alternatives to animals in toxicity testing

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Alternatives to animals used in Teratogenicity testing

Presented by :

Sandhya TallaM.Pharm (Pharmacology)

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TERATOLOGY• Introduction- Teratology is the study of abnormal prenatal development and

congenital malformations induced by exogenous chemical or physical agents, continues to be a burgeoning area of medical research in the quest for the eradication of preventable birth defects.

The term “teratology” was first coined in 1832 by the French physician Isidore Geoffroy Saint-Hilaire to define a field of science dedicated to studying developmental anomalies.

Alternatives to animals uesd in Teratogenicity testing

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Teratogen: An agent which produce a permanat

alteration in structure or function of organism exposed during embryonic or fetal life.


Teratogenesis: Significant occurrence of structural or functional abnormalities in the infant after being administered to either parent before conception , to the mother during pregnancy , or directly to the developing fetus.

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Developmental Toxicity: Involves any adverse effect induced prior

to attainment of adult life.

Reproductive Toxicity: Represents harmful effects on the progeny

and/or an Impairment of male and female reproductive function or

capacity.

NOAEL:Is the abbreviation for No–Observed–Adverse–Effect Level

and is the highest dose level where no adverse effects are observed.Alternatives to animals uesd in Teratogenicity testing

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PRINCIPLES OF TERATOLOGY - • In 1973 , Wilson developed 6 principles of Teratology

1. Susceptibility to teratogenesis depends on the genotype of the conceptus and how it interacts with the environment

2. Susceptibility to a teratogenic agent varies with the developmental stage at which the exposure occurs

3. Teratogenic agents act in specific ways on developing cells and tissues to initiate abnormal embryogenesis (pathogenesis)


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4. The final manifestations of abnormal development are death, malformation, growth retardation and functional disorder.

5. Access of an adverse environmental agent to developing tissues depends on the nature of the agent.

6. The manifestations of deviant development increase in degree as dosage increases from the no-effect to the lethal level.


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Mutational changes in DNA sequences

Choromosomal abnormalities

Alteration/inhibition of intracellular metabolism

Inhibition of DNA/RNA synthesis

Mitotic interference

Cell death through direct cytotoxic effects]]]

Physical disruption of cells/tissues

Interference with cell differentiation

Formation Of Teratogenicity

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.• The following model are used in testing of teratogenisity

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1) Zebrafish Teratogenicity Prediction Model

2) Micromass Teratogen assay

3) The Embryonic Stem Cell Test


Alternative models used in Teratogenicity testing -

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1) Zebrafish Teratogenicity Prediction Model :

• Animal – Zebrafish.• Mating procedure – 2:1• Light cycle – 14/10 hr. light/dark cycle.• Food – 2-3 hr./day.• Water temp. – 26.1-29.4ºC• pH – 6.8 & 7.4


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• Assay Design and Morphological Scoring –


2-4 hrs. post-fertilization (hpf)

Blastula – stage embryos were collected

Dish containing pH 7.2 embryo medium

Chorions removed by enzymatic degradation

Placed in

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2mg/ml pronase E solution in embryo medium for 1 min.

Placed in dish of fresh embryo medium

Embryo transferred into well 24 well culture plate

6 Embryos per untreated control

Wash

Incubate at 28.5ºC for 2-3hrs.

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Larval assessed for viability & functional development

No heart beat, growth retardation, partly/wholly body degraded

Larval length, CVS function , pigmentation

video

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•


Fig-1 Schematic of zebrafish teratogenicity assay method.

Morphology of the 5-dpf zebrafish larva. A: Lateral viewof the 5-dpf zebrafish. B: Close-up of anterior region. C: Larva with normal pigmentation level and melanophore pattern (arrows). D: Larva with reduced pigmentation but normal melanophore pattern (arrows).

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• Evaluation –

1. Morphological and functional scoring.

2. LC25 is determined - LC25 of compound calculated in XLfit by curve-fitting of incidence

data for dead/moribund larvae at 5dpf

3. Teratogenic Index = NOAEL LC25 Ratio ≥ 10 (Teratogen) Ratio ≤ ( Non – Teratogen)

3. LC25 is determined


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• Evaluation of assay parameters - Concordance = % of all compounds tested that were correctly

classified.

Concordance of teratogens = % of invivo mammalian teratogens with +ve result in assay.

Concordance of non-teratogens = % of invivo mammalian non-teratogens with -ve result in assay.

Positive predictivity = % of +ve results among all substances with +ve assay outcome.

Negative predictivity = % of -ve results among all substances with -ve assay outcome.


