Balb/c 3T3 CTA PRP report v1 - 30 July 2010

30 July 2010

Balb/c 3T3 Cell Transformation Assay

Prevalidation study Report

Validation Management Team
Philippe Vanparys
Leonard Schechtman
Marilyn Aardema
Makoto Hayashi
Sebastian Hoffmann
Laura Gribaldo
Raffaella Corvi

ALTOXICON BVBA (Vosselaar, Belgium)Innovative Toxicology Consulting LLC (Lake Worth, Florida, USA)
Marilyn J. Aardema Consulting LLC (Fairfield, Ohio, USA)
Biosafety Research Center, Foods, Drugs and Pesticides (Shizuoka, Japan)
she consulting + services (Köln, Germany)
ECVAM (Ispra, Italy)
ECVAM (Ispra, Italy)

Study Assistants
B. Claire Thomas
Pascal Phrakonkham

ECVAM (Ispra, Italy)
ECVAM (Ispra, Italy)

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

Contents
CONTENTS ............................................................................................................................. 2
LIST OF ABBREVIATIONS ..................................................................................................... 5
SUMMARY .............................................................................................................................. 6
PREFACE................................................................................................................................ 7
1

RATIONALE FOR THE PROPOSED TEST .................................................................... 8
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8

2

INTRODUCTION ............................................................................................................................. 8
INTENDED USE ............................................................................................................................. 9
CURRENT USE ............................................................................................................................ 10
RECENT RESEARCH .................................................................................................................... 10OECD DETAILED REVIEW PAPER ................................................................................................ 11
PUBLISHED DATA ON BETWEEN-LABORATORY REPRODUCIBILITY .................................................... 11
RELEVANT MEETINGS .................................................................................................................. 12
PATENTS .................................................................................................................................... 13

ORGANISATION OF THE STUDY ................................................................................ 14
2.1 VALIDATION MANAGEMENT TEAM ................................................................................................ 14
2.2 LABORATORIES INVOLVED ........................................................................................................... 15
2.3 QUALITY ASSURANCE SCHEMES EMPLOYED BY THE TESTING LABORATORIES .................................. 15
2.4 CHEMICALS TESTED IN THE BALB/C 3T3 PREVALIDATION STUDY .................................................... 15
2.4.1
Chemical selection........................................................................................................... 16
2.4.2
Modules 2 and 3: Within-laboratory reproducibility and transferability (coded and noncoded) ......................................................................................................................................... 17
2.4.3
Module 4: Between-laboratory reproducibility (coded compounds) ................................ 17
2.4.4
Coding/decoding.............................................................................................................. 17
2.5 ASSESSMENT OF WITHIN- AND BETWEEN-LABORATORY REPRODUCIBILITY ...................................... 17
2.6 STUDY TIMELINE ......................................................................................................................... 18

3

MODULE 1: TEST DEFINITION .................................................................................... 20
3.1 SCIENTIFIC BASIS FOR THE PROPOSED TEST METHOD ................................................................... 20
3.2 DESCRIPTION OF THE ENDPOINT PREDICTED AND THE MECHANISTIC BASIS OF THE TEST ................. 20
3.3 BIOLOGICAL TEST SYSTEM: BALB/C 3T3 CELL LINE........................................................................ 20
3.4 BALB/C 3T3 CELL TRANSFORMATION ASSAY................................................................................ 21
3.5 PROTOCOL OPTIMISATION PHASE ................................................................................................. 21
3.5.1
Preliminary experiments .................................................................................................. 22
3.5.2
Results............................................................................................................................. 23
3.5.3
Conclusion of the Validation Management Team on preliminary experiments ............... 26
3.6 PROTOCOL ................................................................................................................................. 26
3.6.1
Balb/c 3T3 cell line........................................................................................................... 26
3.6.2
Serum and medium ......................................................................................................... 26
3.6.3
Controls ........................................................................................................................... 27
3.6.4
Test procedure................................................................................................................. 27
3.6.5
Statistical Analysis of raw data ........................................................................................ 28
3.6.6
Assay acceptance criteria................................................................................................ 30
3.6.7
Assay assessment criteria............................................................................................... 30

4

MODULE 2: WITHIN-LABORATORY REPRODUCIBILITY .......................................... 32
4.1 TRANSFORMATION ASSAY – NON CODED 3-METHYLCHOLANTHRENE .............................................. 32
4.2 TRANSFORMATION ASSAY – CODED 3-METHYLCHOLANTHRENE ..................................................... 33
4.2.1
Concurrent cytotoxicity: Crystal Violet ............................................................................. 35
4.2.2
Concurrent cytotoxicity: Colony Forming Efficiency ........................................................ 35
4.2.3
Morphological transformation .......................................................................................... 362

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
4.2.4
Acceptance criteria .......................................................................................................... 37
4.3 STATISTICAL ANALYSIS ................................................................................................................ 37
4.4 CONCLUSIONS OF THE VALIDATION MANAGEMENT TEAM ON MODULE 2......................................... 38

5

MODULE 3: TRANSFERABILITY.................................................................................. 39
5.1
5.2
5.3

6

GENERAL ASPECTS .................................................................................................................... 39
TRAINING ................................................................................................................................... 39
CONCLUSIONS OF THE VALIDATION MANAGEMENT TEAM ON MODULE 3........................................ 39

MODULE 4: BETWEEN-LABORATORY REPRODUCIBILITY ..................................... 40
6.1 2-ACETYLAMINOFLUORENE ......................................................................................................... 40
6.1.1
Dose-range finding test ................................................................................................... 40
6.1.2
Transformation assay ...................................................................................................... 41
6.1.3
Conclusion ....................................................................................................................... 46
6.2 BENZO(A)PYRENE ....................................................................................................................... 46
6.2.1
Dose-range finding test ................................................................................................... 46
6.2.2
Transformation Assay...................................................................................................... 47
6.2.3
Conclusion ....................................................................................................................... 51
6.3 ANTHRACENE ............................................................................................................................. 52
6.3.1
Dose-range finding test ................................................................................................... 52
6.3.2
Transformation assay ...................................................................................................... 52
6.3.3
Conclusion ....................................................................................................................... 57
6.4 PHENANTHRENE ......................................................................................................................... 57
6.4.1
Dose-range finding test ................................................................................................... 57
6.4.2
Transformation assay ...................................................................................................... 57
6.4.3
Conclusion ....................................................................................................................... 63
6.5 O-TOLUIDINE HCL....................................................................................................................... 63
6.5.1
Dose-range finding test ................................................................................................... 63
6.5.2
Transformation assay ...................................................................................................... 64
6.5.3
Conclusion ....................................................................................................................... 69
6.6 DISTRIBUTIONS OF ALL EXPERIMENTAL CONTROLS ........................................................................ 69
6.7 COMPARISON OF CFE AND CV METHODS FOR CYTOTOXICITY ASSESSMENT................................... 70
6.8 CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON BETWEEN-LABORATORY REPRODUCIBILITYRESULTS .............................................................................................................................................. 71

7

SUMMARY OF RESULTS ............................................................................................. 72
7.1
7.2
7.3
7.4
7.5
7.6

3-METHYLCHOLANTHRENE .......................................................................................................... 72
2-ACETYLAMINOFLUORENE ......................................................................................................... 72
BENZO(A)PYRENE ....................................................................................................................... 73
ANTHRACENE ............................................................................................................................. 73
PHENANTHRENE ......................................................................................................................... 73
O-TOLUIDINE HCL ....................................................................................................................... 73

8

DISCUSSION ................................................................................................................ 74

9

OVERALL CONCLUSION BY THE VALIDATION MANAGEMENT TEAM ................... 76

10

RECOMMENDATIONS.............................................................................................. 78

11

REFERENCES .......................................................................................................... 79

12

CONTACT DETAILS OF PEOPLE INVOLVED IN THE VALIDATION STUDY......... 84

13

ANNEXES.................................................................................................................. 86

13.1
CHEMICALS SELECTED FOR THE PREVALIDATION OF BALB/C 3T3 CTA ...................................... 86
13.2
AMENDED PROTOCOL FOR PHASE 2 ........................................................................................ 87
13.3
EXAMPLE OF STATISTICAL ANALYSIS OF BALB/C 3T3 CTA RESULTS, USING THE GENERALISEDLINEAR MODEL APPROACH WITH A NEGATIVE BINOMIAL DISTRIBUTION ...................................................... 99
13.4
NISHIYAMA TRANSFORMATION ANALYSIS ................................................................................ 106

3

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
13.4.1
Summary of results........................................................................................................ 106
13.4.2
R-Code for the Nishiyama transformation, including comments ................................... 107
13.5
REPEATED EXPERIMENTS...................................................................................................... 109
13.5.1
HRI – Phenanthrene ...................................................................................................... 109
13.5.2
HRI – o-toluidine HCl ..................................................................................................... 109

4

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

List of Abbreviations
CRO
CFE
CTA
CV
DMEM
DMEM/F12
DMSO
DRF
DRP
ECM
ECVAM
ESAC
FBS
GLM
GLP
JaCVAM
MEM
MT
NOEL
OECD
PBS
PC
PE
qRT-PCR
REACH
RCG
rCFE
SHE
SOP
TA
VC
VMT

Contract Research Organisation
Colony Forming Efficiency
Cell Transformation Assay
Crystal Violet
Dulbecco's Modified Eagle Medium
Dulbecco's Modified Eagle Medium / Nutrient mixture F12
Dimethyl Sulfoxide
Dose-Range Finding
Detailed Review Paper
Expert Consultation Meeting
European Centre for the Validation of Alternative Methods
ECVAM's Scientific Advisory Committee
Foetal Bovine Serum
Generalised Linear Model
Good Laboratory PracticeJapanese Centre for the Validation of Alternative Methods
Minimum Essential Medium
Morphological Transformation
No Observed Effect Level
Organisation for Economical Development and Co-operation
Phosphate Buffered Saline
Positive Control
Plating Efficiency
quantitative real time-Polymerase Chain Reaction
Registration, Evaluation, Authorisation and Restriction of CHemicals
Relative Cell Growth
Relative Colony Forming Efficiency
Syrian Hamster Embryonic cells
Standard Operating Procedure
Transformation Assay
Vehicle Control
Validation Management Team5

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

Summary
The potential for a compound to induce carcinogenicity is a crucial consideration when establishinghazard and risk assessment of chemicals and pharmaceuticals in humans. To date, the standard
approach to assess carcinogenicity at a regulatory level is the 2-year bioassay in rodents. The
European legislation on chemicals (REACH, Registration, Evaluation, Authorisation and Restrictionof Chemicals), cosmetics, pesticides and biocides, all limit the use of animals for safety assessment. In
addition, rodent carcinogenicity studies are costly and time consuming and there is a critical need forthe availability and implementation of validated alternative test models that can reduce/replace the useof animals that would otherwise be employed in carcinogenicity assessments. Several in vitro
alternatives have been developed for predicting carcinogenicity. Of these, the in vitro genotoxicity
tests address only one mechanism involved in carcinogenicity, induction of genetic damage. In
contrast, in vitro cell transformation assays (CTAs) have been shown to involve a multistage processthat closely models some stages of in vivo carcinogenesis and have the potential to detect both
genotoxic and non-genotoxic carcinogens. As such, these tests are currently being used by academia,
the chemical, agro-chemical, cosmetic and pharmaceutical industries, and are conducted in-house as
well as at contract research organisations (CROs) to screen for potential carcinogenicity as well as
investigate mechanisms of carcinogenicity. CTAs are not used routinely for regulatory testing but they
are often used for internal safety assessment of chemicals, drugs, etc. and are considered worthwhilefor providing additional useful information to the prevailing tests that are used for assessing
carcinogenic potential.
A recent detailed review paper (DRP) of the Organisation for Economic Co-operation andDevelopment (OECD) on CTAs for the detection of chemical carcinogens (OECD, 2007) concluded
that the performance of Syrian hamster embryo (SHE) and Balb/c 3T3 CTAs were sufficiently
adequate and should be developed into formal OECD test guidelines. Further, the same OECD DRP
recommended that although considerable and sufficient data on the performance of the assays wereavailable, a formal validation of the assays, in particular focusing on development of a standardisedtransferable protocol and further information on assay reproducibility would be important for
preparation of such OECD test guidelines. Based on this and previous European Centre for the
Validation of Alternative Methods (ECVAM) workshops and expert meetings (Combes et al., 1999) aformal prevalidation study of the CTA using the the Balb/c 3T3 A31 cell line protocol was conductedfollowing validation modules 1 to 4 of the ECVAM validation procedure (Hartung et al., 2004) inorder to evaluate the within-laboratory reproducibility, test method transferability, between-laboratory
reproducibility and to develop a standardised state-of-the-art protocol. This prevalidation study is partof a larger program in which two additional variants of the CTA on the SHE cells were assessed: theCTAs using the protocols at pH 6.7 and 7.0, respectively.
In keeping with the objectives of this ECVAM's effort, the Validation Management Team (VMT)
concluded that, on the basis of the outcome of this prevalidation study, an improved Balb/c 3T3 CTA
protocol, incorporating the recommendations made by the VMT, has been developed. Further testingof this improved protocol is recommended in order to confirm its reliability.

6Balb/c 3T3 CTA PRP report v1 - 30 July 2010

Preface
The study presented in this report complements recent Organisation for Economic Co-operation andDevelopment (OECD) activities related to the cell transformation assays (CTAs). The study has beensupervised by a Validation Management Team (VMT) established by the European Centre for the
Validation of Alternative Methods (ECVAM). This report includes a short introduction on the context
and background of the study, the presentation of the results generated in the prevalidation study andthe conclusions and recommendations by the VMT. The conclusions are mainly based on the data
generated in this study, but they also take into account the information and experience on the CTA
publically available to date. It is the intention of this report to provide data and protocols that further
support the consideration of the CTA for use as an alternative method which could contribute to theassessment of the carcinogenic potential of chemicals.

7Balb/c 3T3 CTA PRP report v1 - 30 July 2010

1 Rationale for the proposed test
1.1 Introduction
Development and ultimate utilisation of new chemicals and pharmaceuticals requires, among otherprerequisites, the assessment of human safety. One of the main endpoints in this assessment process isthe determination of potential carcinogenicity. To date, the standard approach to assess carcinogenicity
for regulatory purposes is the 2-year bioassay in rodents (EU Annex V B32, 1998; OECD TG 451,
2008). However, these rodent carcinogenicity assays are associated with technical complexity, highcosts and high animal burden, as well as the uncertainty associated to extrapolating from rodent tohuman. With the entry into force of the new European chemical legislation REACH (Registration,
Evaluation, Authorisation and Restriction of Chemicals) (EU, 2006), the 7th Amendment to the
Cosmetics Directive (EU, 2003), and the EU revised requirements for pesticides and biocides (EU,2009), a need for alternatives to routinely employed full animal methods has arisen. The EU
Regulation on experimental animals also calls for limiting animal experiments to the extent possible(EU, 1986). Among the various in vitro alternatives for carcinogenicity prediction developed, theCTAs have been shown to be a multistage process which closely models key stages of in vivo
carcinogenesis (Landolph, 1985). It is worth mentioning that the CTA is to date the only establishedand promising in vitro assay that has the potential to detect both genotoxic and non-genotoxic
carcinogenic compounds. It also appears that the in vitro CTA can provide some critical evidence
which is specific to the tumourigenic process and that in vitro genotoxicity assays cannot provide.
Moreover, the test is faster and more cost efficient than the in vivo rodent carcinogenicity assay,
providing a useful approach for screening of chemicals with respect to their carcinogenic potential. As
a consequence, data generated using CTAs can facilitate early decision-making as to the need for
and/or experimental design of in vivo carcinogenicity bioassays.
CTAs are currently being used by academia, the chemical, agro-chemical, cosmetic andpharmaceutical industry, and are conducted in-house as well as at contract research organisations(CROs) to screen for potential carcinogenicity as well as investigate mechanisms of carcinogenicity.
CTAs are considered to provide additional useful information to more routinely employed tests forassessing carcinogenic potential and are therefore listed in various guidelines/testing recommendations
for such purposes. Since regulatory agencies receive and review CTA data and these assays are widelyused for internal risk assessment of various chemicals, there is a need within the scientific community
for standardization of these test methods and technical guidance on their conduct and use.
This need was already addressed in 1998 by a workshop organised by ECVAM on CTAs as predictorsof human carcinogenicity (Combes et al., 1999). The workshop concluded that the tests indeed arepromising but required further development, standardisation and verification. In 2007 the OECDpublished a detailed review paper (DRP) on the CTAs (OECD, 2007) concluding that theperformances of the Syrian hamster embryo (SHE) and Balb/c 3T3 CTAs were sufficiently adequate
and that these CTAs should be developed into formal test guidelines. However, considering theamount of available data reported in the literature, study results have been generated using different
test method protocols. In order to provide a basis for the development of CTA OECD test guidelines,
it therefore became important to harmonise and standardise those protocols. Furthermore, as withsome other assays with a long history of use, CTAs have not undergone formal validation inaccordance with current standards (OECD GD 34, 2005). The previous ECVAM workshop and therecent OECD DRP concluded that a formal validation of the assays, in particular focusing on the useof standardised protocols and reproducibility aspects would be necessary.
With that as a basis and following the recommendations of an expert meeting on cell transformation
held at the ECVAM in 2004, ECVAM’s next effort was to organise a prevalidation study of selectCTAs. It was determined that the SHE and Balb/c CTAs would undergo a prevalidation assessment
which would address issues of standardisation of protocols, within-laboratory reproducibility, test
method transferability, and between-laboratory reproducibility. The results of that study should add to8

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
the existing large database of chemicals evaluated over the history of use of these assays (OECD,2007). In particular, a standard protocol for each of the test methods should be defined which could be
used for further development of the sought-after OECD test guidelines. This exercise started in 2005.
In this prevalidation study three variants of the CTA were assessed: CTA with SHE cells at pH 6.7,
CTA with SHE cells at pH 7.0 and CTA using the Balb/c 3T3 cell line. In order to evaluate whether
the tests would meet the criteria stipulated by the ECVAM principles on test validity, the modular
approach of validation was followed (Hartung et al., 2004). In this study the following modules wereassessed:
1) Test definition,2) Within-laboratory reproducibility,3) Transferability,
4) Between-laboratory reproducibility,
Due to the specific objectives of this study and the resources available, a limited number ofcompounds was evaluated as it was not the intention of this study to comprehensively assess thepredictive capacity of the CTAs. That would require an exhaustive evaluation of numerous chemicals
and chemical classes employing the respective standardised multi-laboratory prevalidated protocols,
an effort that was considered beyond the scope of this undertaking. Nevertheless, the data generated bythis effort support the assessment of the predictive capacity of the CTAs, a retrospective analysis ofwhich was previously reported by the OECD (OECD, 2007).
Each CTA was conducted following the same agreed upon protocol in at least three different
laboratories. The laboratories involved encompassed industry, academia, CROs and government
establishments located in the USA, Japan and Europe.
The current report, which was prepared by the ECVAM with the support of the VMT, presents theoutcome of the prevalidation study on the CTA performed using the A31 subclone of the Balb/c 3T3
cell line protocol.

1.2 Intended Use
The possible use of the Balb/c 3T3 CTA is mentioned in various recent testing strategies including the
supplemental data for pharmaceuticals (Jacobson-Kram and Jacobs, 2005) and the guidance on
information requirements and chemical safety assessment for REACH (ECHA, 2008). For chemicals
produced above 1000 tonnes per year, the REACH guidance states that all relevant data from all
toxicity studies should be assessed to see whether a sufficiently reliable assessment about the
carcinogenicity of the chemical is possible, including alternative means if needed: i.e. predictive
techniques such as chemical grouping and read-across, and the use of (quantitative) structure-activity
relationships. On some occasions, it may be proposed to supplement these predictive approaches withshort term tests such as the in vitro CTA, cell proliferation assays or medium-term tests likegenetically engineered (transgenic) or neonatal models in order to circumvent the need for a chronic
carcinogenicity study. This would usually be in the context of adding information to the weight ofevidence that a chemical may be carcinogenic.
Based on performance of the Balb/c 3T3 assay, the OECD Expert Consultation Meeting (ECM) in
Washington DC which convened in October 2006 to finalise the OECD DRP on cell transformation(OECD, 2007), recommended that the Balb/c 3T3 CTA should be developed into an official OECD
test guideline. Although there was insufficient information on mechanism of action and usage specificfor pharmaceuticals, experts at the Washington ECM were of the opinion that the CTA was one
approach (among others) that could be used as a screen in a testing strategy for pharmaceuticals andwasn’t therefore limited to non-pharmaceuticals. In addition to its ability to identify potential
genotoxic rodent carcinogens, the Balb/c 3T3 CTA has shown promise in identifying non-genotoxic
carcinogens. It has been proposed for use as a second level in vitro screening test for carcinogenic

9

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
potential or even as a replacement for the in vitro mammalian cell genotoxicity assays with similar or
lower predictive capacity for chemical carcinogens (OECD, 2007).

1.3 Current Use
The Balb/c 3T3 test is mainly used in Japan by academia, the chemical, agro-chemical, cosmetic,
pharmaceutical and tobacco industries, and CROs to screen chemicals for their potential
carcinogenicity. Some current uses of the CTA are: (a) to provide useful ancillary information when
the biological significance of the bioassay result is uncertain (e.g. in pharmaceutical industry), (b) toclarify in vitro genotoxic positive results by weight of evidence (e.g. in chemical and cosmetic
industries), (c) to screen for non-genotoxic carcinogens (e.g. in agro-chemical industry), (d) todemonstrate differences and similarities across a chemical class (e.g. in chemical companies within
REACH), (e) to screen for efficacy of chemopreventive agents (e.g. in pharmaceutical industry), (f) toinvestigate tumour promotion activity (e.g. in agro-chemical and chemical industries), and (g) for
mechanistic studies of carcinogenicity (e.g. in academia and industry).
Regulatory agencies in general have been reluctant to unconditionally adopt CTA in their routine
safety testing schemes, especially as a full replacement for in vivo carcinogenicity testing, due, for themost part, to the lack of formal validation of such assays which demonstrate that the results obtained
are equal to or better than that generated in vivo. Furthermore, one of the main concerns has been thelack of objective criteria to identify transformed colonies/foci and which could affect the reliability ofthe test.

1.4 Recent researchThis section summarises some of the recent research activities related to the CTA employing theBalb/c 3T3 cell line or Balb/c 3T3-derived cell lines, independently of the protocol used.
Recent publications have suggested that transcriptomics analysis may offer some insight toward
increased understanding of the complex interactions that occur to biological systems when perturbed
by carcinogens. In two recent studies, transcriptomics analysis was applied to Balb/c 3T3 CTA to
identify gene signatures useful for the prediction of risk for carcinogenicity, as well as to determine themolecular mechanism of induction of Balb/c 3T3 cell transformation by specific chemicals. Using
cDNA microarrays Ao et al. (2010) analysed the gene expression to identify chemical associatedprofiles induced by a variety of tumour promoting agents in cells derived from transformed foci.
Characteristic clusters of genes were identified which included genes associated with oxidative stress,
cell proliferation and extracellular matrix, indicating the importance of specific molecular alterations
as potential biomarkers of exposure to tumour promoters (Ao et al., 2010). In the other study genome
wide gene expression analysis and quantitative real time-Polymerase Chain Reaction (qRT-PCR) wereapplied to both untransformed and transformed Balb/c 3T3 cells exposed to several chemicals used in
the present prevalidation study. The data describe selective and commonly regulated carcinogenic
pathways which are associated with carcinogenesis in several human cancers. This indicates that thesegenes are perhaps capable of predicting chemical carcinogenesis in vivo (Rohrbeck, in preparation).
A bottleneck in the conduct of the CTA is that classification of transformed foci relies on lightmicroscopy scoring by a trained human expert employing prevailing conventions. Recently, Poth et al.
(2007) have investigated the alterations in adhesion protein profiles and enzyme activities modulatingcell adhesion and migration that seem to be involved in the process of focus formation. Their data
indicate that these may represent promising candidates for the identification of molecular markers
leading to a more objective scoring of foci and a greater acceptance of cell transformation tests ingeneral (Poth et al., 2007). Another promising approach is the development of a focus classifier basedon image analysis and statistical classification that is under investigation in the CTA conducted with
the C3H10T1/2 cell line (Urani et al., 2009). If this method is proven to be reliable it could also be

10Balb/c 3T3 CTA PRP report v1 - 30 July 2010
explored for the identification of Balb/c 3T3 CTA transformed foci since the foci of the two cell lines
share many common features in their morphology.
An additional approach which aims at avoiding manual scoring, thus increasing the scoring objectivity
and the assay throughput used the soft agar colony formation assay in the screening of anticancer
compounds (Thierbach et al., 2009). The assay is based on the anchorage-independent growth
characteristics of malignantly transformed epithelial cells held in culture (i.e. their ability to grow insemi-solid medium). Currently, efforts are being undertaken to automate this assay.
A short-term CTA has recently been developed, using Bhas 42 cells which were established from
Balb/c 3T3 cells transfected by a v-Ha-ras gene. The Bhas 42 CTA has been reported to be capable ofdetecting both initiating and promoting activities of chemical carcinogens (Asada et al., 2005; Ohmori
et al., 2005; Sakai et al., 2009). The initiation assay involves a two-day treatment of low density cells,
and the promotion assay involves treatment of near-confluent cells with a test chemical for a period of
12 days. The sensitivity of the Bhas 42 CTA was shown to be equivalent to that of the two stage
Balb/c 3T3 CTA for the detection of promoting activities (Murumatsu et al., 2009). Currently,
JaCVAM (the Japanese Centre for the Validation of Alternative Methods) is coordinating thevalidation of the Bhas 42 CTA. This assay promises to be less time and labour intensive than theprevious Balb/c 3T3 CTAs and it has the potential to be used in a high throughput manner.

