You are viewing a javascript disabled version of the site. Please enable Javascript for this site to function properly.
Go to headerGo to navigationGo to searchGo to contentsGo to footer
In content section. Select this link to jump to navigation

Safety evaluation and consideration of 4 Pin Multi-needle for meso-therapy

Abstract

This study was conducted according to the method presented in the Republic of Korea Pharmacopoeia 11th Revision, aseptic test method to evaluate the suitability of sterilization for a sterile needle (4 Pin Multi-needle). In this study, four tests were conducted: sterility test, cytotoxicity test, acute toxicity test, skin sensitization test. First, in the aseptic test, the microorganism was not proliferated in the aseptic test of the medium. As a result of the performance test of the medium, it was confirmed that the microorganism developed within 3 days and the fungus was evident within 5 days. Based on this, it was confirmed that the medium was suitable, and as a result of the aseptic test, the development of microorganisms was not observed during the total culture period. Based on these results, tests were conducted which were confirmed to be suitable for aseptic testing because the development of bacteria on the provided samples was not recognized. For cytotoxicity tests ISO10993-5; 2009 (Biological Evaluation of Medical Devices, Part 5: Test for in vitro Cytotoxicity). As a result, the MEM eluate of the test substance caused very slight cytotoxicity to the fibroblasts of the mouse and was judged to be Grade 1 (Slightly cytotoxic) according to the judgment standard of ISO 10993-5. On the other hand, solvent control, negative control and positive control showed the expected results on the test. Acute Toxicity Test Results: It was judged that there was no systemic toxicity change when ICR mice were treated with 50 mL/kg B.W. of the eluate of sterile injectable needle for 72 hours. Skin sensitization test result: The Hartley guinea pig was evaluated as a substance which is evaluated as a substance which does not induce any skin reaction when skin sensitization is applied to the dissected material of the sterile injectable needle and is weak in skin sensitivity. Based on the above tests, we will study the stability and efficacy of more reliable medical devices based on the verification and performance of medical devices.

1.Introduction

Meso-therapy was developed by the French physician Pistor in 1952. It is a combination of ‘meso’ and ‘therapy’, which means healing, and it is mixed with proven medicines, minerals and vitamins. Injection therapy is injected only at the target site, unlike conventional therapy, so it can maximize the effect with a small amount and a minimum of pain and minimize the systemic side effects of the medicine or drug. Treatment subjects are variously applied to obesity, wrinkles, alopecia, and pain [1]. Drugs suitable purpose of treatment are mixed and injected with dozens to hundreds of injections depending on the treatment site using insulin sensitizer or automatic syringe. The frequency of treatment is usually 12 weeks apart, depending on the response to treatment at 12 months intervals to widen the treatment interval and maintenance treatment is done once every 45 months.

It is a new treatment technology widely applied in 14 countries including Europe, America and some Asian countries.

Using meso-therapy, collagen in the dermal layer of the skin is reduced and denatured to reduce the elasticity and wrinkles caused by the dullness of the skin, thereby promoting healthy collagen production in the dermis without damaging the surface of the skin through the treatment, thereby improving dull and wrinkled skin. Minimal amounts of vitamins and antioxidants are injected directly into the skin to maximize skin regeneration [2, 3, 4, 5].

In combination with a drug that activates blood circulation and lymphatic circulation and a drug that breaks down fat, it can accelerate the decomposition of cellulite using a procedure in a thick cellulite on the side of the hips or thighs.

Vitamin C, antioxidants, decolorizing agents, etc., are added to the skin that looks dark due to the melanin pigment, and it can be treated by injecting it into the epidermis and dermis of the skin. And meso-therapy is more effective than any treatment method on scalp (hair loss) [6]. By injecting drugs with scalp health and hair growth effect directly into the hair follicles, it helps to shorten hair loss period and help hair grow quickly. It is a more effective treatment if you use ancillary interactions with existing hair loss treatments. A syringe used for meso-therapy usually uses a 1 pin-needle syringe. Due to the nature of the procedure, the practitioner who is performing the injection in tens to hundreds of places feels a lot of fatigue.

The 4 Pin Multi-needle was developed to enhance the convenience and efficiency of the practitioner and safety of the device could be secured through safety evaluation, which would improve the efficiency and convenience of the practitioner in meso-therapy.

The present study is expected to improve the safety and safety of the procedure from the safety factors of infection and other factors through the safety evaluation of 4 Pin Multi-needle made for use in meso-therapy.

2.Materials and methods

For the safety evaluation of 4 Pin Multi-needle for meso-therapy, tests such as aseptic test, cytotoxicity test, acute toxicity test, skin sensitization test were conducted. For the four tests, it is the safety evaluation of the medical device conforming to the CE certification [7, 8, 9, 10, 11, 12, 13, 14, 15]. The developed 4 Pin Multi-needle is able to inject drugs more effectively into the skin using mesotherapy.

