Are All Ames Strains in the OECD Mutagenicity Test ... All...The Ames Test Revisited Ames Test =...

Are All Ames Strains in the OECD Mutagenicity Test Guideline 471 Useful and Necessary? An Analysis of Large Mutagenicity Data Sets for the IWGTWHAT IS THE MINIMUM VIABLE STRAIN SET?

Richard WilliamsGTA PresentationMay 3rd 2018

The Ames Test Revisited

Ames Test = Bacterial Reversion Mutation Assay, as described in OECD Test Guideline 471 This guideline was last updated in 1997

Guidelines for many other in vitro genotoxicity assays updated in the meantime

One of the groups looking at the Ames test specifically tasked with the question ‘what is the minimum viable strain profile’?

The Ames Strains Sub-Group

Kevin Cross Leadscope Strains comparisonsDavid DeMarini Univ. of North

CarolinaPhenotypic markers, statistical analysis

Rosie Elespuru Unaffiliated Strain availabilityJon Howe GSK Positive control rangeLeon Stankowski Charles River

LaboratoriesNegative control range

Richard Williams Lhasa Limited Strains comparisons, Lead

& Errol Zeiger after our Tokyo meet

OECD TG471

Minimum Viable Strain Profile

We now have a much larger data set, are the assumptions and analysis on which TG471 was based still true today?

Which strains to compare? TA100 v TA1535 TA102 v E.coli WP2 uvrA v E.coli WP2 uvrA (pKM101) TA97 v TA97a v TA1537

How? Identify compounds producing selective (e.g. TA1535 +ve, TA100 –ve) responses in databases Examine dose response data to confirm

Workflow

Tested inboth strainsundercomparison


Produce selective response

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databases

Confirmed selective responses

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Criteria

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Proportion SelectiveCommon Chemotypes

Statistical AnalysisActivity in other strains

Results

Criteria for Confirming Selectivity

EquivalentProtocol

SimilarMetabolicActivation

Article Purity

Reproducible ResultsHighest Tested Dose

Dose Spacing

Same Study or

Lab

Test Solvent

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TA100 / TA1535

TA100/TA1535

TG471 compliant Ames requires both However

Both detect base-pair mutagens

TA100 engineered to be a more sensitive derivative of TA1535

Is TA1535 needed?

TA100 TA1535Allele/strain hisG46 hisG46DNA target -G-G-G- -G-G-G-uvrB-bio genes Deletion mutation Deletion mutationLPS defect rfa rfaPlasmid pKM101 No plasmid

TA100/TA1535 Previous Publications

Workflow




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Criteria

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Results

TA100 / TA1535 Data Set Sizes

3667 Tested in

Both

4701 Tested in Both

Workflow




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Results

TA100 / TA1535 Selective ResponsesReminder:Extracted data, Identified ‘author call’ selectives,Reviewed data and verified

530 +vein Both

43 Selective +ve in TA1535594 Selective +ve in TA100

43/1167=3.7%

Selective Positives: Further Analysis

43 Selective Positives (TA1535 +ve, TA100 –ve) S9 requirement

16 required rat or hamster S9 (no specificity in species)

8 required hamster S9 specifically

6 Common chemotypes

Activity in other Ames strains 35 Tested in strains other than TA100 & TA1535

30 Not active in other strains ( unique)

5 Active in other strains

Statistical analysis of dose response data Most selective positives produce quantitatively similar response in TA100 and TA1535

Selective Positives: Chemotypes

+ 5 similarRoche cpds

Base Analogues

“Phenobarbital-like” Oximes Nitro/Nitrate Hydrazine N-/O-Allyl

+ 8 Mixed ‘alkyl halides’ + 11 miscellaneous cpds

Are Chemotypes Selective for TA1535? Analysis shows some common chemotypes responsible for majority of selective

TA1535 activity Further analysis required to determine if TA1535 selective activity is common for

the chemotypes

Chemotype # in Data Set

# PositiveAmes

# Selective Positive TA1535

% Selective Positive in

TA1535Base analogue 31 9 3 33.3%Nitro/Nitrate 329 229 3 1.26%Oxime 18 7 3 42.9%Hydrazine 37 20 3 15.0%N-/O-Allyl 100 24 9 37.5%

Statistical analysis

Statistical analysis offers some benefits over human data interpretation Ability to pool data from multiple experiments Objective comparison of dose-response data Quantitative assessment of potency

Of the 42 compounds showing TA1535 selectivity, 27 had dose-response data that could be further analysed