1.5 OECD Detailed Review Paper
Since a number of CTAs have been around for decades and a large number of chemicals have beentested over time using the CTA methods available, the OECD felt it necessary to draft acomprehensive document (“detailed review paper”) that captured as much relevant information as
possible in order to determine whether the data were sufficient and the time was right to develop
appropriate OECD test guidelines for one or more of the CTAs. This DRP, which is an extensive
collection of published data evaluating the performance of the different CTAs, provided an overview
of the three main types of assays, i.e. those which employ (a) primary SHE cells, (b) the Balb/c 3T3mouse fibroblast cell line and, (c) the C3H/10T½ mouse fibroblast cell line (OECD, 2007). The
performance of the Balb/c 3T3 CTA for the prediction of rodent carcinogenicity was reported for 149
compounds as follows: concordance 68%, sensitivity 75%, specificity 53%, positive predictivity 77%,
negative predictivity 50%, false positive 47%, false negative 25% (the prevalence, proportion ofcarcinogens, was 68%). Based on the available data the DRP concluded that the performances of the
SHE and Balb/c 3T3 CTAs were sufficiently adequate and warranted the development of formal
OECD test guidelines. However, to allay any reluctance in drafting such test guidelines and to help
ensure that those guidelines were, in fact, developed based upon validated test methods (OECD,2005), it became apparent that further important information addressing transferability and within- andbetween-laboratory reproducibility was necessary. Moreover, since the data evaluated in the DRP had,in some cases, been produced employing different procedures, there was a clear need, as an ultimate
goal, to develop standardised and reliable protocols from which the OECD test guidelines would be
generated.
In relation to this OECD effort, the development of new test guidelines for SHE and Balb/c 3T3 CTAs
have been included in the OECD work plan for the test guidelines programme. These activities will belead by France and Japan, respectively (OECD, 2009).

1.6 Published data on between-laboratory reproducibility
No formal between-laboratory trial has been previously conducted to fully assess the betweenlaboratory reproducibility of the Balb/c 3T3 CTA performed under the same conditions as thoseevaluated in the present study. However, two inter-laboratory studies on a variant of the Balb/c 3T3
CTA using a modified medium and a two-stage protocol are worth mentioning.

11Balb/c 3T3 CTA PRP report v1 - 30 July 2010
The first study has been published by Tsuchiya et al. (1999) and relates to the comparison of data
produced in 19 laboratories testing 3-methylcholanthrene during the initiation stage and 12-Otetradecanoylphorbol-13-acetate during the promotion stage. The initiating activity of 3methylcholanthrene and the promoting activity of 12-O-tetradecanoylphorbol-13-acetate were 100%
reproducible among laboratories (19/19) in the two-stage protocol. When 3-methylcholanthrene wastested alone, a significant positive response was obtained in 63% of the laboratories (12/19) and when
12-O-tetradecanoylphorbol-13-acetate was tested alone, a significant positive response was obtained in
47 % of laboratories (8/17).
The second study reports the comparison of results for four chemicals tested in four to fivelaboratories for their initiating activity, and for seven chemicals tested in three to five laboratories fortheir promoting activity (Tsuchiya et al., 2010). In the two-stage protocol, concordance was 100% forthree of the chemicals tested during the initiation phase and 50% (2/4 laboratories) for the lastchemical. Results for the chemicals tested during the promotion phase were reproducible among alllaboratories for six test chemicals whereas 2/3 laboratories consistently classified the last chemical.
When chemicals were tested alone (one-stage protocol), the concordance was 100% for 9/11
chemicals tested overall.
The data collected in the OECD DRP for the assessment of the performance of the CTAs enabled anassessment of some measure of reproducibility beyond that suggested by the above studies (OECD,2007). Excluding chemicals with only one reference, consistency between laboratories for the Balb/c
3T3 assay was 68.4% (39/57 chemicals). It should be noted that these results were produced using
different variants of the assay and the lower apparent reproducibility may be attributable to substantial
differences in protocols (e.g. one-stage or two-stages, different cell culture media).

1.7 Relevant meetings
ECVAM Workshop, 1998
A workshop on CTAs as Predictors of Human Carcinogenicity held in Angera, Italy in October 1998
was designed to seek a consensus on the approaches for advancing the use of the in vitro mammalian
CTAs, with the ultimate goal of (a) achieving regulatory acceptance and implementation of themethodology, and (b) reducing the number of animals employed to determine the carcinogenic
potential of agents that would otherwise induce malignant tumours in test animals (Combes et al.,1999). By demonstrating a strong correlation between the transformation of mammalian cells in vitro
and their ability to exhibit neoplasia in vivo, one could, hypothetically, rely solely on the in vitro
endpoint and eliminate animal use and suffering. It is worth noting that the data collected in the OECD
DRP were not available at the time of the conduct of this workshop. Among the conclusions andrecommendations reached by the workshop, the VMT considered the following as the most relevant
ones in relation to this effort:• Positive rodent CTA data should, in general, be considered to be indicative of a highprobability of rodent carcinogenicity, while negative results are indicative of noncarcinogenicity.
• CTAs could provide information which, in combination with data from other testing methods,
could be useful for identifying the carcinogenic potential of physical and chemical agents inhumans.
• CTAs have the potential to detect various types of carcinogens, including those that arethought to act via genotoxic and non-genotoxic mechanisms.
• A more extensive database on the use of CTAs for screening purposes should be set up,
alongside the standard genotoxicity assays (for comparative purposes), by using chemicals
with known activities in rodent bioassays. In the longer term, such information should be used
to add at least one of the established rodent CTAs (SHE, Balb/c 3T3 or C3H/10T½) tostandard carcinogenicity screening packages,

12Balb/c 3T3 CTA PRP report v1 - 30 July 2010••

Consider the need to organise a focused inter-laboratory study involving one or more of therodent cell-based transformation assays, once they are considered to be ready according to the
ECVAM criteria to enter prevalidation,
The suitability of the currently available rodent protocols for independently managed interlaboratory prevalidation studies should be established by ECVAM as a matter of urgency.

ECVAM Expert meeting, 2004
Following the discussions at the OECD and acknowledging the need for alternative methods in thearea of carcinogenicity, the ECVAM Task Force on carcinogenicity recommended to bring together agroup of experts in the field to discuss whether there was a need to validate the CTA and eventuallywhat should be the involvement of ECVAM. The meeting was held at ECVAM on 15-16 April 2004
and the experts agreed that it was valuable to validate CTAs in accordance with current standards. The
funding available at that time for the evaluation of CTAs was only sufficient to conduct theprevalidation of two variants of the assay. For feasibility and practical reasons, the evaluation of the
SHE pH 6.7 and the Balb/c 3T3 CTAs was prioritised. In addition, it was agreed that the SHE pH 7.0
protocol would be evaluated by a single laboratory in parallel to the two main studies, due to theamount of valuable SHE pH 7.0 assay historical data available. However, it was clearly stated that theprevalidation of the SHE pH 6.7 and the Balb/c 3T3 CTAs would not exclude that the SHE pH 7.0
CTA and the C3H/10T½ CTA could be subsequently similarly prevalidated, or undergo a catch-up
validation, after the first two had undergone scientific prevalidation according to modules 1-4(Hartung et al., 2004).

1.8 Patents
The test method and the cells employed in this study are in the public domain and have not been
patented.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

2 Organisation of the Study
The aim of this prevalidation study was to assess the reproducibility of a standardised Balb/c 3T3 CTA
protocol. In order to evaluate whether the test would meet the criteria called for by the ECVAM
principles on test validity, the modular approach of validation was followed (Hartung et al., 2004). In
this study the following modules were assessed: 1) test definition, 2) within-laboratory reproducibility,3) transferability, 4) between-laboratory reproducibility. In addition, the data produced are adding to
the 5th module on predictive capacity which was in part addressed by the OECD DRP (OECD 2007).
Each in vitro test was conducted according to the same agreed-upon protocol in three different
laboratories.
ECVAM entirely coordinated the study and sponsored two of the participating laboratories. The thirdlaboratory was sponsored by Hatano Research Institute and a collaboration agreement was signed with
ECVAM at the beginning of the study.
This study was organised as described below taking into account 1) the objective of the study to assess
reproducibility of the standardised CTA protocol and not its predictive capacity, which is addressed by
the OECD DRP, 2) the high costs and time to perform assays and 3) the limited funding and resources
which could be made available by ECVAM. This allowed to evaluate the CTA using the Balb/c 3T3
prtocol in three laboratories, employing six chemicals.
It is important to note that this study should be viewed as one that complements the OECD DRP(OECD 2007) exercise with ECVAM main goal to development of a protocol that could serve as abasis for an OECD test guideline.

2.1 Validation Management Team
Following the principles for test method validations (OECD 34, 2004) an independent VMT was
established by ECVAM. Its role was to design the study, guide and facilitate the prevalidation process,to evaluate the results and to render subsequent decisions during the progress of the study, and toanalyse the outcome. Philippe Vanparys, being member of the ECVAM Carcinogenicity Task Force,
was appointed as chairman of the VMT.
Chairman
Representative of ICCVAM (until Dec. 2006)
ExpertExpertProject Management (until April 2008)
Project Management
Project Management & contact person(until March 2007)
Statistician
ExpertPhilippe Vanparys (J&J PRD, Beerse, Belgium;
currently ALTOXICON BVBA, Belgium)
Leonard Schechtman (ICCVAM and FDA, USA;currently Innovative Toxicology Consulting, LLC,
USA)
Marilyn Aardema (P&G, USA; currently Marilyn
J. Aardema Consulting, LLC, USA)
MakotoHayashi(NIHS,
Japan;
currently
Biosafety Research Center, Foods, Drugs andPesticides, Shizuoka, Japan)
Thomas Hartung (ECVAM)
Raffaella Corvi (ECVAM)
Daniela Maurici (ECVAM)
Sebastian Hoffmann (ECVAM;
consulting + services, Germany)
Laura Gribaldo (ECVAM)

currentlyseh

The biostatistical analysis of the in vitro data was the responsibility of independent biostatisticians(Sebastian Hoffmann and Andre Kleensang - ECVAM).

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

B. Claire Thomas (ECVAM from May 2007 to May 2009) and Pascal Phrakonkham (ECVAM since
May 2009) assisted ECVAM in the management of the study.

2.2 Laboratories involved
The study included three laboratories, two from Europe and one from Japan. The participating
laboratories are listed below. Laboratories 1 and 3 had extensive expertise with different protocols of
the assay, while Laboratory 2 had limited experience.
Laboratory 1 (Study Director: Enrico Sabbioni)
ECVAM JRC European Commission
Via Fermi 1, TP 580
21027 Ispra (VA), Italy
Tel +39.0332.78.63.42
Fax +39.0332.78.53.36
enrico.sabbioni@cec.eu.int
Retired in September 2007
Laboratory 2 (Study Directors: Albrecht Poth and Susanne Bohnenberger)
Harlan Cytotest Cell Research GmbHIn Den Leppsteinswiesen 19
D-64380 Rossdorf , Germany
Tel +49.(0)6.154.807.266 (Poth) / 270 (Bohnenberger)
Fax +49.(0)6.154.83.399
APoth@harlan.com
SBohnenberger@harlan.com
Laboratory 3 (Study Director Noriho Tanaka)
Hatano Research Institute
Food and Drug Safety Center
729-5 Ochiai- Hadano
Kanagawa 257-8523, Japan
Tel and Fax +81.463.82.0773
tanaka.n@fdsc.or.jp

2.3 Quality assurance schemes employed by the testing laboratories
The present study was conducted under Good Laboratory Practice (GLP)-like conditions by alllaboratories and according to good scientific practice and good cell culture practice (OECD, 2004).
Only Laboratory 2 routinely works under GLP certification and was subjected to regular GLP
inspections while the study was being carried out. Since this was a prevalidation study it was not felt
necessary to conduct this study under GLP.

2.4 Chemicals tested in the Balb/c 3T3 prevalidation study
The chemicals for the prevalidation study were selected using data from the OECD DRP31 document(draft version August 2004) and the publication by Kirkland et al. (2005). Since this prevalidation
study was part of a larger project also involving the analysis of the SHE CTAs, the chemical selection
took into account existing results in both systems as described below. Where possible the samechemicals were selected for the evaluation of the Balb/c 3T3 and SHE CTAs.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

2.4.1 Chemical selection
2.4.1.1 Selection criteria
The chemicals were selected using the following criteria:
1) Positive both in Balb/c 3T3 and in SHE CTAs,
2) Negative both in Balb/c 3T3 and in SHE CTAs,
3) At least two references available for each test chemical (for both Balb/c 3T3 and SHE),
4) If possible, data available using the SHE pH 6.7 and pH 7.0 protocols,5) Clear classification as in vivo carcinogen or non-carcinogen,6) Availability of in vitro genotoxicity data.
Most of the criteria were met for all chemicals, except that only one reference was available for someof the assays: anthracene (only one reference for SHE pH 6.7 and Balb/c 3T3 CTAs), phenathrene(only one reference for Balb/c 3T3 and SHE pH 7.1) and o-toluidine HCl (only one reference for
Balb/c 3T3 CTA and no reference for SHE pH 6.7 CTA).
Four of the chemicals selected were in common with those evaluated in the SHE validation studies,while phenanthrene and 2-acetylaminofluorene were uniquely used in this study instead of 2,4diaminotoluene and phthalic anhydride for which a limited amount of data were available for the
Balb/c 3T3 CTA.
The in vitro genotoxicity, in vivo genotoxicity and carcinogenicity characterisation of the selectedcompounds is reported in Annex 13.1.

2.4.1.2 Chemicals selected
Chemicals selected for the prevalidation study are listed in Table 1.
Table 1: List of chemicals used in the prevalidation study
in vivo
carcinogenenicity(References)(Gold and+
Zeiger, 1997)

Chemical

CAS no.

3-Methylcholanthrene

56-49-5

3-Methylcholanthrene

56-49-5+

2-Acetylaminofluorene

53-96-3+

Benzo(a)pyrene

50-32-8

Anthracene

Suggested dose range
0.5, 1, 2, 4 µg/ml
when used as positive control(Gold and
Zeiger, 1997)(Gold and
Zeiger, 1997)

0.01, 0.03, 0.1, 0.3, 1, 3, 10 µg/ml
when used as coded chemical+(IARC, 2009)

0.1 - 15 µg/ml

120-12-7-(IARC, 2009)na

Phenanthrene

85-01-8-(IARC, 2009)nao-Toluidine HCl

636-21-5+(NTP)

20 µg/ml - 1.2 mg/ml

nana = not applicable

The doses of benzo(a)pyrene, 3-methylcholanthrene and o-toluidine HCl to be used were suggested by
the VMT based on data from the literature to optimise the use of resources (either due to highchemical cost or lack of cytotoxicity) for timely completion of these studies. For the other chemicals

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010
the laboratories were asked to select the dose ranges on their own in order to check their ability toidentify the critical doses for the transformation assay.
3-Methylcholanthrene was chosen as positive control (PC) because it has been generally reported toinduce a strong positive CTA response, in addition to the fact that historical data on this chemical usedas PC were available at the lead laboratory.
All chemicals were purchased from Sigma Aldrich.

2.4.2 Modules 2 and 3: Within-laboratory reproducibility andtransferability (coded and non-coded)
3-Methylcholanthrene was chosen as both a coded and non-coded chemical in this study phase. The
laboratories were unaware of the fact that the same chemical served both purposes. 3methylcholanthrene was later used as the PC, in the subsequent phases of the study. The doses of 3methylcholanthrene to be used were suggested by the VMT based on data from the literature.

2.4.3 Module 4: Between-laboratory reproducibility (coded compounds)
Three chemicals classified as in vivo carcinogens (2-acetylaminofluorene, benzo(a)pyrene, o-toluidine
HCl) and two chemicals classified as non-carcinogens (anthracene, phenanthrene) were selected.
Dose ranges for benzo(a)pyrene, and o-toluidine HCl, were suggested by the VMT based on data fromthe literature. For the other two chemicals, the laboratories had to choose the dose range based on
dose-range finding (DRF) tests. The VMT suggested that DMSO (dimethyl sulfoxide) should be usedas the solvent for all chemicals. Instructions were also sent regarding the order that the chemicals
should be tested in. As the results became available, they were sent to the statistician.

2.4.4 Coding/decoding
All chemicals were coded before sending them to the laboratories. The coding and shipment ofcompounds were performed by J&JPRD and ECVAM. The ECVAM staff involved in the coding andshipment was completely independent from the staff involved in the experimental part of thevalidation study. The coded compounds were sent to the laboratory Safety Officers together with thecorresponding sealed envelopes containing the Safety Data Sheets. These envelopes were to be opened
only in case of accidents and were to be sent back to ECVAM unopened once the experiments were
finished. All sealed envelopes were returned to ECVAM at the end of the prevalidation study. Since
the chemicals were coded, the laboratories did not know their identity and therefore all chemicals weretreated as potential carcinogens.
The identity of the coded chemical used for the within-laboratory reproducibility and transferability
was made known to the study directors at the VMT and study directors meeting of May 2006.
For the between-laboratory reproducibility, the first statistical analysis was conducted before thedecoding. The chemicals used were decoded during the VMT and study directors meeting of May
2007. Subsequent testing after this meeting requested by the VMT was performed non-coded.

2.5 Assessment of within- and between-laboratory reproducibility
Following common practice in genotoxicity studies, both within- and between-laboratory
reproducibility and predictive capacity were evaluated based on concordance of the dichotomous
results (negative or positive) as defined by the assessment criteria listed in section 3.6.7.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Regarding the within-laboratory reproducibility, the concordance of results per laboratory wasdescribed.
Between-laboratory reproducibility was evaluated by comparing results of the three laboratories
obtained for the same substance.
A preliminary assessment of predictive capacity was described by comparing results with the predefined reference results as reported in Table 1, under ‘in vivo carcinogenicity’.

2.6 Study timeline
An important aspect of the initial phase of the study was the training of the laboratory personnel,
including the harmonisation of scoring, the refinement of the protocols and the preparation of Standard
Operating Procedures (SOPs). All laboratories participated in this training phase.
Since the ECVAM and the HRI laboratories had produced their historical data using two slightly
different cell clones and different protocols, it was agreed that both clones and protocols, respectively,
should initially be evaluated. Finally it was decided that the HRI clone and the HRI protocol version
were to be used for the validation study.
Following the preliminary phase of protocol optimisation, both the transferability and the withinlaboratory reproducibility were assessed by evaluating results obtained for a non-coded test chemical
and a coded one. These two chemicals were the same (3-methylcholanthrene), allowing an analysis of
the within-laboratory reproducibility as well as the transferability of the assay. 3-Methylcholanthrene
was then used as PC in the following phases of the study. After the evaluation of these initial resultsby the biostatistician and the conclusion by the VMT that the between-laboratory transfer of the test to
the participating laboratories and the within-laboratory reproducibility analysis were successful, thelaboratories proceeded to the experimental phase on between-laboratory reproducibility. The betweenlaboratory reproducibility was evaluated using five coded chemicals.
The data submission template in Excel was developed for each test, in a collaborative effort between
the laboratories, ECVAM and the biostatistician. The spreadsheets containing the test data generated
by each laboratory were returned to the biostatistician of the VMT.
A final signed report for each of the chemicals tested was provided to ECVAM by the Study Directors
from the participating laboratories. Moreover, the administrative lead laboratory produced a summary
report at the end of the study.
At the completion of the study the laboratories were asked to quality check the data that had been
analysed by the statistician. They received the sheets with the data used by the statistician and wererequested to confirm that the statistician had, in fact, used the correct raw data. The laboratories also
quality checked the data presented in this report.
Table 2 summarises the timeline of the study.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Table 2: Timeline of the study
Date

Location

10-11/02/2005

Kick-off meeting
ECVAM, Ispra, Italy

03/2005-02/2006

Protocol optimisation
All laboratories

4-5/04/2005

21/08/2005

Training week

Study Directors and members of the VMT attended
the kick-off meeting and agreed on the general aspectsof the protocols to be used in the optimisation phase.
Conduct of experimental work by all participating
laboratories to assess best protocol conditions.

ECVAM, Ispra, Italy

Technical staff and study directors met to agree on theharmonisation of protocols and on the criteria forscoring the plates using dishes treated withtransforming and non transforming chemicals.

1st VMT meeting

The initial phase should assess :Berlin, Germanya) both the ECVAM clone used with the ECVAM
protocol, and the HRI clone with the HRI protocol;(in conjunction withthe 5th World Congress b) the within-laboratory reproducibility and theon
Alternative transferability by testing 3-methylcholanthrene as
Methods)
positive control and as a coded chemical.
03/2006

Revisionofthe Protocol was revised based on preliminary data
protocol, ECVAM
produced by the laboratories.

10-11/05/2006

VMT
andstudy Evaluation
anddiscussionofresultsondirectors meeting
standardisation of the protocol, within-laboratory
reproducibility and transferability
ECVAM, Ispra, Italy

20/04/2007

ad hoc expert meeting Discussion on statistical analysis for Balb/c 3T3.
on statistical analysis
ECVAM, Ispra, Italy

30-31/05/2007

VMT
andstudy Evaluation and discussion of results on betweendirectors meetinglaboratory reproducibility and decoding of thechemicals. Additional testing requested by the VMT.
ECVAM, Ispra, Italy

26/09/2007

VMT meeting
ECVAM, Ispra, Italy

01/2009

End of experimental part. Analysis of repeatedexperiments.

VMT
andstudy Final discussion.
directors meeting
ECVAM, Ispra, Italy

03/2010

VMT meeting
ECVAM, Ispra, Italy

Finalisation of the prevalidation report to be submitted
to the ECVAM's Scientific Advisory Committee(ESAC)

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

3 Module 1: Test Definition
The following sections provide information about the scientific purpose of the test and the test
procedure.

3.1 Scientific basis for the proposed test method
The proposed test method has the potential:
1) to detect genotoxic carcinogens,2) to detect non-genotoxic carcinogens,3) to be used for mechanistic studies of multistage carcinogenesis.

3.2 Description of the endpoint predicted and the mechanistic basis of thetest
In vitro cell transformation technology employing cultured mammalian cells has been available forover four decades, since the introduction of the methods for transforming normal diploid hamster cells
to tumour cells by Berwald and Sachs (1963, 1965). Heidelberger et al. (1983) determined that the
majority of cell transformation systems fell into three basic categories:• cell strains (cells with a limited lifespan),• cell lines (cells with an unlimited lifespan),• oncogenic viral-chemical interactions involving cells (Fischer rat embryo cells expressing an
endogenous retrovirus, mouse embryo cells expressing the AKR leukemia virus, chemicalenhancement of a simian adenovirus, SA7 transformation of Syrian hamster or rat embryo
cells).
The phenomenon of morphological transformation is characterised by changes in the behaviour andcell growth of cultured cells in order for progression to the next stage in the transformation process tooccur. A minimum of four phenotypic stages appears to be involved in cell transformation. These
include (from primary to fully malignant cells): (a) a block in cell differentiation; (b) acquisition ofimmortality, characterised by unlimited lifespan, an aneuploid karyotype and decreased genetic
stability; (c) acquisition of tumourigenicity, which is closely associated with the in vitro phenotypes offocus formation, anchorage-independent growth in semi-solid agar and autocrine growth factor
production; and (d) full malignancy, including metastasis when the cells are injected in the suitable
host.
The endpoints used in immortalised cells, such as Balb/c 3T3 cells, are typically the carcinogenconversion from non-tumourigenic immortality to tumourigenicity, with scoring of focus formation
and anchorage-independent growth. Such effects might be caused by changes in the expression of
oncogenes and/or tumour suppressor genes, block in cellular differentiation, increased genetic
instability with progression in vitro, and autocrine growth factor production associated with alteredcell signalling. The Balb/c 3T3 cell line undergoes tumourigenic conversion either spontaneously orafter treatment with a carcinogen (Aarson and Todaro, 1968; Matthews et al., 1993a; Matthews et al.,1993b). Furthermore, the conversion to tumourigenic phenotype of the Balb/c 3T3 cells occurs after
activation or inactivatation of oncogenes and tumour suppressor genes (Nakazawa et al., 1990; Olsonet al., 1993; Silingardi et al., 1994).

3.3 Biological test system: Balb/c 3T3 cell line
A CTA using an established cell line rather than early passage normal diploid cells was developed by
Kakunaga (1973) who obtained a subclone, A31-714, from the Balb/c 3T3 A31 line (Aaronson andTodaro, 1968) and demonstrated its responsiveness in the induction of morphologically transformedfoci with chemical carcinogens. Later, Kakunaga and Crow (1980) isolated several other variants from20

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
the cell line that showed different susceptibility to UV-induced transformation and recommended theA31-1-1 clone as particularly suitable for the cell transformation experiment. The Balb/c 3T3 cells arecontact-inhibited cells with a strong cell-to-substrate adhesion. They show a uniform morphology, a
low saturation density and a high cloning efficiency (50-60%). They are characterised by a shortdoubling time (approximately 19 hours) and a high sensitivity to chemical transformation with a low
incidence of spontaneous transformation.

3.4 Balb/c 3T3 Cell Transformation Assay
A CTA using the Balb/c 3T3 cell line was initially developed as a quantitative transformation assay(TA) for chemical carcinogens employing a subclone, A31-714, of the mouse Balb/c 3T3 A31
embryonic cell line, derived by Aaronson and Todaro (1968) mouse Balb/c 3T3 A31 embryonic cellline, the latter of which was originally established for purposes of studying viral-induced
transformation. Extensive work has been carried out by many researchers using A31-714 and A31-1-1
clones (Heidelberger et al., 1983). In an attempt to explore the utility, advantages, and shortcomings ofdifferent CTAs, an IARC/NCI/EPA Working Group (1985) was convened in which the Balb/c 3T3
cells and C3H/10T½ cells (Reznikoff et al., 1973) systems were considered. This group preparedrecommendations for practical procedures in the performance of CTAs (IARC, 1985).
Despite this and other such efforts, wherein the working group proffered recommendations regarding
the Balb/c 3T3 CTA, that method and CTAs in general, have not been fully exploited for routine
screening of potential carcinogens to the extent that other genotoxicity tests have been. This, in part,may have been due to ignorance regarding the underlying mechanism(s) associated with CTAs
generally and the perception that such assays were technically more difficult, expensive and timeconsuming than other in vitro genotoxicity tests that are also used for screening of potential
carcinogens. Nevertheless, whereas genotoxicity assays provide indirect evidence of potential
carcinogenicity, only CTAs offer the most direct in vitro measure of that potential. Resistance to theroutine use of the Balb/c 3T3 method was also due to the fact that it had not been validated inaccordance with internationally accepted validation procedures (OECD GD34, 2005). In order toovercome some of the limitations associated with the Balb/c 3T3 CTA, improvements to the basic
protocol were proposed that would increase the recognition of induced cell transformation events andshorten the duration of the assay. Some such improvements included 1) enhancing the sensitivity ofcells to the transforming potential of test chemicals by subsequent treatment with known tumourpromoting agents (two-stage method) (Mondal et al., 1976; Sakai et al., 2002), 2) amplification of thenumber of transformed foci by re-plating the culture after the treated cells reached confluence(Schechtman, 1985), 3) increasing the seeding cell number and treating the cells on day 2, i.e. one day
later than the original treatment day (Matthews et al., 1993), 4) using modified cell culture medium(enriched basal medium supplemented with 2% foetal bovine serum FBS) (Tsuchiya and Umeda,
1995), and 5) use of serum-reduced medium in order to increase assay sensitivity (Hayashi et al.,2008) .

