Guideline No: 【H 】G P T 4 – 1

Technical Guideline on Irritation, Sensitization

and Hemolysis

Study for Chemical Drugs

(Second draft)March 1st , 2004Table of Contents1

Introduction.............................................................................................

................................3

2 Overall

principles.................................................................................................

....................3

3 Irritation

test............................................................................................................

..................4

3.1 Definition..................................................................................................................................

4

3.2 Objective...................................................................................................................................

4

3.3 Considerations in irritation test design

........................................................................................ 4

3.4 Skin irritation test

....................................................................................................................... 5

3.5 Irritation test of injection

site...................................................................................................... 5

3.6 Ocular irritation

test.................................................................................................................... 5

3.7 Irritation test for other routes at the administration site

.............................................................. 5

4 Sensitization

test ..........................................................................................................

.............5

4.1 Definition..................................................................................................................................

5

4.2 Objective...................................................................................................................................

6

4.3 Considerations in sensitization test

design................................................................................... 6

4.4 Sensitization test of transdermal administration

.......................................................................... 6

4.5 Sensitization test of injection administration

............................................................................... 6

4.6 Sensitization test of other administration

routes.......................................................................... 7

4.7 Test method

...............................................................................................................................7

5 Hemolysis

test ..........................................................................................................

................7

5.1 Definition..................................................................................................................................

7

5.2 Objective...................................................................................................................................

7

5.3 Scope.........................................................................................................................................

7

5.4 Considerations in hemolysis test design

...................................................................................... 7

5.5 Test method

...............................................................................................................................8

6 Test

report.......................................................................................................

.........................8

7 Evaluation of

results......................................................................................................

...........8

8 Some

considerations ........................................................................................

.........................8

9

References...............................................................................................

.................................8

10

Appendix..................................................................................................

................................9

10.1 Method for irritation test

.......................................................................................................... 9

10.1.1 Skin irritation

test........................................................................................................... 9

10.1.2 Irritation test to injection site

............................................................................................... 11

10.1.3 Eye irritation

test.................................................................................................................. 11

10.2 Method for sensitization

test................................................................................................... 14

10.2.1 Passive cutaneous anaphylaxis

test(PCA)...................................................................... 14

10.2.2 Active systemic anaphylaxis test

(ASA)......................................................................... 14

10.2.3 Guinea pig maximization test（GPMT）and Buehler test（BT） .............................. 15

10.2.4 Skin phototoxicity test..................................................................................................

17

10.2.5 Skin photoallergy test

................................................................................................... 18

10.3 Method for hemolysis

test....................................................................................................... 19

10.3.1 general in vitro tube method (observation with

eyes).................................................... 19

10.3.2 Improved in vitro hemolysis test method(spectrophotometer)

..................................... 20

11 Drafting

notes .......................................................................................................

.................. 21

12 The

author .....................................................................................................

......................... 23

1 Introduction1.1 Definition and ObjectiveThe irritation, sensitization and hemolysis of chemicals is defined as the production oftoxicity at the local site of administration, eg, irritation and sensitization, etc, and/or thesystemic toxicity, eg, sensitization and hemolysis following the unoral application of achemical drug products to eye, ear, nose, mouth, respiratory tracts, joint cavity, derma, recta,vagina, vein, artery, muscle, undermic, vein besides, theca, and so on. It is a part ofpreclinical safety evaluations.

The drug’s active substances and its metabolites, excipient，some

relative components andphysical-chemical properties (PH, osmotic Pressure, etc) are all lead to irritation and/orsensitization/or hemolysis, and serious toxicity may affect safety and efficacy. As a result, it

is necessary to study the local and /or systemic toxicity of the drug product after beingapplied, to suggest the possible toxicity, target organ of toxicity, safety range and parametersof clinical monitoring, further, give reference to clinical detoxifcation or rescuing methodsand guarantee safety and efficacy of the drug in clinic.1.2 ScopeThe Guideline is applied to the study of irritation, sensitization and hemolysis of chemicals.1.3 The main contentThe Guideline’s main content includes the overall/ general principles and commonly usedtest methods of study on irritation, sensitization and hemolysis of chemicals.2 Overall principlesThe study of irritation, sensitization and hemolysis must comply with GLP Nonclinical StudyGood PracticesThe experiment design should adhere to randomized, control, repetitive principles.The study of irritation, sensitization and hemolysis should take account of toxicitymechanism, influent factors, clinical significance, indication and administration method.Selection of relevant animal species should be based on proposed experimental model andobservational parameters. Gender, age, and physiological state should meet the experimentrequirements. The experimental drug should be identical with those applied to human body.The drug delivery, including dose, route and treatment regimen, is determined by experimentmodel and proposed clinical practice. Applying place is expected to be the observationalplace, and peripheral tissue or exposure place should also be considered to be observed atthe same time. If the test substance that will be investigated in clinic is identical or analogouswith other non-clinical safety study, eg, long-term toxicity, and the findings can reflect oneof the toxicity among irritation, sensitization and hemolysis, the corresponding toxicity studymay be omitted.

To study the irritation, sensitization and hemolysis of the chemical, it is best to make clear of

components of the test substance，including the names and

amount of principal drug，

excipient，impurity. If marketed drug product which have the same

route of administrationwith the test substance indicate certain toxicity in the research of irritation and/orsensitization/or hemolysis, it is necessary to conduct compare study, and the toxicity shouldbe equivalent or less compared with the existing ones. The sample size is determined bybasic statistics theory. Due to the limits of animal experiments, irritation, sensitization andhemolysis of the chemical shall be integrally evaluated with pharmacy, pharmacology,toxicology and clinical useto provide the possibility of clinical use of the chemical andconsiderations when used in clinic.3 Irritation test3.1 DefinitionIrritation test means reversible changes at administration site after non-oral drug productdelivery, irreversible tissue damage is named as corrosion.3.2 ObjectiveObservation on irritation after animal locally exposure to the test substance, suggestion onpossible inflammation reaction, denaturalization and necrosis at the administration site whenclinical use.3.3 Considerations in irritation test design3.3.1 Selection of animalThe selection of animal should be based on the observational parameters and the plannedanimal model, in general, the estimation of each experiment is expected to select one speciesof animal.3.3.2 Frequency and duration of administrationThe frequency and duration of administration should be deicide according to proposed

clinical use. Repeated –dose test is conducted everyday, duration is less than 4 weeks. Thetest substance which is administrated casually can be studied by single-dose test.3.3.3 Reversible observationTo investigate the recover after toxicity reaction, convalescence observation after stopping

administration is recommended。Reactions in local and relative

places should be included inreversible evaluation of the toxicity reaction.3.3.4 Test substanceThe test substance should be consistent with that of human body.3.3.5 Control groupThe negative control is adopted menstruum and/or excipient. Active control or marketeddrug preparation shall be added if necessary.3.3.6 DoseDose design shall mainly consider concentration and total dose of the test substance. Ingeneral, one of the concentration should be consistent with that in clinic, and dose can bemodified through changing the frequency of administration. However, it is not feasible toincrease plaster thickness or extend plaster area to increase dose in skin irritation study.Irritation tests of different concentrations should be conducted where necessary.3.3.7 Manner of delivery3.3.7.1 The treatment regimen should be as close as possible to that for clinical use.3.3.7.2 The route and site of administration should be consistent with clinical use.3.3.7.3 Anatomical and physiological characters at the administration site should be takeninto account when design volume, velocity and frequency of administration.3.3.8 Irritation tests of different administration routes can be performed in the same animalas long as no interaction among different routes and the systemic tolerance permitted. Thecontrol test may also be conducted at opposite side.3.3.9 Data Statistic