In addition, it is possible to shorten the procedure and administration time by using four needles. In other words, it can be used effectively by the reduction of time and costs.

2.1Aseptic test

A medical device is a device or device used for a person or an animal with the purpose of treating a disease or the like, and should not be toxic when applied to the body. In addition, medical devices must remain sterile until used to prevent secondary infection by microbial contamination. Sterile testing is widely used as a means of evaluating the suitability of sterilization for a variety of medical devices and test materials. This test was carried out in order to evaluate the proliferation of microorganisms (bacteria and fungi) that were proliferated when the test substances were directly inoculated into liquid thioglycolic acid medium and soybean casein digest medium and cultured at each temperature.

2.1.1Aseptic test materials and methods

1)
  • 1) Test substance: Sterile needle (4 Pin Multi-needle).

  • 2) Aseptic properties of medium.

    (1)
    • (1) Aseptic properties of medium: Microbial growth should not occur when the liquid thioglycolic acid medium is cultured at (3035)C and soybean casein digestion medium is cultured at (2025)C for 14 days.

    • (2) Performance test of the medium: When the culture medium of the following performance test strains or the strains considered to be equivalent thereto is inoculated at a rate of not more than 100 CFU per 1 culture medium, each bacterium must develop clearly within 5 days.

  • 3) Aseptic test (direct method).

    (1)
    • (1) Test method.

      All or part of the specimen was inoculated into the liquid thioglycolic acid medium and the soybean casein digestion medium, respectively. Liquid thioglycolic acid medium (3035)C and soybean casein digestion medium were cultured at (2025)C for 14 days, and the growth of bacteria was observed at 59 and 14 days after culturing. If the medium is turbid depending on the specimen, or if it is otherwise difficult to determine, or if necessary, the appropriate amount is transplanted into the new medium on the 14th day of culture and cultured for 4 days or more with the original medium at the same temperature.

Table 1

Strain for medium performance test

Culture mediumTest strainsCulture
Liquid thioglycolic acid mediumStaphylococcus aureus (ATCC 6538)(3035)C, aerobic culture
Pseudomonas aeruginosa (ATCC 9027)
Clostridium sporogenes (ATCC 11437)
Soybean casein digestion mediumBacillus subtilis (ATCC 6633)(2025)C, aerobic culture
Candida albicans (ATCC 10231)
Aspergillus brasiliensis (ATCC 16404)

2.2Cytotoxicity test

In vitro cytotoxicity testing is commonly used as an early means of assessing biocompatibility for a variety of medical devices and test materials because of the relatively easy access to the test. This test was conducted to evaluate whether the test substance causes cytotoxicity in mouse fibroblasts (NCTC Clone 929 (L-929)) under the culture conditions of the cells. According to ISO 10993-5: 2009 (Part 5: Tests for in vitro cytotoxicity), in order to apply a solid test substance to a cell, it was eluted with MEM medium supplemented with 10% serum A test solution was prepared. This test solution was directly contacted with the cells to examine the effect of the test substance eluate on the cells under a microscope. The MEM medium supplemented with 10% serum used as the elution solvent was selected to maintain the physiological condition most similar to the situation in which the test substance was applied to the human body.

Table 2

ISO 10993-5, 8.5 Determination of cytotoxicity

GradeReactivityConditions of all cultures
0NoneDiscrete intracytoplasmatic granules, no cell lysis, no reduction of cell growth
1SlightNot more than 20% of the cells are round, loosely attached and without intracytoplasmic granules, or show changes in morphology; occasional lysed cells are present; only slight growth inhibition observable
2MildNot more than 50% of the cells are round, devoid of intracytoplasmic granules, no extensive cell lysis; not more then 50% growth inhibition observable
3ModerateNot more than 70% of the cell layers contain rounded cells or are lysed; cell layers not completely destroyed, but more than 50% growth inhibition observable
4SevereNearly complete or complete destruction of the cell layers

2.2.1Cytotoxicity test (materials and methods)

1)
  • 1) Test substance: Sterile needle (4 Pin Multi-needle).

  • 2) Medium and vehicle.

    • Appellation: 1 × MEM (Minimum essential medium) with 10% serum;

    • Manufacturer: Gibco;

    • Lot No.: 1687974;

    • Conditions for keeping: (28)C.

  • 3) Serum.

    • Appellation: Horse serum;

    • Manufacturer: Gibco;

    • Lot No.: 1517711;

    • Conditions for keeping: (-15-20)C.

  • 4) Negative control.

    • Appellation: High density polyethylene Film;

    • Manufacturer: Hatano Research Institute (Food and Drug Safety Center);

    • Lot No.: C-111;

    • Conditions for keeping: Room temperature.

