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Office of the Report on Carcinogens
Gloria D. Jahnke, DVM, DABT
National Toxicology Program
National Institute of Environmental Health Sciences
Read-Across Workshop May 2 & 3, 2019
Oakland, CA
Evaluation of Haloacetic Acids: A test case for chemical grouping
Haloacetic Acids (HAA) Found as Water Disinfection By-Products
Outline
Background on Report on Carcinogens (RoC)
13 HAAs found as water disinfection by-products for evaluation for the RoC, problem formulation, protocol development
Approach as a class, subclasses, or individual chemicals
RoC listing recommendations
• Identifies substances that pose a cancer hazard to people residing in the United States
– Two listing categories: known and reasonably anticipated to be a human carcinogen
• Substance profile is written for each listing
– Listing status, scientific information key to listing and data on properties, uses, production, exposure, and regulations to limit exposure
• Each edition of the report is cumulative
• NTP prepares the RoC for the Secretary of the Department of Health and Human Services using a four-part formal process and established listing criteria
The Report on Carcinogens (RoC) is congressionally mandated
http://ntp.niehs.nih.gov/go/roc
RoC Listing Criteria: Two Categories
• Sufficient evidence of carcinogenicity from studies in humans
Known to be a human carcinogen
• Limited evidence from studies in humansOR
• Sufficient evidence from studies in experimental animalsOR
• Belongs to well-defined structurally related class of substances listed in the RoC or demonstrates convincing mechanistic evidence
Reasonably anticipated to be a human carcinogen
Conclusions based on scientific judgment considering all relevant information
such as chemical structure, metabolism, pharmacokinetics, genetic effects,
and mechanisms of action.
Page viii, https://ntp.niehs.nih.gov/go/rocprocess
• What are water disinfection by-products?
– Formed by reaction of vegetative material or other organic materials, such as chemical pollutants, in water with antimicrobial oxidizing agents such as chlorine, chloramine, chlorine dioxide, or with naturally occurring halides.
– Composition varies with water source, method of disinfection, season of the year.
– Found in public water supply, including swimming pools and spas.
• Over 500 chemicals have been identified.
– Trihalomethanes: 58% by weight of halogenated by-products.
– Haloacetic acids: 36% by weight of halogenated by-products.
Water Disinfection By-Products
Chemicals in red text have animal cancer data
A = acetic acid, C = chloro, B = bromo, I = iodo,
M = mono, D = di, T = tri
Chemical Structures for HAAs Found in Treated Water
Problem Formulation: Literature Review
Systematic literature search
Evidence mapping
Protocol development
Cancer hazard
evaluation
Peer review and final
listing decision
2
4
8
12
12
13
24
40
40
53
64
87
98
Immortalization
Alters DNA repair
Toxicogenomic/gene expression
Electrophilic
Inflammation/immunomodulation
Epigenetic
Animal cancer
Oxidative stress
Receptor mediated
GST-ζ inhibition
Cell proliferation, death, nutrient supply
Genotoxic
ADME & TK
Evidence mappingStudies screened and sorted
for review
• Scientific input
• All 13 HAAs
• Develop read-across
approach
6630 references
for 13 HAAs
Gold: problem formulation steps
Blue: cancer hazard evaluation
Problem Formulation: Protocol Development
Systematic literature search
Evidence mapping
Protocol development
Cancer hazard
evaluation
Peer review and final
listing decision
2
4
8
12
12
13
24
40
40
53
64
87
98
Immortalization
Alters DNA repair
Toxicogenomic/gene expression
Electrophilic
Inflammation/immunomodulation
Epigenetic
Animal cancer
Oxidative stress
Receptor mediated
GST-ζ inhibition
Cell proliferation, death, nutrient supply
Genotoxic
ADME & TK
Evidence mappingStudies screened and sorted
for review
• Scientific input
• All 13 HAAs
• Develop read-across
approach
6630 references
for 13 HAAs
Gold: problem formulation steps
Blue: cancer hazard evaluation
Chemical class and cancer hazard: a test case for class grouping/read across?
• Haloacetic acids are structurally similar.
