Original Article

診療と新薬 2023; 60: 765‑771

The Safety Assessment of
Stable Type Neutral Vitamin C Derivative (MIRAVC)

診療と新薬 Web

Masahiko NAKANO 1)／Atsuyoshi MURAKAMI 2)／Akio YAMAMOTO 1)／Nobuo KUBO 3)
1) Dr s Choice Co., Ltd., Tokyo Japan2) The Information of Biomedical Science and Ultimate Technology Co., Ltd., Tokyo Japan3) The Kitahama TOWER Jibiinkoka Hifuka Clinic, Osaka, Japan● Abstract
Objective: To examine the safety of a newly developed ascorbic acid derivative based on ascorbyl glucoside(MIRAVC), as a raw material for topical preparations.
Methods: A primary skin irritation test, an Ames test, a human patch test, a human stinging test, and an
ocular mucosal irritation test were conducted to evaluate the safety of this compound. In addition, using athree-dimensional cultured skin model, living skin equivalent model (LSE-high), we also evaluated transdermal
absorption using a Franz-type diﬀusion cell system.
Results: Together with the test results of stability and transdermal absorption, all safety tests showednegative results, and no events were observed that would be of concern when used topically by humans.
Key words: Safety assessment, Ascorbyl glucoside, Skin permeability, Topical

1. INTRODUCTION
Ascorbic acid, also known as vitamin C, is a water-soluble
vitamin. Although it is an essential nutrient, it cannot bebiosynthesized by humans, so it must be ingested orapplied topically. It has been widely used as a topicalpreparation for a long time, and is known to have various
functions such as antioxidant, anti-inflammatory,
whitening, and collagen production1)2). Ascorbic acid is a
widely used basic ingredient, but when made into an
aqueous solution, it becomes strongly acidic, easily
browns due to oxygen, heat, light, etc., and is not very
stable. To solve these problems, various ascorbic acidderivatives have been developed, and their functions andsafety have been reported3)-5). Ascorbyl glucoside, which
combines ascorbic acid with glucose, is widely used as awater-soluble derivative 6). We prepared MIRAVC, astable neutral vitamin C derivative raw material, byadding the basic amino acid arginine to this existing
derivative, ascorbyl glucoside. To use this compound as a
topical preparation, it is necessary to confirm sufficient
safety, but since it is a new compound, no safety
evaluation has been performed on this compound to date.
Therefore, in this study, we conducted basic safety tests
to evaluate the safety of this compound, assuming that it
Corresponding author; E-mail address: mnakano@drs-choice.co.jp
Address; Stage Bldg. 17F, 2-7-2 Fujimi, Chiyoda-ku, Tokyo 102-0071, JAPAN

would be used topically on the skin and eyes. In addition,
permeability was evaluated using a Franz-type diffusion
cell system7)8) using a living skin equivalent model (LSEhigh), which is a three-dimensional cultured skin model.

2. MATERALS AND METHODS
2.1. Test materials
Arginine powder was added to ascorbyl glucoside aquasolution, and the pH of the solution was adjusted to 6.4
with arginine. This solution was freeze-dried to obtain
MIRAVC powder, which was used as a test product.
HPLC analysis was performed by LC-2050C (Shimadzu,
Japan) under the conditions shown in Table 1. HPLC
purity was 100% in area%. As a result of measuring withan infrared moisture meter, the moisture content was
2%. In each test, control products were used as
appropriate.
2.2. Safety assessment
2.2.1. Primary skin irritation test
Skin irritation is a skin reaction indexed by changes suchas erythema, edema, and desquamation that occur whena test substance comes into direct contact with the skin.
Skin reactions that are severe and cause irreversible
tissue damage are called skin corrosive reactions. In
addition, the irritation response when a test substancecontacts the skin once is called primary skin irritation,and the irritation response when the test substance14 （766）

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Table 1 HPLC analytical condition
Condition

Description

ColumnInertsil NH2（5μm, 250 × 4.6 mmI.D.）（GL Sciences Inc., Tokyo, Japan）

Guard column

Inertsil NH2（5μm, 20 × 4 mmI.D.）（GL Sciences Inc., Tokyo, Japan）

Mobile phaseA）CH3CN, B）CH3OH, C）0.01M Sodium dihydrogen phosphate aqueous solution,
A/B/C ＝ 60/30/10 v/v/v

