carcinogenecity tests

INTRODUCTION:

The objectives of carcinogenicity studies are to identify a tumorigenic potential in animalsand to assess

the relevant risk in humans, Any cause for concern derived from laboratoryinvestigations, animal

toxicology studies, and data in humans may lead to a need forcarcinogenicity studies. The practice of

requiring carcinogenicity studies in rodents wasinstituted for pharmaceuticals that were expected to be

administered regularly over asubstantial part of a patient's lifetime. The design and interpretation of the

results from thesestudies preceded much of the available current technology to test for genotoxic

potential andthe more recent advances in technologies to assess systemic exposure. These studies

alsopreceded our current understanding of tumorigenesis with non-genotoxic agents. Results

fromgenotoxicity studies, toxicokinetics, and mechanistic studies can now be routinely applied

inpreclinical safety assessment. These additional data are important not only in consideringwhether to

perform carcinogenicity studies but for interpreting study outcomes with respect torelevance for human

safety. Since carcinogenicity studies are time consuming and resourceintensive they should only be

performed when human exposure warrants the need forinformation from life-time studies in animals in

order to assess carcinogenic potential.

HISTORICAL BACKGROUND

In Japan, according to the 1990 "Guidelines for Toxicity Studies of Drugs Manual,"carcinogenicity

studies were needed if the clinical use was expected to be continuously for 6months or longer.If there

was cause for concern, pharmaceuticals generally usedcontinuously for less than 6 months may have

needed carcinogenicity studies. In the UnitedStates, most pharmaceuticals were tested in animals for

their carcinogenic potential beforewidespread use in humans. According to the U.S. Food and Drug

Administration,pharmaceuticals generally used for 3 months or more required carcinogenicity studies.

InEurope, the Rules Governing Medicinal Products in the European Community defined

thecircumstances when carcinogenicity studies were required. These circumstances

includedadministration over a substantial period of life, i.e., continuously during a minimum period of6

months or frequently in an intermittent manner so that the total exposure was similar.

FACTORS TO CONSIDER FOR CARCINOGENICITY TESTING

Duration and Exposure

Carcinogenicity studies should be performed for any pharmaceutical whose expected clinical

use is continuous for at least 6 months. Certain classes of compounds may not be used continuously

over a minimum of 6 months butmay be expected to be used repeatedly in an intermittent manner. It is

1

difficult to determineand to justify scientifically what time represents a clinically relevant treatment

periods forfrequent use with regard to carcinogenic potential, especially for discontinuous treatment

periods. For pharmaceuticals used frequently in an intermittent manner in the treatment ofchronic or

recurrent conditions, carcinogenicity studies are generally needed. Examples ofsuch conditions include

allergic rhinitis, depression, and anxiety. Carcinogenicity studies mayalso need to be considered for

certain delivery systems which may result in prolongedexposures. Pharmaceuticals administered

infrequently or for short duration of exposure (e.g.,anaesthetics and radiolabeled imaging agents) do

not need carcinogenicity studies unless thereis cause for concern.

Cause for Concern

Carcinogenicity studies may be recommended for some pharmaceuticals if there is concern

about their carcinogenic potential. Criteria for defining these cases should be very carefully

considered because this is the most important reason to conduct carcinogenicity studies for

most categories of pharmaceuticals. Several factors which could be considered may include:

(1) previous demonstration of carcinogenic potential in the product class that is considered

relevant to humans;

(2) structure-activity relationship suggesting carcinogenic risk

(3)evidence of preneoplastic lesions in repeated dose toxicity studies; and

(4) long-term tissueretention of parent compound or metabolite(s) resulting in local tissue reactions or

otherpathophysiological responses.

In vitro methods

1. Classical genotoxicity tests

Short description, scientific relevance and purpose

Originally, in vitro genotoxicity tests are used to predict the intrinsic potential of substances toinduce

mutations. The rationale behind using genotoxicity tests for identifying potential carcinogens is that

mutations and/or chromosomal aberrations are strongly associated with thecarcinogenesis process. For

this task only in vitro genotoxicity test which measure a mutationendpoint (gene or chromosomal

mutation) are qualified for this task: the gene mutation test inbacteria (OECD 471), the gene mutation

test in mammalian cells (OECD 487), the chromosomeaberration test (OECD 473) and the in vitro

micronucleus test (OECD 476).The tests rely on the fixation of initial DNA damage (DNA adducts or

chromosomal damage) ordamage to the cellular apparatus like the spindle figure into stable irreversible

2

DNA modificationsor changes in chromosome number. These modifications may again result in the

induction ofdiseases like cancer or genetic inheritable diseases. The tests are used to predict the

potential ofchemical substances to induce the former diseases.

