Derivation of Ingestion-Based Soil Remediation Criterion for Cr+6 Based on the

NTP Chronic Bioassay Data for Sodium Dichromate Dihydrate

Prepared by

Alan Stern, Dr.P.H., DABT

Division of Science, Research and Technology

New Jersey Department of Environmental Protection

for the

Risk Assessment Subgroup

of the

NJDEP Chromium Workgroup

April 8, 2009
Executive Summary

The National Toxicology Program (NTP) chronic bioassay of rats and mice exposed to

sodium dichromate dihydrate in drinking water is the first study that provides data on the

carcinogenicity of hexavalent chromium (Cr+6) by ingestion that is appropriate for

quantitative risk assessment. Sodium dichromate dihydrate is readily soluble, yielding

the dichromate ion that exists in equilibrium in solution with the chromate ion. The

results of the NTP study are, therefore, applicable to the cancer risk assessment of Cr+6

by ingestion in general. NTP concluded that the study provides “clear evidence of

carcinogenicity” in male and female mice and rats, based on benign and malignant

tumors in mouse small intestine and rat oral mucosa. Consistent with the criteria for

carcinogen characterization in the USEPA Guidelines for Carcinogen Risk Assessment,

Cr+6 by ingestion is determined to be “likely to be carcinogenic to humans.” The mouse

was selected as the most sensitive species and the human cancer slope factor was

developed based on assumptions and approaches that are consistent with the 2005

USEPA Guidelines for Carcinogen Risk Assessment. The human cancer slope factor was

estimated to be 0.5 (mg/kg/day)-1 based on the tumor incidence in male mice. Results

from the combined data sets of male and female mice, while more uncertain, are

consistent with these findings. Based on exposure assumptions for the oral exposure

pathway in the NJDEP Soil Remediation Standards, this slope factor corresponds to a

residential direct contact soil remediation criterion for Cr+6 of 1 ppm. Several lines of

evidence support the conclusion that the observed carcinogenicity of Cr+6 did not result

from exceedance of the inherent reduction capacity of the mouse gastrointestinal tract at

the doses used in the NTP (2008) study. While the scientific literature provides ample

data to support the conclusion that Cr+6 can interact with DNA and can act as a mutagen,

the NTP study provides evidence that additional modes of action (MOAs) may have

functioned in the production of the mouse small intestine tumors.

Introduction

In July 2008, the National Toxicology Program (NTP) of the National Institutes of Health

released its Final Technical Report on the Toxicology and Carcinogenesis Studies of

Sodium Dichromate Dihydrate in F344/N Rats and B6C3F1 Mice (NTP, 2008a)

(http://ntp.niehs.nih.gov/files/546_web_FINAL.pdf). This report presents the results of a

two-year chronic drinking water study of a highly soluble form of hexavalent chromium

(Cr+6). The draft final report was peer-reviewed by a panel of outside reviewers in May

of 2007. The peer-review panel voted unanimously to accept the conclusions of “clear

evidence of carcinogenicity” in male and female mice and rats. The final report carries

these conclusions forward without substantive change.

Sodium dichromate dihydrate (Na2Cr2O7 2H2O) is a common soluble compound of

hexavalent chromium (Cr+6). Previous studies on the health effects of the various forms

of Cr+6, including epidemiological studies of occupationally exposed cohorts have

indicated that both common forms of hexavalent chromium, the chromate ion (CrO4

=

)

and the dichromate ion (Cr2O7

=

), are essentially identical in their toxicology. The only

substantive difference is in their stoichiometry. That is, the dichromate ion contains two

moles of Cr+6 for each mole of dichromate, whereas the chromate ion has only a single

2
mole of Cr+6 per mole of chromate. Therefore, the cancer potency and soil remediation

values derived in this analysis and expressed in terms of the dose of Cr+6 apply equally

well to both ions.

Brief Review of Previous Studies of the Carcinogenicity of Cr+6 by Ingestion

The following summary is not intended as a comprehensive review and discussion of the

literature bearing on the carcinogenicity of Cr+6 by ingestion. It is presented to provide

context for the interpretation of the NTP chronic bioassay data and its significance for the

derivation of an estimate of the carcinogenicity of Cr+6 by ingestion.

The carcinogenicity of Cr+6 in the respiratory tract and particularly the lungs has been

known since at least the 1930’s from the experience of workers in the chromate industry.

Cr+6 is currently classified as a known human carcinogen by inhalation by the USEPA

(2007a) and the International Agency for Research on Cancer (IARC) (2007). Despite

some equivocal data that suggest an increased incidence of gastrointestinal tract cancers

among chromate production workers, the earlier epidemiological literature did not

provide a sound basis for assessing the carcinogenicity of Cr+6 by ingestion (reviewed in

NJDEP, 2006). A recent re-analysis of population-based data on stomach cancer in

China among residents in an industrial area whose drinking water was significantly

contaminated by Cr+6 provides a stronger suggestion of the carcinogenicity of Cr+6 by

ingestion (Beaumont et al., 2008). However, Beaumont et al. did not attempt to derive a

quantitative dose-response relationship from their analysis and difficulties in quantifying

exposure and directly linking exposure to cancer incidence make those data unsuitable for

the development of a quantitative estimate of cancer potency.

Prior to the current NTP study, animal data on the carcinogenicity of hexavalent

chromium by ingestion have been sparse. Borneff (1968) exposed three generations of

mice to drinking water containing 500 ppm potassium chromate (K2CrO4). A statistically

significant increase in stomach tumors was observed. However, this study is plagued by

serious methodological problems, the most serious of which is that the mice experienced

a high mortality due to a mouse pox epidemic during the course of exposure. The

increase in tumors was seen almost exclusively in the generation most affected by the

epidemic. This makes it likely that the observed increase in tumors was due, at least in

part, to the infection. This observation makes this study unsuitable for assessment of oral

carcinogenicity and/or for quantitative risk assessment. The Borneff et al. (1968) study is

reviewed in greater detail in NJDEP Chromium Workgroup Report (NJDEP, 2006).

The only other study that directly addresses the oral carcinogenicity of Cr+6 is the study

of Davidson et al. (2004) in which hairless female mice were supplied drinking water

containing 0.1, 0.7, and 1.3 ppm Cr+6 as potassium chromate for 26 weeks and also

exposed to UV light 2-3 times per week during this period. The UV light was in a range

relevant to human exposure and was of sufficient wavelength and intensity to produce

erythema. Comparison mice were exposed to only potassium chromate or to only UV

light. Mice exposed to Cr+6 plus UV light developed significantly more skin tumors

(benign plus malignant) than those exposed only to UV light, while mice with only Cr+6

exposure developed no skin tumors. These results were recently confirmed in male mice

3
(Uddin et al., 2007). This study provides strong evidence that Cr+6 can function as a cocarcinogen within the context of that study design. Of particular note in this study is the

production of tumors at a site remote from the gastrointestinal tract despite the fact that

the doses of Cr+6 in this study can be considered relatively low and potentially subject to

reduction to the non-carcinogenic Cr +3 form within the gastrointestinal tract. This calls

into question the previously posited theoretical ability of the gastrointestinal tract to

completely reduce much larger doses of Cr+6 (Kerger et al., 1996a; De Flora et al., 1989;

Petrilli and De Flora, 1988). Issues relating to reduction of Cr+6 are addressed in detail

in Appendix A of this document. Nonetheless, use of this study as the basis for

quantitative risk assessment is problematic because of its unusual design. The Davidson

et al. (2004) study is reviewed in detail in the NJDEP Chromium Workgroup Report

(NJDEP, 2006).

NTP Two-Year Ingestion Study Design

The NTP study exposed male and female F344/N rats and B6C3F1 mice to a constant

concentration of Cr+6 in their sole source drinking water. Initially, there were 50 animals

of each sex at each dose level. Concentrations of sodium dichromate were selected on

the basis of an estimate of the maximum tolerated dose in earlier, subchronic (90 day)

range finding study conducted by NTP (2007). Male and female rats were supplied with

drinking water containing 0, 14.3, 57.3, 172, or 516 mg/L sodium dichromate dihydrate

for 2 years. Male mice were supplied with drinking water containing 0, 14.3, 28.6, 85.7,

or 257.4 mg/L sodium dichromate dihydrate for 2 years. Female mice were supplied with

0, 14.3, 57.3, 172, or 516 mg/L sodium dichromate dihydrate for 2 years. The drinking

water concentrations and their corresponding time-weighted doses as estimated by NTP

are shown in Table 1.

4
Table 1

Relationship among sodium dichromate dihydrate water concentration, sodium

dichromate dihydrate dose and chromium dose in mice and rats

Rats

Males Females

sodium

dichromate

-dihydrate

water conc.

(mg/L)

Cr+6 water

conc.

