447

Journal of Health Science, 46(6) 447–454 (2000)

Determination of Antimicrobial Agents in NonFormalin Adhesives for Wallpaper
Harunobu Nakashima,* Ichiro Matsunaga, Naoko Miyano, and Mikiya Kitagawa
Osaka Prefectural Institute of Public Health, 3–69 Nakamichi 1-chome, Higashinariku,Osaka, 537–0025, Japan(Received April 26, 2000; Accepted August 15, 2000)
To reduce formaldehyde in indoor environments, non-formalin products for wallpaper adhesives have been
increased in number. In this study, an analysis method using gas chromatography (GC) and gas chromatography
mass spectrometry (GC/MS) specific to each of several antimicrobial agents (2-methyl-4-isothiazolin-3-one (MIT),
5-chloro-2-methyl-4-isothiazolin-3-one (Cl-MIT), 1,2-benzisothiazolin-3-one (BIT), 2-n-octyl-4-isothiazolin-3-one(OIT) and 2,4,5,6-tetrachloro-isophthalonitrile (TPN)) was developed aiming to clarify the actual states of utilization of these agents. These antimicrobials have been reported to induce health impairments such as contact dermatitis. Furthermore, seven non-formalin adhesives for wallpaper sold as building materials were analyzed according
to the established method. The present results demonstrated that these adhesives contained a combination of antimicrobials (MIT, Cl-MIT, OIT and TPN), and one of them contained TPN at a high concentration (2,480 µg/g), which
is highly contact-allergenic.
Key words —–— isothiazolones, tetrachloroisophthalonitrile, gas chromatographic determination, non-formalin adhesive, allergic contact dermatitis, indoor environmental pollution

INTRODUCTION
Health impairments such as multiple chemicalsensitivities and sick-house syndrome caused by thechemical substances in household articles and building materials are becoming important social problems.1–4) In particular, indoor environmental pollution due to formalin is a serious problem at present,
and many attempts to remove the compound fromindoor have been progressing. Therefore, non-formalin adhesives for wallpaper that have safety andecological features as selling points are now increasing. However, the type and content of antimicrobial
agents used as the substitutes for formalin are notknown in most of those adhesives. To clarify theactual states of utilization of these antimicrobials,
we attempted to develop an analytical method foreach of the following antimicrobial agents usable
for wallpaper which have been reported to havecaused health impairments such as contact dermatitis.
Four isothiazolones, including 2-methyl-4isothiazolin-3-one (MIT), 5-chloro-2-methyl-4isothiazolin-3-one (Cl-MIT), 1,2-benzisothiazolin*To whom correspondence should be addressed: Osaka Prefectural Institute of Public Health, 3–69 Nakamichi 1-chome,
Higashinariku, Osaka 537–0025, Japan. Tel.: +81- 6-6972-1321;
Fax: +81-6-6972-2393; E-mail: hrnakaji@iph.pref.osaka.jp

3-one (BIT), 2-n-octyl-4-isothiazolin-3-one (OIT)
and 2,4,5,6-tetrachloro-isophthalonitrile (TPN), werechosen as test chemicals. Several adhesive products for building materials were analyzed according
to the respectively developed methods. It was thus
demonstrated that a combination of four antimicrobial agents (MIT, Cl-MIT, OIT and TPN) were detectable in the respective adhesives, and one of them
contained a high concentration of TPN with highcontact allergenicity (2,480 µg/g). In this report, theanalytical methods and the analytical data are described and the results are discussed from the aspectof toxicity assessment.

MATERIALS AND METHODS
Samples —–— Seven non-formalin adhesive products for wallpaper, sold as building materials, wereused as subjects for analysis. Six samples were prepared in the paste state, and only sample No. 3 wasa solid powder.
Reagents —–— As the standard substances, we used
NS-500W containing 3.2% MIT and 10.3% Cl-MIT(Nagase Chemical Co., Ltd., Hyogo, Japan) for MIT
and Cl-MIT, Ploxcel PL (100% purity, Zeneca Co.,
Ltd., Tokyo, Japan) for BIT, Permachem PD containing 4.75% OIT (Permachem Asia Co., Ltd., To-

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Vol. 46 (2000)

