Trend and Progress of OECD Genotoxicity Testing Guidelines

Masamitsu Honma, Ph.D.National Institute of Health Sciences, Japan

International Conference on Biotechnological Advances in Environmental Health and Biodiversity Conservation (EHBC)& 39th Annual Meeting of Environmental Mutagen Society of India (EMSI)Imphal, India May21-23, 2015

http://www.oecd.org/chemicalsafety/testing/oecdguidelinesforthetestingofchemicals.htm

TG Title Adopted

471 Bacterial reverse mutation test (also named Ames test) 1983 473 In vitro mammalian chromosomal aberration test 1983 474 Mammalian erythrocyte micronucleus test 1983 475 Mammalian bone marrow chromosomal aberration test 1984 476 In vitro mammalian cell gene mutation test 1984

using the hprt or xprt locus 477 Sex-linked recessive lethal test in Drosophila 1984

melanogaster 478 Rodent dominant lethal assay 1984 479 In vitro sister chromatid exchange assay in mammalian cells 1986 480 Saccharomyces cerevisiae, gene mutation assay 1986 481 Saccharomyces cerevisiae, mitotic recombination assay 1986 482 Unscheduled DNA synthesis in mammalian cells in vitro 1986 483 Mammalian spermatogonial chromosome aberration test 1997 484 Mouse spot test 1986 485 Mouse heritable translocation assay 1986 486 Unscheduled DNA synthesis test with mammalian liver cells 1997

in vivo 487 In vitro mammalian cell micronucleus test 2010 488 Transgenic rodent somatic and germ cell gene mutation 2011

assays 489 In vivo alkaline Comet assay 2014

OECD Test Guidelines for Genetic Toxicology

The revision of OECD test guidelines on genotoxicity started from March 2011.

TG Title Adopted Revised Deleted

471 Bacterial reverse mutation test (also named Ames test) 1983 1997 473 In vitro mammalian chromosomal aberration test 1983 1997/2014 474 Mammalian erythrocyte micronucleus test 1983 1997/2014 475 Mammalian bone marrow chromosomal aberration test 1984 1997/2014 476 In vitro mammalian cell gene mutation test 1984 1997/20xx

using the hprt or xprt locus 477 Sex-linked recessive lethal test in Drosophila 1984 2013

melanogaster 478 Rodent dominant lethal assay 1984 20xx 479 In vitro sister chromatid exchange assay in mammalian cells 1986 2013 480 Saccharomyces cerevisiae, gene mutation assay 1986 2013 481 Saccharomyces cerevisiae, mitotic recombination assay 1986 2013 482 Unscheduled DNA synthesis in mammalian cells in vitro 1986 2013 483 Mammalian spermatogonial chromosome aberration test 1997 20xx 484 Mouse spot test 1986 2013 485 Mouse heritable translocation assay 1986 486 Unscheduled DNA synthesis test with mammalian liver cells 1997

in vivo 487 In vitro mammalian cell micronucleus test 2010 2014 488 Transgenic rodent somatic and germ cell gene mutation 2011

assays 489 In vivo alkaline Comet assay 2014

OECD Test Guidelines for Genetic Toxicology

Main Issues of Revised OECD Test Guidelines for Genetic Toxicology

I. In vitro Issues• Top concentration• Measuring cell proliferation and cytotoxicity and choosing treatment

concentrations• Cell lines

II. In vitro/vivo Issues• Number of cells to score• Evaluation and interpretation of results

III. Adaptation of Test Guidelines to Nanomaterials• In vitro micronucleus test for nanomaterials

IV. Other Issues• In vivo Pig-a gene mutation assay

TG473 In vitro mammalian chromosomal aberration test TG474 Mammalian erythrocyte micronucleus testTG475 Mammalian bone marrow chromosomal aberration testTG487 In vitro mammalian cell micronucleus test

Top Concentration in In Vitro Tests

TG473/ TG487/ TG476＜Old＞

＜Revised＞

Options Name Suggested values RationaleOption 1 called “OECD” 10 mM or 5mg/mL, whichever is

lower

current criteria

Option 2 called “ICH” 1 mM or 0.5 mg/mL, whichever is lower,

currently suggested in ICH S2(R1) for pharmaceuticals

Option 3 called “DK” 1 mM or 0.5 mµg/mL, whichever is higher,

mostly based on re-analysis of NTP database and recent experiments (Fowler and Kirkland). This proposal was with drawn by David Kirkland in favour of Option 6)