Appropriate statistical methods should be selected on the basis of models and experimentalmethods.3.4 Skin irritation test3.4.1 Skin irritation test should be performed in terms of transdermally applied drug andnonoral drug product to which skin possibily expose.3.4.2 When skin irritation has been evaluated in acute toxicity test, long term toxicity test orskin sensitization test, and the preparation of the test substance is identical or comparablewith that of the proposed clinical study, a separate skin irritation test may not perform.3.4.3 Test methodSee the commonly used methods and related literature for irritation test in appendix.3.5 Irritation test of injection site3.5.1 For injection, irritation test at injection site should be considered.3.5.2 When irritation at injection site has been evaluated in acute toxicity test, long termtoxicity test, and the formulation of the test drug is identical or comparable with that of theproposed clinical study, a separate skin irritation test at injection site may not be performed.3.5.3 Test methodSee the commonly used methods and related paper for irritation test in appendix.3.6 Ocular irritation test3.6.1 Ocular drugs and the drugs to which eyes are possible exposed should be considered toperform ocular irritation test.3.6.2 When ocular irritation test has been evaluated in acute toxicity test, long term toxicitytest, and the formulation of the test substance is identical or comparable with that of theproposed clinical study, there is no need to perform a separate ocular irritation test.3.6.3 Experimental methodSee the commonly used methods and related paper for irritation test in appendix.3.7 Irritation test for other routes at the administration siteThis sector shall refer to the irritation test on skin, injection site and ocular, under the

guidance presented in overall principals and irritation test design considerations.4 Sensitization test4.1 DefinitionSensitization in this Guideline is synonymous with allergy or hypersensitivity, refers toincreased reactivity to an antigen to which a person (or animal) has been previously exposed,with the specific immunoreaction presenting tissue damage and or physiological disfunction, .It is a kind of abnormal and pathological immunoreaction, for which the most commoncategories are Type I (immediate or rapid) allergy, which is mediated by IgE antibodies,represent urticaria allergic shock, bronchial asthma , allergic coryza, allergy of gastraenterictract; Type II (cytotoxic or cytolytic ), which is mediated by IgG antibodies , representhaemolytic anemia with positive in Coombs’test, agranulocytosis and hematoblastic purpura;Type III(immune complex initiated hypersensitivity or vasculitis ), is mediated by IgG, IgM,represent limited pneumonia, vasculitis, lupus-like response,

glomerular nephritis，etc; Type

IV (delayed hypersensitivity or tuberculin hypersensitivity) is mediated by T lymphocytes,represent contact dermatitis.Photosensitivity includes phototoxicity(light irritation)and photoallergy. Phototoxicity issome kind of non-immunologic response of the skin to sunlight activated by light, drug playsa role in the response directly or indirectly. Photosensitivity is an acquired, immune-mediatedresponse of the skin to sunlight, and photosensitive substances transdermal absorbed orthrough circulation reach skin, and response with absorbed light at epidermal cells layer. It isa special type of the type IV hypersensitivity.4.2 ObjectiveTo observe systemic and local hypersensitivity after the animal is exposed to the testsubstance.4.3 Considerations in sensitization test design

4.3.1 The problem that which kind of sensitization study should be performed should beidentified in conjunct with the self characters, eg. Chemical structure and so on,pharmacological and toxicological findings, especially the long term toxicity test result,indication and the delivery method.4.3.2 The selection of the specified test method is on the basis of the administration routeand the sensitization mechanism, along with the interpretation of rationality. For instance,transdermal administration should consider conduct the guinea pig maximizationtest(GPMT), Buehler test(BT); For injection administration ,the active systemicanaphylaxis(ASA) and the passive cutaneous anaphylaxis test(PCA) should be taken intoaccount; as to inhale administration, the guinea pig inhale sensitization test and irritation testshould be considered.4.3.3 Dose should be designed rationally, and the quality-efficacy relation is also concerned.Active control and passive control is needed.4.3.4 If it has been demonstrated the type of hypersensitivity in other tests, eg, long termtoxicity test, and the test substance is identical or comparable with that of the proposedclinical study, it need not perform the sensitization test separately.

4.3.5 Due to limitation of animal model, eg，there is no appropriate

animal model of type IIand type III, no clear clinical significance of photosensitive model, flexible patterns may beapplied to preclinical sensitization evaluation for some drugs with adequate arguments, andthe potential toxicity should be interpreted in detail in doctor brochure and labeling, as wellas the responding study in clinic.4.3.6 Analysis of data: Appropriate statistical methods should be selected on the basis ofmodels and test methods.4.4 Sensitization test of transdermal administrationSkin sensitization reaction should be investigated, and it is commonly used BT, GPMT or

other rational methods. If drug’s chemical structure is identical or similar with that of otherreported compounds which lead to hypersensitivity, it is appreciated to perform othersuitable methods to investigate whether the drug can cause other hypersensitivity, eg.systemic hypersensitivity.4.5 Sensitization test of injection administrationASA, PCA or other rational methods with the interpretation of rationality are commonlyused to investigate systemic hypersensitivity. If the chemical structure of the drug is identicalor similar with that of a compound reported by literatures that lead to other hypersensitivity,the suitable methods should be considered to investigate whether the drug can cause otherhypersensitivity, eg. skin sensitization reaction, etc.4.6 Sensitization test of other administration routesInhale administration drug should conduct guinea pig inhale sensitization test and irritationtest. Sensitization test of mucous membrane administration drug should consider owncharacters and be reference to transdermal administration test method. If the chemicalstructure of the drug is identical or similar with that of a compound reported by literaturesthat lead to other hypersensitivity, the suitable methods should be considered to investigatewhether the drug can cause other hypersensitivity, eg. photosensitization reaction, etc.4.7 Test methodSee the commonly used methods and related papers for sensitization test in appendix.5 Hemolysis test5.1 DefinitionHemolysis means inner or outer blood vessels hemolysis and hemagglutination caused bydrug products. Hemolysis consists of immunological hemolysis and

non－immunological

hemolysis as well. Immunological hemolysis is defined as the hemolysis produced by specificantibody through immune response, and it belongs to type II and type III hypersensitivity;

