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
Tested inboth strainsundercomparison
Produce selective response
s
Produce selective response
Produce selective
responses in both
databases
Confirmed selective responses
Extract
Refine
MergeApply
Criteria
Refine
Extract
AnalyseD
ata
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
This Photo by Unknown Author is licensed under CC BY-SA
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
Tested inboth strainsundercomparison
Tested inboth strainsundercomparison
Produce selective response
s
Produce selective response
Produce selective
responses in both
databases
Confirmed selective responses
Extract
Refine
MergeApply
Criteria
Refine
Extract
AnalyseD
ata
Proportion SelectiveCommon Chemotypes
Statistical AnalysisActivity in other strains
Results
TA100 / TA1535 Data Set Sizes
3667 Tested in
Both
4701 Tested in Both
Workflow
Tested inboth strainsundercomparison
Tested inboth strainsundercomparison
Produce selective response
s
Produce selective response
Produce selective
responses in both
databases
Confirmed selective responses
Extract
Refine
MergeApply
Criteria
Refine
Extract
AnalyseD
ata
Proportion SelectiveCommon Chemotypes
Statistical AnalysisActivity in other strains
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
Selective Positives: Further Analysis
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
Selective Positives: Further Analysis
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
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