ALTERNATIVES TO ANIMAL IN TOXICITY TESTING

Phototoxicity and Carcinogenecity

Presentation by-Sandhya TallaM.Pharm (Pharmacology)

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IntroductionIntroductionPhototoxicity-Chemically induced skin irritation,

requiring light, that does not involve the immune system.

Types of Phototoxicity- 1) Photodynamic2) Non photodynamic

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PhototoxiPhototoxicitycity

In vitro 3T3 neutral red uptake(NRU) photo toxicity test

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3T3 NRU PT in Phototoxicity evaluation parameter of chemical substance of (OECD TG 432)

abso

rption No absorption

UV absorbanceNo UV

absorbance

Negative

Negativepositive

positive

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Conformation of UV absorbance

Yeast growth inhibition Phototoxicity assay

Red blood cell photohemolysis

Phototoxicity negative end of examination

The examination is over end of examination

Phototoxicity positive end of examination

During 1992-1997, the 3T3 NRU PT test was refined and

evaluated in the EU/COLIPA (European Cosmetic Toiletry

and Perfumery Association) international validation study

on in vitro tests for phototoxic potential.

It was further validated by ECVAM (European centre for

Validation of Alternative Methods)

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The Dermal Phototoxicity of a chemical is defined as-

A toxic response that is elicited after exposure of skin to the

chemical or systemic administration of the chemical, and

subsequent exposure to light.

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Concentration-dependent reduction of the uptake of the

vital dye Neutral Red measured 24 hours after treatment

with the test chemical and irradiation.

The essence of the in vitro 3T3 NRU test phototoxicity test

is to Compare cytotoxicity of the chemical in presence and

absence of the non-cytotoxic UVA/ visible light

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Principle of the 3T3 NRU Test :

According to the first law of photochemistry,

Photoreaction requires sufficient absorption of light quanta.

OECD Test Guideline 101 suggests:

If the molar extinction/absorption coefficient is less than 10

litre/mol-1/cm-1 the chemical is unlikely to be Photoreactive.

Such chemical may not need to be tested in the 3T3 NRU

phototoxicity test or any other biological test for adverse

photochemical effects.

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The test is not designed to predict other adverse effects that

may arise from combined action of a chemical and light.

For example, it does not address:

Photo allergy

Photo carcinogenicity

Or assessment of phototoxic potency.

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Requirements: A permanent mouse fibroblast cell line.

Balb/c 3T3.

Clone 31, (ATCC), US/ (ECACC), UK.

Cells are seeded in culture medium at the appropriate

density so that cultures will not reach confluence by the end

of the test

For Balb/c 3T3 cells grown in 96-well plates, the

recommended cell seeding density is 1 X 104cells per well.

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Test chemicals shall be dissolved in buffered salt solutions.

For example Earle’s Balanced Salt Solution (EBSS).

It must be free from protein components and light absorbing

components (e.g., pH-indicator colors and vitamins) to avoid

interference at Irradiation condition.

Test chemicals of limited solubility in water should be dissolved

in an appropriate solvent.

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Irradiation condition:Light of the UVA and Visible regions is usually associated

with phototoxic reactions.

whereas generally UVB is of less relevance but is highly

cytotoxic.

The cytotoxicity increases 1000-fold as the wavelength goes

from 313 to 280 nm.

Xenon arcs and mercury-metal halide arcs are used as solar

simulators.

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A dose of 5 J/cm2 was determined to be non-cytotoxic to

Balb/c 3T3 cells (when measured in the UVA range).

This dose is sufficiently potent to excite chemicals to elicit

phototoxic reactions.

For example, To achieve 5 J/cm2 within a time period of 50

min, irradiance was adjusted to 1.7 mW/cm2.

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The highest concentration of the test substance should be

within physiological test conditions.

For example, osmotic and pH stress should be avoided.

The maximum concentration of a test substance should

not exceed 1000microg/mL; osmolality should not exceed

10 mM.

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A test meets Acceptance criteria if:

The EC50 + UVA is within : 0.1 -2.0 mg/ml.

The EC50 – UVA is within: 7.0 -90.0 mg/ml.

The factor (PIF) is at least 6.

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Procedure of Procedure of 3T3 NRU3T3 NRU

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Cell viability is expressed as percentage of untreated

solvent controls and is calculated for each test

concentration.

The concentration responses obtained in the presence and

in the absence of irradiation are compared.

Usually at the IC50 level, i.e., the concentration reducing

cell viability to 50 % compared to the untreated controls. 18

Day 1

Day 2: Add drug soln 8 diff Concentrations incubate for 60 min

Incubate in presence and absence of UV light (50 min)

Decant drug soln Incubate overnight

Day 3: Wash the cells .Add Neutral red solution (100 microL of 50 microg/ml) incubate for 3 Hrs

Wash the cells. Decant & remove NBT. Add desorb solution

Read at 540 nm

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The value of PIF is higher if the compound is phototoxic.

i.e. the concentration that kills the cells in presence of UV

light is less that the concentration that kills the cells in

darkness

CarcinogenecityCarcinogenecity

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TYPES OF CARCINOGENSTYPES OF CARCINOGENS

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TEST FOR DETECTION OF GENOTOXIC CARCINOGENSTEST FOR DETECTION OF GENOTOXIC CARCINOGENS

Ame’s assay:Ame’s assay: Measure the mutation at the Histidine regulating gene of

(S.typhy).

