SHE pH 7.0 CTA PRP report v1 - 30 July 2010

30 July 2010

Syrian Hamster Embryonic (SHE) cell
pH 7.0 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)

SHE pH 7.0 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 .................................................. 12
RELEVANT MEETINGS ................................................................................................................ 12
PATENTS .................................................................................................................................. 13

ORGANISATION OF THE STUDY ................................................................................ 14
2.1
VALIDATION MANAGEMENT TEAM ............................................................................................... 14
2.2
LABORATORIES INVOLVED .......................................................................................................... 15
2.3
QUALITY SYSTEMS OF THE PARTICIPATING LABORATORIES ........................................................... 15
2.4
CHEMICALS TESTED IN THE SHE CTA PREVALIDATION STUDY ..................................................... 16
2.4.1
Chemical selection ............................................................................................................ 16
2.4.1.1
2.4.1.2

Selection criteria ........................................................................................................................... 16
Chemicals selected ...................................................................................................................... 16

2.4.2
Modules 2 and 3: Within-laboratory reproducibility and transferability (coded and noncoded chemicals)............................................................................................................................. 17
2.4.3
Module 4: Between-laboratory reproducibility (coded chemicals) .................................... 17
2.4.4
Coding/decoding ............................................................................................................... 18
2.5
ASSESSMENT OF WITHIN- AND BETWEEN-LABORATORY REPRODUCIBILITY..................................... 18
2.6
STUDY TIMELINE ....................................................................................................................... 18

3

MODULE 1: TEST DEFINITION .................................................................................... 21
3.1
SCIENTIFIC BASIS FOR THE PROPOSED TEST METHOD .................................................................. 21
3.2
DESCRIPTION OF THE ENDPOINT PREDICTED AND THE MECHANISTIC BASIS OF THE TEST ................ 21
3.3
BIOLOGICAL TEST SYSTEM: SHE CELLS...................................................................................... 21
3.4
CELL TRANSFORMATION ASSAY VARIANT: SHE PH 7.0 CTA....................................................... 22
3.5
PROTOCOL ............................................................................................................................... 22
3.5.1
SHE cells........................................................................................................................... 22
3.5.2
Medium.............................................................................................................................. 23
3.5.3
Serum................................................................................................................................ 23
3.5.4
Controls ............................................................................................................................. 23
3.5.5
Test procedure .................................................................................................................. 23
3.5.6
Statistical analysis of raw data.......................................................................................... 24
3.5.7
Assay acceptance criteria ................................................................................................. 24
3.5.8
Assay assessment criteria ................................................................................................ 25

4

MODULE 2: WITHIN-LABORATORY REPRODUCIBILITY .......................................... 26
4.1
TRANSFORMATION ASSAY - CODED BENZO(A)PYRENE ................................................................. 26
4.1.1
University of Metz.............................................................................................................. 26
4.1.2
BASF ................................................................................................................................. 28
4.1.3
Harlan CCR....................................................................................................................... 28
4.1.4
BioReliance ....................................................................................................................... 28
4.1.5
Concurrent cytotoxicity (Relative Plating Efficiency)......................................................... 292

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
4.1.6
Morphological transformation frequency........................................................................... 30
4.1.7
Acceptance criteria and assessment ................................................................................ 31
4.2
CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 2 ......................................... 32

5

MODULE 3: TRANSFERABILITY.................................................................................. 33
5.1
5.2
5.3

6

GENERAL ASPECTS ................................................................................................................... 33
TRAINING .................................................................................................................................. 33
CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 3 ......................................... 34

MODULE 4: BETWEEN-LABORATORY REPRODUCIBILITY ..................................... 35
6.1
ANTHRACENE ............................................................................................................................ 35
6.1.1
Dose-range finding test ..................................................................................................... 35
6.1.2
Transformation Assay ....................................................................................................... 36
6.1.2.1
6.1.2.2
6.1.2.3
6.1.2.4
6.1.2.5
6.1.2.6
6.1.2.7

University of Metz ......................................................................................................................... 36
BASF ............................................................................................................................................ 36
Harlan CCR .................................................................................................................................. 37
BioReliance .................................................................................................................................. 37
Concurrent cytotoxicity (Relative Plating Efficiency)..................................................................... 38
Morphological transformation frequency....................................................................................... 38
Acceptance criteria and assessment ............................................................................................ 39

6.1.3
Conclusion ........................................................................................................................ 39
6.2
2,4-DIAMINOTOLUENE ............................................................................................................... 39
6.2.1
Dose-range finding test ..................................................................................................... 39
6.2.2
Transformation assay........................................................................................................ 40
6.2.2.1
6.2.2.2
6.2.2.3
6.2.2.4
6.2.2.5
6.2.2.6
6.2.2.7

University of Metz ......................................................................................................................... 40
BASF ............................................................................................................................................ 41
Harlan CCR .................................................................................................................................. 41
BioReliance .................................................................................................................................. 42
Concurrent cytotoxicity (Relative Plating Efficiency)..................................................................... 42
Morphological transformation frequency....................................................................................... 43
Acceptance criteria and assessment ............................................................................................ 44

6.2.3
Conclusion ........................................................................................................................ 44
6.3
3-METHYLCHOLANTHRENE ......................................................................................................... 44
6.3.1
Dose-range finding test ..................................................................................................... 44
6.3.2
Transformation assay........................................................................................................ 45
6.3.2.1
6.3.2.2
6.3.2.3
6.3.2.4
6.3.2.5
6.3.2.6
6.3.2.7

University of Metz ......................................................................................................................... 45
BASF ............................................................................................................................................ 46
Harlan CCR .................................................................................................................................. 46
BioReliance .................................................................................................................................. 47
Concurrent cytotoxicity (Relative Plating Efficiency)..................................................................... 47
Morphological transformation frequency....................................................................................... 48
Acceptance criteria and assessment ............................................................................................ 49

6.3.3
Conclusion ........................................................................................................................ 49
6.4
O-TOLUIDINE HCL ..................................................................................................................... 49
6.4.1
Dose-Range finding test.................................................................................................... 49
6.4.2
Transformation assay........................................................................................................ 50
6.4.2.1
6.4.2.2
6.4.2.3
6.4.2.4
6.4.2.5
6.4.2.6
6.4.2.7

University of Metz ......................................................................................................................... 50
BASF ............................................................................................................................................ 51
Harlan CCR .................................................................................................................................. 51
BioReliance .................................................................................................................................. 52
Concurrent cytotoxicity (Relative Plating Efficiency)..................................................................... 52
Morphological transformation frequency....................................................................................... 53
Acceptance criteria and assessment ............................................................................................ 53

6.4.3
Conclusion ........................................................................................................................ 54
6.5
PHTHALIC ANHYDRIDE ............................................................................................................... 54
6.5.1
Dose-range finding test ..................................................................................................... 54
6.5.2
Transformation assay........................................................................................................ 55
6.5.2.1
6.5.2.2
6.5.2.3
6.5.2.4
6.5.2.5
6.5.2.6

University of Metz ......................................................................................................................... 55
BASF ............................................................................................................................................ 56
Harlan CCR .................................................................................................................................. 56
BioReliance .................................................................................................................................. 57
Concurrent cytotoxicity (Relative Plating Efficiency)..................................................................... 57
Morphological transformation frequency....................................................................................... 58

3

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
6.5.2.7

Acceptance criteria and assessment ............................................................................................ 59

6.5.3
Conclusion ........................................................................................................................ 59
6.6
OVERVIEW ON VEHICLE AND POSITIVE CONTROLS ........................................................................ 60
6.7
CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 4 ......................................... 60

7

SUMMARY OF RESULTS ............................................................................................. 61
7.1
7.2
7.3
7.4
7.5
7.6

BENZO(A)PYRENE ..................................................................................................................... 61
ANTHRACENE ............................................................................................................................ 61
2,4-DIAMINOTOLUENE ............................................................................................................... 61
3-METHYLCHOLANTHRENE ......................................................................................................... 61
O-TOLUIDINE HCL ..................................................................................................................... 62
PHTHALIC ANHYDRIDE ............................................................................................................... 62

8

OVERALL CONCLUSION BY THE VALIDATION MANAGEMENT TEAM ................... 63

9

RECOMMENDATIONS.................................................................................................. 64

10
CURRENT CONTACT DETAILS OF THE PEOPLE INVOLVED IN THE
PREVALIDATION STUDY..................................................................................................... 65
11

REFERENCES............................................................................................................ 68

12

ANNEXES ................................................................................................................... 72

12.1
12.2
12.3

CHEMICALS SELECTED FOR THE PREVALIDATION OF SHE PH 7.0 CTA......................................... 72
SHE PH 7.0 CELL TRANSFORMATION ASSAY PROTOCOL ............................................................. 73
REPEATED EXPERIMENT: BASF 2,4-DIAMINOTOLUENE ................................................................ 99

4

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

List of Abbreviations
ATR-FTIRCRO
CTA
DMEM-L
DMSO
DRF
DRP
ECM
ECVAM
ESAC
FBS
FDA
GLP
MTF
OECD
PBS
PC
PE
REACH
RPE
SCCP
SHE
SIDS
SOP
TA
VC
VMT

Attenuated Total Reflection Fourier-Transform Infrared
Contract Research Organisation
Cell Transformation Assay
Dulbecco's Modified Eagle Medium with LeBoeuf's modification
Dimethyl Sulfoxide
Dose-Range Finding (test)
Detailed Review Paper
Expert Consultation Meeting
European Centre for the Validation of Alternative Methods
ECVAM's Scientific Advisory Committee
Foetal Bovine Serum
Food and Drug Administration
Good Laboratory Practice
Morphological Transformation Frequency
Organisation for Economic Co-operation and Development
Phosphate Buffered Saline
Positive Control
Plating Efficiency
Registration, Evaluation, Authorisation and Restriction of CHemicals
Relative Plating Efficiency
Scientific Committee on Consumer Products
Syrian Hamster Embryonic cells
Screening Information Data Set
Standard Operating Procedure
Transformation Assay
Vehicle Control
Validation Management Team

5

SHE pH 7.0 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 Restriction ofChemicals), 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/eliminate the use of 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 ofgenetic damage. In contrast, in vitro cell transformation assays (CTAs) have been shown to involve a
multistage process that closely models some stages of in vivo carcinogenesis and have the potential todetect both genotoxic and non-genotoxic carcinogens. As such, these tests are currently being used byacademia, the chemical, agro-chemical, cosmetic and pharmaceutical industries, and are conducted inhouse as well as at CROs to screen for potential carcinogenicity as well as investigate mechanisms ofcarcinogenicity. CTAs are not used routinely for regulatory testing but they re often used for internalsafety assessment of chemicals, drugs, etc. and are considered worthwile for providing additional
useful information to the prevailing tests that are used for assessing carcinogenic potential.
A recent Detailed Review Paper (DRP) of the OECD (Organisation for Economic Co-operation andDevelopment) on CTAs for the detection of chemical carcinogens (OECD, 2007) concluded that theperformance of the 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 SHE cells at pH 7.0 protocol was conducted following
validation modules 1 to 4 of the ECVAM validation procedure (Hartung et al., 2004) in order toevaluate 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 were assessed: the CTA using SHE
cells at pH 6.7 and the CTA based on the Balb/c 3T3 A31 cell line.
In keeping with the objectives of this ECVAM's effort, the Validation Management Team (VMT)
concluded that the SHE pH 7.0 CTA had been prevalidated in accordance with validation modules 1-4(Hartung et al., 2004). It has been demonstrated that the assay is reproducible within- and betweenlaboratories, that it is transferable, and that a standardised protocol is available.

6SHE pH 7.0 CTA PRP report v1 - 30 July 2010

Preface
The study presented in this report complements recent OECD activities related to the celltransformation assays (CTAs). The study has been supervised 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 theresults generated in the prevalidation study and the conclusions and recommendations by the VMT.
The conclusions are mainly based on the data generated in this study, but they also take into accountthe information and experience on the CTA publically available to date. It is the intention of this reportto provide data and protocols that further support the consideration of the CTA for use as an alternative
method which could contribute to the assessment of the carcinogenic potential of chemicals.

SHE pH 7.0 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.
Among the various in vitro alternatives for carcinogenicity prediction developed, the CTAs have beenshown 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 established and promising in vitro assaythat 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 means for initial screening ofchemicals 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, pharmaceuticalindustries, and are conducted in-house as well as at 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 for assessing 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 widely used for internal risk assessment of various
chemicals, there is a need within the scientific community for standardisation of these test methods andtechnical guidance on their conduct and use.
This need was already addressed in 1998 by a workshop organised by the ECVAM on CTAs aspredictors of human carcinogenicity (Combes et al., 1999). The workshop concluded that the tests
indeed are promising but require further development, standardisation and verification. In 2007, theOrganisation for Economical Development and Co-operation (OECD) published a detailed review
paper (DRP) on the CTAs (OECD, 2007) concluding that the performances of the Syrian hamster
embryo (SHE) and Balb/c 3T3 CTAs were sufficiently adequate and these CTA should be developed
into formal test guidelines. However, considering the amount of available data reported in theliterature, study results have sometimes been generated using different test method protocols. In orderto provide a basis for the development of CTA OECD test guidelines, it therefore became important toharmonise and standardise those protocols. Furthermore, as with some other assays with a long historyof use, CTAs have not undergone formal validation in accordance with current standards (OECD GD
34, 2005). The previous ECVAM workshop and the recent OECD DRP concluded that a formal
validation of the assays, in particular focusing on the use of 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 formal prevalidation study of
select CTAs. It was determined that the SHE and Balb/c 3T3 CTAs would undergo a prevalidation

8

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
assessment which would address issues of standardisation of protocols, within-laboratory
reproducibility, test method transferability, and between-laboratory reproducibility. The results of thatstudy should add to the existing large database of chemicals evaluated over the history of use of theseassays (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 the 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 of compounds
was evaluated as it was not the intention of this study to comprehensively assess the predictive
capacity of the CTAs. That would require an exhaustive evaluation of numerous chemicals andchemical classes employing the respective standardised multi-laboratory prevalidated protocols, aneffort 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, contract researchorganisations (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 of the SHE CTA performed with the pH 7.0 protocol, in four
independent laboratories.

1.2 Intended use
The possible use of the SHE CTA is mentioned in various recent testing strategies including theScientific Committee on Consumer Products (SCCP)'s notes of guidance for testing oxidative hair dyes
(SCCP, 2006), as supplemental data for pharmaceuticals (Jacobson-Kram and Jacobs, 2005) and theguidance on information requirements and chemical safety assessment for REACH (ECHA, 2008) andguidance for testing cosmetics (Pfuhler et al., 2010). For chemicals produced above 1000 tonnes/year,
it is stated that all relevant data from all toxicity studies should be assessed to see whether asufficiently 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, andthe use of (quantitative) structure-activity relationships. On some occasions, it may be proposed tosupplement these predictive approaches with short-term tests such as the in vitro CTA, cellproliferation assays or medium-term tests like genetically engineered (transgenic) or neonatal models
in order to circumvent the need for a chronic carcinogenicity study. This would usually be in thecontext of adding information to the weight of evidence that a chemical may be carcinogenic.
Based on the performance of the SHE 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 SHE CTA should be developed into an official OECD Test
Guideline. Although there was insufficient information on mechanism of action and usage specific forpharmaceuticals, experts at the Washington ECM were of the opinion that the SHE assay was one

9SHE pH 7.0 CTA PRP report v1 - 30 July 2010
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 SHE 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
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 SHE CTA is currently being used by academia, chemical, agro-chemical, cosmetic,
pharmaceutical and tobacco industries, and CROs to screen chemicals for their potential
carcinogenicity. Some current uses of the SHE CTA are: (a) to provide useful ancillary information
when the biological significance of the bioassay result is uncertain (e.g. in pharmaceutical industry),(b) to clarify in vitro genotoxic positive results by weight of evidence (e.g. in chemical and cosmetic
industries), (c) to evaluate certain classes of chemicals that have a low predictive capacity in the
traditional in vitro genotoxicity tests (e.g. in chemical and cosmetic industries), (d) to screen for nongenotoxic carcinogens (e.g. in agro-chemical industry), (e) to demonstrate differences and similarities
across a chemical class (e.g. in chemical companies within REACH), (f) to screen for efficacy ofchemopreventive agents (in pharmaceutical industry), and (g) to investigate tumour promotion activity(e.g. in agro-chemical and chemical industries), and (h) for mechanistic studies of carcinogenicity(e.g. in academia and industry).
As part of its safety assessment process, submitters have furnished to the FDA (Food and Drug
Administration) results from SHE CTA testing as part of the data submission package. Such results are
considered as supplemental information in its overall product evaluation (Jacobson-Kram and Jacobs,
2005). However, regulatory agencies in general have been reluctant to unconditionally adopt suchassays in their routine safety testing schemes, especially as a full replacement for in vivo
carcinogenicity testing, due, for the most 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 the lack of objective criteria to identify transformed colonies/fociand which could affect the reliability of the test.

1.4 Recent researchThis section summarises some of the ongoing and recent research activities related to the SHE CTA(pH 6.7 and pH 7.0).
It is recognised that the visual scoring of the colonies, which is still done manually under the
microscope, is one of the greatest weaknesses of this assay. Current developments of the assay include
automation of the scoring in order to speed it up and make it more objective and hence more
reproducible. Emery et al. are currently developing an automated scoring system for SHE pH 6.7 CTA
in collaboration with IMSTAR (a high technology company) through a stepwise approach. This
includes recognition and capture of the colonies, automated scoring, and recognition of transformed
phenotype (presented at the SOT, 2010). In another study, Walsh et al. employed attenuated totalreflection Fourier-transform infrared (ATR-FTIR) spectroscopy to interrogate pH 6.7 SHE colonies, ascomplex biomolecules absorb in the mid-infrared giving vibrational spectra associated with structureand function (Walsh et al., 2009). Further studies are ongoing in this field. Moreover, it has to be
demonstrated whether these approaches are also applicable for the SHE pH 7.0 CTA since colonies
display some differences in morphology.
Pant et al. have demonstrated the feasibility of conducting the SHE pH 6.7 CTA in cells without using
an X-ray irradiated feeder layer, thereby simplifying the test procedure and assisting the scoring ofmorphological transformed colonies. This eliminates the need for an X-ray machine thereby making

10SHE pH 7.0 CTA PRP report v1 - 30 July 2010
the assay more accessible to laboratories, which is an important consideration if for the assay to be
used broadly (Pant et al., 2008; Pant et al., 2010).
The SHE CTA has been used to study the co-effect of different substances applied simultaneously on
cell transformation (Hirose et al., 2007). A variant of the SHE CTA using a two-stage protocol has also
been applied to cigarette smoke particulates which were found to act both at the initiation andpromotion stage of cell transformation (Breheny et al., 2005).
Earlier research suggested that SHE cell transformation involves a block in the in vitro differentiationof a progenitor cell population (partially differentiated stem cell) present in the SHE cell isolate basedon analysis of cellular phenotype, differentiation marker analysis and growth characteristics (Zhang etal., 2004; Nakano et al., 1981; Isfort et al., 1994; Isfort et al., 1996a-b; Kerckaert et al., 1996)..
Studies have been conducted to determine the mechanism of induction of SHE cell transformation by
specific chemicals. Similar to defining the mechanism of a specific rodent or human carcinogen, caseby-case detailed studies are required..For example, diethanolamine-induced morphologicaltransformation in SHE CTA was shown to be due to a non-genotoxic mechanism involving choline
deficiency, consistent with the mechanism of diethanolamine hepatocarcinogenicity in mice (LehmanMcKeeman et al., 2000; Lehman-McKeeman et al., 2002). Acrylonitrile-induced SHE celltransformation appears to be due to oxidative stress and resulting oxidative damage, and is amechanism proposed for acrylonitrile-induced carcinogenicity in rats (Zhang et al., 2000).
More recent work to define the mechanisms involved in the transformation of the SHE cells have been
published and suggest chemical-specific modes of action. Bose et al. (2005) indicated that exposure ofSHE cells to Malachite green led to elevated phosphorylation of ERK1 and JNK1 and an increase in
G2/M phase and apoptotic cells. Maire et al. (2005a-b) reported that changes in bcl-2 and bax
expression and subsequent dysregulation of apoptosis could contribute to the carcinogenic potential ofchemicals such as di(2-ethylhexyl)phthalate and Zinc in SHE cells. Besides, DNA damage andoverexpression of the proto-oncogene c-myc, but without any change in apoptosis, were shown in the
2,4-dichlorophenoxyacetic acid-induced SHE cell transformation (Maire et al. 2007).

