국제 환경규제 대응을 위한 국내 환경독성 시험법 개발 및 적용 방안

다국어 초록 (Multilingual Abstract)

In recent years, global environmental regulatory policies such as the EU's REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals; new chemicals management laws) and environmental regulatory policies of developed countries including the United States and Japan have been introduced and increasingly strengthened. Accordingly, country-by-country GLP (Good Laboratory Practice) management systems and a variety of toxicology test and evaluation technologies have been actively developed and continued to evolve for the chemical substances which cause environmental toxicity. In order to cope with the increasingly strengthened global environmental regulations, there are needed, along with the expansion of the capacity of GLP institutions through the development of domestic environmental toxicity tests, application and utilization of the developed environmental toxicity tests in the industry, systematic training of GLP professional researchers, and the expansion of technical exchanges of experts. Although GLP toxicity assessment technologies of our country started far later than the developed countries, the general toxicity tests among them have become in line with international standards owing to the continued efforts of research institutes and companies. However, environmental toxicity tests have mainly been entrusted to overseas institutions due to immaturity of the domestic market and technologies, and the relevant domestic technologies still remain in a very low level or have not yet been established. Thus, to enable timely responses to the confronting environmental regulations in and outside the country, environmental toxicity tests, which are essential when developing new materials in the domestic biotechnology and chemical companies, were developed in a lump using various substances and established to a level that meets international standards, through the present dissertation. The newly reestablished tests are: the environmental analysis tests of which importance is internationally being emphasized and which accurately determine the homogeneity and stability of test substances; four kinds of microbial biodegradation assays (DOC die-away, CO2 evolution, Closed bottle, and Modified OECD screening) which have not yet been developed and established in this country; avian toxicity tests (acute oral toxicity and dietary toxicity); genotoxicity tests (SOS chromotest); physicochemical assays; and general toxicity tests on rodents. In particular, SOS chromotest was tried as a convenient alternative for the genotoxicity test and, consequently, it was confirmed that six low-molecular-weight chemicals, which had shown IF values of less than 1.5 in the test, have no genotoxicity. Rodent toxicity tests using IVC (Individually Ventilated Cages) system have been established to have high reproducibility and to employ fewer animals. In these tests, extracts of huinmokyi mushrooms (Tremella fuciformis, China) and gardenia (Gardenia jasminoides, Korea) showed no toxicities at their highest doses, and Chlorpyrifos, an organophosphorus insecticide, showed a relatively high LD50 values in mice (Male: 141.42 mg/kg [80.43 - 248.67]; Female: 141.42 mg/kg [-]) and rats (Male: 452.98 mg/kg [-]; Female: 284.24 mg/kg [148.98 - 726.06]), respectively, and the signs of white colored eye, hypersensitivity, dacryohemorrhea, tremor, and hypolocomotion of the test animals were observed depending on the administered concentration. By introducing a new and simple system to the genotoxicity and general toxicity tests, the risk assessment and the application to the prediction screening techniques of environmentally hazardous substances have become easier. In the avian toxicity tests using 10 day-old Japanese quails (Coturnix coturnix japonica), Pyraclofos, an organophosphorus agrochemical which is important in the environmental toxicity assessment, and JCTC-0975 were used as test substances and exhibited LD50 values of 55.00 mg/kg and 9.00 mg/kg, respectively, in the oral toxicity tests and LC50 values of 130.00 mg/kg and 75.00 mg/kg, respectively, in the dietary toxicity tests. The outcome above indicated 35∼100% higher level than any previously reported data, in both domestic and international, which demonstrates that a great difference can be resulted depending on varying conditions for in vivo testing. Further, microbial biodegradation test protocols, 301A, 301B, 301D and 301E, which were formerly unestablished, have been established and microbes suitable for the test protocols have been secured. Furthermore, with respect to the physiochemical characteristics analysis, methods for measuring melting points, boiling points, surface tension, and viscosity, as well as GLP analysis tests including homogeneity and stability tests of which importance are emphasized as a core technology of GLP, globally in recent years.
In this dissertation, those environmental toxicity tests unestablished so far including test substance analysis techniques were developed through a number of comparative and repetitive tests using a variety of toxicity-inducing pesticides and comparative substances. The tests were conducted in accordance with OECD guideline so that the results were drawn with reliability and reproducibility which are consistent with the global standards, and hence the established tests have a significant meaning. In other words, environmental toxicity tests that were previously undeveloped or unestablished for domestic use now became established in standardized GLP protocol model suitable for domestic use.
These developed tests are in line with global test standards, and can be utilized for the assessment of various pesticides, natural materials and environmentally toxic substances and the development of new materials and bioassays. Accordingly, it is expected that the developed tests can be widely utilized in the domestic and international industries and further development of the tests is activated through collaborative researches. In the future, domestic CRO industry should be actively promoted by developing various environmental toxicity tests, activating the domestic application and utilization of the developed techniques, and systematic fosterage of expert consultants and researchers having registration experience and skills, as well as by making it compulsory to domestically perform some test items related to toxicity assessment as the developed countries do.

