초록 ▼

개발 목적 및 필요성
• 연구개발목표
CMIT/MIT의 흡입노출 시 독성학적 영향 평가를 위한 기반 기술 확립과 흡입노출에 따른 독성평가 및 체내 이동 가능성 평가

• 연구개발내용
[1차년도]
1. CMIT/MIT 흡입독성평가를 위한 발생/노출법 확립
2. CMIT/MIT 최내 내성 용량(MTD) 평가 시험 수행
3. 기저질환(랫드 급성 폐염증 및 폐섬유화) 동물모델 확립
4. GLP 기반 생식독성평가 기술을 이용한 CMIT/MIT 입자의 흡입노출 시 태아에서의 독성학적 영향 평가<

개발 목적 및 필요성
• 연구개발목표
CMIT/MIT의 흡입노출 시 독성학적 영향 평가를 위한 기반 기술 확립과 흡입노출에 따른 독성평가 및 체내 이동 가능성 평가

• 연구개발내용
[1차년도]
1. CMIT/MIT 흡입독성평가를 위한 발생/노출법 확립
2. CMIT/MIT 최내 내성 용량(MTD) 평가 시험 수행
3. 기저질환(랫드 급성 폐염증 및 폐섬유화) 동물모델 확립
4. GLP 기반 생식독성평가 기술을 이용한 CMIT/MIT 입자의 흡입노출 시 태아에서의 독성학적 영향 평가
5. CMIT/MIT의 생체 내 분포 및 평가지표 분석법 개발

[2차년도]
1. CMIT/MIT의 13주 반복흡입노출을 통한 독성학적 영향 평가
2. 기저질환 동물모델을 이용한 CMIT/MIT 입자의 흡입노출 시 질환 악화 영향 평가
3. CMIT/MIT의 흡입노출 시 생체 내 분포, 이동, 배출 등에 대한 정량 평가

연구개발결과
1. 흡입노출 동물시험에 의한 CMIT/MIT 흡입독성평가 기술 개발
- CMIT/MIT의 흡입노출 시 최대 내성 용량 수준 자료 확보
- CMIT/MIT의 13주간 반복 흡입노출 시 독성학적 영향 평가 자료 확보
2. 기저질환 유발 모델을 이용한 CMIT/MIT 흡입노출 시 질환 악화 영향 평가
- 랫드에 대한 기저 질환 모델 2종 확보: 급성 폐염증 및 폐섬유화 모델
- 기저질환 유발 모델에서의 CMIT/MIT 흡입독성학적 영향 평가
3. CMIT/MIT 태아 노출에 의한 건강 영향 평가
4. CMIT/MIT의 체내 이동성 평가 기술 개발

성능사양 및 기술개발 수준
- GLP 기반 CMIT/MIT의 아만성 흡입독성평가 자료 확보
- CMIT/MIT의 흡입노출에 따른 폐질환 악화 정보 확보
- GLP 기반 CMIT/MIT의 흡입노출 시 태아에서의 독성학적 영향 평가 자료 확보
- CMIT/MIT의 생체 분포, 이동 등 생체 노출도 평가 자료 확보

활용계획
- 가습기살균제의 흡입노출 시 안전성자료 확보를 통해 정부적인 차원에서 체계적인 관리 및 규제 시스템 확립에 기여할 과학적 근거 자료로 활용
- 가습기살균제 내 유해성분 노출에 의한 독성평가 연구를 통해 호흡기를 통해 흡입되는 여러 대기환경·생활환경 유해물질에 의한 건강영향 평가 가능성 제시
- 태아 시기의 가습기살균제 노출에 의한 건강영향평가 연구를 통해 가습기살균제 유해성분 이외의 산전 위험물질 노출이 태아에 미치는 영향을 평가할 수 있음
- 살균제, 방향제, 탈취제 등 신규 또는 기존 생활화학제품들의 무분별한 사용 방지 및 인체위해성을 조기에 신속하게 파악하여 정책에 반영
- 생체 내 거동 및 분포 등 가습기살균제의 흡입노출 시 생체 내 변화를 탐색할 수 있는 기술 확보를 통한 가습기살균제 성분의 폐질환 유발 기전/원인 규명 연구에활용

( 출처 : 요약서 4p )