Table 1. Operating Conditions of GC and GC/MS
FPD-GC conditions
Column: SPB-5(φ 0.53 mm × 30 m, film thickness 1.5 µm , Supelco)
Column temperature:100◦ C (1 min) – 10◦ C/min – 280◦ C (20 min)Injection inlet temperature: 250◦ C,
Detector temperature: 280◦ C
Carrier gas flow rate: He 20 ml/min,
Injection volume: 2 µl
ECD-GC conditions
Column: PTE-5(φ 0.25 mm × 30 m, film thickness 0.25 µm , Supelco)
Column temperature:60◦ C (1 min) – (20◦ C/min) – 285◦ C (3 min.)Injection inlet temperature: 200◦ C,
Detector temperature: 300◦ C
Carrier gas flow rate
(Injection inlet pressure): He 110 kPa
Injection volume: 1 µl (splitless)
GC/MS conditions
Column: DB-5MS(φ 0.25 mm × 30 m, film thickness 0.1 µm, J & W)
Column temperature:80◦ C (1 min) – 8◦ C/min – 280◦ C (4 min)Injection inlet temperature: 250◦ C,
Interface temperature: 280◦ C
Carrier gas flow rate: He 1 ml/min.,Injection volume: 2 µl (splitless)
Ionization voltage: 70 eV (EI method)kyo, Japan) for OIT, and the standard product (100%purity, Sannopuco, Tokyo, Japan) for TPN. A graphite carbon black cartridge column was ENVI-CARB(1 g/12 ml, Supelco Co., Ltd., Bellefonte, U.S.A.),and the silica cartridge column was Sep-Pak Plus
(Waters Co., Ltd., Massachusetts, U.S.A.). Organic
solvents such as n-hexane were used for residual
pesticide analysis.
GC and GC/MS Operating Conditions —–—FPDGC (5890 Series II, Yokogawa Hewlett Packard
Co., Ltd., Tokyo, Japan) and ECD-GC (GC-17A,
Shimadzu Co., Ltd., Kyoto, Japan) were used forthe quantitative analyses of isothiazolones andTPN, respectively. GC/MS (5890 Series II, 5970,
Yokogawa Hewlett Packard) was used for identification and confirmation of each compound detectedin the samples.
Table 1 shows the operating conditions of GC
and GC/MS.
Preparation of Test Samples
MIT and Cl-MIT: One-half gram of an adhesive

sample was put into a 50 ml centrifuge tube andshaken for 5 min with 20 ml of purified water for
extraction. The mixture was centrifuged for 5 min
at 3000 rpm. The aqueous extract was applied onto
the graphite carbon cartridge column. After runningwith 20 ml of purified water, 20 ml of methanol was
injected to elute MIT and Cl-MIT. After evaporating the eluate, the residue was dissolved in n-hexane and determined by FPD-GC.
BIT: One-half gram of a sample was put into an
80 ml centrifuge tube and shaken for 5 min with
30 ml of purified water and 30 ml of diethyl ether/
cyclohexane mixture (4 : 1) for extraction. The mixture was centrifuged for 5 min at 3000 rpm. The
supernatant (diethyl ether/cyclohexane phase) was
put into a 200 ml round-bottomed flask. A similar
operation was repeated twice after the addition of
30 ml of a diethyl ether/cyclohexane mixture. The
extract was concentrated to 5 ml and applied onto
the silica cartridge column. After running with 20 mlof n-hexane, 20 ml of acetone was injected to elute
BIT, and the eluate was determined by FPD-GC.
OIT: One-half gram of a sample was put into an
80 ml centrifuge tube and shaken for 5 min with
30 ml of purified water and 30 ml of cyclohexane.
The mixture was centrifuged for 5 min at 3000 rpm.
The supernatant (cyclohexane phase) was transferred
into a round-bottomed flask. A similar operation was
repeated twice, with the addition of 30 ml of cyclohexane. The extract was concentrated to 5 ml andapplied onto a silica cartridge column. After running with 20 ml of n-hexane, 20 ml of n-hexane/acetone mixture (1 : 1) was injected to elute OIT, andthe eluate was subjected to FPD-GC analysis.
TPN: One-half gram of a sample was put into an
80 ml centrifuge tube and shaken for 5 min with
30 ml of purified water and 30 ml of cyclohexane.
The mixture was centrifuged for 5 min at 3000 rpm.
The supernatant (cyclohexane phase) of 2 ml wasapplied onto a silica cartridge column. After running with 40 ml of n-hexane, 20 ml of n-hexane containing 1% acetone was injected to elute TPN, andthe eluate was determined by ECD-GC.