  • 5) Positive control.

    • Appellation: ZDEC Polyurethan Film;

    • Manufacturer: Hatano Research Institute (Food and Drug safety Center);

    • Lot No.: A-141;

    • Conditions for keeping: Room temperature.

  • 6) Preparation of experimental material: after aseptically operating at a rate of 20 mL of 4 g 10% MEM medium supplemented with serum per test substance, the cells were eluted in a (5 + 1)% CO2 incubator at (37 ± 1) This solution was used as a test solution. For the solvent control, the MEM medium except for the test substance was eluted under the same elution conditions as the test substance. The negative control and the positive control were assayed in accordance with the cytotoxicity test method of the manufacturer (Hatano Research Institute) at a rate of 10 mL of 1 × MEM medium per 1 g (37 ± 1)C. (24 ± 2) hours in a (5 + 1)% CO2 incubator. The test substance was used for the test within 24 hours after preparation.

  • 7) Test system.

    (1)
    • (1) Test cell line.

      • * Appellation: NCTC Clone 929 (L-929);

      • * Manufacturer: ATCC (American Type Culture Collection, USA).

    • (2) Reason for selecting test system: These cells are known to be highly susceptible to chemicals in cytotoxicity tests and were selected because of the abundance of data related to cytotoxicity studies.

    • (3) Culture conditions.

      Culture medium: 10% Horse serum and Antibiotics [Penicillin (100 Units/mL), Streptomycin (100 ug/mL) include with Minimum Essential Medium (pH 7.4) Condition: (5 ± 1)% CO2 and (37 ± 1)C were transplanted every 3 to 4 days.

  • 8) Test methods.

    (1)
    • (1) Qualitative analysis.

      The monolayer cultured cells were treated with trypsin (EDTA) to adjust the cell concentration to 105 per 1 mL, and inoculated 2 mL into a 10-cm2 6-well plate (35 mm/well). The wells were cultured for 24 hours, monolayer cultures were selected, labeled with each test group and control group, and then the medium was removed. (5 ± 1)% CO2, (37 ± 1)C, and 2 mL of the test substance effluent, solvent control, negative control and positive control in 3 selected wells in cultured for 48 hours. After incubation, the dissolution or morphology of (100x) cells was observed under a microscope.

    • (2) Quantitative analysis.

      After observing the dissolution and morphology of the cells, trypsin (EDTA) was applied to remove the cells from the plate, and the viable cells in each test group were counted.

  • 9) Judgment of cytotoxicity.

    (+) In the presence of a uniform monolayer of cells (Confluent monolayer), or (-) in the absence of a single monolayer of cells. The cell solubility and morphological changes of each well were recorded. The color of the medium of the negative control group and the test group was also observed. When the color of the medium changed to yellow, it was judged that the medium was changed to acid by the eluate. When the color changed to magenta or purple, it was judged that the medium changed to basic.

To demonstrate the efficacy of the test, the solvent control (Reagent control) and negative control should not show cytotoxicity (Grade 0) and positive control should show moderate cytotoxicity(> Grade 2).

If each control does not show the expected result, or if different results are observed in the three test groups, retest is performed.

Table 3

Test group composition

GroupAnimal numberTest materialsDose (mL/kg B.W.)Injection site
G1 negative control11011105Sterile saline50Intravenous
G2 treatment12011205Sterile saline extract50
G3 negative control13011305Cotton seed oil50Intraperitoneal
G4 treatment14011405Cotton seed oil extract50

Table 4

Test group composition

GroupAnimal numberTest materials dose (mL/kg B.W.) injection site
G1 negative control11011105Negative control (sterile saline)Test materials (sterile saline extract)
G2 treatment12011210Test materials (sterile saline extract)Test materials (sterile saline extract)
G3 negative control13011305Negative control (cotton seed oil)Test materials (cotton seed oil extract)
G4 treatment14011410Test materials (cotton seed oil extract)Test materials (cotton seed oil extract)

2.3Acute toxicity test

To investigate the acute toxicity of sterile injectable needles, intravenous and intraperitoneal doses of sterile saline and cottonseed oil were administered intravenously and intraperitoneally to ICR mice at a dose of 50 mL/kg BW. Mortality, general symptoms, weight changes and toxicity respectively.

During the test, no general symptoms, no specific clinical symptoms and no deaths were observed.

The body weight was measured during the test period, and all the body weight changes were normal.

No specific biological response was observed for all animals when observed for 72 hours after administration of the test substance.

From the above results, it was judged that the systemic toxicity change was not observed when ICR mice were treated with 50 mL/kg B.W. of the sterile injectable needle for 72 hours.

2.3.1Animal ethics

This test is based on the Animal Protection Act [Law No. 13023 (2015-01-20, partly revised)] and the Laboratory Animals Act [Act No. 11987 (2013-07-30, partly revised)]. It was approved by the Animal Ethics Committee of Health Care Headquarters.

