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Safety pharmacology 2

 6) Carcinogenicity Studies (Ref: ICH S1A)





Objectives: to identify a tumorigenic potential in animals and to assess the relevant risk in humans.

Factors to consider for carcinogenicity testing

  • Duration and Exposure: Carcinogenicity studies should be performed,
  1. for any pharmaceutical whose expected clinical use is continuous for at least 6 months
  2. for pharmaceuticals used frequently in an intermittent manner in the treatment of chronic or recurrent conditions
  3. for certain delivery systems which may result in prolonged exposures
  • Cause for concern:
  1. previous demonstration of carcinogenic potential in the product class that is considered relevant to humans;
  2. structure-activity relationship suggesting carcinogenic risk;
  3. evidence of preneoplastic lesions in repeated dose toxicity studies; and
  4. long-term tissue retention of parent compound or metabolite(s) resulting in local tissue reactions or other pathophysiological responses
  • Genotoxicity: Such compounds intended to be administered chronically to humans a chronic toxicity study (up to one year) (compound: Unequivocally genotoxic compounds, in the absence of other data, are presumed to be trans-species carcinogens, implying a hazard to humans)
  • Route of Exposure: clinical route
  • Extent of Systemic Exposure: there is cause for concern or unless there is significant systemic exposure, or changes in exposure and consequent toxicity are noted, or on a case-by-case basis
  • Endogenous Peptides and Protein Substances or Their Analogs
  1. long-term carcinogenicity studies in rodent species should be considered
  2. Conduct of carcinogenicity studies may be important in the following circumstances:
    1. For products where there are significant differences in biological effects to the natural counterpart(s);
    2. for products where modifications lead to significant changes in structure compared to the natural counterpart; and
    3. For products resulting in humans in a significant increase over the existing local or systemic concentration (i.e., pharmacological levels).



 7) Reproduction Toxicity Studies. (Ref: ICH S5 (R2)).




Introduction Objective : To reveal any effect of one or more active substance(s) on mammalian reproduction
Animal Criteria Selection species: Animal must be healthy, fertility, fecundity, prevalence of abnormalities, embryofetal deaths and the consistency they display from study to study.
Recommendation concerning treatment
Combination of study
  • Most probable option combination studies for effect on:
    1. Fertility and early embryonic development.
    2. Pre- and postnatal development, including maternal function.
    3. Embryo-fetal development.
  • Study of Fertility and Early Embryonic Development to Implantation.
    1. Test for toxicity effect resulting from treatment from before mating and implantation.
    2. Effect of female; oestrous cycle, tubal transport, implant and development of preimplantation stages of embryo.
    3. Effect on male; functional effect on libido, epididymal (totally rare due to inducing selective effect on male reproduction system).
  • Effect on pre- and postnatal development.
    1. To detect adverse effects on the pregnant/lactating female and on development of the conceptus and the offspring following exposure of the female from implantations thought weaning.
  • Effect on embryo-fetal development.
    1. Effect on the pregnant female and development of the embryo and fetus consequent.
  • Single study (rodents)
  • Dosing period of the fertility study and pre-
  • Two study (rodents)
    1. Consist fertility study and the pre- and postnatal study.



 8Local Tolerance Studies (Refs. EMEA CPMP/SWP/2145/00)





  • Local tolerance may be a part of toxicity studies provided that the medicinal product is administered under adequate conditions as below:

  1. Absorption of the product can be demonstrated to be slow that the possibility of systemic effects can effectively be ruled out.

  2. The product is absorbed, but its systemic toxicity has previously been adequately investigated.


  • To confirm tolerance of medicinal products (both active substances and excipients) at the site of its administration based on clinical use and also at sites which might accidentally or unavoidable exposed into contact with the product. 

General Principles

  • Measurement of mechanical effects of administration and physicochemical actions of the products in its intended formulation to distinguished the effect from toxicological and pharmacodynamic effect.   

Points to consider

  • The physicochemical properties of the product in its intended formulation.

  • The pharmacodynamic, toxicological and pharmacokinetic data for the active substances or combination of active substances and the excipients.

General design of local tolerance test

  • Choice of species: Problem or case by case.

  • Frequency and duration of administration: Based on proposed conditions of administration in clinical use.

  • Reversibility: Included in where relevant.

  • Preparation to be tested: Based on preparation developed to be use in man. Positive controls/reference substances may be included where necessary.

  • Choice of dose: Actual concentration of active substances with reference in humans use. 