Known users

Academics for mechanistic studies, all industries for screening purpose but also for

regulatoryapplication.

Status of validation and/or standardization

With the exception of the in vitro micronucleus test (Corviet al., 2006) none of the genotoxicitytests are

formally validated but nonetheless established, scientifically accepted and used tests. Forall the tests

suggested OECD guidelines exist.

Fields of application and limitations

The problem with in vitro genotoxicity tests, particularly for the tests measuring

chromosomeaberrations, is the high number of misleading positives, i.e. positive test results for known

noncarcinogens, as was discussed before. Improvement of existing in vitro standard genotoxicity testsis

under investigation. Preliminary data generated in a project sponsored by ECVAM andpredominantly

the cosmetic industry show that misleading positive results can be reduced if:

1)p53-competent cell s (e.g. human lymphocytes, TK6) instead of p53-compromised rodent cells

(Fowler et al., in press);

2) cytotoxicity measures based on proliferation during treatment instead ofmeasures based simply on

cell count (Kirkland and Fowler, in preparation);

3) The top testsubstance concentration is reduce from 10mM to 1 mM (Parry et al., in press; Kirkland

and Fowler,in prep). These modifications are in line with the OECD TG, except for the reduction of the

topconcentration which would need revision of the TGs for in vitro genotoxicity testing.It remains

unclear how carcinogenic potency and acceptable human exposure levels will beestimated if a

compound is found to be positive in in vitro genotoxicity tests and no animal tests areallowed as in the

case of the recent ban of animal testing in the cosmetics industry. However,Kirkland demonstrated in a

recent analysis of databases for over 950 compounds that when usingdata from only two in vitro

genotoxicity tests all of the relevant in vivo carcinogens and in vivogenotoxins for which data exist

were detected by the 2-test battery, i.e. the sensitivities of the 2- and3-test batteries are comparable

(Kirkland et al., in prep).

Ongoing developments

3

The role of genotoxicity testing can be both qualitative (hazard assessment) and quantitative

(riskassessment). A preliminary investigation on the applicability of in vivo genotoxicity tests

toestimate cancer potency looked promising. (see also the paragraph on in vivo genotoxicity test

andHernandez et al. 2010, in prep). For a quantitative approach of in vitro genotoxicity tests,

aforeseeable problem is the metrics comparison of the correlation, particularly how the dose of invitro

studies (in mM) translates to a dose in in vivo tests (mg/kg bw/day). For this reason, doseresponse

analysis of both in vitro and in vivo genotoxicity endpoints and carcinogenicity isessential.

Unfortunately, dose-response analyses using sophisticated dose-response software such asPROAST

(RIVM) or the BMDS (USEPA) have never been performed with in vitro genotoxicitytests. Given the

promising results obtained between in vivo genotoxicity and carcinogenicity, it isworthwhile applying

a similar approach to investigate whether in vitro genotoxicity tests arecorrelated to carcinogenic

potency.

2. In vitro Micronucleus test in 3D human reconstructed skin models (RSMN)


The micronucleus test in 3D human reconstructed skin models (RSMN) offers the potential for amore

physiologically relevant approach to test dermal exposure and also including a more relevantexogenous

human metabolizing system. It has been anticipated that these features of thereconstituted skin models

could improve the predictive value of a genotoxicity assessment comparedwith that of existing in vitro

tests and therefore could be used as a follow-up test in case of positiveresults from the standard in vitro

genotoxicity testing battery (Mauriciet al., 2005). Several 3D skinmodels are commercially available

and are suitable for conducting such test, provided that sufficientcell proliferation is available.

Status of validation and/or standardisation

A RSMN protocol using the EpiDermTM (MatTec Corporation, Ashland, MA, USA) model hasbeen

developed and evaluated with a variety of chemicals across three laboratories in the UnitedStates

(Currenet al., 2006; Munet al., 2009; Hu et al. 2009). A multi-laboratory prevalidationstudy was

initiated in 2007 and is sponsored and coordinated by COLIPA. This study aims atestablishing the

reliability of the method and at increasing the domain of chemicals tested forpredictive capacity

(Aardemaet al., 2010). Preliminary results suggest that the RSMN inEpiDermTM is a valuable in vitro

method of dermally applied chemicals.