Table 5

Cranial end

(0.1 mL)(0.1 mL)
(0.1 mL)(0.1 mL)
(0.1 mL)(0.1 mL)

2.3.2Acute toxicity test (materials and methods)

1)
  • 1) Test substance: Sterile needle (4 Pin Multi-needle).

  • 2) Preparation of test materials: Sterile physiological saline (Daihan Pharm CO., LTD, Korea) and cottonseed oil (Junsei Chemical CO., LTD, Japan) were prepared at a temperature of 70C to 24 hours at a ratio of 20 mL per 4 g of the weight of the test substance, which was used as a test substance. Separately, only sterile physiological saline and cottonseed oil were used as negative control substances by reacting under the same conditions.

  • 3) Animal Model information and condition.

    (1)
    • (1) Animal species: Mouse (CrljOri: CD1 (ICR)).

    • (2) The weight range of inject with Syringe, Number of animals used and weeks of age: (17–23 g), Number of 20 (male), 4 weeks old.

    • (3) Reason for selection: The ICR mouse used in this study is a species commonly used in acute toxicity tests and has a relatively rich test base data, so it is easy to interpret and evaluate the test results.

      Table 6

      Caudal end

      GroupAnimal numberTest materialsDose (mL)Injection site
      G1 negative control11011105Sterile saline: FCA (1:1)0.1
      Sterile saline: FCA (1:1)0.1
      Blank (Sterile saline)0.1
      Blank (Sterile saline)0.1
      Blank (Sterile saline): FCA (1:1)0.1
      Blank (Sterile saline): FCA (1:1)0.1
      G2 treatment12011210Sterile saline: FCA (1:1)0.1
      Sterile saline: FCA (1:1)0.1
      Sterile saline extract0.1
      Sterile saline extract0.1
      Sterile saline extract: FCA (1:1)0.1
      Sterile saline extract: FCA (1:1)0.1
      G3 negative control13011305Cotton seed oil: FCA (1:1)0.1
      Cotton seed oil: FCA (1:1)0.1
      Blank (Cotton seed oil)0.1
      Blank (Cotton seed oil)0.1
      Blank (Cotton seed oil): FCA (1:1)0.1
      Blank (Cotton seed oil): FCA (1:1)0.1
      G4 treatment14011410Cotton seed oil: FCA (1:1)0.1
      Cotton seed oil: FCA (1:1)0.1
      Cotton seed oil extract0.1
      Cotton seed oil extract0.1
      Cotton seed oil extract: FCA (1:1)0.1
      Cotton seed oil extract: FCA (1:1)0.1

      FCA : Freund’s complete adjuvant.

    • (4) Purification and Quarantine: After the introduction of the animals, the animals were quarantined and purified under the environment of the animal breeding room of the researcher and the general health condition was observed, and healthy individuals were selected for the test.

    • (5) Breeding environment.

      A.
      • A. Environmental condition: This test was conducted under the conditions of temperature (22 ± 3)C, relative humidity (50 ± 20)%, number of ventilation (1020) times/hr, lighting time 12 hours (08:0020:00) Lux was raised in the animal breeding room of the present researcher.

      • B. Breeding condition: Five animals were housed in Polysulfonate cage [180 (W) mm × 300 (D) mm × 140 (H) mm, ThreeShine, Korea] during the refinement and the test period.

      • C. Rodent Diet 20 5053 [Labdiet, USA], irradiated sterilized feed and feed water, and free R/O water were used for negative feed.

  • 4) Configuration and individual identification of test groups.

    • Object identification: The individual identification was indicated by the individual number using the oil magic on the tail and the animal identification card with the test number, sex, group number, individual number, test period and the person in charge of the test was attached to the breeding box.

  • 5) Test methods.

    Measurement of the body weight of each mouse before administration of tablets, vein using a disposable syringe (26 Gauge) with 5 mice 50 mL/kg B.W. the control substance and the test substance in (sterile saline eluate) and intraperitoneal (cotton seed oil eluate).

  • 6) Progress observation.

    (1)
    • (1) General symptom observation: All animals were observed for changes of general symptoms and presence of dead animals at 4, 24, 48 and 72 hours after injection of the test substance.

    • (2) Weight measurement: Body weight was measured at the time of introduction, on the day of administration and at 24, 48 and 72 hours after administration.

  • 7) Assessment methods.

    If the test group shows no significant biological response compared to the control group for 72 hours after injection of the test substance, it is considered that it does not show acute toxicity. Acute toxicity is also seen when two or more animals die in the test group, abnormal behavior such as seizures or debilitation occurs in two or more animals, or weight loss of more than 10% in three or more animals. If less than one animal exhibits an overall symptom of a biological response or dies, the test is repeated using 10 animal groups. In the repeated tests, 10 animals treated with the test substance are considered not to show acute toxicity if there is no specific biological response than the control.

