researchers: dale hattis, principal investigator rob goble, research professor

Age-Related Differences in Susceptibility to Carcinogenesis—Toward an Improved Analysis of Data on Age-Related Differences in Cancer Sensitivity in the EPA Children’s

Cancer Risk Guidance Document

Researchers:Dale Hattis, Principal Investigator

Rob Goble, Research ProfessorAbel Russ, Research Associate

Jen Ericson and Jill Mailloux, Student Research Assistants

Margaret Chu, EPA Project Monitor

Opinions are mine and do not necessarily reflect EPA policy

INNOVATIVE ASPECTS OF THE ANALYSIS --Paper #1

• Compare measures of potency, rather than uncorrected cancer incidence, among groups.

• Where dosage spans multiple age groups, use dummy variables to represent the observed tumor risk as the sum of cancer contributions from dosing in different periods:– The periods are: fetal (gd 12-19), pre-weaning (1-21 d); weaning - 2 mo;

adult (2 mo - 2 yr).– Where continuous dosing occurs in only a fraction of a period that

fraction is used as the corresponding “dummy” rather than 1.• Use likelihood methods to first derive appropriate statistical

weighting of the different observations, and to avoid bias from excluding “0” points.

• Express dosage for animals of different weights on a metabolically consistent basis (either concentration in air or food, or per unit body weight to the three quarters power).

Paper #2--Monte Carlo Analysis of Uncertainties for Application to Human Risk Assessments

• Uncertainties in the central estimates of the sensitivities of each life stage per dose in mg/kg^3/4, relative to adults

• Uncertainties from chemical-to-chemical differences in life-stage related sensitivities

• Uncertainties in the mapping of comparative ages/times between rodents and humans

• Bottom line:--Overall expected increment to lifetime tumor risks from full lifetime constant exposure per mg/kg^3/4

The Poisson One-hit Transformation--From the Fraction of Animals with at Least One Tumor to The

Number of Tumors Per Animal

Fraction of Animals With Tumors = Ptumor = 1 - PNo Tumor

PNo Tumor = e-m where m = tumor "hits"/animalsolving for m:m =-ln(PNo Tumor) = - ln(1- Fraction of Animals With Tumors)

Effect of the One-Hit Transformation for Various Observations of % Tumors in Animal Groups

Fract Animals with Tumors at

a Site

Tumor Transformations

/Animal0.01 0.0100.1 0.105

0.25 0.2880.4 0.511

0.55 0.7990.7 1.204

0.85 1.8970.9 2.303

0.95 2.996

Use of Part-Period Dummy Variables in Combination To Represent Different Exposure Patterns--Maltoni Vinyl

Chloride Experiments

Exposure GroupBirth-weaning

(21 d) Weaning-2 mo AdultControl 0 0 0.000

5 weeks exposure beginning at birth 1 0.359 0.0005 weeks exposure beginning at 11 weeks of age 0 0 0.04352 weeks of exposure beginning at 13 weeks of age 0 0 0.411

Detailed Model for Statistical Fitting

€

Fraction with tumors = 1 - e- B + A(a + fF + cC + wW) ⎡

⎣ ⎢ ⎤ ⎦ ⎥

Where :B = group background transformations per animalA = group adult transformations per animal at the highest adult dose ratea = fraction of the adult period with dosing at the maximum adult rate (this term reflects an adjustment for cases where a group received less than the full adult dosing rate)f = fraction of the fetal period with dosing at the maximum adult rate (also adjusted for dose rate as needed)F = fetal/adult sensitivity ratioc = fraction of the birth - weaning period with dosing at the maximum adult rate (also adjusted for dose rate as needed)C = birth - weaning/adult sensitivity ratiow = fraction of the weaning - 60 day period with dosing at the maximum adult rate (also adjusted for dose rate as needed)W = weaning - 60 day/adult sensitivity ratio

Summary Results of Analyses for Paper #1

• Central estimate results: 5-60 fold increased carcinogenic sensitivity in the birth-weaning period per dose/(body weight3/4-day) for mutagenic carcinogens--no detectable increase for nonmutagens

• Somewhat smaller increase—about 5 fold—for radiation carcinogenesis per Gray

• Greater increase for mutagens for continuous, rather than discrete dosing protocols

• Greater increase in males than females• Similar increased sensitivity in the fetal period for direct-acting

nitrosoureas, but no such increased fetal sensitivity for carcinogens requiring metabolic activation

• Greater increase in early life sensitivity in liver, and less in lung, than for other tumor sites.