(mg/L)

sodium

dichromatedihydrate

dose

(mg/kg/day)

Cr+6 dose a

(mg/kg/day)

sodium

dichromate

-dihydrate

water

conc.

(mg/L)

Cr+6 water

conc.

mg/L

sodium

dichromatedihydrate

dose

(mg/kg/day)

Cr+6 dose a

(mg/kg/day)

0 0 0 0 0 0 0 0

14.3 5 0.6 0.21 14.3 5 0.7 0.25

57.3 20 2.2 0.77 57.3 20 2.7 0.95

172 60 6 2.1 172 60 7 2.45

516 180 17 5.95 516 180 20 7.00

Mice

Males Females

sodium

dichromate

water conc.

(mg/L)

Cr+6 water

conc.

(mg/L)

sodium

dichromate

dose

(mg/kg/day)

Cr+6 dose a

(mg/kg/day)

sodium

dichromate

water

conc.

(mg/L)

Cr+6 water

conc.

mg/L

sodium

dichromate

dose

(mg/kg/day)

Cr+6 dose a

(mg/kg/day)

0 0 0 0 0 0 0 0

14.3 5 1.1 0.39 14.3 5 1.1 0.39

28.6 10 2.6 0.91 57.3 20 3.9 1.37

85.7 30 7 2.45 172 60 9 3.15

257.4 90 17 5.95 516 180 25 8.75

a. As reported by NTP.

Pathology and histopathology were performed on all major organ systems at

approximately 730 days (~2 years) from the beginning of exposure (following sacrifice)

or at the time of death for animals that died before the end of the study.

Brief Summary of Results

In both rats and male mice, a moderate decreased body weight was observed at the

highest dose in surviving animals compared to controls. Based on the summary data

presented in Table 11 of the NTP report, the time-weighted decreases at the highest doses

averaged over the entire duration of the study were 7.5 and 8.0% for male and female

rats, and 8.2% for male mice. For female mice, the time-weighted decrease in body

weight at the highest dose was 20%. Table 2 summarizes the body weights over time

during the course of the study for both sexes of rats and mice.

NTP stated that the relative decrease in body weight was due, in part, to decreased water

consumption resulting from decreased palatability of the water. NTP further cited an

analysis (unpublished) that examined the water intake of rats and mice in this study as a

function of body weight. They state that through the first 20 weeks of dosing, male and

5
female rats and female mice drank approximately the same quantities of water per gram

body weight as their respective controls. This was also the case for male mice except for

those at the highest dose, which drank less water per gram body weight. In other words,

high-dose male mice appeared to be restricting their normal water intake. Thus, it appears

that palatability may only have been the primary cause of reduced water consumption for

the high-dose male mice. Since water intake was not decreased in the female mice in the

highest dose group, the decreased body weight in this group may, therefore, primarily

reflect an intrinsic adverse effect of Cr+6 exposure. This suggests that at the highest dose,

the female mice appear to have exceeded the maximum tolerated dose (MTD).

Table 2

Body Weight in Relation to Dose

RATS-M bw (g)

weeks fraction of total controls 14.3 mg/L 57.3 mg/L 172 mg/L 516 mg/L

1-13 0.13 261 257 257 252 243

14-52 0.39 457 449 453 441 427

53-101 0.49 523 514 518 502 477

weighted aver 468.43 460.38 463.9 450.73 431.85

% change

from controls

-1.71851 -0.96706 -3.77858 -7.80906

RATS-F bw (g)

weeks fraction of total controls 14.3 mg/L 57.3 mg/L 172 mg/L 516 mg/L

1-13 0.13 163 160 160 158 157

14-52 0.39 245 238 237 233 228

53-101 0.49 326 316 318 311 294

weighted aver 276.48 268.46 269.05 263.8 253.39

% change

from controls

-2.90075 -2.68736 -4.58623 -8.35142

MICE-M bw (g)

weeks fraction of total controls 14.3 mg/L 28.6 mg/L 85.7 mg/L 257 mg/L

1-13 0.13 32.8 33 33.3 32.1 29.4

14-52 0.39 51.6 51.6 51.8 51.1 46.7

53-101 0.49 53.8 53 52.3 52.7 49.8

weighted aver 50.75 50.384 50.158 49.925 46.437

% change

from controls

-0.72118 -1.1665 -1.62562 -8.49852

MICE-F bw (g)

weeks fraction of total controls 14.3 mg/L 57.3 mg/L 172 mg/L 516 mg/L

1-13 0.13 24.6 24.4 23.7 23 22.1

14-52 0.39 50.1 49.9 47.2 42.9 37.2

53-101 0.49 61.9 62.4 60.2 57.1 50.7

weighted aver 53.068 53.209 50.987 47.7 42.224

% change

from controls

0.265697 -3.92138 -10.1153 -20.4342

6
The NTP report specifically addresses the question of whether decreased water

consumption resulted in dehydration. NTP noted that physical signs associated with

dehydration (loss of skin turgor, dry mucous membranes, retraction of eyes, hypoactivity,

poor hair coats) were absent in both species. NTP also noted that hematologic

parameters were measured in male rats at intervals during dosing through the first year of

exposure. Parameters typically associated with dehydration (increases in hematocrit,

serum albumin, total protein, urea nitrogen, and urine specific-gravity) were not

observed. In contrast, significant decreases in hematocrit, serum albumin and total

protein were noted in female mice, particularly at the two highest doses (hematologic

analysis was not carried out on male mice). Based on these observations, NTP concluded

that the observed increases in tumors could not be attributed to dehydration.

Furthermore, we are unaware of any evidence from the scientific literature that suggests

that dehydration can potentiate the development of tumors. No clinical signs of toxicity

were observed in either species.

There was little difference in survival at termination at the highest dose compared to

controls in either rats or mice, with a single animal (3%) comprising the maximum

decrease. Clinical signs were normal at all doses. The only significant toxicity noted in

either species was a statistically significant increase in neoplasms of the mucosa of the

oral cavity and tongue in rats, and of the small intestine (duodenum, ileum, and jejunum)

in mice. Combined neoplasms at these locations at the highest dose in male and female

rats and at the two highest doses in male and female mice were statistically significantly

elevated compared to controls.

The tumors of the oral mucosa seen in exposed rats have not previously been reported in

the NTP database of historic drinking water controls. The combined tumors of the

tongue and oral mucosa seen in exposed rats have a very low incidence among historic

NTP drinking water controls (0.3 and1.2% for males and females respectively). The

combined intestinal lesions seen in the exposed mice also occur with a low incidence in

the NTP database of historic drinking water controls (3.7 and 1.1% for males and

females respectively. NTP notes, however, that comparison to the concurrent (i.e., instudy) controls is the appropriate basis for statistical analysis of dose-response.

The incidence of neoplasms at the doses that were significantly elevated above the

concurrent controls were also significantly elevated above the historic controls. In the

rats, non-neoplastic lesions of the oral mucosa were not seen. This is consistent with the

neoplastic lesions arising independently of necrotic tissue damage. In the mice, a low

incidence of focal epithelial hyperplasia in the small intestine was noted. Its incidence

was not dose related, but was, nonetheless, considered to be pre-neoplastic. Diffuse

epithelial hyperplasia in the duodenum was significantly elevated in both sexes compared

to controls at all doses and at the highest dose in the jejunum in female mice. The diffuse

hyperplasia was characterized by several layers of cells piled up along the long axis of

the intestinal villi. Intestinal crypts were elongated and contained increased number of

cells with increased numbers of mitotic figures. NTP considers this diffuse hyperplasia

to be consistent with regenerative cell growth secondary to tissue injury.

7
Selection of Key Species

Figures 1-4 show the incidence of oral neoplasms (rats) or neoplasms of the small

intestine (mice). To avoid confusion, the tumor incidence in these figures is not adjusted

for the number of animals at-risk. This is explained more fully below in the section,

Calculation of Tumor Incidence and in Table 2. These adjustments do not affect the

selection of the key species for calculation of cancer potency. The tumor incidence in

both species demonstrates a dose-response. However, the response in the rats is mostly

or entirely at the highest dose, whereas the response in the mice is observed at least in the

two highest doses. In addition, the magnitude of the response in the mice at the highest

dose is more than twice that in the rats. It is clear that, in this study, the mouse is the

more sensitive species. The mouse is, therefore, selected as the key species for derivation

of the cancer potency by ingestion and the related soil remediation criterion.

