식품의약품 등의 후세대영향 연구방법론 국립독성연구원 곽 승 준

목차 후세대영향 연구 필요성 국내외 현황 KFDA 후세대영향 연구사업 KFDA 생식발생독성시험 가이드라인 US NTP CERHR RACB 일본 NIHS - BSRC KFDA 후세대영향 연구사업

후세대영향 연구 필요성 식품의약품 산업의 발달 시장개방의 확대 환경오염의 증가 새로운 개념의 식품의약품 신종 유해가능물질 (GMO, 건강기능식품, 바이오의약품 등) 신종 유해가능물질 (신종 마약류, 식품첨가물, 산업환경물질 등)

“식의약품 등의 후세대영향 연구사업” 추진 기존 생식발생독성연구의 문제점 단일세대 또는 차세대에 한정 유전적 요인에 대한 연구 부족 체계적인 DB 관리 부족 국가적 차원의 연구 관리 필요 “식의약품 등의 후세대영향 연구사업” 추진

국내외 현황 KFDA 생식발생독성시험 가이드라인 식품의약품안전청고시 제1999-61호 의약품등의 독성시험기준 제 5조 표준시험법 (The most probable option) 수태능 및 초기배 발생시험 출생 전후 발생 및 모체기능시험 배태자 발생시험 단일시험법 (Single study design) 조합시험법 (Two study design)

US FDA 가이드라인 ICH 가이드라인 Stage A - Premating to Conception Segment I - Fertility and General Reproductive Performance Segment II - Embryo-Fetal Development (“Teratology”) Segment III - Perinatal/Postnatal Evaluation ICH 가이드라인 Stage A - Premating to Conception Stage B - Conception to Implantation Stage C - Implantation to Closure of the Hard Palate Stage D - Closure of the Hard Palate to the End of Pregnancy Stage E - Birth to Weaning Stage F - Weaning to Sexual Maturity

KFDA 가이드라인 평가항목 일반증상 및 사망여부, 체중 및 체중변화량, 사료섭취량 및 음수량 부검 (육안적 관찰) - 육안적 변화시 병리조직검사 황체수, 착상수, 생존배(태)자수, 사망배(태)자수 필요시 정자검사 임신기간 및 분만상태 태자 형태이상, 출산자 및 사산자의 상태 (체중 등) 이유전, 이유후의 생존율, 성장/체중, 성숙 및 수태능 신체적 발달, 감각기능 및 반사, 행동이상 태반의 육안적 관찰

Center for the Evaluation of Risks to Human Reproduction (CERHR) The National Toxicology Program (NTP) Center for the Evaluation of Risks to Human Reproduction (CERHR) http://cerhr.niehs.nih.gov/

CERHR 1998년 NTP와 NIEHS에서 생식발생독성 전문평가센터로 설립 1992년 전쟁후유증 (출산이상 등)을 호소하는 걸프전 참전 군인에 의해 문제 발단 인체노출가능물질이 인간의 생식발생에 미치는 유해영향가능성을 평가 평가결과를 정부규제기관, 관련 연구단체, 일반국민 등에 제공하여 국민보건 향상 및 국가적 차원의 예방

Center Organization

Core Committee (CC) - 구성 CERHR 자문위원회 : NTP 집행위원회 소속 위원들로 구성 Environmental Protection Agency (EPA) Food and Drug Administration (FDA) National Institute of Occupational Safety and Health (NIOSH) /Centers for Disease Control and Prevention (CDC) National Center on Birth Defects and Developmental Disabilities (NCBDDD)/CDC National Center for Environmental Health (NCEH)/CDC US Consumer Product Safety Commission (CPCS) National Institute of Environmental Health Sciences (NIEHS)