Table 7

Magnuson and Krigman grade table

Patch test reactionGrade
No noticeable change0
Separable or refractory erythema1
Moderate erythema and fusiform erythema2
Severe erythema and swelling3

Figure 1.

Four pin-multi needle for Meso-therapy.

Four pin-multi needle for Meso-therapy.

2.4Skin sensitization test (materials and methods)

In order to investigate the skin sensitization to the sterile injectable needle, the Hartley guinea pig was induced to induce sensitization by primary intradermal injection and second transdermal application of sterilized physiological saline and cottonseed oil, and then transdermal application to induce mortality, general symptoms and skin sensitization.

A.
  • A. During the test, no general symptoms, no specific clinical symptoms and no deaths were observed.

  • B. The body weight was measured during the test period, and all the body weight changes were normal.

  • C. After skin sensitization, 24 hours and 48 hours of skin reactions were evaluated. No skin reactions such as erythema and edema were observed.

As a result, it was evaluated that Hartley guinea pigs did not induce any skin reaction when exposed to sensitization of the sterile injectable needle.

2.4.1Animal ethics

This test is based on the Animal Protection Act [Law No. 13023 (2015-01-20, partly revised)] and the Laboratory Animals Act [Act No. 11987 (2013-07-30, partly revised)]. It was approved by the Animal Ethics Committee of Health Care Headquarters.

Table 8

Aseptic testing of media

Culture mediumCulture confirmationJudgment
Liquid thioglycolic acid mediumN.G.Suitable
Soybean casein digestion mediumN.G.Suitable

*G.: Growth, N.G.: No growth.

Table 9

Performance tests of the culture medium

Culture mediumTest strainsInoculationCultureJudgment
amount (CFU)
Liquid thioglycolic acid culture mediumS. aureus 100Exhalation culture (3035)C, 3 daysG.
P. sporogenes 100Exhalation culture (3035)C, 3 daysG.
C. soirigenes 100Exhalation culture (3035)C, 3 daysG.
Soybean casein digestion culturemediumB. subtilis 100Exhalation culture (2025)C, 3 daysG.
C. albicans 100Exhalation culture (2025)C, 3 daysG.
A. brasiliensis 100Exhalation culture (2025)C, 3 daysG.

*G.: Growth, N.G.: No growth.

Table 10

Aseptic test

Culture mediumExp start dateIntermediate inspection dayLast inspection dayJudgment
Liquid thioglycolicSpecimenN.G.N.G.Suitable
 acid culture mediumNegative controlN.G.N.G.
Soybean casein digestionSpecimenN.G.N.G.Suitable
 culturemediumNegative controlN.G.N.G.

*G.: Growth, N.G.: No growth.

Table 11

Result of qualitative analysis

WellConfluent% Growth% Cells without% Rounding% LysisReactivityGrade
monolayerinhibitionintracellular granulation
Test (1)(+)10101010Slight1
Test (2)(+)10101010Slight1
Test (3)(+)10101010Slight1
Negative control (1)(+)0000None0
Negative control (2)(+)0000None0
Negative control (3)(+)0000None0
Reagent control (1)(+)0000None0
Reagent control (2)(+)0000None0
Reagent control (3)(+)0000None0
Positive control (1)(+)100100100100Severe4
Positive control (2)(+)100100100100Severe4
Positive control (3)(+)100100100100Severe4

(+): Present, (-): Absent.

Table 12

Result of qualitative analysis

Group lineResult of cell counting (cells/mL)
Test groupReagent controlNegative controlPositive control
16.6 × 1057.8 × 1057.6 × 1050
26.9 × 1057.5 × 1057.7 × 1050
36.5 × 1057.7 × 1057.4 × 1050
Average6.7 × 1057.7 × 1057.6 × 1050
RCC* (%)87.0100.098.70.0

RCC (relative cell counting, %) = Cell number of test group/Cell number of reagent control group × 100.

Table 13

Mortality and clinical signs

GroupSexAnimal numberClinical signsMortality (dead/total)
4 h24 h48 h72 h
G1 negative control (sterile saline)Male11010/5a (0%)
1102
1103
1104
1105
G2 treatment (sterile saline extract)Male12010/5 (0%)
1202
1203
1204
1205
G3 negative control (cotton seed oil)Male13010/5 (0%)
1302
1303
1304
1305
G4 treatment (cotton seed oil extract)Mael14010/5 (0%)
1402
1403
1404
1405

-: No abnormality detected, a: No. of dead animals/No. of total animals.

1)
  • 1) Test substance: Sterile needle.