Overview of the Data Base

Dose Groups With Exposures in Specific Life Stages (and

numbers of animals*tumor-site observations in parentheses)

DosingProtocol

Number ofChemicals or

RadiationTypes

Total DoseGroups

ControlGroups Fetal Birth-Weaning

Weaning-60Days

Adult (60+Days)

Continuous 9 (5 mut) 151 (103 liver) 29 (2562) 14 (820) 62 (3071) 62 (6128) 85 (7544)Discrete (1-4X) 6 (all mut) 274 (90 liver) 45 (2926) 8 (290) 117 (4681) 85 (3596) 37 (979)Radiation 4 138 (42 liver) 21 (4283) 18 (1323) 18 (1744) 18 (1529) 63 (3668)

Media Concentration or Dose/BW^3/4 Dosimetry

Geometric Mean Ratios of Child/Adult Clearance/BodyWeight and Clearance/Body Weight3/4. Data RepresentRegression Results from 104 Data Groups for 27 Drugs forHumans in Various Age Groups (with ± 1 Standard ErrorRanges in Parentheses)

Form for ExpressingTotal Body Clearance

Prematureneonates

Full termneonates

1 wk - 2mo

2 - 6 mo 6 mo - 2yr

2 -12 yr 12 - 18 yr

Mg/Kg Body Weight 0.52(0.43-0.63)

0.66(0.61-0.73)

0.77(0.71-0.84)

1.21(1.06-1.39)

1.71(1.52-1.92)

1.42(1.31-1.53)

0.97(0.78-1.20)

Mg/(Kg BodyWeight)3/4

0.23(0.19-0.28)

0.31(0.28-0.34)

0.38(0.35-0.42)

0.68(0.59-0.78)

1.03(0.91-1.17)

1.08(1.00-1.17)

0.93(0.74-1.17)

Overall Results--Continuous vs Discrete Dosing Protocols (Caveat: Continuous dosing results include 4/9 nonmutagens)

All Continuous Chemical Dosing Experiments (based on a total of 151group tumor incidence observations for 9 chemicals).

MLE for cancer inductions perdose/(body weight.75 -day) relative

to adults 95% LCL 95% UCL

Fetal Period (8days beginningGD 12) 4.9 0.5 9.3

Birth-Weaning (21days) 8.7 6.5 10.8

Weaning-60 days(39 days) 0.000 0.000 0.24

All Discrete Chemical Dosing Experiments (based on a total of 274 grouptumor incidence observations for 6 chemicals).

MLE for cancer inductions perdose/(body weight.75 -day) relative





Overall Results--Radiation Exposures

All Ionizing Radiation Dosing Experiments (based on a total of 138 grouptumor incidence observations for 4 radiation types).

Maximum likelihood estimate ofcancer inductions per dose in rads

or Gray relative to comparablydosed adults 95% LCL 95% UCL




Age-Related Pharmacodynamic Sensitivity for Carcinogenesis--Mutagens vs Non-Mutagens--Continuous Dosing Protocols

Chemicals Classified by EPA as Mutagenic (5 compounds, 43 tumorincidence observations):

MLE of cancer inductions perdose/(body weight.75 -day) relative


Fetal 8.4 3.5 15.5

Birth-Weaning 24 17.1 34

Weaning-60 days 3.7 0.0 9.1

Chemicals Classified by EPA as Not Mutagenic (4 compounds, 108 tumorincidence observations in animal groups):



Fetal 0.0 0.0 17.4

Birth-Weaning 3.0 0.0 4.7

Weaning-60 days 0.0 0.0 2.0

Different Results for Mutagens by Sex--Continuous + Discrete Dosing Data Combined

Male Animals (9 compounds, 153 tumor incidence observations):Maximum likelihood estimate of cancerinductions per dose/(body weight.75 -day)

relative to comparably dosed adults95%LCL

95%UCL

ArithmeticMean

Fetal 25 15.6 42 27

Birth-Weaning 57 38 90 59

Weaning-60 days 5.0 3.1 8.6 5.3

Female Animals (9 compounds, 153 tumor incidence observations):Maximum likelihood estimate of cancerinductions per dose/(body weight.75 -day)

relative to comparably dosed adults95%LCL

95%UCL

ArithmeticMean

Fetal 1.77 1.05 2.9 1.83

Birth-Weaning 4.4 3.3 6.0 4.5

Weaning-60 days 0.82 0.50 1.29 0.85

Females--Lognormal Plots of Likelihood-Based Uncertainty Distributions for Cancer Transformations Per Daily Dose for Various