RESULTS AND DISCUSSION
Preparation of Standard Solutions
Since BIT and TPN were almost pure, theseproducts were dissolved in acetone to make the stock
solutions, which were diluted with n-hexane to prepare working standards. For data as to the content

No. 6449

of MIT and Cl-MIT, we referred to the package leaflet of the product. Thus, these product data wereused as the standard values. The product was dissolved with acetone and appropriately diluted withn-hexane to prepare the working standards for GC.
For the recovery experiment, the stock solution was
diluted with purified water. For OIT, no data as toproduct content or any product information wereobtainable. So, the concentration of OIT in
Permachem PD was determined first of all. The peakof OIT in Permachem PD was identified by GC/MS
and compared with the peak area of undecane, a samecarbon number compound, by FID-GC to estimate
the concentration of OIT. The concentration of OIT
in Permachem PD was 4.75%. Thus, an acetone
solution of Permachem PD was prepared and appropriately diluted with n-hexane to prepare theworking standards.
Examination of Detection Methods
Since all antimicrobial isothiazolones containsulfur, these compounds were detected by FPD-GC(S filter, 393 nm), and TPN was detected by ECDGC. Figure 1 shows the chemical structures of thosecompounds.
FPD-GC Conditions: There is a report on themeasurement of MIT and Cl-MIT in somemethylisothiazolone formulations (Kathon CG,
Euxyl K 100, etc.) by HPLC.5) However, sincehighly sensitive analysis is needed for the simultaneous measurement of isothiazolones used as antimicrobial agents in adhesives, we investigated theoperating conditions of the GC method for such a
purpose. Since all antimicrobial isothiazolones contain sulfur, FPD-GC (S filter, 393 nm) was employedfor detection. There are few reports on analyticalmethods for isothiazolones using GC. In particular,
BIT was decomposed by adsorption in a GC column, as assumed from its structure, and was lesssensitive than other isothiazolones. Therefore, BIT
was methylated with diazomethane, and two kindsof derivatives (O-methylated and N-methylated derivatives) were induced simultaneously. Althoughdetection sensitivity was raised by the use of thesederivatives, it became necessary to examine the synthetic ratio of the two derivatives. At the concentration used in these test samples, the compound was
detectable without methylation. Therefore, methylation was not conducted in this study because otherisothiazolones scarcely submitted to adsorption decomposition in the GC column. As a result of various GC conditions being examined, discrimination

Fig. 1. Chemical Structures of the Respective Antimicrobial
Agents① MIT: 2-methyl-4-isothiazolin-3-one, ② Cl-MIT: 5-chloro-2methyl-4-isothiazolin-3-one, ③ BIT: 1,2-benzisothiazolin-3-one,④ OIT: 2-n-octyl-4-isothiazolin-3-one, ⑤ TPN: 2,4,5,6tetrachloroisophthalonitrile.

of the four antimicrobial agents and their isolation
became possible using FPD-GC under the conditions
mentioned above. Determination was made using
the respective calibration curves ranging from 0.64–
6.4 ng (0.32–3.2 µg/ml, detection limit 0.32 ng) for
MIT, 1.03–20.6 ng (0.515–10.3 µg/ml, detection
limit 0.25 ng) for Cl-MIT, 8–40 ng (4–20 µg/ml, detection limit 8 ng) for BIT, and 0.38–5.7 ng (0.19–
2.85 µg/ml, detection limit 0.19 ng) for OIT.
ECD-GC conditions: Although a variety of analytical methods for TPN in agricultural products,6–9)
soil,10,11) water12,13) and air14–16) have been reported,no method for analyzing TPN in adhesives wasfound. In the present study, highly sensitive ECDGC was used, and determination was performed
under the above conditions using the calibration
curve in the range of 5–50 pg (5–50 ng/ml, detection limit 3 pg).