non-immunological hemolysis includes drug induced oxidation-caused hemolysis andhemolysis and hemagglutination resulted from changes of blood stable state by drugproduct.5.2 ObjectiveTo investigate if drug can lead to hemolysis in inner or outer blood vessels andhemagglutination reaction.5.3 ScopeAll the injection and other drug products which can lead to immunological hemolysis andnon-immunological hemolysis should be conducted hemolysis test.5.4 Considerations in hemolysis test design5.4.1 The hemolysis mechanism presents complicated, and there is no standard preclinicaltest in vivo to comprehensively assess hemolysis reaction of drug product. As a result,Hemolysis observation of the drug product, to which no marked drug has the sameadministration route, is strongly recommended in long term toxicity study. The relative

parameters and body sign, (eg. reticulocyte，erythrocyte number,

bilirubin, urine protein,nephritis and spleen gore and so on) if hemolysis occurs, study should be furthered.5.4.2 Injection to which the marketed drug has the same route can be evaluated hemolysisthrough general in vitro tube method. If gained a positive result, the comparative research isrecommended between the test drug product and the marketed product, and animalexperiments will be employed if necessary.5.5 Test methodSee the common methods and related literatures for hemolysis test in appendix.6 Test reportThe test report should be in compliance with GLP (Good Laboratory Practice), covering:test method, test substance, test animal, test procedure, parameter, result and resultevaluation.7 Evaluation of results

To explain the methodology, test substance and rational choice of animals and analyse thepossible impacts on the test result. To correctly analyse the test results, well understand thesignificance, justify toxicity and target organ of toxicity. To analyse the integrity of toxicitytest and estimate whether the toxicity has been fully demonstrated by the toxicity study afteradministration, and the possible reasons elicited toxicity. To identify whether the drug can beclinically used and the considerations in clinical use, systemically evaluation should be carriedout in terms of pharmacy, pharmacology, oxicology and clinical practice.8 Some considerationsRegarding the irritation, sensitization and hemolysis studies, considerations should be put inthe following aspects.8.1 Integrated and comprehensive principalDepending upon the drug’s characters, irritation, sensitization, hemolysis studies andevaluations should be conducted together with pharmacy, pharmacology and toxicology andclinical practice.8.2 Analyses case by caseAt present, there are limitations in existing test methods, so new approaches can be adoptedto provide more valuable results, consequently improve the sensitivity and veracity in thestudies of irritation, sensitization and hemolysis. However, rationality should be interpretedfor the other approaches, and specific methods and operational flow should also beexplained.9 References

（1）FDA Guideline for Industry Skin irritation and sensitization

testing of generictransdermal drug products

（2）FDA Guideline for Industry Photosafety testing

（3）FDA Guideline for Industry Immunotoxicology evaluation of

investigation new drug

（4）EPA Health effects test guidelines OPPTS 870.7800

Immunotoxicity

（5）EPA Health effects test guidelines OPPTS 870.2500 Acute

dermalirritation

（6）EPA Health effects test guidelines OPPTS 870.2600 Skin

sensitization

（7）EPA Health effects test guidelines OPPTS 870.2400 Acute eye

irritation

（8）EMEA Non-clinical local tolerance testing of medicinal

products

（9）ISO 10993-10:2002(E) Biological evaluation of medical

devices-Part 10- Tests for irritation and delayed-type hypersensitivity

（10）Office of Pharmacy Policy of Ministry of Health People’s

Republic of ChinaCollection of Guideline of clinical and preclinical study for new drug (Western medicine)

（11）Xu Shuyun, Bian rulian, Chen xiu. Methods of

pharmacologicalexperimentation.

（12）Chen Qi. Methods of pharmacological Study on Chinese

Traditional Medicine.10 AppendixThe test methods collected in appendix only provide references, the researcher should adoptscientific and rational methods accepted by domestic and overseas based on the owncharacters of the test drug, and don’t limit in the test methods collected in appendix.10.1 Method for irritation test10.1.1 Skin irritation test10.1.1.1 DefinitionSkin irritation test means the reversible inflammatory changes on the skin induced by

transdermal administration, inreversible tissue damage is named as skin causticity.10.1.1.2 ObjectiveTo observe of irritation after skin of animal has been exposed to the test substance andprovide possible ADR such as inflammation reaction, denaturalization and necrosis of skin10.1.1.3 Test method

10.1.1.3.1 Animal：Albino rabbit is preferred, 4 to 8 animals, half

females and half males.Excipient control is designed. Right and left sides of each animal serves as its own control.Fur should be removed from the test area 24 h before the test, (generally the dorsal area ofthe trunk of the animals, by clipping or shaving or suitable depilatory). The area of removingfur is approximately 3cm×3cm each side. Skin should not be abraded. Only animals withhealthy intact skin should be used. But if the irritation of abraded skin is like to be observed,using sandpaper to abrade or lacerate “#” until blooding.10.1.1.3.2 Application of the test substance: The test substance 0.5ml should be applied to

the area with no fur, and two layers of gauze patch （2.5

cm×2.5cm）and one piece of

cellophane paper or analog should be covered on the test area, unstimulating adhesiveplaster and bandage should be fixed; Excipient serves as the control at the other side. Therecommended exposure duration is 4 hours at least. At the end of the exposure period,residual test substance should generally be removed, where practicable, using water or anappropriate solvent. Skin irritation test of repeated dose should be performed at the identicalarea with the same time, and the exposure period often less than 4 weeks.10.1.1.3.3 Animal observation: Dermal irritation should be observed in the natural light orholospectrum light. Dermal irritation should be scored according to the grades in the

following Table. 1.As for the single dose skin irritation test, exposure site should be examined and recordedwith eyes for signs of erythema and edema at 30–60 min, 24, 48, and 72 hours afterremoving test drug. For permanent damage, it is necessary to prolong observation period todetermine the recovery and its duration. But the postponement should not exceed 14 days.Histopathology examination should be conducted for animals with moderate or severe skinirritation after observation period.Regarding the repeated dose irritation test, each time after 1 hour of removing the testsubstance and prior to the next exposure ,animals should be examined and recorded forsigns of erythema , edema, pigmentation, hemorrhage, coarse or thin skin and theirbeginning time and ending time. Erythema, edema should also be scored. Animals should beexamined for signs of erythema and edema at 30–60 min, 24, 48, and 72 hours afterremoving the test drug. For permanent damage, it is necessary to extend observation periodto determine the recovery and its duration. But the postponement should not exceed 14 days.Histopathology examination should be conducted for animals with moderate or severe skinirritation after observation period.10.1.1.3.4 Evaluation of results: As for the single dose skin irritation, the mean of skinresponse scores of each test and excipient group at every observation point should becalculated. The mean of the excipient response scores subtract that of skin gets primaryirritation index. Degree of irritation should be evaluated as table 2.The repeated dose skin irritation, at first, the mean of primary irritation scores of each groupat every observation point should be calculated. Then the irritation integral mean score ofeach individual animal (namely, accumulative irritation score) in the observation durationshould be calculated. Irritation degree evaluation shall abide by table 2.

