The document discusses alternatives to animal toxicity testing for phototoxicity and carcinogenicity evaluation. It summarizes the 3T3 neutral red uptake photo toxicity test used to assess phototoxic potential in vitro. It also describes tests to detect genotoxic carcinogens like the Ames assay and mouse lymphoma assay, and non-genotoxic carcinogens using the Syrian hamster embryo cell transformation assay which can identify carcinogens through morphological transformation of cells.

1. ALTERNATIVES TO
ANIMAL IN TOXICITY
TESTING
Phototoxicity and Carcinogenecity
Presentation by-
Sandhya Talla
M.Pharm (Pharmacology)
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2. IntroductionIntroduction
Phototoxicity-
Chemically induced skin irritation,
requiring light, that does not involve
the immune system.
Types of Phototoxicity-
1) Photodynamic
2) Non photodynamic
2

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

4. 4
3T3 NRU PT in Phototoxicity evaluation parameter of chemical
substance of (OECD TG 432)
absorption
No
absorption

5. UV absorbance
No
UVabsorbance
Negative
Negative
positive
positive
5
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

6. 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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7. 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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8.  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 :

9.  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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10.  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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11. 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 104
cells per well.
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12.  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.
12

13. 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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14. 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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15. 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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16.  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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17. Procedure of 3T3Procedure of 3T3
NRUNRU
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18.  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.
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19. 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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20. 20
 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

21. CarcinogenecityCarcinogenecity
21

22. TYPES OF CARCINOGENSTYPES OF CARCINOGENS
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23. 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.
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24. L5178Y Mouse Lymphoma AssayL5178Y Mouse Lymphoma Assay
(MLA)(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.
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25. 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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26. 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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27. ProcedureProcedure
27
Feeder cellTarget
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

28. 28
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

29. Experimental DesignExperimental Design
Day 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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30.  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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31.  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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32. REFERENCESREFERENCES
1) 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 ÁGQTÁs€…€…€‰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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