Flow rate

2 mL/minColumn temperature

40℃

Detector

270 nmInjection volume

10μL

comes into repeated contact with the skin is called
continuous skin irritation.
A cytotoxicity test was conducted on the 3-dimentionalc u l t u r e d s k i n m od e l L a b Cyt e E PI - M O D E L 2 4 i naccordance with the OECD Test Guidelines (OECD TG
439) 9) . The test substance (MIRAVC 30% aqueous
solution), positive control (5% Sodium lauryl sulfate
aqueous solution) and negative control (sterile distilledwater) were exposed to the stratum corneum side of the
3-dimentional skin culture model LabCyte EPI-MODEL24
for 15 minutes, and then cultured for 42 hours. Cell
viability was measured by MTT (3-(4,5-di-methyltiazol-2yl)-2,5-diphenyltetrazolium bromide）assay, and primaryskin irritation was classified based on the cell viability.
This test was conducted at DRC Co., Ltd. (Osaka, Japan)
from March 9 to 15, 2021.
2.2.2. Eye mucosal irritation test
The eye mucosal irritation test is a test to confirm theeye irritation (reversible inﬂammatory changes) and eye
corrosion (irreversible tissue damage) of chemical
substances. Ocular mucosal irritation was evaluatedu s i n g t h e 3 - d i m e n t i o n a l c u l t u r e d c o r n e a m od e l
LabCyteCORNEA-MODEL24 EIT test method10). The
test was conducted in accordance with OECD TG 492
SOP. The test substance (MIRAVC 30% aqueous
solution), positive control (Ethanol), and negative control(sterile distilled water) were used. This study was
conducted at DRC Co., Ltd. (Osaka, Japan) from April 18
to 20, 2023.
2.2.3. Ames Test
The Ames test 11) was conducted to determine thepresence or absence of gene mutagenicity. The test was
carried out by a pre-incubation method using Salmonella
typhimurium TA100, TA1535, TA98, TA1537 andEscherichia coli WP2uvrA under metabolic activation andnon-metabolic activation conditions. Note that water for
injection was used as the solvent for the test substance.
To set the test dose, a dose-finding test was conductedwith the test substance treatment doses of 1.22, 4.88,
19.5, 78.1, 313, 1250, and 5000μg/plate, which werediluted in 6 stages with a common ratio of 4 as follows,with the highest dose at 5000μg/plate. This study was
conducted from April 6, 2021 to May 19, 2021 at Bozo
Research Center Co., Ltd. (Tokyo, Japan).
2.2.4. Human patch test
A 24-hour occlusive human patch test12)13) was conducted
on 24 Japanese adults. Prior to conducting the study,
approval was obtained from the Brain Care Clinic Ethics
Review Committee (Tokyo, Japan). This study was
conducted with consideration to protecting the humanrights of the subjects, based on the ethical standards set
forth in the Declaration of Helsinki (World Medical
Association) and the ethical guidelines for medical
research involving humans (Ministry of Education,
Culture, Sports, Science and Technology, Ministry of
Health, Labor and Welfare). Using a patch test unit, Finn
Chamber on Scanpor Tape purchased from SmartPracticeJapan (Kanagawa, Japan), each test article was
occlusively applied to the upper back (paravertebral
region) of the subject for 24 hours. The number of times
it was pasted was once. MIRAVC 30% aqueous solution
was used as the test substance. In addition, control
substances were white vaseline, physiological saline, anddistilled water for injection. The test judgement wasobserved on the application site one hour after removing
the patch test unit, after the temporary erythema causedby removal had disappeared, and made a judgment after
14 hours based on the criteria. Furthermore, theapplication site was observed again 24 hours after
removal, and judgment was made 48 hours later using thesame criteria. Skin irritation index = ((sum of scores ofthe one with the stronger reaction after 24 hours andafter 48 hours)/number of subjects) x 100. This study was
conducted at DRC Co., Ltd. (Osaka, Japan) from April 15
to 17, 2021.