Option 4 called “BG” 10 mM or 1 mg/mL, whichever is lower,

based on extrapolation made from the use of 1000 mg/kg in vivo, and the need to test higher concentrations for low molecular weight compounds

Option 5 called “BG-GD” 10 mM or 2 mg/mL, whichever is lower,

based on extrapolation made from the use of 2000 mg/kg in vivo for short term treatment, and the need to test higher concentrations for low molecular weight compounds

Option 6 called “VTEL” 4 mM alone when molecular weight is known, and 2 mg/mL, when molecular weight is unknown

Compromise that takes all other options into account, relies on molarity only except when no information is available on molecular weight, applies a 2.5 fold reduction to the current top concentration, and a 4-fold safety factor to “DK” proposal (based on the analysis of existing data)

Option 7 called “Alternative VTEL” (AVTEL)

4 mM or 2 mg/mL, whichever is lower

VTEL proposal with a “cap” at 2000 µg/mL to avoid very high concentration in µg/mL for high molecular weight molecules.

Discussion of Top Concentration in In Vitro Tests in OECD Experts

Oxolinic acid (MW 261)(Antimicrobial drug)

No concern for Clastogenicity Concern for Clastogenicity?

Pharmaceutical for urinary tract infection

Daily dosages : 12-20mg/kg

Agricultural chemical and animal drug ADI : 0.021mg/kg

CA positive in equal or more than 2.5 mM in CHL cells without S9

Negative in CA as a pharmaceutical

(ICH)

Positive in CA as a chemical (OECD)

ICH (1mM) vs. OECD (10mM)

Measuring Cell Proliferation and Cytotoxicity and Choosing Treatment Concentrations

TG473/ TG487＜Old＞

＜Revised＞

Underestimate Cytotoxicity by RCC

Original before treatment

(30 cells/plate)

Neg. Control after treatment Test chemical after treatment

(100 cells/plate) (30 cells/plate; No Growth)

Relative Cell Count (RCC) is calculated to 30%, even though no cell growth is observed.

Underestimate cytotoxicity

Greenwood et al., EMM 43, 36-44 (2004)

PD = - [log(Post-treatment cell number/Initial cell number)]/log2

N RCC (%)

PDRPD (%)

Cont. (0)

10.0100

1.74100

Dose 1

5.050

0.7342

Dose 2

3.535

0.2313

Dose 3

3.030

No calculationNo net growth

55%-RCC is equivalent to 50%-RPD

Calculation of RCC and RPD for Cytotoxicity

RCC: Relative Cell Count

RPD: Relative Population Doubling

RPD (%) =PD in treated culture

PD in control culture

Ori.

3.0

10 2 3 4 5

RCC

RPD

Top conc. by RCC

Top conc. by RPD

RCC vs. RPD for Cytotoxicity

0

10

20

30

40

50

60

70

80

90

100

Cell Lines Used in TG473 (in vitro CA)

＜New＞

＜Old＞

Cell Lines Used in TG487 (In vitro MN)

＜New＞

＜Old＞

47, XY, 13+, t(14; 20), t(21; 3)

TK6 (ATCC No. CRL-8015) (Human lymphoblastoid cell

line)

TK6 Cells

Evaluation and Interpretation of Results

TG473/ TG474/ TG475/ TG476/ TG487＜Old＞

＜New＞

Number of Cells to Score (1)

Expert Group Meeting on the Review of OECD TGs on Genotoxicity20-22 November 2013 Ottawa, Canada

Number of Cells to Score (2)

＜Old＞

＜New＞

TG473

Number of Cells to Score (3)

＜Old＞

＜New＞

TG474

＜Old＞

＜New＞

TG475

Number of Cells to Score (4)

OECD workshop on genotoxicity of manufactured nanomaterials was held on November 2013 in Ottawa, Canada.

• TG 471 (Ames test), as it is not applicable to some types of nanomaterials, e.g. insoluble particles >20 nm in size.

