. For the
sample solution, DMSO-diluted solution of the same density as diluted is used as a reference value.

7-3 Method
3T3 cells propagated in a single layer by trypsinization are exfoliated and 5×104pieces/mL of cell suspensions areprepared. 1mL of cell suspensions is scattered over each well on a tissue culture plate and incubated forapproximately six hours at 5% CO2 at 37°C. After incubation, attachment of the cells to the bottom of the wells is
confirmed and the culture medium is removed. 1mL of sample solution for each sample is added and the samples are
incubated or 48 hours at 5% CO2 at 37°C. The culture medium on each well is removed and 100µL of MTT solution
is added. The samples are then matured for 2 hours at 37°C, 100µL of DMSO is added, and the samples are matured
at room temperature for 5 minutes. Viability is calculated by measuring absorptivity at 570nm with a micro plate
reader (THERMOmax: Molecular Devices, LLC.) and absorptivity rate from a reference value of 100%. All tests arerepeated three times.

8. DNA Synthesis Test
8-1 Performing Cells
3T3 Cells (mouse embryonic fibroblast cells)
8-2 Preparation of Sample Solution
Fluorescent Pigment, Color Pigment
The sample solution is made by adding 1mL of culture medium per 0.1g and extracting at 37°C for 24 hours.
Primer, UDMA, TEGDMA
Because it is difficult to dissolve them into culture medium for the primer, UDMA and TEGDMA are dissolved in
DMSO in advance, with the density prepared as shown below.
P r i m e r : 500µg/mL
U D M A : 286 (1) µg/mL (mM)
TEGDMA: 470 (1) µg/mL (mM)
The density of DMSO in the sample solution here is prepared at less than 1% without toxicity for cultured cells.
For the sample solution, DMSO-diluted solution of the same density as diluted is used as a reference value.
Resin (Test Model)
After washing resin (test model) with tap water and distilled water for injection of the Japanese pharmacopeia (fromHIKARI PHARMACEUTICAL CO., LTD.) and drying, ultraviolet sterilization is performed on both sides for 30
minutes on a clean bench. 1mL of culture medium is added to each 5 cm2 of surface area of the sample and extracted
to make sample solution in incubation at 5% CO2 for 24 hours at 37°C. All of the sample solution is then sterilizedwith a 0.22µm filter and used for the test.

9-1 Performing Cells
3T3 Cells (mouse embryonic fibroblast cells)
9-2 Preparation of Sample Solution
Fluorescent Pigment, Color Pigment
The sample solution is made by adding 1mL of culture medium per 0.1g and extracting at 37°C for 24 hours.

9-3 Method
As with the Cell Growth Inhibition Test, a cell suspension is prepared, 3mL is scattered on a culture medium on atissue culture plate and incubated for approximately 6 hours at 5% CO2 at 37°C. After incubation, attachment of thecells to the bottom of the wells is confirmed and the culture medium is removed. 3mL of sample solution is added toeach test sample (100%) and incubated for 28 hours at 5% CO2 at 37°C. After incubation, the culture fluid including
floating cells is collected into a centrifugal tube (15mL), the plate is washed with 5mL PBS (–), and the PBS (–) iscollected into the same tube. Trypsin solution is added to the plate and kept for 5 minutes at 37°C. After the cells are
released, the fluid is put into a centrifugal tube and centrifuged for 5 minutes at 1,200rpm to perform aspiration forsupernatant. The cells density is set at 1×106pieces/mL with 1 × Binder buffer diluted at 10 times with distilledwater. 500µL of cell-suspension liquid at the appropriate density is then placed into a flow cytometry tube, adding 5µ
L. The fluid is mixed gently, adding 5µL of Annexin V-FITC undiluted solution and 10µL of Propidium iodide
solution in iced water and incubating out of sunlight as a material for flow cytometry. Besides the materials, a tube
sample with only 5µL of Annexin V-FITCT and 10µL of Propidium Iodide Solution added is prepared using-cell-suspension liquid as a reference value for sensitivity adjustment and fluorescence correction of flow cytometry.
The materials are analyzed with a flow cytometry meter.