Core Committee (CC) - 기능 CERHR의 화학물질 평가 등 동 센터 업무 자문 평가대상으로 접수된 화학물질 검토 및 추천 추천된 평가대상물질에 대한 예비검토 및 최종물질 추천 CERHR Expert Registry의 과학자 검토 및 Expert Panel Members 추천 Expert Panel Report 등 CERHR의 각종 보고서 검토

Expert Panels - 구성 각 전문분야별로 화학물질을 평가하는 독립적인 전문가 그룹 CERHR은 화학물질의 생식발생독성 평가시 CC의 자문을 받아 총 10~15명의 전문가로 Expert Panels을 구성 CERHR은 효율적인 Expert Panels 운영을 위해 Expert Registry를 구축 현재 CERHR의 Expert Registry에는 전세계에서 추천된 240여명의 전문가가 등록되어 있음

Expert Panels - 기능 평가대상으로 접수된 화학물질 접수 및 검토 평가대상물질에 대한 생식발생독성 평가자료 작성 Preliminary or full dossiers, draft reports 평가 결과에 대한 Expert Panel Report 작성 및 배포 최종 평가결과 보고서 (NTP-CERHR Monograph) 작성, 발간 및 배포 Workshop 개최 및 각종 제반 사항에 대한 자문

Chemical Evaluation Process

CERHR Reports & Monographs

CERHR Reports & Monographs

CERHR Reports & Monographs

Reproductive Assessment by Continuous Breeding (RACB) The National Toxicology Program (NTP) Reproductive Assessment by Continuous Breeding (RACB) Study Design Task 1 : Dose-range-finding (DRF) portion of an RACB study Task 2 : Main portion of an RACB study Task 3 : Crossover mating trial Task 4 : Evaluation of the second generation

Task 1 : Dose-range-finding (DRF) portion of an RACB study End points : B.W. and food and water consumption In early study be performed for 2 weeks focused exclusively on B.W. and food and water consumption for 5 to 8 animals at each of five dose levels, and controls selected compounds were reproductive toxicants at exposure level all dose levels so high Modified 4-week Task 1 consisting of a 1-week exposure followed by a 3-week cohabitation and exposure period, and birth of the pups more data on weights and consumptions (which can change as the animals acclimate to the exposure) litter data at delivery has proven quite useful

Task 2 : Main portion of an RACB study Animal mice : 10 to 12 weeks old control and three dose levels are used, with 20 male and 20 female per dose level In 14 weeks exposure begins 1 week prior to cohabitation (to allow for any effects on ovulation or sperm motility to manifest) housed as breeding pairs for approximately 14 weeks (continuous chemical exposure, litters are produced approximately 3 to 4 weeks apart) data collected on each litter (day of delivery, number of male pups, number of female pups, aggregate weight of each sex, and number of dead pups) pups are killed; dam enters a postpartum estrous; pregnancy cycle begins anew normally, 4 to 5 litters are delivered adult body weight are taken after each litter female : to avoid confounding effects of pregnancy male : at selected intervals throughout the study

Task 2 : Main portion of an RACB study (continued) After 14 weeks the pair is separated for 6 weeks (delivers and nurses to weaning any last litter) during this time, the litter and B.W. data are summarized and sent to the NTP project officer (PO), who evaluates a adverse effect on reproduction In the presence or absence of reproductive toxicity last litter is nursed by the dam and weaned at postnatal day 21 pups are counted and weighed at this period toxicities presenting during this period late expression of gestational effects lactational transfer of compound or active metabolite reflect compromised milk quality

Task 3 : Crossover mating trial Sex has been affected by treatment be performed after the last litter from Task 2 has been weaned at postnatal day 21 be performed with single exposed group (often, the high dose), and control three groups are formed control males + treated females treated males + control females control + control to obtain 20 pairs in each group, 40 control pairs are needed animals are cohabited for a week without being exposed to the test compound females are subject to vaginal lavage each day, to check for sperm animals are separated when the female is sperm positive or after 1 week delivered litter is evaluated as above females are lavaged again after delivery and resumption of cyclicity to assess the nature of the cycle (normal, altered) Adult F1 animals are then killed and subject to necropsy histopathology is performed at the discretion of the PO

Task 4 : Evaluation of the second generation Exposure to the test compound starts at weaning each pup receiving the same exposure levels as that given its parents B.W. are collected at several times during the growth phase to adulthood when the animals are approximately 74 (mice) or 80 (rats) days of age, they are cohabitated within treatment groups (but avoiding sibling matings) for a week as in Task 3 ………..