The test is aimed for use at chemicals for which there is dermal exposure. The RSMN test must be seen

as an addition to the standard battery of in vitro genotoxicity tests. It will be important todemonstrate if

4

these tests have an equivalent sensitivity and a better specificity of the standard invitro micronucleus

test.

On-going development

Research on the metabolic capacity of the test (Hu et al., 2010) and investigation of the utility ofmore

complex models, such as full-thickness skin models, are ongoing.

3. In vitro Comet assay in 3D human reconstructed skin models


The Comet assay in 3D human reconstructed skin models is considered to be more relevant toevaluate

the genotoxic potential of chemicals than when performed in cell cultures, becausegenotoxic effects

can be evaluated under physiological conditions, especially regarding metabolicproperties (Hu et al.,

2010), and therefore be closer to the human situation than animal testing. Thisassay is a rapid and

sensitive method to evaluate primary DNA damage and it could be used as afollow up test for

chemicals that cause gene mutation in the in vitro standard tests (Mauriciet al.,2005). Several 3D skin

models are commercially available and are suitable for conducting suchassay.


Similarly to the RSMN test, a protocol using the EpiDermTM model has been developed for theComet

assay in 3D human reconstructed skin models and is being optimised and evaluated acrossthree

laboratories in the United States and Europe. This study which aims at establishing thereliability of the

method and at increasing the domain of chemicals tested for predictive capacitywas initiated in 2007

and is sponsored and coordinated by COLIPA.


The test is aimed for use at chemicals for which there is dermal exposure. It must be seen as anaddition

to the standard battery of in vitro genotoxicity tests. Being the endpoint measures verysensitive to DNA

damage, it is crucial that the quality of the tissues and good shipping conditionsare ensured.


Research on the metabolic capacity of the assay (Hu et al., 2010) and investigation of the utility ofmore

complex models, such as full-thickness skin models, are ongoing. Moreover, application ofthe comet

assay to freshly obtained human skin tissue that is generally obtained following cosmeticsurgery is

under investigation.

5

4. GreenScreen HC assay


The GreenScreen HC (Gentronix Ltd, Manchester, UK) is a commercially available assay

forgenotoxicity testing, using human lymphoblastoid TK6 cells transfected with the GADD45a(Growth

Arrest and DNA Damage) gene linked to a Green Fluorescent Protein (GFP) reporter (Hastwellet al.,

2006). This assay is based on the up-regulation of GADD45a-GFP transcriptionand the subsequent

increase in fluorescence, in response to genome damage and genotoxic stress.The test can be performed

with or without metabolic activation by S9 liver fraction.


Standard protocols have been developed for both methods, without (Hastwellet al., 2006) or

with(Jaggeret al., 2009) metabolic activation, and their transferability, within-laboratory

reproducibility(Hastwellet al., 2006; Jaggeret al., 2009), between-laboratory reproducibility (Billintonet

al.,2008; 2010) have been evaluated.


This test is used by the pharmaceutical industry as early screening tool in drug discovery.

However,most pharmaceutical companies are still investigating the utility of the screen in their

strategies, andhow to interpret the data for internal decision making.Some technical aspects have also

to be taken into account for the conduct of the test: the protocol inthe absence of metabolic activation

only requires the use of a microplate spectrophotometer and iscompatible with high throughput

screening, whereas the accessibility and the automation of the S9protocol are both limited by the

necessity of a flow cytometer to avoid interference with the light absorbing and fluorescent properties

of S9 particulates.


A variant of the S9 protocol has been developed, that was adapted for microplate readers by the useof a

fluorescent cell stain and fluorescence (instead of absorbance) measurement to estimate cellnumber.

Although flow cytometry remains the most sensitive method, this variant is more suitablefor non-flow

cytometer users and for high throughput screening.The BlueScreen HC is new assay under

development that uses the same GADD45a reporter gene asthe GreenScreen HC assay but linked to

Gaussia luciferase gene, which leads to a greater signal-tonoise ratio than with GFP and full

compatibility with S9 use and thus with high throughputscreening capability.