General Approach for Calculating the Cancer Potency

The current USEPA Guidelines for Carcinogen Risk Assessment (USEPA, 2005a) state

that: “When the weight of evidence evaluation of all available data are insufficient to

establish the mode of action for a tumor site and when scientifically plausible based on

the available data, linear extrapolation is used as a default approach, because linear

extrapolation generally is considered to be a health-protective approach.” To date, no

mode of action has been unambiguously demonstrated for Cr+6 carcinogenicity. Thus,

under the current USEPA Guidelines, linear extrapolation is the appropriate approach

for calculating Cr+6 oral cancer potency from these data. However, some scientific

evidence suggests that tumors arise from the interaction of Cr+6 with DNA either

directly, or through intra-cellular metabolism to Cr+3 (Kirpnick-Sobol et al., 2006; Dana

Devi et al., 2001; Cohen et al., 1993; Coogan et al., 1991). These data suggest the

possibility of a mutagenic mode of action, and linear extrapolation is also the USEPA

recommended approach when a mutagenic mode of action has been demonstrated.

With regard to linear extrapolation, the USEPA Guidelines also state that: “The linear

approach is to draw a straight line between a point of departure from observed data,

generally as a default, an LED [lower bound on the effective dose] chosen to be

representative of the lower end of the observed range, and the origin (zero incremental

dose, zero incremental response).” Consistent with the USEPA Guidelines for

Carcinogen Risk Assessment (USEPA, 2005a), the point of departure (POD) is

identified here as the lower confidence bound on the benchmark dose (BMDL). This

concept is explained in detail below. The USEPA has used the BMDL as the point of

departure in calculating oral cancer slope factors for several chemicals (e.g., 1,2-

dibromomethane (USEPA, 2004); dichloroacetic acid (USEPA, 2003)). Also consistent

with the USEPA Guidelines, the tumor incidence is based on the sum of benign and

malignant tumors in the same tissue under the assumption that benign tumors have the

potential to progress to malignancy when caused by an agent that also causes malignant

tumors at the same site (USEPA, 2005a).

Determination of Cr+6 Dose

The exposure of the animals in the NTP bioassay was originally expressed in terms of the

concentration of sodium dichromate dihydrate in their drinking water (mg/L). However,

8
cancer potency is expressed in terms of the inverse of dose (i.e., (mg/kg-bw/day)-1). In

addition, the results of the bioassay are used here to derive a generalized cancer potency

estimate and an associated soil remediation criterion for Cr+6 rather than for sodium

dichromate per se. The NTP provided an estimate of the dose of sodium dichromate

dihydrate for each species and sex corresponding to each water concentration. The

corresponding Cr+6 dose is obtained by multiplying the sodium dichromate dihydrate

dose by 0.35, the fraction of the sodium dichromate dihydrate molecular weight

contributed by chromium (see Table 1).

Body Weight

In order to calculate a human risk-specific dose from the animal cancer potency

estimate, it is necessary to consider the animals’ body weight. Since the cancer potency

estimate derived from the animal data integrates dose-response data (including bodyweight) across dose groups, a single representative value for animal body weight is

required. The time-weighted average body weight for the control mice is selected. The

time-weighted value is derived from the summary data reported in Table 11 of the NTP

report. These values are 0.050 kg and 0.053 kg for male and female mice respectively.

The decrease in body weight in male mice at the highest dose and second highest doses

was 8.2% and 1.7% respectively and in the female mice was 20% and 10.2%

respectively. Given these differences between the body weight of control and high-dose

animals, the impact of the choice of the control mice to represent body weight for all

dose groups can be seen in the following sensitivity analysis. For female mice (given

the allometric dose scaling from mice to humans – see below), use of the time-weighted

average body weight from control animals results in a human-specific cancer potency

estimate that is approximately 6% larger than that calculated on the basis of the timeweighted average body weight at the highest dose (depending on the specific

benchmark dose model employed – see below). The difference in the human cancer

potency estimate when comparing the control body weight to the body weight for all

other doses for females and to all doses for male mice would be less than 5%.

Therefore, the derivation of cancer potency and risk-based guidance is not highly

sensitive to the choice of body weight from among the various dose groups and the use

of the time-weighted average control body weight is judged to be appropriate.

Calculation of Tumor Incidence

Dose-response analysis requires data for both dose and incidence. The ultimate goal in

this analysis is the determination of the risk of the occurrence of at least one tumor

occurring in a person as a result of exposure to a given dose of Cr+6. Therefore, in this

analysis, incidence is defined as the number of animals with at least one tumor divided by

the number of animals at risk of developing a tumor. The numerator of this ratio is the

sum of all mice in which a tumor (adenoma or carcinoma) was detected in at least one of

the three sections of the small intestine – the duodenum, ileum, and jejunum. The

denominator of this ratio, the number of animals at-risk of developing a tumor, includes

all mice that survived long enough to have potentially experienced a tumor. Since the

first tumor of the small intestine was recorded at day 451 of exposure, it is assumed that

animals that died prior to that time were not at-risk.

9
NTP has provided both summary information on tumor incidence (Table 13 of the NTP

final report) and individual animal pathology data (http://ntp.niehs.nih.gov/go/29141).

The individual animal pathology data reflect microscopic examination of the small

intestines whereas the summary data reflect both gross and microscopic examination.

Not all segments of the small intestine were available for microscopic examination in all

mice. However, all sections of the intestines of all mice were grossly examined by

multiple examiners. Information supplied by NTP1indicates that nearly all tumors were

identified at least by gross examination and that it is unlikely that any intestinal

neoplasms were missed due to the unavailability of samples for microscopic examination.

NTP therefore recommends that, in general, the denominator of the incidence should be

the number of animals in each dose group (i.e., 50)1. We agree with this

recommendation, with the exception of animals that died prior to day 451 of dosing. In

Table 3, the number of animals at-risk is, therefore, equal to 50 minus the number of

animals that died prior to day 451. The numerator of the incidence ratio is the total

number of mice identified by NTP with small intestinal tumors and reflects the sum of

tumors identified through gross and microscopic examination. That number is used as

reported in Table 13 of the NTP final report.

Table 3

Estimated Tumor Incidence by Dose

at

risk

mice

with

neoplasms

Incidenceat

risk

mice

with

neoplasms

Incidenceat

risk

mice

with

neoplasms

Incidenceat

risk

mice

with

neoplasms

Incidenceat

risk

mice

with

neoplasms

Incidence

sodium dichromate water conc. (mg/L)

mice

M

0 14.3 28.6 85.7 257.4

49 1 0.020 49 3 0.061 49 2 0.041 50 7 0.140 48 20 0.417

mice

F

0 14.3 57.3 172 516

49 1 0.020 50 1 0.020 49 4 0.082 49 17 0.347 49 22 0.449

Determination of the Point of Departure (POD)

As discussed above, the current approach under the USEPA Guidelines for Carcinogen

Risk Assessment (USEPA, 2005a) for linear extrapolation of cancer dose-response

considers that, in general, a POD is a lower effective dose (LED) chosen to be

representative of the lower end of the observed range of response. It is not necessary that

the LED be one of the administered doses. The benchmark dose approach was used to

1 Dr. David Malarkey, NTP - personal communication 3/15/09

10
identify an appropriate POD. The USEPA’s Integrated Risk Information System (IRIS)

defines a benchmark dose as “a dose or concentration that produces a predetermined

change in response rate of an adverse effect (called the benchmark response or BMR)

compared to background (USEPA, 2007b).” For dichotomous data (e.g., tumor

incidence), the BMR is generally chosen to represent the lowest level of response that is

reasonably consistent with the observed data. For well designed bioassays, this value is

often 0.1 or 0.05 (10% or 5% response) depending on the specific data. As can be seen in

Figs. 5 and 6, a response rate of 0.1 (but not 0.05) falls within the ascending portion of

the dose-response in both male and female mice but is close to the lowest dose showing a

positive response. A BMR of 0.1 was, therefore, selected.

In applying the benchmark dose approach to the derivation of risk-based standards and

guidance, the standard approach is to calculate a lower 95% confidence bound on the

dose corresponding to the BMR (i.e., the benchmark dose or BMD). This lower

confidence bound is referred to as the BMDL. For a BMR of 0.1, the corresponding

BMDL is referred to as the BMDL0.1. The use of the lower confidence bound on the

benchmark dose is intended to account for uncertainty in the fit of the dose-response

model to the data (see below).

Benchmark dose modeling was carried out using the USEPA’s BMDS (version 1.4.1)

software package (USEPA, 2007c). The BMDS software offers several possible

mathematical dose-response functions for use with dichotomous data: logistic; gamma

multi-hit; Weibull; quantal linear; probit; and multi-stage cancer. None of these doseresponse functions has a biological basis that is necessarily specific to Cr+6

carcinogenicity. The fit of the data to each model is described in the BMDS software

through the calculation of the chi-squared goodness-of-fit statistic and its corresponding

p-value. As there is no biological basis for selecting any of the models, it could be

argued that the model which best fits the data should be chosen. However, USEPA gives

preference to the multi-stage cancer model because of its historic use as the USEPA’s

default for cancer dose-response modeling prior to POD-benchmark dose approach.