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Table 2 The basic information of the compound
Item

Description

CAS RN

718621-28-4

CAS Index Name

L-Arginine, compd. with 2-O-α-D-glucopyranosylL-ascorbic acid（1:1）
Ascorbyl Glucoside（and）Arginine
530.476
White to light yellow powder
6.43（5％ aqua. solution）＞ 82.5％（room temperature）

INCI name
Molecular weight
Appearance
pH
Solubility to water

Table 3 The stability of the compound
Storage condition

MIRAVC conc. ％Relative conc. ％4℃ for 8 months
Room temperature for 8 months
50℃ for 1 month

31.11
29.26
29.64

100
94.05
95.27

2.2.5. Stinging test
Stinging test is a method to evaluate sensory stimulationsuch as stinging, itching, and hot ﬂashes that occur whenusing cosmetics or cleaning products and is also called
sensory stimulation test. A test was conducted on thestinging irritation of the test product on 10 adults who did
not experience irritation from distilled water for injection(distilled water) but experienced irritation from 0.15%
methylparaben aqueous solution (MP). Prior to
conducting the study, approval was obtained from theBrain Care Clinic Ethics Review Committee (Tokyo,
Japan). This study was conducted with consideration toprotecting the human rights of the subjects, based on the
ethical standards set forth in the Declaration of Helsinki(World Medical Association) and the ethical guidelines for
medical research involving humans (Ministry of
Education, Culture, Sports, Science and Technology,
Ministry of Health, Labor and Welfare). A cotton padsoaked with 1 mL of MP was applied to the subject s skinover left and right cheekbones for 60 seconds. The cotton
pad was removed, and the evaluation site was wiped withcotton soaked in distilled water, dried with a paper towel,
and allowed to rest for 180 seconds. This process was
repeated twice to select the subjects. A cotton padsoaked with 1 mL of the test product was applied near theskin over left and right cheekbones and left to stand for
60 seconds. The irritation felt within 31 to 60 seconds
after the cotton was applied was scored on a five-point
scale. The Sting scores (5 levels from 1 to 5) of the test
product (MIRAVC 30% aqua solution) and the control
product (distilled water or MP) were totaled, andcomparisons between groups were performed using the
Steel-Dwass test. The test was two-sided, and thesigniﬁcance level was 5%. This study was conducted onJune 30, 2021 at DRC Co., Ltd. (Osaka, Japan).
2.2.6. Transdermal absorption test
Permeability to skin of this compound was evaluatedusing a Franz-type diﬀusion cell system using a living skinequivalent model (LSE-high), which is a threedimensional cultured skin model7)8). LSE-high consists ofskin (Subcutaneous and viable epidermis from humankeratinocytes) and a collagen matrix composed of humandermal fibroblasts and a polycarbonate membrane. 5%
MIRAVC in PBS (phosphate buffer pH 7.4) solution or
30% MIRAVC in PBS. After applying 500μL of thesolution as a sample, the MIRAVC concentration in each
receiver solution was analyzed by HPLC under theconditions in Table 1 to evaluate the in vitro skinpermeability of MIRAVC in each test solution. This test
was conducted at Roman Skin Lab Co., Ltd. (Saitama,
Japan) from May 31, 2021 to June 28, 2021.

3. RESULTS
3.1. Physical properties
The basic physical properties are summarized in Table 2.
When dissolved in water, the pH of a 5% aqueous solution
was 6.43, which was within the neutral range. It was
found that the solubility in water was over 82.5%,
indicating that it could be dissolved very well in water.
3.2. Stability
A 30% aqueous solution of MIRAVC was prepared andstored at 4℃ and room temperature for 8 months or at
50℃ for 1 month, and then the MIRAVC concentration
was measured by HPLC in the conditions shown in Table
1. As a result, the concentration after storage at 4℃ for 8

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Table 4 Primary skin irritation test
Cell viability （％）
Test samplen

Negative control（Water*） 3
Positive control（5％ SLS） 3
MIRAVC 30％ aqua soln.
3

Mean
100.0
1.6
97.1

Standard

Classiﬁcation

deviation
3.8
0.1
3.2

−
Category 2
Not classiﬁed* Sterile distilled water.