Life Stages for Mutagenic Chemicals (Relative to Comparable Exposures of Adults)--Discrete + Continuous Dosing Experiments

210-1-2

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

Fem Mut Log(Birth-Wean/Adult)

Fem Mut Log(Fetal/Adult)

Fem Mut Log(Wean-60d/Adult)

Z-Score

Fem Mut Log(Period/Adult Risk Per Daily Dose/BW^3/4)

y = 0.646 + 0.0785x R^2 = 1.000

y = 0.246 + 0.134x R^2 = 1.000

y = - 0.0880 + 0.124x R^2 = 0.999

No difference from adult risk

3X greater than adult risk

95% Lower Confidence Limits

95% Upper Confidence Limits

Males--Lognormal Plots of Likelihood-Based Uncertainty Distributions for Cancer Transformations Per Daily Dose for Various

Life Stages for Mutagenic Chemicals (Relative to Comparable Exposures of Adults)--Discrete + Continuous Dosing Experiments

210-1-2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

Male Mut Log(Birth-Wean/Adult)

Male Mut Log(Fetal/Adult)

Male Mut Log(Wean-60d/Adult)

Z-Score

Male Mut Log(Period/Adult Risk Per Daily Dose/Body Weight^3/4)

y = 1.76 + 0.113x R^2 = 0.999

y = 1.41 + 0.132x R^2 = 1.000

y = 0.705 + 0.133x R^2 = 0.999

10x greater than adult risk

30x greater than adult risk

95% Lower Confidence Limits

95% Upper Confidence Limits

Direct-Acting vs Metabolically-Activated Mutagens--Standard Age Periods, Discrete Dosing Experiments

Direct-Acting--Ethylnitrosourea and Methylnitrosourea (108 tumorincidence observations):



Fetal 11.6 5.4 25

Birth-Weaning 10.2 5.1 21

Weaning-60 days 2.7 1.37 5.6

Metabolically-Activated Mutagenic Carcinogens (Benzo(a)pyrene,dethylnitrosamine, dimethylbenzanthracene, and urethane, 166 tumorincidence observations in animal groups):



Fetal 0.21 0.01 0.90

Birth-Weaning 15.0 8.4 33

Weaning-60 days 1.24 0.76 2.3

Direct-Acting vs Metabolically-Activated Mutagens--Narrower Age Periods, Discrete Dosing Experiments

Direct-Acting Mutagenic Carcinogens--Ethylnitrosourea and Methylnitrosourea (108tumor incidence observations):

MLE of cancer inductions perdose/(body weight.75 -day) relative to

adults 95% LCL 95% UCLFetal 4.4 2.0 12.4Day 1 6.2 3.6 18.0Other Birth-Weaning(except 1 or 21 days) 3.7 1.8 10.0Day 21 2.2 1.44 4.9>21 Weaning-60days 0.92 0.38 2.7

Metabolically-Activated Mutagenic Carcinogens (Benzo(a)pyrene, diethylnitrosamine,dimethylbenzanthracene, and urethane, 166 tumor incidence observations in animalgroups):

MLE of cancer inductions perdose/(body weight.75 -day) relative to

adults 95% LCL 95% UCLFetal 0.13 0.01 0.52Day 1 17.3 10.0 36Other Birth-Weaning(except 1 or 21 days) 10.7 6.2 22Day 21 1.9 1.06 3.7>21 Weaning-60days 0.87 0.54 1.52

Lactational vs Direct Birth-Weaning Exposures (Continuous + Discrete Dosing)

(9 mutagenic carcinogens, 317 tumor incidence observations:MLE of cancer inductions per

dose/(body weight.75 -day) relativeto adults 95% LCL 95% UCL

Fetal 6.0 5.5 8.8Birth-Weaning Direct 11.6 8.5 16.1Birth-Weaning Lactational 21.4 15.3 30Weaning-60 days 1.70 0.77 2.4

Uncertainty From Chemical-to-Chemical Differences in Life-Stage-Specific Sensitivities for Carcinogenesis

• Data are inadequate for separate estimation of 5 model parameters for individual chemicals and sites.

• Approach:– Calculate log differences between observed and model-

predicted cancer transformations per mg/kg^3/4 -day for cases where exposure was confined to a single life stage.

– Average these life-stage-specific log differences within chemicals for each sex.

– Analyze the distribution of average differences among chemicals.