450

Pretreatment Methods
For each antimicrobial agent, pretreatment methods were examined because the polarity was different among the test chemicals.
Extraction and Cleanup for MIT and Cl-MIT:
The adhesives contained starch as a principal ingredient and were water-soluble. Therefore, it was necessary to dissolve a sample in the water in order toextract antimicrobial agents from these adhesives.
However, as the solubilities of two antimicrobial
agents were higher in water than organic solvents,
their extraction from water to an organic solvent wasdifficult. Thus, solid–phase extraction was examined using an ODS column, polystyrene polymer
column and graphite carbon column. The graphite
carbon cartridge column (ENVI-CARB 1 g/12 ml,Supeluco Co., Ltd.) was optimum for extracting both
agents. An aqueous solution of MIT (3.2 or 32 µg)
and Cl-MIT (10.3 or 103 µg) was applied onto thecolumn, then 40 ml of purified water was passed
through. At that time, 99% of MIT and 100% of ClMIT were retained in the column. Then, 20 ml ofmethanol was passed through and the both agentswere completely eluted from the column. Thus, notonly solvent exchange for assay by GC, but also
elimination of polar substances became possible. In
addition, the elimination of non-polar substances was
possible by a liquid–liquid partition using n-hexane
and water (1 : 1). But this process was omitted, because the samples were all water-soluble and did notseem to contain non-polar substances.
Only sample No. 3 was a solid powder. On theleaflet, it was instructed to add the attached antiseptic into the preparation diluted with 7 or 8 volumesof water. Since no antimicrobial agent was present
in the original product, the recovery test was madefor each antimicrobial agent using this product. To
0.5 g of Sample No. 3 prepared with water, MIT (3.2
or 32 µg) or Cl-MIT (10.3 or 103 µg) was added,
and the recovery test was conducted according to
the procedures. The recovery rate was in a range of
95 to 101% (Coefficient of variation CV = 4.0%,
n = 3).
Extraction and Cleanup for BIT: Solvents to extract BIT from aqueous solution of a sample were
examined. To 30 ml of purified water, BIT (10 or
100 µg) was added. After 30 or 15 ml of organic
solvent was added, the mixture was shaken for 5 min,and the partition behavior of BIT between the organic phase and the aqueous phase was observed.
As organic solvents, ① cyclohexane, ② hexane,③ diethyl ether, ④ four diethyl ether/hexane mix-

Vol. 46 (2000)

tures (4 : 1, 3 : 2, 2 : 3 and 1 : 4), ⑤ four diethyl
ether/cyclohexane mixtures (4 : 1, 3 : 2, 2 : 3 and1 : 4) were examined. The mixture containing equalvolumes of water and diethyl ether/cyclohexane mixture (4 : 1 or 3 : 2) gave the highest transfer, at a
rate of 74 to 78%, into the organic phase. Thus, it
was decided to dissolve the adhesive in water andadd an equal volume of diethyl ether/cyclohexane
mixture (4 : 1), followed by repeating this procedure
three times. For this procedure, the recovery rate
was 90 to 98%. In addition, BIT was not extractable with cyclohexane or hexane.
When 2 ml of cyclohexane solution containing
10 or 100 µg of BIT was applied onto the silica gel
cartridge column, and 30 ml of n-hexane was passed
through, BIT was not eluted at all. Then, a total of
100 ml of acetone was passed in portions of 10 ml
each, and 90% of BIT was eluted in the first two
fractions (20 ml) but not in the subsequent fractions.
Thus, it was decided to elute BIT from the column
with 20 ml of acetone.
To 0.5 g of Sample No. 3 prepared with water,
20 or 200 µg of BIT was added, and the recovery
test was conducted according to the procedure. The
recovery rate was in a range of 85 to 90% (CV =3.4%, n = 3).
Extraction and Cleanup for OIT: Solvents to
extract OIT from an aqueous solution of a sample
were examined. To 30 ml of purified water, OIT(11.9 or 119 µg) was added. After 30 ml of organic
solvent was added, the mixture was shaken for 5 min,and the partition behavior of OIT between the organic phase and the aqueous phase was observed.
As organic solvents, ① cyclohexane, ② hexane,③ diethyl ether/cyclohexane mixture (1 : 1) and④ diethyl ether/hexane mixture (1 : 1) were tested.
For a mixture containing equal volumes of water andcyclohexane or n-hexane, the extraction rate into theorganic phase was as high as 90 to 95%. Thus, it
was decided to dissolve the adhesive in water andadd an equal volume of cyclohexane. This was repeated three times. For this procedure, the recovery
rate was 90 to 98%.
When 2 ml of cyclohexane solution containing
11.9 or 119 µg of OIT was applied onto a silica gel
cartridge column and 30 ml of n-hexane were passed
through, OIT was not eluted at all. Then, a total of
100 ml of hexane/acetone mixture (1 : 1) was passedin portions of 10 ml each, and 99% of OIT was eluted
in the first two fractions (20 ml), but not in the subsequent fractions. Thus, it was decided to elute OIT
with 20 ml of n-hexane/acetone mixture (1 : 1). In