  • Animal welfare: Care should be focus  to minimise painful exposure of animals to irritants which termination of tests should be consider where severe adverse reactions are seen and continuation is not expected to provide results essential for risk assessment.

  • Route of administration: Selection is based on administration route for humans use with careful attention on anatomy and physiology of the application site in the selected test model. Investigation of different routes administration in the same selected model is permissible but cannot influence each other parameters.  

  • Evaluation of results: Evaluation of results on the adequacy of the local tolerance test design and significance findings for the clinical use of the product.    

Ocular tolerance testing

  • Testing strategy is greatly influenced by the context in which the eyes are exposed to the product (repeated exposure or accidental exposure).

  • Requirements include:

  1. Ocular tolerance test by single administration in rabbits that include the detection of anaesthetising properties of the administration compound.

  2. Repeated dose ocular test conducted in rabbits with daily administration for four weeks.

  3. Investigations of different tissues in contact with the product (lens, vitreous body, ocular fundus)

  • Single dose ocular tolerance test:

          - The product is applied on area surrounding eyes of only one eye whereas the other eye serves as control. The eyes should be examined for at least 72 hours after administration with suitable evaluation of the reactions are observed.

  • Repeated dose ocular tolerance test:

         -  The design of test dependence on single ocular tolerance test and inclusion of histological examination should be consider.

Dermal tolerance testing

  • Requirement in dermal tolerance testing included:

  • Single dose dermal tolerance test:

        -   In vitro skin corrosion tests can be performed using 24 hour rat skin transcutaneous electrical resistance assay (TER assay) to obtained TER value which indicate the ability of the product to cause a loss of normal stratum corneum integrity and barrier function. The test also can be performed using human skin model assay up to 4 hour to measure the percentage of cell viability that demonstrate the cell death underlying the stratum corneum layer.  

       -    Irritancy test is performed using rabbit, on shaved intact skin and on an equivalent area of shaved and abraded skin to measure the degree of erythema, oedema, desquamation, scab formation and any other lesions. Vehicle control should be included. The observation period will depend on changes at 24, 48 and 72 hours after administration.

  • Repeated dose dermal tolerance test:

       -   The test is conducted in rabbits on shaved intact and abraded skin for four weeks. Histological examination should be considered on a case by case basis and consideration should be given to the type and amount of any decomposition products produced.

Parenteral tolerance testing

  • The test involves route of administration by intravenous, intra-arterial, intramuscular, intrathecal, subcutaneous and paravenous.

  • The test should be done in appropriate selected species with adjustable injection volume and rate while application of product at site should according to the route as below:

  1. suitable veins of ear, the tail or the front of hind limbs

  2. central artery of the ear in rabbits, femoral arteries or other suitable arteries in other species

  3. dorsal or femoral muscles

  4. subcutaneous tissue of the lateral chest wall or other suitable application sites

  5. paravenous tissue near one of the suitable veins mentioned under item (a)

  • Observation period of single administration should be performed during 48-96 hours after administration followed by repeated administration. Mascroscopic examination, histological examination and reversibility studies may be required case by case.

Rectal tolerance testing

  • The test should be performed in selected animal species (rabbits or dogs) with administration of the product once or twice per day for minimum of 7 days in proposed human therapeutic dose volume of the galenic formulation or the maximum applicable volume for the animal species.  Observation should be done on the anal region and anal sphincter, clinical signs and faeces. Necropsy, macroscopic examination and histological examination of rectal mucosa should also be performed.  

Vaginal tolerance testing

  • The test should be performed in selected animal species (rats, rabbits or dogs) with administration of the product once or twice per day for minimum of 7 days in proposed human therapeutic dose volume of the galenic formulation or the maximum applicable volume for the animal species. Observation should be done on the vaginal regions, clinical signs and vaginal secretion. Necropsy, macroscopic examination and histological examination should also be performed.  

Sensitising potential

  • Sensitizing evaluation should be evaluated for products applied to skin (dermal, rectal, vagina).

  • The evaluation of sensitisation is carried out at least in one of these two test systems:

  1. 1. In vivo Guinea Pig Assay

       -     There are two types of test namely Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman which uses adjuvant (potentiated by the injection of Freunds Complete Adjuvant, FCA) and the non-adjuvant Buehler test.

       -      The test is carried out to measure potential of sensitiser.

  1. 2.  In vivo Mouse Models

        -    The Local Lymph Node Assay (LLNA) may be employed as alternative in which positive and negative results generally no longer require further confirmation.