Because neoplasms of the small intestine occur spontaneously to some extent in the

B6C3F1 strain of mouse, it is necessary to account for this background frequency in the

dose modeling. This was addressed by modeling “extra risk.” Extra risk is defined as the

probability of the occurrence of the effect (i.e., a tumor) that can be specifically attributed

to the dose for the animals at risk. Mathematically, this is expressed as PD-ER = (PD -

P0)/(1 - P0) , where PD-ER is the probability of a tumor at dose D under extra risk; PD is the

observed probability of a tumor at dose D; and P0 is the observed probability of a tumor

at zero dose in the in-study controls (the background probability).

Benchmark dose modeling was carried out for the male and female mice separately

(Tables 4a and 4b respectively), as well as for the combined male and female data sets

(Table 4c). In addition, because the highest dose in the female rats appears to exceed the

maximum tolerated dose (MTD), benchmark dose was also carried out for the combined

male and female data sets with the high-dose females removed. This is referred to as the

combined-reduced data set (Table 4d).

11
Results of POD Calculations

The first three columns of Tables 4a-d present the BMDL calculations that are used for

the determination of the POD. For the male mice, all of the models had nearly identical

fits to the data and the BMDL values all fell within a narrow range. In fact, with the

exception of the probit model, all of the values were within 0.01 of each other. For the

female mice, the BMDL values are more variable and none of the models gives a strong

fit to the data as reflected by the low chi-square p-values. For the combined male and

female mouse data (Table 4c), only the multi-stage cancer model gave a marginally

acceptable fit. For the combined-reduced data set (Table 4d), the Weibull, gamma multihit, and quantal linear models each gave marginally acceptable fits. For each of the

models that gave marginally acceptable fits to the data, the BMDL values were in good

agreement with those from for the male mice. Each of the models gave a better fit to the

male-only data than the fit of any of the models to any of the other data sets. Figures 5-8

show the fits of the best fitting model for each data set. The BMDL values obtained for

the male data for each of the models are consistent with the BMDL values for each of the

best fitting model in both of the combined data sets. Therefore, the derivation of the

cancer potency estimate and corresponding soil cleanup value is based on the male mouse

data.

12
.

Table 4a Calculation of cancer potency and soil conc. at 1 x 10-6 cancer risk – Male mice

mice M 2 3 4 5 6 7 8 9 10 11 12

BMD

model

BMDL0.1

mg/kg/d

(extra

risk)

chi-sq

p-value

model

response

at BMDL

model

response

at

0 dose

slope

from

BMDL

to

0 dose

animal

dose at

1x10-6 risk

mg/kg/d

time

weighted

av. study

bw at

0 dose

(kg)

human

dose at

1x10-6 risk

(mg/kg/d)

based on

(bw)3/4

scaling

and 70 kg

bw

human

cancer

potency

slope

(mg/kg/d)-1

mg Cr+6/d

at 1x10-6

risk

for 59 kg

av bw

soil

conc.

Cr+6 at

1x10-6

risk

for 114

mg

soil/d

ppm

logistic 1.17 0.57 0.1 0 0.09 1.17E-05 0.05 1.91E-06 0.52 1.13E-04 0.99

weibulll 1.17 0.57 0.1 0 0.09 1.17E-05 0.05 1.91E-06 0.52 1.13E-04 0.99

probit 1.73 0.57 0.1 0 0.06 1.73E-05 0.05 2.83E-06 0.35 1.67E-04 1.46

gamma

multi hit

1.17 0.56 0.1 0 0.09 1.17E-05 0.05 1.91E-06 0.52 1.13E-04 0.99

quantal

linear

1.17 0.57 0.1 0 0.09 1.17E-05 0.05 1.91E-06 0.52 1.13E-04 0.99

multistage

cancer

1.18 0.60 0.1 0 0.09 1.18E-05 0.05 1.93E-06 0.52 1.14E-04 1.00

Table 4b Calculation of cancer potency and soil conc. at 1 x 10-6 cancer risk – Female mice

mice F 2 3 4 5 6 7 8 9 10 11 12

BMD

model

BMDL

mg/kg/d

(extra

risk)

chi-sq

p-value

model

response

at BMDL

model

response

at

0 dose

slope

from

BMDL

to

0 dose

animal

dose at

1x10-6

risk

mg/kg/d

time

weighted

av. study

bw at

0 dose

(kg)

human

dose at

1x10-6 risk

(mg/kg/d)

based on

(bw)3/4

scaling

and 70 kg

bw

human

cancer

potency

slope

(mg/kg/d)-1

mg

Cr+6/d

at 1x10-6

risk

for 59 kg

av bw

soil

conc.

Cr+6 at

1x10-6

risk

for 114

mg

soil/d

ppm

logisitic 2.64 0.00 0.1 0 0.04 2.64E-05 0.053 4.38E-06 0.23 2.58E-04 2.27

weibulll 0.67 0.06 0.1 0 0.15 6.70E-06 0.053 1.11E-06 0.90 6.56E-05 0.58

probit 2.44 0.00 0.1 0 0.04 2.44E-05 0.053 4.05E-06 0.25 2.39E-04 2.09

gamma

multi hit

0.68 0.06 0.1 0 0.15 6.80E-06 0.053 1.13E-06 0.89 6.66E-05 0.58

quantal

linear

0.67 0.06 0.1 0 0.15 6.70E-06 0.053 1.11E-06 0.90 6.56E-05 0.58

multistage

cancer

1.03 0.13 0.1 0 0.10 1.03E-05 0.053 1.71E-06 0.59 1.01E-04 0.88

13
Table 4c Calculation of cancer potency and soil conc. at 1 x 10-6 cancer risk – Combined Male and

Female Mice

mice F 2 3 4 5 6 7 8 9 10 11 12

BMD

model

BMDL

mg/kg/d

(extra

risk)

chi-sq

p-value

model

response

at BMDL

model

response

at

0 dose

slope

from

BMDL

to

0 dose

(mg/kg/

day)-1

animal

dose at

1x10-6

risk

mg/kg/d

time

weighted

av. study

bw at

0 dose

kg

human

dose at

1x10-6 risk

(mg/kg/d)

based on

(bw)3/4

scaling

and 70 kg

bw

human

cancer

potency

slope

(mg/kg/d)-1

mg

Cr+6/d

at 1x10-6

risk

for 59 kg

av bw

soil

conc.

Cr+6 at

1x10-6

risk

for 114

mg

soil/d

ppm

logisitic 2.67 0.00 0.1 0 0.04 2.67E-05 0.053 4.43E-06 0.23 2.61E-04 2.29

weibulll 1.00 0.20 0.1 0 0.10 1.00E-05 0.053 1.66E-06 0.60 9.79E-05 0.86

probit 2.47 0.00 0.1 0 0.04 2.47E-05 0.053 4.10E-06 0.24 2.42E-04 2.12

gamma

multi hit

1.02 0.21 0.1 0 0.10 1.02E-05 0.053 1.69E-06 0.59 9.98E-05 0.88

quantal

linear

1.00 0.20 0.1 0 0.10 1.00E-05 0.053 1.66E-06 0.60 9.79E-05 0.86

multistage

cancer

1.12 0.3 0.1 0 0.09 1.12E-05 0.053 1.86E-06 0.54 1.10E-04 0.96

Table 4d Calculation of cancer potency and soil conc. at 1 x 10-6 cancer risk – Combined Male and

Reduced Female Mice a

mice F 2 3 4 5 6 7 8 9 10 11 12

BMD

model

BMDL

mg/kg/d

(extra

risk)

chi-sq

p-value

model

response

at BMDL

model

response

at

0 dose

slope

from

BMDL

to

0 dose

(mg/kg/

day)-1

animal

dose at

1x10-6

risk

mg/kg/d

time

weighted

av. study

bw at

0 dose

kg

human

dose at

1x10-6 risk

(mg/kg/d)

based on

(bw)3/4

scaling

and 70 kg

bw

human

cancer

potency

slope

(mg/kg/d)-1

mg

Cr+6/d

at 1x10-6

risk

for 59 kg

av bw

soil

conc.