Abstract ▼

Ⅳ. Results
◦ Total Project
- Development of CMIT/MIT inhalation toxicity assessment technology by inhalation in vivo study
In this study, the maximum tolerated dose (MTD) levels of CMIT/MIT via intra-tracheal instillation and inhalation exposure were evaluated prior to subchronic inhalatio

Ⅳ. Results
◦ Total Project
- Development of CMIT/MIT inhalation toxicity assessment technology by inhalation in vivo study
In this study, the maximum tolerated dose (MTD) levels of CMIT/MIT via intra-tracheal instillation and inhalation exposure were evaluated prior to subchronic inhalation toxicity assessment. The daily dose of 0.08 mg/kg/day was judged to be the MTD level during repeated intra-tracheal instillation in rats. On the other hand,when exposed to 150 mg/m³/day for 1 week repeated whole-body inhalation exposure,clinical symptoms of respiratory system and significant body weight or food consumption changes were observed, and histopathological changes were observed in the larynx. Daily exposure levels of 150 mg/m³/day were determined to be MTD levels exposed to CMIT/MIT particles in the 1 week repeated inhalation exposure to rats.

In order to investigate the toxicological effects of CMIT/MIT on long-term repeated inhalation exposure, the daily exposure concentrations were set at 1.8, 7.8 or 15.5 mg/m³/day and the nose-only inhalation exposure were conducted for 6 hours/day, 7 days/week, 13-week periods. When examining the toxicological influence, no toxicological effects were observed including histopathological examination. However, measurement and analysis of protein or gene levels of toxicological indicators and pulmonary fibrosis-related biomarkers in BAL fluid and lung tissue revealed the possibility of toxicological changes caused by repeated inhalation exposure of CMIT/MIT. In particular, the possibility of toxicological changes including the possibility of pulmonary fibrosis induced by repeated inhalation exposure of CMIT/MIT in males was more evident han in females. The results of male specific studies derived from this study should be further studied. On the other hand, significant changes in pulmonary fibrosis-related indicators at the protein and gene levels did not correlated to histopathological changes. Therefore, we concluded that CMIT/MIT induced pulmonary fibrosis resulted from the repeated inhalation exposure based only on the results of this study is lack, and further studies on this issue should be conducted.

- Assessment of disease severity at CMIT/MIT inhalation exposure using underlying disease induction model
In this study, we developed acute pulmonary inflammation model and pulmonary fibrosis model for rats in order to evaluate disease exacerbation in CMIT/MIT inhalation exposure in the underying disease model. Previously, rodent lung disease models were extensively constructed in mice compared to rats. In this study, we have completed the development of acute pulmonary inflammation and pulmonary fibrosis model in rats by referring to the previous information (dosing substance, dosage,duration of disease, etc.) in mice.

The following two evaluation models were used to evaluate the severity of the disease under the condition of the repeated inhalation exposure of CMIT/MIT. As a result of the evaluation of the severe effect in the acute lung inflammation model, the severity of the lung inflammation, was not confirmed by repeated inhalation exposure of CMIT/MIT. However, it was found that pulmonary hemorrhage was increased when CMIT/MIT was repeatedly inhaled under the condition of acute pulmonary inflammation. In addition, chronic active inflammation was increased in the nasal cavity when repeated inhalation exposure of CMIT/MIT in the condition that acute lung inflammation was continuously induced. Chronic active inflammation observed in the nasal cavity also showed the similar changes in the pulmonary fibrosis model study. Pulmonary haemorrhage in the lungs and chronic active inflammation of the nasal cavity were observed when exposed to CMIT/MIT under the acute lung inflammation induced condition. It was considered to be the changes related to the repeated inhalation exposure of CMIT/MIT.

In the study of the severity effects in the pulmonary fibrosis model, the severity of pulmonary fibrosis and chronic inflammation of alveolar/bronchiole plexus in the 50 mg/m³/day exposure group was relatively increased rather than other exposure group, and the eosinophil infiltration showed a similar tendency. It was the change related to the repeated exposure of CMIT/MIT during the induction of pulmonary fibrosis, which is also attributed to the exacerbation or induction of the target disease or symptoms.

