Summary of antibacterial effecttest results of natural calcined
calcium aqueous solution
Product Name： ＡＢＡＮＴ

Natural calcined calcium aqueous solution 【Product name :ABANT】, developed from Apr. 2010, is a safe product that uses 100%
natural ingredients and has a strong antibacterial deodorizing effect witha wide range of applications.

300ml

30ml
ARROWFIELD CO., LTD.

5-16 Honjo, Koyoen, Nishinomiya, Hyogo 662-0015, Japan
TEL +81-798-75-1100✉ abant@arrowfield.co.jp

With excellent antibacterial properties
and effective against most of food poisoning
bacteria
It is not only effective against general food poisoning bacteria such as
Vibrio parahaemolyticus, Salmonella spp., O-157, etc., but also have agreat effective against viral food poisoning.
Bacteria

Sterilization time

Viable countAt the start

After sterilization

Testing laboratory

E. coli: serotype O157：H710 mins

2.8×10 5＜10Staphylococcus aureus

10 mins

2.7×10 5

40Salmonella

10 mins

2.3×10 5≤ 30 Tokyo Metropolitan Food

Pseudomonas aeruginosa

10 mins

3.2×10 5≤ 30 Technology Research

Listeria monocytogenes

60 mins

6.0×10 5≤ 30 CenterBacillus subtilis

after 3 mins

1.3×10 5≤ 300

Histamine-producing bacteria

5 mins

3.0×10 5≤ 300

Vibrio parahaemolyticus

5 mins

1.2×10 5＜ 10after 3 hours

1.6×10 5＜ 1010 mins

0.82×10 5＜ 10Methicillin-resistant
Staphylococcus aureus
Legionella
Campylobacter

10 mins

10-fold serial
dilution ofbacterial solution

No occurrence

Kyoto Microbio Laboratory
Laboratory of foodenvironment and hygiene
Japan Meat Science &Technology InstituteAdd the bacterial suspension to the natural calcined calcium saturated aqueous solution(0.11%), stir it , and measure the bacterial counts according to the method for measuring
general bacterial counts.
Virus

Virus removal time

Norovirus（Feline calicivirus）

10 mins

Virus infectious titerAt the start
7.0

After sterilization＜ 1.5

Testing laboratory
Japan Food ResearchLaboratories

Although there is no effective drug or vaccine against Norovirus currently,
natural calcined calcium saturated aqueous solution can prevent the onsetof disease with virus removal effect.

Sterilize 99.99%
of coronavirus
Virus inactivation effect test
by natural calcined calcium【 Virus inactivation test】
・Mixed the test solution (0.9 ml) with the virus solution (0.1 ml) andallowed them to act for a predetermined time.
・After the action, sampled 0.1 ml from the mixed solution and diluted it
about 10 times with 0.1 M HEPES buffer (7.0) to stop the action of the test
product against the virus.
・Measured the infectious titer by the TCID50 method using the solution
as a stock solution for the infectious titer measurement sample.
Sample virus：
Feline enteric coronavirus（WSU 79-1683）
Infectious titer(TCID50/ml)

LRVSpecimen0(at the start)
Contrast(PBS)
Natural calcined calcium

after 5 mins

2.4E+05

after 10 mins

after 5 mins

after 10 mins

5.0E+05
1.1E+02＜1.3E+01-0.3
3.3＞4.2

As a control, phosphate buffered saline (PBS) was used.
Detection limit: 1.3E+01 TCID50/ml
LRV (log reduction of infectious titer): log10 (initial infectious titer ÷ infectious titer after each action time)
Testing organization: Kitasato Research Center for
Environmental Science

Based on the above results, natural calcined calcium is proved
to be sterilizable as following
5 minutes later ➡ 99.95%
10 minutes later ➡ 99.99%.

Mold Reproduction Deterrence<Test method>(1) Add a predetermined amount of natural calcined calcium powder to potato dextrose
medium*.
* A medium that makes the molds grow actively on a trial basis by adding potato
extract, glucose, and agar into purified water.
(2) Approximately 10 mold spores are planted in each small schale (approximately 20ml).
(3) Cultivate at room temperature and visually observe the growth of mold over time.