End points for a current RACB study Task 1 Feed and water consumptions Every 3-4 days Bodyweights Bodyweight gains Clinical signs Task 2 Feed and water consumptions during weeks of noncohabitation Pregnancy index by litter and dose Average number litters/pair Average number live pups/litter (male, female, combined) Proportion of pup weight (by sex, and combined) Pup weight adjusted for litter size (by sex, and combined) Cumulative days to deliver each litter Adult body weights at delivery of each litter Feed and water consumption during lactation Task 3 Mating index (% sperm positive female) Pregnancy index (% fertile pairs of cohabited) Fertility index (number of fertile pairs per number sperm-positive females) Number live pups per litter Proportion of pups born alive Sex ratio of liveborn pups Absolute pup weight (by sex and combined) Average dam weight postpartum Average sire weight postpartum Average days to delivery Task 3 necropsy (performed in the presence of reproductive toxicity) Preterminal vaginal cytology for 12 days Terminal B.W. Gross observation of all organs and body cavities Liver & kidney weight (absolute and relative) Weights of other target organs

End points for a current RACB study (continued) Reproductive organ weight Testicular spermatid head count (expressed per mg tissue and per testis) Cauda epididymal sperm count Sperm motility by CASA Sperm morphology Histology as appropriate Task 2 necropsy (performed in the absence of reproductive toxicity) Terminal B.W. Gross observation of all organs and body cavity Task 4 F1 pup B.W. to weaning F1 pup viability to weaning Mating index (% sperm positive female) Pregnancy index (% fertile pairs of cohabited) Fertility index (number of fertile pairs per number sperm-positive females) Number live pups per litter Proportion of pups born alive Sex ratio of liveborn pups Absolute pup weight (by sex and combined) Average dam weight postpartum Average sire weight postpartum Feed and water consumption Task 4 necropsy as for Task 3 necropsy

Integration with other tests Neurotoxicity : rotarod, grip strength, etc. Measure of dominant lethality (DL) : in Task 2, using a uninvolved males Sperm Chromatin Structure Assay (SCSA) measures alterations in chromatin structure (relative abundance of single-stranded DNA vs double-stranded DNA) Sperm morphometry measures of sperm head shape as opposed to shape classification)

National Institute of Health Science 國立醫藥品食品衛生硏究所 National Institute of Health Science (NIHS) http://www.nihs.go.jp/

NIHS – BSRC 1960년대 다양한 화학물질들의 인체 위해성을 평가하기 위해 Biological Safety Research Center (BSRC) 설립 5 division 구성 (Toxicology, Pharmacology, Pathology, Genetics & Mutagenesis, Risk Assessment) Division of Toxicology에서 인체의 생식이상발생과 관련한 연구 진행 (기형유발, 발생유전자 이상 등)

KFDA 후세대영향 연구사업 식의약품 등의 후세대영향 연구사업 사업목적 후세대 유해가능물질에 대한 과학적인 독성영향 평가 후세대 유해가능물질 평가를 위한 제반 관련 기술 개발 정부기관, 관련단체, 일반국민에게 평가결과 및 관련정보를 제공 후세대 유해영향물질에 대한 규제 근거 확보 및 국가적 차원의 예방

