European Archives of Oto-Rhino-Laryngology
https://doi.org/10.1007/s00405-022-07289-8

RHINOLOGY

Studies on safety and efficacy of particles containing a mixtureof hydroxyapatite–argentum–titanium oxide (HAT) and sheets coatedwith HAT particles to be used in masks to improve nasal allergy: II.
Cellular, in vivo, and clinical studies
Narumi Okazaki1 · Dai Yamaki1
Takayuki Shindo4,5· Toshio Takei1· Miyuki Shimizu2· Naoyuki Kamatani3·

Received: 24 October 2021 / Accepted: 24 January 2022
© The Author(s) 2022

Abstract
Purpose We report the manufacture of particles containing a mixture of hydroxyapatite–argentum–titanium oxide (HAT),
followed by attachment to nonwoven polyester fabrics to produce HAT-coated sheets (HATS) for use in masks. The purposeof the present study was to perform cellular, in vivo, and clinical studies to further examine the safety of HATS for use inmasks to improve nasal allergy.
Methods Reverse mutation tests for HAT were performed using five bacterial strains. A cellular toxicity test was performed
using a Chinese hamster cell line incubated with the HATS extracts. Skin reactions after intradermal administration were
examined in rabbits. Skin sensitization tests in guinea pigs were performed using the HATS extracts. HAT was administered
to the nasal cavity and conjunctival sac of the rabbits. An oral administration study was performed in rats. Finally, a humanskin patch test was performed using the HATS.
Results Reverse mutation tests showed negative results. The cellular toxicity test showed that the HATS extract had moderate cytotoxicity. The intradermal skin reaction and skin sensitization tests were all negative. The administration of HAT to
the nasal cavity and intraocular administration showed negative results. No toxicity was observed after oral administrationof HAT powder up to a dose of 2000 mg/kg. Finally, the skin patch test result was negative.
Conclusion Although HAT showed moderate cytotoxicity, in vivo results indicated that HAT is safe because it does notcome in direct contact with cells in normal usage, and HATS is safe when used in masks.
Keywords Hydroxyapatite · Titanium oxide · Nasal allergy · Mite allergy · Cedar pollinosis · Photocatalysis

Introduction* Narumi Okazaki
okazaki@drciyaku.co.jp
1

DR.C Medical Medicine Co. Ltd., Shinjuku, Tokyo, Japan

2

Medical Lab Partners Corporation, Chiyoda, Tokyo, Japan

3

StaGen Co. Ltd., Taito, Tokyo, Japan

4

Department of Cardiovascular Research, Shinshu University
School of Medicine, Matsumoto, Nagano, Japan

5

Department of Life Innovation, Institute for Biomedical
Sciences, Interdisciplinary Cluster for Cutting Edge
Research, Shinshu University, Matsumoto, Nagano, Japan

After World War II, the Japanese cedar tree, an indigenous
tree species, was planted in Japan according to government
policy. As a result, approximately 4.5 million hectares are
now covered by cedar plantations that occupy about 40%of the planted forests in Japan. Approximately 30% of theJapanese population is estimated to be affected by cedar pollinosis, and many Japanese people suffer from severe sneezing and nasal congestion between February and April. Cedar
pollinosis has significant social and economic impacts. In
addition to pollinosis, nasal allergy caused by dust is an
important perennial allergy. Dust containing mite allergens
is an important source of allergies.

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European Archives of Oto-Rhino-Laryngology