Here, we’re looking at multiple methods for data analysis (fold rule & statistical analysis) – Errol will talk more about criteria tomorrow

Mutagenic Potencies (rev/µg) of Agents Indicated to be Mutagenic in Only TA1535 and Not in TA100

TA1535 TA100 + in onlyChemical +S9 -S9 +S9 -S9 TA15351H-Benzotriazole 0.02 0.01 0.02 0.021-Bromotetrafluoro-2-iodoethaneb 6950 7400 - 5500

1,2-Dibromopropane 0.02 0.02 0.2 -2-Bromopropionic acid 0.01 ND 0.02 ND2-(Methylnitroamino)ethyl nitrate 0.001 0.009 - - X

4-Ethylmorpholine 0.02 - - - X6-Mercaptopurine 7.5 2.7 - - XAcetaldehyde oxime ND 0.02 ND - XAcetin 0.01 0.01 - - XBromochlorodifluoromethane 3.8 1.8 - 1.3Carbonochloridothiolic acid 0.05 NDa - ? XCyclohexanone oxime 0.02 - - 0.5Dexlansoprazole ND 0.05 - 0.07Dichloro(methyl)(vinyl) silane 0.05 ND - ND XHydrazine sulfate ND 0.05 ND - XMaltol 0.01 - 0.02 0.02Methyl ethyl ketoxime 0.01 - 0.01 NDN-Butyl-2-nitratoethylnitramine - - - - Non-mut.Oxetane 0.006 - - 0.002Pentaerythritol triallyl ether ND 0.01 ND 0.01Phenobarbital ND 0.02 - - XPrimadone ND 0.003 ND 0.003Roche 47 3555 0.01 0.002 0.02 -Sec-Butyl nitrite 0.01 - 0.01 0.03Thiourea dioxide 0.02 0.02 0.01 -Tricaprylin 0.004 0.004 - - XTrimethylolpropane triacrylate 0.01 - 0.01 -

16/27 Compounds produce quantitatively similar responses in TA100 and TA1535

Only 37% (10/27) are considered to be selectively mutagenic in TA1535 Assessment of the potency values

indicates that 90% (9/10) of these have potencies ≤0.05 rev/µg, suggesting that these are extremely weak mutagens.

Similar results obtained on a proprietary data set of 53 TA1535 selective compounds

TA100/TA1535 Summary

Analysis of the dose response data confirmed only 43 compounds (3.7%) with selective activity in TA1535

Further analysis on selective compounds Common chemotypes observed amongst selective positives. This includes

base analogues such as 6-MP (Ames test not ideal for these cpds) 35/43 compounds also had data for activity in other strains

30/35 are also uniquely positive in TA1535

27/43 compounds had accessible dose-response data Only 10/27 were confirmed selective (quantitatively different responses in TA100 and

TA1535) 9/10 Were low potency mutagens (<0.05rev/µg)

No common chemotypes in this subset

TA97 / TA97a / TA1537

TA97 / TA97a / TA1537

TG471 compliant Ames must include one of these strains However

All detect frameshift mutagens TA97 (and TA97a) engineered to be a more sensitive derivative of TA1537 Do data support use of ‘any’ or is one strain more sensitive than others?

Additionally, low background count in TA1537 can be problematic

TA97 & TA97a TA1537Allele/strain hisO1242, hisD6610 hisC3076

DNA target -C-C-C-C-C-C- (+1 cytosine at run of C’s)

+1 frameshift (near –C-C-C- run)

uvrB-bio genes Deletion mutation Deletion mutationLPS defect rfa rfaPlasmid pKM101 No plasmid

Previous publications

TA97 or TA97a?

TA97a is a ‘reconstruction’ of TA97 from the Ames lab Same phenotype

It would not be possible for current laboratories to know whether they were using TA97 or TA97a Recommend reclassification as

TA97/a

Where data has been classified as TA97 or TA97a, there is very little overlap Data pooled for both for this

analysis

TA97 / TA1537 Selective ResponsesReminder:Extracted data, Identified ‘author call’ selectives,Reviewed data and verified

10/247=4.0%

100 +vein Both

10 Selective +ve in TA1537137 Selective +ve in TA97


10 Selective Positives (TA1537 +ve, TA97 –ve) S9 requirement

4/10 Required metabolic activation

No common chemotypes

All compounds had been tested in other Ames strains 6/10 Not active in other strains ( unique)