6

5. Hens egg test for micronucleus induction (HET-MN)


Another promising system as a follow-up for in vitro positive for cosmetic ingredient is the hensegg

test for micronucleus induction (HET-MN; Wolf et al., 2008). The HET-MN combines the use of the

commonly accepted genetic endpoint “formation of micronuclei” with the well-characterizedand

complex model of the incubated hen's egg, which enables metabolic activation, elimination

andexcretion of xenobiotics, including those that are mutagens or promutagens. This assay procedure

isin line with demands for animal protection. The scientific rationale and methodological aspects

forthis assay as well as results for some well-characterized mutagens and promutagens is

provided.After 8 day of incubation at 37.5°C, the test compounds are applied to the air cell. After

another 2.5days of incubation blood is taken by incising in situ a major vessel. The blood is spread out

onmicroscopic slides, stained and evaluated for micronuclei.


A prevalidation study is planned starting in September 2010 with at least three participatinglaboratories

investigating the transferability and intra-laboratory reproducibility. Results of thisstudy will most

probably be available in 2012.


At present the HET-MN is not frequently used. Only few laboratories have established this test

forscreening purposes. Studies on metabolism indicate that certain important phase I and II enzymesare

active and therefore the detection of liver mutagens is possible. Up to now the transferabilityand intra-

laboratory reproducibility is not provided.

Ongoing developments

An improvement may be the inclusion of flow cytometric analysis where higher cell numbers canbe

evaluated in a shorter time.

6. Cell transformation assay


Mammalian cell culture systems may be used to detect phenotypic changes in vitro induced bychemical

substances associated with malignant transformation in vivo. Widely used cells includeSHE,

C3H10T1/2 Balb/3T3 and andBhas 42 cells. The tests rely on changes in cell colonymorphology and

monolayer focus formation. Less widely used systems exist which detect otherphysiological or

7

morphological changes in cells following exposure to carcinogenic chemicals.Cytotoxicity is

determined by measuring the effect of the test material on colony-forming abilities (cloning efficiency)

or growth rates of the cultures.


In 2007 the OECD published a Detailed Review Paper (DRP31) aiming at reviewing all theavailable

data on the 3 main protocols for cell transformation assays and concluded that theperformance of the

SHE an Balb/c 3T3 were sufficiently adequate and that these assays should bedeveloped into OECD

test guidelines. A prevalidation study including the SHE (pH 6.7 and 7.0) and the Balb/c 3T3 was

organised by ECVAM to address issues of standardisation of the protocols,transferability and

reproducibility. The experimental work was finished in 2009. The datademonstrated that the SHE

protocols and the assays system themselves are transferable betweenlaboratories, and are reproducible

within- and between laboratories. For the Balb/c 3T3 method animproved protocol has been developed,

which allowed to obtain reproducible results. Furthertesting of this improved protocol is recommended

in order to confirm its robustness. Overall, theseresults in combination with the extensive database

summarized in the OECD DRP31 (OECD, 2007)will support the development of the OECD test

guidelines for the assessment of carcinogenicitypotential. This ongoing work should progress in the

coming 3 years.


The in vitro cell transformation assays have been established in order to predict

tumorigenicity(DiPaoloet al., 1969; Isfortet al., 1996; Matthews et al., 1993). Some of the test systems

arecapable of detecting tumour promoters (Rivedal and Sanner, 1982). Some cell types and

substancesmay require an appropriate external metabolic activation system. When primary cell types

are usedthat possess intrinsic metabolic activity, additional metabolic activation is not used. The

scoring oftransformed colonies and foci may require some training and experience.

The cell transformation assays are currently used in the pharmaceutical industry and occasionally inthe

cosmetic industry for clarification of in vitro positive results from genotoxicity assays to beused in the

weight of evidence assessment. Data generated by cell transformation assay can beuseful where

genotoxicity data for a certain substance class have limited predictive capacity (e.g.aromatic amines) or

for investigation of compounds with structural alerts for carcinogenicity(industrial chemicals under the

REACH regulation) or to demonstrate differences or similaritiesacross a chemical category (industrial

chemicals under the REACH regulation). Also the tumourpromoting (non-genotoxic) activity of

8

chemicals (agrochemicals, industrial chemicals, cigaretteindustry) and the chemopreventive activity

(pharmaceuticals) are investigated by the celltransformation assays.