Various attempts have been made to prevent or improvecedar pollinosis. Medicines are undoubtedly effective;
however, there are also other ways of preventing or
improving cedar pollinosis. For example, there are gargles
and masks. Masks are expected to be effective in reducing
the amount of pollen inhaled by one-third to one-sixth
when pollen counts are high, thus reducing nasal symptoms. Furthermore, chemical modification of the mask
material could have an even stronger improvement effect[1].
The photocatalytic action of titanium dioxide has a wide
range of applications, including the purification and disinfection of water and air [2, 3]. Organic substances suchas proteins are decomposed by the active oxygen species
generated by the photocatalytic action of titanium dioxide.
When titanium dioxide is irradiated with ultraviolet light,
free radicals are generated [4]. However, when silver isadded to titanium dioxide, it absorbs both ultraviolet light
below 380 nm and visible light between 700 and 800 nm,
which enhances the photocatalytic effect [5–8]. In contrast,hydroxyapatite (HA) can adsorb organic substances such asproteins and viruses [9]. Nonami et al. reported that HA
coated with titanium dioxide effectively removes airborne
materials [10]. The organic substances that cover the surfaceof HA reduce its adsorption capacity, but the reactive oxygen
species generated by the titanium dioxide decompose theadsorbed organic substances, thereby improving the adsorption capacity of HA [10].
To prevent pollinosis and dust-induced nasal allergies, we
devised a method of producing particles made of a mixtureof HA, silver, and titanium oxide (HAT), which adsorb anddecompose allergens [1]. We attached HAT particles to the
non-woven fabrics used in masks. The HAT attached sheet(HATS) was placed between two sheets of non-woven fabrics and used as a mask to improve nasal allergies [1].
Allergens that cause nasal allergies are trapped by HAT
particles as they pass through the mask and are expected tobe efficiently broken down. In our previous study, we showedthat Cryj1, a major allergen of cedar pollinosis, and Der f1,
a major allergen of mite allergy, were efficiently reduced by
HATS [1]. The reduction of these allergens by HATS wasobserved under light-shielded conditions, but the reduction
was even greater under light irradiation, thereby suggesting
the absorption of major nasal allergens by HATS particles
and their decomposition by the photocatalyst. Therefore,masks with HATS interspersed between non-woven fabrics
are expected to reduce allergens and alleviate nasal allergies before allergens are detected. However, because masks
are worn over the nose and mouth for a long time, some of
the HAT particles attached to the fabric may be exposed.
Therefore, the safety of the HATS and HAT particles needs
to be studied in detail.

13

In general, it has been reported that nanoparticles smaller
than 100 nm can cause harm by oral ingestion [11, 12], inhalation [13], and skin contact [14]. We investigated the particle size distribution of HAT particles and their binding
mode to non-woven fabrics using a laser scattering particle
size analyzer and scanning electron microscopy [1]. As aresult, the particles in our HAT powder showed a unimodal
particle size distribution close to a lognormal distribution,with a median of 371.9 nm. Ninety-five percent of the particle sizes ranged from 199.8 to 1079.9 nm, and no particles
smaller than 131 nm were detected [1]. Scanning electron
microscopy showed that the HAT particles were attached to
the fibers of the non-woven fabric as spherical and flattenedmaterials [1].
Based on the above rudimentary safety data of HAT particles and HATS, various biological and medical tests were
performed in this study. Here, we report the results of mutagenicity tests using bacteria, toxicity tests using cells, andin vivo experiments using animals. In addition, skin patch
tests were conducted in a clinical trial, and safety data wereobtained.

Methods
Reverse mutation tests using bacteria
Reverse mutation tests using HAT were conducted using
five bacterial strains, Salmonella typhimurium TA98 andTA1537 with frame-shift mutations, TA100 and TA1535[15], and Escherichia coli WP2 uvrA with single nucleotide substitutions [16] obtained from the Japan Bioassay
Research Center (Hatano-shi, Japan). The procedures are
essentially based on a previous report [17] and in accordancewith the guidelines of the Japanese government (Guidelines
for the Act on the Evaluation of Chemical Substances andRegulation of Their Manufacture, etc., 1997; Safety and
Health Law Test Guidelines 1988, 1997; Guidelines for
Biological Testing of Medical Devices 1995).
Briefly, after thawing, 20 mL of the cryopreserved bacterial suspension was added to 10 mL of liquid complete
medium made of 25 g nutrient broth No. 2 (Oxid Unipath,
Hampshire, UK) in 1 L distilled water and, after shaking
the medium for 8 h at 37 °C, the bacteria in the medium
were used for the culture. A 35% water suspension of HAT
powder stored in the dark at room temperature (18–23 °C)
was used as the test material. Then, a 0.1 mL solution containing varying concentrations of HAT powder in sterilized
water was mixed with 0.5 mL of either 0.1 M ­NaPO4 (pH7.4) or exogenous metabolic activation system S9 mix (Kikkoman Biochemifa Company, Tokyo, Japan), and 0.1 mL ofthe bacteria-containing solution. Thereafter, the combined
suspension was fully mixed by shaking for 20 min at 37 °C,

European Archives of Oto-Rhino-Laryngology

placed on Tesmedia AN medium (Oriental Yeast Industry;
Tokyo, Japan), and cultured for 48 h at 37 °C. Then, thenumber of colonies was counted using an auto-colony counter or visual inspection.