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 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) the SHE primary cell strain, (b) the Balb/c 3T3 mouse fibroblast cell line and,(c) the C3H/10T½ mouse fibroblast cell line (OECD, 2007). The performance of the SHE pH 7.0 (orhigher pH) CTA for the prediction of rodent carcinogenicity was reported for 204 chemicals as
follows: concordance 85%, sensitivity 92%, specificity 66%, positive predictivity 88%, negative
predictivity 75%, false positive 34%, false negative 8% (the proportion of carcinogens [prevalence]was 74%). Based on the available data the DRP concluded that the performances of the SHE andBalb/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 thatthose guidelines were, in fact, developed based upon validated test methods (OECD, 2005), it became
apparent that further important information addressing transferability and within- and betweenlaboratory reproducibility was necessary. Moreover, since in some cases the data evaluated in the DRP
had been produced with protocols that had some differences, a goal was to develop standardised andreliable protocols from which the OECD test guidelines would be generated.

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010
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 SHE CTA at pH 7.0 performed under the same conditions as thoseevaluated in the present study. However, one review from Isfort et al. (1996c) is worth mentioning,which reports a 82% (92/112 chemical or physical agents) inter-laboratory concordance between
results obtained using the CTA at pH 7.1-7.3.
The data collected in the OECD DRP for the assessment of the performance of the CTAs enabled anassessment of some measure of reproducibility (OECD, 2007). Excluding chemicals with only one
reference, consistency between laboratories for the SHE assay was 87.7% (57/65 chemicals). It should
be noted that these results were produced using different variants of the SHE protocol.

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.
• 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

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010
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 (Hartunget al., 2004).

1.8 Patents
The test method has not been patented.

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SHE pH 7.0 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 SHE CTA using a pH 7.0
standardised protocol. In order to evaluate whether the tests 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.
Each in vitro test was conducted according to the same agreed-upon protocol in four different
laboratories.
The study was entirely coordinated and sponsored by ECVAM.
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 detailed review paper, 2) the high costs and time to perform assays, 3) the limited funding
and resources which could be made available by ECVAM and 4) the study design followed for the
conduct of the SHE pH 6.7 and Balb/c 3T3 studies. This allowed the evaluation of the CTA using theSHE pH 7.0 protocol in four 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 and, in this respect, ECVAM focused on the development of a protocol that
could serve as a basis for an OECD test guideline.

2.1 Validation Management Team
Following the principles for test method validation (OECD 34, 2004) an independent VMT was
established by ECVAM. Its role was to design the study, to guide and facilitate the prevalidation
process, to evaluate the results, and to render subsequent decisions during the progress of the study,
and to analyse 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)
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 statistical analysis of the in vitro data was the responsibility of independent biostatisticians(Sebastian Hoffmann and Andre Kleensang - ECVAM).

14SHE pH 7.0 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 four laboratories from Europe and the USA. The participating laboratories arelisted below. Due to its extensive expertise in the assay under validation, Laboratory 1 acted as
scientific lead laboratory, while Laboratory 2 had a role of administrative coordinator.
Laboratory 1 (Study Director Paule Vasseur)(Scientific lead laboratory)
University of Metz
Laboratoire Interactions Ecotoxicité Biodiversité Ecosystèmes (LIEBE) - CNRS UMR 7146
Campus Bridoux, Rue Général Delestraint
Metz 57070, France
Tel +33.(0)3.873.785.01
Fax +33.(0)3.873.785.12
vasseur@univ-metz.fr
Laboratory 2 (Study Directors: Albrecht Poth and Susanne Bohnenberger)(Administrative coordinator)
Harlan Cytotest Cell Research GmbHIn den Leppsteinswiesen 19
D-64380 Rossdorf,
GermanyTel +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: Kamala Pant)
BioReliance Corporation
14920 Broschart Road
Rockville, MD 20850
USA
Tel +1.301.610.2192
Fax +1.301.738.2362
kamala.pant@bioreliance.com
Laboratory 4 (Study Directors: Markus Schulz and Karl-Rainer Schwind)
BASF Aktiengesellschaft
GV/TB
67056 Ludwigshafen,
GermanyTel +49.(0)6.216.056.108
Fax +49.(0)6.216.051.734
markus.schulz@basf.com
karl-rainer.schwind@basf.com

2.3 Quality systems of the participating laboratories
The present study was conducted under Good Laboratory Practice (GLP)-like conditions by alllaboratories according to good scientific practice and good cell culture practice (OECD, 2004).
Laboratories 2-4 routinely work under GLP certification and were subjected to regular GLP

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010
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 SHE CTA 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 Balb/c 3T3 CTA, the chemical
selection took into account existing results in both systems as described below. The same chemicals
were used for the prevalidation studies of SHE pH 6.7 CTA and SHE pH 7.0 CTA. Where possible thesame chemicals were selected for the evaluation of the SHE and Balb/c 3T3 CTAs.

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 some of
the assays: anthracene (only one reference for SHE pH 6.7 and Balb/c 3T3 CTAs), 2,4-diaminotoluene(only one reference for the Balb/c 3T3 CTA), phthalic anhydride (only one reference for both Balb/c
3T3 and SHE pH 6.7 CTAs) and o-toluidine HCl (only one reference for Balb/c 3T3 CTA and noreference for SHE pH 6.7 CTA).
Four of the chemicals selected were in common with those evaluated in the Balb/c 3T3 CTA study,
while 2,4-diaminotoluene and phthalic anhydride were used in the SHE studies only, instead ofphenanthrene and 2-acetylaminofluorene for which a limited amount of data were available for the
SHE CTAs at pH 6.7 and 7.0.
The in vitro genotoxicity, in vivo genotoxicity and carcinogenicity characterisation of the selectedchemicals is reported in Annex 12.1.

2.4.1.2 Chemicals selected
Chemicals selected for the prevalidation study are listed in Table 1.

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Table 1: List of chemicals used in the prevalidation study
Chemical

CAS no.

In vivo carcinogenicity(References)

Benzo(a)pyrene

50-32-8+(IARC, 2009)

1 or 5 µg/ml
when used as positive control

Benzo(a)pyrene

50-32-8+(IARC, 2009)na
when used as coded chemical

Anthracene120-12-7-(IARC, 2009)na

2,4-Diaminotoluene

95-80-7+(IARC, 2009)na

3Methylcholanthrene

56-49-5+(Gold and Zeiger,
1997)

0.01-10 µg/ml

o-Toluidine HCl

636-21-5+(NTP)

20 µg/ml - 1.2 mg/ml

Phthalic anhydride

85-44-9

-

(NTP)

na

Suggested dose range

na = 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 high chemical
cost or lack of cytotoxicity) for timely completion of these studies. For the other chemicals thelaboratories were asked to select the dose ranges on their own in order to check their ability to identify
the critical doses for the transformation assay.
Benzo(a)pyrene was chosen as positive control (PC) because it has been generally reported to induce astrong positive CTA response, in addition to the fact that historical data on this chemical used as PC
were available at the lead laboratory.
All chemicals were purchased from Sigma-Aldrich.

2.4.2 Modules 2 and 3: Within-laboratory reproducibility and transferability(coded and non-coded chemicals)Benzo(a)pyrene was chosen as a coded chemical in this study phase. Benzo(a)pyrene was later used as
the PC, in the subsequent phases of the study. Dose ranges for benzo(a)pyrene were suggested by the
VMT based on data from the literature.

2.4.3 Module 4: Between-laboratory reproducibility (coded chemicals)
Three chemicals classified as in vivo carcinogens (2,4-diaminotoluene, 3-methylcholanthrene, otoluidine HCl) and two chemicals classified as non carcinogens (anthracene, phthalic anhydride) wereselected. Dose ranges for benzo(a)pyrene, 3-methylcholanthrene and o-toluidine HCl, were suggestedby the VMT based on data from the literature. For the other two chemicals, the laboratories had tochoose the dose range themselves based on dose-range finding (DRF) tests. The VMT suggested thatDMSO (dimethyl sulfoxide) should be used as the vehicle for all chemicals. As the results became
available, they were sent to the statistician.

17SHE pH 7.0 CTA PRP report v1 - 30 July 2010

2.4.4 Coding/decoding
All chemicals were coded before sending them to the laboratories. The coding and shipment ofchemicals were performed by J&JPRD and ECVAM. The coded chemicals were sent to the laboratory
Safety Officers together with the corresponding 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 theend of the prevalidation study, except for BioReliance that returned opened envelopes for two
chemicals. The safety officer of BioReliance officially certified that the envelopes had been opened bytheir administration and had not been shared with the laboratories conducting the study. Since thechemicals were coded, the laboratories did not know their identity and therefore all chemicals weretreated as potential carcinogens.
The statistical analysis of the data was conducted before the decoding. The chemicals were decoded
during the VMT and study directors meeting of January 2009.

2.5 Assessment of within- and between-laboratory reproducibility
Both within- and between-laboratory reproducibility and predictive capacity were evaluated based onconcordance of the dichotomous results (negative or positive) as defined by the assessment criteria
listed in section 3.5.8.
Regarding the within-laboratory reproducibility, the concordance of results per laboratory wasdescribed.
Between-laboratory reproducibility was evaluated by comparing results of the four laboratories.
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
The CTA validation studies in SHE cells at pH 6.7 and in Balb/c 3T3 cell line were conducted in the
period between 2005 and 2007. In addition, a single laboratory (University of Metz) was selected toevaluate the SHE pH 7.0 protocol in parallel to the other studies.
The results of the above studies were presented and discussed during the VMT and Study Directors
meeting and the VMT ECVAM meetings that were held in May 2007 and September 2007,
respectively. The results of the CTAs in SHE cells at pH 6.7 and in Balb/c 3T3 cell line SHE cells at
pH 6.7 and in Balb/c 3T3 cell line are presented in the accompanying reports. Given the good data
produced with the SHE pH 7.0 protocol the VMT recommended a complete assessment of this variantof the test.
A contract for the validation of the SHE pH 7.0 assay between ECVAM and Harlan CCR was signed
in December 2007. This study involved the same laboratories that participated to the SHE pH 6.7
study.
As for the SHE pH 6.7 study, an important aspect of the initial phases of the study was the training of
the laboratory personnel, including the harmonisation of scoring and of procedures amonglaboratories. The Standard Operating Procedure (SOP) followed in all laboratories was the one
initially used by the Lead Laboratory (University of Metz).
Following the preliminary phase of optimisation of the protocols, the transferability and the withinlaboratory reproducibility were assessed by evaluating results obtained for one non-coded test18

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
chemical and a coded one. These two chemicals were the same (benzo(a)pyrene), allowing an analysisof the within-laboratory reproducibility as well as the transferability of the assay. Benzo(a)pyrene wasthen used as PC in the following phases of the study. After the evaluation of these initial results by thebiostatistician and the conclusion by the VMT that the transfer of the tests to the participating
laboratories and the within-laboratory reproducibility analysis were successful, the laboratories
proceeded to the experimental phase on the between-laboratory reproducibility. The betweenlaboratory reproducibility was evaluated using five coded chemicals. The chemicals were the same as
those used in the SHE pH 6.7 CTA prevalidation study, however this was not known to theparticipating laboratories and the coding was different.
The data submission template in Excel was developed for each test, in a collaborative effort between
the laboratories, ECVAM and the statistician. The spreadsheets containing the test data had to be
returned to the statistician 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 coordinator laboratory produced asummary 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 right data. The laboratories also quality
checked the data presented in this report.
Table 2 summarises the timeline of the study.

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Table 2: Timeline of the study
Date

Location

09/200505/2007

University of Metz,
FranceUniversity of Metz assessed the pH 7.0 protocol inparallel to the full prevalidation study carried out on SHE
pH 6.7 CTA (for detailed timelines see report on SHE pH
6.7 CTA prevalidation).

09/2007

VMT meeting

Agreement to initiate a complete assessment of SHE pH
7.0 protocol, based on good results produced by
University of Metz.

ECVAM, Ispra, Italy
18/12/2007-

Contract signature.

01-03/2008

BioReliance, USA

Isolation of cells.

01-06/2008

All laboratories

Protocol optimisation
experiments.

07/2008

Training

Training on the conduct of the test method and on colony
scoring.

University of Metz,
France

and

start

of

preliminary

09-12/2008

All laboratories

Performance of main experiments with coded chemicals.

28-29/01/2009

VMT and study
Directors meeting

Evaluation and discussion of results and decoding ofchemicals. One additional test requested by the VMT.

ECVAM, Ispra, Italy
02-04/2009

Laboratories

Repetition of the test at BASF, finalisation of laboratory
reports.

03/2010

VMT meeting

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

ECVAM, Ispra, Italy

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SHE pH 7.0 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
into 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 SHE CTA is based on the conversion of normal to neoplastic-like colonies of cells having
oncogenic properties and provides a system to detect genotoxic as well as non-genotoxic carcinogens(Berwald and Sachs, 1963; Berwald and Sachs, 1965; DiPaolo et al., 1971).
Transformation in SHE cells is a process which has shown multistage transformation from a normal
cell to a fully malignant cell. A minimum of four phenotypic stages appears to be involved in celltransformation, which include (a) a block in cellular differentiation visualised as morphologicaltransformation in the SHE CTA, (b) the acquisition of immortality expressed by unlimited lifespan, ananeuploid karyotype and genetic instability, (c) the acquisition of tumourigenicity closely associatedwith the in vitro phenotypes of foci formation, anchorage independent growth in semi solid agar andautocrine factor production, and (d) malignant growth when cells are injected into a suitable host
(LeBoeuf et al., 1999). Such effects are caused by changes in the expression of oncogenes and/or
tumour suppressor genes (Isfort and LeBoeuf, 1995), however, the complete mechanisms underliying
these events are not yet fully understood either in CTAs or human/rodent carcinogenesis. The earliest
observation of morphological transformation typically measured in the assay is characterized bychanges in the cellular behaviour and cell growth involving alterations in cellular morphology anddisorganised patterns of cell growth. Considerable effort has been invested over time in further
characterising and describing the use of the SHE CTAs as reviewed in the OECD DRP (2007).

3.3 Biological Test system: SHE cells
SHE cells derived from embryos of Syrian golden hamsters (13-13.5 day gestation) are diploid andgenetically stable cells. The cell population comprises a complex mixture of multiple cell types andcells at various stages in the differentiation process, including progenitor stem cells, determined stem
cells and fully differentiated cells, and hence provides a broad spectrum of cellular targets for theneoplastic response. They possess a competent metabolic system and have a finite lifespan in culture.

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010
SHE cells show a high proliferation rate, good plating efficiency (PE = 20-40%) and a low
spontaneous transformation frequency.

3.4 Cell Transformation Assay Variant: SHE pH 7.0 CTA
The SHE CTA has initially been developed and carried out in medium which exhibited a pH in therange of 7.0-7.35 (Berwald and Sachs, 1963; DiPaolo et al., 1971; Pienta et al., 1977). Thus, a large
amount of historical data available have been generated with protocols at pH 7.0-7.35.
The protocol and the criteria for CTA in SHE cells at pH 7.0 are identical to that of the modified
version of the protocol where the pH was reduced to 6.7 (Leboeuf and Kerckaert, 1987), except thatmedium is adjusted to pH 7.0 and the scoring is perceptibly different. At pH 7.0, control colonies aremonolayered and they show a high cytoplasm/nucleus ratio and their phenotype is conformed to thephenotype of any fibroblastic or epithelial normal cell culture obtained at physiological pH. Therefore,
it is easy for a non-experienced laboratory to discriminate between control and morphologicallytransformed colonies. It is quite pertinent to carry out experiments at pH 7.0 since this is representativeof physiological pH. In case of ionisable chemicals, whose ionisation depends on the pH, it isimportant to have a culture medium close to physiological pH so as to have a bioavailable fraction
which corresponds to the one found at physiological pH.

3.5 Protocol
The detailed protocol is described in Annex 12.2.
The SHE CTA protocol used in this prevalidation study is almost identical to the SHE CTA protocol
at pH 6.7, except that the pH of the medium used needs to be adjusted to pH 7.0 with the adequate
concentration of sodium bicarbonate.

3.5.1 SHE cells
SHE cells were prepared by BioReliance (USA), following the protocol described in detail in Annex
12.2.
These cells (isolate no.021208) were isolated at pH 7.2-7.3 from the embryos from four female
hamsters that were 13-day pregnant on day of arrival (received at BioReliance on 12 February 2008
from Harlan Sprague-Dawley). They were subsequently pooled allowing the preparation of a sufficientnumber of batches with the same quality and performance. After growth in vitro to form sub-confluent
monolayer cultures and subsequent harvest they were cryopreserved in liquid nitrogen (13 February2008). Prior to using this isolate in the transformation assay, the isolate was tested for its
transformation properties. Ampoules of cryopreserved cells were shipped in liquid nitrogen to all thelaboratories involved in the study.
The same cells were used by all laboratories for both phases of the prevalidation study, except for
University of Metz that used target SHE cells isolated at pH 7.0 in its laboratory (batch of cells isolated
in February 2006 and selected in May 2006 employing foetal bovine serum (FBS) Hyclone Perbio
AQL 25 247).
Each laboratory irradiated their feeder cells and froze them. All laboratories used the same machine forirradiating, except University of Metz which used a different one.

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010

3.5.2 Medium
During the whole study, Harlan CCR, BioReliance and BASF used the same DMEM-L (Dulbecco's
Modified Eagle Medium with LeBoeuf's modification) culture medium (not the same batch) from
Quality Biologicals, USA while University of Metz used reconstituted medium from lyophilized
DMEM without phenol red (Invitrogen). The medium was adjusted to pH 7.0 by adding sodium
bicarbonate.

3.5.3 Serum
Harlan CCR, BioReliance and BASF used the same batch of FBS lot no. APB20666, HyClone catalog
no. SH30070.03) for the whole prevalidation study. University of Metz used the batch of serum
Hyclone Perbio AQL 25 247 for the whole prevalidation study. The serum was heat inactivated at
56ºC for 30 minutes for all the tests performed.
Prior to being used, the batches of FBS were tested with cell isolates that have been shown to work
appropriately in previous studies:- The target cell number producing 25 to 45 colonies per dish dish with a lot of serum already shown to
work appropriately in a previous study was seeded on top of the feeder cells using the serum to test.
- The plates were treated with a positive control (PC, 1 µg/ml of benzo(a)pyrene for University ofMetz and 5 µg/ml of benzo(a)pyrene for the other laboratories) and a vehicle control (VC, 0.2%
DMSO) for seven days, the colonies were fixed, and the total number of colonies per plate and themorphological transformation frequency (MTF) were scored.
The results obtained with the serum lot tested were checked to meet the following criteria:- Number of colonies obtained with the VC between 25 and 45- Colonies normal sized- Statistically significant increase in the number of morphologically transformed colonies with the PC(p < 0.05, one-sided Fisher’s exact test) and MTF within historical range for PC- MTF with the VC within the historical range for VC (≤ 0.6%).