Cr+6 at

1x10-6

risk

for 114

mg

soil/d

ppm

logisitic 2.14 0.04 0.1 0 0.05 2.14E-05 0.053 3.55E-06 0.28 2.09E-04 1.84

weibulll 1.11 0.31 0.1 0 0.09 1.11E-05 0.053 1.84E-06 0.54 1.09E-04 0.95

probit 1.98 0.09 0.1 0 0.05 1.98E-05 0.053 3.28E-06 0.30 1.94E-04 1.70

gamma

multi hit

1.13 0.32 0.1 0 0.09 1.13E-05 0.053 1.87E-06 0.53 1.11E-04 0.97

quantal

linear

1.11 0.31 0.1 0 0.09 1.11E-05 0.053 1.84E-06 0.54 1.09E-04 0.95

multistage

cancer

1.07 0.25 0.1 0 0.09 1.07E-05 0.053 1.77E-06 0.56 1.05E-04 0.92

a –The combined male and reduced female data set consists of the entire male mouse data set combined

with the female mouse data set after removal of the high dose females

14
Results of Mouse Cancer Potency Slope Calculation

Column 6 of Table 4a-d gives the slope in (mg/kg/day)-1 of the line between the point at

zero dose-zero response and the point at BMDL0.1-0.1 response. This is illustrated by the

dashed line in Figure 5-8. This is the linear extrapolation approach described by the

USEPA (2005a) cancer guidelines. The potency can also be expressed in terms of the

dose predicted to result in one-in-a-million (1x10-6) cancer risk to the mice in this study.

This is given in column 7 of Table 4a-d.

Calculation of the Human Equivalent Dose

To convert the animal dose corresponding to 1x10-6 risk to the dose corresponding to the

same risk in humans, the USEPA cancer guidelines recommend the allometric conversion

on the basis of body weight to the 3⁄4 power to address differences between species in

metabolism and toxicokinetics related to body mass (USEPA, 2005a). When using this

conversion to scale doses between animals and humans, the appropriate formula is HED

= (ABW/HBW)0.25 x AD where HED is the human equivalent dose (mg/kg/day), ABW is

the animal body weight (kg), HBW is the human body weight (default value of 70 kg),

and AD is the animal dose (mg/kg/day) (Rodricks et al., 2001). The straightforward

calculation of the human equivalent dose requires the assumption of a single animal (and

human) body weight. The time-weighted average body weight of the control mice

(column 8 of Table 3) is taken as the body weight most representative of the overall body

weight across doses. As discussed above, given the exponential nature of the body

weight scaling formula and the relatively small differences in time-weighted average

body weight across doses in the mice, the choice among the time-weighted dose-specific

mouse body weights has a relatively small impact on the human equivalent dose and the

corresponding soil remediation criterion. Column 9 of Table 4a-d gives the human

equivalent dose (mg/kg/day) corresponding to a 1x10-6 lifetime cancer risk. Multiplying

this dose by the assumed human body weight gives the corresponding mass of Cr+6

ingested daily (mg/day) which results in a 1 x 10-6 lifetime cancer risk.

The human lifetime cancer risk can be generalized by dividing the risk of 1x10-6 by the

corresponding dose in column 9. The resulting value is the cancer potency slope

(mg/kg/day)-1), the risk for each mg/kg/day intake. This is given in column 10. For the

slope derived from the male mouse data, the slope ranges from 0.3-0.5 (mg/kg/day)-1, but

all of the models except the probit model give a value of 0.5 (mg/kg/day)-1. This range

can be compared to the slopes of other well known chemicals that are carcinogenic by

ingestion, such as benzo(a)pyrene, (7.3 (mg/kg/day)-1), arsenic (1.5 (mg/kg/day)-1),

carbon tetrachloride (0.13 (mg/kg/day)-1), dimethylnitrosamine (51 (mg/kg/day)-1)

(USEPA 2007a), with higher numbers corresponding to greater potency.

The NJDEP Soil Remediation Standards (final) (NJDEP, 2008) integrate the body weight

from 1 year to 31 years of age in deriving soil cleanup standards for the ingestion/dermal

pathway. This corresponds to a time-weighted average human body weight of 59 kg.

The daily ingested intake of Cr+6 corresponding to a 1x10-6 lifetime human cancer risk

(column 11 of Table 4a-d) is calculated by multiplying the human dose for this risk

(column 9) by 59 kg.

15
Calculation of the Soil Concentration Corresponding to a 1x10-6 Lifetime Cancer Risk

The time-weighted averaging procedure for the amount of soil ingested daily that is

specified in the NJDEP Soil Remediation Technical Regulations yields an integrated

value of 114 mg/day for daily soil ingestion from 1 year to 31 years of age. Dividing the

daily intake of Cr+6 corresponding to a 1x10-6 lifetime cancer risk (column 10) by the

daily soil ingestion gives the concentration of Cr+6 in soil (mg Cr+6/kg soil)

corresponding to the 1x10-6 lifetime cancer risk (column 12 of Table 4a-d).

For a soil remediation criterion of 1 x 10 -6 lifetime cancer risk, the dose-response models

for the male mice all yield soil remediation criteria that converge very closely to a soil

concentration of 1 mg Cr+6/kg. It is noteworthy that the better fitting models in the

combined data sets (Table 4c,d) also yielded the same soil remediation criterion values.

Since most of the models (including the cancer multi-stage model preferred by USEPA)

provide essentially equivalent fits to the data and yield the same soil remediation criterion

value, it is not necessary to select a single model as the basis for the soil remediation

criterion.

Weight of Evidence Considerations and Risk Characterization

Weight of evidence for characterization of carcinogenicity to humans by ingestion - The

results of the NTP study clearly show that ingestion of Cr+6 in drinking water resulted in

tumors in both sexes of rats and mice. The database from the NTP study is judged to be

of high quality. The study was well designed and well executed with no significant

problems that raise questions about the validity of the results. Both the survival and the

overall health of the animals were comparable to control animals at all doses with no

clinical signs of toxicity. The decreased weight of the female mice at the highest dose

(20% less than controls) may partly reflect systemic effects and, as such may indicate

moderate exceedance of the maximum tolerated dose (MTD).

The statistically significant increase in tumors in both rats and mice occurred in the

alimentary system. In both the male and female mice, a clear dose-response was

observable extending through the two highest doses. In the female mice, the response at

the third highest dose was also increased consistent with the overall dose response. As

discussed in Appendix A of this document, the evidence supports a hypothesis that the

observed tumor incidence is relevant to human exposure at reasonably anticipated

environmental levels, and did not occur due to exceedance of the gastrointestinal

reduction capacity for Cr+6. Although the pH of the mouse stomach is higher than the pH

of the human stomach, it appears that pH is not the predominant factor in the reduction of

Cr+6 in the stomach, and that the mouse is a reasonable model for the carcinogenic

potential of ingested Cr+6 in humans. Thus, the mode(s) of action of Cr+6 carcinogenicity

responsible for the observed tumors in the mouse small intestine are likely to be relevant

to the potential for carcinogenicity in the human gastrointestinal system. In addition, the

observed carcinogenicity of Cr+6 by ingestion is consistent with the inhalation

carcinogenicity of Cr+6 observed in studies of occupational exposure.

16
Under the current USEPA Guidelines for Carcinogen Risk Assessment (USEPA, 2005a),

these observations are consistent with the characterization of oral exposure to Cr+6 as

“likely to be carcinogenic to humans.” More specifically, the data are consistent with the

criterion for this characterization of “an agent that has tested positive in animal

experiments in more than one species, sex, strain, site, or exposure route, with or without

evidence of carcinogenicity in humans.”

Weight of evidence for the carcinogenic mode of action (MOA) of Cr+6-

There are considerable data indicating the ability of Cr+6 to react directly and indirectly

with DNA including the production of mutations with in vivo exposure (Kirpnick-Sobol

et al., 2006; Dana Devi et al., 2001; Cohen et al., 1993; Coogan et al., 1991; Knudsen et

al., 1980; Itoh and Shimada, 1998). However, the data on the occurrence of diffuse

hyperplasia in the mouse duodenum suggests that tissue damage and regeneration could

have played a role in the formation of tumors in the mouse small intestine in the NTP

study. The criteria for determination that a carcinogen operates through a mutagenic

MOA with respect to the application of an of an age-dependent adjustment factor to the

cancer potency (ADAF), as described in the USEPA’s Guidelines for Carcinogen Risk

Assessment and Supplemental Guidance for Assessing Susceptibility from Early-Life

Exposure to Carcinogens, are still under development. Thus, the age-dependent

adjustment factor for carcinogens which act through a mutagenic MOA is not applied in

this assessment.

Reliability of the quantitative procedure for calculating the cancer potency estimate and

soil remediation criterion - Although the true shape of the dose-response data below the

POD is not known, the cancer potency estimate (and associated soil remediation

criterion) was derived from the NTP mouse data using a linear-from-POD approach. In

the USEPA Guidelines for Carcinogen Risk Assessment (USEPA, 2005a), this is the

default approach in the absence of sufficient evidence to support a non-linear mode of

action. The fit of all of the mathematical models available in the BMDS package to the

small intestine tumor data from the male mice was strong, and the difference between the

BMD and BMDL doses was less then a factor of two for most of the models. Although

the tumor data from the male mice were significantly better fit by the dose-response

models than were data from the combined data sets, the best fitting models for the

combined data sets resulted in the same potency estimates and soil remediation criterion

values. Thus, the POD is statistically robust. The linear extrapolation procedure for the

calculation of the cancer potency slope from the POD is entirely deterministic and

requires no interpretation. Therefore, within the bounds of the default USEPA

methodology, the quantitative cancer potency slope value derived here is judged to be

robust and reliable.

