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T. Cachet
EFSA / WHO Stakeholder Meeting
2nd December, 2014
Revision of the Cramer
Decision Tree
2
The Cramer/Ford/Hall Decision Tree
Published in 1978 Food Cosmet. Toxicol. (1978) 16, 255-276
Screening for toxicity testing
Classified substances : Structure (key)
Metabolism
Toxicology
Natural occurrence (in body and/or food)
Validated against chemicals with data on biological and toxicological properties (pesticides, drugs, food additives, industrial chemicals, flavorings, fragrances)
3
Heterocyclics
Alicyclics
Open chain
Heteroaromatics
Aromatics
The Original Cramer/Ford/Hall Decision Tree
4
Class I Structures and related data suggest a low order of
oral toxicity. If combined with low human exposure, require a low priority for investigation
Class II Less clearly innocuous than Class I, but no firm
indication of toxicity or the lack thereof
Class III Structure and related data permit no initial
presumptions of safety, or may suggest significant toxicity. These substances deserve the highest priority for investigation
Class Structure
5
Relation of DT Class to Threshold of Toxicological Concern
(TTC) (Munro, 1996)
Structural Class
(examples)
TTC
(µg/person/day)
I
(ethyl butyrate, cinnamaldehyde)
1800
II
(3,6-dimethylpyrazine, pulegone)
544
III
(estragole, anethole)
90
6
Role of the Decision Tree within Flavoring Safety Assessments
• Close to 3000 flavoring substances have been evaluated in
procedures that rely on the Cramer/Ford/Hall DT as critical
component to guide the safety assessment
– JECFA
– EFSA
• US-based Flavor and Extract Manufacturers Association (FEMA)
Expert Panel has utilized the Cramer/Ford/Hall DT as critical part of
safety assessment process for the constituent-based assessment of
natural complex substances used as flavoring ingredients since
publication of a paradigm in 2005
• This has given us the opportunity to better understand the strength
and also some of the weaknesses of the Cramer decision tree
7
Coumarin
DT Class III
Anethole
DT Class III
Carvone
DT Class II
Pulegone
DT Class II
Dihydrocoumarin
DT Class III
Estragole
DT Class III
DT: Reconciling New Knowledge
8
Flavor Industry Work on Revising Cramer/Ford/Hall DT
• In a co-funded project, IOFI and FEMA have developed
suggestions for strengthening the Cramer/Ford/Hall DT
“Package”
• Some proposed reworking of the DT questions/routing
• Updating of the database that supports the application
of the TTC concept to the DT
– Expanded Munro database
– Opportunity to further strengthen and even expand
current TTC classes
9
Revise “trunk” of the tree steps
Steps lacking biochemical basis eliminated
Biological normality Step 1
Common component of food Step 22
Remove need for ‘lookup’ lists
Increase elements
Other than C, H, O, N, divalent S, to include
higher oxidation state S, Cl, F, and P in a
biologically stable oxidation state
Proposed Revisions
10
Factors taken into consideration during revision
• Skeletal structure
• Functional group
• Presence or absence of other functional groups
• Extent of conjugation
• Impact of electron donating groups
• Positional & geometric isomers
11
• Low Concern Substances: predominantly undergo detoxication or
complete metabolism via high capacity enzyme-catalyzed pathways
– oxidation in the TCA cycle, fatty acid pathway, cytosolic carbonyl
reduction, etc.
• Medium Concern Substances: excellent substrates for low capacity
detoxication pathways leading to stable, excretable metabolites
• High Concern Substances: weak substrates for low capacity
detoxication enzyme pathways leading to stable, excretable
metabolites or metabolites of unknown stability
myrcene(FEMA 2762)
CYP450 oxidation/hydroxylationor
epoxidation/hydrolysis
Consideration of Biochemistry
12
• “weak” substrates for low capacity intoxication pathways
leading to reactive metabolites
• “excellent” substrates for low capacity intoxication
pathways leading to reactive metabolites
MeO
MeO
MeO
MeO
OH
OO
Even Higher Biochemical Concern
Could Argue for Additional Structural Classes
13
• Low Concern Substances/metabolites w/functional
moieties
– Substance/metabolites unlikely to react with biomolecules in vivo
leading to adverse response
• Medium Concern/metabolites
– Interact with biomolecules → products unlikely to result in
adverse biological response
OH
O
NH
O
OH
O
O
OH
Consideration of Chemistry
14
8
2
15
7
1
4
2
3
6 5
Class 1Class 3
Class 3
Class 1
Class 3
Class 2
16
2917
18(A-I)
18(J-M)
Class 1
Cl.3 Cl.1
29
Class 3
20
19
21
22 Class 3
Class 217
29 24
23
25
26
Class 3
28
27
Class 3
Class 2
18(A-I)
18(J-M)
Class 1
9A
159C
9B10Class 3
11 Class 3
2912
Class 213
Class 314
29 Class 3
Class 3Class 1
Cl.3
Cl.1Cl.2
29 Cl.3
Cl.1
Cl.3
Cl.1
Class 3
Cl.3
Cl.2
18(A-I)
18(J-M)
Cl.1
Treat hydrolysis products separately
Cl.3 9D
As of 3/2014
Alicyclics
Aromatics
Open chain Heterocyclic
Lactone
Heteroaromatic
Proposed Revisions would lead to Revised Structure
Note that Structure Class
assignments to question
outcome are based on
current tree at this stage
15
Proposed Revisions: Work Continues!
• Relation of new question outcomes to Structure
Class Assignments?
16
Chemical Space:
Foundation: Munro Database (~ 600 chemically defined
compounds)
Additions (~1400 chemically defined compounds): 1. Flavoring Substances
2. Compounds with published studies
National Toxicology Program (NTP) Studies (USA)
Environmental Protection Agency (EPA) Studies (USA)
Scientific Literature
NOEL/NOAEL Values determined for each study are extracted.
Improving and Refining the Munro Database
Munro, I.C. et al. (1996) “Correlation of Structural Class with No-Observed-Effect Levels: A
Proposal for Establishing a Threshold of Concern”, Food and Chemical Toxicology.”, 34, 829 -
867.
17
Studies: Foundation: Munro Database
• Exclusively oral (dietary, gavage) studies
• Mainly chronic studies (short term and acute studies not
included)
Additions:
• Chronic and short term oral studies added (Some studies suggest the validity of using a conversion factor to
equate NOAEL values between studies of differing lengths.)
Improving and Refining the Database
18
Conclusions
• Overall, we trust that both the review of
the Cramer Decision Tree and
strengthening the Munro Database will
increase the robustness of the TTC
concept and we are
working/planning towards sharing our
data with the scientific community
including the WHO/EFSA review project
on TTC