Statistical analysis of dose response data Most selective positives produce quantitatively similar response in TA97 and

TA1537

4/9 Compounds produce quantitatively similar responses in TA100 and TA1535

Only 33% (3/9) are considered to be selectively mutagenic in TA1537

Mutagenic Potencies (rev/µg) of Agents Indicated in TA1537 and TA97 From a Variety of Studies

TA1537 TA97 + in onlyChemical +S9 -S9 +S9 -S9 TA15371-(4-Bromophenyl)-3,3-triazine 1.5 0.05 1.0 1.02-Methylpropanenitritle - 0.04 - 0.009Altertoxin I 1.7 3.2 0 1.3Amiloride hydrochloride NDa 0.1 ND 0.09Chlorambucil 5500 ND - ND XC.I. Basic Red 0.2 0.2 - - XRestrorsine - ND - ND Non-mut.Ro 60-0441 0.1 0.05 - - XZ-2,3-Dimethyloxypropenylbenzene ND - ND - Non-mut.

TA97/a selective mutagens -method Late-breaking idea, so could not review all data

Selected a sample of compounds (16) where A selective response in TA97/a over TA1537 had been reported

i.e. TA97 +ve, TA1537 -ve

Dose-response data were readily available via the NTP website

The sample covered a variety of chemotypes Perhaps an over-representation of aromatic amines

Statistical Analysis on Sample of TA97/a Selective Compounds

Of these 16 compounds• 1 is non-mutagenic• 2 are mutagenic in

both strains; in both cases, the potencies of these agents are 100 times less in TA1537 compared to TA97.

• 13 are selectively mutagenic for TA97/a

TA97/TA1537 Summary

Results pooled for phenotypically identical TA97 and TA97a Analysis of the dose response data confirmed only 10 compounds

(4.0%) with selective activity in TA1537 Further analysis on selective compounds

No common chemotypes observed amongst selective positives All compounds also had data for activity in other strains

6/10 are also uniquely positive in TA1537

9/10 compounds had accessible dose-response data Only 3/9 were confirmed selective (quantitatively different responses in TA97

and TA1537) In a sample of TA97 selective positives 13/16 were confirmed selective by

statistical analysis

E. Coli WP2 uvrA vE. Coli WP2 uvrA pKM101vS. Typh TA102

E.coli WP2 uvra (w/wo pKM101)v TA102 TG471 compliant Ames must include one of these strains However

All detect base-pair mutagens

Do data support use of ‘any’ or is one strain more sensitive than others?

E.coli WP2 uvrA(w/wo)

TA102

Allele/strain trpE56 hisG428

DNA target Ochre TAA (ochre)uvrB-bio genes <uvrA-> Wild typeLPS defect rfaPlasmid w/wo pKM101 pKM101, pAQ1

Previous publications

E.coli WP2 uvraA ±pKM101 v TA102Reminder:Extracted data, Identified ‘author call’ selectives,Reviewed data and verified

12 9 4 21 21 3 427

+ve in E.coli WP2 uvrA pKM101 --- +ve in E.coli WP2 uvrA --- +ve in TA102


4 Selective Positives Positive in TA102 or E.coli WP2 uvrA and negative in E.coli WP2 uvrA pKM101

Includes 2 anthraquinones, bleomycin and mitomycin-C

All compounds had been tested in other Ames strains 2/4 Not active in other strains (bleomycin, mitomycin-C unique)

No statistical analysis of dose response data

TA102/E.coli Summary

Analysis of the dose response data confirmed only 4 compounds not detected by E.coli WP2 uvrA pKM101

Further analysis on selective compounds One common chemotypes observed amongst selective positives

All compounds also had data for activity in other strains 2/4 are also uniquely positive in TA102

Conclusions

Inclusion of TA1535 offered little additional information to a test battery that also included TA100.

More compounds showed selective activity in TA97/a than in TA1537. This suggests that an Ames test battery containing TA1537 would be less sensitive than one containing TA97/a.

More compounds showed selective activity in E.coli WP2 uvrApKM101 than in either E.coli WP2 uvrA or TA102. This suggests that an Ames test battery containing the latter strains would be less sensitive than one containing E.coli WP2 uvrA pKM101.

In the future

Write-up and publish analysis

Recommendations: retain or remove less sensitive strains: TA1535, TA1537, E.coli WP2 uvrA, TA102

Results from focused testing will help with decision making Likely that publication will bring forth additional proprietary data sets

Also, need to consider current Ames test battery

Future recommendations –a comparator Our data suggest that TA1535, TA1537 and E.coli WP2 urvA add relatively little to the

sensitivity of a TG471-compliant Ames test Would it be reasonable to recommend removal from the strain list in TG471?

