1. CTA: Cell Transformation Assay
on BALB/c 3T3
David Ramos
Investigador CERETOX
dramos@pcb.ub.cat
JORNADA TOX®
1 de Febrero de 2013
Parc Científic de Barcelona

2. Principle of the test
Reference
OECD DRP 31
 In vitro test:
 Phenotypic alterations (characteristic of tumorigenic cells) in cultured cells
induced by carcinogens. These cells induce tumours in susceptible animals
(Berwald and Sachs, 1963, 1965)
 Fast
 Cost efficient
 Initial screening for carcinogenic potential
 3 types of CTA:
 Syrian hamster emrbyo cell (SHE pH:6.7)
 Syrian hamster emrbyo cell (SHE pH:7)
 BALB/c 3T3

3. Materials
 Experimental system:
 Clone A31-1-1 derived from BALB/c 3T3
 Transformation: associated with malignancy
 Sensitive to tumour-promoting agents
 Morphological changes related to neoplasia
 immortals, autocrine GF, tumorigenecity…
 Medium: DMEM+10% hiFBS+Ab
 Solvent Control:
 Water, DMSO, acetone and ethanol < 5%, 0.2%, 0.5%, 0.1%
 Positive Control:
 1-stage : 5µg/mL MCA
 2-stage : 0.2µg/mL MCA as initiator + TPA 0.2 µg/mL as tumour promoter
Ref. Kakunaga and Crow (1980)
MCA = 3-methylcholanthrene
TPA = 12-o-tetradecanoylphorbol-13-acetate

4. Experimental design: cytotoxicty test
 Cells seeded at 100 cells/60-mm dish,
3 dishes per treatment, 24 h
 Wide range: 5 concentrations
 Exposure: 72 h + 7 day fresh medium
 Fixed and stained with 20% Giemsa’s solution
Solvent control
 Determined by colony-forming efficiency (CFE)
respect control
Colonies
Concentration Replica 1 Replica 2 Replica 3 Replica 4 CFE (%) Effect
NP 197 200 0 0 0 0 0,00 100,00
NP 197 66,66 3 5 6 14 5,69 94,31
NP 197 22,22 15 18 19 52 21,14 78,86
NP 197 7,4 27 37 33 97 39,43 60,57
NP 197 2,469 60 69 70 199 80,89 19,11
Solvent control 5% H2O 74 92 80 246 100,00 0,00
Positive Control 3% DMSO 30 35 41 106 43,09 56,91
Positive Control, DMSO 3%

5. Experimental design: transformation assay
Cells seeded at 104cells/60-mm dish,
 5 concentrations: 10 dishes/treatment, 4 mL culture medium, 24 h
 High: 80-90% decrease in
CFE 1-stage CTA 2-stage CTA
 3 intermediate doses Non-genotoxic
Genotoxic substance
 Low: NO effect in CFE 72 h
susbtance 72h as MCA 72h as inductor
inductor
 Non toxic substances: Medium x2 / week refilled with fresh
normal medium for 3 days medium for 3 days
Refilled with fresh Refill
 Soluble: ≤ 5 mg/mL during 3½ weeks
 Insoluble: ≤ 2-times Medium x1 / week TPA as promotor Substance as
x2/week promotor x2 / week
visible solubility during 2 weeks during 2 weeks during 2 weeks
Medium x2 / weekmedium 1 week
Mediumx2/ week during x2/ week
during week during week
once a week once a week
during the following week
Medium1x / 2 during 2 following
during the weeks 2
weeks weeks
Fixed and stained with 20% aqueous Giemsa for scoring
focus formation

6. Results
 The average number of foci Type III per dish in a treatment group
(Foci type III: Spindle-shaped markedly basophilic cells. Piling-up and criss-crossing, clear and marked).
 Statistical analysis of treatments relative to the solvent control
2-stage CTA MCA as inductor+TPA as promotor Foci Type III
60
Treatment Concentration(µg/ml) FD/TD* Foci III/dish Foci Type III
50
Foci III/dish
Solvent Control 10,00% 9/10 3,30 40 Foci Type III
30
Substance 78,125 9/10 3,10 20
Substance 156,25 10/10 4,40 10
0
Substance 312,5 9/10 4,90 10,00% 78,125 156,25 312,5 625 1250 0.2
Substance 625 9/9 5,11 Solvent Substance Substance Substance Substance Substance Control MCA
Control
Substance 1250 9/10 3,50
Concentration(µg/ml)
Control MCA 0.2 10/10 52,00
FD/TD: Number of dishes with foci/total number of dishes examined

7. CERETOX
Parc Científic de Barcelona - Edifici Cluster
c/Baldiri Reixac, 10-12
08028 Barcelona
T. 93 403 71 83
www.pcb.ub.cat/ceretox
ceretox@pcb.ub.cat
http://goo.gl/BGLwt
http://goo.gl/VBsu7