Mold type
Mold type

Additiveamount ofnaturalcalcinedcalcium(Approx.0.15%)(Approx.0.15%)

0.3g/200ml(Approx.0.15%)

The No. of the elapsed days

2 days

5 daysControlthegrowthofmany
typesofmolds.

Natural calcined calcium solution isan odorless deodorant.
Blank test
Gas concentration when 5 ml of calcined calcium saturated solution was injected
Gas concentration when 15 ml of calcined calcium saturated solution was injected

Rotten smell of eggs

The smell of soggy socks◇Hydrogen sulfide◇Isovaleric acidGas concentration(ppm)

Test method:Gas injection into the bag and detection
tube method.
Each sample was put in an odor bag,and after applying a heat sheet, 3L of air
was enclosed, and the test gas which
concentration was obtained was added.

Initial stage

Such sample was settled and the gas
concentration in the bag was measured
at each elapsed time using a gas

Vinegar smell

Excreta smell◇Ammonia◇Acetic acidGas concentration(ppm)

Initial stage

Rotten smell of onions

conducted by performing the same
operation without inserting the sample.

Testing laboratory:
Japan Food Research Laboratories

Initial stage

Pungent smell of grass（tobaccoodor components）◇Methyl mercaptan

Initial stage

detector tube. A blank test wasRotten smell of fish

◇Acetaldehyde

Initial stage◇Trimethylamine

Initial stageSafety tests of natural calcined calcium

Natural calcined calcium in the form of powder
has passed the following four safety tests.

1. Acute oral toxicity test

Test No.N17137-1

2.Primary skin irritation test Test No.N17137-2
3.Skin sensitization test

Test No.N17137-3

4.Reverse mutation test

Test No.AN170026

Acute oral toxicity test
Safety was evaluated by acute oral toxicity test using mice and primary skinirritation examined by using rabbits.
An acute oral toxicity test was carried out in mice using a calcium-saturated aqueous
solution of shellfish as a sample.
A 2000 mg/kg dose of the test substance was orally administered to the test group, and water for
injection as a solvent control was orally administered to the male and female mice control grouponce, and observation was performed for 14 days.
（ Testing organization: Japan Food Research Laboratories）

Body Weight Change(Male)
Administration
group

Before administration

After administration（day）
7

14Test group33.5±2.5（5 animals）

37.1±2.740.1±3.7

Control group33.3±1.7（5 animals）

37.9±2.1

40.9±2.5

No abnormalities or deaths were observed
during the observation period in either maleor female administration group.

No difference was found in the body weight
between the test group and the control
group in both males and females 7 and 14
days after the administration.
Body Weight Change (Female)
Administration
group

Before administration

After administration（day）
714

Test group27.7±1.6（5 animals）

30.1±2.1

32.7±3.1

Control group27.8±1.4（5 animals）

30.0±1.432.7±1.5

At the end of the observation period, necropsy
revealed no abnormalities in all test animals ofboth males and females.

Based on the above data, in the acute oral toxicity test using mice, the LD50 value ofthe sample was evaluated to exceed 2000 mg/kg for both males and females.

Skin irritation evaluation category 「non-irritant」

Primary skin irritation test
Three Japanese white rabbits were used as test animals, and theadministration part was the dorsal part of the dehaired animal, and healthyand damaged skin parts were provided.
The administration method was such that 0.5 g of the administration sample
was placed on a 2.5×2.5 cm lint cloth, and a non-permeable adhesiveplaster, an adhesive sponge adhesive plaster, and an adhesive elastic
bandage were used to occlude and apply the adhesive for 24 hours.
The irritative reaction was observed at 3, 24 and 48 hours after removal ofthe patch.
(Testing institution: Yoshimi Research Center,
Drug Safety Testing Center Co., Ltd.)
Body Weight（kg）

General Condition

Animal No. administration final judgement administration
1date

2.29

date

1 day after

2 days after

3 days afterdateadministration administration administration
2.36 no abnormality no abnormality no abnormality no abnormality

22.24

2.29 no abnormality no abnormality no abnormality no abnormality

32.21

2.3 no abnormality no abnormality no abnormality no abnormality

AnimalNo.