10. DNA Fragmentation Test
10-1 Performing Cells
3T3 Cells (mouse embryonic fibroblast cells)
10-2 Preparation of Sample Solution
Fluorescent Pigment, Color Pigment
The sample solution is made by adding 1mL of culture medium per 0.1g and extracting at 37°C for 24 hours.
Primer, UDMA, TEGDMA
Because it is difficult to dissolve them into culture medium for the primer, UDMA and TEGDMA are dissolved in
DMSO in advance, with the density prepared as shown below.
P r i m e r : 500µg/mL
U D M A : 286 (1) µg/mL (mM)
TEGDMA: 470 (1) µg/mL (mM)
The density of DMSO in the sample solution here is prepared at less than 1% without toxicity for cultured cells.
For the sample solution, DMSO-diluted solution of the same density as diluted is used as a reference value.

8-3 Method
3T3 cells propagated in a single layer by trypsinization are exfoliated and 2×105 pieces/mL of cell suspensions areprepared. 1mL of cell suspensions is scattered at 100µL over each well on a tissue culture plate and incubated forapproximately 6 hours at 5% CO2 at 37°C. After incubation, attachment of the cells to the bottom of the wells is
confirmed and the culture medium is removed. 100µL of sample solution for each sample is added and incubated for
48 hours at 5% CO2 at 37°C. Six hours before the incubation, 1µCi of 3H-Tymidine per well is added and theradioactivity of extracted 3H is measured with a liquid scintillation counter.

Resin (Test Model)
After washing resin (test model) with tap water and distilled water for injection of the Japanese pharmacopeia (fromHIKARI PHARMACEUTICAL CO., LTD.) and drying, ultraviolet sterilization is performed on both sides for 30
minutes on a clean bench. 1mL of culture medium is added to each 5 cm2 of surface area of the sample and extracted
to make sample solution in incubation at 5% CO2 for 24 hours at 37°C. All of the sample solution is then sterilizedwith a 0.22µm filter and used for the test.

― 11 ―― 12 ―

《 Results 》
1. Acute Toxicity Test
1-1 Incidence of Death
No incidence of death has been confirmed for either male or female during observation.
1-2 General Condition
No abnormality has been confirmed for either male or female during observation.
1-3 Weight Change
No weight change has been confirmed for either male or female or between control groups in any test group after
either 7 or 14 days from administration.
1-4 Autopsy Findings
No abnormality has been confirmed in main organs for either male or female test animals at autopsy after the periodof observation.

11-1 Performing Cells
3T3 Cells (mouse embryonic fibroblast cells)
11-2 Preparation of Testing Liquid
Fluorescent Pigment, Color Pigment
The sample solution is made by adding 1mL of culture medium per 0.1g and extracting at 37°C for 24 hours.
Primer, UDMA, TEGDMA
Because it is difficult to dissolve them into culture medium for the primer, UDMA and TEGDMA are dissolved in
DMSO in advance, with the density prepared as shown below.
P r i m e r : 500µg/mL
U D M A : 286 (1) µg/mL (mM)
TEGDMA: 470 (1) µg/mL (mM)
The density of DMSO in the sample solution here is prepared at less than 1% without toxicity for cultured cells.
For the sample solution, DMSO-diluted solution of the same density as diluted is used as a reference value.
Resin (Test Model)
After washing resin (test model) with tap water and distilled water for injection of the Japanese pharmacopeia (fromHIKARI PHARMACEUTICAL CO., LTD.) and drying, ultraviolet sterilization is performed on both sides for 30
minutes on a clean bench. 1mL of culture medium is added to each 5 cm2 of surface area of the sample and extracted
to make sample solution in incubation at 5%CO2 for 24 hours at 37°C. All of the sample solution is then sterilizedwith a 0.22µm filter and used for the test.
11-3 Method
As performed in DNA Synthesis Test, cell suspensions are prepared; 1mL is put in place and incubated forapproximately 6 hours at 5% CO2 at 37°C. After incubation, attachment of the cells to the bottom of the wells is
confirmed and the culture medium is removed. 1mL of sample solution (100%) is added to each sample andincubated for 48 hours at 5% CO2 at 37°C. After the incubation, the sample solution is collected from each well into
an Eppendorf tube; sedimentation is performed for 5 minutes at 2,000rpm and aspiration of the supernatant isconducted. 5µL of 2-propanol solution (0.2mM) of Phenyl methyl sulfunyl fluoride and 100µL of mixed solution of
1mL TNE buffer are added, the content fluid of each original well is replaced; the samples are steeped in iced waterfor 15 minutes after thorough pipetting. The cells of each well are propagated by pipetting and collected into a tube,
and sedimentation is then performed for 15 minutes at 12,000rpm, after which the supernatant is transferred into a
tube. 2.5µL of the supernatant is compounded with 200µL of BCA Protein Assay Reagent (PIECE); absorptivity ismeasured at 570nm and protein determination is performed.