Table 1.—Evaluation of Skin Irritation ResponseIrritation Response ScoreErythema formationNo erythema 0Mild erythema (barely perceptible) 1Moderate erythema( Well-defined). 2Severe erythema 3Beet redness erythema to slight escharformation (injuries in depth)4Edema FormationNo edema 0Mild edema (barely perceptible) 1Moderate edema (definite raised edges) 2severe edema (raised approximately 1 mm) 3Severe edema (raised more than 1 mm andextending beyond area of exposure4Maximum total score 8Table 2.—Evaluation Criteria of Skin Irritation DegreeValue Evaluatin0-0.49 No irritation0.5-2.99 Mild irritation3.0-5.99 Moderate irritation6.0-8.0 Severe irritation10.1.1.4 Test reportTest report should be in compliance with GLP, covering: test substance’s name,physical-chemical properties, formulation and dose; animal’s species, strain, sex, age, weight,source (marked by certificate code and animal grade); description of animal raisingconditions (source of feed, ambient temperature, humidity, photoperiod, and certificate codeof facility of test animal); specific operational procedure; skin responses scores for eachindividual animal at each observation point; narrative description of other toxicity; testconclusion, etc.10.1.1.5 Explanation to test resultsThe test system is more sensitive than the human skin irritation test, so the negative resultcan be justified as no irritation and false active response should be excluded. Mild irritation

can be ignorant except that test substance has been used for a long time or with a broadarea.10.1.2 Irritation test to injection site10.1.2.1 DefinitionIrritation test of injection site means the reversible tissue changes in the injection sites afterinjecting test substance, but for irreversible tissue damages, called Corrosion.10.1.2.2 ObjectiveDetermination of the irritant and/or corrosive effects at the injection place is useful to referpossible inflammation reaction, denaturalization and necrosis in clinical practice.10.1.2.3 Test method10.1.2.3.1 Test animal: Albino rabbit is r preferred, at least 3 animals should be used. Salinecontrol is needed. Right and left sides of each animal may serve as its own control. Injection

site shall basis on administration route, e.g. ear edge vein，ear

centra artery （other animanls

can use fore/hand limb vein and femoral artery, etc.），femoral and

dorsal muscles，

hypodermis of lateral chest wall，vein side tissue, etc. .

10.1.2.3.2 Method of administration: test substance is administered depending on the clinicaluse, and drug volume and velocity can be modified according to animal condition.Administration duration is determined by proposed clinical practice, and repeated doseadministration cannot surpass 7 days generally.10.1.2.3.3 Animal observation: in light of single dose irritation test, injection sites andanimals should be examined with eyes; and for repeated dose irritation test, injection sitesand animals should be examined with eyes prior to administration each day and after 48-96hours of the last administration. At the end of observation period, histopathology

examination of some animals should be conducted on the administration site. The rest ofanimals should be further observed in the following 14-21 days depending on the charactersof test substance and the process of irritative response, then conduct histopathologyexamination to learn the irritation reversibility.10.1.2.3.4 Evaluation of results: safety evaluation of test substance is based on the results ofobservation by eyes and pathological examination.10.1.2.4 Test reportTest report should be in compliance with GLP, covering: test substance’s name,physical-chemical properties, formulation and dose; animal’s species, strain, sex, age, weight,source (marking certificate code and animal grade); description of animal raising conditions(source of feed, ambient temperature, humidity, photoperiod, and certificate code of facilityof test animal); specific operational procedure; skin responses scores for each individualanimal at each observation point; narrative description of other toxicity; test conclusion, etc.10.1.3 Eye irritation test10.1.3.1 DefinitionEye irritation is the production of reversible tissue changes in the eye following theapplication of a test substance to the anterior surface of the eye, corrosion is irreversibletissue damage in the eye following application of a test substance to the anterior surface ofeye.10.1.3.2 ObjectiveObservation of the irritant effects on the eye delivered substances may give reference topossible inflammation reaction after clinical usage, denaturalization and necrosis in the eyes.10.1.3.3 Test method10.1.3.3.1 Test animal: Albino rabbit is recommended as the preferred species, At least threeanimals should be used.Saline control should be designed, and right and left sides of each animal serves as its owncontrol. Both eyes of each test animal should be examined within

24 h before testing starts,including Nafluoresecin examination. Animals with eye irritation, corneal defects, orconjunctiva injury should not be used.10.1.3.3.2 Method of administration: A dose of 0.1 ml or 0.1g test substance should beplaced or daubed respectively in one eye of each animal. The lids are then gently heldtogether for about 10 seconds. The eyes of the test animals need not be washed out. Theadministration period is determined by the proposed clinical treatment plan. Normally, forthe repeated dose drug, the period may not exceed 4 weeks.10.1.3.3.3 Observation on eyes: for single dose eye irritation test, the eyes should beexamined at 1, 24, 48, and 72 h after drug delivery. For repeated dose eye irritation test, theeyes should be examined ahead of the administration and at 1, 24, 48, and 72 h after lastadministration. If there is no evidence of irritation at 72h, the study may be ended. Extendedobservation (normally need not exceed 21 days) may be necessary if there is a permanentdamage. Examination of reactions can be facilitated by use of a binocular loupe, handslit-lamp, biomicroscope, or other suitable device. After recorded observation at 24 hours,fluorescein dyeing examination may be further conducted. The grades of ocular reactionusing the table 3 should be recorded at each examination. In addition to the observations ofthe cornea, iris and conjunctivae, any other lesions which are noted should be recorded andreported.10.1.3.3.4 Evaluation of results: As the requirement of table 3, scores of cornea, iris andconjunctivae of each group at every observation point should be summarized to get the totalscores. Summation of each group is divided by the number of animal, then the final score isgotten. Irritation degree is justified using table 4.Table 3.—Evaluation of eye ReactionEye irritation reaction valueCornea

No opacity 0Scattered or diffuse areas of opacity, details of irisclearly visible1Easily discernible translucent area, details of irisslightly obscured2Nacrous area, no details or iris visible, size of pupilbarely discernible3Opaque cornea, iris not discernible through theopacity4IrisNormal 0Markedly deepened rugae, congestion, swellingmoderate circumcorneal hyperemia, or injection, irisstill reacting to light1Hemorrhage / necrosis visible with eyes / noreaction to light (any or all of these)2ConjunctivaeRedness (refers to palpebral and bulbarconjunctivae)Blood vessels normal 0Blood vessels hyperemic, cardinal red 1Blood vessels hyperemic, crimson color, not easilydiscernible2Diffuse beefy red 3Chemosis (refers to lids and/or nictitatingmembranes)No swelling 0Slightly swelling (includes lids) 1Obvious swelling with partial eversion of lids 2Swelling with lids about half closed 3Swelling with lids more than half-closed 4SecretionNo secretion 1A little secretion 2Lids and eyelashes humid or cohesive by secretion 3 `The whole eyes area humid or cohesive by secretion 4Maximum score 16