Procedure : Bacteria co-incubated with drug at different

concentrations and incubated on histidine free medium.

The colonies are counted which show number of bacteria

that have undergone reverse mutation.

The no of colonies growing on the histidine free medium

indicate mutagenicity of the test chemicals.23

L5178Y Mouse Lymphoma L5178Y Mouse Lymphoma Assay (MLA)Assay (MLA)

This mutagenesis test is done in mammalian cells, and therefore

may be a more similar model for chemical mutagenicity in

human cells.

Mutant cells, deficient in TK due to the forward mutation in the

TK locus (from TK+ toTK-).

TK-deficient cells are resistant to the cytotoxic effect of the

lethal analogues.24

The TK-competent L5178Y (TK+/+or TK+/-) cells are treated

with the test agents.

The cells are shifted to a selective medium containing the

lethal analogues.

Only the mutant cells (TK-/-) can survive under the selection

condition.

Mutagenicity of the test compound is evident by the increase

in the number of mutants.

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TEST FOR DETECTION OF NON GENOTOXIC CARCINOGENSTEST FOR DETECTION OF NON GENOTOXIC CARCINOGENS Syrian Hamster Embryo (SHE) Cell Transformation AssaySyrian Hamster Embryo (SHE) Cell Transformation Assay

The SHE cells are normal diploid, metabolically and p53competent primary cells, which retain the ability to bio transform xenobiotic

Exposure to carcinogenic chemicals results in an increase of morphologically transformed (MT) colonies, which are characterised by disorganised growth patterns and considered as an early stage in the carcinogenic process.

SHE cells can be morphologically transformed by treatment with genotoxic and non genotoxic carcinogens.

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ProcedureProcedure

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Feeder cell Target cell

x-ray irradiation

Inactivate their capability to replicate, and seeded as

nutrient base to support metabolic activity.

To assess morphological transformation of colonies.

After 13 days of gestation of hamster SHE cell is isolated (Primary culture)

Target cell seeded over feeder cell allowing development of colonies

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They are exposed to the test substance for 7 days.

cells are washed, fixed and stained

Dishes are coded and colonies are scored for their morphological phenotype by

stereomicroscopy

Cytotoxicity is evaluated by inhibition of cloning efficiency and reduction in

size/density of the colonies

Experimental DesignExperimental DesignDay 0 1 2 9---------------------------------------------- //---------------------------------

Feeder Target Treatment Fixing cells cells with test substance staining(2 mL) (2 mL) (4 mL)

Timeline of the SHE CTA assay (the volumes are per each 60 ml culture dish)

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Morphological transformation The morphological transformation frequency (MTF) should be

calculated for each concentration level

MTF= number of transformed colonies x 100 total number of scorable colonies Cytotoxicity The plating efficiency (% PE) and the Relative plating efficiency (%

RPE) will be calculated as follows: PE = total number of colonies per dish x 100 total number of target cells seeded per dish RPE = PE of dose group x 100 PE of the vehicle control group

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Test chemicals are scored as positive if :

At least two dose groups showed a statistically significant

increase in morphological transformation frequency OR

One dose group showed a statistically significant increase and

the trend test was significant at P>=0.05

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REFERENCESREFERENCES1) Long ME. Predicting carcinogenicity in humans : The need to supplement animal-based

toxicology. 2008;553–9. 2) Carcinogenicity V, Embryo SH, Transformation C, Introduction A, Dna S. In Vitro

Carcinogenicity : Syrian Hamster Embryo ( SHE ) Cell Transformation Assay. 2012;(June). 3) Ohmori K. In Vitro Assays for the Prediction of Tumorigenic Potential of Non-genotoxic

Carcinogens. 2009;55(1):20–30. 4) Holzh H. In Vitro Phototoxicity Testing : Development and Validation of a New

Concentration Response Analysis Software and Biostatistical Analyses Related to the Use of. 2002;415–32.

5) Law G, Guideline OT. S ÁGQTÁs€…€…€‰tht…Á…p„…Á. 2004;(April):1–15. � �6) Bouvier M, Bremer S, Casati S, Ceridono M, Coecke S, Corvi R, et al. CHAPTER 10 ECVAM

AND NEW TECHNOLOGIES FOR TOXICITY TESTING. 2012;154–80.

7) Eisenbrand G, Pool-zobel B, Baker V, Balls M, Blaauboer BJ, Boobis A, et al. Methods of in

vitro toxicology. 2002;40:193–236.

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