The derivation of the ingestion-based remediation criterion value from the cancer potency

estimate follows NJDEP-SRP procedures and exposure assumptions as specified in the

Soil Remediation Standards (final) (NJDEP, 2008), and involves no additional data

interpretation.

17
Characterization of uncertainty - While it seems clear that the oral cavity tumors in the

rats and the small intestine tumors in the mice both resulted from ingestion of Cr+6, it is

unclear why there was a lack of concordance in the location of the tumors in these

species. It should be noted, however, that the USEPA Guidelines for Carcinogen Risk

Assessment (2005a) state that, “Target organ concordance is not a prerequisite for

evaluating the implications of animal study results for humans.”

It is known that the gastrointestinal tract has a reserve capacity for the reduction of Cr+6

to Cr+3. It has been argued that this capacity is sufficient to reduce the relatively large

doses of Cr+6 that could reasonably be anticipated to be encountered under extreme

conditions of environmental contamination (De Flora et al., 1987, 1997). It might,

therefore, be hypothesized that the tumors observed in the NTP study reflect a threshold

mechanism that functions only after this reduction capacity is exceeded, and that such a

mechanism is not relevant to human environmental exposure. As discussed in detail in

Appendix A of this document, analysis of the estimated maximum mouse intake rate of

Cr+6 in comparison to the estimated reduction capacity of mouse gastric fluid suggests

that even under the assumption that the mouse stomach is a closed system with respect to

reduction of Cr+6, the reduction capacity of the mouse gastrointestinal system would only

potentially be exceeded at the highest dose in female mice. However, as explained further

in Appendix A, the mouse stomach is not a closed system and the kinetics of gastric

emptying make it likely that even at very low doses a significant fraction of ingested Cr+6

will reach the small intestine without being reduced. Even under the limiting assumption

of a closed gastric reduction system, the possibility of exceedance of the reduction

capacity at the highest dose in female mice cannot explain the overall dose-response

pattern showing significant increases in tumor incidence in both male and female mice at

the two highest doses and a non-significant, but consistent increase in female mice at the

third highest dose. A separate analysis of the rate of accumulation of Cr in various

mouse tissues and biological media as a function of dose (see Appendix A) is, likewise,

inconsistent with a hypothesis of a threshold for exceedance of the reduction capacity at

the doses in the NTP study. In addition, the observation of diffuse hyperplasia in the

duodenum of both sexes of mice at the lowest dose is also inconsistent with the

exceedance of the reduction capacity at any dose in the NTP study. Overall, there is no

evidence of a threshold for tumor production, including exceedance of the gastrointestinal

reduction capacity of Cr+6 within the dose range of the NTP study. In addition, there are

no data to support a hypothesis that assumes a threshold for exceedance of the Cr+6

reduction capacity at doses below those in the NTP study. Such a hypothesis is,

furthermore, inconsistent with the evidence for a substantial reserve capacity of the

gastrointestinal system for reduction of Cr+6 and also inconsistent with evidence of

adverse systemic effects of ingested low dose Cr+6.

NTP observed a decrease in hematocrit, particularly at the two highest doses in female

mice (hematologic analysis was not carried out on male mice). Dehydration would be

expected to decrease blood fluid volume and, therefore, increase the hematocrit. NTP

thus noted the decrease in hematocrit as evidence that dehydration did not occur in these

groups despite significantly decreased body weight. The decrease in hematocrit was

18
accompanied by microcytosis. It, therefore, appears that this decrease in hematocrit

resulted from a systemic effect of Cr+6. It is theoretically possible that the systemic

decrease in hematocrit could have masked a decrease in blood fluid volume from

dehydration. However, the lack of physical signs of dehydration argues against this

possibility.

Based on kinetic, chemical, and toxicological considerations, it appears that the doses of

Cr+6 in the NTP study did not exceed the reduction capacity of the gastrointestinal

system. It also appears likely that even at lower doses, a significant fraction of ingested

Cr+6 will escape reduction in the gastrointestinal tract. This raises the possibility that

ingested Cr+6 could cause tumors at sites distant from the gastrointestinal tract. There

was no evidence of such tumors, however, in the NTP study. Furthermore, although the

Davidson et al. (2004) study suggested that ingested Cr+6 could be co-carcinogenic with

UV light in the production of skin tumors, there is currently no evidence in the literature

of non-gastrointestinal cancer resulting solely from Cr+6 ingestion. Blood is known to

have a significant reduction capacity for Cr+6 as do other organs (De Flora et al., 1997).

Nonetheless, the potential for ingested Cr+6 to cause tumors at other locations remains an

uncertainty.

The USEPA default procedure for calculation of cancer potency that was employed

herein linearly extrapolates across 5 orders of magnitude of cancer incidence from the

data-based benchmark incidence rate (BMR) of 0.1 to estimate the dose at 1 x 10-6 (onein-a million) cancer incidence. The shape of the dose-response function is not known

below the range of the observed data, and the linear extrapolation across so large a range

carries significant uncertainty. Although at the present time, there is no way to further

reduce this uncertainty, this derivation of cancer potency for Cr+6 is entirely consistent

with the approaches used for other cancer potency estimates calculated according to

USEPA methodology.

Comparison of the suggested and current soil remediation criteria- The bases for the

suggested soil remediaton criterion for Cr+6 ingestion derived from the NTP data (1 ppm)

and the soil remediation criterion for Cr+6 inhalation cancer risk (20 ppm) are essentially

unrelated. They reflect different conditions and routes of exposure and are supported by

different cancer endpoints in different organs that may result from different toxicological

modes of action.

Other ingestion cancer risk assessments based on the NTP data - The USEPA Office of

Prevention, Pesticides and Toxic Substances (OPPTS) has developed a cancer potency

based on the NTP chronic bioassay. This is discussed in Appendix B.

19
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Cohen MD, Kargacin B, Klein CB, Costa M (1993). Mechanisms of chromium

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Coogan TP, Motz J, Snyder CA, Squibb KS, Costa M (1991). Differential DNA-protein

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Dana Devi K, Rozati R, Saleha Banu B, Jamil K, Grover P (2001). In vivo genotoxic

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evaluated to date

Accessed at: http://monographs.iarc.fr/ENG/Classification/crthalllist.php, 7/24/07.

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treated with hexavalent chromium. Mutat Res. 412:63-7.

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Kirpnick-Sobol Z, Reliene R, Schiestl RH (2006). Carcinogenic Cr(VI) and the

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Knudsen I. (1980). The mammalian spot test and its use for the testing of potential

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http://www.state.nj.us/dep/dsr/chromium/report.htm, 7/24/07.

NJDEP (2008). Ingestion-Dermal Exposure Pathway. Soil Remediation Standards

Basis and Background. June, 2008.

http://www.nj.gov/dep/srp/regs/srs/bb_ingest_dermal.pdf.

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Petrilli FL, de Flora S (1988). Metabolic reduction of chromium as a threshold

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22
Appendix A

Issues Relating to the Capacity of the Gastrointestinal Tract to Reduce Cr+6

It has been known for some time, based on in vitro studies with human gastric fluid, that

reduction of at least some portion of ingested Cr+6 to Cr+3 occurs in the human stomach

(De Flora et al., 1987, 1997). However, no data on the capacity of the stomach of rodents

to reduce chromium are available. De Flora et al. (1997) estimated that the total Cr+6

reduction capacity of human gastric fluid is 10 mg between meals and up to 35 mg in the

2-4 hours following meals. Based on this, they have argued that the reduction capacity of

the human stomach is sufficiently large to maintain an excess reduction capacity even

with high doses of Cr+6. Given this assertion, it is appropriate to ask whether the

observed increase in intestinal tumors at the doses employed in the NTP study results

from an exceedance of the reduction capacity of the mouse gastrointestinal system. If the

tumors occur only because the reductive capacity of the mouse stomach was exceeded,

they may be less relevant to human risk at the lower doses that are more likely to be

encountered under environmental conditions.