  • 2) Preparation of test materials: Sterile physiological saline (Daihan Pharm CO., LTD, Korea) and cottonseed oil (Junsei Chemical CO., LTD, Japan) were prepared at a temperature of 70C to 24 hours at a ratio of 20 mL per 4 g of the weight of the test substance, which was used as a test substance. Separately, only sterile physiological saline and cottonseed oil were used as negative control substances by reacting under the same conditions.

  • 3) Animal model information and condition.

    (1)
    • (1) Animal species: Guinea pig, SPF.

    • (2) The weight range of inject with Syringe, Number of animals used and weeks of age: (300–500 g), Number of 30 (female), 6 weeks old.

    • (3) Reason for selection: The guinea pigs used in this study were selected because they were generally used for skin sensitization tests and had a relatively rich test basis data, which made it easy to interpret and evaluate the test results.

    • (4) Purification and quarantine: After the introduction of the animals, the animals were quarantined under the environment of the animal breeding room of the researcher, and the general health condition was observed while being purified, and healthy individuals were selected for the test.

  • 4) Breeding environment.

    (1)
    • (1) Environmental condition: This test was conducted under the conditions of temperature (20 ± 3)C, relative humidity (50 ± 20)%, number of ventilation (1020) times/hr, lighting time 12 hours (08:0020:00)) Lux was raised in the animal breeding room of the present researcher.

    • (2) Breeding condition: Stainless breeding box of the refining period and the test period [540 (W) mm × 610 (D) mm × 200 (H) mm, MJ., Ltd.] was put in 5 rats bred on.

    • (3) Animal feed and drinking water: Feeds were fed free of SAFE 114 (Scientific Animal Food & Engineering (SAFE), Route de Saint Bris-89290 Augy, France) containing radio-sterilized vitamin C and negative water using R/O water.

    Table 14

    Body weights (SEX: Male, Unit: g)

    GroupAnimal numberDay(s) after injection
    0 day3 days
    G1 negative control (sterile saline)110118.0822.78
    110218.5624.43
    110319.3624.00
    110419.5624.82
    110518.0223.89
    Mean18.7223.98
    S.D.0.710.77
    G2 treatment (sterile saline extract)120118.1722.81
    120218.3323.05
    120317.7721.84
    120417.9021.78
    120520.5425.59
    Mean18.5423.01
    S.D.1.131.55
    G3 negative control (cotton seed oil)130118.5023.62
    130217.8522.15
    130318.8824.24
    130419.7224.83
    130520.2525.28
    Mean19.0424.02
    S.D.0.961.22
    G4 treatment (cotton seed oil extract)140120.2826.14
    140220.7424.82
    140319.7923.94
    140418.9724.44
    140519.1423.82
    Mean19.7824.63
    S.D.0.750.93

    Table 15

    Mortality and clinical signs

    GroupNo. ofMortalityAnimal numberClinical signs
    animalsDays0123456789101112131415161718192021222324
    G1 negative50/5a (0%)1101
    control1102
    (sterile1103
    saline)1104
    1105
    G2 treatment100/10 (0%)1201
    (sterile1202
    saline)1203
    1204
    1205
    G3 negative50/5a (0%)1301
    control1302
    (cotton1303
    seed oil)1304
    1305
    G4 treatment100/10 (0%)1401
    (cotton1402
    seed oil)1403
    1404
    1405
    1406
    1407
    1408
    1409
    1410

    a: No. of dead animals/No. of total animals, –: Normal.

    Table 16

    Body weights (unit: g)

    GroupAnimal numberFirst induction phaseSecond induction phaseChallenge phase
    G1 negative control (sterile saline)1101309.89327.44417.61
    1102333.87358.21447.56
    1103316.61330.66417.81
    1104337.15353.37425.45
    1105340.32391.86492.10
    Mean327.57352.31440.11
    S.D13.4825.9231.53
    G2 treatment (sterile saline)1201309.89333.29440.24
    1202320.98365.25432.66
    1203335.56349.40411.43
    1204335.33368.79416.53
    1205318.89337.88414.83
    1206326.35366.59401.43
    1207323.57363.7944.654
    1208335.59378.17442.85
    1209363.59399.64443.88
    1210314.17368.85432.27
    Mean328.39363.17428.27
    S.D15.3019.3115.96
    G3 negative control (cotton seed oil)1301309.84323.10409.03
    1302325.35370.74447.08
    1303305.59341.84427.95
    1304336.21366.96417.46
    1305350.03399.71474.93
    Mean325.40360.47465.29
    S.D18.429.2926.32
    G4 treatment (cotton seed oil)1401317.60355.92412.42
    1402342.08382.55448.72
    1403350.20417.88481.60
    1404319.77392.00444.66
    1405311.98350.08460.98
    1406335.74357.76453.20
    1407342.28359.87442.27
    1408352.06398.57477.25
    1409332.47399.08485.34
    1410326.05360.59461.93
    Mean33.02377.43456.84
    S.D13.9023.5221.85