Table 4.—Evaluation of Skin Reaction DegreeValue Evaluatin0-3 No irritation4-8 Mild irritation9-12 Moderate irritation13-16 Severe irritation10.1.3.4 Test reportTest report should be in compliance with GLP laboratory administrant criterion, thefollowing specific information should be reported: name , physical-chemical properties,formulation and dose; Species, strain, sex, age ,weight, source(marked by identification codeand animal grade) of test animal; Description of caging conditions including source of feed,ambient temperature, humidity, photoperiod, and identification code of facility of test animal;Specific operational procedure; Eyes response scores for each individual animal at eachobservation time point; Narrative description of other toxicity; test conclusion, etc.10.1.3.5 Explanation of resultsThe test system is more sensitive than the eye irritation test of human, so the negative resultcan be justified no irritation, but false active response should be excluded when got activeresults. Mild irritation can be ignorant except that test substance has been used for a longtime or in a broad area.10.2 Method for sensitization test10.2.1 Passive cutaneous anaphylaxis test(PCA)10.2.1.1 Elementary principleNormal animal is given a intraderma injection of IgE-rich serum from an allergen-sensitizedanimal. IgE binds to Fc3 acceptors of mast cells in the skin, the latter is sensitized passively.Local mast cells release allergied mediators, followed by increased local vascularpermeability . The injected dye can leak from wheal, making a bule. Sensitization degree isjustified according to the range of blue or spectrophotometer measurement.10.2.1.2 Test animal: Rats are the commonly used animal for PCA reaction, and mice are

also employed. Albino rabbit is used to meet the test requirements. Because PCA reactionsof all the animals are mediated by IgE.10.2.1.3 Groups of test: groups should be designed as negative group, active group and testsubstance group in different dose. Negative control group should be administered samevolume vehicle, and positive control group is given ovalbumin or allergen sensitized drug. 4animals in each group are available at least.10.2.1.4 Route and manner of sensitization: The route of administration is in compliancewith that of in proposed clinical practice. Sensitization is employed every other day, 5 timesin total. Blood is collected at 10 days approximately after the last sensitization, Aftercentrifugation (2000 rpm) for 10 min, and serum is separated and stored at -20℃ until usedwithin 2 weeks.10.2.1.5 Excitation: The above anti-serum is commonly diluted in saline to 1:2, 1:8, 1:32. furshould be removed from the dorsal area of the trunk of the animals for 3cm×4cm2. After24 or 48 hours, each group is injected iv with excitation antigen which is an equal amountwith the sensitization dose and mixed with 0.5-1% evan’s blue dye, the total amount is 1 ml.10.2.1.6 Test and evaluation of results: After 30 min, all animals are sacrificed byanaesthesia, skin of the dorsal area of the trunk are clipped .A reaction was scored as positiveif the spot of the skin was more than 0.5 cm in diameter.10.2.2 Active systemic anaphylaxis test (ASA)10.2.2.1 Elementary principleA venous injection of antigen is given to allergen-sensitized animal to observe systemicanaphylaxis resulted from degranulation of mast cell and basophil, active medium releaseafter antigen binding with IgE.10.2.2.2 ObjectiveTo observe anaphylaxis induced by systemic administration of the test substance.10.2.2.3 Test method10.2.2.3.1 Test animal

Male Guinea pig of Hartley strain, 300-400g of weight, is commonly used10.2.2.3.2 Dose GroupNegative group, active group, high dose group and low dose group. At least 4 animals ineach group.10.2.2.3.3 Sensitization10.2.2.3.3.1 Route of sensitization: follow the route of clinical administration.10.2.2.3.3.2 Times of sensitization: every other day, 5 times in total.10.2.2.3.3.3 Dose of sensitizationNegative control group: Vehicle diluting drug is administrated with the equal volume.Active control group: 1-5mg BSA or valbumin /per animal or allergen sensitized substance

Low dose group: clinically maximum dose（/kg or m2）

High dose group: Several multiple amount of low dose group10.2.2.3.4 Excitation10.2.2.3.4.1 Route of excitation: A venous injection commonly.10.2.2.3.4.2 Time of excitation: single excitation at the 10-14 days after last injection.10.2.2.3.4.3 Dose of excitation: generally 2-5 times of sensitization dose.10.2.2.3.5 Observation variable10.2.2.3.5.1 Sensitization period: Symptoms of each animal is observed everyday. Weight ofindividual animal in each group should be weighed at the initial and terminal sensitizationdays and excitation day.10.2.2.3.5.2 Excitation: Animal responses and appear /disappear time of symptoms shouldbe closely observed according to the table 5 from right after injection through 30 min afterinjection. It should persist 3 hours at most.Table 5 symptoms of anaphylaxis0 normal 7 increased inspiratory rate 14 unsteady gait1 dysphoria 8 emiction 15 jump2 pilar erecti 9 defecation 16 wheeze3 tremor 10 watering 17 convulsion4 scratch around the nose 11 labored repiratory 18 turn5 sneeze 12 rale 19 wavy respiration6 tussis 13 purpura 20 death

Table 6 evaluation of systemic sensitization0 – negative anaphylaxis1-4 symptom + weak positive1-10 symptom ++ positive1-19 symptom +++ strong positive20 ++++ highly strong positive

10.2.3 Guinea pig maximization test（GPMT）and Buehler

test（BT）

10.2.3.1 DefinitionThe test animals are initially exposed to a sensitization dose through intradermal and/orepidermal application. After that, a period of 10 to 14 days induction period develops animmune response. Then the animals are exposed to an excitation dose to observe whetheranaphylaxis occurs. The response and degree of skin reaction to the sensitization andexcitation exposure should be compared, as well as compared with sham treatment group.10.2.3.3 Test method10.2.3.3.1 Test animal10.2.3.3.1.1 Species and strain: it is better the young adult guinea pig and commonly usedstrains in laboratory.10.2.3.3.1.2 Housing and feeding. The temperature should be 20±3℃ with the relativehumidity 30–70 percent. Where artificial light, the sequence should be alternate 12 h light/12h dark. Conventional laboratory diets may be used without limitation of water. Guinea pigsshould receive an adequate amount of ascorbic acid.10.2.3.3.1.3 Number and sex The number and sex depends on the choice of method,either male or female. If females are used, they should be nulliparous and no pregnant. TheBuehler test recommends a minimum of 20 animals in the treatment group and no lessthan10 in control treatment. For GPMT, no less than 10 animals in the treatment group and5 in the control group should be used. If the test result fails to suggest the test substance is asensitizer, the test should be continued, for which no less than 20