Relatively little sensitivity loss is not the same as no sensitivity loss: what would be acceptable?

Similar decisions have precedence How many strain positives are from non-std strains

i.e. not: TA98, TA100, TA97, TA1535, TA1537, TA102, E. coli WP2 urvA, E. coli WP2 uvrA pKM101?

Examination of 10,423 compounds in the Leadscope database with bacterial mutation studies 5764/10423 compounds called negative in the std battery

1356/5764 of these negatives were also tested in non-std strains (e.g. TA104 or TA1538) 40/1356 of these tested positive in at least one non-std strain

So, 1%-3% of all ‘known mutagens’ are not positive in the current Ames test battery

The end, for nowQUESTIONS?

Extra slides

Example: Selectivity ConfirmedDichloro(methyl)(vinyl)silane, 124-70-9, C[Si](C=C)(Cl)Cl

Condition Pos in other strains

Other in vitro data

In vivo data

Carcinogen?

Ham S9 No MLA (neg) - -

TA1535 + Ham S9TA100 + Ham S9

Data from NCI

Example: Selectivity Disputed

https://manticore.niehs.nih.gov/cebssearch/test_article/110 69 0

Butanal Oxime, 110-69-0, CCC/C=N/O

Inconsistent intra-strain results

https://manticore.niehs.nih.gov/cebssearch/test_article/110-69-0

Results

Not selective

Selective, but not potent

Selective & potent

Butanal Oxime

Butanal OximeTA100 10% Rat S9

Dose (µg/plate)

rev/

plat

e

0 500 1000 15000

50

100

150

200

R2 = 0.95Slope = 0.07 ± 0.01 rev/µg

P = 0.02

Butanal OximeTA1535 5% Rat S9

Dose (µg/plate)

rev/

plat

e

0 1000 2000 3000 40000

10

20

30

40Exp 1Exp 2

R2 = 0.85 and 0.72Slope = 0.006 and 0.005 rev/µgP = 0.008 and 0.15

Butanal oximeTA1535 10% Rat S9

Dose (µg/plate)

rev/

plat

e

0 1000 2000 3000 40000

10

20

30

40

50Exp 1Exp 2Exp 3

R2 = 0.70 to 0.97Slope = 0.005 rev/µg

P = 0.01 to 0.04

1 + and 1 – Exp inTA100

Replicated + resultsin 5% and 30% S9 In TA1535

Mutagenic Potency of Butanal Oxime (Rev/µg)

TA100 TA1535

10% Rat 5% Rat 10% Rat 30% Rat

0.06 & Neg. 0.006 Neg 0.005

6-Mercaptopurine6-Mercaptopurine monohydriate

TA1535 -S9

Dose (µg/plate)

rev/

plat

e

0 20 40 60 800

50

100

150

200

-S9 2-S9 3

R2 = 0.89 to 0.98

P = 0.005 to 0.01slope = 2.7 rev/µg

6-Mercaptopurine monohydriateTA1535 + Rat S9

Dose (µg/plate)

rev/

plat

e

0 10 20 30 400

100

200

300

400 Exp 1Exp 2

R2 = 0.94 to 0.99slope = 7.5 rev/µg

P = 0.001 to 0.004

6-Mercaptopurine monohydrateTA100

Dose (µg/plate)

rev/

plat

e

0 10 20 30 40100

120

140

160

180

200

Hamster S9

Rat S9

-S9

P = 0.1 to 0.8

Mutagenic Potency of 6-Mercaptopurine (Rev/µg)

TA100 TA1535-S9 Rat Ham -S9 Rat Ham Neg Neg Neg 3.0 7.3 - / 2.6

Clearly mutagenicOnly in TA1535

Acetaldehyde Oxime –S9Acetaldehyde oxime -S9

Dose (µg/plate)re

v/pl

ate

0 2000 4000 60000

50

100

150

TA1535

TA100

R2 = 0.91slope = 0.02 rev/µgP < 0.0001

Mutagenic Potency of Acetaldehyde Oxime (Rev/µg)

TA100 TA1535

Neg 0.2

Clearly positive Only in TA1535

TA1537 Selective Positives:No Common Chemotypes

Are All Ames Strains in the OECD Mutagenicity Test ... All...The Ames Test Revisited Ames Test =...

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