    Table 17

    Evaluation of skin response

    GroupAnimal numberScore per observation timesMean scoreSensitization rate (%)
    G1 negative control (sterile saline)1101000.00.0
    110200
    110300
    110400
    110500
    G2 treatment (sterile saline)1201000.00.0
    120200
    120300
    120400
    120500
    120600
    120700
    120800
    120900
    121000
    G3 negative control (cotton seed oil)1301000.00.0
    130200
    130300
    130400
    130500
    G4 treatment (cotton seed oil)1401000.00.0
    140200
    140300
    140400
    140500
    140600
    140700
    140800
    140900
    141000

  • 5) Configuration and individual identification of test groups.

    The individual identifications were indicated by individual numbers using saturated picric acid solution and individual identification cards were attached to the breeding boxes with the test number, sex, group number, individual number, duration of the test and the person responsible for the test.

  • 6) Test methods.

    (1)
    • (1) Primary induction (Intradermal induction induction): An intradermal injection the day before the three types of test substances following hair removal around the shoulder blade in the area of 4 cm × 6 cm, such as areas of the skin (2 cm × 4 cm) was intradermally injected by 0.1 mL.

    • (2) Secondary induction (local induction): After 6 days (± 1 day) of primary intradermal injection, intradermal injection site was epilated and 10% SLS (Sodium Lauryl Sulfate)/white vaseline was applied. On the next day, each test material was applied to sterile gauze (2 cm × 4 cm), attached to the primary induction site of the animal, and stained with non-magnetic tape (Tegarderm, 3 M) and Corban (3 M) I kept it for a while.

    • (3) Cause sensitization: Sensitization second induction 14 days (± 1 days), after depilation the flank of the animal left patch containing the test material (2 cm × 2 cm), a non-irritating tape (Tegarderm, 3 M) and koban (Corban, 3 M) For 24 hours.

  • 7) Progress observation.

    (1)
    • (1) General symptom observation: General symptoms and presence or absence of dead animals were observed daily for all animals.

    • (2) Weight measurement: All animals were measured at the time of introduction, first induction day, second induction day, and sensitization day.

    • (3) Application site observation: After the induction of the test substance (for 24 hours), the patch at the application site was removed and the skin reaction was evaluated at 24 hours and 48 hours.

  • 8) Assessment methods.

    In the control group, the magnitude of the Magnuson and Cligman test is less than 1, and in the test group of 1 or more, it is generally considered to indicate sensation. If the control group shows grade 1 or higher, the response of the test group showing a more severe reaction considered to be due to sensitization. If the reaction is not clear, the material is subjected to induction to verify the results of the first test result. The test results are expressed as the frequency of the positive results of the test animals and the control animals.

3.Results

The following is a list of sequential results for the experiments in this study.

A.
  • A. Aseptic test results: The aseptic test results are shown in Tables 911. When the liquid thioglycolic acid culture medium and the soybean casein digestion medium were cultured at the respective temperatures for 14 days, the microorganisms did not proliferate and the asepticity of the medium was confirmed. As a result of the performance test of the medium, the bacteria were clearly grown within 3 days and the fungi within 5 days when cultured at each temperature. When the specimens were inoculated into liquid thioglycolic acid medium and soybean casein digest medium and cultured at (3035)C, (2025)C, no growth of bacteria was observed during total culture period.

  • B. Cytotoxicity test results: The results of the qualitative analysis showing the morphological changes and the degree of dissolution of the cells are shown in Table 12. After incubation in the cells to which the eluate of the test substance was administered, a uniform monolayer was maintained but about 10% proliferation inhibition was observed. Therefore, the eluate of the test substance may have very slight reactivity to the cultured cells. In addition, quantitative analysis of the viable cell counts revealed that the effluent of the test material had a very slight effect on the number of viable cells (Table 12). The solvent control and negative control also did not show any toxicity to the cultured cells, and the positive control caused cytotoxicity in more than 75% of the total cells as expected.

  • C. Acute toxicity test results: Mortality and general symptoms Results No clinical signs and spermatozoa associated with the administration of the test substance were observed in all the treated animals for 4, 24, 48 and 72 hours after the injection (Table 13). In the case of weight change, normal weight change was observed in all test animals in the control group and the administration group on the day of administration and at 24, 48 and 72 hours after administration (Table 14).

  • D. Skin sensitization test results: No clinical signs or deaths due to the application of the test substance were observed during the skin sensitization test (Table 15). In the case of weight change, normal weight change was observed in both the test and control groups (Table 16). Skin responses were evaluated at 24 and 48 hours after the end of the test. Skin responses such as erythema and edema were not observed in the control group and the test group, and the solvent specific index was 0.0 and 0.0, respectively (Table 17).