Control Groups

Color Pigment

40

Fluorescent Pigment

Primer

45

Male

30
20
10
0

Female40

Weight (g)Weight (g)11. Protein Synthesis Test

45

30
20
10

0 Days

7 Days0

14 Days0 Days

14 Days

2. The Colony-Forming Assay
No reduction in the number of colonies at fluorescence pigment extraction at any density (Fig.2). No reduction in
the colony-formation rates for negative control group sample solution against empty extract was confirmed at thistime. For color pigment extract, a reduction in the number of colonies appeared with an increase in the sample
solution density, and a 50% inhibitory density is approximately 39%. For primer, a reduction in the number ofcolonies appeared with an increase in the testing liquid density, but the value for IC50 did not increase.

120
100
80
60
Fluorescent Pigment

40
20
0

0

Color Pigment

Empty Extraction

Primer

Negative Control Groups

12.5

25

50

Sample Solution Density (%)
Fig. 2: Result of the Colony-Forming Assay― 13 ―

7 DaysFig. 1: Result of Acute Toxicity Test (Weight Change)

Colony-Formation Rates (%)

10-3 Method
Exfoliate 3T3cells (Chinese Hamster Lung) propagated in a single layer by trypsinization are exfoliated and 1×15
pieces/mL of cell suspension is prepared using culture medium. Cell suspension is prepared, 3mL is scattered on
culture medium on a tissue culture plate and incubated for approximately 6 hours at 5% CO2 at 37°C. Afterincubation, attachment of the cells to the bottom of the wells is confirmed and the culture medium is removed. 3mLof sample solution is added to each sample and incubated for 48 hours at 5% CO2 at 37°C. Using a cell scraper, theculture cells are collected into an Eppendorf tube, with sedimentation for 5 minutes; 1mL of TORIZOL Reagent isthen added to dissolve the cells. 0.2mL of chloroform is added to the cell lysate, it is stirred for 15 seconds, andsedimentation takes place for 15 minutes (4°C) at 12,000rpm; it is then left for 2-3 minutes at room temperature.
Aspiration of clear supernatant is performed, 300µL of 100% ethanol is added to the rest of the suspension and it isleft for 2-3 minutes; sedimentation is then performed for 5 minutes at 2,000rpm. After performing aspiration of thesupernatant, 500µL of 0.1M sodium citrate (in 10% ethanol) is added, it is left for 30 minutes at room temperature,and sedimentation is performed for 5 minutes at 2,000rpm to re-perform aspiration of the sedimentation. 500µL of
0.1M sodium citrate (in 10% ethanol) is again added and aspiration performed on the supernatant also, adding 1mLof 75% ethanol. This is left for 20 minutes and DNA is extracted by performing 5-minute sedimentation at 2,000 rpm
and aspiration of supernatant. The fixed quantity of DNA is calculated from 260mm absorptivity measured byadding 20µL of TE buffer after dissolving the DNA. Based on the fixed quantity result, DNA solution is collected
and compounded with electrophoretic buffer (DNA Solution: Electrophoretic-6) to set the quantity at 0.05µg.
SeaKem ME agarose is used as electrophoretic agarose gel, diluted with TAE buffer at 1.8%. Electrophoresis isperformed for 1 hour with Mupid-2Plus at a constant voltage of 100V.

― 14 ―

100

4. Skin Sensitization Test

No dose-dependent change and no more than a double increase or decrease in the number of reverse mutationcolonies was confirmed compared to negative control groups for all samples.

No skin reaction was confirmed on any application site 48 and 72 hours after application in test groups; sensitivity
was 0% both 48 hours and 72 hours after induction (Table 3). For negative control groups, no skin reaction was
confirmed on any application site both 48 and 72 hours after application in test groups. On the other hand, forpositive control groups, clear red spots were confirmed within 24 hours from induction and medium to severe
inflammation appeared within 48 hours; necrosis and scabbing were confirmed within 72 hours. Sensitivity was
100% at 24, 48, and 72 hours.