Healthy Skin
Erythema・Scab

Damaged Skin

Edema

Edema

Erythema・Scab

3 hours 48 hours 3 hours 48 hours 3 hours 48 hours 3 hours 48 hours

100

000

0

00

200000000

3000000000

Average000

Strength
P.1.1000No skin reaction was observed in both normal and damaged skin,and P.I.I was 0. No abnormalities were observed in the generalconditions during the observation period, and the gain in bodyweight was also shown.
Based on the above results, it was concluded that this test
substance had P.I.I=0, and the skin irritation evaluation category
was non-irritant.

Reaction Category P.I.I

Evaluation Category0non-irritant

Skin sensitization test in guinea pigs
Natural Calcined Calcium was examined by Guinea Pig Maximization Test method using
guinea pig.
As test animals, 4 animals for preliminary tests, 10 animals for sensitization groups, and5 animals for control groups, 19 animals in total were used.
For intradermal sensitization, a: Freund's complete adjuvant (FCA) and physiologicalsaline equivalent emulsion, b: 0.05 w/w% solution of test substance (vehicle:physiological saline solution), c: 0.1 w/w% solution of test substance(vehicle:physiological saline solution) and equivalent emulsion of FCA were intradermallyadministered to neck skin at 0.1 ml×2 places (however, shortening the administrationinterval of administration samples a and b). For contact sensitization, 0.2 g of the test
substance drug substance (100%) was placed on a 2×4 cm lint cloth on the 7th day, andthe test substance was occluded and applied on the intradermal administration site for48 hours. The control group was treated in the same manner by impregnating 2×4 cmof lint cloth with 0.2 ml of water for injection. And, since the drug substance of this test
substance was non-irritant, about 0.5ℊof the IO 3/4 SDS vaseline mixture was applied
openly to the intradermal administration site where hair was removed on the sixth day.
On the 21st day after the sensitization test, 0.1g of the drug substance of this test
substance was placed on a 1.5×1.5 cm lint cloth and occluded on the flank for 24 hours.
The skin reaction was observed 24 and 48 hours after the removal of the adhesive patch.
As a result, no skin reaction was found in both the test substance sensitized group andthe test substance control group. There were no abnormalities found under its generalconditions and the body weight also gained."(Testing institution: Yoshimi Research Center,
Drug Safety Testing Center Co., Ltd.)

Based on the above results,
no skin sensitization was found in this test substance.

Reverse mutation test
As part of the safety assessment of natural calcinedcalcium using bacteria, we aimed to clarify thepresence or absence of gene mutagenicity.
"To examine the presence or absence of gene mutagenesis of natural calcined calcium,Salmonella typhimurium (hereinafter abbreviated as S. typhimurium ) TAIOO, TAl535, TA98,
TAl 537 and Escherichia coli (hereinafter abbreviated as E. coli) WP2 uvrA were used by thepreincubation method under the conditions of metabolic activation and metabolic inactivation.
Water for injection was used as the solvent for the test substance.

In order to set the dose for this study, a dose setting study was conducted with 5 doses of

5000，1250, 313, 78.1, and 19.5 µg/plate, which were serially diluted at a common ratio of 4
with 5000 μg/plate as the highest dose. According to the results, the lowest dose showing

growth inhibition was taken as the highest dose. The test of this study was carried out with all
strains under the conditions of metabolic inactivation, and with S. typhimurium TA strains

under the conditions of metabolic activation in 6 doses of 313, 156, 78.1, 39.1, 19.5, 9.77 µg/plate, and with E. coli WP2 uwpA under the conditions of metabolic activation in 6 doses of1250, 625, 313, 156, 78.1, 39.1 µg/plate.

Regardless of the presence or absence of metabolic activation in both the dose setting study

and this study,in both the base pair substitution type and frameshift type strains, no increasein the number of revertant colonies, which is more than double the negative control value, wasobserved. No dose response was observed.

(Testing institution: Yoshimi Research Center, Drug Safety
Testing Center Co., Ltd.)

Based on the above test results, it was determined that natural calcined
calcium does not have the ability to induce gene mutation in bacteria(negative) under the conditions of this test.