- Assessment of toxicological effects via inhalation exposure of CMIT/MIT on fetal
Twenty pregnant female rats were exposed for 6 hours per day with concentration of 0, 25, 50 and 100 mg/m³/day from 6th day to 20th day of gestation period to investigate the prenatal developmental toxicity of CMIT/MIT. As a result, body weights of dam and fetus were decreased in 50 or 100 mg/m³/day exposure groups.
The body weight loss of dams was considered to be due to the decrease of the food consumption, and the body weight loss of fetus was considered to be due to uterine growth retardation as the indirect effect from dams. The decrease of the number of ossification points in the cervical vertebrae was also considered to be due to the developmental delay. Under the conditions of this study, No-Observed-Adverse-Effect Levels (NOAEL) of CMIT/MIT was established to be 25 mg/m³ in prenatal developmental toxicity.

- Assessment of CMIT/MIT migration in the body
In this study, we used MSPD extraction method and GC/MS based analytical method to confirm the distribution and/or behavior of CMIT/MIT. CMIT/MIT was not detected from quantitative analysis of the bio-samples (lung, kidney, liver, blood, urine) obtained by inhalation exposure by the developed extraction and analytical method. It is considered that CMIT/MIT compounds absorbed through the respiratory system might be rapidly reacted with the tissues, and formed other degradation products or adducts. It is reported that CMIT/MIT has high reactivity with the materials having nucleophilic properties in vivo. In this study, rapid reaction between CMIT/MIT and GSH was showed by the reaction products production. When CMIT/MIT is penetrated into the body through the respiratory system, it is assumed that the produced metabolite may be systemically circulated after the reaction occurred in the primary contact body tissues. In order to evaluate the possibility of CMIT/MIT migration in the body, it is essential to confirm metabolites that can prove this hypothesis.

◦ Unit Research Project 1
- Based on lung fibrosis AOP mode, the effect on pulmonary fibrosis of CMIT/MIT was assessed using human normal lung epithelial cells(BEAS-2B), human lung cancer epithelial cells(A549, NCI-H358), human monocyte(THP-1), and human lung fibroblast cells(MRC5), tested with Kathon™ CG, which have the same composition as SKYBIO FG^® used as humidifier disinfectant product.

- Exposing the cells to CMIT/MIT at concentrations ranging from 0 to 25.2 ㎍ / mL(0to 2000 ㎍ / mL as Kathon™ CG) depending on the cells to be tested, the appropriate concentrations were selected. The test chemicals were mixed in culture medium and exposed short-term(3-96 hours), long-term(7 days) to cells based on cell line and experimental method.

* Concentration selection: Based on cell line and experimental methods, considering 50% inhibitory concentration(IC₅₀) of cell growth, a concentrations suitable for confirming toxicity development were selected.

- In observation of inductin of ROS(oxidative stress) corresponding to initial signaling and the markers related to epithelial mesenchymal transition(EMT), which is one of activation of key cells in fibrosis, no significant changes were observed.

- In macrophage, when observing the markers in initial signaling and pro-inflammatory mediators, NO and NFkB increased significantly, but the indicators related to inflammasome were not affected in initial signaling. IL-1β, TNF-α secretion increased in the index corresponding to the pro-inflammatory mediator stage. Therefore, it seems that the induction of inflammatory response was mediated by the NFkB signaling mechanism. In addition, further mechanism was investigated using siRNAs,which suggests that the inflammatory response of Kathon CG triggers inflammation via TLR signaling mediated by NFkB.

- As a result of observing indices corresponding to distal signaling stage and activation of key cells in fibrosis stage of pulmonary fibrosis in lung fibroblasts, protein expression of PAI-1, an index related to differentiation of lung fibroblasts showed weak increase, however, there was no change in the level of the gene expression. In addition, significant change in the Smad activation reaction was not observed, which is an index corresponding to the distal signaling stage.

- Therefore, considering the overall effects of Kathon CG in pulmonary fibrosis steps in vitro based on lung fibrosis AOP, partial inflammatory effect in macrophage was confirmed. However, after the increase of the inflammatory response substance, the markers such as cell signaling and cell activation of pulmonary fibroblast and epithelial were not changed, which suggests that the possibility of pulmonary fibrosis induction Is very low.

- Meanwhile, as a result of evaluating the effect of Kathon CG by combined treatment with fibrogenic substances such as TGF-β and PHMG-p, no significant change due to concentration-dependent Kathon CG treatment was observed.

- However, the inflammatory responses can be classified into acute and chronic inflammation, and chronic inflammation may progress into pulmonary fibrosis by a kind of tissue scar, so further studies are required to confirm the possibility of fibrogenesis under sustained inflammation. In addition, although there was no concentration dependence, in some cells, additional experiments with more diverse in vitro test conditions are required for the part where the pulmonary fibrosis related index slightly changed.