Chemical-to-Chemical Mean Log Predicted vs Observed Differences

Data for Observations in Male Animals—Mean Log(Observed/ModelPredicted Ca Transformations/Animal Relative to Adults:

Chemical Fetal PeriodBirth-Weaning

PeriodWeaning-60 Day

PeriodBenzidine 0.004 0.163 -0.367Benzo(a)pyrene 0.017 -0.045DEN Diethylnitrosamine -0.961 -0.045 0.132DMBADimethylbenz(a)anthracene -0.108 -0.166ENU Ethylnitrosourea 0.103 -0.135 0.423N-Nitroso-N-Methylurea(NMU) -0.257 0.142Safrole -0.486 0.084 -0.236Urethane 0.008 -0.023

Standard Deviation 0.490 0.132 0.249

Data for Observations in Male Animals—Mean Log(Observed/ModelPredicted Ca Transformations/Animal Relative to Adults:

Chemical Fetal PeriodBirth-Weaning

PeriodWeaning-60 Day

PeriodBenzidine -0.104 -0.111Benzo(a)pyrene 0.037 0.098DEN Diethylnitrosamine -1.086 -0.025 -0.172DMBADimethylbenz(a)anthracene -0.087 -0.038ENU Ethylnitrosourea 0.416 0.006 -0.142N-Nitroso-N-Methylurea(NMU) -0.184 0.038Safrole -0.720Urethane 0.183

Standard Deviation 0.763 0.269 0.115

Uncertainties in the mapping of comparative ages/times between rodents and humans

• Find comparable developmental benchmark for calibrating similar ages across species--current approach based on times of sexual maturity

• Find interspecies comparisons of other ages based on the fraction of body weights attained relative to the weight at the time of sexual maturity.

• Derive distributional treatment of uncertainty in interspecies time mapping from separate results for rat/human and mouse/human projections.

Population-weighted differences in mean height for NHANES III subjects of different

ages (2-90 years)

9080706050403020100

80

100

120

140

160

180

Male Ht (cm)

Female Ht (cm)

Age (yrs)

Mean Height (cm)

Age 15

Age 16

Population- weighted differences in Log(Mean Weight in kg) for NHANES III subjects of

different ages (2-90 years)

9080706050403020100

1.0

1.2

1.4

1.6

1.8

2.0

Log(Male Body Weight kg)

Log(Female Body Weight kg)

Age (yrs)

Log(Male Body Weight kg)

Post-natal growth of Sprague-Dawley Rats, based

on data compiled for EPA

100806040200

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Male Log(g Body Weight)

Female Log(g Body Weight)

Age (Days)

Log(g Body Weight)

Post-natal growth of ICR/Jcl mice, based on data of Nomura (1976)

706356494235282114700

0.0

0.5

1.0

1.5

2.0

Male Mouse Log(BW g)

Female Mouse Log(BW g)

Days Postnatal

Mouse Log(BW g)

Birth

Species Differences in Times of Sexual Maturity

Species (and Time Units) Male FemaleMouse (months) 1.5 1.0Rat (months) 1.8-2.1 1.8-2.1

Human (Years) 11.5 10.5

Source: Kilborn et al. (2002) Contemp Top Lab Animal Sci 41(5):21-26.

Inferences of Corresponding Human Ages from Weight-Based Comparisons Relative to the Times

of Sexual Maturity--Female Mice

Female MouseTime/Event

Fraction ofFemale Mouse

Weight atSexual

MaturitySource of Human Weight

DataCorrespondingHuman Ages

Units ofHuman Age

Begin Fetal Dosing(GD12) 1.8E-03 Potter and Craig (1975) 112

Daysgestation

Birth (GD20) 0.092 Sunderman and Boerner, 1949 14Dayspostnatal

Weaning (PND21) 0.677 NHANES III 7.40Yearspostnatal

60 day postnatal ="adult" 1.435 NHANES III 15.1

Yearspostnatal

Inferences of Corresponding Human Ages from Weight-Based Comparisons Relative to the Times

of Sexual Maturity--Female Rats

Female Rat Time/Event

Fraction ofFemale Rat

Weight at SexualMaturity

Source of HumanWeight Data

CorrespondingHuman Ages

Units ofHuman Age

Begin Fetal Dosing(GD12) 6.3E-05 Potter and Craig (1975) 66

Daysgestation

Birth (GD22) 0.029 Potter and Craig (1975) 30Weeksgestation

Weaning (PND21) 0.250 NHANES III 0.90Yearspostnatal

60 day postnatal ="adult" 1.025 NHANES III 10.6

Yearspostnatal

Estimated Lengths of Various Life Stages in Humans Inferred from the Ages of Sexual Maturity in Mice, Rats, and Humans, and