Table 5 Eye mucosal irritation test
Cell viability （％）
Test samplen

Negative control（water*） 3
Positive control（Ethanol） 3
MIRAVC 30％ aqua soln.
3

MeanStandard
deviation

Classiﬁcation

100.0
3.4
90.4

7.1
0.9
1.8

−
Can not predict
Not classiﬁed* Sterile distilled water.

months was 29.26%, which was a relative concentrationof 94.05% compared to the initial concentration. The
concentration after storage at 50℃ for one month was
29.64%, which was a relative concentration of 95.27%
compared to the initial concentration (Table 3). From
this, it was considered that the stability of this compound
was suﬃciently enough for topical application.
3.3. Safety
3.3.1. Primary skin irritation test
The cell survival rates for the test substance, positive
control, and negative control were 97.1 ± 3.2, 1.6 ± 0.1,
and 100.0 ± 3.8%, respectively. The cell viability of thepositive control was less than 40%, the measured valuesof the negative control were 0.7 or more and 2.5 or less,and the standard deviations of the cell viability were all
18 or less. It was confirmed that the test met the test
acceptance criteria. Based on the UN GHS (United
Nation Globally Harmonized System of Classiﬁcation andLabeling of Chemicals) classification of RhE(reconstructed human epidermis) method prediction
model, the cell viability relative to the negative control isgreater than 50% and outside the classification. The
substance was determined to be non-irritating to the skin
(Table 4).
3.3.2. Eye mucosal irritation test
As a result of this test, the cell viability of the positive
control was 3.4%, which was less than 40%, and themeasured value of the negative control was 0.940, which
was 0.5 or more and 1.6 or less. The standard deviations
were 1.8 for the test substance, 0.9 for the positive
control, and 7.1 for the negative control, all of whichwere below 18, confirming that the test acceptancecriteria were met. The cell viability of the test substancewas 90.4%, which was higher than 40%, so the ocularmucosal irritation of the test substance was classiﬁed as
not classiﬁed (Table 5).
3.3.3. Ames Test
Since no precipitation or growth inhibition was observedin the dose-finding test, this test was conducted at fivedoses ranging from 313-5000μg/plate for all strains,regardless of the presence or absence of metabolic
activation. No precipitation on the plate due to the test
substance was observed at any dose, regardless of thepresence or absence of metabolic activation. As a resultof observing bacterial growth inhibition, no growth
inhibition was observed in any bacterial strain, regardlessof the presence or absence of metabolic activation. With
this test substance, regardless of the presence or absenceof metabolic activation, no increase in the number ofrevertant colonies, which was more than twice that of thenegative control, was observed in any of the strains, andno dose response was observed (Table 6). From theabove test results, it was determined that MIRAVC does
not have the ability to induce genetic mutations inbacteria (negative) under the test conditions.
3.3.4. Human patch test
The subjects were 3 men and 21 women, aged 23-58
years (mean age 44 years). Table 7 shows the skinirritation index of each test product. MIRAVC 30%
aqueous solution had a skin irritation index of 0.0, and no
irritation was observed.

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Table 6 Ames testGroup

S9Mix（−）

S9Mix（＋）

Positive
Control

Base pair substitution type

Dose（μg/plate）

S9Mix
（−）
S9Mix
（＋）

TA100

Frame shift typeTA1535

WP2uvr

TA98

TA1537

Negative control

106, 121（114）

313
625
1250
2500
5000

105, 121（113）146, 147（147）
130, 132（131）130, 122（126）126, 133（130）8, 12（10）14, 8（11）8, 9（9）7, 6（7）7, 9（8）
10, 6（8）24, 22（23）17, 14（16）17, 18（18）20, 27（24）26, 20（23）14, 19（17）18, 22（20）24, 23（24）17, 19（18）14, 10（12）14, 26（20）23, 20（22）8, 11（10）Negative control
313
625
1250
2500
5000