451

No. 6

addition, only 30% of OIT was extracted with nhexane containing 20% acetone in the first two fractions.
To 0.5 g of Sample No. 3 prepared with water,
23.8 or 238 µg of OIT was added, and the recovery
test was conducted according to the procedures. The
recovery rate was 95 to 99% (CV = 2.9%, n = 3).
Extraction and Cleanup for TPN: Solvents to extract TPN from an aqueous solution of a sample were
examined. To 30 ml of purified water, TPN (20 or
200 µg) was added. After adding 30 ml of organic
solvent, the mixture was shaken for 5 min, and thepartition behavior of TPN between the organic phaseand the aqueous phase was observed. As organic
solvents, ① cyclohexane and ② diethyl ether/cyclohexane mixtures (4 : 1 and 2 : 3) were examined. Ahigh transfer rate of 95 to 97% was obtained for all
solvents. Therefore, it was decided to extract thecompound from an equivolume mixture of water andcyclohexane (1 : 1) because of the simplicity of themethod.
When 2 ml of cyclohexane solution containing
2 or 20 µg of TPN was applied onto the silica gel
cartridge column and 40 ml of n-hexane was passed
through, no TPN was eluted. Then, 20 ml of n-hexane containing 1% acetone was passed through, and
99% of TPN was eluted. TPN was not eluted withan additional 20 ml of n-hexane containing 1% acetone. Thus, it was decided to elute TPN with 20 mlof n-hexane containing 1% acetone.
To 0.5 g of Sample No. 3 prepared with water,
200 µg of TPN was added, and the recovery test was
conducted according to the procedures. The recovery rate was in a range of 98 to 103% (CV = 3.0%,
n = 3).
Analytical Results
Table 2 shows the analytical results of the adhesives by the procedures mentioned above. These
antimicrobial agents were detected in most of the
products. OIT and TPN were detected at a high concentration in Sample No. 7, especially, TPN was
detected at the order of mg/g. Analysis of the antiseptics attached to Sample No. 3 revealed that theantiseptics were composed of MIT and Cl-MIT.
Figure 2(A) shows FPD-GC for the standard
solutions of four isothiazolone antimicrobial agents,
while Fig. 2(B) shows an FPD-GC for Sample No. 1(a mixture of test solutions prepared for the quantitative analysis of each compound). Figure 3 showsan ECD-GC of Sample No. 7. Figure 4 shows themass spectra of each antimicrobial agent in the

Table 2. Analytical Results of Antimicrobial Agents in NonFormalin Adhesives for Wallpaper (µg/g)
Sample No
No. 1
No. 2
No. 3
No. 4
No. 5
No. 6
No. 7

MIT
10.4
4.4
N.D.
8.1
8.9
7.6
5.9

Cl-MIT
26.5
13.7
N.D.
20.3
14.6
17.7
18.2

BIT
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.

OIT
11.0
5.9
N.D.
N.D.
N.D.
N.D.
133.0

TPN
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
2480

N.D.: Not detected. The unit of the measurement concentration is µg/g.

samples.
Although MIT and Cl-MIT have been used as
antiseptics for cosmetics for a long time and the negative data on contact allergenicity have been published,17) many cases of skin impairments due to thesecompounds have been reported.18–23) While there isa report on the analysis of methylisothiazolone formulation (Kethon C.G., etc.) by HPLC,5) analyticalmethods for commercial products such as cosmetics containing these chemicals have hardly been reported. Therefore, the actual states of their utilization and concentrations are not known. The present
study clarified that these chemicals are actually used
as antiseptics for wallpaper adhesives.
An antimicrobial agent, BIT, is used as an antiseptic or a fungicide in paints, leathers, cutting oils
and printing ink.24) This agent is known to have skinirritability and skin-sensitization activity.25–27) Numerous cases among paint manufacturers28) and theemployees in the printing industry, wall repapering,
metal processing and chemical industries29–35) havereported allergic symptoms, probably due to BIT.
In the study of subacute toxicity test in rats, a weightdecrease in the liver and pituitary gland,36) thickening of the anterior stomach and thymus atrophy37)
have been reported. However, there are few reportsof analytical methods for this chemical.
OIT is a compound widely used as an antiseptic
in paints, timbers and plastics,38) and we have detected it qualitatively in commercially available
household paints.39) Although no data on contact
allergenicity of OIT has been reported,38) there aresome case reports on skin damage.40–43) But there
are no reports on analytical methods, and it was difficult to obtain a standard product of OIT. As mentioned above, the concentration of OIT in the antiseptics sold for industrial use was used as the standard reference.
One of the organic chlorine antiseptics, TPN, has452Vol. 46 (2000)