7. In vitro toxicogenomics


Since the introduction of genomic technologies ca 10 years ago, their application in toxicology –

toxicogenomics – has developed enormously (Ellinger-Ziegelbaueret al., 2009; Guyton et al., 2009;

Waters et al., 2010, in press) The unbiased analyses of global perturbations by chemicals incells and

organisms at the level of genes, transcripts, proteins and metabolites, in combination withpowerful

bioinformatic tools, provides an unprecedented wealth of information about the molecularprocesses and

mechanisms that can be affected. This knowledge can be used for elucidating themode-of-action of

compounds, prediction of toxic properties, cross-species and in vitro-in vivocomparison, and even in

epidemiological settings for assessment of exposure and (adverse) effectsin humans. Predominantly

transcriptomics (gene expression analyses at the level of mRNA) hasreceived most attention and has

proven to be promising. For in vitro hazard assessment in the areaof genotoxicity and carcinogenicity

TK6, HepG2, and liver cells are mostly used as cell models(Ellinger-Ziegelbaueret al., 2009; Li et al.,

2007; Tsujimuraet al., 2006; Le Fevreet al., 2007; Huet al., 2004; Mathijset al., 2010). These

toxicogenomics approaches reach 80-90% accuracy forpredicting in vivo toxicity in rodents.

Known users

Academics. Pharmaceutical and cosmetic industry in screening purposes only.


No formal validation of a method has been performed. For some tests based on gene

expressionanalyses standard protocols are being developed and optimised (see “Ongoing

developments”). Thisongoing work will progress in the coming 3 years; depending on the results and

conclusions ofthese studies, some tests might be ready to enter prevalidation.


Various tests based on gene expression analyses can be foreseen for the near future, which can beused

for screening purposes and labelling of compounds, thus only for hazard assessment. Tests

forgenotoxicity and carcinogenicity in general, or for specific mechanisms therein, are

underdevelopment Thetranscriptomic biomarkers will be complex,consisting of profiles for multiple

9

genes. As many genes have been annotated with respect to theirfunction and sometimes to

toxicological pathways (e.g. the DNA damage pathway), this willprovide mechanistic information as

well. Some cell types and substances may require anappropriate external metabolic activation system,

but in general cells derived from liver are usedthat possess intrinsic metabolic activity and additional

metabolic activation is not used. Thelimitations are many, such as risk assessment is problematic, each

assay focuses on a specific aspect.

In vivo methods

1. In vivo genotoxicity tests

For the same reason as for in vitro tests, also in vivo genotoxicity tests may be a tool to predict cancer

risk assessment. In vivo tests may even be a better predictor of carcinogenicity tests since in these test

the role ADME is implicated. As for the in vitro genotoxicity tests, also for in vivogenotoxicity test the

specificity and predictivity have to be at a level that the prediction of carcinogenicity is justified. This

again may lead to modifications of test protocols, i.e. species, (top) doses among others. But then a

positive result in an in vivo genotoxicity test may point to a carcinogenic potency of the compound

under investigation and further carcinogenicity testing may not be needed. As for the in vitro tests

predominantly tests which measure irreversible genotoxicdamage may be considered: the chromosome

aberration test (OECD 475), the micronucleus test (OECD 474) and the gene mutation test with

transgenic animals (OECD guideline in prep). However, as an exception the Comet assay (OECD

guideline in prep), measuring single and double strand breaks may be considered. The role of

genotoxicity testing can be both qualitative (hazard assessment) and quantitative (risk assessment). A

preliminary investigation on the applicability of genotoxicity tests to estimate cancer potency was

undertaken in the RIVM using the benchmark dose approach and positive correlations between in vivo

genotoxicity (micronucleus test and transgenic rodent mutation test) and carcinogenic potency were

found (Hernandez et al. 2010, in prep). Dose-response analyses using sophisticated dose-response

software such as PROAST (RIVM) or the BMDS (USEPA) wasused. The results suggest that in vivo

genotoxicity tests may be used to estimate carcinogenic potency.

2. Transgenic mouse models

Short-term tests with transgenic mouse models (p53+/-, rasH2, Tg.AC, Xpa-/- and Xpa-/-p53+/) are a

good alternative for the classical 2-year cancer bioassay (Ashby 2001). The rationale for using

transgenic mice in regulatory carcinogenicity testing is that transgenic mouse models may be more

10

sensitive predictors of carcinogenic risk to humans. Indeed these transgenic mouse models had a

reduced tumor latency period (6-9 months) to chemically-induced tumors (Marx 2003). The increased

sensitivity to tumor formation in transgenic mouse models is primarily due to modifications in the

mouse genome by either removing or adding specific genetic material (Tennant 1995; 1999). Although

not a complete replacement to the rodent 2-year cancer bioassay, transgenic mouse models are a

refinement and result in a significant reduction in the use of experimental animals.