Cytotoxicity test using colony formation test
All procedures were performed in accordance with ISO
10993-5:2009(E) and “Japanese Drug and Food Safety
Bureau Notification No. 0301-20, March 1, 2012: Basic
Biological Safety Assessment Required for Application forApproval of Manufacture and Sale of Medical Devices”.
M05 medium was prepared by adding nonessential aminoacids for minimum essential medium (ICN Biochemicals,
Cranberry Township, PA), 0.11 g/L sodium pyruvate (ICN
Biochemicals) and 5 vol% of fetal bovine serum (Sanko
Junyaku in Tokyo) to Eagle MEM medium (Thermo Fisher
Japan, Tokyo). Then, 35% HAT suspension in water waslyophilized and autoclaved, and 10 mL of M05 medium wasadded to 1 g of HAT. After incubating the HAT-containing
medium for 24 h, only the medium was recovered and usedas the 100% HAT extract. Polyurethane films containing
0.1% zinc diethyldithiocarbamate (standard material A),
polyurethane film containing 0.25% zinc dibutyldithiocarbamate (standard material B), and high-density polyethylene
film (negative control material) were obtained from the Foodand Drug Safety Center (Hatano-shi, Japan) and cut into
small pieces and autoclaved. Thereafter, the samples weretreated as described for HAT, and the extracts were used as100% extracts.
Cells from the V79 Chinese hamster cell line [18]obtained from RIKEN (Yokohama, Japan), cultured in M05,
were used. Aliquots (5 mL) of the cell suspension (adjusted
to 40 cells/mL) were seeded onto 60 mm plates and incubated in a C­ O2 incubator for 24 h at 37 °C. After removalof the culture medium, 5 mL of each extract was added to aplate. Three plates were used per concentration per extract(HAT, standard material A, standard material B, or negative control material). In addition, three plates to which only
medium was added were used as negative control plates.
After the addition of the extracts, the plates were incubatedin a ­CO2 incubator for six days.
After incubation, the media were discarded from allplates, and the plates were washed with 5 mL of phosphatebuffered solution. Then, 5 mL of methanol was added toeach plate and left for 10 min to fix the cells. After fixation,
the methanol was discarded, and 5 mL of 5% Giemsa solution was added to the plates and incubated for 10 min. Plateswith stained colonies were washed with sterile water. Aftercolony staining, the number of colonies on each plate wascounted under a microscope, and those with more than 50
cells were considered as colonies.

The colony formation rate (%) was calculated by dividing
the average number of colonies for each extract concentration by the average number of colonies for the negative control plate and multiplying by 100. Based on the relationshipbetween the concentration of the extract and the colony formation rates, the concentration of the extract that inhibits thenumber of colonies by 50% was determined using the probit
model [19] and designated as IC50. If the colony formation
rate exceeded 100%, the rate was considered to be 100%.

Intradermal administration of the extract in rabbits
All animal studies described in this manuscript were performed in accordance with the Principles of Laboratory
Animal Care (NIH publication No. 86-23, revised 1985),
as well as specific Japanese guidelines, including “Basic
Guidelines for the Implementation of Animal Experiments,
etc. at Institutions under the Jurisdiction of the Ministry of
Health, Labour and Welfare of Japan" established in 2006.
Two sheets of HATS (surface area of one sheet:
2 cm × 15 cm) were placed in an extraction vessel (glass
bottle), and 20 mL of either saline or sesame oil (Wako
Pure Chemical, Tokyo, Japan) was added to the vessel.
Extraction was carried out at 120 °C for 1 h, and the extract
without sheets was used after cooling to room temperature(18–23 °C). To prepare control extracts in saline and sesameoil, the same procedure as that used to prepare the HATS
extract was performed except for the addition of HATS.
Four different extracts were used—HATS extract insaline, HATS extract in sesame oil, control in saline, andcontrol in sesame oil. Each extract (0.2 mL) was injected atfive points in the back of each rabbit.
In a separate experiment, extracts were prepared using
non-woven fabric instead of HATS in saline and sesame oil,and the effect of the intradermal injection of the extracts
of non-woven fabric was examined. The procedures were
almost the same as those for the HATS extracts, except forthe conditions for the extraction. Thus, in the procedure forthe non-woven fabric, extraction was performed for 24 h at
70 °C.
The skin regions where the extracts were injected wereobserved at 24, 48, and 72 h after injection. Scoring for erythema and crusting was as follows: no erythema (0), mild
erythema (1), obvious erythema (2), moderate to severe erythema (3), and severe erythema to slight crusting (4); edema
scoring was as follows: no edema (0), very mild edema (1),mild edema (2), moderate edema (3), and severe edema (4).