3.5.4 Controls
Positive Control: benzo(a)pyrene (5 µg/ml dissolved in 0.2% DMSO) was used as the PC in alllaboratories, except University of Metz which used 1 µg/ml of benzo(a)pyrene.
Untreated Control: The cell culture medium served as the concurrent negative control.
Vehicle Control: The cell culture medium containing 0.2% DMSO served as the concurrent VC.

3.5.5 Test procedure
The CTA is composed of two phases:• An initial dose-range finding (DRF) cytotoxicity test to determine the experimental treatment
doses that will be used for the transformation assay (TA),• The TA, which represents the main experiment and which includes the measurement ofcytotoxicity, the morphological evaluation of individual colonies, and the determination ofmorphological transformation frequency (MTF), in the same dish.
The DRF tests are carried out by measurement of the Plating Efficiency (PE = [total number ofcolonies/total number of target cells seeded] × 100) and subsequent assessment of the relative plating
efficiency (RPE = [PE of treated cells/PE of control cells] × 100).

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010
The measurement of cytotoxicity during the TA includes RPE and density/size measurements. MTF is
calculated as follows: MTF = [number of transformed colonies/total number of colonies] × 100.
The testing procedure for the DRF and for the TA is similar:
Briefly, early passage SHE cells were seeded on the feeder layer of irradiated SHE cells into 40 dishes(Ø 60 mm) per dose, so as to obtain between 25-45 colonies per dish and to score at least 1000
colonies, per treatment group. Twenty-four hours after seeding, the cells were treated with 4 ml ofcomplete medium containing the test chemical. The cells were exposed to the test chemical for sevendays. At the end of the exposure period the medium was removed and the cells were washed withphosphate-buffered saline (PBS), fixed with absolute ethanol and stained with 10% aqueous Giemsa.
After rinsing with tap water, the dishes were air dried before being scored. Each dish was coded andscored blindly. The colonies were examined under a stereomicroscope for scoring normal ormorphologically transformed phenotypes. The morphologically transformed cells are characterised bya spindle shape, an increased nuclear/cytoplasm ratio and a higher basophilic affinity. These cells havea criss-cross orientation pattern and may be multilayered compared to normal cells.
Around 1000 colonies were scored per concentration for PE, RPE and MTF determinations, in control
groups and in each treatment group.
At least five concentrations were scored, so typically seven to eight concentrations were tested in orderto ensure having the adequate number of scorable (i.e. fulfilling the assay acceptance criteria listed in
section 3.5.7) concentrations. Definitive assay doses should include if possible: a high dose causing atleast a 50% decrease in RPE and/or ≥ 50% reduction in relative colony density/size (by visualappearance), and at least one dose which has no effect on PE. If the test chemical was non-toxic, thenat least five concentrations were selected up to a maximum of 5 mg/ml or 10 mM (whichever islower), solubility permitting. For non-toxic and insoluble test chemicals, the highest dose level tested
was within 2-times the visible solubility limit in complete medium. For toxic and insoluble test
chemicals, the highest dose level tested caused an approximate 50% decrease in RPE or relativecolony density, regardless of the number of insoluble dose levels.

3.5.6 Statistical analysis of raw data
The data were analysed using methods established previously as described most recently in Custer etal., 2000. Results were analysed using the one-sided Fisher’s exact test to determine if an increase inmorphological transformation occurred compared to VC (significance level: p < 0.05, uncorrected for
multiple testing).
The Cochran-Armitage trend test (Armitage, 1955) for a positive dose-related response was performed
when only one chemical concentration was statistically significant (significance level: p < 0.05).

3.5.7 Assay acceptance criteria
The following assessment criteria were discussed and agreed by the VMT, although it is important tonote that some modifications are described in the Recommendations section (section 9) of this reportbased upon the outcome of the studies conducted:•

The PE of the untreated/vehicle control should be > 20%,•

No colony formation should be observed in the feeder cell control dishes. Feeder cells must be
visible in the chemical treatment groups except if they are affected selectively by the chemical,

24

SHE pH 7.0 CTA PRP report v1 - 30 July 2010•

≥ 1000 colonies per treatment group should be available for MTF assessment (less than 1000
colonies is acceptable if the dose group shows a statistically significant increase in thetransformation rate),•

There should be 25-45 colonies per dish for each treatment group. However, in the case ofnegative results < 25 colonies is acceptable and in the case of a positive result > 45 colonies
per dish are acceptable,•

Transformation frequency in the negative controls (untreated and vehicle) must be within therange of historical controls. 0.6% has been chosen as upper limit. This value was based on
published data and was consistent with the historical data of the laboratories,•

The PC chemical must lead to a statistically significant increase of morphological celltransformation,•

There should be at least five scorable concentrations.

3.5.8 Assay assessment criteria
The following assessment criteria were agreed by the VMT based on laboratories experience and theliterature. These criteria were the same as those used in the SHE CTA pH 6.7 study (most recently
Custer et al., 2000):•

A test chemical will be considered "negative/non-transforming" if the following criterion ismet:- the percentage of morphologically transformed colonies in the test chemical-treated groups is
not significantly different from that of the concurrent VC or it is less than or equal to 0.6%.

•

A test chemical will be considered "positive" if the following criteria are met:
- a statistically significant increase in transformation frequency (above 0.6% MTF) at at least
two dose levels compared to the concurrent VC, or- a statistically significant increased frequency in morphologically transformed colonies(above 0.6% MTF) only at a single dose level but with a general positive trend.

25

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

4 Module 2: Within-laboratory reproducibility
In order to evaluate the initial within-laboratory reproducibility, the PC chemical, benzo(a)pyrene, wastested in each laboratory as a coded chemical. This section of the report provides these results,
including the data of the DRF tests and the TAs of all laboratories.
In addition, benzo(a)pyrene was used as the PC for the assay conducted with coded chemicals and thewithin-laboratory reproducibility of benzo(a)pyrene was further evaluated (Figure 18).
Figure 1-Figure 2 and Table 3-Table 6 show the results of the TAs. Sparse colonies were not scored for
MTF assessment, but were included in the total number of colonies for the calculation of the PE.
Therefore the column “scorable colonies” is the total number of colonies minus the sparse colonies.
This number is used in the Fisher's exact test to calculate the significance of the increase in MTF.
The cytotoxicity of benzo(a)pyrene is shown by the RPE (%) and colony density/size (normal, slightly
or greatly reduced) columns.
The other columns indicate the number of morphologically transformed colonies, the percentage ofmorphologically transformed colonies compared to the total number of scorable colonies and theresults (p-value) of the Fisher’s exact test.

4.1 Transformation Assay - coded benzo(a)pyrene
Benzo(a)pyrene was assessed for its potential to induce morphological transformation in early passage
SHE cells after an exposure time of seven days. Each laboratory carried out initial DRF cytotoxicity
tests to determine the experimental doses. The VC (DMSO) gave transformation frequencies within the
expected range for the SHE cells under the assay conditions employed (≤0.6%): 0.26-0.45%(University of Metz), 0.08% (BASF), 0.34% (Harlan CCR) and 0.42% (BioReliance). The PC
chemical (benzo(a)pyrene) led to the expected increase in morphologically transformed colonies.
Further evidence of within-laboratory reproducibility is seen in the results for benzo(a)pyrene used as
the PC in the studies described in Module 4 below.

4.1.1 University of Metz
The within-laboratory reproducibility of the SHE pH 7.0 CTA protocol was initially assessed by
University of Metz only (2005-2006), in parallel to the evaluation of the SHE pH 6.7 CTA protocol.
Since at that time University of Metz was the only laboratory to assess the pH 7.0 protocol, the VMTrequested that the laboratory should perform three independent assays using coded benzo(a)pyrene.
Upon the demonstration that the assays performed with the pH 7.0 protocol were reproducible, thelaboratory could proceed to the next phase of the prevalidation.
In the preliminary phase of optimisation, University of Metz used the cells that had been isolated by
BioReliance, grown and stored at pH 6.7. These cells showed not to be appropriate for the conduct of
the assay at pH 7.0 indicating the importance of isolating the cells at the pH that will be used for the
conduct of the assay. Therefore, University of Metz subsequently used cells isolated by their own
laboratory at pH 7.0 and modified the SOP (Standard Operating Procedure) to include the protocol ofcell isolation and preparation.
In the following table (Table 3) the measurements of RPE and MTF of the experiments are
summarised. Based upon the results of the preliminary DRF test, the doses selected for the evaluationof the induction of morphological transformation ranged from 0.01 to 25 µg/ml. The MTF values in thethree independent experiments (TA1, TA2 and TA3) ranged from 0.39% to 6.41%, 0.34% to 6.11%,

26

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
and 0.45% to 5.13%, respectively. All experiments reproducibly showed significant increases in MTF
compared to the VC (p < 0.01) at all test chemical concentrations equal to or higher than 0.1 µg/ml.
Results of TA1 for 1 µg/ml were not taken into consideration for the final assessment since the average
number of colonies per dish was slightly below the limit for a positive result (24.8).
Table 3: Transformation assay results from University of Metz, testing coded benzo(a)pyrene
Uni Metz
Coded
benzo(a)pyrene(µg/ml)TA1VC(DMSO 0.2%)
0.01
0.1
1
5
10
15
25
Uni Metz
Coded
benzo(a)pyrene(µg/ml)TA2VC(DMSO 0.2%)
0.01
0.1
1
5
10
15
25
Uni Metz
Coded
benzo(a)pyrene(µg/ml)TA3VC(DMSO 0.2%)
0.01
0.1
1
5
10
15
25

Scorablecolonies

RPE (%)

1576

100.0

+

1281
1043
993
1018
1359
1297
1213

81.0
66.1
62.5
68.7
87.1
83.5
80.6

+
+
+
+
+
+
+

Scorablecolonies

RPE (%)

1559

100.0

+

1476
1103
1100
1129
1241
1196
1178

94.7
71.1
70.7
73.3
79.9
77.2
75.8

+
+
+
+
+
+
+

Scorablecolonies

RPE (%)

1543

100.0

+

1556
1195
1210
1174
1224
1190
1190

100.8
77.4
78.6
76.7
80.1
78.0
85.2

+
+
+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

6

0.38

-

5
33
26
43
35
73
77

0.39
3.16
2.62
4.22
2.58
5.63
6.41

>0.5
<0.01**<0.01**<0.01**<0.01**<0.01**<0.01**

MTF (%)

Fisher's exact
test p-value(one-sided)

4

0.26

-

5
22
41
69
45
36
47

0.34
1.99
3.73
6.11
3.63
3.01
3.99

>0.5
<0.01**<0.01**<0.01**<0.01**<0.01**<0.01**

MTF (%)

Fisher's exact
test p-value(one-sided)

7

0.45

-

7
21
35
52
61
61
58

0.45
1.76
2.89
4.43
4.98
5.13
4.43

>0.5
<0.05**<0.01**<0.01**<0.01**<0.01**<0.01**

ColonyTransformed
density/sizecolonies

ColonyTransformed
density/sizecolonies

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation Frequency, + = normal
density/size.
** = p < 0.01 (one-sided Fisher's exact test)27

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

4.1.2 BASF
The doses selected ranged from 1.25 to 10 µg/ml and the MTF ranged from 0.88% to 1.87% (Table 4).
All test chemical concentrations induced significant increases in MTF compared to the VC (p < 0.01).
Table 4: Transformation assay results from BASF, testing coded benzo(a)pyrene
BASF
Coded
benzo(a)pyrene(µg/ml)VC(DMSO 0.2%)
1.25
2.5
5
7.5
10

Scorablecolonies

RPE (%)

1277

100.0

+

1018
1018
1016
1024
1139

78.7
81.1
79.0
80.6
87.2

+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

1

0.08

-

19
9
16
18
16

1.87
0.88
1.57
1.76
1.40

<0.0005**
0.004**<0.0005**<0.0005**<0.0005**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation Frequency, + = normal
density/size.
** = p < 0.01 (one-sided Fisher's exact test)

4.1.3 Harlan CCR
The test doses ranged from 1.25 to 10 µg/ml and the MTF ranged from 1.45% to 3.29% (Table 5). All
test chemical concentrations induced significant increases in MTF compared to the VC (p < 0.01), witha dose-dependent effect.
Table 5: Transformation assay results from Harlan CCR, testing coded benzo(a)pyrene
Harlan CCR
Coded
benzo(a)pyrene(µg/ml)VC(DMSO 0.2%)
1.25
2.5
5.0
7.5
10.0

Scorablecolonies

RPE (%)

2337

100.0

+

2424
2002
2138
1896
1960

85.7
91.5
81.1
84.1
91.0

+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

8

0.34

-

29
56
57
58
70

1.45
2.62
3.01
2.96
3.29

<0.0005**<0.0005**<0.0005**<0.0005**<0.0005**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation Frequency, + = normal
density/size.
** = p < 0.01 (one-sided Fisher's exact test)

4.1.4 BioReliance
The test doses ranged from 1 to 10 µg/ml and the MTF ranged from 1.38% to 2.17% (Table 6). All test
chemical concentrations induced significant increases in MTF compared to the VC (p < 0.01).

28

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Table 6: Transformation assay results from BioReliance, testing coded benzo(a)pyrene
BioRelianceCoded
benzo(a)pyrene(µg/ml)VC(DMSO 0.2%)
1.0
2.5
5.0
7.5
10.0

Scorablecolonies

RPE (%)

1203

100.0

+

1372
1406
1522
1472
1487

114.2
117.4
126.8
122.4
124.1

+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

5

0.42

-

19
20
33
27
24

1.38
1.42
2.17
1.83
1.61

0.008**
0.006**<0.0005**<0.0005**
0.002**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation Frequency, + = normal
density/size.
** = p < 0.01 (one-sided Fisher's exact test)

4.1.5 Concurrent cytotoxicity (Relative Plating Efficiency)
Cytotoxicity of benzo(a)pyrene was evaluated by RPE assessment in all laboratories. University ofMetz repeated the experiment three times (TA1, TA2 and TA3), whereas BASF, Harlan CCR and
BioReliance performed a single experiment (Figure 1).
130

A

120
110
100

RPE [%]

90
80
70
60
50
40

Uni Metz, TA1

30

Uni Metz, TA2

20

Uni Metz, TA3

10
0
0

5

10

15

20

25

30

Benzo(a)pyrene [µg/ml]

29

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

130

B

120
110
100

RPE [%]

90
80
70
60
50
40
30

BASF

20

Harlan CCR

10

BioReliance

0
0

5

10

Benzo(a)pyrene [µg/ml]

Figure 1: Relative Plating Efficiency (RPE) compared to vehicle control in all laboratories
testing coded benzo(a)pyrene: University of Metz repeated the experiment three times (A), whereas
BASF, Harlan CCR and BioReliance performed a single experiment (B)

4.1.6 Morphological transformation frequency
MTF results with benzo(a)pyrene are shown in Figure 2. Benzo(a)pyrene induced a statisticallysignificant increase in morphological transformation compared to VC in all laboratories and at every
concentration tested. The shapes of the curves of University of Metz repeated experiments show aclearer dose-dependent increase of MTF in comparison to the curves of the other laboratories, which isin part due to testing at lower doses.

65

MTF [%]

**

**

A

**

4

**

3

**

2

**
**

**
** **
**
**
**
**
**
**

**

Uni Metz, TA1**

Uni Metz, TA2

1

Uni Metz, TA3

0
0.001

0.01

0.1

1

10

100

Benzo(a)pyrene [µg/ml]

30

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6

B

BASF
Harlan CCR

5

BioReliance

MTF [%]

4
3

**

**

**
**

**

**
**
**
**
**
**
** **
**
**

2
1
0
0.001

0.01

0.1

1

10

100

Benzo(a)pyrene [µg/ml]

6

C

BASF
Harlan CCR

5

BioReliance

MTF [%]

4
3

**

**

**

**
**
**

2
1

**

**

**
**

**

**

**

**

**
0
0

1

2

3

4

5

6

7

8

9

10

11

Benzo(a)pyrene [µg/ml]

Figure 2: Morphological Transformation Frequency (MTF) shown as a percentage oftransformed colonies compared to scorable colonies, for all laboratories testing coded
benzo(a)pyrene: University of Metz repeated the experiment three times (A), whereas BASF, Harlan
CCR and BioReliance performed a single experiment (B). For a better visualisation of the data, resultsfrom BASF, Harlan CCR and BioReliance were also represented with a linear scale (C)** = p < 0.01 (one-sided Fisher's exact test)

4.1.7 Acceptance criteria and assessment
All acceptance criteria were met in all experiments performed by the four laboratories. These aresummarised in Table 7.

31

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Table 7: Acceptance criteria and assessment of benzo(a)pyrene results
Benzo(a)pyrene
Laboratory
Criteria
TA1

Uni Metz
TA2

BASF

HarlanCCR

BioRelianceyes(≥1018)yes(≥1896)yes(≥1203)yes(26.0-33.1)

yes(47.4-60.7)

yes(30.1-38.1)

TA3

Number of scorable colonies
yes
yes
yes
per treatment group ≥1000(≥1018)(≥1100)(≥1174)or <1000 if positive result
Average number of 25-45
yes
yes
yes
colonies per dish or>45 colonies if positive result or (27.2-39.6) (27.6-39.0) (29.6-38.9)<25 colonies if negative result
Plating Efficiency of vehicle
control >20%

yes
(26.4%)

yes(26.0%)

yes(25.8%)

yes(30.1%)

yes(53.2%)

yes(25.1%)Morphological Transformation
Frequency of vehicle control
<0.6%

yes
(0.38%)

yes(0.26%)

yes(0.45%)

yes(0.08%)

yes(0.34%)

yes(0.42%)

Number of scorable
concentrations ≥5

yes
(6#)

yes
(7)

yes
(7)

yes
(5)

yes
(5)

yes
(5)

Fisher’s test p-value of positive
control <0.05

na

na

na

na

na

na

Fulfilment of all assay
acceptance criteria

YES

YES

YES

YES

YES

YES

+

+

+

+

+

+

Assessment

na = not applicable#
results for 1 µg/ml were not taken into consideration. See section 4.1.1 for details.

4.2 Conclusion of the Validation Management Team on Module 2
The data generated by University of Metz in the three experiment repeats showed a good withinlaboratory reproducibility of the results for coded benzo(a)pyrene. .
Although no dose-dependent cytotoxicity could be observed (due to insolubility of the test chemical athigher concentrations), benzo(a)pyrene induced a reproducible increase in MTF. Results from thisphase of the study were in agreement with published data for benzo(a)pyrene (Rivedal and Sanner,1980).
In addition, these results showed reproducibility for benzo(a)pyrene between the laboratories and overtime (since the experiments of University of Metz were performed prior to the main study) anddemonstrated that the method had been successfully transferred to all laboratories.
Overall, the VMT agreed that the data generated were satisfactory and reproducible.

