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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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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.
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