Skin sensitization test in guinea pigs
A skin sensitization test was conducted on guinea pigs using
saline and sesame oil extracts of HATS in accordance with
ISO 10993-10. Extracts of HATS in saline and sesame oil

13European Archives of Oto-Rhino-Laryngology
Table 1  Skin sensitization testGroup

Primary sensitization

Extract in saline
Control in saline
Extract in sesame oil
Control in sesame oil
Positive controlabSecondary sensitization

A

B

C

D

Water + ­FCAa
Water + FCAWater + FCAWater + FCAWater + FCA

100% saline extract
Saline control
100% sesame oil extract
Sesame oil control
0.1% ­DNCBb

100% saline extract + FCASaline control + FCA
100% sesame oil extract + FCA
Sesame oil control + FCA
0.2% DNCB + FCA

100% saline extract
Saline control
100% sesame oil extract
Sesame oil control
0.1% DNCB

Number ofanimals
10
5
10
5
5

Freund’s complete adjuvant
1-Chloro-2,4-dinitrobenzene

as well as the control extracts were prepared according to
the same procedure as for the previous experiment, that is,
intradermal administration of the extract using rabbits. On
day 0, 0.1 mL of the solutions indicated in Table 1 wereintradermally injected at six sites (primary sensitization A,
B, and C shown in Table 1) within a 2 × 4 cm square area
(Fig. 1) at the back of animals for groups as described inTable 1.
On the day 7, a 2 × 4 cm filter paper permeated with thetest solution shown in Table 1 (secondary sensitization) was
attached to site D (Fig. 1), and the paper was occluded with
polyethylene film tape for 48 h. On the 22nd day, a lint cloth
patch permeated with the solution shown in Table 2 was
attached to sites E, F, G, and H (Fig. 1) of the skin andoccluded with polyethylene film tape for 24 h. Thereafter,
skin reactions were observed according to the Magnusson
and Kligman scale [20] at 24 and 48 h after patch removal.

Cephalic side2 x 4 cm
A
B
C

D

A
B
C

1.5 x 1.5 cm
E

G

F

H

Administration of HAT to the nasal cavity of rabbits

Caudal side
Fig. 1  Sites of intradermal injection (A–D) and areas where lint cloth
patches were attached (E–H) to the back of a guinea pig

The intranasal administration test in rabbits was performedin accordance with the OECD Guidelines for the Testing ofChemicals (No. 417, 2010). HAT powder was suspended inwater (10 mg/mL weight/volume), and 50 mL of the suspension was administered intranasally to the left nasal cavity ofeach rabbit. The right nasal cavity was used as the control.
Six animals were used and nasal mucosal samples were collected from three rabbits each, after 7 and 14 days of administration for pathological study (hematoxylin–eosin staining

Table 2  Induction testGroup

Extract in saline
Saline control
Extract in sesame oil
Sesame oil control
Positive control