32

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

5 Module 3: Transferability
5.1 General Aspects
In general, the proposed test method 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 alaboratory furnished with the appropriate test protocol and supporting SOPs could be expected toeffectively 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. Access to facilities for theirradiation of feeder cells is necessary.
The preparation of primary cells is more laborious and requires the isolation of cells from pregnant
Syrian hamsters and the evaluation for their suitability for use in this assay. However, primary SHE
cells are commercially available, though, as with any such preparation, the cells would need to betested for their suitability.
Scoring of transformed colonies 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 Quality Control tissue culture laboratories.
The operator should, in particular, be trained in the scoring of transformed colonies. The training
requirements for a person to be competent in scoring the plates are quite rigorous.
Three of the laboratories participating to this prevalidation study (Harlan CCR, BioReliance andBASF) had also been involved in the prevalidation study of the CTA in SHE cells at pH 6.7 where theygained experience in working with these cells. However, it became clear that the scoring of colonies
produced with the protocol at pH 7.0 was slightly different from that at pH 6.7. 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 was held at the University of Metz, France, in July 2008.
Representatives from all laboratories involved participated in the training. Agreement was reached on
criteria for scoring the plates, using dishes treated with both the vehicle and positive controls. Overall,
the training was extremely useful for harmonising the procedures among the laboratories for theprevalidation experiments.
As part of this prevalidation exercise a photo catalogue was produced by the lead laboratory(University of Metz) with the aim of standardising the scoring. The catalogue includes pictures of both
non-transformed and transformed colonies. Examples of clearly scorable colonies, recognisablytransformed colonies, colonies with questionable or mixed morphology, as well as examples of alteredcolonies that should not be scored were included in the catalogue to obtain an overview of the different
types of colonies that can be encountered during a CTA experiment. Such a catalogue was found to bevery useful in establishing consistency in assessing colony morphology and for the scoring of theexperiments performed to assess the between-laboratory reproducibility.

33

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

5.3 Conclusion of the Validation Management Team on Module 3
It should be noted that three of the laboratories (BASF, Harlan CCR and BioReliance) had noexperience in conducting the SHE CTA at pH 7.0, prior to starting this prevalidation study.
Basic cell culture experience and training in the conduct and scoring of the assay are important. In
addition, the photo catalogue produced was found to be very useful in establishing consistency inassessing colony morphology and for the scoring of the experiments performed to assess the betweenlaboratory reproducibility.
The VMT agreed on the success of the method transfer.
This was also subsequently confirmed by the satisfactory results for the between-laboratory
reproducibility (sections 4 and 6 on within- and between-laboratory reproducibility, respectively).

34

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6 Module 4: Between-laboratory reproducibility
For the between-laboratory reproducibility assessment, the same conditions (medium, serum, SHE
cells, controls and protocol) used for the within-laboratory reproducibility assessment weremaintained. The following coded chemicals were tested (Table 1): anthracene, 2,4-diaminotoluene, 3methylcholanthrene, o-toluidine HCl and phthalic anhydride.
The results of the between-laboratory reproducibility are summarised by chemical and laboratory. The
data are shown in tables for each individual laboratory. Each table includes information on the total
number of scorable colonies, RPE, colony density and size, the number of transformed colonies, MTF,
and the result of the Fisher’s exact test (p-value), for VC, test chemical at different concentrations andthe PC.
An initial DRF test was performed by all laboratories to determine the experimental doses to be used in
the TA, for each chemical (Figure 3, Figure 6, Figure 9, Figure 12 and Figure 15). Subsequently, thecomplete TA including concurrent cytotoxicity tests (Figure 4, Figure 7, Figure 10, Figure 13 andFigure 16) and MTF assay were performed (Figure 5, Figure 8, Figure 11, Figure 14 and Figure 17).
The statistical analysis was performed by the laboratories and recalculated by the statistician. The
laboratories and the statistician conclusions showed complete concordance, which can also be
considered as a data quality control check.

6.1 Anthracene
6.1.1 Dose-range finding test
Figure 3 shows the results of the DRF tests with anthracene. The common range of concentrations for
DRF was 0.6-13 µg/ml. At the concentrations tested, limited cytotoxicity (IC80) was observed byHarlan CCR, whereas the other laboratories saw some cell proliferation.
130
120
110
100

RPE [%]

90
80
70
60
50
30

BASF
Harlan CCR

20

BioReliance40

Uni Metz

10
0
0.001

0.01

0.1

1

10

100

Anthracene [µg/ml]

Figure 3: Relative plating efficiency (RPE) compared to vehicle control in all laboratories testingcoded anthracene, for the Dose-Range Finding

35

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.1.2 Transformation Assay
VCs gave transformation frequencies within the expected range (≤0.6%): 0.40% (University of Metz),
0.25% (BASF), 0.27% (Harlan CCR) and 0.21% (BioReliance). The PC chemical benzo(a)pyrene led
to the expected increase in morphologically transformed colonies: 2.85% (University of Metz), 1.36%(BASF), 1.85% (Harlan CCR) and 1.03% (BioReliance). It can be seen from Figure 4 that anthracene
was not cytotoxic in any laboratory, nor did it induce a statistically significant increase inmorphological transformation (Figure 5).

6.1.2.1 University of Metz
Anthracene was dissolved in DMSO. 13 µg/ml was selected as the top dose on the basis of the DRF
test and based on the solubility of the chemical. The MTF values of the test chemical doses ranged
from 0.24% to 0.89%. None of the test chemical concentrations induced a significant increase in MTF
compared to the VC (p ≥ 0.05). The results evaluated by University of Metz are shown in Table 8.
Table 8: Transformation assay results from University of Metz, testing coded anthracene
Uni Metz
Anthracene(µg/ml)VC(DMSO 0.2%)
1.0
2.5
5.0
7.5#
10##
13##
PC

Scorablecolonies

RPE (%)

1238

100.0

+

1206
1280
1272
1330
1344
1390
1193

96.6
103.0
101.8
106.7
108.5
111.9
96.0

+
+
+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

5

0.40

-

10
7
3
9
12
7
34

0.83
0.55
0.24
0.68
0.89
0.50
2.85

0.13840.40970.34830.25280.097240.778<0.0001**

ColonyTransformed
density/sizecolonies#= a slight opalescence was observed in the premix and the test medium, ## = opalescence was observed in the premix andthe test medium
VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size.
** = p < 0.01 (one-sided Fisher's exact test)

6.1.2.2 BASF
Anthracene was dissolved in DMSO. 25 µg/ml was selected as the top dose on the basis of the DRF
test and based on the solubility of the chemical. The MTF values of the test chemical doses ranged
from 0.00% to 0.25%. None of the test chemical concentrations induced a significant increase in MTF
compared to the VC (p ≥ 0.05). The results evaluated by BASF are shown in Table 9.
Table 9: Transformation assay results from BASF, testing coded anthracene
BASF
Anthracene(µg/ml)VC(DMSO 0.2%)
2.5
5.0
10.0
15.0
20.0
25.0
PC

Scorablecolonies

RPE (%)

1575

100.0

+

1607
1569
1696
1731
1686
1606
735

100.1
96.7
105.0
107.3
105.4
99.0
82.9

+
+
+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

4

0.25

-

0
4
3
3
4
4
10

0.00
0.25
0.18
0.17
0.24
0.25
1.36

1.000
0.635
0.803
0.811
0.673
0.648
0.003**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size.
** = p < 0.01 (one-sided Fisher's exact test)

36

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.1.2.3 Harlan CCR
Anthracene was dissolved in DMSO. 20 µg/ml was selected as the top dose on the basis of the DRF
test and based on the solubility of the chemical. The MTF values of the test chemical doses ranged
from 0.18% to 0.26%. None of the test chemical concentrations induced a significant increase in MTF
compared to the VC (p ≥ 0.05). The results evaluated by Harlan CCR are shown in Table 10.
Table 10: Transformation assay results from Harlan CCR, testing coded anthracene
Harlan CCR
Anthracene(µg/ml)Scorablecolonies

RPE (%)

1498

100.0

+

1418
1524
1546
1438
1556
1626
1458

94.6
101.7
103.2
96.1
103.9
108.5
97.5

+
+
+
+
+
+
+

VC
(DMSO 0.2%)
0.63
1.25
2.5
5.0
10.0
20.0
PC

MTF (%)

Fisher's exact
test p-value(one-sided)

4

0.27

-

3
3
4
3
4
3
27

0.21
0.20
0.26
0.21
0.26
0.18
1.85

>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
<0.001**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size.
** = p < 0.01 (one-sided Fisher's exact test)

6.1.2.4 BioReliance
Anthracene was dissolved in DMSO. 100 µg/ml was selected as the top dose on the basis of the DRF
test and based on the solubility of the chemical. The MTF values of the test chemical doses ranged
from 0.20% to 0.36%. None of the test chemical concentrations induced a significant increase in MTF
compared to the VC (p ≥ 0.05). The results evaluated by BioReliance are shown in Table 11.
Table 11: Transformation assay results from BioReliance, testing coded anthracene
BioReliance
Anthracene(µg/ml)VC(DMSO 0.2%)
2.5
5.0
10
25
50
100
PC

Scorablecolonies

RPE (%)

1463

100.0

+

1391
1331
1532
1474
1396
1512
1546

95.6
92.0
105.3
101.0
96.1
103.9
105.7

+
+
+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

3

0.21

-

5
3
3
4
4
4
16

0.36
0.23
0.20
0.27
0.29
0.26
1.03

0.336
0.611
0.678
0.504
0.474
0.518
0.003**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size.
** = p < 0.01 (one-sided Fisher's exact test)

37

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.1.2.5 Concurrent cytotoxicity (Relative Plating Efficiency)
Cytotoxicity of anthracene was evaluated by RPE assessment in all laboratories (Figure 4). The
common range tested was 2.5-40 µg/ml. No evident signs of cytotoxicity were observed by any
laboratory.
130
120
110
100

RPE [%]

90
80
70
60
50
40

BASF

30

Harlan CCR

20

BioReliance

10

Uni Metz

0
1

10

100

Anthracene [µg/ml]

Figure 4: Relative plating efficiency (RPE) compared to vehicle control in all laboratories testingcoded anthracene

6.1.2.6 Morphological transformation frequency
MTF results with anthracene are shown in Figure 5. Anthracene did not induce a statisticallysignificant increase in morphological transformation in any of the laboratories.
6

BASF
Harlan CCR
BioReliance

5

Uni Metz

MTF [%]

4
3
2
1
0
1

10

100

Anthracene [µg/ml]

Figure 5: Morphological Transformation Frequency (MTF) shown as a percentage oftransformed colonies compared to total colonies, for all laboratories testing anthracene

38SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.1.2.7 Acceptance criteria and assessment
All acceptance criteria were met in all laboratories. Anthracene was assessed to be negative by alllaboratories (Table 12).
Table 12: Acceptance criteria and assessment of anthracene results
Anthracene
Laboratory

Criteria
BASF

Harlan CCR

BioReliance

Uni Metzyes(≥1569,735#)

yes(≥1418)yes(≥1331)yes(≥1193)yes(33.6-43.5)

yes(34.6-40.7)

yes(33.7-38.7)

yes(30.0-35.0)

Plating Efficiency of vehicle control >20%

yes
(36.9%)

yes(57.7%)

yes(30.5%

yes(20.9%)Morphological Transformation Frequency ofvehicle control <0.6%

yes(0.25%)

yes(0.27%)

yes(0.21%)

yes(0.40%)

yes
(6)

yes
(6)

yes
(6)

yes
(6)

Fisher’s test p-value of positive control <0.05

yes(0.003)yes(<0.001)

yes(0.003)yes(<0.0001)

Fulfilment of all assay acceptance criteria

YES

YES

YES

YES

-

-

-

-

Number of scorable colonies per treatmentgroup ≥1000
or <1000 if positive result
Average number of 25-45 colonies per dishor >45 colonies if positive resultor <25 colonies if negative result

Number of scorable concentrations ≥5

Assessment#

735 colonies for the positive control

6.1.3 Conclusion
Treatment with anthracene did not produce a statistically significant increase in morphologicallytransformed colonies in any laboratory at any dose tested. As such, anthracene is considered to be nontransforming in the SHE pH 7.0 CTA. These results agree with published data (Heidelberger et al.,
1983; Tennant et al., 1985).

6.2 2,4-Diaminotoluene
6.2.1 Dose-range finding test
Figure 6 shows the results of the DRF tests with 2,4-diaminotoluene. All laboratories observed a dosedependent cytotoxicity induced by 2,4-diaminotoluene at the concentrations tested.

39

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

130
120
110
100

RPE [%]

90
80
70
60
50
40

BASF
Harlan CCR
BioReliance
Uni Metz

30
20
10
0
0

10

20

30

40

50

60

70

80

90

100

2,4-Diaminotoluene [µg/ml]

Figure 6: Relative plating efficiency (RPE) compared to vehicle control in all laboratories testingcoded 2,4-diaminotoluene, for the Dose-Range Finding

6.2.2 Transformation assay
VCs gave transformation frequencies within the expected range (≤0.6%): 0.39% (University of Metz),
0.28% (BASF), 0.20% (Harlan CCR) and 0.21% (BioReliance). The PC chemical benzo(a)pyrene led
to the expected increase in morphologically transformed colonies: 1.82% (University of Metz), 1.70%(BASF), 2.34% (Harlan CCR) and 1.03% (BioReliance).

6.2.2.1 University of Metz
2,4-Diaminotoluene was dissolved in DMSO. The concentrations were selected on the basis of the
DRF test. The MTF values of the test chemical doses ranged from 1.03% to 3.41% and the VC value
was 0.39%. Three test chemical concentrations induced a significant increase in MTF compared to theVC (p < 0.05) (Table 13). At the two highest concentrations no significant increase in MTF wasobserved, most probably due to the cytotoxicity caused by the test chemical. 50 and 75 µg/ml
concentrations were not considered scorable because the total number of colonies was below the limit(<1000) with a negative result.

40

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Table 13 : Transformation assay results from University of Metz, testing coded 2,4diaminotoluene
Uni Metz
2,4-diaminotoluene(µg/ml)VC(DMSO 0.2%)
0.1
1
5
10
25
50
75
PC

Scorablecolonies

RPE (%)

1291

100.0

+

1159
1218
1189
1257
1230
967
170
1265

89.6
94.2
94.6
98.1
84.7
56.9
9.9
98.3

+
+
+
+
+
+++
+++
+

MTF (%)

Fisher's exact
test p-value(one-sided)

5

0.39

-

14
38
28
35
42
10
3
23

1.21
3.12
2.35
2.78
3.41
1.03
1.76
1.82

0.01794*<0.0001**<0.0001**<0.0001**<0.0001**
0.05430.055
0.0003**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size, ++ = slightly reduced density/size, +++ = highly reduced density/size.
* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)

6.2.2.2 BASF
The first experiment (TA1) did not fulfill the acceptance criterion for the number of scorable
concentrations and the number of transformed colonies for one test chemical concentration (10 µg/ml)
was in the same range as for the PC (see section 12.3 for details). BASF was thus asked by the VMT torepeat the experiment (TA2).
2,4-Diaminotoluene was dissolved in DMSO. The concentrations were selected on the basis of the
DRF test. The MTF values of the test chemical doses ranged from 0.85% to 2.70% and the VC value
was 0.28%. All test chemical concentrations, except the lowest, induced a significant increase in MTF
compared to the VC (p < 0.05) (Table 14).
Table 14: Transformation assay TA2 results from BASF, testing coded 2,4-diaminotoluene
BASF
2,4-diaminotoluene(µg/ml)TA2VC(DMSO 0.2%)
5
10
20
30
40
PC

Scorablecolonies

RPE (%)

1080

100.0

+

1173
1116
1080
1075
1091
1178

108.6
103.3
100.0
99.5
67.3
109.2

+
+
+
+
++
+

MTF (%)

Fisher's exact
test p-value(one-sided)

3

0.28

-

10
12
10
29
16
20

0.85
1.08
0.93
2.70
1.47
1.70

0.061
0.002**
0.045*<0.001**0.002**<0.001**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size, ++ = slightly reduced density/size.
* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)

6.2.2.3 Harlan CCR
2,4-Diaminotoluene was dissolved in DMSO. The concentrations were selected on the basis of the
DRF test. The MTF values of the test chemical doses ranged from 0.87% to 3.51% and the VC value
was 0.20%. All test chemical concentrations induced a significant increase in MTF compared to theVC (p < 0.05) (Table 15).

41

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Table 15: Transformation assay results from Harlan CCR, testing coded 2,4-diaminotoluene
Harlan CCR
2,4-diaminotoluene(µg/ml)VC(DMSO 0.2%)
20.0
40.0
60.0
80.0
100.0
PC

Scorablecolonies

RPE (%)

1519

100.0

+

1729
1469
1168
1202
1009
1625

113.9
96.7
79.0
78.2
51.3
107.1

+
+
+
++
+++
+

MTF (%)

Fisher's exact
test p-value(one-sided)

3

0.20

-

15
31
41
31
23
38

0.87
2.11
3.51
2.58
2.28
2.34

0.008**<0.001**<0.001**<0.001**<0.001**<0.001**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size, ++ = slightly reduced density/size, +++ = highly reduced density/size.
** = p < 0.01 (one-sided Fisher's exact test)

6.2.2.4 BioReliance
2,4-Diaminotoluene was dissolved in DMSO. The concentrations were selected on the basis of the
DRF test. The MTF values of the test chemical doses ranged from 0.10% to 0.93% and the VC value
was 0.21%. Three test chemical concentrations induced a significant increase in MTF compared to theVC (p < 0.05). At the highest concentration no significant increase in MTF was observed, mostprobably due to the cytotoxicity caused by the test chemical (Table 16).
Table 16: Transformation assay results from BioReliance, testing coded 2,4-diaminotoluene
BioReliance2,4-diaminotoluene(µg/ml)VC(DMSO 0.2%)
5.0
10
25
50
100
PC

Scorablecolonies

RPE (%)

1463

100.0

+

1720
1759
1781
1609
1004
1546

117.6
120.3
121.9
110.3
68.6
105.7

+
+
+
+
+++
+

MTF (%)

Fisher's exact
test p-value(one-sided)

3

0.21

-

13
13
10
15
1
16

0.76
0.74
0.56
0.93
0.10
1.03

0.023*
0.026*
0.091
0.007**
0.877
0.003**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size, +++ = highly reduced density/size.
* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)

6.2.2.5 Concurrent cytotoxicity (Relative Plating Efficiency)
Cytotoxicity of 2,4-diaminotoluene was evaluated by RPE assessment (Figure 7). The common range
tested by all laboratories was 20-40 µg/ml and 2,4-diaminotoluene was shown to be dose-dependently
cytotoxic in all laboratories. Although the shapes of the curves were similar in all laboratories, thechemical resulted more toxic in the BASF and University of Metz laboratories.

42

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

130
120
110
100

RPE [%]

90
80
70
60
50
40

BASF
Harlan CCR
BioReliance
Uni Metz

30
20
10
0
0

10

20

30

40

50

60

70

80

90

100

2,4-Diaminotoluene [µg/ml]

Figure 7: Relative plating efficiency (RPE) compared to vehicle control in all laboratories testingcoded 2,4-diaminotoluene

6.2.2.6 Morphological transformation frequency
MTF results with 2,4-diaminotoluene are shown in Figure 8. 2,4-Diaminotoluene induced astatistically significant increase in morphological transformation compared to the VC in alllaboratories.
6

BASF
Harlan CCR
BioReliance
Uni Metz

5

MTF [%]

4
3

**

**

**
**

2
1

*

**

*

* *

0
0

**

**

**

10

20

**

**
**

**

**
30

40

50

60

70

80

90

100

2,4-Diaminotoluene [µg/ml]

Figure 8: Morphological Transformation Frequency (MTF) shown as a percentage oftransformed colonies compared to total colonies for all laboratories testing coded 2,4diaminotoluene* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)

43

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.2.2.7 Acceptance criteria and assessment
All acceptance criteria were met in all laboratories. 2,4-diaminotoluene was assessed to be positive by
all laboratories (Table 17).
Table 17: Acceptance criteria and assessment of 2,4-diaminotoluene results
2,4-Diaminotoluene
Criteria
Uni Metz
Number of scorable colonies per treatmentgroup≥1000
or <1000 if positive result
Average number of 25-45 colonies per dishor >45 colonies if positive resultor <25 colonies if negative result

BioRelianceyes(≥1159)yes(≥1075)yes(≥1009)yes(≥1004)yes(29.1-32.4)

yes(26.9-29.5)

yes(26.0-43.3)

yes(25.1-44.6)

Plating Efficiency of vehicle control >20%yes
(21.6%)

yes(24.5%)

yes(50.7%)

yes(30.5%)Morphological Transformation Frequency ofvehicle control <0.6%

yes(0.39%)

yes(0.28%)

yes(0.20%)

yes(0.21%)

yes
(5#)

yes
(5)

yes
(5)

yes
(5)

Fisher’s test p-value of positive control <0.05

yes(<0.0003)

yes(<0.001)

yes(<0.001)

yes(0.003)Fulfilment of all assay acceptance criteria

YES

YES

YES

YES

Assessment

+

+

+

+

Number of scorable concentrations ≥5#

Laboratory
BASF
Harlan CCR(TA2)results for 50 and 75 µg/ml were not taken into consideration. See section 6.2.2.1for details.