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2. In vitro micromass Teratogen Assay by INVITTOX protocols no.122 & no.114 –

Objective- This method is intended to identify substances which

induce malformation resulting in embryotoxicity.The micromass test involving culture of differentiating rat, mouse or chicken embryo limb and midbrain cells exposed to test agents may be usefull of a battery of in vitro tests for teratogens.


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• Limbic bud (LB) & midbrain (MB) cell cultures -


Cultured according to INVITTOX protocols no.122 & no.114 respectively

Embryos transferred to Hank’s balanced salt solution

Fore & hind LB & MB isolated

Killed by cervical dislocation

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Incubated in Ca++ & Mg++ free phosphate buffered saline (PBS) for 20 min

PBS replaced with 0.5% trypsin in PBS for 10 min

Suspension pass through stainless steel 200 mesh filter

Single cell suspension

Cells counted in haemocytometer

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LB – 2 x 107 cells / ml MB – 5 x 106 cells / ml

5µl drop of cell in well (96 well microplate)

300µl medium with/without test chemicals add in well

Incubate 2-3 hrs.

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Incubate culture again for 5 days

•Assessment of Emryotoxicity -

LB cell culturesStain

1% Alcian blue in 0.1 N HCl overnight

Absorbance at 620 nm

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MB cell cultures are fixed by 4% formaldehyde

Harries haematoxylin

Images of foci of differentiated cells captured by camera linked microscope & analysed by computer image analysis

software image

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• Evaluation – IC50 calculation -

IC50 – P = Proliferation cytotoxicity

IC50 – D = Differentiation Embryotoxicity


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• Flint’s rule -

• IC50 – D < 10µg/ml (strong teratogen)• IC50 – D > 10µg/ml ( +ve potential teratogen)


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• 3. Cytotoxicity test using Blastocyst-derived euploid Embryonal stem cells:

Objective- Pluripotent embryonal stem cells (ESC) derived from

mouse blastocysts were established. ESC are able to differentiate into a variety of embryonal

tissues, retain the euploid chromosome constitution, and proliferate very rapidly. Therefore, they may provide an ideal tool for testing developmental toxicity after exposure of teratogen.


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Embryonal stem cells culture-

Mice strain – Swiss mice. Tempreture – 220 C Housed atmosphere Humidity – 60 %


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Cultured 6 to 7 day using DMEM with supplememented 10% Fetal Calf Serum

Blastocyte-stage Embryo were flushed on Day 3 of pregancy

Inner mass cell was isolated Mechanically from trophoblast cell

Mass cell clump was trypsinised

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Sufficient no of cell were frozen and store in liquid nitrogen

Stem cell were subcultivated by repeating the Disaggregation procedure

Collnies were inspected a few day latter

Disaggregated and transfer dish containing Feeder cell

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• Fibroblast culture –


Suspension was pippette out & transfer to tube contain growth medium

Placed in fresh dish containing trypsin EDTA solution

Embriyo dissected on day 14 of pregancy

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• Experimental Design –


Cells were prepared in Parallel

70% of fibroblast & 50% ES cell are incubated for 24 hr

96 wells were incubated 0.1 ml medium containing

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After 24 hr removed medium by decanting and cells refed with medium containing 5% FCS &

Test agnt and Incubated for another 24 hr

video

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• Evaluation –

• 1) The kenacid blue (KB) Method for cytotoxicity testing was performed.

• 2) The Neutral red (NR) assay.

% Absorbance obtained which indicate the cytotoxicity. No of cell servive which shows the teratogen potential to produced toxicity.


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• Reference – • Brannen, K.C. et al., 2010. Development of a zebrafish embryo teratogenicity assay and

quantitative prediction model. Birth Defects Research Part B - Developmental and Reproductive Toxicology, 89(November 2009), pp.66–77.

• Garfield, E., 1986. Teratology Literature and the Thalidomide Controversy. Essays of an Information Scientist, pp.3–11. Available at: http://garfield.library.upenn.edu/essays/v9p404y1986.pdf.

• Knight, A., Balcombe, J. & Bailey, J., 2005. The future of teratology research is in vitro. Biogenic Amines, 19, pp.97–145.

• Laschinski, G., Vogel, R. & Spielmann, H., 1991. Fibroblasts Cytotoxicity tests. , 5(June 1990), pp.57–64.

• Memon, S. & Pratten, M., 2013. Teratogenic effects of two known teratogens ( Nicotine and Cadmium ) and prevention of such effects by addition of antioxidants in chick embryos : An evaluation of two culture systems ( Micromass and in ovo culture ). , 7(5), pp.27–38.

• Smith, D.W., 1973. Teratology. , pp.1–4.• Standard, T. et al., 2009. The Micromass Test - Method of Brown DB-ALM Protocol n ° 122

Data Analysis / Prediction Model. , pp.1–16.


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