Several studies (ILSI/HESI ACT 2001; Eastin 1998; Bucher 1998; Pritchard 2003; de Vrieset al., 2004)

demonstrate that in all transgenic models a limited number of animals 20 – 25 animals/sex/treatment

group can be used and that an exposure of 6 – 9 months is sufficient. However, wild type animals

should be included in test battery to demonstrate that no genetic drift may affect interpretation of the

results. Transgenic mouse models showed a high specificity given that all non carcinogens tested gave

negative results in all 5 transgenic models. These findings provide evidence against “oversensitivity”

concerns associated with transgenic mouse models due to modifications in cancer-related genes.

Transgenic models were able to discriminate not only between carcinogens and non-carcinogens but

even between rodent carcinogens and putative human non-carcinogens to a high degree of accuracy.

v-Ha-ras

MICROINJECTED

Occasionally, it becomes necessary to discontinue live production of Taconic Transgenic Models due

to lack of interest. The v-Ha-ras mouse is one such model. Live production of this model is ending. If

you have a need for this model, please contact your Area Technical Representative to discuss available

options.

Model Description

Carries the activated v-Ha-ras oncogene fused to a mouse zeta-globin promoter

Exhibits characteristics of genetically initiated skin as a target for tumorigenesis, with a predisposition

to papillomas

11

http://www.taconic.com/wmspage.cfm?parm1=262

Useful in rapid screening of tumor promoters, non-genotoxic carcinogens and for assessing anti-tumor

and anti-proliferative agents

Hemizygous TG.AC mice are under evaluation as alternatives to the 2-year carcinogenicity bioassay

Origin: The v-Ha-ras mouse was developed in the laboratory of Philip Leder at Harvard University.

The model was created by microinjecting the v-Ha-ras oncogene fused to a mouse zeta-globin promoter

gene into FVB zygotes. Taconic received hemizygous male mice from Philip Leder on behalf of the

NIEHS in 1988. The mice were mated to inbred FVB females and then derived by caesarean transfer in

December 1988. The mice were then backcrossed three generations (N3) to FVB prior to intercrossing

hemizygous mice. Frank Sistare of the FDA indentified the responder genotype that correlates with

TPA-induced tumor formation in March 1998. This responder genotype is detected by Southern blot

using a zeta-globin probe. The hemizygous responder mice are maintained by backcrossing proven

homozygous responder TG.AC mice to FVB mice. Homozygous mice were transferred in July 1997 to

maintain continuity to the NIEHS Foundation Colony.

Color: Albino

Genetics: Wildtype for Nnt mutation; carries Pde6brd1

mutation

Initial Publication: Leder A, Kuo A, Cardiff RD, Sinn E, Leder P. (1990) v-Ha-ras Transgene

Abrogates the Initiation Step in Mouse Skin Tumorigenesis: Effects of Phorbol Esters and Retinoic

Acid. Proc Natl Acad Sci USA, 87:9178-82.

Animal Diet: NIH #31M Rodent Diet

2.TSG-p53®

Model Description

Contains a disruption of the p53 tumor suppressor gene, the most commonly mutated gene in human

cancers

12


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Retrieve&list_uids=91067670&dopt=Citation#_blank

Useful for studying p53 gene function or screening potential cancer intervention therapies

Primary use is for evaluation studies as an alternative to the 2-year carcinogenicity bioassay

Homozygous TSG-p53® mice are totally deficient in p53 protein and prone to the development of

spontaneous tumors

Heterozygous TSG-p53® mice carry one normal p53 allele and have a low rate of spontaneous tumor

development

Origin:

The TSG-p53® mouse was developed in the laboratory of Lawrence A. Donehower and Allan Bradley

at the Baylor College of Medicine. The model was created by targeting the Trp53 gene in AB1 ES cells

and injecting the targeted cells into C57BL/6 blastocysts. Resultant chimeras were backcrossed to

C57BL/6J for two generations (N2). Taconic received stock in 1991. The mice were backcrossed to N3

for caesarean derivation and to N4 immediately after derivation. The homozygous colony is maintained

at N4 through mating of heterozygous females with homozygous males. The heterozygous colony is

maintained at N5 through mating of N4 male homozygotes to C57BL/6NTac females. The wild type

control colony is maintained at N5 through mating of N4 wild types to C57BL/6NTac mice.