        -    The principle of test is primary proliferation of lymphocytes in the lymph node draining the site of test items that is examined as a dose-response expressed in Stimulation Index,SI.  

Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman

Facility system

  • Temperature of experimental animal room : 20ºC (±3ºC)

  • Humidity: 30-70%

  • Lighting (artificial): Sequence should be 12 hours light, 12 hours dark.

  • Conventional laboratory diets applicable for feeding with unlimited supply of drinking water.

Test animals

  • Male and/or female (nulliparous and non-pregnant) healthy young adult. The number of animals should not be fewer than 20 animals in treatment group and at least 10 animals in control group.


  • The process is conducted for at least 5 days prior to the test. Removal of hair is by clipping, shaving or possibly by chemical depilation, depending on the test method. 

Dose level

  • Induction exposure should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. Concentration to be use in challenge exposure should be the highest non-irritant dose.

  • Appropriate concentration may be determined from pilot study using two or three animals. 

Route of administration

  • Intradermal injection and/or epidermal application in induction exposure and topical application in challenge exposure.

Study design

  • The induction period following 10 to 14 days observation is conducted to allow the immune response develop. The product is applied in a midline of cleared hair shoulder region by three series of intradermal injection (Day 0) followed by induction with topical application (Day 5-8).

  • The sensitisation is challenged (Day 20-22) by topical application of the product on the flanks of both treated and control animals that are cleared of hair.


  • Observations is done in a classified period and record is based on Magnussan and Kligman scale as below:

           -          0 = no visible change

           -          1 = discrete or patchy erythema

           -          2 = moderate and confluent erythema

           -          3 = intense erythema and swelling

  • Clinical observations and unusual finding including systemic reactions resulting from induction and challenge procedures should be observed and recorded.

Buehler Test

Dose level

  • Induction exposure should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. Concentration to be use in challenge exposure should be the highest non-irritant dose.

  • Water soluble test items are appropriately use in water or a dilute non-irritating solution of surfactant as the vehicle. Water-insoluble test item may be used in 80% ethanol/water for induction and acetone for challenge.

Route of administration

  • Topical application

Study design

  • The test patches system in induction (Day 0-15) and challenge period (Day 27-29).


  • Observations is done in a classified period and record is based on Magnussan and Kligman scale as below:

           -          0 = no visible change

           -          1 = discrete or patchy erythema

           -          2 = moderate and confluent erythema

           -          3 = intense erythema and swelling

  • Clinical observations and unusual finding including systemic reactions resulting from induction and challenge procedures should be observed and recorded

Data reporting (GPMT & Buehler Test)

  • Test substance

  • Vehicle

  • Test animals

  • Test conditions

  • Reliability check

      -  Should be assessed every 6 months by use of the substance. A response of at least 30% in an adjuvant test and at least 15% in a non-adjuvant test should be expected for mild/moderate sensitisers. 

  • Results

  • Discussion

Local Lymph Node Assay (LLNA)

Facility system

  • Temperature of experimental animal room: 22ºC (±3ºC)

  • Relative humidity: 50-60%

  • Lighting (artificial): Sequence should be 12 hours light, 12 hours dark.

  • Conventional laboratory diets applicable for feeding with unlimited supply of drinking water.

Test animals

  • Young adult female mice (8-12 weeks old) of CBA/Ca or CBA/J (nulliparous and non-preganant) with weight variation should be minimal and not exceed 20% of the mean weight. The number of animals should not be fewer than four animals per dose group.

  • The positive controls should produce a positive LLNA response (stimulation index > 3 over negative control). 


  • The process is conducted for at least 5 days prior to the test.

Dose level

  • Minimum of three concentrations with dose and vehicle selection are selected from 100, 50, 25, 10, 5, 2.5, 1, 0.5% etc. while considering existing acute toxicity and dermal irritation data.

  • Vehicle should be selected based on maximising the test concentrations and solubility while producing a solution/suspension suitable for application of the test substance.

Study design

  • No induction-challenge period is required. The test items, vehicle and positive controls are applied to the dorsum of each ear. 

  • The draining auricular lymph nodes are excised and pooled (pooled treatment group approach/individual animal approach).

  • Single suspension of lymph node cells (LNC) is prepared and incorporated with radioisotope for detection. Incorporation will be expressed as disintegrations per minute (DPM)/treatment group or DPM/animal. 