119, 119（119）123, 117（120）124, 124（124）123, 109（116）134, 131（133）134, 122（128）11, 11（11）13, 13（13）8, 6（7）
11, 9（10）13, 10（12）7, 10（9）27, 28（28）19, 18（19）23, 167（20）28, 17（23）30, 20（25）22, 19（21）25, 34（30）Compound
Dose

AF-2
0.01

SAZ
0.5

AF-2
0.01

AF-2
0.1

Colony count/plate

605, 621（613）200, 212（206） 113, 122（118） 423, 444（434） 1765, 1778（1772）

CompoundDose

B［a ］P
5.0

2AA
2.0

Colony count/plate

999, 1003（1001） 187, 178（183） 750, 778（764） 256, 267（262） 88, 87（88）ICR-1911.0

B［a ］P
5.0

2AA
10.0

B［a ］P
5.0

The values in brackets show the mean values（n ＝ 2）.

Table 7 Human patch test
SampleJudgement time
Reaction
−
±
＋
++
+++
Skin irritation index

MIRAVC
30％ aqua soln.

White vaseline

24-hr

48-hr

24-hr

48-hr

24-hr

48-hr

24-hr

48-hr

24/24
0/24
0/24
0/24
0/24

24/24
0/24
0/24
0/24
0/24

24/24
0/24
0/24
0/24
0/24

24/24
0/24
0/24

24/24
0/24
0/24
0/24
0/24

24/24
0/24
0/24
0/24
0/24

24/24
0/24
0/24
0/24
0/24

24/24
0/24
0/24
0/24
0/24

0.0

3.3.5. Stinging test
The subjects were 10 adult men and women, 2 men and 8
women, aged 25-56 years (average age 43 years). Since
MP had a significantly higher Sting score than distilledwater, it was confirmed that it was valid as a Sting
stimulation evaluation test. A signiﬁcant diﬀerence test of
the Stinging score revealed that the test product had asignificantly higher Stinging score than distilled waterand a signiﬁcantly lower Stinging score than MP (Table
8).
3.4. Transdermal absorption test
For both 5% and 30% test specimen, the Q value, whichis the cumulative permeation amount per unit area,

0/24
0/24

Saline0.0

0.0

Distilled water
for injection

0.0

increased over time. After 12 hours, the Q value of 5%
and 30% of aqua solution was 72.7 ± 46.0μg/cm2, 1117
± 360μg/cm2, respectively (Figure 1).

4. DISCUSSION
We evaluated the safety of a stable neutral vitamin C
derivative (MIRAVC) for topical use. In the primary skinirritation test, the cell survival rate was 97.1 ± 3.2%,
indicating that there was almost no primary skin irritationcompared to the negative control. In addition, MIRAVC
was judged to be a substance that does not show irritation
to the skin based on the out-of-classiﬁcation evaluation.
The cell survival rate in the eye mucosal irritation test

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Table 8 Stinging test
MIRAVC

No.

Sex

Age

Distilled water

MP*

1
2
3
4
5
6
7
8
9
10

F
F
F
F
F
F
F
M
F
M

56
26
36
50
49
49
50
51
25
34

1
1
1
1
1
1
1
1
1
1

2
2
3
3
2
3
2
4
3
2

1
2
1
1
2
2
2
2
2
1

1.0

2.6
＜ 0.001

1.6
0.014
0.011

Averagep value against distilled waterp value against MP

30％ aqua soln.

* 0.15％ Methyl Paraben aqua. solution.

30 w/v％

5 w/v％
1500

Q（μg/cm2）

Q（μg/cm2）

150

100

50

1000

500

0

0
0

2

4

6

8

10

0

12

2

4

Time（h）

6

8

10

12

Time（h）

Lag time

Q

Q（μg/cm2）

Permeation rate
（μg/cm2/h）

Lag time（h）（h）（μg/cm2）

Permeation rate
（μg/cm2/h）

3.09 ± 0.82

72.7 ± 46.0

7.88 ± 4.29

4.06 ± 0.611117 ± 360

141 ± 47.3

Dates are mean ± standard deviation（n ＝ 3）.