3.5 Protocol optimisation phase
An experimental phase was considered necessary in order to develop an optimised version of the
protocol to be used in the subsequent prevalidation study.
During the kick-off meeting it became clear that the ECVAM and HRI laboratories were using thesame clone of the Balb/c 3T3 cell line (clone A31-1-1), but two different cell lineages of this clone,
and two different versions of the cell transformation protocol. In view of the distinctive nature of boththe cell lineages and the protocols used in the two laboratories, and the fact that the historical data
available from the two laboratories were likewise disparate, it was decided to initially test andcompare both cell lineages with the respective protocols. Moreover, it was agreed to organise a“training week” for the participant laboratories before the initiation of the study. The aim of training
week was to: 1) compare the different protocols, 2) agree on a standard protocol and provide training

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010
on how to perform the assay, 3) compare the results derived from the ECVAM and HRI protocols andthe two cell lineages used, 4) ensure that all laboratories were using the same criteria for scoring.

3.5.1 Preliminary experiments
Several preparatory experiments were performed before the training week.
For the optimisation phase of the study, two different lineages of the Balb/c 3T3 A31-1-1 clone wereused (ECVAM and HRI). The ECVAM lineage was used at passage 91 and was routinely cultured in
Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetalclone III serum (FCIII,
Hyclone, USA), while HRI’s lineage (passage 17) was cultured in Minimum Essential Medium(MEM) supplemented with 10% FBS (Moregate, Australia) and 1% penicillin 10,000 U/ml
/streptomycin 10 mg/ml (PS) solution (M10F). It should be noted that the method used for determining
passage number was also dissimilar in the two laboratories, thereby accounting for the dramatic
numerical difference in this property.
ECVAM compared CTA in Petri dishes of different sizes (ø 60 vs. 90 mm), determined the best fixing
and staining conditions for the Colony Forming Efficiency (CFE) test, and performed the CTA withboth the ECVAM and HRI cell lineages. HRI performed the CFE test and the CTA with the HRI celllineage and using cell culture medium supplemented with the synthetic serum employed by ECVAM.
The analysis of cytotoxicity and morphological transformation (MT) at ECVAM was carried out using
two inorganic compounds (NaAsO2, CisPt) and one organic compound (3-methylcholanthrene).
The results from the experiments performed during the training week suggested that the cell lineages
behaved differently. They differed in plating efficiencies (PEs), cytotoxicity responses, and themorphology of colonies, cell monolayers and type III foci. Interestingly, in these studies, the HRI celllineage showed a higher number of spontaneous type III foci in the negative control compared to thatroutinely detected in the HRI historical controls. It was hypothesised that the HRI cell lineage used at
ECVAM had undergone some transformation during cell culture, although this was not verified.
Subsequently, HRI compared the ECVAM and HRI sera using the HRI cell lineage and protocol. They
concluded that the ECVAM FCIII synthetic serum is not suitable for use with the HRI protocol andcell lineage.
HRI performed some additional experiments. The results, which were reported in July 2005, were as
follows:
1) The ECVAM clone was resistant to 3-methylcholanthrene induction of transformation,2) The HRI and ECVAM clones exhibited different morphologies,3) The different media tested yielded different cellular responses,
4) The plating efficiency (PE) of the ECVAM clone was lower than that of the HRI clone,5) CTA and soft agar experiments with the two clones and different media showed that 3methylcholanthrene did not induce type III foci in the ECVAM cell lineage (only type I and II foci)and no colonies were observed in soft agar.
At the SHE kick-off meeting, which was held in Berlin on the 21 August 2005, in conjunction with the
5th World Congress on the Alternatives and Animal Use in Life Sciences, the VMT also discussed
some major issues regarding the Balb/c 3T3 validation study and the following decisions were taken:
1) Clones
HRI was to re-ship the Balb/c 3T3 clone to ECVAM since the clone used during the training week wasconsidered transformed by the HRI expert. Starting from October 2005, all three laboratories were to
perform the cytotoxicity experiments with both cell lineages.
2) Protocols
The HRI cell lineage was to be used with the HRI SOP. The ECVAM clone was to be used with the
ECVAM SOP.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010
3) Dose-range finding (cytotoxicity)
CFE (ECVAM SOP) and Crystal Violet (CV) (HRI SOP) cytotoxicity assays were used to determine
the appropriate test concentrations in the initial phases of the study. For the CFE assessment cells weregrown for 9 days, while for CV two different time points were investigated (4 day- and 7 daycultures). Non-coded and coded 3-methylcholanthrene were to be tested. The laboratories were notaware that these two chemicals were the same. The concentrations tested ranged from 0.01 to 10 µg/ml
for non-coded 3-methylcholanthrene and from 0.01 to 25 µg/ml for coded 3-methylcholanthrene.
The data were circulated and reviewed by the VMT in February 2006.

3.5.2 Results
Figure 1 and Figure 2 show the results of the CFE and CV analyses of the HRI and ECVAM celllineages treated with coded and non-coded 3-methylcholanthrene. Complete data sets are included in
the laboratory reports of phase 1.

150

CV (4-day culture)

125
100
75
50
25
0

100
75
50
25

0

0.01

0.1

1

10

3-Methylcholanthrene
MCA (µg/ml) [µg/ml]

150
Relative cell growth(% of control)

CFE

125
Relative PE(% of control)

Relative cell growth(% of control)

150

0

0

0.01

0.1

1

10

3-Methylcholanthrene
MCA (µg/ml) [µg/ml]

CV (7-day culture)

125

ECVAM (HRI cells)
ECVAM (ECVAM cells)
RCC (HRI cells)
RCC (ECVAM cells)
HRI (HRI cells)

100
75
50
25
0

0

0.01

0.1

1

10

3-Methylcholanthrene
MCA (µg/ml) [µg/ml]

Figure 1: Crystal Violet (CV) and Colony Forming Efficiency (CFE) results of ECVAM and
HRI cell lineages treated with non-coded 3-methylcholanthrene. CV was performed at 4 and 7
days of culture, whereas CFE was assessed on day 9.
PE = Plating Efficiency

23

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

125

150

CV (4-day culture)

125
Relative PE(% of control)

Relative cell growth(% of control)

150

100
75
50
25
0

Relative cell growth(% of control)

125

100
75
50
25

0 0.001 0.01 0.1

1

10 1003-Methylcholanthrene[µg/ml]Compound X (µg/ml)

150

CFE

0

0 0.001 0.01 0.1

1

10 1003-Methylcholanthrene[µg/ml]Compound X (µg/ml)

CV (7-day culture)
ECVAM (HRI cells)
ECVAM (ECVAM cells)
RCC (HRI cells)
RCC (ECVAM cells)
HRI (HRI cells)

100
75
50
25
0

0 0.001 0.01 0.1

1

10 1003-Methylcholanthrene[µg/ml]Compound X (µg/ml)

Figure 2: Crystal Violet (CV) and Colony Forming Efficiency (CFE) results of ECVAM and
HRI cell lineages treated with coded 3-methylcholanthrene. CV was performed at 4 and 7 days ofculture, whereas CFE was assessed on day 9.
PE = Plating Efficiency

The ECVAM cell lineage did not show any cytotoxicity to treatment with 3-methylcholanthrene, while
the HRI cell lineage exhibited dose-dependent cytotoxicity when treated with the same compounds.
This difference in responses of the two cell lineages was reproducible by the different laboratories.
HRI and RCC laboratories reported difficulties in growing the ECVAM clone. Therefore, data for the
ECVAM cell lineage were not available for all laboratories. Overall, the experiments were considered:
1) reproducible within laboratories when the results for non-coded 3-methylcholanthrene werecompared with those for coded 3-methylcholanthrene in each laboratory and 2) reproducible between
laboratories when the data produced by the three laboratories were compared.
Additional differences of the two cell lineages were reported by the laboratories. Preliminary CTA
experiments showed that the morphology of the ECVAM and HRI cell lineages was different (Figure
3-Figure 4). The ECVAM cell lineage showed small colonies of different size and density, while the
HRI cell lineage yielded more homogeneous colonies. Moreover, the monolayer of the HRI celllineage showed cells with a uniform morphology, whilst cultures of the ECVAM cell lineage showed
irregular growth with many clusters and islands of cells in the control culture. The PE of the ECVAM
cell lineage was also lower.

24Balb/c 3T3 CTA PRP report v1 - 30 July 2010

Figure 3: Colony Forming Efficiency with Balb/c 3T3 ECVAM and HRI cell lineages.

DMSO

MNNG

MCA

ECVAM

HRI

Figure 4: Morphological Transformation with Balb/c 3T3 ECVAM (top row) and HRI (bottomrow) cell lineages treated with 0.5% DMSO, 1.5 µg/ml N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), or 10 µg/ml 3-methylcholanthrene (MCA).
Although the ECVAM and HRI cell lineages seemed to had ostensibly originated from a common
Balb/c 3T3 parental cell line, it is clear that some phenotypic and morphological modifications haveoccurred during the independent in vitro cultivation of the respective progeny cells over time.
The analysis of data produced with CV showed that experiments conducted with 7-day cultures
performed better than those with 4-day cultures. A longer cell proliferation phase allowed a more
precise discrimination between the cytotoxicity responses. The protocol was modified accordingly.
The comparison between CFE and CV was difficult due to the different sensitivities of the two
methods, CFE being the most sensitive showing cytotoxicity at lower doses.
Harlan CCR and HRI reported good CTA results with the HRI cell lineage. However, no results could
be provided for the ECVAM cell lineage since all the cells detached from the plastic Petri dishes after25

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
3 weeks. ECVAM reported similar results with the ECVAM cell lineage by using Falcon Petri dishes.
However, ECVAM reported good CTA results with the same clone when COSTAR dishes were used.
No CTA was carried out by ECVAM with the HRI clone.

3.5.3 Conclusion of the Validation Management Team on preliminary
experiments
Based on the data provided by the three laboratories and previous preliminary data, the VMT agreed
that the HRI cell lineage together with the HRI SOP should be used for the completion of thevalidation (minutes of the teleconference of 23/02/2006 available at ECVAM).
Since the HRI cell lineage gave a higher background of type III foci the acceptance criteria in theStatement of the Work which stated “not more than one focus type III in the whole set of control
dishes” was revised to read “the maximum number of Type III foci in the entire set of vehicle control
dishes should not exceed five”.
The data showed that the two cytotoxicity methods (CFE and CV) may have different sensitivities,
CFE appears to be more sensitive in some cases. In the CV method the cell density undergoingchemical treatment is closer to that used in the MT, suggesting that the CFE might overestimate thecytotoxicity due to the different number of cells seeded. However, since CFE is the method that has
been used historically to assess cytotoxicity in cell transformation experiments it was determined thatits use would allow for a better comparison of data generated by this validation study with historical
data. This would also be important in case a further retrospective validation study is performed in the
future. Due to the difficulty of choosing the optimal cytotoxicity assay, it was agreed that thelaboratories should use both cytotoxicity assays in order to allow for a more complete comparison at
the conclusion of the study. However, it was agreed that the appropriate treatment doses for MT were
to be determined based on the CFE data.
The protocol was revised in March 2006. The Moregate FBS necessary for the medium to be usedwith the HRI clone was received by ECVAM and Harlan CCR at the beginning of April 2006.

3.6 Protocol
The detailed test protocol used in this study is described in Annex 13.2.

3.6.1 Balb/c 3T3 cell line
HRI’s cell lineage (passage 17) of the Balb/c 3T3 cell line was cultured in M10F. The batch of serum
used was preselected by testing its cell PE (>30%), its ability to maintain a uniform contact-inhibitedmonolayer and to its ability to support 3-methylcholantrene-induced transformation (IARC/NCI/EPA,
1985).
The cells were distributed by HRI to the participating laboratories and used for the CTA within 3 to 4
passages. In the transformation experiments all laboratories used the same culture medium (not thesame lot) from GIBCO: M10F was used during the growing stage of the cells (until day 7) andDF2I2F was used during the confluent stage of cells.

3.6.2 Serum and medium
All the laboratories used the same batches of FBS (Moregate, Australia) during the different phases ofthe validation study. Batch No. 2430013 serum was used during the optimisation and the withinreproducibility phases, while batch No. 94300107 serum was used for the between-laboratory
reproducibility assessment. The latter was tested and selected by Harlan CCR among six serum26

Balb/c 3T3 CTA PRP report v1 - 30 July 2010batches prior to use in this exercise (Report 31/10/2006, available at ECVAM), based on its ability to
induce a high cloning efficiency (CFE = 52.1%), a relatively low spontaneous transformation
frequency with the vehicle control VC ( mean number of 0.3 transformed focus/dish under DMSO)
and a high transformation frequency with a PC (mean number of 12.8 transformed foci/dish under 4µg/ml of 3-methylcholanthrene).
The laboratories used the same media from GIBCO (MEM and Dulbecco's Modified Eagle
Medium/Nutrient F12 DMEM/F12), but from different batches.

3.6.3 Controls
Positive Control: 3-methylcholanthrene (4 µg/ml dissolved in 0.5% DMSO) was used as the PC.
Untreated Control: The cell culture medium served as the concurrent untreated control.
Vehicle Control: The cell culture medium containing 0.5% DMSO served as the concurrent VC.

3.6.4 Test procedure
The CTA is composed of two phases: A preliminary dose-range finding (DRF) experiment to determine the appropriate dose-rangethat will be used for the transformation assay (TA), The TA, which represents the main experiment and which includes the assessment ofmorphological transformation (MT) and cytotoxicity in separate dishes.
The DRF tests are carried out by measurement of the Colony Forming Efficiency (CFE = [total
number of colonies formed in the treatment dishes / total number of colonies formed in the control
dishes] × 100). In parallel, the Crystal Violet (CV) cytotoxicity test (Saotome et al., 1989) was also
performed to calculate the Relative Cell Growth (RCG = [(Absorbance of treated well - Absorbance of
medium blank well) / (Absorbance of solvent control well - Absorbance of medium blank well)] ×
100).
The measurement of cytotoxicity during the TA includes both the CFE and CV cytotoxicity tests,
concurrently with the MT assay. Eight doses are chosen for the TA on the basis of the DRF CFE
results to cover the range from no toxicity up to 90% reduction in CFE. A PC (4 µg/ml of 3methylcholanthrene) and a VC are included in each experiment (MT, CFE and CV). Briefly, 10 ml ofcell suspension (2 × 103 cells/ml) are seeded into 100 mm-diameter dishes. Seventy-two hours after
exposure (day 4), the treatment medium is removed and replaced with fresh complete medium. From
day 7 to day 24 or 25 the medium is removed and replaced with DMEM/F12 medium with 2 µg/ml
insulin, 2% FBS and 1% PS (DF2I2F) twice a week. One week after the last medium change, themedium is removed and the cells are washed with Phosphate Buffered Saline (PBS), fixed withmethanol and then stained with 10% Giemsa solution.
For the analysis of the MT, foci consisting of more than 50 cells or more than 2 mm in diameter are
evaluated using a stereomicroscope. Only type III foci are recorded, which are characterised by thefollowing morphological criteria: deep basophilic staining of spindle-shaped cells which aremorphologically different from the background monolayer cells, dense multi-layering of cells (pilingup) and random orientation and invasive growth of cells at the edge of foci (criss-cross pattern)(IARC/NCI/EPA, 1985). The number of type III foci per dish is counted and this value is used for thestatistical analysis.

27

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

3.6.5 Statistical Analysis of raw data
While a recommended protocol of the assay has been published by an IARC/NCI/EPA Working
Group (IARC/NCI/EPA, 1985), no common statistical approach had been agreed on. Many different
approaches to data evaluation have been proposed and applied for the Balb/c 3T3 CTA in
toxicological literature. To clarify this issue, the VMT recommended that ECVAM would: 1) collectdata from the literature on the different statistical methods that had been used to evaluate Balb/c 3T3
CTA results; and 2) organise an ad hoc expert group to evaluate the data and come up with asuggestion on the most suitable statistical method to use for this assay.
The experiments conducted for the assessment of the within-laboratory reproducibility were initiallyanalysed by comparing the number of foci formed from the surviving seeded cells, i.e. adjusted bycytotoxicity results, in control and treated dishes using Fisher’s exact test,. As this approach wasconsidered not appropriate by the VMT, the respective experiments as well as those conducted for theassessment of the between-laboratory reproducibility were evaluated using the approaches
recommended by the ad hoc expert group (section 3.6.5.2).
The determination of the NOEL (No Observed Effect Level) concentration was based on the ANOVA
with Dunnett post test comparing each concentration with the VC with an overall α of 0.05, using
Prism v5.01 (GraphPad Software Inc., La Jolla, CA, USA). The highest concentration not significantly
different from the negative control was considered as NOEL concentration.
To further compare CFE and CV cytotoxicity tests, a rough comparison of the IC50 obtained with eachmethod was made, based on the cytotoxicity data of the TA experiments. The IC50 were calculated by
non-linear regression using the "Log[inhibitor] vs. normalised response" model with variable slope of
Prism v5.01 (GraphPad Software Inc., La Jolla, CA, USA).

3.6.5.1 Report on “Analysis of statistical methods and experimental designs used in
Balb/c 3T3 transformation assay”
A report on the “Analysis of statistical methods and experimental designs used in Balb/c 3T3transformation assay” was commissioned by ECVAM. This was an extension of a former report, inwhich an extensive database on Balb/c 3T3 CTA results has been developed, compiling existing dataon 414 chemical compounds retrieved from 250 references (ECVAM unpublished reports by M.
Fischbach, available at ECVAM).
The objectives of this study were: 1) to provide a qualitative and quantitative analysis of the statisticalmethods used to evaluate data obtained in published Balb/c 3T3 assays, and 2) to provide an analysisof the experimental practices employed. To this end, 169 experimental references have been
considered, encompassing data for 365 compounds.
A variety of statistical approaches were found in the literature. Some of them analysed the number offoci per dish quantitatively, e.g. by means of linear models (analysis of variance and t-test) after datatransformation (Matthews, 1993), by means of the non-parametric Mann-Whitney test (Colacci et al.,1996) or by assuming a modified Poisson distribution for count data. Others analysed the data as
qualitative outcomes considering the proportion of dishes showing foci, e.g. by applying Fisher’s exact
test (Lubet et al., 1990; Sakai, 1997). The main statistical approaches used were: Student’s test (52%),
modified Poisson distribution (19%), Fisher’s exact test (7%), a method reported by Perocco et al. in
1991 (7%), dose-response trend analysis and Chi2 analysis (2%). Seven percent of the publications
evaluated were devoid of statistical analyses.
Regarding the experimental aspects of the studies, data were collected on (a) the Balb/c 3T3 derived
cell clone used, (b) parallel cytotoxicity assays performed, (c) supplemental metabolic activation used,(d) a two-stage initiation/promotion procedure, and (e) a replating/amplification method (ECVAM
unpublished report by M. Fischbach available at ECVAM). The most widely used cell strains were the28Balb/c 3T3 CTA PRP report v1 - 30 July 2010
clone A31-1-13 used to test 92% of the chemicals (334 chemicals), followed by the cell clone A31-1-1
for 27 % of chemicals (100 chemicals). Some chemicals were examined in more than one cell strain. A
concurrent cytotoxicity study was performed in 98% of the studies. Metabolic activation using an S9
fraction was performed in 24% of the studies, representing 67 tested compounds (18%). The two-stage
initiation/promotion procedure is described in 26 studies (16%) for 66 compounds (18%). Finally, thereplating/amplification method, which is a variation of the initial protocol, was performed in 17
studies (10%).

3.6.5.2 Ad hoc expert meeting on statistical analysis for Balb/c 3T3 CTA
Based on the above analysis and on the recommendation by the VMT, it was decided that a statistician
ad hoc expert meeting should take place in order to discuss the most appropriate method for statisticalanalysis of the CTA in Balb/c 3T3 cells. This meeting was held at ECVAM on 20 April 2007.
The participants were: L. Edler (German Cancer Research Centre, Heidelberg, Germany), L. Hothorn(University of Hannover, Germany), M. Suzuki (Biosafety Research Center, Shizuoka, Japan), R.
Corvi (ECVAM), S. Hoffmann (ECVAM), B.C. Thomas (ECVAM). S. Hoffmann chaired themeeting.
Reviewing the most commonly used data evaluation methods (Fischbach, unpublished data available
at ECVAM), the experts concluded that none of the previously suggested methods adequately
addressed the particular statistical properties of the Balb/c 3T3 CTA endpoint of number oftransformed foci type III per dish. In this context, the experts discussed several statistical evaluation
methods. It was agreed that the Nishiyama transformation (Nishiyama et al., 2003) and an approachbased on the negative binomial distribution combined with William’s-type (Bretz et al., 2003)
protected tests were the preferred methods. They match the particular statistical properties of the
Balb/c 3T3 CTA test and were recommended to be used in the present validation study.
Empirically comparing the results of the data analyses using both approaches, i.e. Nishiyamatransformation and the approach based on negative binomial distribution, suggested that the latter was
more suited for the data at hand as it better reflected the dose-concentration curve (Manuscript in
preparation, minutes of the meeting available at ECVAM). Therefore, the results presented in thisreport have been generated with the approach based on negative binomial model (Annex 13.3). For
completeness, the respective results when applying the Nishiyama transformation and thecorresponding R-code are reported in Annex 13.4.
It was anticipated that for these approaches with complex multiple comparisons conventional p-values,
such as 0.05, would not be appropriate. In this study, a p-value of 0.01 was considered. However, theexpert group recommended to retrospectively determine an empirical threshold p-value for theassessment criteria based on the p-values obtained in the study.

3.6.5.3 Statistical method based on the negative binomial distribution
A Generalised Linear Model (GLM) with a negative binomial distribution and identity as link function
was fitted. Based on that GLM downturn-protected Williams contrast tests based on unweighted
combinations of concentrations were calculated and adjusted p-values based on the joint normal
distribution of the linear function were reported.
The GLM could not be fitted in case at least one concentration and/or the VC induced the same
number of foci in every dish, i.e. showed no variability in focus numbers. In such cases, the pragmatic
solution of arbitrarily adding one focus to one of the respective dishes was applied resulting in an
empirical estimation of the variability of larger than 0 (nine cases). The impact of these modifications
was considered negligible.

29

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Furthermore, the GLM could only be fitted to a balanced data set. Therefore, focus numbers of thesame number of dishes for each concentration and control in a given experiment were required. In
cases when less than ten dishes were available, e.g. due to contamination or the presence of daughter
foci, the amount of dishes was harmonised either by random exclusion of dishes (one case only) or byplug-in of the median number of foci of the remaining dishes (seven cases). Impact of thesemodifications was considered negligible.
This approach is publicly available and implemented into R (R Development Core Team, 2009;
Hothorn et al., 2008; Venables and Ripley, 2002). For the specific conduct of the analysis, see Annex
13.3. The original R-codes were kindly provided by Prof. Hothorn (University of Hannover,Germany).

3.6.6 Assay acceptance criteria
The following criteria were employed in these studies, although it is important to note thatmodifications are described in the recommendations section (section 10) of this report based upon theoutcome of the study conducted.

3.6.6.1 Concurrent cell growth assays•

Uniform cell growth on culture wells should be observed,•

At least one NOEL concentration should be used,•

Cell growth curves should cover the range between NOEL and IC90 (ideally six concentrations
between NOEL and IC90) based on CFE,•

For CFE, a minimum PE of 30% in the negative control should be achieved.

3.6.6.2 Transformation assay•

The maximum number of Type III foci in the entire set of VC dishes (10 dishes) should notexceed five,•A minimum of six analysable concentrations is required, and they must cover the range ofcytotoxicity from NOEL to IC90,•

A minimum of nine Petri-dishes/concentration is required.

Transformation dishes must be stored and archived in each laboratory until the end of the validation
study, in the event that results need further analysis.

3.6.7 Assay assessment criteria
The following criteria were generated based on the data indicating that the negative binomial approach
was the most appropriate (see Section 3.6.5). The results presented in this report were evaluated withthe following criteria:•

A test chemical was considered "negative” (non-transforming) if no downturn-protected
Williams contrast showed a p-value < 0.01.

•

A test chemical was considered "positive" (transforming) if the downturn-protected Williams
contrast with the lowest p-value:- (I)included at least two consecutive concentrations, and30Balb/c 3T3 CTA PRP report v1 - 30 July 2010
- (II)•

showed a p-value < 0.01.

A test chemical was considered "inconclusive" if the downturn-protected Williams contrast
with the lowest p-value:- (I)included only one concentration or several non consecutive concentrations, and- (II)
showed a p-value < 0.01.

31

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

4 Module 2: Within-laboratory reproducibility
This phase of the study assessed the within-laboratory reproducibility and transferability of the
protocol by testing 3-methylcholanthrene as the PC and as a coded chemical. The HRI clone was usedwith the revised SOP (March 2006). This section provides the results of the TAs for all laboratories.
Figure 5-Figure 7 and Table 3-Table 8 show the results of the TAs. The doses for the TAs were chosenon the basis of the CFE experiments. The cytotoxicity determination was conducted in parallel by CFE
and CV. Table 3-Table 8 report the cytotoxicity results of 3-methylcholanthrene as assessed by the
relative Colony Forming Efficiency (rCFE) and the RCG in the CV experiment. The other columns of
the tables indicate the total number of morphologically transformed foci for each concentration tested,
and the mean number of foci per dish corresponding to each concentration tested.
The VC (DMSO 0.5%) gave spontaneous transformation frequencies within the expected range for the
Balb/c 3T3 cells under the assay conditions employed. The mean number per plate of Type III
transformed foci was 0-0.50 for ECVAM, 0.30 for Harlan CCR and 0 for HRI experiments. Further
evidence of within-laboratory reproducibility is seen in the results for 3-methylcholanthrene used as
the PC in the studies described in Module 4.
When the experiments did not meet the acceptance criteria, the laboratories were requested to repeatthem.