• Published QSARs on ex vivo study of neural tube defects
• Available studies on ADME/TK
• Cancer studies in experimental animals
• Available data on10 Key Characteristics of Carcinogens (KCs) and relative chemical potencies
• Comparative test array data across HAAs for concurrent measurements of properties, oxidative stress, toxicokinetic parameters, genetic toxicity.
Protocol Development
Cancer Hazard Evaulation
Systematic literature search
Evidence mapping
Protocol development
Cancer hazard
evaluation
Peer review and final
listing decision
2
4
8
12
12
13
24
40
40
53
64
87
98
Immortalization
Alters DNA repair
Toxicogenomic/gene expression
Electrophilic
Inflammation/immunomodulation
Epigenetic
Animal cancer
Oxidative stress
Receptor mediated
GST-ζ inhibition
Cell proliferation, death, nutrient supply
Genotoxic
ADME & TK
Evidence mappingStudies screened and sorted
for review
• Scientific input
• All 13 HAAs
• Develop read-across
approach
6630 references
for 13 HAAs
Gold: problem formulation steps
Blue: cancer hazard evaluation
Approach for Evaluation of 13 HAAs
Figure 2
Endpoint 3 mono HAAs
6 di HAAs
4 tri HAAs
Properties Electrophilicity (ELUMO), pKa
TK Metabolism, clearance, trends
KCs/other Comparative potencies, trends
Animal cancer data
Species/tumor sites, BMDLs
•Define class
•Predicts toxicity
•Inform read-across
Properties
•Inform MOA
•Inform read-across ADME and
TK
•Inform cancer hazard of 6 HAAs
•Inform read-across
Animal cancer studies
•Inform strength of mechanistic evidence
•Inform read-across
•Inform MOA
Key carcinogen character-
istics
Evaluate evidence of 13 HAAs Conduct "read-across" approaches Integrate evidence & reach cancer hazard conclusions
Listing
Recommendations
RoC Listing Criteria
Animal cancer studies
ADME and TK
Read across
KC
•Data with animal cancer data: MCA, DCA, BCA ,DBA, TCA, DBCA
13 HAAs as a class
•Based on numbers and/or types of halogens
7 sub-classes
•Targets: Two brominated haloacetic acids (CDBA, TBA)
Specific HAAs
; Study quality
Approach for Evaluation of 13 HAAsFigure 2
Endpoint 3 mono HAAs
6 di HAAs
4 tri HAAs
Properties Electrophilicity (ELUMO), pKa
TK Metabolism, clearance, trends
KCs/other Comparative potencies, trends
Animal cancer data
Species/tumor sites, BMDLs
•Define class
•Predicts toxicity
•Inform read-across
Properties
•Inform MOA
•Inform read-across ADME and
TK
•Inform cancer hazard of 6 HAAs
•Inform read-across
Animal cancer studies
•Inform strength of mechanistic evidence
•Inform read-across
•Inform MOA
Key carcinogen character-
istics
Evaluate evidence of 13 HAAs Conduct "read-across" approaches Integrate evidence & reach cancer hazard conclusions
Listing
Recommendations
RoC Listing Criteria
Animal cancer studies
ADME and TK
Read across
KC
•Data with animal cancer data: MCA, DCA, BCA ,DBA, TCA, DBCA
13 HAAs as a class
•Based on numbers and/or types of halogens
7 sub-classes
•Targets: Two brominated haloacetic acids (CDBA, TBA)
Specific HAAs
Class
• All 13 HAAs as a potential class
Subclasses
• 7 Potential subclasses
Analogues (metabolism)
• Individual untested HAAs (carcinogenicity)
Approach for Evaluation of 13 HAAsFigure 2
Endpoint 3 mono HAAs