The relative gastrointestinal absorption of Cr+6 and Cr+3

Based on their relative levels in urine or organs following oral or gastric administration,

it is known that Cr+6 is absorbed more readily from the gastrointestinal tract of rodents

than Cr+3 by a factor of about 1.8-60 in different studies (Donaldson and Barreras, 1966;

Maruyama, 1982; MacKenzie et al., 1959). This mirrors the difference in absorption

between Cr+3 and Cr+6 in humans, based on area-under-the-curve for urinary Cr, by a

factor of 53 (Kerger et al.,1996b). NTP conducted a 2-year bioassay with the Cr+3 dietary

supplement, chromium picolinate, formulated to maximize the generally low

bioavailability of Cr+3 (NTP, 2008b). This bioassay was conducted in parallel with

NTP’s study of sodium dichromate and employed the same strains of rats and mice.

As part of that study, the concentration of total Cr that was retained in plasma,

erythrocytes, liver, kidney, glandular stomach and forestomach of male rats and female

mice was measured at 25 weeks. This concentration was compared for similar doses of

Cr+3 and Cr+6 (15.18 and 8.95 mg/kg/day as Cr, respectively in male rats; and 36.73 and

13.2 mg/kg/day as Cr respectively in female mice). These data are presented, in part, in

Fig. 7 of the NTP final report for sodium dichromate dihydrate (NTP 2008a) and were

supplemented by personal communication from NTP.2 Despite the fact that the Cr+3 dose

was 1.8 and 2.8 times larger than the Cr+6 dose in rats and mice respectively, the

concentration of total Cr in these tissues was 1.4-16.7 times larger for the rats ingesting

Cr+6, and 2.1-38.6 times larger for the mice ingesting Cr+6. The lower end of these ranges

was found in the plasma, which was inconsistent with the Cr concentrations found in the

other tissues. The reason for this difference between plasma and the other tissues is that

there was a 48 hr “washout period” between the end of dosing and the collection of the

tissue samples. Cr was largely cleared from the plasma during this period and the

remaining Cr in the plasma mainly reflects redistribution from other tissues. For the

other tissues, the Cr concentration was 5.3-16.7 and 6.6-38.6 times larger for the rats and

2 Dr. Mathew Stout, NTP - personal communication 3/23/09.

23
mice (respectively) ingesting Cr+6 compared to animals ingesting Cr+3. It seems clear

that despite the assumed capacity of the gastrointestinal tract to reduce Cr+6, at least at

this dose, ingested Cr+6 was absorbed as Cr+6 rather than Cr+3.

Do the NTP pharmacokinetic data provide evidence that the Cr+6 reduction capacity of

the mouse gastrointestinal tract was exceeded?

If the reduction capacity of the mice was exceeded at the higher Cr+6 water

concentrations of sodium dichromate that were also associated with increased intestinal

tumors, there would be a threshold concentration at which unreduced Cr+6 would become

available for absorption. Given the significantly greater rate of Cr+6 absorption, such a

threshold would be evidenced by an increased rate of accumulation of total Cr in the

blood and organs. An increased rate of absorption in conjunction with a threshold

concentration would appear as a positive change in the slope of tissue Cr concentration

versus drinking water concentration. This hypothesis can be investigated using the

detailed animal-specific data, summarized in Appendix J, “Chromium Tissue Distribution

Study” of the NTP 2-year bioassay (animal-specific data provided as a personal

communication1), as well as data in the NTP short-term toxicokinetic study (NTP, 2007)

conducted in conjunction with its 2-year bioassay. In the first study, female mice from

among the exposure groups in the overall chronic bioassay were sacrificed at different

time points and the total Cr concentration in various tissues and biological media was

measured. In the second study, 6-7 week old male mice (the same strain used in the 2-

year study) were provided with drinking water ad libitum containing 1, 3, 10, 30, 100 or

300 mg/L Cr+6 as sodium dichromate dihydrate for 21 days. Animals were sacrificed and

total Cr concentration was measured in blood and kidney. Figure A-1a-g presents these

data for female mice for all drinking water concentrations. Figure A-2 presents the data

from the 21-day study in male mice.

We investigated this hypothesis through statistical analysis of the NTP pharmacokinetic

data. For the female mouse data, this hypothesis was tested for each of the tissues and

biological media by determining whether the slope of a linear function of tissue Cr

concentration versus water concentration of Cr+6 fit to a portion of the data was

significantly different from the linear slope fit to the entire data set. The portions of the

data set from 0 mg/L sodium dichromate water concentration to the first concentration

(14.3 mg/L) and from 0 mg/L to the second concentration (57.3 mg/L) were selected for

this test based on examination of the full data set. The hypothesis was tested at each of

the four time points at which tissue Cr concentrations were determined. For all tissues

and biological media, there was no significant difference between the slope for the partial

data set and the slope of the full data set at any of the time points. For the male mouse

data, it is clear from visual examination of Fig. A-2 that the trend of Cr accumulation

with increasing dose is supralinear (i.e. convex) across all doses. That is, changes in the

slope reflect a decrease in the rate of Cr accumulation with increasing dose rather than

the increase that would be expected if there were an exceedance of the reductive capacity

of the gastrointestinal tract. These findings do not support the hypothesis that the

reduction capacity of the mouse gastrointestinal tract was exceeded at some dose in the

NTP study. In this respect, it is interesting to note that diffuse hyperplasia was seen in

1 Dr. Bradley Collins, personal communication, 6/18/08

24
the duodenum at all doses in both sexes of mice. NTP attributed these lesions to

regenerative cell growth secondary to tissue injury. It is not clear whether this response

is causally related to the development of the observed neoplasms. This response is,

nonetheless, associated with Cr+6 exposure. The lack of an observed threshold for this

response, including at the lowest doses where the reductive threshold is not expected to

be exceeded, likewise does not support the hypothesis that the mice exceeded a threshold

for reduction of Cr+6 in this study.

Comparison of Cr+6 intake and the reduction capacity of mouse gastric fluid

Another approach to evaluating whether the Cr+6 exposures in the NTP study

overwhelmed the reductive capacity of the mice is to assess their estimated reduction

capacity compared to their rate of Cr+6 intake. There are no data on the reduction

capacity of mouse gastric fluid. However, the reduction capacity of mouse gastric fluid

can be estimated from data on the reduction capacity of human gastric fluid. Based on

experiments in which aspirated human gastric fluid was reacted ex-vivo with Cr+6, and

data on the total daily volume of human gastric fluid, De Flora et al. (1987, 1997)

estimated the Cr+6 reduction capacity of human gastric fluid at >84-88 mg Cr+6/day,

although they also indicate that the procedures used in preparation of the gastric fluid

likely resulted in a underestimation of its reductive capacity (De Flora et al., 1997). They

also estimated an additional 11-24 mg/day reduction capacity from intestinal bacteria, but

it is unclear how much of this capacity resides in the small intestine. Ingested Cr+6 is not

resident in the stomach for an entire day, but is likely to be either rapidly absorbed (see

below) or passed on to the small intestine as a function of gastric emptying time (T1/2 for

gastric emptying in humans is reported as 127 min by Hellmig et al.,2006). De Flora et

al. (1997) reported that the reduction capacity of gastric fluid reached a maximum in

conjunction with meals and was sustained for 2-3 hr following the meal. This is

consistent with the above estimate for T1/2 for gastric emptying. The meal-associated

reduction was 25.1 mg Cr+6/meal. This gives a meal-associated reduction rate of 25.1 mg

Cr+6/2.5 hr = 10 mg Cr+6/hr. This value can be scaled to the mouse gastric fluid on the

basis of (body-weight)3/4. This method of interspecies scaling takes metabolic and

physiological factors (e.g., food consumption rate, gastric fluid secretion rate) into

account. While the actual chemical process governing the reduction of Cr+6 is probably a

physico-chemical interaction and thus not a function of body weight, the circumstances

governing the conditions under which this chemical interaction occurs are likely to be

under metabolic and physiological control. These include the production and secretion of

the chemicals involved in the reduction reaction, the gastric mixing, and the gastric and

small intestine emptying time. Therefore, (body-weight)3/4 scaling was applied in the

calculation of the human cancer potency slope in Table 3 of the main document. Using

the human reduction rate of 10 mg Cr+6/hr and assuming an adult human body-weight of

70 kg, adjustment on the basis of (body-weight)3/4 gives a generalized (body-weight)3/4

gastric fluid reduction rate which should be applicable to any mammalian species of

0.4132 mg/hr/kg3/4. This rate can be applied to the mice in the NTP study by multiplying

by the control mouse (body weight)3/4 (i.e., (0.050)3/4 and (0.053)3/4 for males and

females respectively), giving values of 0.044 mg/hr and 0.046 mg/hr for males and

females.