- As a result of investigating the skin irritation of CMIT/MIT through the existing literature survey, it was reported that there are skin irritancy in many literatures. It was presumed that Kathon CG may induce skin irritation. Regarding eye irritancy of Kathon CG, short-term exposure test carried out based on OECD TG 491 and classified as "unpredictable substance", which suggests that additional in vivo testing is required.

◦ Unit Research Project 2
- Development of radiolabeling method for PHMG: Preparation of radiolabeled PHMG　 using In-111 in high radiochemical yield and purity (>99%)
- Biodistribution study and in vivo imaging of In-111 labeled polyhexamethyleneguanidine phosphate (PHMG) solution via various administration routes
▪ Oral administration of In-111 labeled PHMG in SD rats: rapid excretion in 24 h, negligible adsorption of PHMG from gastrointestinal tract to other internal organs such as lungs, liver
▪ Intravenous injection of In-111 labeled PHMG in SD rats: high accumulation in liver and lungs, slow excretion (liver 43.2% ID, lungs 7.7 %ID after 168 hours injection)
▪ Intratracheal instillation of In-111 labeled PHMG in SD rats: high accumulation in lungs, slow excretion, high retention in lungs (75% ID after 0.5 hours; 45% ID after 168 hours injection, approximately 60% of PHMG retained after 1 week compared to that of initial value)
- Installation of radioisotope-inhalation chamber system for biodistribution study of PHMG aerosol particles
- Biodistribution study of radiolabeled polyhexamethyleneguanidine phosphate (PHMG) aerosol particles via inhalation exposure
▪ PHMG aerosol particle concentration in inhalation chamber: approximately 0.11 mg/m³
▪ PHMG aerosol particles size in inhalation chamber: less than 0.56 μm size (>96% of aerosol particles)
▪ Inhalation exposure of In-111 labeled PHMG aerosol particle in SD rats: high accumulation in lungs, slow excretion, high retention in lung (approximately 74% of PHMG retained after 1 week compared to that of initial value)
▪ Translocation of PHMG to other organ (liver/lungs = 5.2% 168 hours after exposure)
▪ Excretion of PHMG in gastrointestinal tract in 48 hours post exposure
- Conclusion: Because PHMG was exposed as nano-sized aerosols, the biodistribution data from radiolabeled PHMG aerosol particles will be more reliable results to investigate in vivo behavior of PHMG than those of radiolabeled PHMG solution.

( 출처 : SUMMARY 25p )

목차 Contents

• 표지 ... 1
• 제 출 문 ... 3
• 요 약 서 ... 4
• 요 약 문 ... 11
• SUMMARY ... 22
• 목차 ... 32
• 표목차 ... 34
• 그림목차 ... 37
• 제1장 서 론 ... 44
• 제1절 연구개발과제의 개요 ... 46
• 1. 연구개발의 목적 및 필요성 ... 46
• 2. 연구개발대상 기술의 차별성 ... 51
• 제2절 연구개발의 국내외 현황 ... 54
• 제3절 연구개발의 내용 및 범위 ... 62
• 1. 연구개발의 최종목표 ... 62
• 2. 연도별 연구개발 목표 및 평가방법 ... 63
• 3. 연도별 추진체계 ... 66
• 제2장 연구개발 수행내용 및 결과 ... 70
• 제1절 연구개발 결과 및 토의 ... 72
• 제2절 연구개발 결과 요약 ... 226
• 제3장 목표 달성도 및 관련분야 기여도 ... 234
• 제1절 연도별 연구개발목표의 달성도 ... 236
• 제2절 관련분야의 기술발전 기여도 ... 239
• 제4장 연구개발결과의 활용계획 등 ... 241
• 제1절 연구개발 결과의 활용계획 ... 243
• 제2절 연구개발과정에서 수집한 해외 과학기술정보 ... 244
• 제3절 연구개발결과의 보안등급 ... 245
• 제4절 NTIS에 등록한 연구시설·장비현황 ... 245
• 제5장 참고문헌 ... 247
• 1. 국내문헌 ... 249
• 2. 국외문헌 ... 249
• 3. 기 타 ... 253
• 부 록 ... 254
• 끝페이지 ... 301