Long-term Toxicity Study
? Objective
? Procedures
? Problems
?Mechanic? Innovative?
Objective
? Toxic dosage
? Safety margin
? Toxic reaction
? Target organ
? Reversibility
Procedures
Animals Housing Test article
Route Dosage and group
Administration duration Observation
Procedures 1--- animals
rodent non-rodent
rat beagle dog
mouse mongrel dog
monkey
Procedures 2---- housing
? Diet,T,humidity,light,ventilation
? Rat,≤5 /cage,separated，
acclimation ≥1w
? Dog,TB test,anthelmintic
acclimation,1-2w / 1m
Procedures 3---- test article
test article / solvent / excipient
name,batch No.,
source,specification,
purity,preserve condition,etc
same quality criterion
Procedures 4---- route
? The same as intended route
? iv ? ip (rat)
im or sc ?change injection sites
vd ? iv
? Change route if necessary
Procedures 5---- dosage and group
Low Median High Vehicle /Control
no slight or obvious solvent /excipient
toxicity median or severe normal control
?♂♀ randomized separately
?same volume,different concentration
Procedures 5---- dosage and group
rat ≤3m rat >3m non-rodent
20/group 40/group 4-6/group
?♂,?♀ ?♂,?♀ ?♂,?♀
?Limit test,LD50,ig > 5 g/kg,iv > 2g/kg,one
group,>=50ACD (rat),>=30ACD(non-rodent)
Procedures 6--- ad,duration
Tab,Correspond duration of clinical ad,with animal ad.
clinical ad,duration animal ad,duration
1～ 3d 14d
1w 1m
4w 3m
≥1m 6m
? duration ≥3m ad,6 day weekly(mon,to sat,)
Procedures 7---- Observations
Clinical Examinations
rodent non-rodent
signs of ill health +pupil,T
behavioral changes breath,sialosis
reaction to treatment vomiting
?Dead or moribund animal,necropsy within 1h
Procedures 7---- Observations
Hematology
Red blood cell count(RBC)
Hemoglobin(Hb)
Erythrocyte indices (MCV,MCH,MCHC)
Hematocrit(Hct)
Platlet count
White blood cell count and percent differential
Coagulation time(CT)
Reticulocyte count,Bone marrow smear
Procedures 7---- Observations
Clinical chemistry
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Alkaline phosphatase (ALP)
Total bilirubin（ Tbill）
Blood urea nitrogen（ BUN）
Creatinine（ Cr）
Total protein（ TP）
Albumin（ Alb）
Glucose (Glu)
Total cholesterol （ Tch）
Procedures 7---- Observations
Urinalysis
Color and appearance pH
glucose ketones
urobilinogen blood
specific gravity nitrite
protein bilirubin
WBC
microscopy of centrifuged deposit
Procedures 7---- Observations
Pathology
Organ weight assessment:
heart liver thyroid lobes and
spleen lung parathyroids
kidneys brain (lobes weighed
pituitary adrenals together)
ovaries testes
? paired organs weigh together,calculate organ
weights relative to body weight
Procedures 7---- Observations
Pathology
Histopathological examination:
heart liver spleen lung
kidney brain adrenals pancreas
stomach duodenum jejunum ileum
colon cecum pituitary thymus
spinal cord prostate testes uterus
ovaries optic nerve injection site
bone and marrow(sternum) urinary bladder
lymph nodes(mesenteric)
thyroid lobes( and parathyroids)
Procedures 7---- Observations
Pathology
tissues
(c+h embedding
dead+ sectioning
target) staining
examination
Procedures 7---- Observations
Cardiovascular studies
? ECG:Limb lead II for non-rodents
? Parameters,heart rate,voltage of P
wave,P-R interval,voltage of R wave,
QRS time,ST segment,voltage of T
wave,Q-T interval
Procedures 7---- Observations
Recovery
? Observation, 1/3～ 1/2 animal,2～ 4w
? ?Late toxic reaction and reversibility
? Items, same as treatment period
Procedures 7---- Observations
Frequency of lab,investigations
rat<=3m rat > 3m Non-rodent
last ad,interim twice pretreatment
recovery end last ad,once every 1-1.5m
recovery end last ad.
recovery end
Problems
Pretest Dosage
Animal & Housing Route
Duration Observation
Parameters Data handling
Archives
Problems 1--- pretest
? What depend on?
?Overseas pre-clinical / clinical data
?Acute toxicity,pharmacodynamics
and pharmacokinetics or intended
clinical dosage
? How long? 2w -- 1m
? Recombinant article,antibody?
