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Analysis of published data for top concentration of cytotoxicity testing in mammalian genotoxicity testing

J. Parry, E. Parry, P. Phrakonkham, R. Corvi

In vitro Methods/European Centre for the Validation of Alternative Methods (ECVAM)

IWGT, Basel, August 2009

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European Centre for the Validation of Alternative MethodsECVAM

• Validation

• Research

• Communication

EU Policy support

• 7th Amendment to Cosmetics Directive

• REACH

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CARCINOGENOMICSCOMICS

ECVAM key area

genotoxicity / carcinogenicity

Validation

Reduction

Policy support (eg. REACH ITS)

Research support

Refine

Reduce

Replace

Taskforce

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Genotoxicity testing requirements for REACH

In vitro gene mutation study in bacteria 1t-10t

In vitro studyin mammalian cells (2 tests) > 10t

In vivo somatic cell genotoxicity study

if positive

if positive classified as mutagen

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ECVAM Workshop Italy, April 2006

Objectives:

1. Review data from currently available systems

2. Review the performance of new test systems

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ECVAM Workshop on false positivesFollow up activities

• Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: A follow-up to an ECVAM WorkshopD. Kirkland, Mut Res, 653 (2008), 99-108

• Collaboration with COLIPA and NC3Rs on improvement of current in vitro tests

• Analysis of published data for top concentration and upper limit of cytotoxicity in mammalian cell genotoxicity testing.(ECVAM commissioned this analysis to J. & L. Parry)

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Analysis of published data for top concentration of cytotoxicity in mammalian genotoxicity testing

Objectives• Review of existing data to determine whether concentration as 10

mM (or 5000 µg/ml) are needed to detect in vivo genotoxins and DNA-reactive, mutagenic carcinogens using in vitro mammalian cell tests or whether a lower level can be justified.

• Identified chemicals positive in mammalian cell tests at > 1mM

• Data from Ames tests were reviewed to see if chemicals would be detected in other parts of the standard battery if high concentration was needed to produce positive results in the mammalian cell tests.

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Screen Carcinogenicity Genotoxicity eXpert (CGX)*

database of rodent carcinogens

Consider chemicals with genotoxicity data

Analysis of top concentration of cytotoxicity testing in mammalian genotoxicity testing

Data for CA, MLA, MNT, SCE, Ames

Focus on CA and MLA

Ames

Establish databases*Kirkland et al., Mutation Research 584 (2005) 1-256

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Content of the databases

Whenever possible available original data were consulted in the original publications

CA• NTP DB• Ishidate et al., 1988• Additional publications

MLAMost of original papers reviewed because of concerns about criteria

used for interpretation of data Mitchell et al., 1997 (for 2/3 chemicals original data reviewed)• NTP DB• Additional publications

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General Criteria used for building up the databases

• Follow the OECD and current Guidelines • In cases were several publications available, most

reliable were taken into consideration, the study that fulfilled current guideline criteria overruled the other studies

• For positive results lowest effective concentration was reported

• For negative and/or equivocal results highest concentration available was reported

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Criteria used for interpretation of CA dataconservative approach

• Available raw data (i.e. NTP DB and additional publications)

• Criteria used in the NTP considered

• Ishidate et al., 1988– Authors conclusions reported

– Ishidate criteria for a positive call were considered quite conservative, therefore adequate for this analysis

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Criteria used for interpretation of MLA dataApplied to most of the chemicals reported

• Available raw data For a positive call:

– Exclusion of RTG data < 10%– Fulfil GEF– Dose-response or trend– Considered also datasets with negative controls with

lower mutant frequency than currently accepted, in case other criteria were fulfilled

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Considerations related to this retrospective analysis

• For the purpose of this analysis as many positive compounds as possible were included– Included equivocal compounds, when clear positive results were

available for those compounds– MLA positive results with a negative control with low frequency

mutants

• A subset of data not checked in original papers

• Chemicals classified as carcinogens, classification as in vivo genotoxins lacking

• In the long term analysis can be improved dependent upon availability of further data

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Chemicals with genotoxicity data

404

CA

338

MLA

228

+

197

–

105

i

36 +

148

–

44

i

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Overview of data for CA and MLA