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Inducing test
E

F

G

H

100% saline extract

50% saline extract

25% saline extract

Saline control

100% sesame oil extract

50% sesame oil extract

25% sesame oil extract

Sesame oil control

0.1% DNCB

Acetone––European Archives of Oto-Rhino-Laryngology

and toluidine blue staining). The plasma concentrations of
the test substances (silver and titanium) were determined inthree animals 7 days after administration. The concentrations of both silver and titanium were determined by inductively coupled plasma mass spectrometry (ICP-MS, ELAN
DRC II, PerkinElmer SCIEX, No. 1). The mucosa near theentrance of the nasal cavity where the HAT suspension was
administered was visually inspected daily, and the conditionsof the mucosa were judged based on erythema and edema
according to Draize scoring system for skin and mucosal
lesions [21].
In a separate experiment, HAT was administered daily for
14 or 28 days. A 50-mL suspension of HAT powder (10 mg/
mL, weight/volume) was administered daily to the left nasal
cavity for 14 or 28 days to determine whether HAT has acumulative effect on irritation of the nasal mucosa. The rightnasal cavity was used as the control. Pathological examination of the samples collected from the nasal cavity was
performed in three rabbits each for the 14-day and 28-day
groups, as described in the single administration study. The
plasma concentrations of the test substances (silver and titanium) were examined in three animals in the 28-day group.

Intraocular administration study using rabbits
Intraocular administration tests in rabbits were performed
according to the OECD Guideline for the Testing of Chemicals (No. 405, 2012). HAT suspension (0.1 mL) (34.5 weight%) was dropped once into the conjunctival sac of a totalof three rabbits. Corneal, iris, and conjunctival changes
in the treated eyes were observed grossly at 1, 24, 48, and
72 h after dosing, followed by detailed observation using
a slit lamp, and judged according to the evaluation criteria
described in the OECD guidelines [22]. The extent of corneal opacity and conjunctival effusion was judged according
to Draize evaluation criteria [23]. Upon observation 24 h orlater after the administration, after the observation using aslit lamp, approximately 0.1 mL of saline was added to thesodium fluorescein-containing portion of 0.7 mg Flores Eye
Test Paper (Lot No. 6181 V, Ayumi Pharmaceutical Co.,
Ltd.) and the paper was attached to the cornea. After 2–3 s,
the eye was rinsed with approximately 20 mL of purified
water and checked for damage to the corneal epithelium
using a slit lamp.

Oral administration study using rats
The HAT suspension was prepared by mixing 35% HAT
powder with 65% water. In the first experiment, a single doseof 2000 mg/kg of HAT was orally administered to five maleand five female Sprague Dawley (SD) rats (obtained fromCharles River, Yokohama, Japan) using a feeding tube, andthey were observed for 14 days after administration. Animal

death, general conditions, and weight were monitored daily.
Necropsies were performed after the experiment.
In the next experiment, 36 male and 36 female rats wererandomly assigned to 6 groups, each with 6 female and 6
male rats, for HAT powder doses of 0, 30, 100, 300, 1000,
and 2000 mg/kg. HAT powder was mixed with water in asyringe and orally administered to each rat. For 14 days after
the administration, animal death was checked at least oncea day, and body weight was measured on Day 1 (before dosing), Day 4, Day 8, and Day 15; food intake was measured
on Days 1, 4, 8, and 15 (before the end of the study), andboth gross necropsy and histological examinations were performed on Day 15, followed by hematoxylin–eosin staining
and microscopic examination.

Human patch test using HATS
The human patch test was conducted by the Japan Hair Science Association (Tokyo, Japan), a contract testing organization. Written informed consent was obtained from eachsubject, and the study was approved by the Ethics Committee of the Japan Hair Science Association (Tokyo, Japan)
and conducted in accordance with the ethical standards ofthe Declaration of Helsinki. Details that might disclose theidentities of the subjects under study were omitted.
A small piece of HATS, cut into a 10 × 10 mm area, wasapplied to the flexor side of the subject’s upper arm using atape for Finn Chamber (EPITEST Ltd., Fessenheim, France).
The sample was removed 48 h after the application, and theskin was observed and judged by a dermatologist at 1 h and
24 h after removal.
The following International Contact Dermatitis ResearchGroup (ICDRG) criteria were used for judgment [24]:Negative: − (no reaction), ± (faint erythema only).
Positive: + (erythema, infiltrates, possibly papules), ⧺(erythema, infiltrates, papules, vesicles), ⧻ (bullae).