6 di HAAs
4 tri HAAs
Properties Electrophilicity (ELUMO), pKa
TK Metabolism, clearance, trends
KCs/other Comparative potencies, trends
Animal cancer data
Species/tumor sites, BMDLs
•Define class
•Predicts toxicity
•Inform read-across
Properties
•Inform MOA
•Inform read-across ADME and
TK
•Inform cancer hazard of 6 HAAs
•Inform read-across
Animal cancer studies
•Inform strength of mechanistic evidence
•Inform read-across
•Inform MOA
Key carcinogen character-
istics
Evaluate evidence of 13 HAAs Conduct "read-across" approaches Integrate evidence & reach cancer hazard conclusions
Listing
Recommendations
RoC Listing Criteria
Animal cancer studies
ADME and TK
Read across
KC
•Data with animal cancer data: MCA, DCA, BCA ,DBA, TCA, DBCA
13 HAAs as a class
•Based on numbers and/or types of halogens
7 sub-classes
•Targets: Two brominated haloacetic acids (CDBA, TBA)
Specific HAAs
Class
• All 13 HAAs as a potential class
Subclasses
• 7 Potential subclasses
Analogues (metabolism)
• Individual untested HAAs (carcinogenicity)
Mono-haloacetic acids
MCA
MBA
MIA
Di-haloacetic acids
DCA
DBA
DIA
BCA
CIA
BIA
Tri-haloacetic acids
TCA
TBA
BDCA
CDBA
Experimental Animal Cancer Studies on Six of the HAAs
Chemicals in blue have sufficient cancer data to meet RoC listing criteria
A = acetic acid, C = chloro, B = bromo, I = iodo,
M = mono, D = di, T = tri
Neoplasm or tissue
DCA DBA BCA TCA BDCA
Rats Mice Rats Mice Rats Mice Rats Mice Rats Mice
M F M F M F M F M F M F M F M F M F M F
Liver X X X X X X X X X X X
Mononuclear-cell
leukemia X
Malignant
mesothelioma X X X
Mammary gland X X
Lung X
Skin X
Harderian gland X
Large intestine X X
DCA = dichloroacetic acid, DBA = dibromoacetic acid, BCA = bromochloroacetic acid, TCA = trichloroacetic acid, BDCA =
bromodichloroacetic acid.
Evidence of Cancer in Experimental Animals
• MCA had no evidence of cancer in rats or mice in a 2-yr cancer bioassay.
• Brominated HAAs were associated with multiple cancer sites.
Endpoint
Mono-
HAAs
Di-
HAAs
Tri-
HAAs
Properties
(reactivity)
Electrophilicity (ELUMO), pKa
Metabolism &
Toxicokinetics
Comparative data
Mechanistic data Comparative data: potency
Animal
cancer data
Br-HAAs more cancer sites
than Cl-HAAs
BMDLs for quantitative
assessment
• Category approach
– What are physical/chemical and biological properties?
– Can these be grouped as a class or subclasses based on structure?
– Used comparative data across mono-, di-, tri- HAAs from individual laboratories to help determine what compounds have similar properties.
– Heat map in supplemental slides have data evaluated.
Biological effects varied with number and type of halogens
Conduct “Read-Across” Analysis
* Graphical representation (Table 7-1) in the Monograph captures all data evaluated
• Multiple modes of action (MOAs)
– Metabolism via reductive dehalogenation major triHAApathway (free radical intermediate)
– Disruption of energy metabolism, mitochondrial stress
– Metabolism via GST-ζ major diHAA pathway – leads to GST-ζinhibition (minor pathway reductive dehalogenation)
– Activate Nrf2/ARE pathway
– Oxidative damage DNA
– Lipid peroxidation
– PPARα activation
• Positive correlation with genotoxicity