6.2.3 Conclusion
Treatment with 2,4-diaminotoluene produced a statistically significant increase in morphologicallytransformed colonies in all laboratories. As such, 2,4-diaminotoluene was considered to be a positivetransforming agent in the SHE pH 7.0 CTA. These results agree with published data (Holen et al.,1990).

6.3 3-Methylcholanthrene
6.3.1 Dose-range finding test
Figure 9 shows the results of the DRF tests with 3-methylcholanthrene.

44SHE pH 7.0 CTA PRP report v1 - 30 July 2010

130
120
110
100

RPE [%]

90
80
70
60
50
40
30
20
10
0
0.001

BASF
Harlan CCR
BioReliance
Uni Metz

0.01

0.1

1

10

3-Methylcholanthrene [µg/ml]

Figure 9: Relative plating efficiency (RPE) compared to vehicle control in all laboratories testingcoded 3-methylcholanthrene, for Dose-Range Finding

6.3.2 Transformation assay
VCs gave transformation frequencies within the expected range (≤0.6%): 0.51% (University of Metz),
0.25% (BASF), 0.32% (Harlan CCR), and 0.38% (BioReliance). The PC chemical benzo(a)pyrene led
to the expected increase in morphologically transformed colonies: 2.46% (University of Metz), 1.36%(BASF), 2.04% (Harlan CCR), and 1.59% (BioReliance). Figure 10 shows that 3-methylcholanthrene
was not cytotoxic at the doses tested by Harlan CCR and BioReliance whereas some cytotoxicity wasobserved in the other two laboratories. Despite the differences in cytotoxicity, all laboratories showed
3-methylcholanthrene to induce a statistically significant increase in morphological transformation at
all doses tested (Figure 11).

6.3.2.1 University of Metz
3-Methylcholanthrene was dissolved in DMSO. The concentrations were selected on the basis of thedose range suggested by the VMT and the DRF experiment, and the results evaluated by University ofMetz are shown in Table 18. The MTF values of the test chemical doses ranged from 0.77% to 1.43%.
Four test chemical concentrations induced significant increases in MTF compared to the VC(p < 0.05).
The three highest test chemical concentrations were not considered valid because the average numberof colonies per dish was below the limit for positive results (<25 colonies) for 0.1 and 1 µg/ml (20.9
and 24.4 colonies per dish, respectively), and because the total number of colonies for 2 µg/ml was
below the limit (<1000) with a negative result.

45

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Table 18: Transformation assay results from University of Metz, testing coded 3methylcholanthrene
Uni Metz
3-methylcholanthrene(µg/ml)VC(DMSO 0.2%)
0.0005
0.001
0.005
0.01
0.1
1
2
PC

Scorablecolonies

RPE (%)

1373

100.0

+

1445
1332
1117
1099
976
796
775
1344

105.1
96.8
85.6
71.4
63.3
49.2
45.7
100.5

+
+
+
++
++
++
++
+++

MTF (%)

Fisher's exact
test p-value(one-sided)

7

0.51

-

13
13
15
15
14
11
6
33

0.90
0.98
1.34
1.36
1.43
1.38
0.77
2.46

0.156
0.116
0.023 *
0.021 *
0.017*
0.030*
0.312<0.0001**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size, ++ = slightly reduced density/size, +++ = highly reduced density/size.
* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)

6.3.2.2 BASF
3-Methylcholanthrene was dissolved in DMSO. The concentrations were selected on the basis of thedose range suggested by the VMT and the DRF experiment, and the results evaluated by BASF are
shown in Table 19. The MTF values of the test chemical doses ranged from 0.38% to 1.14%. Threetest chemical concentrations induced significant increases in MTF compared to the VC (p < 0.05).
Table 19: Transformation assay results from BASF, testing coded 3-methylcholanthrene
BASF
3-methylcholanthrene(µg/ml)VC(DMSO 0.2%)
0.31
0.63
1.25
2.50
5.00
10.00
PC

Scorablecolonies

RPE (%)

1575

100.0

+

1041
1001
1054
1486
1582
1634
735

66.7
65.4
72.5
64.3
66.2
68.5
82.9

+
+
+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

4

0.25

-

4
4
12
12
6
15
10

0.38
0.40
1.14
0.81
0.38
0.92
1.36

0.401
0.379
0.005**
0.029*
0.379
0.012*
0.003**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size.
* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)

BASF used for this experiment the same VC and PC that were used to test anthracene.

6.3.2.3 Harlan CCR
3-Methylcholanthrene was dissolved in DMSO. The concentrations were selected on the basis of thedose range suggested by the VMT and the DRF experiment, and the results evaluated by Harlan CCR
are shown in Table 20. The MTF values of the test chemical doses ranged from 1.12% to 2.99%. All
test chemical concentrations induced significant increases in MTF compared to the VC (p < 0.05), witha dose-dependent effect.

46

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Table 20: Transformation assay results from Harlan CCR, testing coded 3-methylcholanthrene
Harlan CCR
3-methylcholanthrene(µg/ml)VC(DMSO 0.2%)
0.16
0.31
0.63
1.25
2.5
5.0
10.0
PC

Scorablecolonies

RPE (%)

1565

100.0

+

1345
1438
1515
1428
1551
1407
1569
1470

86.1
92.1
95.9
91.4
97.6
89.9
99.3
64.2

+
+
+
+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

5

0.32

-

15
22
20
26
42
42
39
30

1.12
1.53
1.32
1.82
2.71
2.99
2.49
2.04

<0.0005**<0.0005**<0.0005**<0.0005**<0.0005**<0.0005**<0.0005**<0.0005**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size.
** = p < 0.01 (one-sided Fisher's exact test)

6.3.2.4 BioReliance
3-Methylcholanthrene was dissolved in DMSO. The concentrations were selected on the basis of thedose range suggested by the VMT and the DRF experiment, and the results evaluated by BioRelianceare shown in Table 21. The MTF values of the test chemical doses ranged from 1.29% to 2.24%. All
test chemical concentrations induced significant increases in MTF compared to the VC (p < 0.05).
Table 21: Transformation assay results from BioReliance, testing coded 3-methylcholanthrene
BioReliance
3-methylcholanthrene(µg/ml)VC(DMSO 0.2%)
1.0
2.5
5.0
7.5
10
PC

Scorablecolonies

RPE (%)

1047

100.0

+

1008
1009
1005
1025
1008
1132

96.4
96.7
96.3
98.8
96.6
108.3

+
+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

4

0.38

-

18
22
16
23
13
18

1.79
2.18
1.59
2.24
1.29
1.59

0.002**<0.0005**
0.004**<0.0005**
0.020*
0.004**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size.
* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)

6.3.2.5 Concurrent cytotoxicity (Relative Plating Efficiency)
Cytotoxicity of 3-methylcholanthrene was evaluated by RPE assessment in all laboratories (Figure 10).
3-Methylcholanthrene was not cytotoxic at the doses tested by Harlan CCR and BioReliance whereassome cytotoxicity was observed in the other two laboratories. University of Metz tested a wider dose
range and a clear dose-dependent cytotoxicity curve can be seen.
BASF reported that the shift in the RPE was most probably due to the seeding procedure employed.
This has been observed when several experiments were performed using a single cell preparation in
one day, and the cells employed in later experiments were maintained in the medium for extended
periods of time before seeding.

47

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

130
120
110
100

RPE [%]

90
80
70
60
50
40

BASF

30

Harlan CCR

20

BioReliance

10

Uni Metz

00.0001

0.001

0.01

0.1

1

10

100

3-Methylcholanthrene [µg/ml]

Figure 10: Relative plating efficiency (RPE) compared to vehicle control in all laboratories
testing coded 3-methylcholanthrene

6.3.2.6 Morphological transformation frequency
MTF results with 3-methylcholanthrene are shown in Figure 11. The concentrations tested by alllaboratories were in the range of 1-2 µg/ml. All laboratories showed 3-methylcholanthrene to induce astatistically significant increase in morphological transformation at most of if not all doses tested, witha clear dose-dependent response for Harlan CCR.
6

BASF
Harlan CCR

5

BioReliance
Uni Metz

MTF [%]

4
3

**

2

**
****

*

*

**

*

1

**

**

*

**

*

**
**
**
**

*
*

0
0.001

0.01

0.1

1

10

3-Methylcholanthrene [µg/ml]

Figure 11: Morphological Transformation Frequency (MTF) shown as a percentage oftransformed colonies compared to total colonies for all laboratories testing 3methylcholanthrene.
* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)48

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.3.2.7 Acceptance criteria and assessment
All acceptance criteria were met in all laboratories, except for University of Metz which failed to meetthe criteria on the number of scorable concentrations (four instead of five). University of Metz showed
clearly positive results (a statistically significant increase in MTF was observed at two valid
concentrations), although only four concentrations fulfilled all the acceptance criteria. Therefore the
VMT considered these results acceptable for the overall evaluation of the study.
3-methylcholanthrene was assessed to be positive by all laboratories (Table 22).
Table 22: Acceptance criteria and assessment of 3-methylcholanthrene results
3-Methylcholanthrene
Laboratory

Criteria
Uni Metz

BASF

Harlan CCR

BioRelianceyes(≥1099)yes(≥1001,735*)

yes(≥1345)yes(≥1005)

Average number of 25-45 colonies per dishor >45 colonies if positive resultor <25 colonies if negative result

yes(27.5-36.3)

yes(26.5-41.7)

yes(33.7-39.2)

yes(25.2-28.4)

Plating Efficiency of vehicle control >20%yes
(20.3%)

yes(36.9%

yes(52.2%

yes(21.8%)Morphological Transformation Frequency ofvehicle control <0.6%

yes(0.51%)

yes(0.25%)

yes(0.32%)

yes(0.38%)

Number of scorable concentrations ≥5

no
(4#)

yes
(6)

yes
(7)

yes
(5)

Fisher’s test p-value of positive control
<0.05

yes
(<0.0001)

yes(0.003)yes(<0.0005)

yes(0.004)

Fulfilment of all assay acceptancecriteria

NO

YES

YES

YES

+

+

+

≥1000 scorable colonies per treatmentgroup or <1000 if positive result

Assessment+##* 735 colonies for the positive control#
results for for 0.01, 0.1, and 2 µg/ml were not taken into consideration (not valid). See section 6.3.2.1 for details.
##considered acceptable for the overall study evaluation as there were 2 statistically significant positive concentrations.

6.3.3 Conclusion
Treatment with 3-methylcholanthrene produced a statistically significant increase in morphologicallytransformed colonies in all laboratories. As such, 3-methylcholanthrene was considered to be a positivetransforming agent in the SHE pH 7.0 CTA. These results agree with published data (Heidelberger etal., 1983).

6.4 O-toluidine HCl
6.4.1 Dose-Range finding test
Figure 12 shows the results of the DRF tests with o-toluidine HCl. Harlan CCR’s results showed little
cytotoxicity whereas the results from the other laboratories, having tested through a larger range,
showed complete dose-dependent cytotoxicity curves.

49

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

130
120
110
100

RPE [%]

90
80
70
60
50
30

BASF
Harlan CCR

20

BioReliance40

10

Uni Metz

0
10

100

1000

o-Toluidine HCl [µg/ml]

Figure 12: Relative plating efficiency compared to vehicle control in all laboratories testingcoded o-toluidine HCl, for Dose-Range Finding

6.4.2 Transformation assay
VCs gave transformation frequencies within the expected range (≤0.6%): 0.49% (University of Metz),
0.26% (BASF), 0.35% (Harlan CCR), and 0.30% (BioReliance). The PC chemical benzo(a)pyrene led
to the expected increase in morphologically transformed colonies: 3.02% (University of Metz), 1.43%(BASF), 2.29% (Harlan CCR) and 1.37% (BioReliance).

6.4.2.1 University of Metzo-Toluidine HCl was dissolved in DMSO. The concentrations were selected on the basis of thesuggested dose range by the VMT, from findings from solubility testing and the DRF test. The results
evaluated by University of Metz are shown in Table 23. The MTF values of the test chemical doses
ranged from 1.47% to 3.69%. All the test chemical concentrations induced statistically significant
increases in MTF compared to the VC (p < 0.01).
Table 23: Transformation assay results from University of Metz, testing coded o-toluidine HCl
Uni Metzo-toluidine HCl(µg/ml)VC(DMSO 0.2%)
20
40
80
120
160
300
PC

Scorablecolonies

RPE (%)

1416

100.0

+

1192
1272
1268
1155
1215
1180
1358

84.0
89.6
91.0
83.4
85.8
83.7
95.7

+
+
+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

7

0.49

-

44
47
20
17
26
35
41

3.69
3.69
1.58
1.47
2.14
2.97
3.02

<0.001**<0.001**0.004**
0.009**<0.001**<0.001**<0.001**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size.
** = p < 0.01 (one-sided Fisher's exact test)

50

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.4.2.2 BASF
o-Toluidine HCl was dissolved in DMSO. The concentrations were selected on the basis of thesuggested dose range by the VMT, from findings from solubility testing and the DRF test. The results
evaluated by BASF are show in Table 24. The MTF values of the test chemical doses ranged from
0.42% to 1.54%. All the test chemical concentrations dose-dependently induced statistically significant
increases in MTF compared to the VC (p < 0.01).
Results for 200 and 400 µg/ml were not taken into consideration since the average number of colonies
per dish was beyond the limit for a negative result (>45).
Table 24: Transformation assay results from BASF, testing coded o-toluidine HCl
BASF
o-toluidine HCl(µg/ml)Scorablecolonies

RPE (%)

1524

100.0

+

2123
2109
1879
1649
1752
1396

139.1
139.8
124.2
110.8
77.9
92.0

+
+
+
++
++
+

VC
(DMSO 0.2%)
100
200
400
600
800
1 000
PC

MTF (%)

Fisher's exact
test p-value(one-sided)

4

0.26

-

9
9
17
25
27
20

0.42
0.43
0.90
1.52
1.54
1.43

0.305
0.301
0.013*<0.001**<0.001**<0.001**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size, ++ = slightly reduced density/size.
* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)

6.4.2.3 Harlan CCR
o-Toluidine HCl was dissolved in DMSO. The concentrations were selected on the basis of thesuggested dose range by the VMT, from findings from solubility testing and the DRF test. The results
evaluated by Harlan CCR are shown in Table 25. The MTF values of the test chemical doses ranged
from 0.91% to 2.54%. All the test chemical concentrations induced statistically significant increases in
MTF compared to the VC (p < 0.01), with a dose-dependent effect.
Table 25: Transformation assay results from Harlan CCR, testing coded o-toluidine HClHarlan CCR
o-toluidine HCl(µg/ml)VC(DMSO 0.2%)
80.0
100.0
130.0
160.0
200.0
260.0
PC

Scorablecolonies

RPE (%)

1442

100.0

+

1323
1471
1425
1321
1338
1494
1487

91.8
102.0
97.6
91.8
91.6
103.6
103.5

+
+
+
+
+
+
+

MTF (%)

Fisher's exact
test p-value(one-sided)

5

0.35

-

12
14
19
23
31
38
34

0.91
0.95
1.33
1.74
2.32
2.54
2.29

<0.001**<0.001**<0.001**<0.001**<0.001**<0.001**<0.001**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size.
** = p < 0.01 (one-sided Fisher's exact test)

51

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.4.2.4 BioRelianceo-Toluidine HCl was dissolved in DMSO. The concentrations were selected on the basis of thesuggested dose range by the VMT, from findings from solubility testing and the DRF test. The results
evaluated by BioReliance are shown in Table 26. The MTF values of the test chemical doses ranged
from 0.47% to 1.15%. Three test chemical concentrations induced statistically significant increases in
MTF compared to the VC (p < 0.01).
Table 26: Transformation assay results from BioReliance, testing coded o-toluidine HCl
BioRelianceo-toluidine HCl(µg/ml)Scorablecolonies

RPE (%)

1330

100.0

+

1476
1651
1475
1588
1540
1388

111.7
124.1
111.0
119.4
116.8
104.4

+
+
+
+
++
+

VC
(DMSO 0.2%)
25
50
100
200
300
PC

MTF (%)

Fisher's exact
test p-value(one-sided)

4

0.30

-

7
12
17
17
15
19

0.47
0.73
1.15
1.07
0.97
1.37

0.336
0.090
0.007**
0.011*
0.021*
0.002**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size, ++ = slightly reduced density/size.
* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)

6.4.2.5 Concurrent cytotoxicity (Relative Plating Efficiency)

RPE [%]

The cytotoxicity curves for Harlan CCR, BioReliance and University of Metz show a similar pattern
within the same dose range. In contrast, the results from BASF show a higher, dose-dependent
cytotoxicity due to the fact that this laboratory tested at higher concentrations (Figure 13).
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
10

BASF
Harlan CCR
BioReliance
Uni Metz

100

1000

O-toluidine HCl [µg/ml]

Figure 13: Relative plating efficiency (RPE) for all laboratories, for o-toluidine HCl

52

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.4.2.6 Morphological transformation frequency
MTF results with o-toluidine HCl are shown in Figure 14. o-Toluidine HCl induced a statisticallysignificant increase in morphological transformation in all laboratories, with a clear dose-dependent
effect for Harlan CCR and BASF. University of Metz showed a statistically significant increase in
MTF already at very low concentration (20 µg/ml), while for BASF MTF was observed at a higher
concentration (600 µg/ml).

6

BASF
Harlan CCR

5

MTF [%]

4

BioReliance**

Uni Metz

**
**

3

**
2
1

**

**
**

**

**
** *

**
** **
*

*

** **
0
10

100

1000

O-toluidine HCl [µg/ml]

Figure 14: Morphological Transformation Frequency (MTF) shown as a percentage oftransformed colonies compared to total colonies, for all laboratories, for o-toluidine HCl* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)

6.4.2.7 Acceptance criteria and assessment
The acceptance criteria were fulfilled by all laboratories. o-Toluidine HCl was assessed to be positiveby all laboratories (Table 27).