Color: Black

Genetics: Wildtype for Nnt mutation

Initial Publication: Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA, Jr, Butel

JS, Bradley A. (1992) Mice deficient for p53 are developmentally normal but susceptible to

spontaneous tumors. Nature, 356:215-221.


rasH2

MICROINJECTED

BALB/cBy x C57BL/6 F1 Hybrid Background

Model Number Zygosity Nomenclature

13


http://www.ncbi.nlm.nih.gov/pubmed/1552940#_blank

1178-F Hemizygous CByB6F1-Tg(HRAS)2Jic

1178-M Hemizygous CByB6F1-Tg(HRAS)2Jic

1178-F Wild Type CByB6F1-Tg(HRAS)2Jic

1178-M Wild TypeCByB6F1-Tg(HRAS)2Jic

Model Description

Carries the human homolog of the Harvey rat sarcoma virus oncogene (c-Ha-ras, now identified as

HRAS) with its own promoter region in addition to the endogenous murine Ha-ras oncogene Total ras-

encoded p21 protein expressed at 2-3 times normal levels, in all tissues, with somatic mutational

activation found only in tumor tissue transgenesSignificantly more rapid onset and higher incidence of

more malignant tumors after treatment with genotoxic carcinogens compared to wild-type controls

Very low incidence of spontaneous tumors, with no indication of pre-neoplastic cell stages or tumors at

6 months of age Approximately 50% of transgenic rasH2 mice develop spontaneous tumors by 18

months of age, predominantly angiosarcomas and lung adenocarcinomas Useful as a rapid, cost-

effective, and sensitive carcinogenicity testing model for detection of nongenotoxic and genotoxic

carcinogens, as supported by studies conducted by ILSI Also useful in the study of oncogene-initiated

tumorigenesis and can be used to evaluate tumor treatment compounds In Japan, contact CIEA for

purchase of rasH2 mice Please note that the model nomenclature has been updated. The CIEA

designation for the rasH2 model, CB6F1/Jic-TgrasH2@Tac was used previously.

Origin: The rasH2 mouse was developed in the laboratory of Tatsuji Nomura of the Central Institute for

Experimental Animals (CIEA) in Kawaski, Japan. The model was created by microinjecting the human

c-Ha-ras gene into C57BL/6 x BALB/c F2 zygotes. Hybrid transgenes were constructed from two

human HRAS (c-Ha-ras) genes isolated from malignant melanoma and bladder carcinoma tumors. The

transgene integrated into the murine transgenic genome as 5-6 copies in tandem array. The resultant

mice were backcrossed to C57BL/6J. Taconic received stock in March 2000 and again in December

2005. The mice were derived by embryo transfer and are maintained by intercrossing C57BL/6JJic

Tg(HRAS)2Jic hemizygous male mice with BALB/cByJJic female mice.

Color: Black Agouti

14

Genetics: Carries Nnt mutation

Initial Publication: Saitoh A, Kimura M, Takahasi R, Yokoyama M, Nomura T, Izawa M, Sckiya T,

Nishimura S, Katsuki M. (1990) Most tumors in transgenic mice with human c-Ha-ras gene contained

somatically activated transgenes. Oncogene, 5(8):1195-1200.


2. In vivo toxicogenomics

In the paragraph on in vitro toxicogenomics an introduction is given on relevance of genomics

technologies and their application in toxicology. Also in case of in vivo toxicogenomics, gene

expression analyses (transcriptomics) has been developed furthest. Most in vivo toxicogenomicsstudies

on assessment of carcinogenicity, focus – but do not limit – on short term rat studies and non-

genotoxichepato-carcinogenicity These toxicogenomics approaches reach 80-90% accuracy for

predicting rodent carcinogenicity. Pharmaceutical and sometimes the chemical industry are the main

users in screening purposes. In rare cases the pharmaceutical industry also uses in vivo toxicogenomics

for mechanistic purposes. Various assays based on gene expression analyses can be foreseen for the

near future, which can be used for screening/prioritization purposes and for labelling of compounds.

These assays can also be helpful for the understanding of modes of action. Especially assays for non-

genotoxic hepato1003 carcinogenicity are under development Like for in vitro toxicogenomics, the

limitations are many, such as quantitative risk assessment is in its infancy, strong focus on non-

genotoxic carcinogens (mainly pharmaceuticals) and on the liver as target organ, limited public

accessibility of raw data, the mechanism of not all genes in the prediction sets are understood, lack of

uniformity in study design (e.g. rodent species and strain, dose setting criteria, time points, repeats,

etc.) and bio-informatic analyses, and the requirement of expensive equipment and specialized staff.