A. TA100

B. TA1535

120

120

Number of Colonies Formed

Number of Colonies Formed

3. Reverse Mutation Test

100
80
60
40
20
0

0

1000

2000

3000

4000

5000

100

Table.3: Result of Skin-Sensitization Test

80
60

Test Group

10％

20
0

Fluorescent
Pigment
0

2000

3000

4000

5000

100
80
60
40
20
2000

3000

4000

5000

Quantity of Sample (μg)

Number of Colonies Formed

0.1％

Color Pigment

120

9

1％
0.1％

100

10％

80
60

Primer

9

40

1％
10％

20
0

0

1000

2000

3000

4000

Observation
Duration(hrs)

Positive Rate
(%)

48
72
48
72
48
72
48
72
48
72
48
72
48
72
48
72
48
72

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

Test GroupThe Numberof Test Animals

Density
10％

Negative
Control Groups

9

1％
0.1％
0％

Positive
Control Groups

6

0.1w/v ％Observation
Duration(hrs)

Positive Rate
(%)

48
72
48
72
48
72
48
72
24
48
72

0
0
0
0
0
0
0
0
100
100
100

5000

Quantity of Sample (μg)

5. Oral Mucosa Irritation Test

E. TA1537
120
Sodium Phosphate Buffer Solution (Fluorescent Pigment)

100

Sodium Phosphate Buffer Solution (Color Pigment)

80

None of 1) Thickening of horny layers and epithelium2) Carcinogenesis 3) Infiltration of inflammatory cells and 4)
Vasodilatation were confirmed in any of the samples. (Pic.1-3)

Sodium Phosphate Buffer Solution (Primer)

60

S9 Mix (Fluorescent Pigment)

40

S9 Mix (Color Pigment)

20
0

1％

10％

D. TA98
Number of Colonies Formed

Number of Colonies Formed

C. WP2 urvA

1000

1000

9

Quantity of Sample (μg)

1200Density

40

Quantity of Sample (μg)

0

The Numberof Test Animals

S9 Mix (Primer)
0

1000

2000

3000

4000

5000

Quantity of Sample (μg)

Fig. 3: Result of Reverse Mutation Test using Bacteria
Pic.1: Fluorescent Pigment― 15 ―

Pic.2: Color Pigment― 16 ―

Pic.3: PrimerNo cytostatic effect was confirmed in fluorescent pigment at the time of extraction. A slight reduction in viability
was confirmed for resin (test model) 168 hours after extraction. A reduction in viability was confirmed for color
pigment as time after extraction elapsed, and viability was reduced by 30% 168 hours after extraction. The time
period after extraction during which viability reduced by 50% was ascertained at 95 hours on this occasion. No
cytostatic effect was confirmed in primer at any sample solution density. In TEGDMA and UDMA, viability was
reduced by 30% and 15% respectively.

No exceptional occurrence of cell death was confirmed in any of the samples.

140
120
100
80
60
40
20
0

0

50

100

150

Viability (% of Reference Value)

8. Cytotoxicity Test Using Cultured Cells

Viability (% of Reference Value)

6. Cell Growth Inhibition Test

200

140
120
100
80
60
40
20
0

14.2

84.4

Color Pigment Elute

16.3

83.6

M05 Culture Medium (Reference Value)

14.1

82.2

Primer0

Extraction Time (hrs)
Fluorescent Pigment

Fluorescent Pigment Elute

200

400

600

PrimerColor Pigment

63.8

UDMA

20.9

79.0

TEGDMA

20.2

79.7

800 1000 1200

Sample Solution Density (µg/mL)

Resin (Test Model)

30.8

DMSO (Reference Value)

30.3

0%

TEGDMA

UDMA

69.6

20%

40%

60%

80%

Number of Necrotic Cells

Living Cells

Latter-Stage Apoptosis

Early-Stage Apoptosis

Fig. 4: Result of the in vitro Cytotoxicity Test

100%

Fig. 6: Result of Cytotoxicity Test

7. DNA Synthesis Test

9. DNA Fragmentation Test

Radiation activity volume of resin (test model) was the same as the reference value, but it increased by 78% in
fluorescent pigment, reduced by 38% in color pigment, and increased by 23% and 98% in primer and UDMA andTEGDMA respectively.

DNA degradation was confirmed in all of the samples; however, the difference between reference values and specific
DNA fragmentations in apoptosis were not measured (Pic. 4).