53

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Table 27: Acceptance criteria and assessment of o-toluidine HCl resultso-Toluidine HCl
Laboratory

Criteria
Uni Metz

BASF

Harlan CCR

BioRelianceyes(≥1155)yes(≥1409)yes(≥1321)yes(≥1330)yes(29.9-35.7)

yes(35.2-47.6)

yes(33.1-37.4)

yes(33.3-41.3)

Plating Efficiency of vehicle control >20%yes
(21.0%)

yes(34.8%)

yes(55.6%)

yes(27.8%)Morphological Transformation Frequency ofvehicle control <0.6%

yes(0.49%)

yes(0.26%)

yes(0.35%)

yes(0.30%)

yes
(6)

no
(3#)

yes
(6)

yes
(5)

Fisher’s test p-value of positive control <0.05

yes(<0.001)

yes(<0.001)

yes(<0.001)

yes(0.002)

Fulfilment of all assay acceptance criteria

YES

NO##

YES

YES

+

+

+

+

Number of scorable colonies per treatmentgroup ≥1000
or <1000 if positive result
average of 25-45 colonies per dishor >45 colonies if positive resultor <25 colonies if negative result

Number of scorable concentrations ≥5

Assessment#

results for 200 and 400 µg/ml were not taken into consideration. See section 6.4.2.2 for details.
considered acceptable for the overall study evaluation as there were 3 statistically significant positive concentrations.

##6.4.3 Conclusion
Treatment with o-toluidine HCl produced a statistically significant increase in morphologicallytransformed colonies in all laboratories. As such, o-toluidine HCl was considered to be a positivetransforming agent in the SHE pH 7.0 CTA. These results agree with published data (Jones et al.,1988).

6.5 Phthalic anhydride
6.5.1 Dose-range finding test
Figure 15 shows the results of the DRF tests with phthalic anhydride. All laboratories produced very
similar cytotoxicity curves.

54

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

130
120
110
100

RPE [%]

90
80
70
60
50
40

BASF

30

Harlan CCR

20

BioReliance

10

Uni Metz

0
0.1

1

10

100

Phthalic anhydride [µg/ml]

Figure 15: Relative plating efficiency compared to vehicle control in all laboratories testingcoded phthalic anhydride, for Dose-Range Finding.

6.5.2 Transformation assay
VCs gave transformation frequencies within the expected range (≤0.6%): 0.56% (University of Metz),
0.26% (BASF), 0.44% (Harlan CCR), and 0.52% (BioReliance). The PC chemical benzo(a)pyrene led
to the expected increase in morphologically transformed colonies: 1.60% (University of Metz), 1.43%(BASF), 2.66% (Harlan CCR), and 1.41% (BioReliance). It can be seen from Figure 16 that phthalicanhydride was shown to be cytotoxic in three laboratories (BASF, Harlan CCR and BioReliance). It
did not induce statistically significant increase in morphological transformation in most of thelaboratories, except at BASF where a statistically significant increase in morphological transformation
was observed at a single concentration (120 µg/ml).

6.5.2.1 University of Metz
Phthalic anhydride was dissolved in DMSO. The concentrations were selected on the basis of the DRF
test and the chemical solubility and the results evaluated by University of Metz are shown in Table 28.
The MTF values of the test chemical doses ranged from 0.34% to 1.11%. None of the test chemical
concentrations induced a significant increase in MTF compared to the VC (p ≥ 0.05).
The two highest test chemical concentrations were not considered valid because the total number ofcolonies was below the limit (<1000) with a negative result.

55

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Table 28: Transformation assay results from University of Metz, testing coded phthalicanhydride
Uni Metz
Phthalic anhydride(µg/ml)Scorablecolonies

RPE (%)

1441

100.0

+

1455
1446
1479
1560
1327
883
631
1687

100.2
99.8
101.9
106.9
91.2
60.7
49.6
116.9

+
+
+
+
+
+
+
+

VC
(DMSO 0.2%)
0.2
1
5
25
125
300
560
PC

MTF (%)

Fisher's exact
test p-value(one-sided)

8

0.56

-

12
10
5
13
10
6
7
27

0.82
0.69
0.34
0.83
0.75
0.68
1.11
1.60

0.258
0.409
0.272
0.244
0.339
0.451
0.139
0.004**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size.
** = p < 0.01 (one-sided Fisher's exact test)

6.5.2.2 BASF
Phthalic anhydride was dissolved in DMSO. The concentrations were selected on the basis of the DRF
test and the chemical solubility and the results evaluated by BASF are shown in Table 29. The MTF
values of the test chemical doses ranged from 0.07% to 0.93%. Treatment with the highest test
chemical concentration induced a statistically significant increase in MTF compared to the VC (p <0.05) and the trend test was positive.
Table 29: Transformation assay results from BASF, testing coded phthalic anhydride
BASF
Phthalic anhydride(µg/ml)Scorablecolonies

RPE (%)

Colonydensity/size

Transformed
colonies

MTF (%)

Fisher's exact
test p-value(one-sided)

1524

100.0

+

4

0.26

-

1513
1412
1487
1417
1606
1396

98.8
94.2
65.0
62.2
52.6
92.0

+
+
+
+
++
+

2
1
7
3
15
20

0.13
0.07
0.47
0.21
0.93
1.43

0.889
0.962
0.260
0.743
0.013*< 0.0005**

VC(DMSO 0.2%)
40
60
80
100
120
PC

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size, ++ = slightly reduced density/size.
* = p < 0.05, ** = p < 0.01 (one-sided Fisher's exact test)

6.5.2.3 Harlan CCR
Phthalic anhydride was dissolved in DMSO. The concentrations were selected on the basis of the DRF
test and the chemical solubility and the results evaluated by Hralan CCR are shown in Table 30. The
MTF values of the test chemical doses ranged from 0.00% to 0.38%. None of the test chemical
concentrations induced a significant increase in MTF compared to the VC (p ≥ 0.05).

56

SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Table 30: Transformation assay results from Harlan CCR, testing coded phthalic anhydride
Harlan CCR
Phthalic anhydride(µg/ml)Scorablecolonies

RPE (%)

1368

100.0

+

1303
1321
1033
1024
1036
1430

95.4
96.6
73.7
64.9
56.2
104.8

+
++
++
+++
+++
+

VC
(DMSO 0.2%)
27.5
41.25
55.00
82.5
110.0
PC

MTF (%)

Fisher's exact
test p-value(one-sided)

6

0.44

-

3
5
0
1
0
38

0.23
0.38
0.00
0.10
0.00
2.66

>0.5
>0.5
>0.5
>0.5
>0.5
< 0.0005**

ColonyTransformed
density/sizecolonies

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size, ++ = slightly reduced density/size, +++ = highly reduced density/size.
** = p < 0.01 (one-sided Fisher's exact test)

6.5.2.4 BioReliancePhthalic anhydride was dissolved in DMSO. The concentrations were selected on the basis of the DRF
test and the chemical solubility and the results evaluated by BioReliance are shown in Table 31. The
MTF values of the test chemical doses ranged from 0.33% to 0.62%. None of the test chemical
concentrations induced a significant increase in MTF compared to the VC (p ≥ 0.05).
Table 31: Transformation assay results from BioReliance, testing coded phthalic anhydride
BioReliancePhthalic anhydride(µg/ml)VC(DMSO 0.2%)
2.5
5.0
10
25
50
100
PC

Scorablecolonies

RPE (%)

Colonydensity/size

Transformed
colonies

MTF (%)

Fisher's exact
test p-value(one-sided)

1347

100.0

+

7

0.52

-

1425
1406
1286
1255
1200
1197
1417

106.3
104.3
95.8
93.9
89.9
44.4
105.6

+
+
+
+
+
++
+

7
5
8
7
7
4
20

0.49
0.36
0.62
0.56
0.58
0.33
1.41

0.646
0.827
0.464
0.552
0.518
0.845
<0.013*

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size, ++ = slightly reduced density/size.
* = p < 0.05 (one-sided Fisher's exact test)

6.5.2.5 Concurrent cytotoxicity (Relative Plating Efficiency)
Cytotoxicity of phthalic anhydride was evaluated by RPE assessment in all laboratories (Figure 16).
Phthalic anhydride was shown to be dose-dependently cytotoxic in all laboratories, with the mostpronounced effects for BASF, Harlan CCR and BioReliance.

57

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

130
120
110
100
90

RPE [%]

80
70
60
50
40

BASF
Harlan CCR
BioReliance
Uni Metz

30
20
10
0
0

20

40

60

80

100

120

140

Phthalic anhydride [µg/ml]

Figure 16: Relative plating efficiency (RPE) compared to vehicle control in all laboratories
testing coded phthalic anhydride

6.5.2.6 Morphological transformation frequency
MTF results with phthalic anhydride are shown in Figure 17. The common range tested by alllaboratories was from 40 to 100 µg/ml. Phthalic anhydride did not induce a statistically significant
increase in morphological transformation in any laboratory, except for BASF, where a statisticallysignificant increase in morphological transformation was observed at the highest concentration only(120 µg/ml).

6

BASF
Harlan CCR
BioReliance
Uni Metz

5

MTF [%]

4
3
2

*

1
0
0

20

40

60

80

100

120

140

Phthalic anhydride [µg/ml]

Figure 17: Morphological Transformation Frequency (MTF) shown as a percentage oftransformed colonies compared to total colonies for all laboratories testing coded phthalicanhydride* = p < 0.05 (one-sided Fisher's exact test)

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.5.2.7 Acceptance criteria and assessment
The acceptance criteria were fulfilled by all laboratories (Table 32):
Table 32: Acceptance criteria and assessment of phthalic anhydride results
Phthalic anhydrideLaboratory

Criteria
Uni Metz

BASF

Harlan CCR

BioRelianceyes(≥1327)yes(≥1396)yes(≥1024)yes(≥1197)yes(33.4-42.7)

yes(28.6-40.3)

yes(25.2-35.9)

yes(29.9-35.8)

Plating Efficiency of vehicle control >20%

yes
(24.4%)

yes(34.8%)

yes(52.6%)

yes(28.1%)Morphological Transformation Frequency ofvehicle control <0.6%

yes(0.56%)

yes(0.26%)

yes(0.44%)

yes(0.52%)

yes
(5#)

yes
(5)

yes
(5)

yes
(6)

Fisher’s test p-value of positive control <0.05

yes(0.004)yes(<0.0005)

yes(<0.0005)

yes(0.013)Fulfilment of all assay acceptance criteria

YES

YES

YES

YES

-

+

-

-

Number of scorable colonies per treatmentgroup ≥1000
or <1000 if positive result
Average number of 25-45 colonies per dishor >45 colonies if positive resultor <25 colonies if negative result

Number of scorable concentrations ≥5

Assessment#

results for 300 and 560 µg/ml were not taken into consideration (not valid). See section 6.4.2.1 for details.

6.5.3 Conclusion
Treatment with phthalic anhydride did not produce a statistically significant increase inmorphologically transformed colonies in three of the laboratories at any dose tested. In one laboratory
a statistically significant increase in morphologically transformed colonies was induced at one
concentration only and the trend test was statistically significant, so this chemical was concluded to bepositive. As such, under the conditions of the test as performed, phthalic anhydride was considered to
be a non-transforming agent in the SHE pH 7.0 CTA in three of the four laboratories. These results
agree with published data (Elias et al., 1996).

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.6

Overview on vehicle and positive controls

Vehicle and positive control data for all experiments performed for the between-laboratory
reproducibility assessment are summarised in Figure 18. It can be seen that the responses are all within
the acceptable limits, i.e. below 0.6% for the VCs and above 1.0% for the PCs.

6
5

MTF [%]

4
3
2
1
0

DMSO [0.2%]

Benzo(a)pyrene

Figure 18: Morphological Transformation Frequency (MTF) of vehicle (■) and positive controls
for all cell transformation experiments, in all laboratories. Benzo(a)pyrene was used as positive
control at 1 µg/ml by University of Metz (▲) and at 5 µg/ml by the other laboratories (●).

6.7 Conclusion of the Validation Management Team on Module 4
The between-laboratory reproducibility was shown to be satisfactory. All chemicals tested as well as
vehicle and positive controls gave reproducible results in the four laboratories, but for phthalicanhydride results which were reproducible in three laboratories out of four.
Although it did not impact the overall reproducibility of the results, it can be noted that the cells from
University of Metz were in general slightly more sensitive to cytotoxicity and morphologicaltransformation than those from the other laboratories, inducing a statistically significant MTF at lower
doses. This could be related to the fact that University of Metz used a different batches of cells andserum than the other laboratories.
Furthermore, for uniformity in testing, all the laboratories were asked by the VMT to dissolve theirchemicals in DMSO. In some cases this was not followed and the chemical was dissolved intoaqueous medium (DMEM-L). In those cases, a different outcome in results was obtained. In DMSO,
the bioavailability of the chemicals towards DNA may have been increased. This emphasizes theimportance of vehicle choice in the CTA.

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010

7 Summary of results
An optimised and standardised protocol was initially produced and assessed by University of Metz
laboratory and agreed upon by all laboratories before the initiation of the prevalidation study. In
addition, training for the participating laboratories and availability 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 colony morphology and in scoring experimental results, especiallyin this study where three of the laboratories had no previous experience with this CTA variant. Using
the standardised protocol, all laboratories showed good reproducibility within as well as between
laboratories. Table 33 summarises the results obtained with each chemical.
Table 33: Summary table of between-laboratory reproducibility results
Laboratory
Expected
result#BASF

Harlan CCR

BioReliance

Uni Metz

Benzo(a)pyrene

+

+

+

+

+

Anthracene

-

-

-

-

-

2,4-diaminotoluene

+

+

+

+

+

3- Methylcholanthrene

+

+

+

+

+

o-Toluidine HCl

+

+

+

+

+

Phthalic anhydride

-

+

-

-

-

Chemical#Based on previous results from the literature (see sections 2.4.1.1 and 2.4.1.2)

7.1 Benzo(a)pyrene
The results for benzo(a)pyrene, tested as the PC, showed a good reproducibility within and between
laboratories and resulted to be within the acceptable limits requested for the PC in all studies. The
experiment using coded benzo(a)pyrene was repeated three times in University of Metz laboratory andperformed once by the other laboratories, showing both a good within- and between-laboratory
reproducibility.

7.2 Anthracene
The results for anthracene were reproducible between the laboratories and anthracene was shown to be
negative in the CTA, which was the expected result.

7.3 2,4-Diaminotoluene
The results for 2,4-diaminotoluene were reproducible between the laboratories and 2,4-diaminotoluene
was shown to be positive in the CTA, which was the expected result.

7.4 3-Methylcholanthrene
The results for 3-methylcholanthrene were reproducible between the laboratories and 3methylcholanthrene was shown to be positive in the CTA, which was the expected result.

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010

7.5 o-Toluidine HCl
The results for o-toluidine HCl were reproducible between the laboratories and o-toluidine HCl was
shown to be positive in the CTA, which was the expected result.

7.6 Phthalic anhydride
The results for phthalic anhydride were reproducible in three laboratories out of four. Theselaboratories showed that phthalic anhydride was negative in the CTA, which was the expected result,whereas BASF observed a significant increase in MTF at a single concentration only, with a positive
trend test.

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010

8 Overall conclusion by the Validation Management TeamThe aim of the study was to prevalidate the SHE CTA at pH 7.0, in a formal inter-laboratory study,
following the modular approach (Hartung et al., 2004) and concentrating on the modules 1-4: test
definition, within-laboratory reproducibility, transferability and between-laboratory reproducibility.
Table 34 summarises the conclusion by the VMT on the assessment of the SHE pH 7.0 CTA.
Table 34: Conclusions of the Validation Management Team for the different modules
ModuleSummary & Conclusion

Module 1

Test definition- Clear definitions of the scientific basis- description of the endpoint induced by genotoxic andnon-genotoxic mechanisms- improved protocol available

yes

Within-laboratory
reproducibility

The within-laboratory reproducibility was shown to besatisfactory in all laboratories for:- the vehicle control- the positive control- the test chemicals (tested in a single laboratory)

yes

Transferability- The test method is transferrable between laboratories- Basic cell culture experience is needed- Training in the conduct and scoring of the assay isimportant- Photo catalogue produced as a useful aid for scoring

yes

Between-laboratory
reproducibility

The between-laboratory reproducibility was shown to besatisfactory for:- the vehicle control- the positive control- the test chemicals

yes

Module 2

Module 3

Module 4

The VMT concluded that in keeping with the objectives of this CTA effort, the SHE pH 7.0 CTA had
been prevalidated in accordance with modules 1-4 (Hartung et al., 2004). It has been demonstrated thata standardised protocol is available that should be the basis for future use. This protocol and the assay
system itself are transferable between laboratories, and reproducible within- and between-laboratories.
This conclusion is substantiated by the existing body of knowledge related to this assay. In particular,by 1) the reproducibility evaluations of similar protocols as reported in the literature (Isfort et al.,1996c) and, 2) the overall evaluation of the SHE data contained in the OECD DRP, which reported
consistent results for 87.7% (57/65) of chemicals which had been tested in more than one laboratory(OECD, 2007). Moreover, the VMT concluded that with the appropriate training and the use of thephoto catalogue, colony scoring was not problematic despite the concerns raised in the past.
In addition, the data produced add to the understanding of the predictive capacity (module 5) of the
CTA, which was previously addressed by the OECD DRP evaluation (OECD, 2007).
The VMT supports the conclusions of the OECD DRP and the generation of an OECD SHE celltransformation test guideline.

63SHE pH 7.0 CTA PRP report v1 - 30 July 2010

9 Recommendations
Taking into account clarifications and minor modifications introduced by the VMT and theparticipating laboratories, the VMT agreed that a standardised, transferable and reproducible protocolhas been established. This protocol will be published by ECVAM and used in the future to produce
new data and an OECD test guideline. Based upon the experience gained in this effort, points that need
to be taken into consideration in the future conduct of the assay and generation of the OECD guideline
include the following:•Considering the limited differences between the protocols for the SHE CTA at pH 6.7 and pH
7.0, it is recommended that both CTAs be incorporated into a single protocol since they only
differ by the pH used to culture the cells and the morphology of the transformed colonies.

•

Since there is a certain degree of subjectivity associated with the identification of transformed
colonies in the SHE CTA and correct scoring of transformed colonies is critical, training isnecessary to ensure scoring which is as consistent and objective as possible. It needs to be
noted that the SHE protocol at pH 7.0 produces colonies with slightly different morphologies
from those produced with the protocol at pH 6.7. Nevertheless, the successful assessment of
the between-laboratory reproducibility demonstrated that if the laboratories are well trained,
the manual scoring of colonies and the potential subjectivity in identifying transformed
colonies are not problematic issues. It is therefore recommended that appropriate training anda photo catalogue for this specific pH protocol variant be made available to laboratories
conducting the SHE CTA. It is intended that such a photo catalogue will be published by
ECVAM in the near future for that purpose.

•

It is important to note that cells for this assay need to be isolated at pH 7.0.

•

For results that do not meet the criteria for a clear positive or a clear negative call(inconclusive results) the experiment should be repeated, as is normal practice in assays ingeneral.

•In case of at least 2 statistically significant concentrations, a positive call could be concluded
regardless of the number of scorable concentrations (i.e. those that fulfil the assay acceptancecriteria) as opposed to a specific requirement for a set number of concentrations (see also point
below).

•

Based upon the experience of the participating laboratories and the results obtained, the VMTrecommends that one of the acceptance criteria be revised as follows: "At least four valid test
chemical concentrations should be available to evaluate a negative call", instead of arequirement for five concentrations.

•

It is recommended that clarifications on terminology be made as required (for instance,negative control should be understood as untreated control) and will be taken into account in
the published protocol and for the drafting of the OECD guidelines.