Patterns of Growth of Body Weight for Rodents Through 60 Days of Age, and for Humans Through Age 16

Rodent Life Stage Equivalent and Gender

Mouse-BasedEstimate(days)

Rat-BasedEstimate

(days)Geometric

Mean

GeometricStandardDeviation

Gestation Day 12 –Birth (Fetal), Males 150 134 142 1.105Birth-Weaning, Males 1184 235 527 3.945Weaning-60 Days, Males 3514 4128 3809 1.146Gestation Day 12 –Birth (Fetal), Females 175 142 157 1.195Birth-Weaning, Females 2689 392 1027 5.115Weaning-60 Days, Females 2827 3560 3172 1.216

Monte Carlo Simulations

• All done in Microsoft Excel• 3 Uncertainty distributions are lognormal, except that

the length of the human equivalents to the birth-weaning and weaning-60 day periods are limited to 15 years in females and 16 years in males

• Distributional results are the means at each percentile for three simulations of 5000 trials each

Detailed Results by Life Stage For Males--Uncertainty Distributions of Risks for Full Lifetime Exposures to a Generic Mutagenic

Carcinogen at a Constant Dose Rate Per Kg of Body Weight3/4 (The numbers represent the increment to lifetime relative risk/dose where

the risk from treatment for the adult period is 1)

Percentile ofUncertaintyDistribution

Male Fetal PeriodRisk Relative to

Adult Period

Male Birth-WeaningPeriod Risk Relative to

Adult PeriodMale Weaning-60d Period

Risk Relative to Adult Period1 0.011 0.054 0.167

2.5 0.018 0.084 0.2175 0.026 0.135 0.27310 0.039 0.220 0.36525 0.078 0.565 0.56350 0.173 1.44 0.88275 0.392 3.77 1.3890 0.764 7.79 2.0395 1.20 10.7 2.53

97.5 1.72 13.2 3.1899 2.89 17.4 3.91

Arithmetic Mean 0.351 2.92 1.09

Detailed Results by Life Stage For Females--Uncertainty Distributions of Risks for Full Lifetime Exposures to a Generic Mutagenic Carcinogen at a Constant Dose Rate Per Kg of Body

Weight3/4 (The numbers represent the increment to lifetime relative risk/dose where the risk for the full adult period is 1)


Female Fetal PeriodRisk Relative to

Adult Period

Female Birth-WeaningPeriod Risk Relative to

Adult PeriodFemale Weaning-60d PeriodRisk Relative to Adult Period

1 0.000 0.004 0.0122.5 0.000 0.007 0.0215 0.001 0.012 0.03410 0.001 0.023 0.04925 0.004 0.069 0.07450 0.014 0.210 0.10775 0.047 0.564 0.15090 0.137 1.09 0.19995 0.278 1.57 0.233

97.5 0.505 2.10 0.27399 0.961 2.79 0.323


Overall Bottom Line--Population Expected Risks from Lifetime Constant Exposure to a Mutagenic Carcinogen per Body Weight^3/4 Relative to Adult-Only Exposure


Male Full LifetimeRisk Relative to

Adult Period Only

Female Full LifetimeRisk Relative to Adult

Period Only

Male and Female PopulationAverage Risk Relative to

Adult Period Only1 1.66 1.09 1.49

2.5 1.81 1.11 1.595 1.97 1.13 1.6910 2.21 1.15 1.8425 2.81 1.23 2.1950 4.06 1.40 2.8475 6.46 1.73 4.0690 10.2 2.25 5.9395 13.3 2.73 7.40

97.5 16.2 3.19 8.9099 19.7 3.91 10.6


Take-Home Conclusions From the Analysis of the Current Data Base

• Improved life-stage specific analyses of risks from mutagenic carcinogens are possible using current information.

• These involve appreciable uncertainties, particularly in the mapping of rodent exposure periods to human equivalents.

• The current analysis suggests that early-life exposure could make important contributions to full-life cancer risks. The mean estimate is a 3.5 fold increment to the risks for full life exposure per body weight^3/4 relative to adult-only exposure, with 5%-95% confidence limits of 1.7 - 7.4 fold. The increments will be somewhat less for constant daily dosage expressed on a mg/kg body weight basis.

researchers: dale hattis, principal investigator rob goble, research professor

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