– Treatment with antioxidants reduced genotoxicity
All HAAs Induced Oxidative Stress
Potency decreases with the number of halogens
Oxidative Stress: Nrf2/ARE assay
Source: Stalter et al. 2016
AREc32 = MCF-7 breast cancer cell line
ARE-bla = HepG2 hepatocellular carcinoma cell line
0
0.5
1
1.5
2
2.5
3
MCA MBA MIA DCA BCA DBA CIA BIA TCA BDCA CDBA TBA
log(1/EC+1)(m
M;IR=1.5)
AREc32 ARE-bla
Mono-HAAs Di-HAAs Tri-HAAs
Potency increases with the type of halogen: I > Br >>Cl
Oxidative Stress: Nrf2/ARE assay
Source: Stalter et al. 2016
AREc32 = MCF-7 breast cancer cell line
ARE-bla = HepG2 hepatocellular carcinoma cell line
0
0.5
1
1.5
2
2.5
3
MCA MBA MIA DCA BCA DBA CIA BIA TCA BDCA CDBA TBA
log(1/EC+1)(m
M;IR=1.5)
AREc32 ARE-bla
Mono-HAAs Di-HAAs Tri-HAAs
Brominated analogues more potent than clorinated
Oxidative Damage: In Vivo Mouse Liver
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
DCA DBA BCA TCA BDCA
FoldIncrease
TBARs 8-OHdG
Sources: Larson and Bull 1992, Austin et al. 1996
Di-HAAs Tri-HAAs
(nmol/g liver) /105 dG liver
0.000
1.000
2.000
3.000
4.000
5.000
6.000
MCA MBA MIA DCA BCA DBA CIA BIA DIA TCA BDCA CDBA TBA
log1/GP(M
)
p53-bla Comet
Potency decreases with the number of halogens
DNA Damage in Mammalian Cells
Sources: Stalter et al. 2016, Plewa et al. 2010
p53-bla = HCT-116 human colon carcinoma cell line
Comet assay = CHO cells
Mono-HAAs Di-HAAs Tri-HAAs
KCs Mono- Di- Tri-
Electrophilic All All All
Ox stress All All All
Genotoxic All All All (*TCA)
Hypomethyl-
ation
- DCA
DBA
TCA
GAPDH
inhibition
All - -
PDK inhibition - DCA -
PPARα - - TCA
Cell trans-
formation
MIA DBA -
Summary
• Associated with many of the characteristics of carcinogens (KCs); no single MOA for carcino-genicity
• General trends observed for several relevant endpoints, e.g., oxidative stress, genotoxicity, electrophilicity
– I > Br >> Cl
– Mono > Di > Tri- HAAs
• Soft electrophiles, bind to proteins
and nucleic acids
Biological Effects Varied with Number and Type of Halogens
HAAs associated with various
characteristics of carcinogens (KCs)
- = no studies found; *TCA was
negative for genotoxicity
• Published Benchmark Dose Low (BMDL, mg/kg/day)
• 95% lower confidence limit on dose = 10% extra risk response relative to background
• TCA > DCA > BCA > BDCA > DBA
• Predicted Toxic Dose 50 (TD50, mg/kg/day)
• Chronic dose-rate in mg/kg body wt/day which would induce tumors in half the test animals at the end of a standard lifespan for the species.
• (TCA = DCA) > (TBA = DBA = BA) > (IA = DIA)
• MCA predicted among most potent in rats and mice
• Trends in cancer potency using chronic cancer data:
• Brominated HAA forms had more cancer sites than DCA or TCA
• CA was negative in chronic cancer bioassay.
• TCA has one site (mouse liver) most likely due to a separate mechanism (PPAR-alpha activation)
QSAR Attempts Failed to Predict Carcinogenicity
HAAs as a Class
• Lack of a clear trend in cancer potency; estimates did not follow known cancer outcomes
– Estimates do not account for number of tumor endpoints
• Lack of carcinogenicity of MCA
• Lack of a common MOA among HAAs
– Differences in metabolism with number of halogen substitutions.