Date Reporting (LLNA)

  • Test substance

  • Vehicle

  • Test animals

  • Test conditions

  • Reliability check

       -  Preferred positive controls substances are cinnamic aldehyde and mercaptobenzothiazole.

       -  Historic positive control data are acceptable provided showing consistency of satisfactory response over 6 months or more extended period.

       -  Possible interaction between positive control and vehicle should be tested.

  • Results

  • Discussion



 9) Photosafety Evaluation of Pharmaceutical (Ref: ICH S10 (2)).




  • It is standard for photosafety assessment, to harmonize and support human clinical trial, marketing authorization for pharmaceutical. Include the criteria for initiation and trigger for additional photosafety testing.
  • in vitro alternative method or clinical data for photosafety assessment could reduce the animal accordance with the 3R principle.
  • An initial assessment of phototoxic potential be conducted, if appropriate, an experimental evaluation be undertaken before exposure of large number of subjects (Phase III).
Scope of the Guideline
  • Generally applies to new active pharmaceutical ingredient and new excipient.
General Principle
  • Involving an evaluation of photochemical characteristics, data from nonclinical studies and human safety information.
  • Use to determine adequate risk minimization measure in human.
  • Four effect upon the photosafety testing: phototoxicity, Photoallergy, Photogenotoxicity and Photocacinogenicity.
  • Phototoxicity and photoallergy effect define below:
    1. Phototoxicity (photoirritation) : An acute light-induced tissue response to a photoreactive chemical
    2. Photoallergy : An immunologically mediated reaction to a chemical, initiated by the formation of photoproducts (e.g, protein adducts) following a photochemical reaction.
    3. Chemical demonstrate phototoxicity and/or photoallergy, the following characteristic are critical:
      1. Absorbs light within the range of natural sunlight (290-700 nm);
      2. Generates a reactive species following absorption of UV/visible light; and
      3. Distributes sufficiently to light-exposed tissue (e.g., skin, eye).
Factors to consider in the Photosafety Evaluation          Application of GLP/GMP
  • Photochemical Properties.
    1. Photoreactive potential is whether a compound that absorb wavelengths between 290-700 nm.
    2. Excitation of molecules by light can lead to generation of Reactive Oxygen Species (ROS), including superoxide and singlet oxygen via energy transfer mechanism.
  • Tissue Distribution/Pharmacokinetics.
    1. Concentration of photoreactive in tissue at the time light exposure is a very important parameter for determining the phototoxic reaction.
    2. Several factor can affect the concentration; plasma concentration, perfusion of the tissue, partitioning from vascular to interstitial and cellular compartment, binding, retention and accumulation of chemical in the tissue.
  • Conducted under high-quality scientific standard and compliance with GLP/GMP regulation.
  • Metabolite Cosideration.
    1. Generally do not warrant separate photosafetyevaluation as metabolism does not typically create new chromophores.
  • Pharmacological Properties.
    1. Most cases, drug-induced phototoxicity is due to their chemical structure, and not their pharmacology.
    2. Some can enhance susceptibility to light-induced effect; skin irritation to carcinogenesis.
Nonclinical Photosafety Testing
  • General consideration.
    1. Consider both model system and exposure to a relevance radiation spectrum is critical in clinical testing. Ideally, nonclinical assay should exhibit both high sensitivity and specificity, but to support the integrated assessment, it most important that nonclinical photosafety assays show high sensitivity.
    2. Both in vitro and in vivo assay are focused primarily on detecting potential phototoxicity, which might be not translated into clinically relevant phototoxicity. False positive rate for an assays still be considered when deciding whether or not to use an assay.
  • Using chemical assays
    1. Assays should be qualified using pharmaceutical agents under appropriate conditions to demonstrate assay sensitivity.
    2. One of the assays that is subject of a validation exercise is a ROS assay.
  • Using in vitro assays
    1. Widely model used in vitro assays for phototoxocity is the “in vitro 3T3 Neutral Red Uptake Phototoxicity Test” (3T3 NRU-PT) which a guideline is available.
    2. Most appropriate method for soluble compounds that are not exclusively UVB absorbers.
    3. European Centre for the Validation of Alternative Methods (ECVAM) validation exercise conducted on this assay indicated a sensitivity of 93% and a specificity of 84%.
    4. Organisation for Economic Co-operation and Development (OECD) protocol has been proposed to address the low specificity observed with drug substance.
  • Using in vivo assays and systemic administration.
    1. Today, no nonclinical in vivo phtotoxicity or photoallergy assay has been formally validated.
    2. No standardize study design has been enstablished and thus the following criteria might be considered as best practices.
    3. For species selection, irradiation sensitivity, heat tolerance, and performance of references should be considered and both pigmented and non-pigmented model available.
    4. Testing drugs for in vivo nonclinical phototoxicity, studies a maximum doseslevel that complies with the recommended for general toxicity stusdies in ICH M3. If negative result were obtained at the maximum dose, testing of lower dose is usually not warranted. However, positive result obtain, additional dose group can support NOAEL-based risk assessment.
    5. PK study should be done first to ensure that irradiation of the animals is conducted at the appropriate Tmax. As part in vivo, the relevant systemic exposure data (e.g, Cmax) should be collected as part of it.
    6. Sensitive sign of compound-induced phototoxicity usually erythema and followed edema at normally-sub-erythemogenic irradiation dose.each respon might vary with the compound.
  • Using in vivo assays and dermal administration.
    1. Generally, clinical formulation should be tested first. Irritation of the exposed area should take place at specific time after application, interval between application and irradiation should be justified on specific formulation tested.
    2. Acute and contact phototoxicity are often been investigate in conjunction with nonclinical skin sensitization testing.
    3. However, no formal validation method has been performed and their predictivity still unknown.
  • Using in vivo assays and ocular administration.
    1. No standardised nonclinical in vivo approaches for assessing phototoxicity following ocular administration.
Clinical Photosafety assessment
  • Case-by-case basis.