4.1 Transformation Assay – non coded 3-methylcholanthrene
The non-coded 3-methylcholanthrene was initially assessed in each laboratory. The doses of 3methylcholanthrene to be used were suggested by the VMT based on data from the literature andranged from 0.5 to 4 µg/ml. Harlan CCR tested only the doses recommended by the VMT, while theother laboratories tested additional concentrations in order to reach NOEL. The TA results arepresented in Table 3-Table 5. It should be noted that Harlan CCR and HRI used the same VC when
testing non-coded and coded chemicals.
Table 3: Transformation assay results from ECVAM, testing non-coded 3-methylcholanthrene
ECVAM
3-Methylcholanthrene(µg/ml)Untreated control
VC (DMSO 0.5 %)
0.01
0.03
0.1
0.3
1
3
10

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
95.6
94.2
64.8
62.8
51.2
83.1
4.1

na
100
82.4
92.6
83.9
85.5
61.5
42.2
39.2

1
0
3
6
14
10
12
47
94

0.10
0
0.30
0.60
1.40
1.00
1.20
4.70
9.40

VC = Vehicle Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV = Crystal Violet, na =not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

32Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Table 4: Transformation
methylcholanthrene
Harlan CCR
3-Methylcholanthrene(µg/ml)Untreated control
VC (DMSO 0.5 %)
0.5
1
2
4

assay

resultsfromHarlanCCR,testing

non-coded

3-

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
7.2
3.5
2.3
1.3

na
100
314
25.8
27.4
20.0

2
3
16
44
39
67

0.20
0.30
1.60
4.40
3.90
6.70

VC = Vehicle Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV = Crystal Violet, na =not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

Table 5: Transformation assay results from HRI, testing non-coded 3-methylcholanthrene
HRI
3-Methylcholanthrene(µg/ml)Untreated control
VC (DMSO 0.5 %)
0.01
0.03
0.1
0.3
1
3
10

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
66.6
54.6
26.6
18.0
10.3
6.6
5.4

na
100
95.1
90.4
83.2
68.6
43.7
29.7
19.7

0
0
5
7
10
15
34
119
224

0
0
0.50
0.70
1.00
1.50
3.40
11.90
22.40

VC = Vehicle Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV = Crystal Violet, na =not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

4.2 Transformation Assay – coded 3-methylcholanthrene
The coded 3-methylcholanthrene was assessed in each laboratory. The doses of 3-methylcholanthrene
to be used had been suggested by the VMT and ranged from 0.01 to 10 µg/ml.
The TA results are presented in Table 6-Table 8.

33

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Table 6: Transformation assay results from ECVAM, testing coded 3-methylcholanthrene
ECVAM
Coded
3-methylcholanthrene(µg/ml)Untreated control
VC (DMSO 0.5%)
0.01
0.03
0.1
0.3
1
3
10

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
106.1
63.6
23.6
27.9
17.0
10.9
7.3

na
100
70.1
35.4
24.8
16.9
46.3
13.5
15.2

5
5
2
4
2
6
13
15
41

0.50
0.50
0.20
0.40
0.20
0.60
1.30
1.50
4.10

VC = Vehicle Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV = Crystal Violet, na =not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

Table 7: Transformation assay results from Harlan CCR, testing coded 3-methylcholanthrene
Harlan CCR
Coded
3-methylcholanthrene(µg/ml)Untreated control
VC (DMSO 0.5%)
0.01
0.03
0.1
0.3
1
3
10

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
72.0
53.0
38.5
11.2
5.9
2.0
1.3

na
100
92.0
69.4
45.6
29.3
22.6
19.9
11.9

2
3
5
2
12
26
46
50
59

0.20
0.30
0.50
0.20
1.20
2.60
4.60
5.00
5.90

VC = Vehicle Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV = Crystal Violet, na =not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

Table 8: Transformation assay results from HRI, testing coded 3-methylcholanthrene
HRI
Coded
3-methylcholanthrene(µg/ml)Untreated control
VC (DMSO 0.5%)
0.01
0.03
0.1
0.3
1
3
10

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
76.9
48.6
31.7
16.3
10.6
6.3
6.0

na
100
86. 7
82.0
72.3
60.1
39.3
24.8
19.4

0
0
2
3
10
9
39
89
211

0
0
0.20
0.30
1.00
0.90
3.90
8.90
21.10

VC = Vehicle Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV = Crystal Violet, na =not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

4.2.1 Concurrent cytotoxicity: Crystal Violet

RCG [%]

Cytotoxicity of 3-methylcholanthrene was evaluated by the CV method in all laboratories (Figure 5).
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
0.01

ECVAM: non-coded
ECVAM: coded
Harlan CCR: non-coded
Harlan CCR: coded
HRI: non-coded
HRI: coded

0.1

1

10

3-Methylcholanthrene [µg/ml]

Figure 5: Relative cell growth (RCG) measured with the crystal violet assay compared to vehicle
control in all laboratories testing non-coded and coded 3-methylcholanthrene

4.2.2 Concurrent cytotoxicity: Colony Forming Efficiency
Cytotoxicity of 3-methylcholanthrene was evaluated by CFE assessment in all laboratories (Figure 6).
The results show a dose-dependent cytotoxicity with non-coded and coded chemicals in alllaboratories. The cytotoxicity curves are similar with the exception of the non-coded 3methylcholanthrene tested by ECVAM, which showed in general a lower level of cytotoxicity and an
unexpected result at the dose of 3 µg/ml, suggesting that a mistake in the chemical dilution for thisconcentration had been made.
In general CFE seemed to be more sensitive than CV, except for ECVAM.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

120

ECVAM: non-coded
ECVAM: coded
Harlan CCR: non-coded

110
100

Harlan CCR: coded
HRI: non-coded
HRI: coded

Relative CFE [%]

90
80
70
60
50
40
30
20
10
0
0.01

0.1

1

10

3-Methylcholanthrene [µg/ml]

Figure 6: Relative Colony Forming Efficiency (CFE) compared to vehicle control in alllaboratories testing non-coded and coded 3-methylcholanthrene

4.2.3 Morphological transformation
MT results with non-coded and coded 3-methylcholanthrene are shown in Figure 7. Although thedose-range tested by Harlan CCR for the non-coded 3-methylcholanthrene was limited, the trend in
the formation of transformed foci by non-coded and coded 3-methylcholanthrene was similar in alllaboratories. In HRI laboratory the overall response was higher than in other laboratories.

Mean number of foci per dish

40
35
30

ECVAM: non-coded
ECVAM: coded
Harlan CCR: non-coded
Harlan CCR: coded
HRI: non-coded
HRI: coded

25
20
15
10
5
0
0.0001

0.001

0.01

0.1

1

10

3-Methylcholanthrene [µg/ml]

Figure 7: Number of transformed foci for all laboratories testing non-coded and coded 3methylcholanthrene.
Results of the negative binomial statistical analysis are shown: concentrations that contributed to the statistically significant
result, i.e. which were present in the downturn-protected Williams contrast that resulted in the lowest p-value for a positive
response, are indicated by a horizontal line next to the corresponding laboratory name in the legend. The assessment criteria
are described in section 3.6.7.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

4.2.4 Acceptance criteria
Most of the acceptance criteria were met in the three laboratories (Table 9), except for the PE criteriaof coded 3-methylcholanthrene in ECVAM laboratory, which did not reach 30%. However thisexperiment was considered acceptable by the VMT since a clear positive response in morphologicaltransformation could be observed. In this phase of the validation study, the criteria that one NOEL
concentration and at least six analysable concentrations should be tested were not taken into
consideration, since the doses to be tested were fixed and had been previously recommended by the
VMT.
Table 9 : Acceptance criteria and assessment of non-coded and coded 3-methylcholanthrene
results
3-Methylcholanthrene
ECVAM
noncoded

ECVAM
coded

Laboratory
HarlanHarlannoncoded
coded

HRI
noncoded

HRI
coded

Concentration range(rCFE%)

96-4%

106-7%

7-1%

72-1%

67-5%

77-6%

PE of untreatedcontrol

41.4

22.8

32.6

32.6

41.6

41.6

PE of vehicle control
>30%

yes
(43.0)

no(20.6)

yes(38.0)

yes(38.0)

yes(43.8)

yes(43.8)

Vehicle used

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

Number of foci ofvehicle control <6

yes
(0)

yes
(5)

yes
(3)

yes
(3)

yes
(0)

yes
(0)

Number of analysable
concentrations

7

7

4

7

7

7

YES

NO#

YES

YES

YES

YES

Criteria

CFE

MT

Fulfillment of all assay acceptancecriteria
Assessment(Fisher’sCallexact test)
Concentrations in theAssessment
contrast [µg/ml]*(negative

+

+

+

+

+

+

0.01-10(7 conc.)

3-10(2 conc.)

0.5-4(4 conc.)0.1-10(5 conc.)0.01-10(7 conc.)0.01-10(7 conc.)

Call

+

+

+

+

+

+

binomial)

CFE = Colony Forming Efficiency, rCFE = relative Colony Plating Efficiency, MT = Morphological Transformation, PE =Plating Efficiency.
# considered acceptable for overall study evaluation.
* for positive assessment, the concentrations that contributed to the statistically significant result, i.e. which were present in
the downturn-protected Williams contrast that resulted in the lowest p-value, are indicated. The assessment criteria aredescribed in section 3.6.7.

4.3 Statistical analysis
The statistical analysis of these data was performed using the Fisher’s exact test, by comparing
number of foci formed in the surviving seeded cells, i.e. adjusted by cytotoxicity results, of control andtreated dishes. This analysis is based on the assumption that the relative cell survival in the CFE test is
the same as in the MT component of the assay. However, this is not completely correct since thesurvival in CFE may depend, in part, on the density of the cells seeded on the dish, which is different
in CFE and in MT, since the experiments are carried out independently. Moreover the cell survival ismeasured at a different time point than the MT. Though other statistical approaches had been used in
the past, no agreement was reached on the most appropriate statistical method for this analysis.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Consequently the VMT (at the meeting of May 2006) recommended that ECVAM would organise an
ad hoc expert group to evaluate the data and come up with a suggestion on the most suitable statisticalmethod to use for this assay (see section 3.6.5). Accordingly, the above data were re-analysed aposteriori using the negative binomial approach and showed a statistically significant increase intransformed foci in all experiment. Note that for the data generated with 3-methylcholanthrene, both
the Fischer's exact test and the negative binomial approach gave comparable results and led to thesame conclusions.

4.4 Conclusions of the Validation Management Team on Module 2
The data generated showed satisfactory within-laboratory reproducibility between the non-coded andthe coded 3-methylcholanthrene. Moreover the results demonstrated that the method had beensuccessfully transferred to all laboratories and provided an initial indication of between-laboratory
reproducibility for 3-methylcholanthrene.
The curves of cytotoxicity tests for non-coded and coded 3-methylcholanthrene were similar for
Harlan and HRI, whereas they varied for ECVAM. Overall, the CFE test was more sensitive than the
CV test, although the ranges of their separation differed among the laboratories.
Since all laboratories reproducibly induced a positive response in morphological transformation with
non-coded and coded 3-methylcholanthrene, even though there was some variance in response rates,
the VMT agreed that the laboratories were ready to proceed to the between-laboratory reproducibility
phase of the validation study. These results agree with published data (Heidelberger et al., 1983;
Kajiwara Y. and Ajimi S., 2003)

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

5 Module 3: Transferability
5.1 General Aspects
In general, the Balb/c 3T3 CTA can be performed in a laboratory that is experienced in routine cell
culture techniques. Thus, given the level of experience in general cell/tissue culture, such a laboratory
furnished with the appropriate test protocol and supporting SOPs and adequately trained to identify
morphological transformation events could be expected to effectively conduct the CTA.
General cell culture laboratory equipment and instruments are sufficient to perform the proposed test
method. All supplies and reagents are readily available commercially.
Scoring of transformed foci is at the moment still done manually using the microscope, thoughmethods for automation are being worked on. Proper training is therefore essential to ensure uniform
and objective scoring to the extent possible.

5.2 Training
The CTA requires personnel trained for general cell biology and cell culture techniques (e.g. aseptic
operations). Such expertise is available in most if not all QC-tissue culture laboratories. The operator
should, in particular, be trained in the scoring of transformed foci. The training requirements for a
person to be competent in scoring the plates are quite rigorous.
In order to ensure that all laboratories participating in this prevalidation study would use a harmonised
protocol and would be able to score appropriately, a training week was held at ECVAM in April 2005.
Representatives from all laboratories involved in the study participated in the training. Agreement wasreached on criteria for scoring the plates, using dishes treated with both the vehicle and reference
chemicals. In addition, a comparison was conducted between two methods of cytotoxicity, the CFE
and CV. Moreover, historical data produced by the ECVAM and HRI laboratories were also
evaluated. The experimental team agreed on the scoring of morphologically transformed foci asfollows: 1) only type III foci would need to be scored; 2) a detailed description of the morphology oftype III foci would be produced; 3) foci would to be scored using a stereomicroscope. Overall, thetraining was extremely useful for harmonising the procedures among the laboratories to be used in theprevalidation experiments. A report of the training week is available at ECVAM.
As part of this validation exercise a photo catalogue was produced by the HRI laboratory, incollaboration with the other participating laboratories, with the aim of standardising the scoring.
Although the classification of foci into distinct morphological categories can be difficult since theirdevelopment is a continuous process, three types of foci have been reported and defined. Although thiscategorisation was originally defined for the C3H/10T½ CTA (Reznikoff et al., 1973), it has since
been applied to the Balb/c 3T3 CTA. The catalogue includes pictures of both non malignantly
transformed foci (type I, type II) and malignantly transformed foci (type III). Examples of clearlyscorable foci, foci with questionable or mixed morphology, as well as examples of altered foci thatshould not be scored as transformed were included in the catalogue to obtain an overview of thedifferent types of foci that can be encountered during the Balb/c 3T3 CTA experiment.

5.3 Conclusions of the Validation Management Team on Module 3
Basic cell culture experience and training in the conduct and scoring of the assay are important. In
addition, the photograph catalogue of foci was found to be very useful in establishing consistency inassessing focus morphology and for the scoring of the experiments performed to assess the betweenlaboratory reproducibility.
The VMT agreed on the success of the method transfer.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6 Module 4: Between-laboratory reproducibility
The between-laboratory reproducibility experiments were conducted using the “SOP_rev_June_06”
version (Annex 13.2), which is the resulting version that was previously revised after the withinlaboratory and transferability phases of the validation study had been completed. The following codedchemicals were tested (Table 1): 2-acetylaminofluorene, benzo(a)pyrene, anthracene, phenanthrene, otoluidine HCl. The Safety Officers identified by the laboratories received the instructions to dissolve
all coded chemicals in DMSO. HRI laboratory did not follow these instructions and used insteadwater, therefore the laboratory was requested to repeat some of the experiments using DMSO as thesolvent. HRI laboratory stated that they had not received this information. Most probably the Safety
Officer had not passed the information to the laboratory staff.
The results of the between-laboratory reproducibility studies are presented in this section and aresummarised by compound and laboratory. The data are shown in tables for each individual laboratory.
Each table includes information on: the measures of cytotoxicity used shown by the rCFE (%), and the
RCG (%) in the CV experiment; the total number of morphologically transformed foci, and the mean
number of foci per dish corresponding to each concentration of chemical tested, to vehicle and positive
controls.
An initial DRF test using the CFE was performed by all laboratories to determine the experimental
doses to be used in the TA, for each chemical (Figure 8, Figure 13, Figure 18, Figure 23 and Figure28). Subsequently, the complete TA including concurrent cytotoxicity tests (Figure 9, Figure 10,
Figure 14, Figure 15, Figure 19, Figure 20, Figure 24, Figure 25, Figure 29 and Figure 30) and MT
assays were performed (Figure 11, Figure 16, Figure 21, Figure 26 and Figure 31). The concurrent
cytotoxicity tests were performed in parallel by CFE and CV in order to allow a comparison between
the two methods.
PE of vehicle and negative controls were similar in all the experiments performed.
VCs gave levels of transformed foci within the acceptable range dictated by the acceptance criteria
established for this Balb/c 3T3 CTA, except for four experiments where the level of transformed fociwas higher than five. The PC chemical 3-methylcholanthrene induced an increase in morphologicallytransformed foci in all experiments. The average number of transformed foci per dish in eachexperiment was always higher than 10 foci per dish (section 6.6).
All results were sent to the statistician as soon as they became available. A VMT meeting was held on
the 30 May 2007 to discuss the between-laboratory results.
Due to costs and resources needed to perform such an assay it was necessary to minimise therepetition of experiments. Therefore, some experiments were still considered by the VMT although
they did not fulfill all acceptance criteria. Justifications are given in these cases.

6.1 2-Acetylaminofluorene
6.1.1 Dose-range finding test
Figure 8 shows the results of the DRF tests with 2-acetylaminofluorene. The results were reproducible
in all laboratories and showed a narrow cytotoxic concentration range. ECVAM and HRI laboratories
performed the DRF twice.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

120
110
100

Relative CFE [%]

90
80
70
60
50
40
30
20
10
0
0.001

ECVAM
Harlan CCR
HRI

0.01

0.1

1

10

100

1000

2-Acetylaminofluorene [µg/ml]

Figure 8: Relative Colony Forming Efficiency (CFE) compared to vehicle control in alllaboratories testing coded 2-acetylaminofluorene, for the Dose-Range Finding. ECVAM and HRI
repeated the experiment.

6.1.2 Transformation assay
The following tables and figures show the MT and concurrent cytotoxicity test results upon treatment
with 2-acetylaminofluorene at the test concentrations which were selected based on solubility and
DRF tests.

6.1.2.1 ECVAM
2-Acetylaminofluorene was dissolved in DMSO. The concentrations were selected on the basis of the
DRF results and evaluated by ECVAM (Table 10). This chemical induced a statistically significant
positive response in morphological transformation.
Table 10: Transformation assay results from ECVAM, testing coded 2-acetylaminofluorene
ECVAM
2-Acetylaminofluorene(µg/ml)Untreated control
VC (DMSO 0.5%)
0.05
0.1
10
15
20
25
30
35
PC

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
nd
104.5
nd
54.2
26.3
17.4
10. 8
4.5
1.8

na
100
nd
108.9
nd
95.4
37.0
21.5
10.6
9.5
15.9

4
12
6
7
3
5
12
36
123
114
182

0.44
1.20
0.60
0.78
0.33
0.50
1.20
4.00
12.30
12.67
20.22

VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable, nd = not determined.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.1.2.2 Harlan CCR
2-Acetylaminofluorene was dissolved in DMSO. The concentrations were selected on the basis of the
DRF results and evaluated by Harlan CCR (Table 11). This chemical induced a statistically significant
positive response in morphological transformation at a single dose. As such, the result was considered
inconclusive.
Harlan CCR was requested to repeat the assay since it gave an inconclusive call, however this was notdone due to lack of resources.
Table 11: Transformation assay results from Harlan CCR, testing coded 2-acetylaminofluorene
Harlan CCR
2-Acetylaminofluorene(µg/ml)Untreated control
VC (DMSO 0.5%)

1
2.5
5
10
15
20
25
30

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
91.0
82.7
90.4
77.8
44.4
12.3
5.9
1.2
1.5

na
100
96.3
100.7
94.5
87.8
79.8
31.1
22.5
4.8
22.0

3
3
2
4
5
3
3
3
7
24
113

0.30
0.30
0.20
0.40
0.50
0.30
0.30
0.30
0.78
2.40
11.30

PC
VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable.
Concentration that contributed to the statistically significant result, i.e. which was present in the downturn-protected Williams
contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values arehighlighted in bold.

6.1.2.3 HRI
2-Acetylaminofluorene was dissolved in ultrapure water by HRI. The concentrations were selected on
the basis of the DRF results and evaluated by HRI (Table 12). This chemical induced a statisticallysignificant positive response in morphological transformation.
Although this chemical was tested in water, HRI was not asked to repeat the experiment in DMSO
because 1) HRI results on cytotoxicity are similar to cytotoxicity curves from the other laboratories, 2)2-acetylaminofluorene produces a statistically significant positive response related to multiple doses
and, 3) to avoid a waste of resources.
Table 12: Transformation assay results from HRI, testing coded 2-acetylaminofluorene
HRI
2-Acetylaminofluorene(µg/ml)Untreated control
VC (water)

10
25
50
75
100
150

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
98.6
67.2
34.7
21.4
11.8
2.1
11.0

na
100
104.6
47.3
6.6
4.1
5.0
6.6
-3.6

5
2
1
32
16
10
17
32
161

0.50
0.20
0.10
3.20
1.60
1.00
1.70
3.20
16.10

PC
VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.1.2.4 Concurrent cytotoxicity: Colony Forming Efficiency
Cytotoxicity of 2-acetylaminofluorene was evaluated by CFE assessment in all laboratories (Figure 9).
All laboratories showed cytotoxicity within a narrow concentration range: ECVAM (10-30 µg/ml),
Harlan CCR (6.8-27.3 µg/ml) and HRI (25-100 µg/ml).

120

ECVAM
Harlan CCR
HRI

110
100

Relative CFE [%]

90
80
70
60
50
40
30
20
10
0
0.01

0.1

1

10

100

1000

2-Acetylaminofluorene [µg/ml]

Figure 9: Relative Colony Forming Efficiency (CFE) compared to vehicle control in alllaboratories testing coded 2-acetylaminofluorene

6.1.2.5 Concurrent cytotoxicity: Crystal Violet
Cytotoxicity of 2-acetylaminofluorene was evaluated by the CV method in all laboratories (Figure 10).
All laboratories showed cytotoxicity within a narrow concentration range. For this chemical no clear
differences were noted between the cytotoxicity curves of the CFE and CV tests.

43

Relative cell growth [%]Balb/c 3T3 CTA PRP report v1 - 30 July 2010

140
130
120
110
100
90
80
70
60
50
40
30
20
10
0.01

ECVAM
Harlan CCR
HRI

0.1

1

10

100

1000

2-Acetylaminofluorene [µg/ml]

Figure 10: Relative cell growth compared to vehicle control in all laboratories testing coded 2acetylaminofluorene

6.1.2.6 Morphological transformation
MT results with 2-acetylaminofluorene are shown in Figure 11. Both HRI and ECVAM showed astatistically significant increase in the number of transformed foci, while Harlan CCR showed astatistically significant increase of transformed foci related to the highest dose tested only. The
transformed foci appeared at doses corresponding to IC80 and more.

mean number of foci per dish

20

ECVAM
Harlan CCR
HRIInconclusive

10

100

15

10

5

0
0.01

0.1

1

1000

2-acetylaminofluorene [µg/ml]

Figure 11: Mean number of foci per dish for all laboratories testing coded 2acetylaminofluorene.
Results of the negative binomial statistical analysis are shown. For positive assessment the concentrations that contributed tothe statistically significant result, i.e. which were present in the downturn-protected Williams contrast that resulted in thelowest p-value, are indicated by a horizontal line next to the corresponding laboratory name in the legend. Assessment for
Harlan CCR was reported inconclusive since the downturn-protected Williams contrast with the lowest p-value showed a pvalue < 0.01 but included only one concentration (30 µg/ml). The assessment criteria are described in section 3.6.7.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

MT results for the different control treatments are summarised in Figure 12. The untreated, vehicle andpositive controls were reproducible across the laboratories.

Number of transformed colonies

30
ECVAM
Harlan CCR
HRI

20

10

0
untreated

vehiclepositive

Controls to 2-acetylaminofluorene

Figure 12: Number of transformed colonies (mean + standard deviation) for untreated, vehicle
and positive controls in all laboratories testing coded 2-acetylaminofluorene

6.1.2.7 Acceptance and assessment criteria
All acceptance criteria were fulfilled upon treatment with 2-acetylaminofluorene, except for thenumber of transformed foci in the VC in the ECVAM laboratory (Table 13).
Although the ECVAM experiment did not fulfil all acceptance criteria, it was considered acceptable
for the overall study evaluation since a clear dose-response transforming effect was observed.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Table 13: Acceptance criteria and assessment of 2-acetylaminofluorene results
2-Acetylaminofluorene

ECVAM

Laboratory
Harlan CCR

HRI

1 NOEL [µg/ml]

yes
(0.1)

yes
(5)

yes
(10)

PE of untreated control

44.8

38.3

62.9

PE of vehicle control >30%

yes
(41.7)

yes(40.5)

yes(64.8)

Vehicle used

DMSO

DMSO

water

Number of foci of vehicle
control <6

no
(12)

yes
(3)

yes
(2)

Number of analysable
concentrations ≥6

yes
(8)

yes
(8)

yes
(6)

NO#

YES

YES

20 - 35(4 conc.)30(1 conc.)

25 - 150(5 conc.)

+

i**

+

Criteria

CFE

MT

Fulfilment of all assay acceptance criteria
Assessment

Concentrations in the contrast[µg/ml]*Call

CFE = Colony Forming Efficiency, MT = Morphological Transformation, NOEL = No Observed Effect Level, PE = Plating
Efficiency, i = inconclusive.
#
considered acceptable for overall study evaluation.
* for positive assessment, the concentrations that contributed to the statistically significant result, i.e. which were present in
the downturn-protected Williams contrast that resulted in the lowest p-value, are indicated.
** for Harlan CCR assessment was reported inconclusive since the downturn-protected Williams contrast with the lowest pvalue showed a p-value < 0.01 but included only one concentration (30 µg/ml).
The assessment criteria are described in section 3.6.7.

6.1.3 Conclusion
Treatment with 2-acetylaminofluorene produced a statistically significant positive response inmorphologically transformed foci in ECVAM and HRI laboratories, and a clear increase in the numberof foci at the highest concentration only in Harlan laboratory. Since the assessment criteria for apositive call for MT required at least two concentrations to be in the downturn-protected Williams
contrast with the lowest p-value, the resulting call for Harlan CCR was inconclusive and should havebeen repeated. Due to a lack of resources and time reasons Harlan CCR could not repeat thisexperiment.
It can be concluded that, according to the assessment criteria established for this study, theexperiments were reproducible in two laboratories, and that 2-acetylaminofluorene was considered to
be a positive transforming agent in the Balb/c 3T3 CTA. These results agree with published data
(Hedelberger et al., 1983; Matthews et al., 1993c).