test animals in treatmentgroup and 10 animals in control group.10.2.3.3.1.4 Control animals10.2.3.3.1.4.1 Sensitivity and reliability of the method should be examined by knownmild-to-moderate positive substances every 6 months. Mild-moderate sensitizers withadjuvant shall at least response by 30%, without adjuvant by 15%. Recommended positivesubstances are mercaptobenzothiazole , benzocaine , dinitro-chloro-benzene ,or DER 331epoxy resin. Other sensitizers substances may also be used.10.2.3.3.1.4.2 A separate sham-treated group which only receive vehicle should be designedto ensure the excitation response arising from sensitization rather than irritation. The solventshould not interfere or change the judgement of results.10.2.3.3.1.5 Dose Dose depends on the test method. In the Buehler test, the sensitizationdose should be high enough to cause mild irritation, and the excitation dose is the highestnon-irritating dose. In the GPMT, the sensitization dose should be well toleratedsystemically, and should be high enough to cause mild-to-moderate skin irritation; theGPMT excitation dose is the highest non-irritating dose.10.2.3.3.1.6 Observation to animals10.2.3.3.1.6 .1 Skin reactions should be graded and recorded after the excitation exposures atthe time specified in the methodology, usually at 24 and 48 hours. Time adjustment shouldbe made as necessary to describe abnormal responses.10.2.3.3.1.6.2 Body weights at initial and terminal should be recorded10.2.3.3.1.7 Test procedure10.2.3.3.1.7.1 The Buehler test uses topical administration via a closed patch on days 0, 6–8,and 13–15 for sensitization, with topical excitation of the untreated flank for 6 hours on day27–28. Readings are made approximately 24 hours and 48 hours after removing the patch. Ifthe results are equivocal, the animals may be re-excitated one week later, using either theoriginal control group or a new control group.

10.2.3.3.1.7.2 The GPMT uses intradermal injection to topically sensitize for 5–8 days, withand without adjuvant. After that, topical excitation will be given for 24 hours on day 20–22.Readings are made approximately 24 hours and 48 hours after removing the excitation dose.As same as the Buehler test, if the results are equivocal, the animals may be reexcitationd 1week later. If only 10 animals were used initially and lead to equivocal results, 10 treatmentanimals and 5 control animals shall be added.10.2.3.3.1.7.3 Blind access to both test and control animals10.2.3.3.1.7.4 Removal of the test material by water or an appropriate solvent, withoutaltering epidermis’ existing response and integrity.10.2.3.3.1.7.5 Hair is removed from the site of application by clipping, shaving, or depilating,depending on the test method.10.2.3.4 Test reportTest report should comply with GLP, covering: test substance’s name, physical-chemicalproperties, formulation and dose; animal’s species, strain, sex, age, weight, source (markingcertificate code and animal grade); description of animal raising conditions (source of feed,ambient temperature, humidity, photoperiod, and certificate code of facility of test animal);specific operational procedure; the skin response results of each individual animal subjectedto sensitization exposure at 1 and 24 hours, and the excitation exposure at 24 hours and 48hours. As a minimum, the record should include erythema and edema grad, any abnormalfinding, the proportion of sensitization in each group and the extent (slight, moderate, severe)of the sensitization reaction in each individual animal, as well as test conclusion, etc.10.2.3.5 Explanation of resultsThe test system is more sensitive than human sensitization test, so the strong active responseon Guinea pig possibly means sensitization on human body, and week active response onGuinea pig may means week or non-sensitization on human body. .10.2.4 Skin phototoxicity test

10.2.4.1 DefinitionPhototoxicity reaction means the release of ultraviolet energy embodied in drug to skinresults in skin damage. Namely, a skin toxicity reaction generated when skin or wholebody first exposure to chemical, and subsequently to ultraviolet radiation. It is a mostcommon adverse reaction, and has dose dependency. The clinical symptoms are similar

with sunburn，showing erythema , edema, prutitis, pigmentation,

and local necrosis,canker or desquamation of epidermis in severe condition. Once occur, symptoms developvery quickly, usually around 24 hours light irradiation or even

shorter。

10.2.4.2 Test method10.2.4.2.1: Test animals: Albino or hairless Guinea pig, healthy, 300-500g of weight, 14animals at least, half females and half males.10.2.4.2.2 Test groups:, Guinea pig are randomizly divided into 2 groups depending uponweight and sex, 4 in control group, 10 in treatment group, half females and half males. Thecontrol group should not expose to light after administration of test substance, but activecontrol drug expose to light.10.2.4.2.3 Drug and dose: 8- methoxypsoralen is preferred as an active control substance; thedose of test substance is determined by deliquescence, required concentration, chemical/physical properties, local/systemic tolerance of animals and their rationalities. Twoconcentrations are commonly selected; the low concentration is same as the clinically usedconcentration.10.2.4.2.4 Method of administration: the back should be removed hair and divided into four

parts. 0.025ml（g）/cm2 drug delivered to each part. The

application as following:Group Site Treatment IrradiationControl group 1 solvent or excipient no

2 test substance concentration 1 no3 test substance concentration 2 no4 active control noTreatment group 1 solvent or excipient yes2 test substance concentration 1 yes3 test substance concentration 2 yes4 active control yesAfter administration, the animals are placed in the given detents to limit activity. Beforeexposure to light, the test substance and control substance should have enough time(60 min

is preferred) to permeate into cuticle，and then interact with

epidermal cells. At the propertime after administration, generally 0.5-4 hours, residual test substance and excipient at thetest site of control group should be removed, using water or an appropriate solvent, theanimal are then placed back into cages. The treatment group does as the control group didafter irradiation.10.2.4.2.5 Method of irradiation: wave band should be sensitive to the test substance.Irradiation dose, light intensity, irradiation period and distance should be carefullydetermined through Pre-test. During the experiment, the animals are fixed at the irradiationdesks with the heads covered, and irradiated for appropriate time, using proper light source,eg, water-cooled xenon arc lamps. It is generally considered that wavelengh should cover280-450nm, and irradiation period is within 0.5-2 hours, and irradiation intensity is proper,which should be measured before and after irradiation.10.2.4.2.6 Test items: Individual animal in each group should be weighed when initiate andterminate the test. Skin responses should be observed and recorded by photos at 24 and 48hours. Skin responses should be scored according to the grades in the table 7.Table 7.—Evaluation of Skin Responses DegreeSkin response valueNo erythema 0Mild erythema 1

Moderate erythema 2

Severe erythema（with or without edema） 3

10.2.4.2.7 Judgement and evaluation of results: compared with control group, if score ofindividual animal in the control group is less than 1, those in treatment group whose score isequal with or more than 1 is regarded as positive; if score of individual animal in the controlgroup is equal with or more than 1, those in treatment group whose score is more than thehighest in the control group is justified as positive. Positive ratio can be further obtained.10.2.5 Skin photoallergy test10.2.5.1 DefinitionThe drug becomes active state after absorbing light energy, and binds to protein in the skin,

in a manner of hapten, to become drug-protein complex（complete

atigen）which is

transferred to immune active cells by Langerhans cells in epidermis, inducing supersensitivereaction. Photoallergy belongs to type IV bradyanaphylaxis. It undergoes latent period. Thereaction occurs after a relatively long time. In general, continued administration for 5-10days and irradiation can induce immune system response. When

the drug is applied again，

photoallergy happens within 24-48 hours after drug and irradiation treatment.10.2.5.2 Method of test10.2.5.2.1 Preparation: removing hairs of animals ( 2×4cm2 area). Freund’s completeadjuvant is injected to the four corners of the hairless area, 0.1ml respectively.10.2.5.2.2 Sensitization: 20% Sodium dodecyl sulfate is applied to the hairless area, followedby the test substance. The residual drug should be removed after some time. The positivedrug can chose salicylamide bromide.

