evaluation of haloacetic acids: a test case for chemical ... · of health and human services using...

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


Evidence mapping


Cancer hazard

evaluation


listing decision

2

4

8

12

12

13

24

40

40

53

64

87

98

Immortalization

Alters DNA repair


Electrophilic


Epigenetic

Animal cancer

Oxidative stress

Receptor mediated

GST-ζ inhibition


Genotoxic

ADME & TK


for review


• All 13 HAAs


approach

6630 references

for 13 HAAs



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


Evidence mapping


Cancer hazard

evaluation


listing decision

2

4

8

12

12

13

24

40

40

53

64

87

98

Immortalization

Alters DNA repair


Electrophilic


Epigenetic

Animal cancer

Oxidative stress

Receptor mediated

GST-ζ inhibition


Genotoxic

ADME & TK


for review


• All 13 HAAs


approach

6630 references

for 13 HAAs



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


Animal cancer studies

•Inform strength of mechanistic evidence


•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


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


Properties

•Inform MOA

•Inform read-across ADME and

TK

•Inform cancer hazard of 6 HAAs



•Inform strength of mechanistic evidence


•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


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


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


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/



Rats ✔ ✔ ✔

MCL ⎼ ✔ ⎼

Mesothelioma ✔ ✔ ✔

Mammary ✔ ⎼ ✔

Skin ⎼ ⎼ ✔

Mice ✔ ✔ ✔

Liver ✔ ✔ ✔

Lung ⎼ ✔ ⎼


CDBA and TBA are metabolized to rodent carcinogens; TBA and CDBA have similar properties to BDCA

Metabolism


Species/



Rats ✔ ✔ ✔ Predicted Predicted

MCL ⎼ ✔ ⎼

Mesothelioma ✔ ✔ ✔ Likely site Likely site

Mammary ✔ ⎼ ✔

Skin ⎼ ⎼ ✔

Mice ✔ ✔ ✔ Predicted Predicted

Liver ✔ ✔ ✔ Very likely

site

Very likely

site

Lung ⎼ ✔ ⎼


CDBA and TBA are predicted to be rodent carcinogens

Prediction


• 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

https://ntp.niehs.nih.gov/go/790113

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

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

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