– TCA non-genotoxic, possibly causing liver tumors by an independent mechanism (PPAR alpha)
• Limitations of dataset:
– Few data on iodinated HAAs; few in vivo genetic toxicity data
– Relying on predictions of in vitro data, albeit tested concurrently
Challenges – HAAs as a Class
Subclass Members*
Confidence for read across based
on cancer outcomes and properties
Mono-HAAs CA, BA, IA No
Di-HAAs DCA, DBA, BCA, DIA,
CIA, BIA
Low
Tri-HAAs TCA, BDCA, CDBA,
TBA
Low
Cl-HAAs CA, DCA, BCA, CIA,
TCA
No
Br-HAAs BA, DBA, BCA, BIA,
TBA, BDCA, CDBA
Low/moderate
I-HAAs IA, DIA, CIA, BIA No
Br-Di-/Tri-HAAs DBA, BCA, BIA, BDCA,
CDBA, TBA
Moderate BEST POTENTIAL
-but no cancer data on iodinated HAAs
Seven subclasses evaluated, but no common MOA and inconsistencies in cancer data among most subclasses
HAA Subclasses
* Red = rodent carcinogens, Blue = not carcinogenic, black = not tested
HAAs as a subclass
• Challenges similar to those of class evaluation
• Lack of cancer data for iodinated HAAs and mono HAAs
• TCA was nongenotoxic and may cause cancer by separate mechanism (PPAR-alpha)
• Data gaps we considered for testing:
– Validate key cancer events across HAAs such as for iodinated HAAs, e.g., short-term cell transformation or immortalization assays; short-term in vivo assays (e.g., genotoxicity, oxidative stress ); subchonic toxicity data or transcriptomic data.
Challenges
• Br substitution for Cl enhanced metabolism
– TCA DCA
– BDCA DCA
– CDBA BCA
– TBA DBA
Red = rodent carcinogens; Black = untested
• TBA and CDBA metabolized to animal carcinogens
– No other rat or microsomal metabolites identified; same metabolites found with human microsomes.
– Br loss from Tri-HAA corresponded 1:1 to Di-HAA formation.
Tri-HAAs with both Cl and Br always lose a Br
Evaluation of Individual HAAs Using Analogue Approach:
Hypothesis Based on Cell Transformation to a Carcinogen
Species/
Tumor typeTested chemicals Untested chemicals
BCA DBA BDCA CDBA TBA
Rats ✔ ✔ ✔
MCL ⎼ ✔ ⎼
Mesothelioma ✔ ✔ ✔
Mammary ✔ ⎼ ✔
Skin ⎼ ⎼ ✔
Mice ✔ ✔ ✔
Liver ✔ ✔ ✔
Lung ⎼ ✔ ⎼
Harderian gland ⎼ ⎼ ✔
All Tested Br-HAAs are Rodent Carcinogens
✔ = tumor site, ⎼ = not a tumor site
Species/
Tumor typeTested chemicals Untested chemicals
BCA DBA BDCA CDBA TBA
Rats ✔ ✔ ✔
MCL ⎼ ✔ ⎼
Mesothelioma ✔ ✔ ✔
Mammary ✔ ⎼ ✔
Skin ⎼ ⎼ ✔
Mice ✔ ✔ ✔
Liver ✔ ✔ ✔
Lung ⎼ ✔ ⎼
Harderian gland ⎼ ⎼ ✔
CDBA and TBA are metabolized to rodent carcinogens; TBA and CDBA have similar properties to BDCA
Metabolism
✔ = tumor site, ⎼ = not a tumor site
Species/
Tumor typeTested chemicals Untested chemicals
BCA DBA BDCA CDBA TBA
Rats ✔ ✔ ✔ Predicted Predicted
MCL ⎼ ✔ ⎼
Mesothelioma ✔ ✔ ✔ Likely site Likely site
Mammary ✔ ⎼ ✔
Skin ⎼ ⎼ ✔
Mice ✔ ✔ ✔ Predicted Predicted
Liver ✔ ✔ ✔ Very likely
site
Very likely
site
Lung ⎼ ✔ ⎼
Harderian gland ⎼ ⎼ ✔
CDBA and TBA are predicted to be rodent carcinogens
Prediction
✔ = tumor site, ⎼ = not a tumor site
• Peer review panel agreed with NTP conclusions that
– DCA, DBA, CBA, BDCA are reasonably anticipated to be human carcinogens based on sufficient evidence in experimental animals and supporting mechanistic data
– CDBA and TBA are reasonably anticipated to be human carcinogens based on metabolism studies that provide convincing evidence that CDBA is metabolized to CBA and TBA is metabolized to DBA and supporting mechanistic data that demonstrate biological plausibility of its carcinogenicity in humans.