Figure 1 Transdermal absorption test

was 90.4%, and it was classiﬁed as Not classiﬁed . The
positive control survival rate in this test was less than
40%, conﬁrming the reliability of the test. In the Ames
test, MIRAVC was judged to be negative because it does
not have the ability to induce gene mutagenesis.
Furthermore, no dose response or growth inhibition wasobserved. In a 24-hour occlusive human patch test, theskin irritation index was low in 24 adults. In addition, itsscore in the stinging test was signiﬁcantly lower than thatof methylparaben. The results of the transdermal
absorption test conﬁrmed that MIRAVC absorbs into theskin, and it was confirmed that it is not less irritating
because it does not penetrate the skin.
MIRAVC has a molecular weight of 530, is relatively
small, and contains amino acids, which arebiocomponents, so it is thought to have highbiocompatibility. When glucose is added, the timerequired for permeation becomes longer, so it is thoughtthat MIRAVC permeates slowly and has sustained releaseproperties. Although the test conditions are notcompletely the same as this study, there is a report on
ascorbyl glucoside using LSE-high8). It has been reported

診療と新薬・第 60 巻

that ascorbyl glucoside has strong hydrophilicity, so its
penetration is relatively slow 6). The penetration rate
when using 1% ascorbyl glucoside solution is 0.91 ± 0.15μg/cm 2/h, while 7.88 ± 4.29μg/cm 2/h, which is thepenetration rate for 5% MIRAVC in this study. If theconcentration is reduced to 1/5, it is calculated as 1.576
μg/cm2/h, which suggests that MIRAVC has a higher
penetration rate than that of ascorbyl glucoside.
All the results of the safety tests were negative, and noadverse events were observed in all the tests conducted.
From these facts, there are no safety concerns for human
topical use. Even in the eye mucosal irritation test, whichis thought to be easily irritating, there was no irritation
at all even at a high concentration of 30%, so thiscompound is considered to be extremely non-irritating.
This is because the pH of the aqueous solution of thiscompound is extremely close to the pH of the epidermis
or dermis of the skin, and it is presumed that irritation isextremely suppressed compared to ascorbic acid or
ascorbyl glucoside.
This compound has arginine added to ascorbyl glucoside,in which glucose is bound to the 2nd-position of ascorbic
acid. Arginine is probably bound to the 3rd-position of
ascorbic acid. More specifically, it is considerednecessary to determine the structure of this compound by
NMR (Nuclear Magnetic Resonance) or the like. HPLC
analysis of transdermally absorbed receiver ﬂuid detectedglucose and arginine in a bound state. In this threedimensional skin model, the enzyme system, etc. may beslightly diﬀerent from that of a living body. It is assumed
that the chemical form is stable even within the skintissue, and this is thought to contribute to its low
irritation. Future research will be needed to further
investigate the dynamics and chemical forms within theskin.
These results suggest that MIRAVC has low irritation
to the skin and eyes, and moreover has no ability to
induce genetic mutations. It was conﬁrmed that there isalmost no skin irritation in humans, and the irritation islow. This indicates that MIRAVC can be safely used for
topical applications such as on the skin and eyes. In thisstudy, we conﬁrmed the basic safety of MIRAVC throughseveral safety tests, but we did not examine sensitization,
phototoxicity tests, or the safety of repeated or long-term
use. It is likely that future research will accumulate evenmore data. Through future research and application, it isexpected that MIRAVC will be widely used around theworld and make a major contribution to society.

5. CONCLUSION
In this study, we conducted a comprehensive test on thesafety of a stable neutral vitamin C derivative (MIRAVC).
As a result, MIRAVC was negative for primary skin第 12 号（2023 年 12 月）

19 （771）

irritation, ocular mucosal irritation, and genemutagenicity, and no significant irritation was observedin human patch tests or stinging tests. Judging from theresults of these safety tests, MIRAVC was conﬁrmed tobe safe for human topical use even at a concentration as
high as 30%, and to have low irritation to the skin andeyes.
Therefore, MIRAVC is a safe and promising compound,and is expected to be used in future applications anddevelopment of various topical products. As further
research and clinical trials progress, it is believed that
our understanding of its safety and utility will further be
examined.
CONFLICT OF INTEREST STATEMENT
Dr s Choice Co., Ltd. provided the funding for this study. There was no
particular conﬂict of interest.

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