Problems 2--- dosage
LD50 MTD MBS ACD
3m 6m
1/10 1 10Cmax 50～ 100 30～ 50
1/50 1/3 5Cmax 30～ 50 15～ 25
1/100 1/10 Cmax 10～ 20 5～ 10
? half for dog
Problems 3--- animal
rodent non-rodent
Wistar Beagle dog(4-12m)
Sprague Dewley mongrel dog (<5y)
6-8w monkey (<3y)
body weight CV<20%
?Duration ≥6m,natural death ( rat,1y-15%,
1.5y-50%,2y-70-80%)
Problems 3--- housing
main factor animal speciesmice,rat guinea pig & rabbit dog
T(℃ ) 20-26 18-28 18-28
humidity 40-60% 40-60% 40-60%
ventilation 10-15 /h 10-15/h 10-15 /h
airflow velocity 13-18cm/s 13-18cm/s 13-18cm/s
pressure positive positive negative
clean degree 100000 grade 100000 grade
falling bacteria ＜＝ 3 / vessel ＜＝ 3 / vessel
light 150-300 Lux 150-300 Lux
noise ＜ 60 db ＜ 60 db
odor amonia＜ 20 ppm amonia＜ 20 ppm
Problems 4--- route
? Intended clinical ad,route
? iv,pH,stimulation,osmotic pressure?
prevent local damage or necrosis
? Rat iv ? ip
im or sc injection ?change injection sites
? vd,?iv
? Change route if necessary
Problems 5--- duration
Western Chinese Recombinant
3～ 4 times ≥2 times antibody?
≤6m
?everyday or 6 days a week?
Problems 6--- observations
Western Chinese Recombinant
3～ 4 times ≥2 times antibody
≤6m
Toxic reaction,properties,degree,dose-toxicity
relationship,initiating time,peak time,lasted
time,late reaction toxicity,accumulating toxi-
city,tolerance
Problems 7--- parameters
Possible Toxic Reactions
pharmaco- chemical pretesting published
logical effcts structure results data
Problems 7--- parameters
? Overloading
? Reticulocyte count
? AST ALP BUN Alb
Problems 7--- parameters
? Tissue,free of fat
? half rationed：
no changes(-) 0,suspected （ ± ） 0.5，
mild（＋）１,median（ ++）２，
severe （ +++)３
? Local inflammation,WBC infiltration,local
bleeding,haemostasis,suspected cell
degeneration? self-infected,sacrifice induced
Problems 8--- data handling
? Ration data,x± s,international unit
? vs control and dosed group,vs d0
? Statistics,t test or F test,Ridit analysis
? P<0.05,significance?
Problems 9--- archives
? raw data,documentation,records,
? protocol (and amendments thereto),
? specimens,reserve samples,
? control article,
? final report(signed)
Innovative
Animal Dosage
Parameters
Route Data handling
?确实做好预试 pretest
?周密设计剂量 dosage
?精心饲养动物 housing
?细心观察反应 observation
?认真测定指标 examination
?实事求是总结 conclusion
Summary
? Importance
? 5 objectives
? Good references
? 9 problems
? Standard & innovative
? Objective
? Procedures
? Problems
?Mechanic? Innovative?
Objective
? Toxic dosage
? Safety margin
? Toxic reaction
? Target organ
? Reversibility
Procedures
Animals Housing Test article
Route Dosage and group
Administration duration Observation
Procedures 1--- animals
rodent non-rodent
rat beagle dog
mouse mongrel dog
monkey
Procedures 2---- housing
? Diet,T,humidity,light,ventilation
? Rat,≤5 /cage,separated，
acclimation ≥1w
? Dog,TB test,anthelmintic
acclimation,1-2w / 1m
Procedures 3---- test article
test article / solvent / excipient
name,batch No.,
source,specification,
purity,preserve condition,etc
same quality criterion
Procedures 4---- route
? The same as intended route
? iv ? ip (rat)
im or sc ?change injection sites
vd ? iv
? Change route if necessary
Procedures 5---- dosage and group
Low Median High Vehicle /Control
no slight or obvious solvent /excipient
toxicity median or severe normal control
?♂♀ randomized separately
?same volume,different concentration
Procedures 5---- dosage and group
rat ≤3m rat >3m non-rodent
20/group 40/group 4-6/group
?♂,?♀ ?♂,?♀ ?♂,?♀
?Limit test,LD50,ig > 5 g/kg,iv > 2g/kg,one
group,>=50ACD (rat),>=30ACD(non-rodent)
Procedures 6--- ad,duration
Tab,Correspond duration of clinical ad,with animal ad.