Fig. 2. FPD Gas Chromatograms of a Standard Solution of Isothiazolones (A) and Sample No. 1 (B)① MIT: 2-methyl-4-isothiazolin-3-one, ② Cl-MIT: 5-chloro-2-methyl-4-isothiazolin-3-one, ③ BIT: 1,2-benzisothiazolin-3-one, ④ OIT: 2-n-octyl4-isothiazolin-3-one, Sample No. 1 (a mixture of the testing solutions prepared for the quantitative analysis of each compound). Operating conditions of
FPD-GC are given in Table 1.

been widely used as an antiseptic or fungicide for
agriculture, paints and timber under the market
names of Chlorothalonil or Daconil.44,45) The lethal
dose of TPN by oral administration in rats was 10 g/
kg, and the chemical was toxic to fishes and irritable to human eyes and skin.45) The International
Agency for Research on Cancer (IARC) has classified TPN as Group 3 (materials of which carcinogenicity have not been reported in man), and the Environment Agency has defined it as a substance subject to the Pollutant Release and Transfer Register(PRTR). The occurrence of allergic symptoms inwoodworkers,46) painters47) and gardeners48) has beenreported. In a recent study of contact allergenicity
in a guinea pig maximization test (GPMT),49) TPN
was reported to be an extremely strong contact sensitizer.50)
There are many reports on analytical methods
for TPN in agricultural products,5–9) soils,10,11) water12,13) and air.14–16) TPN is classified as a semivolatileorganic compound by WHO criteria,51) and its detection in air has been reported.14–16) Therefore, there
is a possibility that workers in wall repapering might
be exposed to a high concentration of TPN, not only
through the skin but also the respiratory tract if theFig. 3. ECD Gas Chromatogram of Sample No. 7⑤ TPN: 2,4,5,6-tetrachloroisophthalonitrile. Operating conditionsof ECD-GC are given in Table 1.

compound is used at a high concentration, as seen
for Sample No. 7. Moreover, residents may be exposed to TPN in the air since wallpapers are used inlarge areas. Allergic contact dermatitis is known
to be inducible even by exposure to a low concentration, once the skin is sensitized by a high concentration of the compound. Multiple chemical sensitivities are also reported to occur by slight re-expo-

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No. 6

Fig. 4. Mass Spectra of Each Confirmed Antimicrobial Agent in the Samples by GC/MS.
(1) Mass spectrum of MIT detected from Sample No. 1. (2) Mass spectrum of Cl-MIT detected from Sample No. 1. (3) Mass spectrum of OIT
detected from Sample No. 1. (4) Mass spectrum of TPN detected from Sample No.7. Operating conditions of GC/MS are given in Table 1.

sure after previous exposure at a high concentration.1–4) Although there is no specification for thechemicals in the package insert of Sample No. 7,
except description that chemicals of safety and low
toxicity should be used, it is not likely that the safetyof TPN is higher than that of formalin. Therefore, it
is recommended to avoid the use of TPN at such ahigh concentration as seen in the case of Sample
No. 7.
These results suggest that, in the use of non-formalin adhesives as building materials with safety
and ecological properties, isothiazolone antimicrobial agents and TPN were mixed as substituting
chemicals. It is not likely that these are more safe
than formalin in some conditions of exposure. Thus,
it is necessary to specify the kind and the concentration of drug used. Furthermore, for infants, the elderly and hypersensitive patients who are more susceptible to agents, the application must be made ateach appropriate concentration in consideration of
exposure conditions (area of contact, exposure timeand distance to contact, etc.). In the future, it will

be necessary to evaluate the toxicities of these drugs
as well as their exposure effects.

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