식의약품 등의 후세대영향 연구사업 사업내용 후세대 유해가능물질에 대한 국내·외 정보수집 및 분석 후세대영향 독성평가기술 개발 및 확립 후세대 유해가능물질에 대한 동물 및 인체 모니터링 실시 및 관련 기초조사 연구 후세대 유해영향물질 데이타베이스 구축 국제학술심포지움 개최 등 국제협력 네트워크 구축 후세대 유해영향물질에 관한 단기기술연수 후세대 유해영향물질 관련 자료집 발간

사업추진절차 정책반영 식의약품 등의 후세대영향 연구사업 국민보건 향상 국가기술경쟁력 확보 새로운 식품·의약품 등 개발지원 및 사업계획 연구사업 방향 설정 연구사업 수행부서 구성 예산확보 시험연구 및 평가 독성연구 평가기술연구 모니터링실시 DB 구축 평가모델 개발 연구기반 구축 정보수집 국제협력연구 전문인력 확보 용역연구과제 식의약품 등의 후세대영향 연구사업 식품의약품안전청 국립독성연구원 정책반영 국민보건 향상 국가기술경쟁력 확보 새로운 식품·의약품 등 개발지원 및 규제근거 마련

식의약품 등의 후세대영향 연구사업 2005년도 수행 연구사업 자체사업 용역사업 엑스타시(MDMA)의 후세대에 미치는 영향 연구 엑스타시의 생식발생독성 자료 확보 용역사업 후세대영향 연구사업의 발전방안 및 연구방법론에 관한 연구 중장기 발전방안 연구 연구 대상물질 선정 기준 제시 후세대영향 안전성평가기술 개발 (I) : TRC 유전자 발현 관련 유전자 네트워크 신호전달 체계

식의약품 등의 후세대영향 연구사업 향후 우선대상물질 신종 마약류 기호식품류 생(한)약재류 MDMA (Ecstasy) 등 알코올 카페인 니코틴 생(한)약재류 육두구, 산조인, 인삼성분 등

3,4-Methylenedioxymethamphetamine (MDMA) Synonyms : Ecstasy, Adam, X, XTC Molecular formula : C11H15NO2 Molecular weight : 193.25

MDMA - Basics A standard oral dose : 80 - 150mg A single tablet bought at a rave can cost as much as $50 though more commonly $10-$25 MDMA is illegal in the USA and in most other countries MDMA generally takes 30-60 minutes (although sometimes as long as 2 hours) The primary effects of MDMA last approximately 3-4 hours when taken orally

MDMA - History MDMA was first synthesized and patented by Merck pharmaceuticals in 1912 and patented in 1914 (slimming pill) But it wasn’t until the mid 1970s that articles related to its psychoactivity began showing up in scholarly journals In the late ’70s and early ’80s MDMA was used as a psychotherapeutic tool and also started to become available on the street Its growing popularity led to it being made illegal in the USA in 1985

MDMA - Effects The primary effects are emotional openness, euphoria, stimulation, reduction of critical and cynical thoughts, and decrease of inhibitions Negative effects include overheating, nausea, vomiting, jaw-clenching, eye-twitching, and dizziness, as well as depression and fatigue in following days There have been problems with MDMA users experiencing : dehydration, hyperthemia, hyponatremia, exhaustion, blackouts, and few cases of death generally while using MDMA at clubs or raves MDMA has the potential to be psychologically addicting

후세대영향 안전성평가기술 개발 (일반, 생식, 유전, 면역, 독성병리 등) 연구사업 추진 로드맵 단기 (2005～2007년) 중기 (2008～2010년) 장기 (2011～2014년)   후세대영향 안전성평가기술 개발 (일반, 생식, 유전, 면역, 독성병리 등) 유전체 관련 연구 단백체 관련 연구 독성동태동력 관련 연구 안전관리 대책 수립을 위한 기초조사 연구 유해가능물질의 모니터링 연구 국제협력 네트워크 구축 안전성평가 데이터베이스 구축 후세대영향 연구평가센타 설립 후세대 유해물질 관리체계 구축

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