6.2 Benzo(a)pyrene
6.2.1 Dose-range finding test
Figure 13 shows the results of the DRF tests with benzo(a)pyrene. Harlan CCR and HRI laboratories
observed a dose-dependent cytoxicity induced by benzo(a)pyrene at the concentrations tested. HRI
repeated the experiment twice.

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010

120
ECVAM
Harlan CCR
HRI

110

Relative CFE [%]

100
90
80
70
60
50
40
30
20
10
0
0.0001

0.001

0.01

0.1

1

10

100

Benzo(a)pyrene [µg/ml]

Figure 13: Relative Colony Forming Efficiency (CFE) compared to vehicle control in alllaboratories testing coded benzo(a)pyrene, for the Dose-Range Finding.

6.2.2 Transformation Assay
The following tables and figures show the MT and concurrent cytotoxicity test results upon treatment
with benzo(a)pyrene at the test concentrations which were selected based on solubility and DRF tests.

6.2.2.1 ECVAM
Benzo(a)pyrene was dissolved in DMSO. The concentrations were selected on the basis of the DRF
results and evaluated by ECVAM (Table 14). This chemical induced a statistically significant positive
response in morphological transformation.
Table 14: Transformation assay results from ECVAM, testing coded benzo(a)pyrene
ECVAM
Benzo(a)pyrene(µg/ml)Untreated control
VC (DMSO 0.5%)
0.0005
0.001
0.005
0.05
0.125
0.625
3.125
15

CFE (%)RCG (%)
CV

Total number offociMean number offoci per dish

na
100
85.8
nd
62.1
0.0
0.8
0.4
nd
0.0
7.1

na
100
83.7
nd
89.0
49.3
10.4
3.2
nd
2.6
25.0

5
4
6
2
15
69
91
110
75
13
131

0.50
0.40
0.60
0.20
1.50
6.90
9.10
11.00
7.50
1.30
13.10

PC
VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable, nd = not determined
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

47

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.2.2.2 Harlan CCR
Benzo(a)pyrene was dissolved in DMSO. The concentrations were selected on the basis of the DRF
results and evaluated by Harlan CCR (Table 15). This chemical induced a statistically significant
positive response in morphological transformation.
Table 15: Transformation assay results from Harlan CCR, testing coded benzo(a)pyrene
Harlan CCR
Benzo(a)pyrene(µg/ml)Untreated control
VC (DMSO 0.5%)
0.01
0.015
0.02
0.03
0.04
0.06
0.08
0.1
0.12
0.2
PC

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
65.3
49.6
37.5
21.5
4.5
1.8
1.3
0.8
0.8
0.5
6.3

na
100
76.3
79.4
70.4
89.8
73.0
58.2
34.3
27.1
23.5
9.2
25.9

4
0
5
7
19
21
21
33
39
45
48
72
135

0.40
0
0.50
0.70
1.90
2.10
2.10
3.30
3.90
4.50
4.80
7.20
15.00

VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

6.2.2.3 HRI
Benzo(a)pyrene was dissolved in DMSO. The concentrations were selected on the basis of the DRF
results and evaluated by HRI (Table 16). This chemical induced a statistically significant positive
response in morphological transformation.
Table 16: Transformation assay results from HRI, testing coded benzo(a)pyrene
HRI
Benzo(a)pyrene(µg/ml)Untreated control
VC (DMSO 0.5%)
0.003
0.010
0.030
0.100
0.300
1.000
PC

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
90.2
70.1
15.5
3.5
1.4
0.4
8.8

na
100
96.4
83.3
23.6
6.1
4.4
4.4
9.2

2
5
6
7
20
17
27
65
250

0.22
0.50
0.67
1.00
2.22
1.70
3.38
6.50
25.00

VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

48

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.2.2.4 Concurrent cytotoxicity: Colony Forming Efficiency
Cytotoxicity of benzo(a)pyrene was evaluated by measuring CFE in all laboratories (Figure 14).
Benzo(a)pyrene was shown to be cytotoxic in all laboratories as a function of concentration.
Cytotoxicity was similar in all laboratories.
120

ECVAM
Harlan CCR
HRI

110
100

Relative CFE [%]

90
80
70
60
50
40
30
20
10
0
0.0001

0.001

0.01

0.1

1

10

100

Benzo(a)pyrene [µg/ml]

Figure 14: Relative Colony Forming Efficiency (CFE) compared to vehicle control in alllaboratories testing coded benzo(a)pyrene

6.2.2.5 Concurrent cytotoxicity: Crystal Violet

Relative cell growth [%]Cytotoxicity of benzo(a)pyrene was evaluated by the CV method in all laboratories (Figure 15).
The results suggest that CV may be less sensitive than CFE in the assessment of cytotoxicity ofbenzo(a)pyrene.
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
0.0001ECVAM
Harlan CCR
HRI

0.001

0.01

0.1

1

10

100

Benzo(a)pyrene [µg/ml]

Figure 15: Relative cell growth compared to vehicle control in all laboratories testing coded
benzo(a)pyrene

49Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.2.2.6 Morphological Transformation
MT results with benzo(a)pyrene are shown in Figure 16. All three laboratories clearly demonstrated
that benzo(a)pyrene induced a positive response in a relatively broad concentration range: from 0.005µg/ml to 15 µg/ml for ECVAM, from 0.02 µg/ml for Harlan, and from 0.03 µg/ml to the highest
tested concentration of 1.0 µg/ml for HRI. The decrease in the number of foci at the two highest
concentrations for ECVAM can be explained by the high cytotoxicity induced at these doses.

Mean number of foci per dish

20
ECVAM
Harlan CCR
HRI

15

10

5

0
0.0001

0.001

0.01

0.1

1

10

100

1000

Benzo(a)pyrene [µg/ml]

Figure 16: Mean number of foci per dish for all laboratories testing coded benzo(a)pyrene.
Results of the negative binomial statistical analysis are shown: for positive assessment, the concentrations that contributed tothe statistically significant result, i.e. which were present in the downturn-protected Williams contrast that resulted in thelowest p-value, are indicated by a horizontal line, next to the corresponding laboratory name in the legend. The assessment
criteria are described in section 3.6.7.

MT results for the different control treatments are summarised in Figure 17. The untreated, vehicle andpositive controls were reproducible across the laboratories. HRI shows a higher value for the PC.

Number of transformed colonies

35
30

ECVAM
Harlan CCR
HRI

25
20
15
10
5
0
untreated

vehiclepositive

Controls to benzo(a)pyrene

Figure 17: Number of transformed colonies (mean + standard deviation) for untreated, vehicle
and positive controls in all laboratories testing coded benzo(a)pyrene

50Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.2.2.7 Acceptance and assessment criteria
ECVAM met all the assay acceptance criteria (Table 17). Although one of the assay acceptancecriteria was not met in Harlan (absence of NOEL) and HRI (number of analysable concentrations),
benzo(a)pyrene induced a clearly positive response in transformed foci in all laboratories. Therefore
despite these limitations, these experiments were considered acceptable for the overall study
evaluation.
Table 17: Acceptance criteria and assessment of benzo(a)pyrene results
Benzo(a)pyrene
Criteria
ECVAM

MT

no

yes
(0.003)PE of untreated control

32.5

49.9

62.6

PE of vehicle control >30%yes
(31.6)

yes(47.6)

yes(61.1)

Vehicle used

DMSO

DMSO

DMSO

Number of foci of vehicle
control <6

yes
(4)

yes
(0)

yes
(5)

Number of analysable
concentrations ≥6

yes
(8)

yes
(10)

no
(4*)

YES

NO#

NO#

0.0005 - 15
(All conc.)

0.01 - 0.2(All conc.)

0.03 - 1(4 conc. *)

+

+

+

Fulfilment of all assay acceptance criteria
Assessment

HRI

yes
(0.0005)

1 NOEL [µg/ml]
CFE

Laboratory
Harlan CCR

Concentrations in the contrast[µg/ml]**Call

CFE = Colony Forming Efficiency, MT = Morphological Transformation, NOEL = No Observed Effect Level, PE = Plating
Efficiency.
#
considered acceptable for overall study evaluation.
* only concentrations with at least 9 dishes available were used for the assessment (i.e. 0.003, 0.03, 0.1 and 1 µg/ml).
** for positive assessment, the concentrations that contributed to the statistically significant result, i.e. which were present in
the downturn-protected Williams contrast that resulted in the lowest p-value (negative binomial analysis), are indicated. The
assessment criteria are described in section 3.6.7.

6.2.3 Conclusion
Treatment with benzo(a)pyrene produced a positive response in morphologically transformed foci inall laboratories.
It can be concluded that, according to the assessment criteria established for this study, theexperiments were reproducible in all laboratories, and that benzo(a)pyrene was considered to be apositive transforming agent in the Balb/c 3T3 CTA. These results agree with published data
(Heidelberger et al., 1983, Sheu et al., 1994).

51

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.3 Anthracene
6.3.1 Dose-range finding test

Relative CFE [%]Figure 18 shows the results of the DRF tests with anthracene. This chemical does not show clear signsof toxicity up to the limits of solubility. ECVAM performed the DRF twice. HRI tested a different
concentration range since they used water as a solvent.

140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
0.0001ECVAM
Harlan CCR
HRI

0.001

0.01

0.1

1

10

100

1000

10000

Anthracene [µg/ml]

Figure 18: Relative Colony Forming Efficiency (CFE) compared to vehicle control in alllaboratories testing coded anthracene, for the Dose-Range Finding. ECVAM repeated theexperiment.

6.3.2 Transformation assay
The following tables and figures show the MT and concurrent cytotoxicity test results upon treatment
with anthracene at the test concentrations which were selected based on solubility and DRF tests.

6.3.2.1 ECVAM
Anthracene was dissolved in DMSO by ECVAM. The concentrations were selected on the basisof the DRF results and evaluated by ECVAM (Table 18). The chemical was tested up to precipitating
doses. No positive response in morphological transformation was observed upon treatment with thischemical.

52

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Table 18: Transformation assay results from ECVAM, testing coded anthracene
ECVAM
Anthracene(µg/ml)Untreated control
VC (DMSO 0.5%)

0.1
0.2
0.4
1
2
10
20
40

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
104.5
nd
94.9
nd
108.6
98.8
100.8
91.9
9.8

na
100
96.5
nd
84.1
nd
90.5
83.5
74.1
86.6
38.7

4
5
3
4
15
8
7
16
14
13
156

0.40
0.50
0.30
0.40
1.50
0.80
0.70
1.60
1.40
1.30
15.60

PC
VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable, nd = not determined

6.3.2.2 Harlan CCR
Anthracene was dissolved in DMSO by Harlan CCR. The concentrations were selected on the basis of
the DRF results and evaluated by Harlan CCR (Table 19). The chemical was tested up to precipitating
doses. No positive response in morphological transformation was observed upon treatment with thischemical.
Table 19: Transformation assay results from Harlan CCR, testing coded anthracene
Harlan CCR
Anthracene(µg/ml)Untreated control
VC (DMSO 0.5%)

0.7
1.4
2.8
5.6
11.2
22.5
45

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
83.3
95.8
96.1
79.4
101.1
88.9
88.6
0

na
100
94.8
100.0
94.9
92.1
92.7
90.6
92.9
23.9

4
4
1
5
5
1
5
2
0
137

0.40
0.40
0.11
0.50
0.50
0.10
0.50
0.20
0
13.70

PC
VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable.

6.3.2.3 HRI
A suspension of anthracene in water was tested by HRI. The concentrations were selected on the basisof the DRF results and evaluated by HRI (Table 20). No positive response in morphologicaltransformation was observed upon treatment with this chemical.
Although this chemical was tested in water, HRI was not asked to repeat the experiment in DMSO, thesolvent requested by the VMT, because 1) anthracene was clearly non cytotoxic in DMSO and waterand 2) anthracene was clearly negative in DMSO and water.

53

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Table 20: Transformation assay results from HRI, testing coded anthracene
HRI
Anthracene(µg/ml)rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
98.6
104.0
100.2
98.0
98.6
101.2
15.0

na
100
85.3
123.4
77.2
94.0
105.4
140.2
0.3

9
6
5
2
3
3
2
0
207

0.90
0.60
0.50
0.20
0.30
0.30
0.20
0
20.70

Untreated control
VC (water)

100
300
500
1000
3000
5000

PC
VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable.

6.3.2.4 Concurrent cytotoxicity: Colony Forming Efficiency
Cytotoxicity of anthracene was evaluated by CFE assessment in all laboratories (Figure 19) and wasnon cytotoxic at the concentrations tested (up to the limits of solubility). HRI curve is shifted to theright because water instead of DMSO has been used as solvent. For the DMSO the upper limit was 4045 µg/ml for ECVAM and Harlan CCR due to precipitation of the compounds into the medium, while
HRI tested up to 5000 µg/ml. In none of the conditions cytotoxicity was reached.
120
110
100

Relative CFE [%]

90
80
70
60
50
40
30

ECVAM
Harlan CCR
HRI

20
10
0
0.01

0.1

1

10

100

1000

10000

Anthracene [µg/ml]

Figure 19: Relative Colony Forming Efficiency (CFE) compared to vehicle control in alllaboratories testing coded anthracene

6.3.2.5 Concurrent cytotoxicity: Crystal Violet
Cytotoxicity of anthracene was evaluated by CV in all laboratories (Figure 20). HRI curve is shifted to
the right because water instead of DMSO has been used as solvent.

54

Relative cell growth [%]Balb/c 3T3 CTA PRP report v1 - 30 July 2010

150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
0.01

ECVAM
Harlan CCR
HRI

0.1

1

10

100

1000

10000

Anthracene [µg/ml]

Figure 20: Relative cell growth compared to vehicle control in all laboratories testing coded
anthracene

6.3.2.6 Morphological transformation
MT results with anthracene are shown in Figure 21. A negative response was observed in alllaboratories. HRI curve is shifted to the right because water instead of DMSO has been used assolvent.

M ean number of foci per dish20

15

Negative

ECVAM

Negative

Harlan CCR

Negative

HRI

100

1000

10

5

0
0.01

0.1

1

10

10000

Anthracene [µg/ml]

Figure 21: Mean number of foci per dish for all laboratories testing coded anthracene.
Results of the negative binomial statistical analysis are shown. Negative = no downturn-protected Williams contrast resulted
in a statistically significant p-value for any laboratory. The assessment criteria are described in section 3.6.7.

MT results for the different control treatments are summarised in Figure 22. The untreated, vehicle andpositive controls were reproducible across the laboratories.

55

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

Number of transformed colonies

30
25

ECVAM
Harlan CCR
HRI

20
15
10
5
0
untreated

vehiclepositive

Controls to anthracene

Figure 22: Number of transformed colonies (mean + standard deviation) for untreated, vehicle
and positive controls in all laboratories testing coded anthracene

6.3.2.7 Acceptance and assessment criteria
For ECVAM and Harlan CCR, all acceptance criteria were fulfilled. For HRI, all acceptance criteria
were met except that the number of transformed foci in the VC was slightly above the limit: six fociwere observed instead of five as stated by the acceptability criteria (Table 21). Since the number offoci in the treatment groups was within the VC range, the data were accepted and a negative call was
assigned.
Table 21: Acceptance criteria and assessment of anthracene results
Anthracene
ECVAM

Laboratory
Harlan CCR

HRI

yes
(40 = max. conc.)yes(45 = max. conc.)

yes(5000 = max. conc.)

PE of untreated control

63.4

38.1

62.4

PE of vehicle control >30%

yes
(63.6)

yes(44.9)

yes(62.0)

Vehicle used

DMSO

DMSO

water

Number of foci of vehicle
control <6

yes
(5)

yes
(4)

no
(6)

Number of analysable
concentrations ≥6

yes
(8)

yes
(7)

yes
(6)

YES

YES

NO#

Concentrations in the contrast[µg/ml]*

na

na

na

Call

-

-

-

Criteria
1 NOEL [µg/ml]
CFE

MT

Fulfilment of all assay acceptance criteria
Assessment

CFE = Colony Forming Efficiency, MT = Morphological Transformation, NOEL = No Observed Effect Level, PE = Plating
Efficiency, na = not applicable.
#considered acceptable for overall study evaluation* The assessment criteria are described in section 3.6.7.

56

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.3.3 Conclusion
Treatment with anthracene produced negative response in morphologically transformed foci in alllaboratories.
It can be concluded that, according to the assessment criteria established for this study, theexperiments were reproducible in all laboratories, and that anthracene was considered to be a nontransforming agent in the Balb/c 3T3 CTA. These results agree with published data (Heidelberger etal., 1983).

6.4 Phenanthrene
6.4.1 Dose-range finding test
Figure 23 shows the results of the DRF tests with phenanthrene. The DRF experiments show thatphenanthrene induced cytotoxicity in a very narrow concentration range, in all laboratories. ECVAM
laboratory performed the DRF twice.
120
110
100

Relative CFE [%]

90
80
70
60
50
40
30
20
10
0
0.001

ECVAM
Harlan CCR
HRI

0.01

0.1

1

10

100

1000

10000

Phenanthrene [µg/ml]

Figure 23: Relative Colony Forming Efficiency (CFE) compared to vehicle control in alllaboratories testing coded phenanthrene, for the Dose-Range Finding. ECVAM repeated theexperiment.

6.4.2 Transformation assay
The following tables and figures show the MT and concurrent cytotoxicity test results upon treatment
with phenanthrene at the test concentrations which were selected based on solubility and DRF tests.

6.4.2.1 ECVAM
Phenanthrene was dissolved in DMSO. The concentrations were selected on the basis of the DRF
results and evaluated by ECVAM (Table 22).

57

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Table 22: Transformation assay results from ECVAM (1st experiment), testing codedphenanthrene
ECVAM
Phenanthrene(µg/ml)TA1
Untreated control
VC (DMSO 0.5%)
0.01
0.1
1
10
20
30
50
75
PC

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
nd
106.5
nd
85.7
76.3
59.1
19.2
4.2
2.9

na
100
nd
103.0
nd
91.3
84.8
64.5
9.9
1.3
14.9

4
4
0
5
7
4
1
5
22
0
114

0.40
0.40
0
0.50
0.70
0.40
0.10
0.50
2.20
0
11.40

TA1 = Transformation Assay 1, VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency,
RCG = Relative Cell Growth, CV = Crystal Violet, na = not applicable, nd = not determined

According to the statistical analysis the assessment of phenanthrene for the ECVAM laboratory was
negative. However, an increase in the number of transformed foci was observed at the highest testable
dose (50 µg/ml), suggesting that phenanthrene could have a transforming potential. To verify thissuspicion the VMT requested the laboratory to retest the same chemical (TA2) focusing on moreclosely spaced concentrations (Table 23). A retest with this chemical showed a statistically significant
positive response in morphological transformation.
Table 23: Transformation assay results from ECVAM (2nd experiment), testing codedphenanthrene
ECVAM
Phenanthrene(µg/ml)TA2
Untreated control
VC (DMSO 0.5%)
10
20
30
40
45
50
60
75
PC

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
85.1
nd
62.2
42.3
17.7
14.1
nd
0.0
25.7

na
100
86.7
nd
90.5
75.1
49.2
32.2
nd
11.7
41.8

4
6
2
4
7
16
4
16
32
80
161

0.40
0.60
0.20
0.40
0.70
1.60
0.40
1.60
3.20
8.00
16.10

TA2 = Transformation Assay 2, VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency,
RCG = Relative Cell Growth, CV = Crystal Violet, na = not applicable, nd = not determined
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

6.4.2.2 Harlan CCR
Phenanthrene was dissolved in DMSO. The concentrations were selected and evaluated by Harlan
CCR on the basis of the DRF results (Table 24).

58

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Table 24: Transformation assay results from Harlan CCR (1st experiment), testing codedphenanthrene
Harlan CCR
Phenanthrene(µg/ml)TA1
Untreated control
VC (DMSO 0.5%)
1
2.5
5
7.5
10
30
60
100
PC

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
102.0
88.2
89.7
118.1
86.8
52.9
22.5
1.0
2.9

na
100
105.4
105.9
106.8
99.1
95.8
74.8
38.7
15.0
18.3

2
3
1
1
2
4
2
5
28
74
115

0.20
0.30
0.10
0.10
0.20
0.40
0.20
0.50
2.80
7.40
11.50

TA1 = Transformation Assay 1, VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency,
RCG = Relative Cell Growth, CV = Crystal Violet, na = not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

Although, the first experiment did not fulfil one of the assay acceptance criteria (PE <30%), the data
showed a statistically significant positive response at the two highest dose levels. As such, the VMTconsidered the test to be acceptable. The VMT requested a repeat study to confirm these results (Table
25). A retest with phenanthrene, in which all of the assay acceptance criteria were fulfilled, confirmed
this result, showing a statistically significant positive response in morphological transformation.
Table 25 : Transformation assay results from Harlan CCR (2nd experiment), testing codedphenanthrene
Harlan CCR
Phenanthrene(µg/ml)TA2
Untreated control
VC (DMSO 0.5%)

10
30
40
50
60
70
80
90
100

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
86.2
77.1
47.1
30.3
12.8
3.4
1.7
1.0
0.7
0.7

na
100
101.2
79.5
53.6
30.0
20.7
12.5
14.8
15.29
16.4
15.8

3
1
3
2
14
15
17
28
39
48
51
136

0.30
0.10
0.30
0.20
1.56
1.50
1.70
2.80
3.90
4.80
5.10
13.60

PC
TA2 = Transformation Assay 2, VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency,
RCG = Relative Cell Growth, CV = Crystal Violet, na = not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

59

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.4.2.3 HRI
Phenanthrene was initially dissolved in water (TA1) and according to the statistical analysis theassessment of phenanthrene was negative (see Annex 13.5.1). The VMT asked the laboratory to retest
using DMSO which was the solvent originally requested (TA2). The concentrations were selected andevaluated by HRI on the basis of the DRF results (Table 26). A statistically significant positive
response in morphological transformation was observed at two concentrations.
Daughter foci were observed in a single dish of the PC, but were not considered during scoring.
Table 26: Transformation assay results from HRI (2nd experiment), testing coded phenanthrene
HRI
Phenanthrene(µg/ml)TA2
Untreated control

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
108.2
99.8
83.5
68.0
50.6
26.3
6.8
0.9
21.4

na
100
114.9
105.6
104.0
84.1
58.5
38.5
34. 8
16.0
36.4

0
0
0
0
0
1
1
2
32
16
93

0
0
0
0
0
0.10
0.10
0.20
3.20
1.60
10.33

VC (DMSO 0.5%)

10
15
22
32
45
70
100
150

PC
VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

6.4.2.4 Concurrent cytoxicity: Colony Forming Efficiency
Cytotoxicity of phenanthrene was evaluated by CFE assessment in all laboratories (Figure 24).
Cytotoxicity was induced in very narrow concentration range in all laboratories.
120
110
100

Relative CFE [%]

90
80
70
60
50
40
30
20
10
0
0.01

ECVAM TA1
ECVAM TA2
Harlan CCR TA1
Harlan CCR TA2
HRI TA2

0.1

1

10

100

Phenanthrene [µg/ml]

Figure 24: Relative Colony Forming Efficiency (CFE) compared to vehicle control in alllaboratories testing coded phenanthrene. ECVAM and Harlan CCR repeated the experiment.

60

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.4.2.5 Concurrent cytotoxicity: Crystal Violet
Cytotoxicity of phenanthrene was evaluated by the CV method in all laboratories (Figure 25).

Relative cell growth [%]The cytotoxicity of phenanthrene measured by the CV method gave similar results to that measured by
the CFE method.
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
0.01

ECVAM TA1
ECVAM TA2
Harlan CCR TA1
Harlan CCR TA2
HRI TA2

0.1

1

10

100

Phenanthrene [µg/ml]

Figure 25: Relative cell growth compared to vehicle control in all laboratories testing codedphenanthrene. ECVAM and Harlan CCR repeated the experiment.

6.4.2.6 Morphological transformation
MT results with phenanthrene are shown in Figure 26.

Mean number of foci per dish

20

ECVAM TA1
ECVAM TA2
Harlan CCR TA1
Harlan CCR TA2
HRI TA2

15

Negative

10

5

0
0.001

0.01

0.1

1

10

100

Phenanthrene [µg/ml]

Figure 26: Mean number of foci per dish for all laboratories testing coded phenanthrene.
ECVAM and Harlan CCR repeated the experiment.
Results of the negative binomial statistical analysis are shown: for positive assessment, the concentrations that contributed tothe statistically significant result, i.e. which were present in the downturn-protected Williams contrast that resulted in thelowest p-value, are indicated by a horizontal line, next to the corresponding laboratory name in the legend. The assessment
criteria are described in section 3.6.7.

61

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

MT results for the different control treatments are summarised in Figure 27. The untreated, vehicle andpositive controls were reproducible across the laboratories.

Number of transformed colonies

20
ECVAM
Harlan CCR
HRI

15

10

5

0
untreated

vehiclepositive

Controls to Phenanthrene

Figure 27: Number of transformed colonies (mean + standard deviation) for untreated, vehicle
and positive controls in all laboratories testing coded phenanthrene

6.4.2.7 Acceptance and assessement criteria
Two experiments did not fulfil all the acceptance criteria for phenanthrene TA results (Table 27),
however in both cases a clear transforming response was observed and the data were considered
acceptable for the overall study evaluation.

62

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Table 27: Acceptance criteria and assessment of phenanthrene results
Phenanthrene
ECVAM
TA1

ECVAM
TA2

Laboratory
Harlan
CCR TA1

Harlan
CCR TA2

HRI
TA2

1 NOEL [µg/ml]

yes
(0.1)

yes
(10)

yes
(10)

yes
(10)

yes
(15)

PE of untreated control

36.1

47.9

25.8

38.4

55.8

PE of vehicle control >30%

yes
(38.5)

yes(45.3)

no(25.5)

yes(37.1)

yes(53.1)

Vehicle used

DMSO

DMSO

DMSO

DMSO

DMSO

Number of foci in vehicle
control < 6

yes
(4)

no
(6)

yes
(3)

yes
(1)

yes
(0)

Number of analysable
concentrations ≥6

yes
(8)

yes
(8)

yes
(8)

yes
(9)

yes
(8)

YES

NO

#

NO

YES

YES

Concentrations in thecontrast [µg/ml]*na

60 - 75(2 conc.)