Reference: Nonclinical Guidelines (specifically)


1) Drug Registration Guidance Document (DRGD), First Edition, January 2013

2) The Asean Common Technical Dossier (ACTD) for the Registeration of Pharmaceuticals for Human Use – Part III: Nonclinical Document; adopted from ICH-CTD on Nonclinical Overview.


1) ICH M3 (R2) Guideline: Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorisation for Pharmaceuticals; June 2009.
2) ICH S6(R1) Guideline: Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals; June 2011
3) ICH S7A Guideline: Safety Pharmacology Studies for Human Pharmaceuticals; Nov 2000
4) ICH S3B Guideline: Pharmacokinetics: Guidance for repeated dose tissue distribution studies; Oct 1994
5) ICH S3A Guideline: Toxicokinetics: the assessment of systemic exposure in toxicity studies; Oct 1994
6) ICH S2 (R1) Guideline: Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use; Nov 2011
7) ICH S1A Guideline: Guideline On The Need For Carcinogenicity Studies Of Pharmaceuticals; Nov 1995
8) ICH S5(R2) Guideline: Detection of toxicity to reproduction for medicinal products including toxicity to male fertility; Nov 2005
9) ICH S8 Guideline: Immunotoxicity Studies for Human Pharmaceuticals; Sep 200
10) ICH S10 Guideline: Photosafety Evaluation of Pharmaceuticals; Nov 2012


11) Q&A on the withdrawal of the “Note for guidance on single dose toxicity” EMA/CHMP/SWP/81714/2010;  Jun 2010

12) Guideline on Repeated dose toxicity CPMP/SWP/1042/99 Rev. 1 Corr; Nov 2010

13) Non-clinical local tolerance testing of medicinal products CPMP/SWP/2145/00; Feb 2001

14) Guideline on Non-Clinical Documentation For Herbal Medicinal Products in Applications for Marketing Authorisation (Bibliographical and Mixed Applications) and in Applications for Simplified Registration; Jul  2006

15) Guideline on Selection of Test Materials for Genotoxicity Testing for Traditional Herbal Medicinal Products/ Herbal Medicinal Products; Jul 2010

16) Guideline on The Assessment of Genotoxicity ofSubstances/Preparations; Dec 2008

17) Guideline on The Clinical Assessment of Fixed Combinations of Herbal Substances / Herbal Preparations; Jan 2006

18) Guideline on The Assessment Of Clinical Safety And Efficacy in The Preparation of Community Herbal Monographs for Well-Established and of Community Herbal Monographs / Entries to The Community

List for Traditional Herbal Medicinal Products / Substances / Preparations; Sep 2006



19) Guidance For Industry – Botanical Drug Products; Jun 2004

20) Guidance for Industry - Complementary and Alternative Medicine Products and their Regulation by the Food and Drug Administration; Dec 2006
21) Review of Botanical Drug Products; Feb 2012





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