Results
Reverse mutation test using bacteria
Five different bacterial strains, Salmonella typhimurium
TA98, TA1537, TA100, TA1535, and Escherichia coli
WP2 uvrA, were used to perform reverse mutation tests as
described in the Methods section. Culturing was performed
for each strain in duplicate plates, and the same experiment
was performed twice. The number of revertant colonies
induced by the test substance in the absence or presence of
S9 mix never exceeded twice the control value for each test
bacterium, and thus the mutagenicity of HAT was negative.

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European Archives of Oto-Rhino-Laryngology

Three rabbits were used for the intradermal injection of
the HATS extracts, and the skin was observed at 24, 48, and
72 h. The extract of HATS, neither in saline nor in sesameoil, showed any irritation. Thus, the extracts from HAT had
neither cytotoxic nor inflammation-inducing activity on rabbit skin.

Cytotoxicity test using colony formation
Cytotoxicity tests using colony formation were performed
using V79 cells obtained from a Chinese hamster cell line.
In a preliminary study using an extract of non-woven fabrics
without HAT, no decrease in the colony formation rate wasobserved, indicating that the non-woven fabrics were notcytotoxic. Then, HAT extract, standard material A (polyurethane film containing 0.1% zinc diethyldithiocarbamate),
standard material B (polyurethane film containing 0.25%
zinc dibutyldithiocarbamate), and negative control material (high-density polyethylene film) were tested. V79 cells
were cultured with various concentrations of HAT extract,
and the colony formation rate decreased with increasing
concentrations of the extract (Table 3). The concentrationof the test substance that inhibited the number of colonies
formed by V79 cells by 50% (IC50) was 53.5% (95% CI
47.3–63.0%). Data from standard materials A and B showeda dose-dependent inhibition of colony formation (Table 3).
Based on these results, the HATS extract, but not the nonwoven fabric extract, had moderate cytotoxicity.

Skin sensitization test in guinea pigs
The extracts of non-woven fabrics were obtained without
HATS, and a skin sensitization test with the extract was
conducted using guinea pigs. No positive reactions wereobserved in any of the conditions except for the positive
control, when non-woven fabrics were used.
Then, a skin sensitization test by HATS extract using
guinea pigs was conducted. As a result, no skin reaction
was observed at any site at 24 and 48 h after patch removal
in any animals except for the positive control. Thus, positive reactions were observed only when the positive control (2,4-dinitrochlorobenzene) was used. Regardless of theextraction solvent (saline or sesame oil), the HATS extracts
did not induce positive reactions. Indeed, the negative controls (saline control and sesame oil control) did not show
positive reactions.

Intradermal administration of the extract in rabbits
Extracts were prepared using non-woven fabric instead of
HATS in saline and sesame oil. Each extract was injected atfive points in each rabbit. The observation of the skin after
24, 48, and 72 h indicated that the results were all negative.
That is, the extract of the non-woven fabric did not induce
any irritation.
Table 3  Cytotoxicity test

The HAT suspension was applied once to the nasal cavityof rabbits. The results showed no changes in general condition, body weight, or the conditions of the mucosa at the

Test material

Concentration of
extract (%)

Mean colony
count

Colony forming
rate (%)

IC50 (%) (95% CI)

HAT extract

100
33.3
11.1
3.7
5
2.5
1.3
0.9
100
71.4
51
36.5
100
0d

0
147.3
160.0
169.3
0
62.3
146.0
161.7
0
81.0
144.3
164.7
167.0b
162.7

0
90.5
98.3
100
0
38.3
89.7
99.4
0
49.8
88.7
100
100
ND

53.5 (47.3–63.0)

Standard ­Aa

Standard ­Ba

Negative control ­materiala
Negative control
a
b
c
d

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Administration of HAT to the nasal cavity of rabbits

2.3 (2.1–2.4)

69.7 (67.1–72.4)

NDc
ND

Details of Standard A, Standard B, and negative control material are described in the Methods section
There was no significant difference between negative control material and negative control
ND not determined
Only medium was added to the plate

European Archives of Oto-Rhino-Laryngology

site of administration throughout the observation period.
In the mucosal samples collected after seven or 14 days ofadministration, no changes were observed by pathologicalexamination of the nasal cavity. In the plasma, no silver ortitanium was detected at any of the timepoints of the experiment. These results indicate that under the conditions of
this study, HAT powder was not considered to be a primary
irritant to the nasal mucosa.
In a separate experiment, HAT was administered dailyfor 14 or 28 days. Pathological examination of the nasal cavity collected at the end of the 14-day and 28-day treatment
periods showed no changes attributable to the administrationof the test substances. No silver or titanium was detected in
the plasma at any timepoint. Therefore, under the conditionsof this study, HAT was not considered to have a cumulative
effect as an irritant to the nasal mucosa.