M1: Marker (1 kb DNA Ladder)

Radiation Activity Volume

60,00

M2: Marker (100kb DNA Ladder)

50,00

a: Fluorescent Pigmentb: Color Pigment

40,00

c: Resin Test Model

30,00

d: Reference Value

20,00

e: Primerf : UDMA

10,00
0

M1 M2

t
t
l)
en
en
de
o
m
m
g
M
ig
Pi
st
tP
lor
en
(Te
o
c
C
es
sin
or
Re
Flu

EM
DM

er
im
r
P

A
M
UD

A
M
GD
E
T

SO
DM

a

b

c

d

e

f

g: Reference Value of Test Primer MaterialPic. 4: DNA Fragmentation Test

Fig. 5: Result of DNA Synthesis Test

― 17 ―g― 18 ―

《 Discussion 》

10. Protein Synthesis Test
No reduction of the quantity of protein synthesis in fluorescent pigment and resin (test model) was confirmed
compared to the reference value (Fig.7). However, it reduced by 23% in color pigment compared to the reference
value. Also, it reduced by 6% in primer compared to the reference value. It reduced by 52% and 25% in UDMA andTEGDMA respectively.

1. Acute Toxicity

Since no weight change in mice and no shape changes in organs were confirmed in any sample, it becomes clear that
there is no acute toxicity in the substance.

2. Cytotoxicity
Quantity of Protein Synthesis (%)

140
120
100
80
60
40
20
0

nt

Fl

r
uo

e

e
sc

t

t

en

P

or

l
Co

m
ig

P

sin

)

el

en

m
ig

st

(Te

M

od

er

im
Pr

Re

A

M

UD

ue

A

M

T

D
EG

Fig. 7: Result of Protein Synthesis Test

ce

en

r
fe
Re

l
Va

There are some reports of cytotoxicity for UDMA and TEGDMA in biological evaluation reports of resin materials.
According to the reports, the value of IC50 is 0.12-0.26mM and 0.06-0.47mM respectively. In the fluorescentpigment, color pigment, resin (test model), prime, UDMA, and TEGDMA used in the test, color pigment inparticular showed cytostatic effect. It is considered that such cytostatic effect is caused either by cell death or delay
in progress of the cell cycle. To clarify these possibilities, a cytotoxicity test to observe whether or not there isapoptosis and necrosis was performed; the test results confirmed neither. From this fact, it is considered thatcolony-forming inhibitory action is caused not because cell death is caused by color pigment but because the cell
cycle influences and delays it in some manner. In addition, it is reported that while cytostatic effect was shownparticularly on uncured materials, almost no cytostatic effect was shown on cured materials, according to the resultof the in vitro cytotoxicity test on resin materials.13) The resin (Test Model) used this time was cured and hardened
as a test material. As a result, it is considered to have no impact on safety for color pigment on which cytostatic effectwas confirmed in the test from the fact that no abnormality was confirmed in any in vitro cytotoxicity test; the same
result as in the biological evaluation reports of resin materials.

3. Genetic Toxicity

In the reverse mutation test using bacteria, no increase or decrease in the number of reverse mutation colonies was
confirmed compared to the negative control group for any of the samples. Therefore, mutation of the sample isconsidered to be negative and there is no special risk of cancer being caused by DNA mutation under the test
conditions.

4. Skin Sensitization

In the result of the skin sensitization test using guinea pigs, no skin reaction was confirmed on any application site
either 48 or 72 hours after application in test groups, and sensitivity was 0%. Comparing these testing results withthe result of negative control groups and positive control groups, no skin reaction was confirmed on any applicationsite in negative control groups, and sensitivity was 0% at both 48 hours and 72 hours; therefore, the substances usedin this test have no risk of causing skin sensitization.

5. Oral Mucosa Irritation

No irritation response in any of the samples and solvent control cheek pouches was confirmed in observation with
the naked eye. In histopathological observation, no irritation response was confirmed in any of the samples offluorescent pigment, primer, and solvent control cheek pouches. In the color pigment, thickening of horny layers was
confirmed in 8 out of 15 of the cheek pouches for the test sample. However, the affected areas were topical and very
weak. Therefore, the substances used in this test are considered to have no irritation risk for oral mucosa of
hamsters.