clinical ad,duration animal ad,duration
1～ 3d 14d
1w 1m
4w 3m
≥1m 6m
? duration ≥3m ad,6 day weekly(mon,to sat,)
Procedures 7---- Observations
Clinical Examinations
rodent non-rodent
signs of ill health +pupil,T
behavioral changes breath,sialosis
reaction to treatment vomiting
?Dead or moribund animal,necropsy within 1h
Procedures 7---- Observations
Hematology
Red blood cell count(RBC)
Hemoglobin(Hb)
Erythrocyte indices (MCV,MCH,MCHC)
Hematocrit(Hct)
Platlet count
White blood cell count and percent differential
Coagulation time(CT)
Reticulocyte count,Bone marrow smear
Procedures 7---- Observations
Clinical chemistry
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Alkaline phosphatase (ALP)
Total bilirubin（ Tbill）
Blood urea nitrogen（ BUN）
Creatinine（ Cr）
Total protein（ TP）
Albumin（ Alb）
Glucose (Glu)
Total cholesterol （ Tch）
Procedures 7---- Observations
Urinalysis
Color and appearance pH
glucose ketones
urobilinogen blood
specific gravity nitrite
protein bilirubin
WBC
microscopy of centrifuged deposit
Procedures 7---- Observations
Pathology
Organ weight assessment:
heart liver thyroid lobes and
spleen lung parathyroids
kidneys brain (lobes weighed
pituitary adrenals together)
ovaries testes
? paired organs weigh together,calculate organ
weights relative to body weight
Procedures 7---- Observations
Pathology
Histopathological examination:
heart liver spleen lung
kidney brain adrenals pancreas
stomach duodenum jejunum ileum
colon cecum pituitary thymus
spinal cord prostate testes uterus
ovaries optic nerve injection site
bone and marrow(sternum) urinary bladder
lymph nodes(mesenteric)
thyroid lobes( and parathyroids)
Procedures 7---- Observations
Pathology
tissues
(c+h embedding
dead+ sectioning
target) staining
examination
Procedures 7---- Observations
Cardiovascular studies
? ECG:Limb lead II for non-rodents
? Parameters,heart rate,voltage of P
wave,P-R interval,voltage of R wave,
QRS time,ST segment,voltage of T
wave,Q-T interval
Procedures 7---- Observations
Recovery
? Observation, 1/3～ 1/2 animal,2～ 4w
? ?Late toxic reaction and reversibility
? Items, same as treatment period
Procedures 7---- Observations
Frequency of lab,investigations
rat<=3m rat > 3m Non-rodent
last ad,interim twice pretreatment
recovery end last ad,once every 1-1.5m
recovery end last ad.
recovery end
Problems
Pretest Dosage
Animal & Housing Route
Duration Observation
Parameters Data handling
Archives
Problems 1--- pretest
? What depend on?
?Overseas pre-clinical / clinical data
?Acute toxicity,pharmacodynamics
and pharmacokinetics or intended
clinical dosage
? How long? 2w -- 1m
? Recombinant article,antibody?
Problems 2--- dosage
LD50 MTD MBS ACD
3m 6m
1/10 1 10Cmax 50～ 100 30～ 50
1/50 1/3 5Cmax 30～ 50 15～ 25
1/100 1/10 Cmax 10～ 20 5～ 10
? half for dog
Problems 3--- animal
rodent non-rodent
Wistar Beagle dog(4-12m)
Sprague Dewley mongrel dog (<5y)
6-8w monkey (<3y)
body weight CV<20%
?Duration ≥6m,natural death ( rat,1y-15%,
1.5y-50%,2y-70-80%)
Problems 3--- housing
main factor animal speciesmice,rat guinea pig & rabbit dog
T(℃ ) 20-26 18-28 18-28
humidity 40-60% 40-60% 40-60%
ventilation 10-15 /h 10-15/h 10-15 /h
airflow velocity 13-18cm/s 13-18cm/s 13-18cm/s
pressure positive positive negative
clean degree 100000 grade 100000 grade
falling bacteria ＜＝ 3 / vessel ＜＝ 3 / vessel
light 150-300 Lux 150-300 Lux
noise ＜ 60 db ＜ 60 db
odor amonia＜ 20 ppm amonia＜ 20 ppm
Problems 4--- route
? Intended clinical ad,route
? iv,pH,stimulation,osmotic pressure?
prevent local damage or necrosis
? Rat iv ? ip
im or sc injection ?change injection sites
? vd,?iv
? Change route if necessary
Problems 5--- duration
Western Chinese Recombinant
3～ 4 times ≥2 times antibody?
≤6m
?everyday or 6 days a week?
Problems 6--- observations
Western Chinese Recombinant
3～ 4 times ≥2 times antibody
≤6m
Toxic reaction,properties,degree,dose-toxicity
relationship,initiating time,peak time,lasted
time,late reaction toxicity,accumulating toxi-
city,tolerance
Problems 7--- parameters
Possible Toxic Reactions
pharmaco- chemical pretesting published
logical effcts structure results data
Problems 7--- parameters
? Overloading
? Reticulocyte count
? AST ALP BUN Alb
Problems 7--- parameters
? Tissue,free of fat
? half rationed：
no changes(-) 0,suspected （ ± ） 0.5，
mild（＋）１,median（ ++）２，
severe （ +++)３
? Local inflammation,WBC infiltration,local
bleeding,haemostasis,suspected cell
degeneration? self-infected,sacrifice induced
Problems 8--- data handling
? Ration data,x± s,international unit
? vs control and dosed group,vs d0
? Statistics,t test or F test,Ridit analysis
? P<0.05,significance?
Problems 9--- archives
? raw data,documentation,records,
? protocol (and amendments thereto),
? specimens,reserve samples,
? control article,
? final report(signed)
Innovative
Animal Dosage
Parameters
Route Data handling
?确实做好预试 pretest
?周密设计剂量 dosage
?精心饲养动物 housing
?细心观察反应 observation
?认真测定指标 examination
?实事求是总结 conclusion
Summary
? Importance
? 5 objectives
? Good references
? 9 problems
? Standard & innovative