60 - 100(2 conc.)

10 - 100(All conc.)

100 - 150
(2 conc.)

Call

-

+

+

+

+

Criteria

CFE

MT

Fulfilment of all assay acceptance criteria
Assessment#

CFE = Colony Forming Efficiency, MT = Morphological Transformation, NOEL = No Observed Effect Level, PE = Plating
Efficiency, na = not applicable.
#considered acceptable for overall study evaluation.
* for positive assessment, the concentrations that contributed to the statistically significant result, i.e. which were present in
the downturn-protected Williams contrast that resulted in the lowest p-value (negative binomial analysis), are indicated. The
assessment criteria are described in section 3.6.7.

6.4.3 Conclusion
Treatment with phenanthrene in the repeated tests performed by ECVAM and in all other studies
produced a positive response in morphologically transformed foci. As such, phenanthrene wasconsidered to be a positive transforming agent in the Balb/c 3T3 CTA.
Surprisingly, the reproducible positive call for phenanthrene is in contrast with previously publishedresults (Heidelberger et al., 1983). Although phenanthrene has been classified by IARC as in Group 3(not classifiable as to its carcinogenicity to humans), there is some evidence that phenanthrene ismutagenic in the Ames test and in human cells in culture when S9 fraction was prepared by Aroclor
1254 induction (Oesch et al., 1981, IARC, 1983) and that may produce papillomas when applied athigh doses to mouse skin (Scribner and Suss, 1978).

6.5 o-Toluidine HCl
6.5.1 Dose-range finding test
Figure 28 shows the results of the DRF tests with o-toluidine HCl.

63

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

120
110
100

Relative CFE [%]

90
80
70
60
50
40
30

ECVAM
Harlan CCR
HRI

20
10
0
1

10

100

1000

10000

o-Toluidine HCl [µg/ml]

Figure 28: Relative Colony Forming Efficiency (CFE) compared to vehicle control in alllaboratories testing coded o-toluidine HCl, for the Dose-Range Finding

6.5.2 Transformation assay
The following tables and figures show the MT and concurrent cytotoxicity test results upon treatment
with o-toluidine HCl at the test concentrations which were selected based on solubility and DRF tests.

6.5.2.1 ECVAM
o-Toluidine HCl was dissolved in DMSO. The concentrations were selected on the basis of the DRF
experiments and evaluated by ECVAM (Table 28). This chemical induced a statistically significant
positive response in morphological transformation.
Table 28: Transformation assay results from ECVAM, testing coded o-toluidine HCl
ECVAM
o-Toluidine HCl(µg/ml)Untreated control
VC (DMSO 0.5%)

20
100
200
500
800
1000
1200
1750

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
nd
87.9
nd
52.0
22.3
10.9
7.1
1.9
11.3

na
100
nd
111.1
nd
103.3
105.1
57.0
37.6
22.7
45.0

3
8
5
3
4
8
15
35
37
26
119

0.30
0.80
0.50
0.30
0.40
0.80
1.50
3.50
3.70
2.60
11.90

PC
VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable, nd = not determined.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

64Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.5.2.2 Harlan CCR
o-Toluidine HCl was dissolved in DMSO. The concentrations were selected on the basis of the DRF
experiments and evaluated by Harlan CCR (Table 29). This chemical induced a statistically significant
positive response in morphological transformation.
Table 29: Transformation assay results from Harlan CCR, testing coded o-toluidine HClHarlan CCR
o-Toluidine HCl(µg/ml)Untreated control
VC (DMSO 0.5%)

5
15
75
150
300
450
600
800
1000
1200

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
105.9
101.7
102.5
83.1
60.1
28.4
17.7
5.6
1.4
2.0
11.5

na
100
99.2
98.6
103.2
105.2
104.1
78.0
52.5
23.1
15.3
12.6
39.9

5
3
3
8
4
0
3
8
12
32
48
52
109

0.50
0.30
0.30
0.80
0.40
0
0.30
0.80
1.20
3.20
4.80
5.20
10.90

PC
VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

6.5.2.3 HRI
o-Toluidine HCl was initially dissolved in water (TA1) and according to the statistical analysis theassessment of o-toluidine was negative (Annex 13.5.2). The experiment was repeated using DMSO,
which was the solvent suggested by the VMT (TA2). The concentrations were selected on the basis of
the DRF experiments and evaluated by HRI (Table 30).
Daughter foci were observed in two dishes of the PC, one dish at 500 µg/ml, and one dish at 800µg/ml, but were not considered during scoring (see section 3.6.5.3 for analysis of the data).
Table 30: Transformation assay results from HRI (2nd experiment), testing coded o-toluidine
HCl
HRI
o-Toluidine HCl(µg/ml)TA2
Untreated control
VC (DMSO 0.5%)
63
125
250
500
630
800
1000
1200
PC

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
106.0
98.1
91.0
71.5
48.8
27.1
17.4
4.9
22.0

na
100
115.5
109.8
99.5
82.0
63.9
45.5
43.6
11.2
37.5

1
0
1
0
0
1
1
2
0
17
90

0.10
0
0.10
0
0
0.11
0.10
0.22
0
1.70
11.25

TA1 = Transformation Assay 1, VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency,
RCG = Relative Cell Growth, CV = Crystal Violet, na = not applicable.

65Balb/c 3T3 CTA PRP report v1 - 30 July 2010

According to the statistical analysis the assessment of o-toluidine HCl for the HRI laboratory was
negative. However, an increase in the number of transformed foci was observed at the highest dosetested (1200 µg/ml), suggesting that o-toluidine HCl could have a transforming potential. To verify
this suspicion the VMT requested the laboratory to retest the same chemical (TA3) focusing on moreclosely spaced concentrations (Table 31). A retest with o-toluidine confirmed this result, showing astatistically significant positive response in morphological transformation.
Table 31: Transformation assay results from HRI (3rd experiment), testing coded o-toluidine
HCl
HRI
o-Toluidine HCl(µg/ml)TA3
Untreated control
VC (DMSO 0.5%)
100
200
400
600
800
900
1000
1100
1200
PC

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
95.5
93.4
58.5
36.2
24.4
18.0
14.3
12.2
7.9
30.0

na
100
95.7
82.6
42.8
20.7
15.3
15.7
11.4
9.8
4.1
9.8

3
0
1
0
1
2
15
27
27
29
20
161

0.30
0
0.10
0
0.10
0.20
1.50
2.70
2.70
2.90
2.00
16.10

TA2 = Transformation Assay 2, VC = Vehicle Control, PC = Positive Control, CFE = Colony Forming Efficiency,
RCG = Relative Cell Growth, CV = Crystal Violet, na = not applicable.
Concentrations that contributed to the statistically significant result, i.e. which were present in the downturn-protected
Williams contrast that resulted in the lowest p-value for a positive response (negative binomial analysis), and related values
are highlighted in bold.

6.5.2.4 Concurrent cytotoxicity: Colony Forming Efficiency
Cytotoxicity of o-toluidine HCl was evaluated by CFE assessment in all laboratories (Figure 29).
120
110
100

Relative CFE [%]

90
80
70
60
50
40
ECVAM
Harlan CCR
HRI TA1
HRI TA2

30
20
10
0
1

10

100

1000

o-Toluidine HCl [µg/ml]

Figure 29: Relative Colony Forming Efficiency (CFE) compared to vehicle control in alllaboratories testing coded o-toluidine HCl. HRI repeated the experiment.

66

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

6.5.2.5 Concurrent cytotoxicity: Crystal Violet

Relative cell growth [%]Cytotoxicity of o-toluidine HCl was evaluated by the CV method in all laboratories (Figure 30).
Due to the quantitative variability of the responses in the above experiments, it was difficult to clearlycompare CFE with CV results.
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0

ECVAM
Harlan CCR
HRI TA1
HRI TA2

1

10

100

1000

o-Toluidine HCl [µg/ml]

Figure 30: Relative cell growth compared to vehicle control in all laboratories testing coded otoluidine HCl. HRI repeated the experiment.

6.5.2.6 Morphological transformation
MT results with o-toluidine HCl are shown in Figure 31. The results showed that o-toluidine induced
an increase in the number of transformed foci in a narrow concentration range in all laboratories.

Mean number of foci per dish

20
ECVAM
Harlan CCR
HRI TA1
HRI TA2

15

Negative

10

5

0
1

10

100

1000

o-Toluidine HCl [µg/ml]

Figure 31: Mean number of foci per dish for all laboratories testing coded o-toluidine HCl. HRI
repeated the experiment.
Results of the negative binomial statistical analysis are shown. For positive assessment, the concentrations that contributed tothe statistically significant result, i.e. which were present in the downturn-protected Williams contrast that resulted in thelowest p-value, are indicated by a horizontal line, next to the corresponding laboratory name in the legend. Negative = No
downturn-protected Williams contrast resulted in a statistically significant p-value in the corresponding laboratory. The
assessment criteria are described in section 3.6.7.

67

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

MT results for the different control treatments are summarised in Figure 32. The untreated, vehicle andpositive controls were reproducible across the laboratories.

Number of transformed colonies

18
16

ECVAM
Harlan CCR
HRI

14
12
10
8
6
4
2
0
untreated

vehiclepositive

Controls to o-toluidine HCl

Figure 32: Number of transformed colonies (mean + standard deviation) for untreated, vehicle
and positive controls in all laboratories testing coded o-toluidine HCl

6.5.2.7 Acceptance and assessment criteria
Harlan CCR and HRI experiments fulfilled all the assay acceptance criteria for o-toluidine HCl TA
results, while the ECVAM experiment did not fulfil the criterion for the number of foci in the VC(Table 32). The ECVAM experiment was still considered acceptable by the VMT for the overall
evaluation since a clear positive response was observed.

68Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Table 32: Acceptance criteria and assessment of o-toluidine HCl resultso-Toluidine HCl
Criteria
ECVAM

CFE

MT

HRI TA3

1 NOEL [µg/ml]

yes
(100)

yes
(75)

yes
(250)

yes
(200)

PE of untreated control

67.0

44.8

54.9

58.6

PE of vehicle control >30%

yes
(65.1)

yes(44.5)

yes(54.0)

yes(58.4)

Vehicle used

DMSO

DMSO

DMSO

DMSO

Number of foci of vehicle
control < 6

no
(8)

yes
(3)

yes
(0)

yes
(0)

Number of analysable
concentrations ≥6

yes
(8)

yes
(10)

yes
(8)

yes
(9)

NO#

YES

YES

YES

1000-1200(2 conc.)

600-1200(4 conc.)na

800-1200(5 conc.)

+

+

-

+

Fulfilment of all assay acceptance criteria
Assessment

Laboratory
Harlan CCR
HRI TA2

Concentrations in the contrast[µg/ml]*Call

CFE = Colony Forming Efficiency, MT = Morphological Transformation, NOEL = No Observed Effect Level, PE = Plating
Efficiency, na = not applicable.
#considered acceptable for overall study evaluation.
* for positive assessment, the concentrations that contributed to the statistically significant result, i.e. which were present in
the downturn-protected Williams contrast that resulted in the lowest p-value (negative binomial analysis), are indicated. The
assessment criteria are described in section 3.6.7.

6.5.3 Conclusion
Treatment with o-toluidine HCl in the repeated tests of HRI and in the other studies conducted by theother participating laboratories produced a positive response in morphologically transformed foci. As
such, o-toluidine HCl was considered to be a positive transforming agent in the Balb/c 3T3 CTA.
These results agree with published data (Matthews et al., 1993c).

6.6 Distributions of all experimental controls
The negative and vehicle controls gave in general levels of transformed foci within the acceptable
range expected for Balb/c 3T3 cells. The average number of foci was 0.3 ± 0.2 in the negative controls
and 0.4 ± 0.3 in the VCs. The average number of transformed foci per experiment was less than 1.0
per dish, except in one experiment where it was 1.2 ± 1.8. However, in four experiments the number oftransformed foci in the VC exceeded five, which was the acceptance criterion. A revision of thisacceptance criterion for future work is recommended.
The PC chemical 3-methylcholanthrene (at 4 µg/ml) always induced a positive response. The average
focus number was 14.7 ± 4.0. The average number of transformed foci in each experiment was always
higher than 10 per dish. At the beginning of this study no acceptance criteria for a PC were
established. Based on the data collected in this study, additional criteria for the acceptability of the PC
might be recommended for future application of the Balb/c 3T3 CTA.

69

Number of transformed foci type III per dishBalb/c 3T3 CTA PRP report v1 - 30 July 2010

50

40

30

20

10

0
Untreated control

Vehicle control

Positive control

Figure 33: Untreated, vehicle and positive controls for all experiments in all laboratories for celltransformation

6.7 Comparison of CFE and CV methods for cytotoxicity assessment
In this study, the appropriate dose-range for the TA was based on the data produced by the CFE,
which is the principal cytotoxicity method that has been used in published studies. In order toinvestigate whether the CV cytotoxicity method was also suitable to identify the appropriate doses for
the TA, the CV was run in parallel with the CFE during the TA and the IC50 obtained with bothmethods were compared. The CV method is less laborious than the CFE method and uses a moresimilar cell density (350 cells/cm2) to the TA (500 cells/cm2). The CFE test uses a much lower celldensity than the TA (approximately 10 cells/cm2) and it was thought this may influence the measure ofcytotoxicity.
When the results from the two methods were compared, a larger difference between the twocytotoxicity measures was observed for 3-methylcholanthrene and benzo(a)pyrene (Table 33).
Although the CFE test has a tendency to be more sensitive than the CV test (based on the IC50), thiswas not always the case.
Moreover, we compared the different cytotoxicity measures assessed with CFE and CV in the TAexperiments. This qualitative analysis suggests that if the doses would have been chosen based on the
CV method alone, the range of concentrations tested according to the protocol would have included
the concentrations that contributed to the positive results in the present study.
No clear conclusions on the difference between the two methods can be drawn from the data producedin this study and further studies in this area are needed to determine whether CV is an appropriate
alternative to CFE.

70

Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Table 33: Summary table of IC50 values measured by CFE and CV
CFE
IC50 (µg/ml)

CV
IC50 (µg/ml)

ECVAM

0.063

0.016

Harlan CCR

0.037

0.11

HRI

0.036

0.52

ECVAM

16

20

Harlan CCR

14

14

Chemical

3-Methylcholanthrene

2-Acetylaminofluorene

Laboratory

HRI
Benzo(a)pyrene

Anthracene

Phenanthreneo-Toluidine HCl

37

25

ECVAM

0.0066

0.047

Harlan CCR

0.015

0.060

HRI

0.014

0.019

ECVAM

na

na

Harlan CCR

na

na

HRI

na

na

ECVAM

31

33

ECVAM (TA2)34

45

Harlan CCR (TA1)

32

49

Harlan CCR (TA2)

39

42

HRI

44

63

ECVAM

506

1126

Harlan CCR

329

622

HRI (TA1)

624

785

HRI (TA2)

480

366

na = not applicable because compound was not toxic.

6.8 Conclusion of the Validation Management Team on betweenlaboratory reproducibility results
The between-laboratory reproducibility was shown to be satisfactory when the repeated experiments
were taken into consideration. Two of the experiments required repetition because a biological effectwas observed although the statistical analysis was negative.
Overall, according to the assessment criteria established for this study and considering the repeatedexperiments, all chemicals tested (except 2-acetylaminofluorene that produced an inconclusive resultin one laboratory and could not be repeated) as well as vehicle and positive controls gave reproducible
results in the three laboratories.

71Balb/c 3T3 CTA PRP report v1 - 30 July 2010

7 Summary of results
An optimised Balb/c 3T3 CTA protocol was produced and agreed upon by the different laboratories
during the initial phase of the study. In addition, training for the participating laboratories andavailability of a comprehensive photo catalogue proved to be useful in facilitating the proper conduct
and prevalidation of the CTA. These aids helped to ensure consistency in assessing foci morphology
and in scoring experimental results. Using the improved protocol and providing that repeated tests
were considered, test reproducibility was shown to be satisfactory within as well as between
laboratories. Table 34 summarises the results obtained with each chemical.
Table 34: Summary table of the reproducibility results. The assessment was made using thenegative binomial statistical approach, p < 0.01.

Chemical

Expected
result#Laboratory
Harlan CCR

ECVAM

HRI

TA1

TA2

TA1

TA2

TA1

TA2

$

+

+$

+

+$

+

3-Methylcholanthrene++

2-Acetylaminofluorene

+

+

i*

+

Benzo(a)pyrene

+

+

+

+

Anthracene

-

-

-

-

Phenanthrene

-

- **

o-Toluidine HCl

+

+

+

+
+

+

nc

+

nc

- **

TA3

+

#

Based on previous results from the litereature (see sections 2.4.1.1 and 2.4.1.2)
i = inconclusive call; + = positive call; - = negative call; nc = not considered.
$
Chemical tested non coded in module 2* Assessment was reported inconclusive since the downturn-protected Williams contrast with the lowest p-value showed a pvalue < 0.01 but included only one concentration (30 µg/ml). This study should have been repeated. See section 6.1.2.2 for
explanation.
** An increase in the number of foci was observed at the highest dose tested, but the result was considered negative according
to the assessment criteria.

7.1

3-Methylcholanthrene

The results of 3-methylcholanthrene, tested as a coded and a non-coded chemical were reproducible
within and between the laboratories. Moreover, the within- and between-laboratory reproducibility of
3-methylcholanthrene was demonstrated when this chemical was used as PC in the different
experiments.

7.2

2-Acetylaminofluorene

According to the assessment criteria established for this validation study, the results were reproduciblein two laboratories and an experiment was found inconclusive in one laboratory. 2Acetylaminofluorene was considered to be a positive transforming agent in the Balb/c 3T3 CTA,
which was the expected result.
Since the assessment criteria for a positive call required two concentrations to be in the downturnprotected Williams contrast with the lowest p-value, the resulting call for Harlan CCR was
inconclusive and should have been repeated using a narrower concentration range around the positive
concentration. However, Harlan CCR could not repeat the experiment due to time restrictions andother commitments.

72

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

7.3

Benzo(a)pyrene

The final results of benzo(a)pyrene were reproducible between the laboratories and benzo(a)pyrene
was shown to be a positive transforming agent in the Balb/c 3T3 CTA, which was the expected result.

7.4 Anthracene
The final results of anthracene were reproducible between the laboratories and anthracene was shown
to be negative in the Balb/c 3T3 CTA, which was the expected result.

7.5 Phenanthrene
The results of phenanthrene were reproducible in all laboratories when repeated tests were considered.
All laboratories showed a reproducible dose-dependent increase in the number of transformed foci andthe statistical analysis showed phenanthrene to be positive in all laboratories. Surprisingly, the positive
call for phenanthrene is in contrast with previously published results.
The IARC classification of phenanthrene is Group 3, not classifiable as to its carcinogenicity tohumans (IARC, 1983). In spite of this, there is some evidence that phenanthrene is mutagenic in theAmes test and in human cells in culture when S9 fraction was prepared by Aroclor 1254 induction(Oesch et al., 1981, IARC, 1983). It also induced sister chromatid exchange in Chinese hamster bonemarrow cells in vivo (IARC, 1983). Moreover, Scribner and Suss (1978) described that papillomas
were obtained when applying phenantrene at high doses to mouse skin. In the present study all threelaboratories produced positive calls at doses above 50 µg/ml (but still within acceptable cytotoxicity
ranges). The data reported in the literature resulted from using lower concentrations, which may
explain the CTA results reported herein.

7.6 o-Toluidine HCl
The experiments were reproducible in all laboratories and statistically significant MT was seen in alllaboratories above 800 µg/ml of o-toluidine HCl when the repeated tests were considered. It can beconcluded that, o-toluidine HCl was shown to be a positive transforming agent in the Balb/c 3T3 CTA,
which was the expected result.

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Discussion

In the present study some experiments did not fulfil all acceptance criteria, ideally triggering theirrepetition. However for reason of costs, resources and time needed for the conduct of the CTA, therepetition of experiments was kept at a minimum. Justifications for considering the experiments that
did not fulfil all acceptance criteria were given and were based on biological relevance, such as thepresence of a clear transforming dose-response.
For uniformity in testing, all the laboratories were requested by the VMT to dissolve their compounds
in DMSO. This recommendation was not initially adopted in all cases and in those situations thecompounds had been dissolved in water. In some of those cases, a different outcome was obtained. It
is possible that the bioavailability of compounds dissolved in DMSO may be increased, explaining thedifferent results obtained. Water causes a shift in cytotoxicity influencing the choice of the dose range
used in the CTA. This variance in outcome depending on the solvent used suggests that further careshould be taken that the same solvent is used when comparing data from different studies. It alsoemphasizes the fact that the choice of solvent is critical to the outcome of a given assay.
The Balb/c 3T3 prevalidation results clearly demonstrated that it is critical to test the correct dose
range in order to produce the accurate results. This is especially important for chemicals that show acytotoxic response in a very narrow concentration range.
The training of laboratory personnel is important to ensure consistency in assessing the morphology offoci both within a given laboratory and between laboratories. Moreover, the photo catalogue proved to
be an invaluable aid during the scoring. Overall, the results produced in this study demonstrated that if
the laboratories are well trained, the manual scoring of foci and the potential subjectivity in identifying
transformed foci are not problematic issues.
The CFE test has a tendency to be more sensitive than the CV test in some cases and a larger
difference between the two cytotoxicity measures (based on IC50 values) was observed for 3methylcholanthrene and benzo(a)pyrene. The results presented here and historical data from theliterature were based on the use of CFE for dose selection. It can be noted that in the present study theconcentrations that contributed to the statistically significant results for the positive chemicals would
also have been tested if the DRF had been based on the cytotoxicity values obtained with CV insteadof CFE. However, the limited dataset generated in the present study doesn't allow a conclusion to bemade on the equivalence or superiority of one or the other method and further studies are needed toaddress this important aspect.
The data produced during this study suggest that the acceptance criteria initially established were notalways appropriate for the conduct of this protocol. In some experiments the following criteria were
not fulfilled: the number of foci in the VC (exceeded five foci in four experiments), the use of a NOEL
concentration (one experiment), the PE (less than 30% in two experiments), the number of analysable
concentrations (one experiment). From the data collected, it is clear that under these experimental
conditions the acceptable number of foci in the VC needs to be increased, but there are insufficient
data to define what the limit should be. In most cases the effect of a slight increase in the Type III fociin the VC will be only marginal and does not affect the overall call when there is a large response witha test article. Moreover, it should be noted that in the case of a clear positive call with a statisticallysignificant dose-dependent increase of foci, experiments were not repeated in the present study. In
fact, seven experiments which did not fulfil one of the assay acceptance criteria each showed a clear
positive response and as such the chemicals were considered to be positive transforming agents.
Various statistical approaches had been used in the past to evaluate the Balb/c 3T3 CTA data, while
their appropriateness has never been assessed in detail. Consequently the VMT (at the meeting of May
2006) recommended that ECVAM organise an ad hoc expert group to evaluate the CTA data and thevarious related statistical methods previously employed and to come up with a recommendation on the74Balb/c 3T3 CTA PRP report v1 - 30 July 2010
most suitable statistical method to use for analysing data typically generated with this assay (seesection 3.6.5). The experts concluded that none of the statistical methods used in the past wereappropriate for this kind of analysis and proposed using an analysis based on the so-called Nishiyamatransformation and an approach based on the negative binomial distribution to evaluate the data
produced in this study. From a statistical point of view, the approach based on the negative binomial
distribution seemed more appropriate. However due to the limited amount of data produced in thisstudy it is premature to draw conclusions on whether the assessment criteria based on this approachprovide the properties required for the analysis of the CTA. Further use of this statistical approach willallow for the generation of more data with which to evaluate its relevance and applicability.
When a single concentration induced an increase in transformed foci but the statistical analysis
indicated the result was negative, repetition of the experiment with additional concentrations thatbracketed the concentration yielding the increase in foci resulted in a clear positive result. Thisoccurred in two experiments where an increase in transformed foci was observed at the highest
concentration only, although it was not significant as demonstrated by the statistical approach used. In
both cases the repetition of the experiments proved that the chemicals tested had indeed a transforming
effect. Based on this observation, it is recommended that when an increase in foci is observed at asingle concentration, a repeat study is conducted with more doses covering the concentration range
where the increase was observed.
As for any other statistical approach, the choice of the p-value defines the statistical significance of theresults. As the significance is directly linked to the interpretation (significant = positive; not significant= negative), the p-value impacts on the sensitivity and specificity of the method. Since it was the firsttime that the proposed statistical method was used for the given assay, the data generated were used tostart to empirically derive a suitable p-value. Based on the very limited set of chemicals from thisprevalidation study there are too few carcinogens and non-carcinogens to define an appropriate pvalue for an optimised balance between sensitivity and specificity. Therefore, it is recommended thatfurther formal evaluation of the performance of the new proposed statistical method, including an
optimisation of the p-value that should be used as a decision criterion, be performed.
At the beginning of this prevalidation study no acceptance criteria for a PC had not been established
due to the lack of historical data produced with the same optimised and agreed upon protocol used inthis study. Since the formal definition of an acceptable range of transformed foci for the PC is an
essential part of every assay, based on the data collected in this study, it is recommended that such acriterion for the PC be defined and incorporated into the protocol for future Balb/c 3T3 CTA studies.
During the conduct of the study some concerns were raised regarding the formation of “daughter” foci,which represent secondary foci developing from cells that break away from the “parent” focus.
Although this is a rare phenomenon, it was suggested that dishes exhibiting such secondary foci not becounted for the statistical analysis.