목차 (Table of Contents)

• I. General introduction
• I. General introduction 1
• II. 국제적 환경규제정책 현황과 동향 및 대응 전략
• A. 서론 3
• (1) 국제적 환경규제 정책의 변화 3
• (2) 기술환경변화에 대한 외국의 대응 사례 4
• (3) 국제적 GLP 기술 동향과 전망 13
• (4) 국내 GLP 독성평가 기관의 현황과 문제점 20
• B. 본론 29
• (1) 환경독성 GLP 기관 확충의 기술적 배경 29
• (2) 선행결과의 활용방안 31
• (3) 연구개발 목표 34
• C. 결론 37
• III. 환경규제 대응을 위한 OECD 독성시험법 확립
• A. Introduction 39
• B. Materials and methods 41
• (1) Materials 41
• (2) Methods 43
• a. 유전독성시험 (SOS chromotest) 43
• a.1. Enzyme assay 43
• a.1.1. ß-galactosidase assay 43
• a.1.2. Alkaline phosphatase assay 43
• a.2. 시험 평가 방법 44
• b. 일반독성 (General toxicology) 44
• b.1. SD rat와 ICR mouse를 이용한 일반독성 시험법 44
• b.2. ICR mouse를 이용한 Limit test 46
• c. 시험물질 농도 분석 (Bioanalysis) 47
• d. 조류 (Bird) 독성 시험 47
• d.1. 섭식독성시험 (Dietary toxicity) 48
• d.2. 경구독성시험 (Oral toxicity) 48
• e. 미생물 분해성 시험 49
• e.1. 배지 49
• e.2. 접종원 51
• e.3. DOC die-away test (OECD TG 301A) 51
• e.4. CO2 evolution test (OECD TG 301B) 51
• e.5. Closed bottle test (OECD TG 301D) 52
• e.6. Modified OECD screening test (OECD TG 301E) 52
• f. 이화학적 특성 분석 53
• f.1. 녹는점 (Melting point) 및 끓는점 (Boiling point) 53
• f.2. 점도 (Viscosity) 55
• f.3. 표면장력 (Surface tension) 56
• g. 통계분석 56
• C. Results 57
• (1) 유전독성시험 (SOS chromotest) 57
• a. DBF-12080, K-56756 58
• b. DBH-129, DBI-3204, K-15403 59
• c. Cadusafos 59
• (2) 설치류 일반독성 65
• a. 임상증상 65
• b. 치사 및 체중 변화 68
• (3) 설치류 Limit test 71
• a. 임상증상 71
• b. 치사율 및 체중의 변화 71
• c. 사료 섭취량과의 상관관계 72
• d. 부검 및 병리학적 소견 72
• (4) 시험 농약물질의 분석 (Bioanalysis) 76
• (5) 조류 독성시험 79
• a. 임상증상 79
• b. 치사 79
• c. 체중, 사료 섭취량 및 부검 80
• (6) 미생물 분해성 시험 (Biodegradation test) 84
• a. 301A DOC die-away test 86
• b. CO2 evolution test 88
• c. Closed bottle test 90
• d. Modified OECD screening test 92
• (7) 이화학적 특성 분석 94
• a. 녹는점 94
• b. 끓는점 94
• c. 점도 측정 97
• d. 표면장력 측정 99
• D. Discussion 101
• 참고문헌 106
• 부록 119