10.2.5.2.3 Irradiation: Wave band should be sensitive to the test substance. Irradiation dose,

light intensity, irradiation period and distance should be carefully determined through thepilot test to select wave and the site of application for UV irradiation. The evident eythema istaken as the selection standard. The operational procedure of application of 20% Sodiumdodecyl sulfate and then the test substance which is followed by irradiation should berepeated for 5 times.10.2.5.2.4 Excitation: two weeks after the last sensitization, drug is applied to the preparedskin area, for which the concentration lower than that of sensitization to prevent irritationand phototoxicity. The test substance and control substance should have enough time(60

min is preferred) to permeate into cuticle，and then to interact

with epidermal cells. UVirradiation is charactered by sub-erythema (1/2-2/3 of minimum amount of erythem) whichdoesn’t elicit erythema reaction.10.2.5.2.5 Observation of results: Skin responses should be observed and presented byphotoes within 1-72 hours after irradiation. The test substance which is exposed repeatedlyand subject to UV irradiation, and then lead to skin response even systemic response isregarded to cause photoallergy.10.3 Method for hemolysis test10.3.1 general in vitro tube method (observation with eyes)10.3.1.1 Objective: Whether the state of erythrocyte is affected by test substance.10.3.1.2 Test method

10.3.1.2.1 Preparation of blood cell suspension：put rabbit (or

sheep) blood of milliliters(about 20 ml ) into a triangular conical flask with a beading for vibration 10 minutes, orstirring blood by a Glass rod, to remove away fibrinogen, then become decellulose blood.10-fold amount of 0.9% Nacl solution is added, shake, and centrifugation (2000 rpm) for 10min. The supernatant is removed, and precipitated red cells should be washed for 2-3 times

until the supernatant shows no red color in the 0.9% Nacl solution according to the methoddescribed above. 2% mixed suspension is prepared with RBC dissolved in 0.9% Naclsolution, which is prepared for test.10.3.1.2.2 Preparation of test substance: Except additional requirements, for non bloodvessel injection, the clinical use concentration in drug labeling should be diluted as 1:3 by0.9% saline solution to be test solution; in terms of in blood vessel injection, the testsolution is made in the concentration recommended in drug labeling.10.3.1.2.3 Test method: 7 clean test tubes are numbered, number 1-5 are the tubes for testsubstance. Number 6 is for negative control, number 7 for positive control. 2% RBCsuspension, 0.9% NaCl solution or distilled water are added in sequence as the followingtable, after mixed, immediately incubated at 37℃±0.5℃ in incubator. Observation isconducted every other 15min at the beginning, one hour later, observing every other onehour. The total observation period lasts 3 hours. Various solutions are added in thefollowing sequence:…………………………………………….tube number 1 2 3 4 5 6 7...

2％RBC suspension (ml) 2.5 2.5 2.5 2.5 2.5 2.5 2.5

Nacl(ml) 2.0 2.1 2.2 2.3 2.4 2.5

Distilled water（ml） 2.5

Test substance（ml） 0.5 0.4 0.3 0.2 0.1…………

10.3.1.2.4 Observation of results: When the solution is red and there is no cell residual orfew RBC residual in the tube bottom, it demonstrates hemolysis occurred; When RBC are allcompletely precipitated, and the supernatant is colorless and pellucid, it demonstrates nohymolysis occurred. When there is red brown or brown red floccule which does not dispersewhen shaked, it means RBC have agglutinated. If there is a agglutination phenomenon,

distinguishing true agglutination from the false should be further justified as this method: ifthe agglutinated substance is dispersed uniformly after shaking, or put on the microscopeslide. Two drops of 0.9% Nacl is added at the verge of coverslips to observe under themicroscope. If the agglutinated RBC is dispersed, false agglutination can be concluded,conversely, true agglutination.10.3.1.2.5 Judgement of results: When there are hemolysis and agglutination in the positivecontrol tube but no in the negative control tube, and there are/is no hemolysis and/oragglutination happening in solution in the test substance tube within 3 hours, the testsubstance can be used as injection, otherwise, it can not be used as injection.10.3.1.3 Test reportTest report should be in compliance with GLP, covering: name, physical-chemical properties,formulation and dose of the test substance; test method; specific operational procedure;observation results of individual tube at each observation point; test conclusion and so on.10.3.2 Improved in vitro hemolysis test method(spectrophotometer)10.3.2.1 Objective: observation on whether the test substance affects erythrocyte state10.3.2.2 Test method

10.3.2.2.1 Preparation of blood cell suspension：put rabbit (or

sheep) blood of milliliters(about 20 ml ) into a triangular conical flask with a beading for vibration 10 minutes, orstirring blood by a Glass rod, to remove away fibrinogen, then become decellulose blood.10-fold amount of 0.9% Nacl solution is added, shake, and centrifugation (2000 rpm) for 10min. The supernatant is removed, and precipitated red cells should be washed for 2-3 timesuntil the supernatant shows no red color in the 0.9% Nacl solution according to the methoddescribed above. 2% mixed suspension is prepared with RBC dissolved in 0.9% Nacl

solution, which is prepared for test.10.3.2.2.2 Preparation of test substance: Except additional requirements, for non bloodvessel injection, the clinical use concentration in drug labeling should be diluted as 1:3 by0.9% saline solution to be test solution; in terms of in blood vessel injection, the testsolution is made in the concentration recommended in drug labeling.10.3.2.2.3 Test method: 7 clean test tubes are numbered, number 1-5 are the tubes for testsubstance. No. 6 is for negative control, no. 7 for positive control. 2% RBC suspension,0.9% NaCl solution or distilled water are added in sequence as the following table, aftermixed, immediately incubated at 37℃±0.5℃ in incubator. Observation is conducted everyother 15min at the beginning, one hour later, observing every other one hour. The totalobservation period lasts 3 hours. Various solutions are added in the following sequence:tube number 1 2 3 4 5 6 7

2％RBC suspension (ml) 2.5 2.5 2.5 2.5 2.5 2.5 2.5

0.9% Nacl(ml) 2.0 2.1 2.2 2.3 2.4 2.5

Distilled water（ml） 2.5

Test substance（ml） 0.5 0.4 0.3 0.2 0.1…………...

10.3.2.2.4 Observation of results: Solution in individual tube is put into dried centrifugationtube for centrifugation, and supernatant fluid is obtained. At 545nm of spectrophotometer,OD value is read base on distill water is taken as the blank.10.3.2.2.5 Judgement of result: hemolysis ratio(%) can be calculated using the followingformula:

Hemolysis ratio（％）＝（ODt－ODnc）/(ODpc－ODnc) X 100％

In the formula: ODt：Absorbency of the tube for test substance;

ODnc：Absorbency of negative control tube；

ODpc：Absorbency of positive control tube.