75Balb/c 3T3 CTA PRP report v1 - 30 July 2010

9 Overall conclusion by the Validation Management TeamThe aim of the study was to prevalidate the Balb/c 3T3 CTA, in a formal inter-laboratory study,
following the modular approach (Hartung et al., 2004) and concentrating on modules 1-4: test
definition, within-laboratory reproducibility, transferability and between-laboratory reproducibility.
Table 35 summarises the conclusion by the VMT on the assessment of the Balb/c 3T3 CTA.
Table 35: Conclusions of the Validation Management Team for the different modules
ModuleSummary & Conclusion- Clear definitions of the scientific basis- Description of the endpoint induced by genotoxic andnon genotoxic mechanisms

Module 1

Test Definition- Protocol available with improvement in the interpretationof data and clearer definition of a valid study (e.g. some
acceptance and assessment criteria have been refined)yes- A new statistical approach was applied but needs to befurther evaluated
The within-laboratory reproducibility was shown to besatisfactory in all laboratories for
Module 2

Within-laboratory
reproducibility- the vehicle control

yes- the positive control- the test chemical *- Test method is transferable between laboratories- Basic cell culture experience is needed

Module 3

Transferability- Training in the conduct and scoring of the assay isimportantyes- Photo catalogue produced as a useful aid for scoring
The between-laboratory reproducibility was shown to besatisfactory for:
Module 4- the vehicle control
Between-laboratory- the positive control
reproducibility- the test chemicals, when considering repeated tests **, #yes &Confirmation needed with further testing* One experiment did not fulfil one of the acceptance criteria but was considered valid by the VMT for the overall evaluation.
** Seven experiments did not fulfil one of the acceptance criteria but were considered valid by the VMT for the overall
evaluation since they all showed a statistically significant dose-response effect.
#
Between-laboratory reproducibility was initially not considered satisfactory, leading to the revision of the criteria for
repetition of a test to achieve reliable results. Two experiments were repeated because a biological effect was observed
although the statistical analysis was negative.
&
According to revised protocol.

On the basis of the outcome of this prevalidation study, an improved protocol, incorporating therecommendations made by the VMT has been developed. The recommended changes to the protocol
relate only to data interpretation (refinement of acceptance and assessment criteria). Moreexperimental data are required to allow for further refinement and evaluation of the statistical method

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Balb/c 3T3 CTA PRP report v1 - 30 July 2010
which may impact future study design (number of plates, concentrations tested, requirements for
repeat studies, etc).
If the repeated experiments and the modifications to the data interpretation of the improved protocol
are taken into consideration, including the importance of considering biological relevance, it can beconcluded that the assay is transferable between laboratories and, reproducible within and between
laboratories. Moreover, this study demonstrated that with the appropriate training and the use of thephoto catalogue, the scoring of foci was not problematic despite the concerns raised in the past. It is
recommended that this improved protocol be used in the future in order to confirm its utility.
Furthermore, although limited, these prevalidation data add to the fifth module i.e. predictive capacity,which has been addressed by the OECD DRP evaluation.

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10 Recommendations
Taking into account clarifications and modifications introduced into the protocol by the VMT and theparticipating laboratories, an improved protocol has been generated and it is recommended that thisprotocol be used in the future to produce new data in order to expand the body of evidence on assay
reproducibility, and to address remaining issues described below such as appropriate statisticalanalysis. Based upon the experience gained from this effort, points that should be taken into
consideration in the future conduct of the assay include the following:•

Check that the cell lineage and the culture conditions are suitable to be used in the CTA. Thisshould be done by establishing that the PC induces a clear and reproducible increase and the
VC exhibits a low and reproducible response.

•

Carefully assess the correct dose range to be used for chemicals that show a cytotoxic
response in a very narrow concentration range. In such cases, more closely spaced doses may
be required.

•

Since there is a certain degree of subjectivity associated with the identification of transformedfoci in the Balb/c 3T3 CTA and correct scoring is critical, training is necessary to ensure
scoring which is as consistent and objective as possible. The photo catalogue produced during
this study has proven to be an invaluable aid in establishing consistency in assessing
morphology of the foci and for scoring of the experiments performed to assess the betweenlaboratory reproducibility. It is therefore recommended that appropriate training and a photocatalogue for this protocol be made available to laboratories conducting the Balb/c 3T3 CTA.
It is intended that such a photo catalogue will be published by ECVAM in the near future forthat purpose.

•

An acceptable background of spontaneous foci in the VC could not be defined. Eachlaboratory needs to develop its own database and criteria for an acceptable number of foci in
VC, which should be as low as possible.

•

An acceptance criterion for the PC should be added to the protocol. For data that have similar
properties to those observed in our study, especially regarding the number of foci in VC andPC, the average number of Type III foci induced by the PC chemical should always exceed 10
per dish. However, the criterion should be adjusted in relation to the historical data of thelaboratory. Alternatively, either an approach based on the definition of an absolute number offoci above the VC or a statistical approach may be developed for the PC.

•

If in the case of a negative call, one or more acceptance criteria are not fulfilled, theexperiment should be repeated. This proviso should be added in the recommended protocol.

•

An experiment need not be repeated if a clear positive result is obtained even if one of theacceptance criteria is not fulfilled.

•

An experiment that does not produce a statistically significant positive response, but shows amarked increase in foci at a single dose needs to be repeated.

•

It is recommended that further formal evaluation of the performance of the new proposed
method, including an optimisation of the p-value that should be used as a decision criterion, be
performed.

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Y., Kajiwara Y., Kasahara Y., Kato Y., Kawabata M., Kitada E., Kaneko K, Kizaki Y., Kubo K.,
Miura D., Mashiko K., Mizuhashi F., Muramatsu D., Nakajima M., Nakamura T., Oishi H., Sasaki T.,
Shimada S., Takahashi C., Takeda Y., Wakuri S., Yajima N., Yajima S. and Yatsushiro T. Applicationof the improved BALB/c 3T3 cell transformation assay to the examination of the initiating andpromoting activities of chemicals: the second interlaboratory collaborative study by the non-genotoxic
carcinogen study group of Japan. Altern. Lab. Anim., 38 (2010) 11-27.
Urani C., Stefanini F.M., Bussinelli L., Melchioretto P. and Crosta G.F.. Image analysis and automaticclassification of transformed foci. J. Microsc., 234 (2009) 269-79.
Venables W.N. and Ripley B.D. Modern Applied Statistics with S. Fourth Edition. (Springer U.S.,
New York) (2002) p. 495.

83

Balb/c 3T3 CTA PRP report v1 - 30 July 201012 Contact details of people involved in the validation study
Validation Management Team:- Philippe Vanparys
ALTOXICON BVBA
Boskant 101
B2350 Vosselaar, Belgium
Tel +32.14.61.55.88
Fax +32.14.61.55.88
Mobile +32.479.22.91.66
philippe.vanparys@altoxicon.com

CHAIRMAN- Marilyn Aardema
Marilyn Aardema Consulting, LLC
5315 Oakbrook Dr.
Fairfield OH 45014, USA
Tel +1.513.253.2683
Fax +1.513.939.3269
mjaardema@me.com- Makoto Hayashi
Biosafety Research Center, Foods, Drugs and Pesticides
582-2, Shioshinden, Iwata,
Shizuoka 437-1213, JAPANTel +81.538.58.1593
Fax +81.538.58.1293
hayashi@anpyo.or.jp- Leonard M. Schechtman
Innovative Toxicology Consulting, LLC
9735 Via Verga St.
Lake Worth, FL 33467-6162, USA
Tel +1.561.304.4356
Fax +1.561.649.6663
Lschechtman@InnovativeToxicologyConsulting.com- Sebastian Hoffmannseh consulting + services
Gottfried-Hagen-Str. 60-62
51105 Köln, GermanyTel+49 (0)221.89060516
Mobile +49 (0)171.1976159
sebastian.hoffmann@seh-cs.com- Daniela Maurici (until January, 2007)
Unit of the Scientific Committee & Advisory Forum
European Food Safety Authority (EFSA)
Largo Palli 5/A,
I-43100 Parma, Italy
Tel +39.0521.036.435
Fax +39.0521.036.535
Daniela.MAURICI@efsa.europa.eu

84Balb/c 3T3 CTA PRP report v1 - 30 July 2010

- Thomas Hartung (until April, 2008)
Johns Hopkins University
Center for Alternatives to Animal Testing (CAAT)
615 N Wolfe Street, W7032
Baltimore, MD 21205, USA
Tel +1.410.614.4990
Fax +1.410.614.2871
thartung@jhsph.edu

ECVAM staff that are/were part of the VMT:
Raffaella Corvi, Sebastian Hoffmann, Daniela Maurici, Laura Gribaldo, Thomas Hartung.
ECVAM staff involved in the management and statistical analysis of the study:
B. Claire Thomas, Pascal Phrakonkham, Andre Kleensang.
Laboratories Involved:- Study Director Laboratory 1 (Scientific Lead Laboratory)
Enrico Sabbioni

Retired September 2007
- Study Directors Laboratory 2
Albrecht Poth & Susanne Bohnenberger
Harlan Cytotest Cell Research GmbHIn den Leppsteinswiesen 19
D-64380 Rossdorf , Germany
Tel +49.(0)6.154.807.266 (Poth) / 270 (Bohnenberger)
Fax +49.(0)6.154.83.399
APoth@harlan.com
SBohnenberger@harlan.com- Study Director Laboratory 3
Noriho Tanaka
Hatano Research Institute
Food and Drug Safety Center
729-5 Ochiai- Hadano
Kanagawa 257-8523, Japan
Tel and Fax +81.463.82.0773
tanaka.n@fdsc.or.jp85Balb/c 3T3 CTA PRP report v1 - 30 July 201013 Annexes
13.1 Chemicals selected for the prevalidation of Balb/c 3T3 CTA
Chemicals were selected based on the genotoxicity and carcinogenicity data compiled by the OECD
DRP31 (2007) and Kirkland et al. (2005). The in vitro genotoxicity, in vivo genotoxicity andcarcinogenicity characterisation of the selected compounds is reported in Table 36.
Table 36: Genotoxicity and carcinogenicity data on the chemicals selected for the Balb/c 3T3
CTA prevalidation study

Chemical

3-MethylcholanthreneCASnumber

Genotoxic profile
in vitro
Ames MLA MNT CA

56-49-5

+

+

+

+

2-Acetylaminofluorene 53-96-3

+

+

+

+

Benzo(a)pyrene

50-32-8

+

+

+

+

Anthracene120-12-7

+/-

+

nd

-

Phenanthrene

85-01-8

+/-

nd

nd

o-Toluidine HCl

636-21-5

+/-

+/-

+/-

Genotoxic
in vivoi

IARC
in vivoclass carcinogenicity
nd

+*

nd

+*

1

+

i

3

-

nd

i

3

-

+/-

+/-

2A

+

+
(gene mutation,
MN)+(gene mutation,
MN)* source: Gold and Zeiger (1997)+: positive; -: negative; +/-: diverging results inside a database; nd: not determined; i: inconclusive result;
MLA: Mouse Lymphoma Assay; MNT: Micronucleus Test; CA: Chromosome Aberration; MN: micronucleus.

86Balb/c 3T3 CTA PRP report v1 - 30 July 201013.2 Amended protocol for Phase 2
Pre-validation study on Balb/c 3T3 cell transformation assay
to detect carcinogenic potential of chemicals
Phase II
The following Standard Operating Procedures (SOPs) describe the protocols used for the
pre- validation of the Balb/c 3T3. SOPs are provided for dose-range findings (cytotoxocity)and for morphological transformation assay induced by chemicals in mouse fibroblast Balb/c
3T3 clone A31-1-1 cell line provided by Hatano Research Laboratory (Japan).
Materials and reagents used and their catalogue numbers are listed in Appendix 1.
This document amends the previous ones.
Following the recommendations of the Validation Management Team during the meeting of
the 10th-11th May 2006 in Ispra, minor modifications were made to the original protocols
before proceeding to phase 2.

Media, Reagents and Supplements(Appendix 1 contains all the information regarding the suppliers)
Cell line. Balb/c 3T3 clone A31-1-1 cells (1,2): Mouse fibroblasts, free from bacteria, fungi
and mycoplasma, distributed by HRI Cell Bank at the passage number 12.
Culture media: prepared as follows and stored at 4ºC for no longer than 1 month.
The following media, reagents and supplements will be prepared.
All the media and reagents will be labeled with the date of the preparation.
M10F: MEM (Minimum essential medium) + 10% FBS (Fetal Bovine Serum).
M10F is used for the routine passage, the cell storage and the early stage of transformation
experiments.
- MEM (500 ml): Minimum essential medium (GIBCO, CAT No.: 11095-080 (Japan),
31095-029 (Europe), liquid, with low glucose (1000 mg/l), L-glutamine (292 mg/l) and sodium
bicarbonate (2200 mg/l) is stored at 4ºC.
- FBS (55.5 ml): Fetal bovine serum (Moregate Lot N. 94300107), stored below -20ºC
upon arrival, is thawed and added to MEM to reach 10% final concentration of FBS.
- PS (5.5 ml): Penicillin (10000 units/ml) - Streptomycin (10 mg/ml) solution from GIBCO(CAT No.: 15140-122 (Japan and Europe) is stored at -20ºC until needed. 5.5 ml of thesolution are added to the culture medium to reach a final concentration of 1%.
DF2I2F: DMEM/F12 (Dulbecco's modified Eagle's medium/F12) + 2 µg/ml insulin + 2% FBS.
DF2I2F is used for the later stage of transformation experiments (3,4).
- DMEM/F12 (500 ml): Dulbecco's modified Eagle's medium/F12 (GIBCO, CAT No.:11330-032 (Japan), 31330-038 (Europe)), liquid, with high glucose (3151 mg/l), L-glutamine(365 mg/l) and sodium bicarbonate (1200 mg/l) is stored at 4ºC.
- FBS (10.2 ml): Fetal Bovine Serum (Moregate Lot N. 94300107), stored below -20ºC
upon arrival, is thawed and added to DMEM/F12 to reach a final concentration of 2%.
- PS (5.1 ml): Penicillin (10000 units/ml) - Streptomycin (10 mg/ml) solution from GIBCO(CAT No.: 15140-122) is stored at -20ºC until needed. 5.1 ml of the solution are added to themedium to reach a final concentration of 1%.
- 0.5 ml of insulin (2 mg/ml) is added to reach a final concentration of 2 µg/ml.

87Balb/c 3T3 CTA PRP report v1 - 30 July 2010

Serum: FBS is stored at -20ºC upon arrival. To prepare aliquots, 500 ml bottle is thawed at
4ºC in a refrigerator, and aliquots of 55.5 ml or 10.2 ml are prepared aseptically and stored at-20ºC. One aliquot of 55.5 ml is added to 500 ml of MEM bottle to reach a final concentrationof 10% FBS, and one aliquot of 10.2 ml is added to a 500 ml bottle of DMEM/F12 to reach a
final concentration of 2% FBS.
Insulin stock solution (2 mg/ml): Insulin (SIGMA I5500) is dissolved in 0.1N HCl to reachthe concentration of 2 mg/ml. The solution is filtered with 0.22 µm filter. Aliquots of 5 ml areprepared and stored at -20 ºC. Once thawed, the aliquot is used within one month.
0.5 ml of the solution is added to DMEM/F12 to a reach final concentration of 2 µg/ml.
Freezing solution (M10F containing 10% DMSO): Freezing solution is prepared freshly
every time needed by the addition of 10% DMSO to the M10F medium.
2% EDTA-2Na solution: 2 g of EDTA-2Na is dissolved in 100 ml of distilled water, and thesolution was sterilized by autoclaving (121ºC, 10 min). It is used under sterile conditions anddiscarded after 6 months from the day of the preparation.
Washing solution (PBS containing 0.02% EDTA-2Na): 5 ml of 2% EDTA-2Na solution
were added to 500 ml bottle of PBS (GIBCO, CAT NO.: 14190-144 (Japan and Europe). It is
used under sterile conditions, stored at room temperature and discarded after 6 months fromthe day of the preparation.
Fixing solutions: (stored at room temperature and discarded after 6 months from the day ofthe preparation)•
Pure methanol is used for cell fixation in the transformation assay and the Colony
Forming Efficiency (CFE) assessment.
•
10% formalin (3.7% formaldehyde): 100% formalin (37% formaldehyde solution) is
diluted with distilled or MilliQ water to make a concentration of 10%. This is used for fixationof cell growth assay.
Staining solutions: (stored at room temperature and discarded after 6 months from the dayof the preparation)•
0.04% Giemsa solution: One volume of Giemsa solution contained 0.4% Giemsa isdiluted with 9 volumes of distilled or MilliQ water. This is used for staining in thetransformation assay and the CFE assessment. (Note: Giemsa solution from Merck (Cat.
No.: 1.09204) is 0.4% Giemsa).
•
0.1% crystal violet (CV) solution: Crystal Violet is dissolved in MilliQ water to make a
1% solution. The 1% solution is diluted in distilled or MilliQ water to a final concentration of
0.1%. This is used for cell staining in the CV cell growth assay.
Extraction solution (for crystal violet): 50 volumes of ethanol and 1 volume of 1 mol/l HCl
are added to 49 volumes of distilled water. Extraction solution consists of 50% ethanol
containing 0.02 mol/l HCl (final concentration 0.01 mol/l HCl) and is used for extracting CVfrom stained cells.
Trypsin: Trypsin (GIBCO, Cat No. 15050-065) is stored at -20°C upon arrival. To prepare
aliquots, trypsin is thawed and aliquots of 10 ml are prepared aseptically and re-stored at 20°C. When needed, aliquots are thawed and used. After use, always in sterile conditions,trypsin can be stored at 4°C up to one month from the date of thawing. Special care should
be taken to avoid leaving trypsin at room temperature for a long time.

88Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Test article solution: Test chemicals are dissolved or suspended in an appropriate solvent
or vehicle and diluted with M10F complete medium. The solvent/vehicle should not interact
with test chemicals and affect survival and focus formation of the cells. Final concentration ofsolvent/vehicle in medium is < 10 vol% with distilled water or saline, and < 0.1 vol% withDMSO (maximum dose of 0.5 vol% is permissible when test chemicals do not dissolve).

Methods for cell culture
Cell freezing:
Preparation of MASTER STOCK and WORKING STOCK is performed the same way. The
cells should be stocked in liquid nitrogen at an early passage generation. One of the frozen
vial is thawed in warm water (approximately 37ºC). The thawed tube must be immediately
put in ice cold water. The thawed cells are suspended in 20 ml of M10F complete medium
and seeded onto 2 culture dishes and cultured in a humidified 5% CO2 incubator at 37ºC until
subconfluence (60 to 70% confluence). Then, the cells are dislodged, and plated in 16 petri
dishes (Ø90 mm) with 10 ml of fresh complete medium. After the cultures reach 70%
confluence, the cells are dislodged, and cell number is adjusted to 1 × 106 cells/ml with
M10F. 0.5 ml of cell suspension is transferred into freezing vials, then 0.5 ml of freezing
solution is added and the vials are frozen in a deep freezer at -70 to -80ºC for 1 day, andthen transferred into liquid nitrogen.
One working stock tube should be used for one transformation assay within 2 passages. For
the cell growth assay, cells can be used several times within 9 passages.
Note: When cells are seeded in the petri dish, cell suspension should be mixed in theappropriate amount of medium, and should not be distributed before adding medium into thedishes. This is to allow the cells to attach homogeneously to the petri dish.
Cell thawing and culturing: One vial of frozen cells is thawed in warm water (approximately
37ºC). The thawed tube must be immediately put in ice cold water. The thawed cells are
suspended in 20 ml M10F complete medium and seeded onto 2 culture dishes and cultured
in a humidified 5% CO2 incubator at 37ºC until subconfluence (70% confluence).
Cell passage and maintenance: When the cultures reach about 70% confluence, medium
is removed and petri dishes are washed with washing solution, then treated with 0.25%
trypsin solution until the cells dislodge from the culture surface. Trypsin is inactivated by theaddition of M10F complete medium. The number of cells in the cell suspension is counted.
The cells are adjusted to make a required cell density with M10F complete medium andseeded onto Ø90 mm dish(es). The cells are cultured in M10F complete medium in ahumidified 5% CO2 incubator at 37ºC. The cells should be harvested when they havereached 60 to 70% confluence.
Cell countOnly cells with whole cell membrane are counted using Bürker chamber and trypan blue
solution (two dilutions can be prepared depending on the cell density/concentration: 10 µl
cells + 90 µl trypan blue or 50 µl cells + 50 µl trypan blue).
Calculate the number of cells with the formula N = ( a / b) × 104 × DF
where “N” is the number of cells/ml; “a” is the number of cells counted in minimum 3
squares; “b” = number of squares considered (minimum 3); 104 is the conversion factor ofchamber volume and “DF” is the Dilution Factor (usually equal to 2 or 10).
Record the cell count and passage number in the ad hoc working sheet.

89Balb/c 3T3 CTA PRP report v1 - 30 July 2010

Experimental procedures
Cell growth assays for the determination of test concentrations
CV method
Day 0: Cells at 60 to 70% confluence are trypsinized and suspended in M10F complete
medium at 2 x 103 cells/ml. 0.5 ml of the cell suspension is transferred to each well of 24-wellplates (1 x 103 cells/well). At least 3 wells of 24-well plates are used for each concentration.
Three wells for medium alone (medium control), 3 wells for medium plus solvent controls(solvent control), and 3 wells for blank (medium without cells) must be included.
In order to obtain homogeneously growing cells in 24-well plates, it is necessary to keepplates without moving for 10 to 15 minutes after cell inoculation. Meanwhile, cells sediment to
the plate surface and don’t move thereafter under carrying the plates.
Day 1: Media containing various concentrations of test chemical are prepared, and used for
medium change. Cells are exposed to the treatment medium for 72 hours.
Day 4: Medium is replaced with fresh complete medium. Check cell growth under
microscope.
Day 7: Medium is removed. Cells are fixed with 10% formalin for 30 min, washed with water,
air dried, and then stained with 0.1% CV solution for 15 min. After rinsing with water, theplates are air dried.
The staining dye in each well is extracted with 0.5 ml/well of extraction solution for 10
minutes (under gentle shaking) and the optical density of each well is measured at 540-570
nm (depending on the photometer filters available). Growth rates relative to the control
culture are calculated from the absorbance with the formula:
Relative cell growth (% of control) = (Absorbance of treated well – Absorbance of medium
blank well) / (Absorbance of solvent control well – Absorbance of medium blank well) x 100

Colony Forming Efficiency (CFE) method
1) SEEDING
Cells at 60 to 70% confluence are trypsinized and resuspended in M10F complete medium(as already mentioned before). Cells are seeded at the density of 200 cells/dish in 4 ml of
M10F complete medium (60x15 mm petri dish, 4 dishes/dose).
Note: Cell suspension should be mixed in the appropriate amount of medium, and seeded
into petri dish where the medium has already been placed. This will allow the cells to attachhomogeneously to the petri dish.
2) TREATMENT
Day 0: 200 cells/dish (60x15 mm petri dish) are seeded, 4 dishes/dose
Day 1: Replace culture medium with 4 ml treatment medium (72 hours of exposure)Day 4: (72 hours after exposure): Remove treatment medium without washing with PBS andreplace with 4 ml of complete fresh medium
Day 7: Replace culture medium with fresh complete medium

90Balb/c 3T3 CTA PRP report v1 - 30 July 2010
Day 9: Remove medium and fix petri dishes with methanol (1 ml/ dish) for 3 minutes. Stain
petri dish with filtered Giemsa 0.04% solution for 20 minutes. Then remove the solution andair dry the dishes.
The colonies that have been grown are counted under stereomicroscope. Only colonies witha well defined centre and more than 50 cells/colony are scored (5)3) DATA COLLECTION
Relative CFE and plating efficiency (PE) are recorded.
Results are expressed as Relative CFE (%) mean ± SD.
Relative CFE (%) = total number of colonies formed in the treatment dishes x 100 / total
number of colonies formed in the control dishes
For PE (%) = number of colonies formed in the control x 100 / 200,
Where 200 is the total number of cells seeded in the CFE dishes.
Suggested templates to be used for data collection are presented in Appendix 2.

Transformation assay and concurrent cell growth assays (CV and
CFE)
For the second part of Phase 1, CV and CFE are performed in parallel to the morphologicaltransformation according to the above-mentioned protocols.
Day 0: Cells at 60 to 70% confluence are trypsinized and harvested in M10F complete
medium at 2 x 103 cells/ml. Ten ml (10 ml) of the cell suspension are plated into each Ø90
mm petri dish (2 x 104 cells/dish, 10 dishes/dose).
Note: Homogeneously growing cells can be obtained if dishes are gently moved back andforth and right and left a few times, and then placed in the incubator.
Day 1: Medium containing various concentrations of test chemical is prepared. 10 ml oftreatment medium is replaced in each dish. Cells are exposed to the treatment solution for 72
hours.
Day 4: Treatment medium of Ø90 mm dishes is replaced with 10 ml of fresh M10F complete
medium.
Day 5 through Day 24 or 25: Medium is changed with 10 ml of fresh DF2I2F complete
medium twice a week.
Day 31 or 32: One-week after the last medium change, cells are fixed with methanol for 10
min and stained with 10% Giemsa solution for 30 min. Then staining solution is removed andPetri dishes are air dried.
Transformed foci type III are colonies with the following characteristics: deep basophilicstaining, dense multilayered, cells randomly orientated at focus edge, cells spindle-shaped
different from the background monolayer cells, multi-layering (piling up) cells (5). Fociconsisting of more than 50 cells are scored. Only foci type III are considered tumourigenic
because it has been shown that they are the only ones inducing neoplastic transformation innude mice with a frequency of 85% (5, 8).
Number of foci in each dish is scored. Average number and standard deviation in each groupare calculated and recorded. (To be further clarified with the photo catalogue)
Transformation dishes must be stored and archived in each laboratory until the end of the
pre-validation, in case that a not clear result needs clarification.