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010

10 Current contact details of the people involved in theprevalidation 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
Director General
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@comcast.net- 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- Thomas Hartung (until April 2008)
Johns Hopkins University
Center for Alternatives to Animal Testing (CAAT)
615 N Wolfe Street, W7032
Baltimore, MD 21205, USA

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Tel +1.410.614.4990
Fax +1.410.614.2871
thartung@jhsph.edu- 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
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)
Paule Vasseur
University of Metz
Laboratoire Interactions Ecotoxicité Biodiversité Ecosystèmes (LIEBE) - CNRS UMR 7146
Campus Bridoux, Rue Général Delestraint
Metz 57070, France
Tel +33.(0)3.87.37.85.01
Fax +33.(0)3.87.37.85.12
vasseur@univ-metz.fr- Study Directors Laboratory 2
Susanne Bohnenberger & Albrecht PothHarlan 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
Kamala Pant
BioReliance Corporation
14920 Broschart Road
Rockville, MD 20850, USA
Tel +1.301.610.2192
Fax +1.301.738.2362
kamala.pant@bioreliance.com

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SHE pH 7.0 CTA PRP report v1 - 30 July 2010
- Study Directors Laboratory 4
Markus Schulz and Karl-Rainer Schwind
BASF Aktiengesellschaft
GV/TB
67056 Ludwigshafen, Germany
Tel +49.(0)6.216.056.108
Fax +49.(0)6.216.051.734
markus.schulz@basf.com
karl-rainer.schwind@basf.com

67SHE pH 7.0 CTA PRP report v1 - 30 July 2010

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Rivedal E. and Sanner T. Synergistic effects of morphological transformation of hamster embryo cells
initiated by nickel sulphate and benzo(a)pyrene. Cancer Lett., 17 (1980) 203-8.
SCCP, SCCP/0971/06: Updated Recommended Strategy for Testing Oxidative Hair Dye Substances
for Their Potential Mutagenicity/Genotoxicity, adopted by the SCCP during the 7th plenary meeting of
28 March 2006. http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_s_02.pdf
Tennant R.W., Stasiewicz S. and Spalding J. Comparative evaluation of three mammalian celltransformation assay systems. In Carcinogenesis, J.C. Barrett and R. Tennant, Eds (1985) 399-410.
Walsh M.J., Bruce S.W., Pant K., Carmichael P.L., Scott A.D. and Martin F.L. Discrimination of atransformation phenotype in Syrian golden hamster embryo (SHE) cells using ATR-FTIR
spectroscopy. Toxicology, 258 (2009) 33-8.
Zhang H., Kamendulis L.M., Jiang J., Xu Y. and Klaunig J.E. Acrylonitrile-induced morphologicaltransformation in Syrian hamster embryo cells. Carcinogenesis, 21 (2000) 727-33.
Zhang H., Borman H.C. and Myhr B.C. Enhancement of the morphological transformation of Syrian
hamster embryo (SHE) cells by reducing incubation time of the target cells. Mutat. Res., 548 (2004) 17.

71

SHE pH 7.0 CTA PRP report v1 - 30 July 201012 Annexes
12.1 Chemicals selected for the prevalidation of SHE pH 7.0 CTA
Chemicals were selected based on the genotoxicity and carcinogenicity data compiled by the OECD
DRP31 (2004; 2007) and Kirkland et al. (2005).The in vitro genotoxicity, in vivo genotoxicity andcarcinogenicity characterisation of the selected compounds is reported in Table 35.
Table 35: Genotoxicity and carcinogenicity data on the chemicals selected for the SHE pH 7.0
CTA prevalidation study

ChemicalCASnumber

Genotoxic profile
in vitro
Ames MLA MNTCAGenotoxic
in vivoIARC
in vivoclass carcinogenicity

Benzo(a)pyrene

50-32-8

+

+

+

+

+
(gene mutation,
MN)

1

+

Anthracene120-12-7

+/-

+

nd

-

i

3

-

2B

+

95-80-7

+

+

nd

+

+
(UDS, transgenic
mutant, comet)(MN)

3-Methylcholanthrene 56-49-5

+

+

+

+

i

nd

+*

o-Toluidine HCl

636-21-5

+/-

+/-

+/-

+/-

+/-

2A

+

Phthalic anhydride

85-44-9

-

+

nd

+

(gene mutation)

nd

- **

2,4-Diaminotoluene* source: Gold and Zeiger (1997); ** source: NTP database+: positive; -: negative; +/-: diverging results inside a database; nd: not determined; i: inconclusive result;
MLA: Mouse Lymphoma Assay; MNT: Micronucleus Test; CA: Chromosome Aberration; UDS: unscheduled DNA synthesis; MN:
micronucleus.

72

SHE pH 7.0 CTA PRP report v1 - 30 July 201012.2 SHE pH 7.0 Cell Transformation assay protocol

SOP

PREVALIDATION STUDY ON
SYRIAN HAMSTER EMBRYO (SHE) IN VITRO
CELL TRANSFORMATION ASSAY

SHE ASSAY pH 7.0

Written by LIEBE CNRS UMR 7146 - UPV Metz

73

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

1. CONTENTS
1.

CONTENTS

2

2.

INTRODUCTION

43.

PRINCIPLE

54.

MATERIALS AND METHODS

6

4.1.

Preparation of SHE cells

6

4.2.

Test chemical: provision and handling

6

4.3.

Culture media and reagents for testing

6

4.4.

Preparation and cryopreservation of SHE cell stocks

7

4.5.

Irradiation of SHE cells

8

4.6.

Equipment

95.

EXPERIMENTAL DESIGN

10

5.1.

Time schedule

10

5.2.

Preparation of test cultures

10

5.3.

Preliminary Cytotoxicity Assay / Dose range finding study

11

5.4.

Dose selection for the main experiment

11

5.5.

Controls

12

5.6.

Cell Transformation Assay

12

5.7.

Determination of Solubility, pH and Osmolality

12

6.

EVALUATION

13

6.1.

Morphological Cell Transformation

13

6.2.

Evaluation criteria

13

6.3.

Cytotoxicity

13

6.4.

Statistics

14

6.5.

Acceptance Criteria and historical controls

14

6.6.

Assessment criteria

14

7.

DOCUMENTATION AND DEVIATION

158.

REFERENCES

15

9.

ANNEX I

16

9.1.

Test Chemical Receipt Page

16

74

SHE pH 7.0 CTA PRP report v1 - 30 July 201010.

ANNEX II

17

10.1. Test Reporting Template for Chemical Dilution

17

11.

18

ANNEX III11.1. Test Reporting Template for Pre-validation of SHE assay

18

12.

19

ANNEX IV

12.1. Template for preparation of feeder cells

1912.2. Template for preparation of target cells

20

13.

21

ANNEX V13.1. Template for Test Substance Preparation

21

13.2. Template for Seeding and Treatment

22

13.3. Template for Treatment Conditions

23

14.

24

ANNEX VI

14.1. Template for Preliminary Cytotoxicity Assay24

14.2. Template for Morphological Transformation – Raw data

25

14.3. Template for Morphological Transformation – Table Results

26

75SHE pH 7.0 CTA PRP report v1 - 30 July 2010

2. INTRODUCTION
Syrian Hamster Embryo (SHE) cells have been used to study transformation in vitro since Berwald
and Sachs (1963) confirmed the key observation by Earle (1943) that morphological changes in cell
cultures were associated with oncogenicity of these cells in vivo. These authors demonstrated
oncogenicity of SHE cells which presented a transformed phenotype after exposure to chemical
carcinogens in vitro.
SHE cells are primary and normal diploid cells, which derive from mid-gestation embryos (13 - 13.5
days gestation). The cells are genetically stable, metabolically competent and have a finite life span.
The cell population isolated from embryos of pregnant Syrian Hamster comprises of multiple cell
types and cells at various stages in the differentiation process and hence provides a broad spectrum ofcellular targets for neoplastic transformation. SHE cells show a high proliferation rate, good plating
efficiency (20 - 40 %) and a low spontaneous transformation frequency. Exposure to carcinogenic
agents results in an increase in the percentage of morphologically transformed (MT) colonies
compared to controls. MT colonies are characterized by a random growth pattern, which expresses aloss of growth inhibition and cell-cell orientation at confluency.
The Syrian Hamster Embryo in vitro cell transformation assay provides a system to detect
carcinogenic chemicals that act via genotoxic and non-genotoxic mechanisms. The assay is proposed
as an alternative to long term rodent experiments for carcinogenicity.
Study of morphological transformation in vitro in SHE cells is in agreement with the OECD draft
proposal (In vitro Syrian Hamster Embryo (SHE) Cell Transformation Assay, 1996). In the celltransformation assay (CTA), SHE cells are used to estimate cytotoxicity and morphologicaltransformation. The endpoint of cytotoxicity is the formation of colonies (Plating efficiency, PE) andthe endpoint of the carcinogenic potential is the presence of morphologically transformed colonies.
The treatment time is 7 days and duration of the test is 9 days in total.
This protocol describes the procedure of the SHE assay carried out at pH 7.0 for phase I of theprevalidation study. The test should meet the acceptance criteria on reproducibility as defined by
ECVAM principles for test validity (Hartung et al., 2004).
The test reproducibility will be assessed using benzo[a]pyrene (B[a]P) which will be tested as a test
substance in three independent assays. B[a]P tested had been provided as chemical Y by ECVAM in
November 2005.Assays will be conducted on batch of SHE cells isolated in complete growth medium
at pH 7.0 in February 2006 and checked for their performances as cell reagents in CTA in May 2006.
Number of target cells is 150 cells/plate, number of feeder cells is 50 000 cells/plate.

76

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

3. PRINCIPLE
SHE cells are obtained from primary culture of individual Syrian hamster embryos at 13 days ofgestation. After enzyme tissue digestion, cells are collected and stored in liquid nitrogen. One part ofcryopreserved SHE cells will be used as feeder cells, the other part as target cells. These ‘feeder cells’
will be X-ray irradiated to be no longer capable of replication and seeded as nutrient base and support
for metabolic activity.
The principle of the test consists in seeding target cells at clonal density onto a feeder layer of X-ray irradiated cells. Twenty-four hours after seeding feeder cells, the target cells are seeded onto the feeder
layer at a density appropriate to obtain 25-45 colonies per plate (60 mm diameter) and treated 24 hours
later. After 7 days necessary for clonal expansion, cells are washed, fixed and stained with Giemsa.
Dishes are coded and colonies are scored for their morphological phenotype under stereomicroscope.
Cytotoxicity is evaluated by inhibition of cloning efficiency. The number of MT colonies reported tothe total number of scorable colonies is calculated for each concentration tested. A statistically higher
percentage of MT colonies at two concentration levels compared to control vehicle will conclude topositivity.
The test medium is the Dulbecco’s modified Eagle’s medium (DMEM) containing 1 g/l of glucose,
without phenol red, supplemented with fetal calf serum (15%) and pH of the test medium is 7.0.

The test performances of the protocol of SHE cell transformation assay at pH 7.0 will be assessed on
three independent assays, conducted using:B[a]P as test substance (chemical Y provided by ECVAM in November 2005)
SHE target cells isolated at pH 7.0 (batch T1) : 150 cells/plate, seeded on a feeder layer of
50 000 cells/plate
DMEM (low glucose, without phenol red, pH 7.0) added with
15% fetal bovine serum (Hyclone Perbio AQL 25 247)

77

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

4.

MATERIALS AND METHODS

4.1. Preparation of SHE cells
For this prevalidation study phase I, SHE cells are prepared from embryos as described in section 4.4. The
cell batch selected is No T1. SHE cells were isolated from one embryo in February 2006. The SHE cells ofbatch T1 were checked for their performances - spontaneous transformation rate, plating efficiency and its
ability to form morphologically transformed colonies after exposure to B[a]P (7 days at 1 µg/ml) - in May
2006.

4.2. Test chemical: provision and handling
Benzo[a]pyrene has been selected as the test substance for the assays/Phase 1. The test chemical is thecoded chemical Y provided by ECVAM in November 2005.
Appropriate routine safety procedures will be followed in handling the test chemicals, unless otherwise
specified. Test facility personnel has been instructed to treat B[a]P and all other coded test chemicals asvery hazardous and potentially carcinogenic and to dispose laboratory wastes as toxic wastes.

4.3. Culture media and reagents for testingCulture media and reagents

DMEM : Dulbecco’s Modified Eagle’s Medium containing 1 g/l of glucose, 4 mM of glutamineand 110 mg/l of sodium pyruvate, without phenol red (Invitrogen, Gibco 31600). Lyophilized.



FBS: Fetal Bovine Serum (Perbio, Hyclone SH3-0070. Lot AQL 25 247). FBS is inactivated at
56°C for 30 minutes.



PBS-CMF: calcium and magnesium-free phosphate buffer saline.

Trypsin 2.5% (×10) liquid (Invitrogen, Gibco No.15090-46)

Sodium bicarbonate NaHCO3 (Sigma, No S-5761).



Dissociation solution

Dispase (0.5 U) (Roche No. 165859) used at concentration 1.2 U/ml in PBS-CMF.
The dissociation solution for cell isolation is prepared and used freshly in each case.


CGM : Complete Growth Medium

The lyophilized DMEM medium (DMEM) is reconstituted with ultrapure water, then adjusted to pH 7.0
with 1.5 g/l of NaHCO3 and sterilized by membrane filtration (0.1 µm porosity). The culture medium isstored at 4° C during a period not exceeding 2 weeks. The complete culture medium is prepared withaddition of 15% FBS.
The pH of the culture medium is checked so as to be 7.0 after incubation at 37.0 ± 0.1°C with 10.0 ± 0.5%
CO2 in a humidified incubator for at least 4 hours.


Detachment solution

The cell detachment solution consists of calcium/magnesium-free phosphate buffer saline (PBS-CMF)
containing 0.25% trypsin .

78

SHE pH 7.0 CTA PRP report v1 - 30 July 2010 Cryopreservation Medium
DMEM pH 7.0, added with :- 10% FBS and- 10% DMSO (Sigma D-8779)
 Cell isolation medium (CIM)
The cell isolation medium is constituted with DMEM (1.5 g/l of NaHCO3) added with 15% FBS and 1%
antibiotics (penicillin 5000 U/ml ; streptomycin 5000 µg/ml)
 Wash solution for cell isolation
The wash solution is PBS-CMF with 1% antibiotics (5000 U/ml ; streptomycin 5000 µg/ml)
 Stain Solutions
Trypan Blue Stain (0.5% in PBS) (ICN Biomedicals, No. 195532).
Giemsa Stain (10% Giemsa in water) (Sigma, No. 48900).

4.4. Preparation and cryopreservation of SHE cell stocks
Sacrifice of hamster to obtain embryosSacrifice a pregnant hamster at 13 days of gestation by guillotine.
Swab ventral surface of hamster with betadin.
Incise and retract abdominal skin and open peritoneal cavity in sterile conditions.
Remove both uterine horns containing embryos and place them in sterile ø100 mm culture dishes
containing wash solution.

Dissociation of embryonic tissue













Remove embryos from uterus and transfer to sterile ø100 mm culture dishes containing wash
solution. After two washings in wash solution, embryos are transferred in culture dishes containing
cell isolation medium (CIM). The differentiated organs (eyes, heart and viscera) are removed fromeach embryo.
Cut tissue into thin pieces with sterile curved scissors.
Transfer minced tissue to erlenmeyers (one erlenmeyer for one embryo) containing a magnetic stirbar on a stir plate and rinse the minced tissue with wash solution at slow stirring speed to remove
as many blood cells as possible at 37.0 ± 0.1°C. Allow tissue to settle then pipette off and discard
wash solution.
Add dissociation solution to the flask and gently stir tissue for 5 minutes at 37.0 ± 0.1°C.
Allow tissue to settle and pipette off dissociation solution and discard.
Add dissociation solution to the flask and gently stir tissue for 10 minutes at 37.0 ± 0.1°C.
Put FBS to a series of sterile plastic centrifuge tubes.
After 10 minutes with dissociation solution, allow tissue to settle and carefully pipette off
supernatant (dissociation solution and cells) and after filter through the sterile gauze into the
prepared centrifuge tubes.
Add fresh dissociation solution to the tissue and gently stir for 10 minutes. Collect the solution andcells as describe above.

79

SHE pH 7.0 CTA PRP report v1 - 30 July 2010






Repeat dissociation 2-4 times.
Centrifuge the cell suspension at 250×g for 10 minutes at 4°C. Discard the supernatant, resuspend
it and pool cells in CIM.
Count the viable cells by Trypan Blue Dye exclusion and seed 2×106 viable cells/culture dish.
Place culture dishes in incubator (37.0 ± 0.1°C and 10.0 ± 0.5% CO2).
24 hours after seeding, cryopreserve cells when at 60-80% confluency.
If 60-80% confluency is not reached within 24h, remove growth medium and refeed culture disheswith CIM. Return culture dishes to incubation and collect cells when appropriate confluency isreached.

Cryopreservation of the cells








When cells are 60-80% confluent, remove growth medium from the culture dishes and rinse cell
layers with PBS. Detach cells with detachment solution.
Add CIM to culture dishes and pool cells in centrifuge tubes.
Collect cells by centrifugation at 250×g for 10 minutes.
Discard supernatant and add CGM.
Count the viable cells by Trypan Blue Dye exclusion. Centrifuge at 250×g for 10 minutes andresuspend the pellet with the cryopreservation medium at 1×106 cells/ml.
Distribute 1.5 ml of cell suspension into storage vials (1.5×106 cells/vials).
Step freeze cells by placing vials into ice for 30 minutes, -20°C for 4 hours and -80°C for onenight, prior to transfer and store under liquid nitrogen.

Checking of the SHE cells
Before use, each new cell batch is checked for:
 Spontaneous transformation rate Plating efficiency (= colony forming ability) Morphological transformation with the standard carcinogen B[a]P

4.5. Irradiation of SHE cells










Reconstitute cryopreserved SHE cells by gently thawing (37°C) frozen vials and transferring cells
into centrifuge tubes with fresh CGM. Centrifuge at 250×g for 10 minutes to eliminate DMSO.
Resuspend the pellet in 10 ml fresh CGM and transfer 1.5×106 cells in each ø100 mm culture
dishes.
Grow cells in incubator for 2-3 days to achieve 50-90% confluency.
On the day of irradiation, rinse each plate with 10 ml of PBS and cover cells briefly with 1 ml ofdetachment solution. Observe detachment with a phase microscope.
Immediately after detachment, resuspend cells in 460 ml CGM in a 175 cm² growth flask
on wet ice.
Expose cells to X-ray irradiation (5000 rads) so that they are still viable, yet no longer capable ofreplication.
Transfer cells to centrifuge tubes and centrifuge in a refrigerate unit to form a pellet. Remove
supernatant medium and resuspend pellet in 50 ml of CGM on wet ice.
Count the viable cells using the Trypan Blue dye exclusion test.
Distribute 1.5 ml of cells suspension into storage vials (5×106 cells/vials) on wet ice, then at 4°C
for 30 minutes, -20°C for 4 hours and -80°C for one night, prior to transfer and storage underliquid nitrogen.

80SHE pH 7.0 CTA PRP report v1 - 30 July 2010

4.6. Equipment-

Laminar flow hood (biohazard type and restricted to cell culture assays)
Cell culture incubators (37.0 ± 0.1°C; 10.0 ±0.5% CO2; 90 ± 5% humidity)
Low-speed centrifuge
Water bath (37°C)
Inverse phase microscope
Micropipettes
ComputerRefrigerator (4°C)
Freezers (-20°C)
Containers for storage in liquid nitrogen
Autoclave (for instruments and for bio-hazardous waste materials)
BalancepH meter
Osmometer
Cell counting system
General cell culture laboratory equipment (e.g. glassware, filtration systems, cell culture plasticware, etc...)X-ray machine (Philips SL25)

All equipment maintenance and calibration shall be routinely performed in GLP spirit.