Intraocular administration study in rabbits
The HAT suspension was administered in the conjunctival
sac of the eye of each rabbit. The index of acute ocular irritation was two, and the degree of irritation was classified
as non-irritant. No corneal damage was observed in any of
the rabbits during the observation period using fluorescein
sodium. Therefore, under the conditions of this study, HAT
was not considered to be acutely irritating or corrosive tothe eyes.

Oral administration study in rats
In the first experiment, 2000 mg/kg of HAT was administered to five male and five female SD rats, which wereobserved for 14 days. No abnormalities in general condition
or weight were observed. Animal death was not observed,
and necropsy after the experiment showed no abnormalities.
In the next experiment, HAT was administered at doses
of 0, 30, 100, 300, 1000, and 2000 mg/kg, and no deaths
were observed in rats treated with these concentrations. During the study period, no evidence of toxicity related to thetest substance was observed in any rat with respect to mean
body weight, mean body weight gain, food intake, organ
weight, and clinical findings. Gross necropsy revealed nogross lesions associated with the test substance in any of
the study groups. Histopathological evaluation indicated noabnormalities associated with the test substance. In conclusion, no toxicity was observed with HAT powder up to adose of 2000 mg/kg.

Human patch test using HATS
A total of 25 healthy subjects, 12 males (age47.2 ± 10.1 years) and 13 females (age 38.4 ± 9.6 years) aged
27–61 years, were subjected to the patch test. Skin lesions

were observed and judged at 1 h and 24 h after the removalof the samples. No positive judgment was made for any subject at any timepoint.

Discussion
Since HATS absorbs and breaks down nasal allergens, masks
using HATS are expected to be more effective against nasal
allergies than masks using only non-woven fabrics.
In our previous paper, we reported an initial basic study toconfirm the safety of HATS by examining the size distribution of HAT particles and their adsorption onto non-woven
fabric fibers [1]. However, the present study is the first reportof a biological safety study.
There have been reports on the safety of titanium dioxide,silver, and HA as individual substances. For example, manysunscreens use nanoparticles of titanium dioxide, which areconsidered safe because they are not absorbed [25]. On theother hand, ultrafine titanium dioxide dust has been reported
to cause respiratory cancer in rats when exposed to inhalation and intratracheal administration [26]. However, thisresult can be applied only to subjects exposed to high dosesof titanium dioxide, such as workers manufacturing titanium dioxide. To date, human studies have not suggested
an association between exposure to titanium dioxide andan increased risk of cancer. The U.S. National Institute for
Occupational Safety and Health recommends an exposure
limit of 2.4 mg/m3 for fine titanium dioxide particles and an
exposure limit of 0.3 mg/m3 for ultrafine titanium dioxide
particles as a time-weighted average concentration for up
to 10 h per day in a 40-h work week [27]. We measured theamounts of titanium released from HATS and found that itwas considerably lower than the limits (data not shown).
There have also been reports on the safety of silver inhumans. Medical applications of silver include wound dressings, creams, and antibiotic coatings for medical devices[28–30]. Wound dressings containing silver sulfadiazine orsilver nanomaterials are sometimes used to treat external
infections [31–33]. Silver coatings on endotracheal tubes
have been used to reduce the incidence of ventilator-associated pneumonia [34]. Silver is generally of low toxicity,and the risk is considered to be negligible when it is used forapproved medical applications [35].
On the other hand, HA is a physiological component.
Seventy percent of human bone is composed of HA by
weight. In addition, it is also a major component of teeth
and is sold as an ingredient in toothpaste [36]. Therefore,
it is considered safe in many cases, including oral intake.
However, there have been no reports on the safety of HAT
powder, which is a mixture of titanium dioxide, silver, and
HA.