91

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

2 x 104 cells/dish10 φ90 mm dishes/group

D1
D0 D4

D7

D14

D21

D28 D31 or 32: MEM + 10% FBS (M10F): Test chemical treatment:DMEM/F12 + 2 µg/mL insulin + 2% FBS (DF2I2F):Medium change
Fig. BALB/c 3T3 Cell Transformation Assay Protocols

General experimental design
Note: Following the suggestions of the Management Team after the teleconference of the
23rd February 2006, the concentrations for the morphological transformation and theconcurrent CV and CFE assays (MCA and Compound X) have been chosen:
0.01 µg/ml, 0.03 µg/ml, 0.1 µg/ml, 0.3 µg/ml,1 µg/ml, 3 µg/ml, 10 µg/ml
This has been applied only to the second part of Phase 1.
For Phase 2, the general experimental design is specified below.
Crystal Violet method
MCA has been chosen as a positive control.
Fixed dose of MCA to be tested for the transformation test is 4.0 µg/ml- 3 wells/dose. A
concurrent growth assessment must be done.
Test chemicals are dissolved or suspended in an appropriate solvent or vehicle anddiluted with the medium. The solvent/vehicle should not interact with test chemicals andaffect survival and focus formation of the cells. Final concentration of solvent/vehicle inmedium is < 10 vol% with distilled water or saline, and < 0.1vol% with DMSO (maximum
dose of 0.5% is permissible when test chemicals do not dissolve).
The highest concentration is 5 mg/ml or 10 mM, whichever is the lowest (6,7), or isdetermined based on historical studies.
Five or more concentration levels are set by an appropriate serial dilution factor such as
2 or square root 10.
Each transformation experiment (for each compound) in Phase I will be performed withuntreated and solvent controls.
It is desirable that cell growth curve covers the range between NOEL and >IC90.
Colony Forming Efficiency (CFE)MCA has been chosen as positive control.
- Fixed doses of MCA to be tested for the transformation test is 4.0 µg/ml - 4 dishes/dose.
A cell growth assessment must be done.
- The second compound will be coded and a dose-curve for cell growth assay will be
performed before transformation assay.
- Test chemicals are dissolved or suspended in an appropriate solvent or vehicle anddiluted with medium. The solvent/vehicle should not interact with test chemical and affectsurvival and focus formation of the cells. Final concentration of solvent/vehicle in medium is <10 vol% with distilled water or saline, < 0.1vol% with DMSO (maximum dose of 0.5% is92Balb/c 3T3 CTA PRP report v1 - 30 July 2010
permissible when test chemicals do not dissolve).
- Each transformation experiment (for each compound) in Phase II will be performed withuntreated and solvent controls.
- The doses that should be assessed for the selected compounds (ideally) are:
NOEL……………………………IC50 …………………………………………IC90
One dose in between

Two doses in between

Morphological transformation assay
MCA will be used as a positive control and tested at 4.0 µg/ml.
Test concentrations of the chemicals to be tested after a dose-curve for cell growth willbe (ideally) as follows;…………………………NOEL………………………………IC50 ……………………...……IC90

One dose-

Two doses in

Two doses inTen (Ø90 mm) Petri dishes (20000 cells/dish) are used for each concentration.

Acceptance Criteria
Concurrent cell growth assays
Homogeneous cell growth on culture wells should be observed.
At least one NOEL-concentration.
Cell growth curves cover the range between NOEL and IC90 (ideally six concentrations
between NOEL and IC90).
For CFE, minimum plating efficiency 30% in the negative control.
Transformation assay
The maximum number of Type III foci in the entire set of solvent control dishes should
not exceed 5.
If, while performing experiment, one entire set-concentration is lost, the experiment isstill OK. If 2 concentrations are lost, this might still be acceptable if the remaining
concentrations still cover the range of toxicity.
If one Petri-dish is lost (out of the 10 dishes/concentration) by accident or
contamination, the experiment is still acceptable.

93Balb/c 3T3 CTA PRP report v1 - 30 July 2010

References(1) T. Kakunaga, A quantitative system for assay of malignant transformation by chemical
carcinogens using a clone derived from BALB-3T3, Int. J. Cancer, 12 (1973) 463-473.
(2) T. Kakunaga, J.D. Crow, Cell variants showing differential susceptibility to ultraviolet lightinduced transformation, Science, 209 (1980) 505-507.
(3) T. Tsuchiya, M. Umeda, Improvement in the efficiency of the in vitro transformation assay
method using BALB/3T3 A31-1-1 cells, Carcinogenesis, 16 (1995) 1887-1894.
(4) T. Tsuchiya, M. Umeda, H. Nishiyama, I. Yoshimura, S. Ajimi, M. Asakura, H. Baba, Y.
Dewa, Y. Ebe, Y. Fushiwaki, S. Hamada, T. Hamamura, M. Hayashi, Y. Iwase, Y. Kajiwara,
Y. Kasahara, M. Kawabata, E. Kitada, K. Kudo, K. Mashiko, D. Miura, F. Mizuhashi, F.
Mizuno, M. Nakajima, Y. Nakamura, N. Nobe, T. Oishi, E. Ota, A. Sakai, M. Sato, S.
Shimada, T. Sugiyama, C. Takahashi, Y. Takeda, N. Tanaka, C. Toyoizumi, T. Tsutsui, S.
Wakuri, S. Yajima, N. Yajima, An interlaboratory validation study of the improved
transformation assay employing BALB/c 3T3 cell: Results of a collaborative study on the twostage cell transformation assay by the non-genotoxic carcinogen study group, ATRA, 27(1999) 685-702.
(5) IARC/NCI/EPA Working Group, Cellular and molecular mechanisms of celltransformation and standardization of transformation assays of established cell lines forprediction of carcinogenic chemicals: Overview and recommended protocols, Cancer Res.,45 (1985) 2395-2399.
(6) OECD Guidelines for the Testing of Chemicals. Test No. 473: In vitro Mammalian
Chromosome Aberration Test.
(7) OECD Guidelines for the Testing of Chemicals. Test No. 476: In vitro Mammalian Cell
Gene Mutation Test.
(8) U. Saffiotti, M. Bignami, F. Bertolero, E. Cortesi, C. Ficorella, M.E. Kaighn, Studies on
chemically induced neoplastic transformation and mutation in the BALB/3T3 Cl A31-1-1 cellline in relation to the quantitative evaluation of carcinogens, Toxicol. Pathol., 12 (1984) 383390.

94

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

Appendix 1

Culture reagents
Minimum Essential Medium
Fetal bovine serum Phase 1
Fetal Bovine serum Phase 2
Dulbecco’s modified Eagle's medium/F12
Penicillin (10000 U/ml) - Streptomycin (10mg/ml)
Insulin
Other reagents
Phosphate buffered saline
EDTA
TrypsinDMSO
Fixation and staining solutions
MethanolGiemsa solution *Giemsa solution (0.4%)
Formalin (37% formaldehyde)
Crystal violet
Ethanol
HCl
Culture vessels **60 x 15 mm dishes
100 x 20 mm dishes
24-well microplates

Supplier

Cat. No.

GIBCO

GIBCO

11095-080 (Japan)
31095-029 (Europe)
Lot No. 24300133
Lot No. 94300107
11330-032 (Japan)
31330-038 (Europe)
15140-122

Sigma

I5500

GIBCO
SIGMA
GIBCO
SIGMA

14190-144
E5134
15050-065
D8418

SIGMA
MERK
Sigma
SIGMA
SIGMA

15490-3
1.09204
GS 500
F8775
C3886

Moregate
Moregate
GIBCO

Falcon or
Costar can beusedFalcon or
Costar can beusedFalcon or
Costar can beused* Giemsa solution purchased from Merck is 0.4%** for phase 1 FALCON vessels were used95

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

Appendix 2
Colony forming efficiency with COMPOUND XX

COLONY FORMING EFFICIENCY (CFE)
Coded compound
Untreated control1
Solvent control

Conc.
(µg/mL)

1. dish

-

2

-

3

4.00

Positive control

Number of colonies
2. dish3. dish4. dishMean

SD

Relative PE Relative(%)
CFE (%)-

P

Precipitation occurred at the end of treatment
Complete culture medium
2
DMSO 0.5 % (v/v)
3
MCA
1

96

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

Crystal violet determination with COMPOUND XX

CRYSTAL VIOLET (CV)Coded compound
Untreated control
Solvent control

2

Positive control3

1

Conc.
(µg/mL)

Number of colonies
1. well-

2. well

3. well

4. well

Mean

SD

Relative
growth (%)
-

4.00

P

Precipitation occurred at the end of treatment
Complete culture medium
2
DMSO 0.5 % (v/v)
3
MCA
1

97

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

Morphological transformation with COMPOUND XX

Coded compound
Untreated control1
Solvent control

Conc.
(µg/mL)

1. dish2. dish3. dish4. dish5. dish6. dish7.dish8.dish9.dish

10. dish

Mean

SD

Sum

-

2

-

3

4.00

Positive control

Number of Type III fociP

Precipitation occurred at the end of treatment
Complete culture medium
2
DMSO 0.5 % (v/v)
3
MCA
1

98

Balb/c 3T3 CTA PRP report v1 - 30 July 201013.3 Example of statistical analysis of Balb/c 3T3 CTA results, using theGeneralised Linear Model approach with a negative binomial
distribution
Following the conclusions of the ad hoc expert group meeting held at ECVAM in April 2007
to discuss the most suitable statistical method to use for the Balb/c 3T3 CTA, a Generalised
Linear Model (GLM) with a negative binomial distribution and identity as link function was
designed.
That GLM is applied for the calculation of downturn-protected Williams contrast tests basedon unweighted combinations of concentrations and for the calculation of adjusted p-values
based on the joint normal distribution of the linear function.
This approach is publicly available and implemented into R (http://www.R-project.org). The
original R-codes were kindly provided by Pr. Hothorn (University of Hannover, Germany).
Reported below is one example of the analysis of morphological transformation results(number of foci per dish), using the statistical method developed for this validation study.

1/ Data File
Filename:

BALB TB Hatano 3nd_exp.txt

Original data
NC
0
0
SC
0
0
PC
17
20
c100 01c200 00c400 10c600 01c800 02c900 82c1000 65c1100 65c1200 2
0

0
0
13
0
0
0
0
1
1
1
2
1

0
0
18
0
0
0
0
0
3
3
6
2

0
0
18
0
0
0
0
1
3
1
4
3

0
0
11
0
0
0
0
6
3
3
2
3

0
0
15
0
0
0
0
1
2
2
2
2

0
0
18
0
0
0
0
3
2
3
0
3

0
0
14
0
0
0
0
1
2
1
1
2

0
0
17
0
0
0
1
0
1
2
1
2

Modified data (used for the prediction)
NC
0
0
0
0
0
SC
0
1
0
0
0
PC
17
20
13
18
18
c100 01000c200 00000c400 10000c600 01000c800 021
01c900 82133c1000 65131c1100 6
5
2
6
4

0
0
11
0
0
0
0
6
3
3
2

0
0
15
0
1
0
0
1
2
2
2

0
0
18
0
0
0
0
3
2
3
0

0
0
14
0
0
0
0
1
2
1
1

0
0
17
0
0
0
1
0
1
2
1

99

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

c1200 2

0

1

2

3

3

2

3

2

2

columnsrows

Æ dishes (ten in total)Æ NC negative control, SC solvent control, PC positive control
and test concentrations

RemarkBecause of mathematical reasons (at least one event per row is necessary Æ probability > 0),"zero" values for solvent control (dish 2) and concentration 200 µg/ml (dish 7) were changed
into one (negative control values were not modified although all "zero" since they are notused in the analysis)

100Balb/c 3T3 CTA PRP report v1 - 30 July 2010

2/ R-Code for the Generalised Linear Model with a negative binomial distribution, includingcomments################################################################ BALB Phase II#### Andre Kleensang## European Commission, JRC, IHCP, Systems Toxicology Unit#
# Code developed by Prof. Dr. Hothorn## University of Hannover## 07 November 2008#
# Filename: BALBanalysis TB Hatano 3nd.R## Remark: If applied to new data the code needs to be adapted ##based on the number of test concentrations and##their values as given in the comments!
################################################################# Libraries
library(multcomp)library(MASS)# Function for defining the contrasts for the downturn-protected Williams tests
protwill <- function(n){k <- length(n)CM <- c()if (!is.null(names(n))) varnames <- names(n) else varnames <- 1:length(n)for (j in 1:(k-1)) {for (i in 1:(k - j)) {helper <- c(-1, rep(0, k - i - j), n[(k - i + 1):k]/sum(n[(k - i + 1):k]), rep(0, j-1))
CM <- rbind(CM, helper)}}
rownames(CM) <- paste("C", 1:nrow(CM))
colnames(CM) <- varnames
CM}# Read file, second line needs to be adapted based on the number of test concentrations and their values

101Balb/c 3T3 CTA PRP report v1 - 30 July 2010

CTA1 <- read.table(file("BALB TB Hatano 3nd_exp.txt", encoding="latin1"))compound <- c("control","solvent","positive","100","200","400","600","800","900","1000","1100","1200")# Transforming the matrix into a flat-file format:
CTA1stk <- stack(CTA1[,2:ncol(CTA1)])
compound <- rep(compound, ncol(CTA1)-1)
CTA <- data.frame(foci = CTA1stk[,1], dish = CTA1stk[,2], compound)# Arranging factor levels# First line needs to be adapted based on the number of test concentrations and their values
lcmpd <- c("control","solvent","100","200","400","600","800","900","1000","1100","1200","positive")
CTA$compound <- factor(CTA$compound, levels=lcmpd)# Omitting P and C by sub-setting the dataset and redefinition of factor levels# First line needs to be adapted based on the number of test concentrations and their values
lcmpdsub <- c("solvent","100","200","400","600","800","900","1000","1100","1200")
CTAs <- subset(CTA, compound != "control" & compound != "positive")
CTAs$compound <- factor(CTAs$compound, levels=lcmpdsub)# Defining the contrasts for the downturn-protected Williams testsn <- tapply(CTAs$foci, CTAs$compound, length)
K <- protwill(n)K# Fitting GLM with negative binomial distribution and identity link function
nb.CTAs <- glm.nb(foci ~ compound, data=CTAs, link = identity)# Calculation of p-values for the Williams downturn-protected contrasts tests# adjusted for multiple testing based on the joint normal distribution of the linear function
MCP.CTAs <- glht(nb.CTAs, linfct = mcp(compound = K), alternative="greater")pval <- summary(MCP.CTAs)$test$pvalues
pval

102

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

3/ Results
pval (Variable with p-values)[1] 8.867174e-04 1.307175e-08 1.296629e-12 5.551115e-16 0.000000e+00[6] 1.110223e-16 1.110223e-15 1.443290e-14 1.533218e-13 3.663518e-05[11] 1.180222e-09 8.404388e-14 2.442491e-15 1.409983e-14 1.115774e-13[16] 1.249667e-12 1.994904e-11 6.550699e-05 1.781550e-09 1.818046e-11[21] 8.806822e-11 9.012645e-09 5.546341e-09 6.336666e-08 5.676274e-05[26] 2.016288e-07 2.119999e-06 1.537223e-05 3.443053e-05 1.465107e-04[31] 7.091067e-03 1.258352e-02 3.490272e-02 7.941265e-02 1.467802e-01[36] 8.926942e-01 9.403999e-01 9.575313e-01 9.656967e-01 9.858827e-01[41] 9.858841e-01 9.858818e-01 9.858830e-01 9.858835e-01 9.858843e-01
attr(,"error")[1] 0.0007979342
K (Variable with downturn-protected Williams contrasts)
C 1
C 2
C 3
C 4
C 5
C 6
C 7
C 8
C 9
C 10
C 11
C 12
C 13
C 14
C 15
C 16

solvent
100
200
400
600
800
900
1000
1100
1200
-1 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 1.0000000-1 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.5000000 0.5000000-1 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.3333333 0.3333333 0.3333333-1 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.2500000 0.2500000 0.2500000 0.2500000-1 0.0000000 0.0000000 0.0000000 0.0000000 0.2000000 0.2000000 0.2000000 0.2000000 0.2000000-1 0.0000000 0.0000000 0.0000000 0.1666667 0.1666667 0.1666667 0.1666667 0.1666667 0.1666667-1 0.0000000 0.0000000 0.1428571 0.1428571 0.1428571 0.1428571 0.1428571 0.1428571 0.1428571-1 0.0000000 0.1250000 0.1250000 0.1250000 0.1250000 0.1250000 0.1250000 0.1250000 0.1250000-1 0.1111111 0.1111111 0.1111111 0.1111111 0.1111111 0.1111111 0.1111111 0.1111111 0.1111111-1 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 1.0000000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.5000000 0.5000000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.3333333 0.3333333 0.3333333 0.0000000-1 0.0000000 0.0000000 0.0000000 0.0000000 0.2500000 0.2500000 0.2500000 0.2500000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.2000000 0.2000000 0.2000000 0.2000000 0.2000000 0.0000000-1 0.0000000 0.0000000 0.1666667 0.1666667 0.1666667 0.1666667 0.1666667 0.1666667 0.0000000-1 0.0000000 0.1428571 0.1428571 0.1428571 0.1428571 0.1428571 0.1428571 0.1428571 0.0000000

103

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

C 17
C 18
C 19
C 20
C 21
C 22
C 23
C 24
C 25
C 26
C 27
C 28
C 29
C 30
C 31
C 32
C 33
C 34
C 35
C 36
C 37
C 38
C 39
C 40
C 41
C 42
C 43
C 44
C 45

-1 0.1250000 0.1250000 0.1250000 0.1250000 0.1250000 0.1250000 0.1250000 0.1250000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 1.0000000 0.0000000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.5000000 0.5000000 0.0000000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.0000000 0.3333333 0.3333333 0.3333333 0.0000000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.2500000 0.2500000 0.2500000 0.2500000 0.0000000 0.0000000-1 0.0000000 0.0000000 0.2000000 0.2000000 0.2000000 0.2000000 0.2000000 0.0000000 0.0000000-1 0.0000000 0.1666667 0.1666667 0.1666667 0.1666667 0.1666667 0.1666667 0.0000000 0.0000000-1 0.1428571 0.1428571 0.1428571 0.1428571 0.1428571 0.1428571 0.1428571 0.0000000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 1.0000000 0.0000000 0.0000000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.0000000 0.5000000 0.5000000 0.0000000 0.0000000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.3333333 0.3333333 0.3333333 0.0000000 0.0000000 0.0000000-1 0.0000000 0.0000000 0.2500000 0.2500000 0.2500000 0.2500000 0.0000000 0.0000000 0.0000000-1 0.0000000 0.2000000 0.2000000 0.2000000 0.2000000 0.2000000 0.0000000 0.0000000 0.0000000-1 0.1666667 0.1666667 0.1666667 0.1666667 0.1666667 0.1666667 0.0000000 0.0000000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.0000000 1.0000000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.0000000 0.0000000 0.0000000 0.5000000 0.5000000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.0000000 0.0000000 0.3333333 0.3333333 0.3333333 0.0000000 0.0000000 0.0000000 0.0000000-1 0.0000000 0.2500000 0.2500000 0.2500000 0.2500000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.2000000 0.2000000 0.2000000 0.2000000 0.2000000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.0000000 0.0000000 0.0000000 1.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.0000000 0.0000000 0.5000000 0.5000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.0000000 0.3333333 0.3333333 0.3333333 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.2500000 0.2500000 0.2500000 0.2500000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.0000000 0.0000000 1.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.0000000 0.5000000 0.5000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.3333333 0.3333333 0.3333333 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.0000000 1.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000-1 0.5000000 0.5000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000-1 1.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000 0.0000000

104

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

4/ Prediction

The downturn-protected Williams contrast with the lowest p-value (the 5th contrast) (I)includes at least two consecutive concentrations (concentrations 800 – 1200; in total 5), and(II) showed a p-value of < 0.01 (p-value <1e-16) Æ positive prediction105Balb/c 3T3 CTA PRP report v1 - 30 July 201013.4 Nishiyama transformation analysis
The ad hoc expert group discussed several statistical evaluation methods for the analysis of the Balb/c
3T3 CTA results. The Nishiyama transformation (Nishiyama et al., 2003) and an approach based on
the negative binomial distribution combined with William’s-type (Bretz et al., 2003) protected tests
were recommended for use in this validation study since they both match the particular statisticalproperties of the Balb/c 3T3 CTA test. The latter method was suggested to be more suited for the dataat hand as it better reflected the dose-concentration curve (Manuscript in preparation, minutes of themeeting available at ECVAM).
Nevertheless, for completeness, the results obtained when applying the Nishiyama transformation andthe corresponding R-code are reported below.

13.4.1 Summary of results
The predictions based on Nishiyama transformation and ordinary least squares linear model in
combination with downturn-protected Williams contrasts are presented for each chemical in Table 37.
The results were evaluated with the following criteria:• A test chemical was considered "negative” (non-transforming) if no downturn-protected
Williams contrast showed a p-value < 0.01.
• A test chemical was considered "positive" (transforming) if the downturn-protected Williams
contrast with the lowest p-value:- (I)included at least two consecutive concentrations, and- (II)
showed a p-value < 0.01.
• A test chemical was considered "inconclusive" if the downturn-protected Williams contrast
with the lowest p-value:- (I)included only one concentration or several non consecutive concentrations, and- (II)
showed a p-value < 0.01.
Table 37: Summary table of the predictions based on Nishiyama transformation and ordinary
least squares linear model in combination with downturn-protected Williams contrasts (p <0.01).

Chemical

Expected
result#Laboratory
Harlan CCR

ECVAM

HRI

TA1

TA2

TA1

TA2

TA1

TA2

i

+$

+

+$

+

3-Methylcholanthrene++$

2-Acetylaminofluorene

+

+

i

i

Benzo(a)pyrene

+

+

+

i

Anthracene

-

-

-

-

Phenanthrene-io-Toluidine HCl

+

+

i

i
+

+

nc

i

nc

i

TA3

+

#

Based on previous results from the litereature (see sections 2.4.1.1 and 2.4.1.2)
i = inconclusive call; + = positive call; - = negative call; nc = not considered.
$
Chemical tested non coded in module 2

All inconclusive assessments reported were as such because the downturn-protected Williams contrast
with the lowest p-value showed a p-value < 0.01 but included only one concentration.

106

Balb/c 3T3 CTA PRP report v1 - 30 July 2010

13.4.2 R-Code for the Nishiyama transformation, including comments# Libraries
library(multcomp)library(MASS)# Function for defining the contrasts for the downturn-protected Williams tests
protwill <- function(n){k <- length(n)CM <- c()if (!is.null(names(n))) varnames <- names(n) else varnames <- 1:length(n)for (j in 1:(k-1)) {for (i in 1:(k - j)) {helper <- c(-1, rep(0, k - i - j), n[(k - i + 1):k]/sum(n[(k - i + 1):k]), rep(0, j-1))
CM <- rbind(CM, helper)}}
rownames(CM) <- paste("C", 1:nrow(CM))
colnames(CM) <- varnames
CM}# Read file, second line needs to be adapted based on the number of test concentrations and their values
CTA1 <- read.table(file("BALB TB Hatano 3nd_exp.txt", encoding="latin1"))compound <- c("control","solvent","positive","100","200","400","600","800","900","1000","1100","1200")# Transforming the matrix into a flat-file format:
CTA1stk <- stack(CTA1[,2:ncol(CTA1)])
compound <- rep(compound, ncol(CTA1)-1)
CTA <- data.frame(foci = CTA1stk[,1], dish = CTA1stk[,2], compound)# Arranging factor levels# First line needs to be adapted based on the number of test concentrations and their values
lcmpd <- c("control","solvent","100","200","400","600","800","900","1000","1100","1200","positive")
CTA$compound <- factor(CTA$compound, levels=lcmpd)# Omitting P and C by sub-setting the dataset and redefinition of factor levels# First line needs to be adapted based on the number of test concentrations and their values

107Balb/c 3T3 CTA PRP report v1 - 30 July 2010

lcmpdsub <- c("solvent","100","200","400","600","800","900","1000","1100","1200")
CTAs <- subset(CTA, compound != "control" & compound != "positive")
CTAs$compound <- factor(CTAs$compound, levels=lcmpdsub)# Defining the contrasts for the downturn-protected Williams testsn <- tapply(CTAs$foci, CTAs$compound, length)
K <- protwill(n)K# Square root transformation of Nishiyama et al. 2003
CTAt <- CTAs
CTAt$foci <- sqrt(CTAt$foci) + sqrt(CTAt$foci + 1)
# Fitting ordinary least squares linear model
fit.CTAt <- lm(foci ~ compound, data=CTAt)# Calculation of p-values for the Williams downturn-protected contrasts tests# adjusted for multiple testing based on the joint normal distribution of the linear functionn <- tapply(CTAt$foci, CTAt$compound, length)
K <- protwill(n)K
MCP.CTAt <- glht(fit.CTAt, linfct = mcp(compound = K), alternative="greater")pval <- summary(MCP.CTAt)$test$pvalues
pval

108

Balb/c 3T3 CTA PRP report v1 - 30 July 201013.5 Repeated experiments
13.5.1 HRI – Phenanthrene
In the first experiment by HRI (TA1), phenanthrene was dissolved in water. Table 38 shows the test
concentrations which were evaluated. The VMT asked the laboratory to retest using DMSO which was
the solvent originally requested (see section 6.4.2.3).
Table 38: Transformation assay results (1st experiment) from HRI, testing coded phenanthrene
HRI
Phenanthrene(µg/ml)TA1
Untreated control
VC (water)

250
500
750
1000
1500
2000

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
76.1
78.0
23.7
9.3
14.4
5.1
12.4

na
100
10.7
9.0
9.9
6.5
5.8
5.3
8.0

5
3
7
6
15
12
8
8
139

0.50
0.30
0.70
0.60
1.50
1.20
0.80
0.80
13.9

PC
VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency, RCG = Relative Cell Growth, CV= Crystal Violet, na = not applicable.

13.5.2 HRI – o-toluidine HCl
In the first experiment by HRI (TA1), o-Toluidine HCl was dissolved in water (TA1). Table 39 shows
the test concentrations which were evaluated. The VMT asked the laboratory to retest using DMSO
which was the solvent originally requested (see section 6.5.2.3).
Table 39: Transformation assay results (1st experiment) from HRI, testing coded o-toluidine HCl
HRI
o-Toluidine HCl(µg/ml)TA1
Untreated control
VC (water)
50
100
250
500
750
1000
PC

rCFE (%)

RCG (%)
CV

Total number offociMean number offoci per dish

na
100
96.3
99.0
87.4
76.6
59.5
28.3
11.8

na
100
62.1
62.0
64.0
60.9
43.8
44.7
8.6

3
7
4
0
2
3
2
9
98

0.30
0.70
0.40
0
0.20
0.30
0.20
0.90
9.80

TA1 = Transformation Assay 1, VC = Vehicle Control, PC = Positive Control, rCFE = relative Colony Forming Efficiency,
RCG = Relative Cell Growth, CV = Crystal Violet, na = not applicable.

109