4.Conclusions

In this study, the safety of 4 Pin Multi-needle for meso-therapy was evaluated [16]. In the safety evaluation, sterility test, cytotoxicity test, acute toxicity test and skin sensitization test were carried out. The aseptic test was judged to be suitable for the aseptic test. In the cytotoxicity test, the MEM eluate of 4 Pin Multi-needle did not change the acidity of the culture medium, but showed very weak proliferation inhibition in mouse fibroblasts. Therefore, it is considered that the eluate of the test substance used in the cytotoxicity test has very slight cytotoxicity, and it is judged to be Grade 1 (Slightly cytotoxic) according to the criteria of the cytotoxicity of ISO 10993-5. The test substance also appears to meet the criteria for cytotoxicity in general medical devices that should be below grade 2 (Mild). The test results of the solvent control, negative control and positive control were as expected, demonstrating the effectiveness of the cytotoxicity test results. In the acute toxicity test, mortality, general symptoms, weight change and toxic response were evaluated within 72 hours after intravenous and intraperitoneal administration of 50 mL/kg B.W. dose to ICR mice. After administration of the test substance, no deaths or specific clinical symptoms were observed, and normal weight changes were observed in all animals. From the above results, it was judged that ICR mice were treated with 4 Pin Multi-needle eluate at 50 mL/kg B.W. for 72 hours and no systemic toxicity was observed. Finally, the skin sensitization test was conducted by inducing sensitization with a primary intradermal injection and a second transdermal application of sterile physiological saline and cottonseed oil extract to a Hartley guinea pig, and then evaluated the mortality, general symptoms, and skin sensitization induced by transdermal application. No deaths or abnormal findings were observed during the test period, and no skin reaction was observed in all test groups after 24 hours and 48 hours after the completion of sensitization. As a result, the 4 Pin Multi-needle eluate in the Hartley guinea pig was evaluated as a weak skin sensitizer because it did not cause any skin reaction after sensitization exposure. In this study, the safety of 4 Pin Multi-needle was shown to be safe, and based on this, it is possible to provide safety and efficiency of operation and ease of operation for meso-therapy.

Acknowledgments

This study specifies that the results of the service test conducted by the Korea Testing and Research Institute (KTR), This work was supported by research fun of Catholic Kwandong University (CKURF-201407130001), This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Research Foundation of Korea (NRF-2015H1C1A1035925).

Conflict of interest

None to report.

References

[1] 

Sugawara J, Kou S, Kolkubo K, Kuroda A, Hashizume Y, Kobayashi S, Maegawa J, Satake T. Application for lower facial fat reduction and tightening by static type monopolar 1-MHz radio frequency for body contouring. Lasers in Surgery and Medicine 2017.

[2] 

Prikhnenko S. Polycomponent mesotherapy formulations for the treatment of skin aging and improvement of skin quality. Dove Medical Press (Open Access to Scientific and Medical Research) 2015; 7(8): 151-7.

[3] 

Kim S, Kye J, Lee M, Park B. Evaluation of mesotherapy as a transdermal drug delivery tool. Skin Research & Technology 2016; 22(2): 158-63.

[4] 

Gazitaeva ZI, Drobintseva AO, Chung Y, Polyakova VO, Kvetnov IM. Cosmeceutical product consisting of biomimetic peptides: antiaging effects in vivo and in vitro. Clinical, Cosmetic and Investigationaal Dermatology 2017.

[5] 

Chen DL, Cohen JL, Green JB. Injectable agents affecting subcutaneous fats. Seminars in Cutaneous Medicine and Surgery 2015; 34(3): 134-7.

[6] 

El-Komy M, Hassan A, Tawdy A, Solimon M, Hady MA. Hair loss at injection sites of mesotherapy for alopecia. Journal of Cosmetic Dermatology 2017.

[7] 

Food and Drug Regulations in Korea Statute 2014-115, Common criteria for Biological Safety of Medical Devices.

[8] 

Food and Drug Regulations in Korea Statute 2014-155, Medical Device Standard.

[9] 

ISO 10993-12 (2012), Biological evaluation of medical devices – Part 12: Sample preparation and reference materials.

[10] 

ISO 10993-5 (2009), Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity.

[11] 

USP 36 (2013), <87> Biological reactivity tests, in vitro.

[12] 

ISO 10993-11 (2006), Acute Systemic Toxicity.

[13] 

ISO 10993-10 (2010), Gulnea pig maximization test (GMPT).

[14] 

ISO 10993-11(2006), Information on material-mediated pyrogens.

[15] 

USP 36, <151> Pyrogen Test.

[16] 

Phillips KS, Wang Y. FDA authors publihc articles on dermal filler materials, injection methods and skin preparation. Plastic and Reconstructive Surgery 2017.