81SHE pH 7.0 CTA PRP report v1 - 30 July 2010

5. EXPERIMENTAL DESIGN
5.1. Time schedule
Day 0
1
2
9
---------------------------------------------- //--------------------------------↑↑↑↓
FeederTargetTreatment
Fixingcells
cells
with test substance
Staining(2 ml)(2 ml)(4 ml)

5.2. Preparation of test cultures
5.2.1. Feeder cells
On day 0 (feeder cells day), the cryopreserved irradiated SHE cells are thawed (37°C) and transferred into acentrifuge tube with 10 ml of fresh complete growth medium. Centrifuge at 250×g for 10 minutes toeliminate DMSO and resuspend the pellet in 10 ml fresh CGM.
Count the viable cells using the Trypan Blue dye exclusion test and adjust the cell concentration to 25000
cells/ml in CGM and add 2 ml into each ø60 mm culture dish.
The culture dishes are incubated at 37.0 ± 0.1°C and 10.0 ± 0.5% CO2 in humidified air for 24 hours before
adding target cells.
For each test, at least 5 dishes filled with feeder cells only will be used concurrently to check the inability
to replicate and to form colonies. No colony must form in these dishes.
5.2.2. Target cells
Cryopreserved SHE cells are thawed and seeded for growth in culture flasks. After an incubation period of
24 hours, the target cells will be detached, counted with a hemocytometer and adjusted with the complete
growth medium to a concentration allowing us to obtain approximately 25 - 45 colonies/dish at the end of
the test. Two ml of the target cell suspension will be added into each culture dish containing 50000 feeder
cells. Dishes will be incubated in a humidified incubator (37.0 ± 0.1°C; 10.0 ± 0.5% CO2) for 24 hours
prior to treatment.
In case of cytotoxicity at certain dose groups, the target cell number is adjusted so as to obtain the required
number of colonies at the end of the test. The target cell number is determined on the basis of the results ofthe cytotoxicity assay.
5.2.3. Preparation of test solutions and treatment of cultures
The solutions of the test substance will be prepared 24 hours after the seeding of target cells, on the day oftreatment. Each dosing solution will be prepared at a concentration corresponding to 2x the final
concentration expected and 4 ml of each dosing solution will be transferred to culture dishes (ø60 mm)
already containing 4 ml of complete medium with feeder and target cells (final volume 8 ml). The cultures
will be incubated for 7 days in a humidified incubator (37.0 ± 0.1°C; 10.0 ± 0.5% CO2).
A series of solutions of the test substance (500x the final concentration) in the appropriate vehicle will be
prepared so as to obtain the final desired concentration x in the test medium, as follows:82

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

Test substancesolubilised in

Vehicle*Dosing solution(4 ml test medium)

Final test medium(8 ml)

Concentration of
the test substance

500x

2x

x

Concentration of
the vehicle

100%

0.4%

0.2%

*For non hydrosoluble test substances, concentrated solutions (500x) will be prepared in DMSO

5.3. Preliminary Cytotoxicity Assay / Dose range finding study
The maximum dose of the test substance will be determined taking into account the solubility and any
relevant cytotoxicity information available for the test substance. A range of at least 10 concentrations toachieve a wide toxicity range will be tested in parallel to the vehicle. Ten dishes will be seeded perconcentration tested. At this stage, the number of target cells seeded is the same in all dose groups. The
conditions of testing (test medium, incubation conditions and time) are the ones described for the main
experiment for cell transformation (see section 5.6).
The highest dose level tested for soluble test substance will be 5 mg/ml or 10 mM. Test substance solutions
will be prepared fresh on the day of treatment.
The cultures will be incubated for a period of 7 days to allow colony development. The relative cytotoxicity
of each treatment group will be measured by the reduction in plating efficiency and/or colony density andsize of the treated SHE cells compared to the vehicle control group.

5.4. Dose selection for the main experiment
The relative cytotoxicity of each treatment group will be assessed from the dose range-finding study. The
results will be evaluated by the study Director for selecting doses to be used in the SHE cell transformation
assay. Unless there is evidence of MT, definitive assay doses should include, if possible: A high dose causing an approximate 50% decrease in relative plating efficiency and/or ≥ 50%
reduction in relative colony density/size (by visual appearance).
 At least one dose which has no effect on plating efficiency.
 3 or 4 intermediary doses
If the test substance is essentially non-toxic, then at least five concentrations will be selected up to a
maximum of 5 mg/ml or 10 mM. For non-toxic and insoluble test substances, the highest dose level tested
will be within 2-times the visible solubility limit in complete medium. For toxic and insoluble test
substance, the highest dose level tested should cause an approximate 50% decrease in relative plating
efficiency or relative colony density, regardless of the number of insoluble dose levels.

5.5. Controls
5.5.1. Vehicle control
In case the test substance is not hydrosoluble, the vehicle control selected will be DMSO used at aconcentration that will not exceed 0.2%. The final concentration of DMSO will be the same in all vehicle
control and treated dishes: 0.2%.

83SHE pH 7.0 CTA PRP report v1 - 30 July 2010
5.5.2. Positive control
Benzo[a]pyrene (B[a]P) will be tested at a concentration of 1.0 µg/ml to demonstrate the sensitivity of theassay. B[a]P will be dissolved in DMSO.

5.6. Cell Transformation Assay
A sufficient number of target cells (around 150 cells/dish) to produce an average of 25 - 45 colonies will bedispensed in 2 ml of complete medium per ø60 mm culture dish, each of which was seeded approximately
24 hours earlier with 5×104 feeder cells in 2 ml of complete medium. In case of cytotoxicity, the cellnumber should be adjusted: e.g. a reduction of approximately 30% needs a 1.5x adjustment; a reduction of
approximately 50% needs a 2x adjustment.
The transformation assay may be a single trial that results in at least 1000 colonies per treatment group(total of 40 dishes) to establish a negative result. The assay should include at least 5 scorable concentrationsof the test compound and the appropriate vehicle and positive control1.
The dose cell cultures will be incubated for a period of 7 days in a humidified incubator (37.0 ± 0.1°C;
10.0 ± 0.5% CO2) following treatment initiation. The culture dishes will be labelled with a code/assay
number, trial number and dose level.
After the incubation period of 7 days, the medium is aspirated off and the dishes are rinsed with 3-4 ml
PBS-CMF. After removal of PBS, cells are covered with 5 ml of ethanol for 10 minutes. Ethanol is thenremoved and plates stained for 20 minutes with 3-5 ml of Giemsa stain (10% in pure water). Stain is poured
off and plates rinsed under tap water before cells are air-dried.

5.7. Determination of Solubility, pH and Osmolality
The solubility of the test article in the vehicle and in the test culture (medium) will be observed anddocumented.
The pH of the test article dosing solutions will be checked after at least four hours of undisturbed
incubation in humidified atmosphere at 37.0 ± 0.1°C and 10.0 ± 0.5 % CO2. Prior to performing thepreliminary cytotoxicity assay, the test article will be dissolved in an appropriate solvent and diluted incomplete medium at a concentration equal or greater than the highest concentration to be tested. The pH
will be determined using a standard pH meter both at the time of preparation and after at least 4 hours of
incubation.
The osmolality will be determined using a suitable osmometer and may be measured prior to or at the timeof performing the preliminary cytotoxicity determination or the main experiment.

1

In this Phase I study, as the test substance is B[a]P (compound Y) no other positive control will be performed.

84

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6. EVALUATION
6.1. Morphological Cell Transformation
The stained colonies are blindly scored under stereomicroscope for plating efficiency and morphologicaltransformation.
Morphologically-transformed colonies are characterized by a multi-layered, criss-cross pattern of growththroughout the colony and piling up of cells.
Sparse colonies are not scored for MT, however included in the total number of colonies for plating
efficiency determination. If the colony contains less than 50 cells, it is not counted and recorded. Colonies
at the edge of the plates can be scored for MT if clearly morphologically transformed.
Generally, for each test group ≥ 1000 colonies will be evaluated for morphological cell transformation.
A differentiation will be made between normal (non-transformed) colonies and transformed colonies.

6.2. Evaluation criteria
Morphologically transformed colonies contain cells in an extensive, random-oriented, three-dimensional
growth pattern with cell stacking and criss-crossing both at the colony centre and on the perimeter.
Individual cells within the colony are more basophilic relative to their normal counterparts and have adecreased cytoplasm-to-nucleus ratio.
The morphological transformation frequency (MTF) will be calculated for each test group as follows:
MTF (%) =number of transformed colonies
total number of scorable colonies

x 100

6.3. Cytotoxicity
6.3.1. Relative Plating Efficiency (RPE) Relative plating efficiency
The average number of colonies per dish, the plating efficiency and the relative plating efficiency (RPE)
will be determined for each test group.
The plating efficiency and the relative plating efficiency will be calculated as follows:
PE (%) =total number of colonies per dishtotal number of target cells seeded per dishx 100

The relative plating efficiency :RPE (%) =PE of dose group
PE of the vehicle control groupx 100

 Colony size and density
In addition to the RPE, the colony size and density will be recorded as parameters of cytotoxicity.
The size and density is observed visible and given in three categories:- Normal (+)- Slightly reduced (++ ; 20 - 30 % reduction )- Greatly reduced (+++ ; 40 - 60 % reduction)

85

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

6.4. Statistics
The data of one or several trials are pooled for each treatment group. Results are analysed using the onesided Fisher’s exact test (Armitage, 1955) to determine if an increase in morphological transformation
occurred compared to vehicle control.
The Cochran-Armitage trend test for a positive dose-related response is performed when only one chemical
concentration shows a statistically significant response.

6.5. Acceptance Criteria and historical controls











1000 colonies per treatment group should be available for morphological transformation (less than
1000 colonies is acceptable in case of significant increase in morphological transformation rate).
However the average number of colonies per plate should not be less than 25.
An average of 25-45 colonies per dish for each treatment group (a colony number beyond theselimits is acceptable in the case of negative results with < 25 colonies or positive results with > 45
colonies per dish).
Cloning efficiency of the negative/vehicle control should be ≥ 20%.
No colony formation should be observed in the feeder cell control dishes. Feeder cells must be
visible in the chemical treatment groups except if they are affected selectively by the compound.
Transformation frequency in the negative controls (untreated and vehicle) within historicalcontrols.
Increase in morphological transformation frequency of the positive control (this criterion can be
discussed and should not be required in case of significant positivity of the test substance).
The positive control substance must lead to a statistically increase of morphological celltransformation.
There should be at least 5 scorable concentrations.

Historical controls are : 0.28 ± 0.22 with negative control and 0.36 ± 0.31 with DMSO,
 1.93 ± 0.78 with 1µg/ml B[a]P

6.6. Assessment criteria
A test chemical is declared positive if it causes a significant increase in morphological transformation
frequency either in two successive concentrations compared to the vehicle control or if one concentrationshows a statistically significant increase and the trend test is significant.
Otherwise, a test chemical is declared negative, i.e. no significant increase in morphological transformationor only at one dose without a significant trend test.

7. DOCUMENTATION AND DEVIATION
The prevalidation study will be conducted in the spirit of GLP.
All the data will be recorded in the study workbook and results registered in labelled files which will be
provided in the draft report.
The culture dishes of all the experiments will be archived until the end of the prevalidation study.

86SHE pH 7.0 CTA PRP report v1 - 30 July 2010
Deviations from the Statement of work or these SOP will be documented in the study workbook andcountersigned by the study director. They will be indicated in the draft report.

8. REFERENCES
Armitage P. (1955) Test for linear trend in proportions and frequencies. Biometrics 11, 375-386.
Berwald Y. and Sachs L. (1963) In vitro cell transformation with chemical carcinogens. Nature, 200, 11821184.
Earle W.R. (1943) Production of malignancy “ in vitro ”. IV. The mouse fibroblast cultures and changes
seen in the living cells. JNCI, 4, 165-212.
Hartung T., Bremer S., Casati S., Coecke S., Corvi R., Fortaner S.; Gribaldo L., Halder M., Hoffmann S.;
Roi A.J., Prieto P.; Sabbioni E., Scott L., Worth A., Zuang V. (2004) A modular approach to the ECVAM
principles of test validity. ATLA, 32, 467-472

87

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

9. ANNEX I
9.1. Test Chemical Receipt Page
Test Facility
Test Chemical
Identification
NumberTest Chemical
PhysicalDescription

Storage
Conditions

Test
ChemicalReceipt
Date

Test
ChemicalReceived By

Comments

88

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

10. ANNEX II
10.1. Test Reporting Template for Chemical Dilution

Testing Facility___________________
Test Chemical Code__________
Physical Description_____________________________Liquid Density_________
Date______________
Solvent

Amount of
Test
Chemical

Total
VolumeFinal Concentration

Comments

RoutineCultureMediumDMSO

89

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

11. ANNEX III
11.1. Test Reporting Template for Pre-validation of SHE assay

TEST CHEMICAL
Test Facility

Technician responsible

Chemical CodeExperiment ID

PREPARATION OF TEST CHEMICAL
Solvent_____Culture Medium

Positive Control

Vehicle Control_____DMSO(0.2%)

solvent

CELL ISOLATE
Cell batch

Supplier

CELL CULTURE CONDITIONSName of Medium

Supplier/ID

Lot No./Lab I.D.

Name of Serum

Supplier/ID

Lot No.

Serum Concentration

During Growth:

During Exposure:

TIMELINE

90

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

12. ANNEX IV
12.1. Template for preparation of feeder cells
FEEDER CELLS
Project ID.: RING TEST CTA

Experiment No.:

Cell count( mL cell susp. +count 1

mL trypan blue solution. 0.5%; Mallassez chamber, 1 big square)count 2

Seeding in petridishes 60mm (mean(x 4000) = cells / mL

cells / 2 mL =/ mL)Preparation of cell suspension for seeding
x-irrad. cell suspension [mL]in [mL] complete medium

91SHE pH 7.0 CTA PRP report v1 - 30 July 201012.2. Template for preparation of target cells
TARGET CELLS
Project ID.:

Experiment No.:Count of cell suspension A :(0.1 mL cell susp. + 0.1 mL trypan blue solution. 0.5%; Mallassez chamber, 1 big square)count 1 count 2

mean(x 8000) = cells /mL

Dilution of the cell suspension A := cell suspension B

Preparation of cell suspension for seeding
Nb of cells
Preparation/dish = /mL Cell suspension B (mL) Medium (mL)

Nb pools

Seeding1
Seeding2
Seeding
3

92

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

13. ANNEX V
13.1. Template for Test Substance Preparation
Project ID.:

Experiment No.:

Test substance mixed before removal
Vehicle:Dose[µg/mL cult.]

DMSO
weighted sample / filled with vehicle[mg]

Test substance solution mixed with:[mL]

shaker ; ultrasonicfor about min.

Dose[µg/mL cult.]

solubility; by pipetting;

Dilution
[mL]

from
dose

+ vehicle[mL]

solubility

Solubility: s = solution, e = emulsion; su = suspension

93SHE pH 7.0 CTA PRP report v1 - 30 July 2010

13.2. Template for Seeding and Treatment
TIME SCHEDULE, SEEDING AND TREATMENT
Project ID.:

Experiment No.:(PT = pretest; MT = maintest)

SeedingDose[µg/mL cult.]

numberofdishesfeedercells
2 mL

Test substancetarget cells
numberApplication2 mL

4 mL

wash andrefeed8 mLFixinganddying[time]

94

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

13.3. Template for Treatment Conditions
TREATMENT CONDITIONS
Project ID.:
Experiment No.:(PT = pretest; MT = maintest)

Dose[µg/mL
culture]pHvalueosmolarity

solubility in culture
at treatment[mOsm]

after about 4 h

begin

end

Vehicle control

Date / Signs = soluble; p = precipitation

95

SHE pH 7.0 CTA PRP report v1 - 30 July 2010

14. ANNEX VI
14.1. Template for Preliminary Cytotoxicity Assay
Project ID.:
Experiment No.:
Test groups

Colonies
per dish

Plating efficiency %

Doses

Mean

Absolute

Density / sizeof colonies

Feeder cells

Relative

Vehicle control
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
P
x
a
+
++
+++

Precipitation
Feeder cells present
Feeder cells absent
Cell density / size normal
Cell density / size slightly reduced
Cell density / size greatly reduced

96SHE pH 7.0 CTA PRP report v1 - 30 July 2010

14.2. Template for Morphological Transformation – Raw data
Project-ID
Experiment-No.

B[a]P 5.0

Vehiclecontrolµg/mL

Feeder control

Test
goup
TCS
D/S
FC

Test itemMT

Col MT NSC Col MT NSC Col MT NSC Col MT NSC Col MT NSC Col MT NSC Col MT NSC Col MT NSC Col MT NSC Col
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Date/sign:
Legend:
TCS
Col
MT
NSC
FC
x
-

Target cells seeded
Colonies (all)Transformed colonies
Non-scorable colonies
Feeder cells
Feeder cells present
Feeder cells not present

D/S
+
++
+ ++
c
B[a]P

Colony density / size
D/S normal
D/S slightly reduced
D/S greatly reduced
ContaminatedNot done / present
Benzo[a]pyrene

97SHE pH 7.0 CTA PRP report v1 - 30 July 2010

14.3. Template for Morphological Transformation – Table Results
Project ID.:
Experiment No.:
Test groups

TCS

Colonies
Total

Average

PE %
abs.
± SD

Transformed Colonies
RPE
%

d/s

Total

MTF
%

p value

Vehicle control
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
B[a]P 5.0

P
B[a]P
TCS
SD
RPE
PE
MTF
d/s
+
++
+++
*
**

µg/mL

Not evaluated
Precipitation
Positive control benzo[a]pyrene
Target cells seededStandard deviation
Relative plating efficiency
Plating efficiency
Morphological transformation frequency
Density / size of colony
Cell density / size normal
Cell density / size slightly reduced
Cell density / size greatly reduced
p < 0.05
p < 0.01

98

SHE pH 7.0 CTA PRP report v1 - 30 July 201012.3 Repeated experiment: BASF 2,4-diaminotoluene
In the first experiment by BASF (TA1), 2,4 diaminotoluene was dissolved in DMSO. Table 36 shows
the test concentrations which were evaluated based on solubility and cytotoxicity tests. The MTF
values of the test chemical treated doses ranged from 0.15% to 1.08%. None of the test chemical
concentrations had a significant increase in MTF compared to the VC (p ≥ 0.05).
Only four scorable concentrations were available. In addition, based on the relatively weak positive
response obtained with the PC and on the fact that results for one test chemical concentration (10µg/ml) were in the same range as for the PC, the VMT considered this experiment worth repeating andasked BASF to repeat the experiment (see TA2, section 6.2.2.2).
Table 36: Transformation assay TA1 results from BASF, testing coded 2,4-diaminotoluene
BASF
2,4-diaminotoluene(µg/ml)TA1
VC (DMSO 0.2%)
5
10
20
40
60
80
100
PC

Scorablecolonies

RPE (%)

1121
1228
1295
1139
1120
670
1287

100.0
109.8
112.9
99.3
65.1
41.0
112.2

ColonyTransformed
density/sizecolonies
+
+
+
+
++
++
+++
+++
+

5
9
14
6
9
1
15

MTF (%)

Fisher's exact
test p-value(one-sided)

0.45
0.73
1.08
0.53
0.80
0.15
1.17

0.265
0.061
0.511
0.211
0.940
0.041*

VC = Vehicle Control, PC = Positive Control, RPE = Relative Plating Efficiency, MTF = Morphological Transformation
Frequency, + = normal density/size, ++ = slightly reduced density/size, +++= highly reduced density/size.
* = p < 0.05 (one-sided Fisher's exact test)99