《 Conclusion 》
In the result of the in vitro cytotoxicity test, color pigment mainly showed cytostatic effect. However, neither
apoptosis nor necrosis appeared in the cytotoxicity test. From these facts, the cytostatic effects observed in thecolony-forming assay and the cell growth inhibition test are considered to be caused by delay in progress of the cell
cycle, not by induction of cell death in color pigment.
In the reverse mutation test using bacteria, no colony formation was confirmed; it is clear that DNA mutation ofbacteria is not induced. Therefore, there is considered to be no risk of incidence of disease related to DNA mutation,such as cancer.
In the skin sensitization test using guinea pigs, no sensitization was confirmed. From this fact, the substances usedin the test are considered to have risk which is lower than the standard level for sensitization.
These tests were conducted in collaboration with the Oral Tumor Control Science, Tumor Pathology Course of Kochi
Medical School, Kochi University.

― 19 ―― 20 ―

《References》
1) Yumiko NISHIMOTO, Takeshi HOSHIKAWA, Teruo ANRAKU, Hirohisa YAMAMOTO: Refractive Index andLight Transmission of the SiO2-ZrO2 Filler Series in Composite Resin for Crowns. Journal of Nippon Academy of
Dental Technology, 22(1): 106-111, 2001
2) Yoshinori KISHIMOTO, Takeshi HOSHIKAWA, Hirohisa YAMAMOTO, Teruo ANRAKU: Development of Hard
Resin for Crown and Bridge Containing Spherical and Irregular Inorganic Filler Prepared by Sol-Gel method I.
Mechanical Properties. Journal of Nippon Academy of Dental Technology, 23(1): 88-92, 2002
3) Ai MIYAZAKI, Yoshinori KISHIMOTO, Takeshi HOSHIKAWA, Teruo ANRAKU, Hirohisa YAMAMOTO:
Development of Hard Resin for Crown and Bridge Containing Spherical and Irregular Inorganic Filler Preparedby Sol-Gel method II. Opal Characteristic, Journal of Nippon Academy of Dental Technology, 23(1): 93-97, 2002
4) Yoshinori KISHIMOTO, Takeshi HOSHIKAWA, Teruo ANRAKU, Hirohisa YAMAMOTO: Development of
Composite Resin for Crown and Bridge: Toothbrush Abrasion, Journal of Nippon Academy of Dental Technology,
24(1): 61-66, 2003
5) Yoshinori KISHIMOTO, Takeshi HOSHIKAWA, Hirohisa YAMAMOTO, Teruo ANRAKU: Study on
Processability of Composite Resin for Crown and Bridge by Means of Dynamic Viscoelastic Analysis, Journal of
Nippon Academy of Dental Technology, 24(1): 67-71, 2003
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Resin Composites for Crown and Bridge Containing Thiol Compound, Journal of Nippon Academy of Dental
Technology, 24(1): 79-83, 2003
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8) Hirohisa YAMAMOTO, Takahiro KATO, Ai MIYAZAKI, Takeshi HOSHIKAWA, Teruo ANRAKU: Synthesis of
SiO2-ZrO2 Fillers by Emulsion Method and Optical Properties of Composite Resins with Fillers, PROCEEDINGSOF THE 19TH KOREA-JAPAN INTERNATIONAL SEMINAR ON CEAMIC: 21-23, 30-308, 2002
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International Journal of Dental Technology, 32: 263-268, 2004
10) Jiro TANAKA, Hidekazu TAKAHASHI, Masaaki NAKAMURA, Kazuomi SUZUKI: Development of HighStrength, High Modulus, High Toughness Dental Matrix Resin without Environmental Endocrine Disruptor,
Journal of the Japanese Association for Dental Science, 23: 70-75, 2004
11) Dr. Angelika RZANNY Dr. Roland GOBEL, Prof. Dr. Dieter WELKER: Today’s Resin for Veneer
Crown-Scientific Research, QDT, 29: 75-83, 2004
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century: Especially, in relevance to endocrine disrupting effects, Dental Materials & Appliances, 21: 220-260,
200413) Hirokazu IMAI, Akihiro UEDA, Masaaki NALAMURA: P-54 Cell viability of photo fabrication resins, Dental
Materials & Appliances, 23: 148, 2004

《Series on Safety Reports》
Vol.1 The Pursuit of International Standards in Quality and Safety, December 2004
Vol.4 Biological Evaluation of “Luna-Wing”, June 2006
Vol.8 Biological Evaluation of “TWiNY” Hybrid Composite Resin, June 2010― 21 ―― 22 ―