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European Archives of Oto-Rhino-Laryngology

In this study, we conducted mutagenicity tests of HAT
extract using bacteria and showed that HAT was not mutagenic. In general, titanium is not water-soluble; therefore, if
an effect is observed in the HAT suspension, it is thought tobe due to the action of eluted silver or HAT particles. Precipitates were observed in plates containing high concentrations of HAT. Next, we conducted a cytotoxicity test using
the extract obtained from HATS using the colony formation
method. The results showed moderate cytotoxicity, suggesting that inflammatory reactions may occur in the mucosal
tissues.
Based on the results of the above cellular level studies,
we conducted in vivo experiments in mammals. Intradermal
administration of HATS extract in rabbits showed no irritation. The results of skin sensitization tests on guinea pigs
using saline and sesame oil extracts of HATS were negative,
indicating that the HATS extracts were not skin sensitizers.
Intranasal administration of the HAT powder suspension torabbits showed no abnormalities. In addition, no silver ortitanium was detected in the plasma, and the administrationof HAT in the conjunctival sac of rabbits also showed noacute eye irritation/corrosion. Next, we administered HAT
powder orally to rats. No toxicity was observed up to a single
dose of 2000 mg/kg, and the minimum lethal dose of HAT
by oral administration was over 2000 mg/kg.
Finally, the results of a patch test conducted on 25 subjects showed that HAT did not induce irritation reactions.
HATS is used in a mask sandwiched between two sheetsof non-woven fabrics that do not contain HAT. Therefore,
under normal usage, HATS will not come into contact withhuman skin. However, if the mask is damaged, it is possible for the HATS to touch the skin. In addition, some HAT
particles in the HATS may be exposed through the nose.
Therefore, the safety of HAT particles and HATS should be
carefully examined.
In this study, HATS was not mutagenic in bacterial tests
and was relatively safe when administered to the skin, nasal
cavity, and eye mucosa of animals. There were no adverse
events, including pathological findings, when administeredorally to rats, and neither titanium dioxide nor silver was
detected in the plasma after nasal administration. There is apossibility of toxicity if HAT comes into direct contact withcells. However, neither silver nor titanium was detected in
the plasma after nasal administration, and no toxicity wasobserved in dermal, ocular, nasal, or oral administration.
Therefore, it is unlikely that HAT comes into direct contact
with cells through these routes.
We are currently conducting a clinical trial in Japan under
the guidance of the Ministry of Health, Labor, and Welfare
to evaluate the safety and efficacy of the drug in patientswith allergic rhinitis.
In conclusion, although HAT showed moderate cytotoxicity, in vivo results indicated that HAT is safe because it does13

not come in direct contact with cells in normal usage, and
HATS is safe when used in masks.

Limitations
This study has the following limitations. First, the experiments were conducted primarily on bacteria, mice, rabbits,
guinea pigs, and Chinese hamster cells, and it is not clear
whether these results are applicable to humans. Second,although titanium dioxide and silver are insoluble, it is possible that a small amount of dissolution has occurred, andwe did not measure the dissolved substances in each experiment. Third, of the hydroxyapatite, silver, and titanium dioxide that make up HAT, hydroxyapatite is a physiologicalsubstance and exists in the body in abundance. Therefore,
there may be more hydroxyapatite in the body than what
was used in the experiment, but its effects have not beenstudied. Lastly, in our separate study, we showed that HAT
reduced Cryj1, a major allergen in cedar pollinosis, and Der
f1, a major allergen in dust mite allergy in an in vitro study[1], but whether HATS really reduces nasal allergies such as
cedar pollinosis and dust mite allergy needs to be determinedby clinical trials.
Acknowledgements We are grateful to Hiroyuki Yokote and Shigeo
Kamitsuji for their assistance in preparing the manuscript. We would
like to thank Editage (www.​edita​ge.​com) for English language editing.
Funding This study was funded with the Research and Development
expenses of DR.C Medical Medicine Co., Ltd.

Declarations
Conflict of interest N.O., T.T., and D.Y. are the CEO, employee, andstaff, respectively, of an affiliated company of DR.C Medical Medicine
Co., Ltd. N. K. is the CEO of StaGen Co., Ltd.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as longas you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article areincluded in the article's Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view acopy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.

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