• Other in vitro TGs could be useful tests with adaptation. However, the current draft TGs 473 (Chrom Ab) and 487 (in vitro MN) exclude from their applicability domain insoluble chemicals and particulate materials.

• The group agreed that a direct inclusion of some adaptations to the TGs at this stage might be misleading. Further work is still needed to confirm some recommendations for adaptations. It was agreed that the recommendations developed by the workshop would be included in the Guidance Document.

Summary in Ottawa Meeting

OECD expert meeting on the adaptation of the genotoxicity in vitro micronucleus assay TG487 testing of nanomaterials was held on

October 2014 in Paris.

Summary in Paris Meeting (1)-General Issues-

• A Guidance Document addressing the necessary adaptations of current genotoxicity TGs for nanomaterials testing should be developed.

• Scientific information available to date is not sufficient to fully support a common version of the in vitro micronucleus test protocol. A ring trial aimed at the standardization of the micronucleus in vitro assay for testing of nanomaterials will be organized under the coordination of JRC where five selected cell types (primary human lymphocytes, TK6 cells, Caco-2 cells, A549 cells, V79 cells) will be included and 3 nanomaterials (gold NPs, silver NPs, silica NPs) will be tested. Italy, France, Poland, Belgium, Norway, UK, BIAC and Japan already agreed to join the study.

• The expert group decided that a Standard Project Submission Form (SPSF) for this project, including the proposal for the inter-laboratory comparison study and the development of the Guidance Document on the use and modifications of genotoxicity TG for NMs testing will be submitted to the OECD Secretariat by 15th November 2014.

Summary in Paris Meeting (2)-Technical Issues-

1. Duration of exposure to NMs: there was a general agreement that treatment with NMs for 3-6 hours may be too short, as it may not allow the NMs to enter in contact with the cells and to be internalized

2. Choice of relevant cell system: The most sensitive cell system, preferably of human origin and p53 positive should be used, regardless the expected route of exposure to the nanomaterial in the final consumer product.

3. Dosing (concentration ranges, top dose): the selection of the highest concentration to be tested is dependent on NMs cytotoxicity.

4. Use of serum in the exposure media: Exposure should be done in conditions when cells divide and are healthy for the whole length of the treatment.

5. Treatment with cytochalasin B (cyto B): There was a general view that delayed co-treatment (i.e. at least 6h exposure to NMs before addition of cyto B) or no treatment with cyto B are the most suitable protocols for NMs.

6. Physico-chemical characterization in the in vitro: This issue was considered to be of fundamental importance

7. Nanomaterial in vitro dosimetry (cellular vs administered dose): Exposure should take into account sedimentation/dispersion and aggregation state of NMs.

8. Use of S9 mix: the use of S9 mix was not recommended.

In vivo Pig-a gene mutation assay

TG Title Adopted Revised Comments

471 Bacterial reverse mutation test (also named Ames test) 1983 1997 473 In vitro mammalian chromosomal aberration test 1983 2014 474 Mammalian erythrocyte micronucleus test 1983 2014 475 Mammalian bone marrow chromosomal aberration test 1984 2014 476 In vitro mammalian cell gene mutation test 1984 2015 Final round

using the hprt or xprt locus 478 Rodent dominant lethal assay 1984 2015 Final round483 Mammalian spermatogonial chromosome aberration test 1997 2015 Final round485 Mouse heritable translocation assay 1986 486 Unscheduled DNA synthesis test with mammalian liver 1977

cells in vivo 487 In vitro mammalian cell micronucleus test 2010 2014 488 Transgenic rodent somatic and germ cell gene mutation 2011 2013

assays 489 In vivo alkaline Comet assay 2014 49x In vitro mammalian cell gene mutation test 2015 Final round

using the TK locus

In vivo Pig-a gene mutation assay SPSF submitted

Guidance document on the adaptation of genotoxicity SPSF submittedTGs for testing of manufactured nanomaterials

Summary-Current Status of OECD Test Guidelines for Genetic Toxicology (November, 2014)-

Thank you!

http://www.oecd.org/chemicalsafety/testing/oecdguidelinesforthetestingofchemicals.htm