The in vivo toxicogenomics assays could be very helpful for hazard assessment and by that may lead to

a reduction in the number of bioassays and the number of animals in the remaining in vivotests. The

number of animals required for toxicogenomics based assays are at least 10-fold smaller then for the

rodent bio-assay, and the exposure periods last maximally four weeks instead of two years. hepato-

carcinogenicity is being investigated for some aspects of reliability. This ongoing work will progress in

the coming 3 years; depending on the results and conclusions of these studies, some tests may might be

15

ready to enter pre-validation. The Predictive Safety Testing Consortium of the Critical Path Institute

evaluated the predictivity of two published hepatic gene expression signatures when sharing the data.

Based on the results, a QPCR-based signature has been derived and is currently evaluated for inter-

laboratory precision, sensitivity and specificity, timedependency, and non-genotoxic versus genotoxic

mechanisms.

Conclusion:

As a general limitation, cancer is a long-term process that is to day despite best efforts impossible to

mimic with relatively short-term in vitro tests. An in vivo testing is no longer possible, the safety of

many potential new cosmetic ingredients will not be able to be substantiated and will therefore not be

allowed. The process of carcinogenesis is recognised as resulting from a sequence of stages and

complex contribute to the carcinogenic process and that even for one chemical, the mode of action can

bedifferent in different target organs, or in different species. Despite best efforts, the modeling of such

complex adverse effects cannot fully be accomplished at present by the use of non-animal tests.For

genotoxic chemicals, a number of in vitro genotoxicity tests are available that are currently used to

screen chemicals for activity that is considered to be predictive of potential carcinogenicity. While

these tests have good sensitivity, some (especially the in vitro mammalian cell tests) also have a high

rate of misleading positives. Prior to 2009, a positive finding in an in vitro study was commonly

followed by an in vivo study that was of critical importance for clarifying the in vitroresults. Indeed,

the vast majority of compounds tested in vivo were negative. Because of the 2009 ban on in vivo

genotoxicity testing, the situation is now problematic and many potential cosmetic ingredients may be

lost because of an inability to clarify misleading positive results from an in vitrogenotoxicity

16

REFERENCE

1.Aardema, M.J., Barnett, B., Khambatta, Z.S., Reisinger, K., Ouedrago-Arras, G., Faquet, B.,

Ginestet A., Mun, G.C., Dahl, E.L., Hewitt N.J., Corvi R. & Curren, R.C.

2. International pre validation studies of the EpiDerm TM 3D human reconstructed skin micronucleus

(RSMN) assay:

3.Ames, B.N. & Gold, L.S. (1991). Science 251, 607-608.

Ao, L., Liu, J.Y., Liu, W.B., Gao, L.H., Hu, R., Fang, Z.J., Zhen, Z.X., Huang, M.H., Yang, M.S. &

Cao, J. (2010). Comparison of gene expression profiles in BALB/c 3T3 transformed foci exposed to

tumor promoting agents. Toxicol. In Vitro, 24, 430-8.

4.Ashby, J. (2001). Expectations for transgenic rodent cancer bioassay models. Toxicol Sci 64, 7-

Baylin, S.B. & Ohm, J.E. (2006). Epigenetic gene silencing in cancer – a mechanism for early

oncogenic pathway addition? Nat. Rev. Cancer 6, 107-116.

5.Benfenati, E., Benigni, R., DeMarini, D.M., Helma, C., Kirkland, D., Martin, T.M., Mazzatorta,

P., Ouédraogo-Arras, G., Richard, A.M., Schilter, B., Schoonen, W.G.E.J., Snyder, R.D. & Yang

C (2009). Predictive Models for Carcinogenicity and Mutagenicity: Frameworks, State-of-the-Art,

and Perspectives. J Environ Sci Health C 27, 57-90

6.Benigni, R. & Bossa,C. (2008). Predictivity of QSAR. Journal of Chemical Information and

Modeling 48, 971-980.

7.Benigni, R., Bossa, C., Tcheremenskaia, O. & Giuliani, A. (2010). Alternatives to the

carcinogenicity bioassay: in silico methods, and the in vitro and in vivo mutagenicity assays. Expert

Opinion on Drug Metabolism & Toxicology 6, 809-819.

8.Bercu, J.P., Morton, S.M., Deahl, J.T., Gombar, V.K., Callis, C.M., van Lier, R.B. (2010). In silico

17

carcinogenecity tests

Documents

Transcript of carcinogenecity tests