• The Final Monograph on HAA found as drinking water disinfection by-products is on the NTP ORoC webpage at: https://ntp.niehs.nih.gov/go/790113
• Next Steps: The substance profile will be submitted to the Secretary DHHS for approval. If approved, listing will be in the 15th RoC.
Conclusions and Next Steps
Acknowledgments
Monograph Preparation
NTP/ORoC
Gloria Jahnke, Co-Project Lead
Ruth Lunn, Director ORoC
Suril Mehta
Amy Wang
ILS, Inc.*
Stan Atwood, Co-Project Lead
Sandy Garner, PI
Whitney Arroyave
Ella Darden
Andy Ewens
Jessica Geter
Alton Peters
Tracy Saunders
Technical Advisors and SupportRon Melnick, Consultant
Grace Patlewicz, US EPA/NCCT
Michael Plewa, Univ. of IL (emeritus)
Susan Richardson, Univ. of SC
Jane Ellen Simmons, US EPA
Scott Auerbach DNTP, NIEHS
Michael Devito, DNTP, NIEHS
Steve Ferguson, DNTP, NIEHS
Andy Shapiro, DNTP, NIEHS
*Contract Support
Questions?
Supplemental Slides
Relative HAA Potencies for Mechanistic and Chemical Endpoints*
*Table 7-1 HAA Monograph
Relative HAA Potencies for Mechanistic and Chemical Endpoints*
*Table 7-1 HAA Monograph
Endpoint
Monohaloacetic acids Dihaloacetic acids Trihaloacetic acids
CA BA IA DCA DBA DIA BCA CIA BIA TCA TBA BDCA CDBA
Toxicokineticsa
Oral bioavailability (%) 100 81 30 47 100 62 96 100
Unbound fraction (%) 27 94 89 93 53 18 49 55
Total clearance
(mL/kg/h)
262 267 491 1037 92.5 754 286 486
Renal (% of total) 59 1.1 2.6 3.6 45.5 22.7 31.1 37.4
Non-renal (% of total) 41 98.9 97.4 96.4 54.5 77.2 68.9 62.6
Chemical propertiesb
pKa 2.97 2.96 2.95 1.41 1.39 1.4 1.47 1.67 0.66 0.03 0.05 0.04
ELUMO (deprotonated) 9.43 8.68 7.18 8.44 7.51 7.78 6.40 6.46 7.13 6.12 6.65 6.42
TRGSH 0.71 2.18 2.15 1.68 1.48 1.93 1.35 0.62 1.01 2.26 1.56 1.1
TRDNA 0.92 0.84 0.81 0.5 1.9 3.18 1.17 1.47 0.99 2.61 0.73 0.95
Oxidative stress in vitroc
AREc32 3.7 192 278 0.17 8.3 7.1 45.5 38.5 N 2.3 0.5 0.2
ARE-bla 4 90.9 196 0.06 4 2.2 10 18.9 N 1.5 0.25 0.46
Oxidative stress in vivod
8-OHdG 1.4 2.9 2.9 1.2 1.7
TBARS 129 250 290 67 240
Genetox in vitro
SOS-umuCc,e 60 2400 15400 180 2564 2941 5263 9091 60 142860 9091 9091
Ames TA100 (-S9)f 44 6588 14129 36 183 N N
Ames TA100 (-S9)g 5.2 61.9 60.6 N 1.2 31.6 1.7
Comet CHO cellsh 2439 58820 114900 N 556 500 333 313 N 400 N 71
HGPRT CHO cellsi 8.7 14.6 836 2.8 66.2 N
P53-blac 5882 105260 212770 N 3846 4348 9091 9091 N N N N
Genetox in vivoj 0.5 1 N N N
PPARα in vitrok 2 20 100 1 1 2 1
PPARα in vivol N 2 3.5 N 4.3 N
GST-ζ inhibitionm 45 83 81 N
Animal carcinogenicity
Species/tumor siten N 2 3 3 1 3
BMDLo 0.49 0.04 0.08 0.67 0.06
Relative HAA Potencies for Mechanistic and Chemical Endpoints*
Darker to lighter shade = high to low value or potency, white = negative (N), gray = no data.
*Table 7-1 HAA Monograph