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Distribution of concentrations which produced positive results in CA and MLA

Positive concentration (mM)

CAn° of chemicals (%)

MLAn° of chemicals (%)

≤ 1 139 (70.6%) 110 (74.3%)

1 to 10 46 (23.3%) 32 (21.6%)

>10 12 (6.1%) 6 (4.1%)

Total n° of chemicals 197 148

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2

17

26Ames +

Ames e

Ames -

Ames data for CA positive at 1-10 mM

Tot. n. chemicals = 46

17Chemicals positive at 1-10mM in CAequivocal/negative Ames

mM mM

Clofibrate 1.03 Methapyrilene hydrochloride 2.51

3-Chloro-2-methylpropene Technical grade

1.32 N-Methylolacrylamide 2.94

Caffeic acid 1.4 Furfural 3.12

Furan 1.47 Methimazole 3.2

Phenylbutazone 1.63 Hydroquinone 4.1

2-Mercaptobenzothiazole 2.1 Methylphenidate HCl 4.63

Allyl isovalerate 2.11 Furosemide 6

Ethionamide 2.4 3-(p-Chlorophenyl)-1-1-dimethylurea (AKA monuron)

6.54

Styrene 2.4 alpha-Methylbenzyl alcohol 8.15

Chlorendic acid 2.5

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4

12

16 Ames +

Ames e

Ames -

Ames data for MLA positive at 1-10 mM

Tot. n. chemicals = 32

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Chemicals positive at 1-10mM in MLAequivocal/negative Ames

mM mM

Methapyrilene hydrochloride 1.01 FD & C red 1 (Ponceau 3R) 3.44

Caffeic acid 1.11 Benzaldehyde 3.77

Chlorobenzene 1.11 Chlorendic acid 3.86

Benzofuran 1.27 Acetaldehyde 3.98

C.I. Direct blue 15 1.51 alpha-Methylbenzyl alcohol 4.1

Furfural 2.1 Furosemide 4.55

Toluene 2.44 Isophorone 5.79

Allyl isovalerate 2.81 Acrylamide 9.8

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Chemicals that require >1mM in mammalian cell tests (CA or MLA) for a +ve response

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CA +

16

MLA +

22

CA or MLA +

Ames -/e

Ames -/e

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Compounds detected positive at >1mM in one test, but positive < 1mM in the other

These compounds were removed from final list

• Benzaldehyde• Acetaldehyde• Acrylamide• 2-chloro-2-methylpropene• 2-mercaptobenzothiazole• Hydroquinone

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 CA CA +ve (mM) MLA +ve (mM)

Methapyrilene hydrochloride 2.94 1.01

Caffeic acid 1.4 1.11

Allyl isovalerate 2.11 2.81

Chlorendic acid 2.5 3.86

Furfural 3.12 2.1

Furosemide 6 4.55

alpha-Methylbenzyl alcohol 8.15 4.1

Chlorobenzene 1.11

Benzofuran 1.27

Furan 1.47

Phenylbutazone 1.63

C.I. Direct blue 15 1.51

Ethionamide 2.4

Styrene 2.4

Clofibrate 2.51

N-Methylolacrylamide 1.03

Methimazole 3.2

Isophorone 5.79

Methylphenidate HCl 4.63

3-(p-Chlorophenyl)-1-1-dimethylurea (AKA monuron) 6.54

Toluene 2.44

FD & C red 1 (Ponceau 3R) 3.44

Chemicals that require >1mM in mammalian cell tests

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Initial evaluation on MNT(not yet checked)

MNT

50All MNT +

at < 1mMMNT +

45

Ames +

26

Ames -

19

MNT -

5

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Conclusions

Identified 22 (out of 404) chemicals that are Ames negative/e and require >1mM in mammalian cell tests (CA or MLA) for a +ve response

• Are these chemicals all relevant positive?• Are they supposed to be picked up as genotoxic

carcinogens?• How many irrelevant chemicals would we avoided if top

concentration reduced to 1mM?• Work in progress, analysis can be improved in the future,

but let’s start discuss!!

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Aknowledgements

James M. Parry

Elizabeth Parry

Pascal Phrakonkham, ECVAM

David Kirkland, Kirkland Consulting