When the hemolysis ratio is more than 5% (>5％), it demonstrates

there is hemolysishappening, and the test substance should not be used as injection.10.3.2.3 Test reportTest report should be in compliance with GLP, the following specific information should bereported: name, physical-chemical properties, formulation and dose of the test substance;test method; Specific operational procedure; observation results of individual tube at eachobservation time point; test conclusion and so on.11 Drafting notesBased on the original New Drug Review Regulation, skin irritation, sensitization of dermaladministration, eye irritation, drops irritation and inhalational product irritation are appliedto the study of irritation of rectal and vaginal products. In terms of the study and evaluationon sensitization, hemolysis of vein administration, there is a principle requirement. With thedevelopment of science and technology, the understanding of mechanism, influent factorsand clinical meaning of irritation, sensitization and hemolysis is deepen, so is the position inthe nonclinical safety evaluation. We revise technical Guideline principles about irritation,sensitization and hemolysis of chemicals according to ‘Drug Registration Regulation’,referring to relative technical Guideline principles of each country of three sides under theICH framework and the research development in domestic and foreign countries onirritation, sensitization and hemolysis of nonoral administration drug, incorporating ingeneral rule of nonclinical safety evaluation for drugs and the working practice in drugresearch technic. It is intended to provide technical reference for study and estimation andapproval of irritation, sensitization and hemolysis of chemicals.The technical Guideline principle only reflects the present understanding, viewpoint and

advice on irritation, sensitization and hemolysis of chemicals, and will be revised andimproved with the development of science and technology.The revised guidline is the inheriting and development of the original one. Compared withthe printed Guideline, it pays much attention on expounding how to design the study ofirritation, sensitization and hemolysis of chemicals scientifically and rationally. The testsamples are the proposed clinical drug products. It includes the local and/or systemictoxicity after administration of various routes covering eyes, ears, noses, mouth, respiratorytracts, joint cavity, skin, recta, vagina, vein, artery, muscle, derma, vein besides, theca, and soon.It emphasizes the study of irritation, sensitization and hemolysis of chemicals should executethe ‘Quality Administrative Criterion of Drug Nonclinical Study. It also stresses that the testdesign should be based on mechanism of toxicity and clinical pertinence, combined withpharmacy eg, Structure-activity relationships and/or physical-

chemical properties，

pharmacology and toxicology eg acute toxicity, log term toxicity, pharmacokinetics andPharmacodynamics, and clinical use to justify how to study and evaluate irritation,sensitization and hemolysis of chemicals. However, it should be noted that specific issue

should be ，and represented integrated, compositive principle.

Considering the close relationship between drug products and drug toxicity, whereas newdrug research in our country is mainly imitated, and drug preclinical animal safety evaluationis usually lack of systematization and integrity, therefore, to guarantee its safety and efficacyin clinical use, in the revised technical Guideline, it suggests marketed drug products shouldcompare with those of the marketed products which have the same administration route,when the former showed positive results in toxicity study.

The study on the toxicity in the revised technic Guideline principle has improved a lotcomparing with the printed one, eg, sensitization includes systemic sensitization, skinsensitization and photoallergy; hemolysis covers immunological and nonimmunologicalhemolysis. In addition to evaluate the irritation and skin sensitization of transdermal drugproducts, if it is possible to have other toxicity, eg systemic sensitization, hemolysis and soon, appropriate test method should be performed to fully investigate its possible relatedtoxicity during clinical use. In terms of injection drug products, it also should be consideredto adopt suitable methods to study the responding toxicity if the drug products maybe causeother toxicity, eg, skin sensitization, in spite of irritation systemic sensitization, hemolysis.Irritation and/or sensitization and/or hemolysis of the drug products with other routes ofadministration should be investigated in conjunction with their own characters.The study on the toxicity in the revised technic Guideline principle also has big differencewith the printed one. For instance, the investigation of skin sensitization commonly uses BTor GPMT; Systemic sensitization is performed using ASA and PCA. Irritation and/orsensitization and/or hemolysis can be considered when other toxicity tests eg, long termtoxicity test are being performed, but the test substance must be identical or comparablewith the proposed clinical drug products, and the test result should completely show thepossible irritation and/or sensitization and/or hemolysis which maybe occur in clinical use.In the appendix of the Guideline, commonly used test methods are embodied. But they areonly viewed as a reference. Owing to the differences of chemical structure and drugproducts, clinical use, mechanism, character, intensity of toxicity response, the researchershould select recognized method to perform the test according to the characters of the drug.

In this Guideline, irritation is defined as the production of reversible inflammatory changesat the administration site, whereas the production of irreversible tissue damage is defined ascorrosion, which are referred to the ‘Methods of pharmacological Experimentation’, EPT,OECD and the Guideline for skin and eye irritation response to cosmetic.In the present, the sensitivity and veracity of nonclinical safety evaluation of irritation,sensitization, hemolysis are not highly predicted, it is thought to be related to: 1. The testsaren’t designed with the consideration of mechanism of toxicity, effected factors, clinicalmeaning and indication symptoms in clinical practice and administration, eg, ① in the skinsensitization test, active skin anaphylaxis is often used, but the method is designed based onthe mechanism of type I hypersensitivity, the test results only can predict the possiblesystemic anaphylaxis, to the transdermal administration, skin sensitization response is moreimportant to the safety and efficacy of administration. Therefore, the sensitization testshould be designed with the consideration of pathogenesis, eg, BT or GPMT isrecommended.② in the test design, the test substance, dose, route, period, times of administration and so on, can

not

reflect the condition in clinical use. 2. there are limits in the present methods, eg, ① there are not

ideal animal models for II and III type hypersensitivity at present; ② there is not definite clinical

meaning about the model of photoallergy; ③ the reliability of measuring small molecular

substances

using PCA and ASA is not certain. 3. The observation of some toxicity reactions is neglected in

the

test design. eg, ① with regard to the hemolysis test, only routine test tube method in vitro is

adopted,

but this test can not predict immunological hemolysis and oxidation-caused hemolyis induced by

drug

as an inducer; ② for vein injection, blood vessel irritation is taken into account merely, and it is a

pity to ignore the irritation response to muscle when the drug leaks to the out of blood vessel. 4.

Sensitivity of the test method is not high enough, eg, when the hemolysis is observed using the

routine

test tube method, the hemolysis is often justified with eyes, thus subjective error is bigger. It is

also

affected by the drug’s color and obscuration facors. 5. The evaluation methods and standards is

not

perfect, eg, there are not semi-quantitative and/or quantitative evaluation tests. Due to the

unceasing

progress and development of scientific research, other methods which provide more valuable

results

and enhance the prediction on irritation, sensitization and hemolysis are encouraged. But when

other

methods are used, their rationale and specific methods and operational flow should be interpreted.

12 AuthorThe project research group of Technical Guideline on Irritation, Sensitization and Hemolysis

Study for

Chemical Drugs