25
The Cr+6 intake rate of the mice can be estimated from the rate of water consumption in

mice. Ho and Chin (1988) reported that daily water consumption in mice was 4.4 +/- 0.3

ml. Water consumption was closely linked temporally to eating, and 86% of water

consumption occurred during the 12 hr dark period. Thus, the hourly dark period water

consumption rate can be estimated as (4.4 ml x 0.86)/12 hr or 0.32 ml/hr. Toya and

Clapp (1972) measured 5.7 ml of water consumption by mice during a 17 hr overnight

period giving a nighttime consumption rate of 0.34 ml/hr. Given this close agreement,

the average rate of 0.33 ml/hr is assumed. This rate of water consumption for the

maximum period of water intake (i.e., night), can be multiplied by the Cr+6 drinking

water concentrations in the NTP study to give an estimate of the maximum rate of Cr+6

intake for the mice (mg/hr). The estimated maximum Cr+6 intake rate for the mice at

each dose is presented in Table A-1.

Comparing the estimated capacity of gastric reduction of Cr+6 of 0.044 and 0.046 mg/hr

for male and female mice respectively, to the estimated maximum Cr+6 intake rates in

Table A-1, shows that only the intake rate for female mice at the highest concentration of

Cr+6 in drinking water (0.059 mg/hr) exceeds the estimated reduction capacity. The next

highest intake rate in female mice is 43% of the estimated reduction capacity. The

highest intake and second highest rate in male mice were 68% and 23% of the estimated

reduction capacity. While only the highest dose in females exceeded the estimated

reduction capacity in this analysis, the observed significant increase in tumor rates

occurred at the two highest doses in males and the two highest doses in female mice

(along with a non-significant, but consistent increase at the third highest dose).

Therefore, the observed increase in tumor rates is not consistent with the hypothesis that

the mouse intestinal tumors resulted from the intake rate of Cr+6 exceeding the reduction

capacity.

26
Table A-1

Estimated Cr+6 intake rates for male and female mice as a function of Cr+6 drinking

water concentration

Cr+6 water

conc.

(mg/L)

Mean peak period

Cr+6 intake rate

(Assuming water

consumption at 0.33

ml/hr)

(mg/hr)

Male mice

0 0

5 0.0017

10 0.0033

30 0.0099

90 0.030

Female

mice

0 0

5 0.0017

20 0.0066

60 0.020

180 0.059

This comparison assumes that with respect to the reduction of Cr+6, the mouse stomach

can be viewed as a closed system. That is, that the mass of ingested Cr+6 is all present in

the stomach during the entire period under consideration. In fact, even if the reduction

capacity of the mouse stomach is not exceeded, the extent of reduction is limited by the

kinetics of gastric emptying. The half-time for gastric emptying of liquids in the mouse

has been reported as <5-9 min (Moretó et al., 1982; Symonds et al., 2007). This means

that even when the hourly rate of Cr+6 reduction greatly exceeds the hourly rate of Cr+6

intake, a substantial fraction of the ingested Cr+6 can be expected to escape reduction by

being transported from the stomach to the small intestine.

Effect of pH on reduction capacity for Cr+6

There is a difference in the pH of human and mouse gastric fluids. In the normal human

gastric fluid evaluated by De Flora et al. (1987, 1997), the pH varied from approximately

1.0-3.5. In the mouse, the pH of the stomach varies from 3.1-4.5 (Kararli, 1995;

McConnell et al., 2008). While there is some relationship between pH and Cr+6 reductive

capacity, the correlation is only moderate. This can be seen in the study of De Flora et al.

(1987) in which aspirated gastric fluid from individuals collected hourly for 24 hours was

reacted with Cr+6. These individuals included those with normal gastric fluid as well as

those with gastric fluid with higher than normal pH resulting from anti-secretory

27
medication or duodenal reflux. In normal subjects, the pH of the gastric fluid increased at

the start of a meal from the fasting pH of approximately 1.0 to 3-3.5, probably due to the

buffering capacity of the food. While the pH at the time of maximum reduction was

approximately 1.5-2.0, the increased pH associated with the meal resulted in only about a

50% decrease in the reduction capacity (about 6 times the fasting capacity). Increasing

the pH to 7.0 from an initial value of 0.8-1.5 resulted in a decrease in reduction by a

factor of approximately 5. However, decreasing the pH to 1.0 from subjects with an

initial pH of 5.1-7.2 resulted in only a slight and non-statistically significant increase in

reduction. Thus, it appears that reduction capacity is affected to some extent by pH, but

is largely under control of other gastric factors associated with eating, possibly including

gastric secretions and food itself. Reduction appears to be accomplished by small

molecules such as ascorbate rather than by thermo-labile substances such as enzymes (De

Flora et al., 1987). Therefore, it seems unlikely that Cr+6 reduction capacity is

significantly affected by potential inter-species differences in enzyme type or function or

pH. Rather, it seems likely that interspecies differences in Cr+6 reduction capacity are a

function of their underlying metabolic rate.

Additional evidence of the role of pH on the reduction capacity of gastric juice is

provided by Kerger et al. (1996a). Cr+6 (5 mg) added to 10 ml of orange juice with a pH

of 3.74 was 100% reduced within 15 minutes. In 10 ml of lemonade, with a pH of 3.01,

40% of 5 mg of Cr+6 was reduced in approximately 170 minutes. The difference in

reduction in these two solutions, with the lower pH resulting in significantly less

reduction, illustrates that the chemical nature of the gastric fluids rather than their pH is

the critical factor in determining the extent of Cr+6 reduction. In addition, it should be

noted that the orange juice, whose pH is within the range of the mouse stomach (3.1-4.5),

rapidly and completely reduced a relatively large mass of Cr+6. These lines of reasoning

suggest that the validity of the above comparison of the Cr+6 reduction rate of human

gastric fluid and the intake rate of Cr+6 in the NTP mice is not dependent on the pH

difference between the human and mouse stomachs. Therefore, it is reasonable to

assume that differences between humans and mice in the reduction capacity of their

gastrointestinal tracts stem largely from differences in their metabolic rates rather than

from differences in pH or biochemistry. Thus, inter-species differences in this function

are appropriately adjusted on the basis of a (body-weight)3/4 adjustment.

When the metabolic rate is adjusted on this basis, the mouse appears to be a reasonable

model for the human gastrointestinal carcinogenicity of Cr+6.

Comparison of the reduction capacity of the mouse and rat

The rats exposed in the NTP study had an elevated incidence of tumors in the oral cavity

but not in the small intestine or elsewhere in the gastrointestinal tract. This raises the

possibility that the absence of an elevated incidence of gastrointestinal tumors in the rat

results from a more efficient capacity for reduction of Cr+6 in the rat gastrointestinal tract

than in the mouse. This can be investigated by comparing the rate of increase of Cr in

urine as a function of Cr+6 drinking water concentrations in male rats and female mice at

day 371. Urine is an appropriate medium for this comparison since urine integrates the

total body absorption of Cr. Day 371 reflects the maximum accumulation for both

species. The NTP data on accumulation of Cr do not permit the comparison of these data

28
for the same sex in rats and mice. For rats and mice, the linear slope of the relationship

between mean urine Cr concentration and drinking water concentration is 3.00 and 0.94

g Cr/g urine per mg Cr/L drinking water respectively. In other words, the rate of uptake

of Cr from the rat gastrointestinal tract as a function of concentration of Cr+6 in drinking

water is more than 3 times that of the mouse. Since the tumors in the mouse small

intestine must have resulted from the absorption of Cr+6 into the intestinal tissue, the even

greater rate of absorption of Cr by the rat must, likewise, reflect an even greater exposure

of the intestinal tissues to Cr+6. Thus, while it is not clear why the mice, but not the rats

developed gastrointestinal tract tumors, the evidence does not support the hypothesis that

the lack of gastrointestinal tumors in the rats reflects a more efficient reduction capacity

for Cr+6.

Human gastric reduction capacity and exposure to Cr+6

The lack of evidence to support the hypothesis that the observed tumor incidence results

from exceedance of the reduction capacity of the mouse gastrointestinal tract raises the

question of whether similar considerations would also apply to human environmental

exposures. O’Flaherty et al. (2001) analyzed the data from a series of related studies of

intentional human exposure to Cr+6 in drinking water (Paustenbach et al., 1996; Kerger et

al., 1996a, 1996b; Finley et al., 1997) in which the daily Cr+6 dose ranged from 0.001

mg/kg to approximately 0.2 mg/kg. In one of these studies (Kerger, 1996b), both Cr+3

and Cr+6 (5 mg each) were ingested each by four subjects (with one subject separately

ingesting both) and the rate of appearance of each in the urine (as total Cr) was

compared. Consistent with the studies discussed above, both the rate of appearance and

the overall area-under-the-curve of total Cr in urine were much larger for the ingestion of

Cr+6 than for ingestion of Cr+3. For Cr+3, the mean peak urinary concentration was 8.9

g/g creatinine and a total of 0.13% of the dose was recovered in the urine. For Cr+6, in

contrast, the peak concentration was 209 g/g creatinine and 6.9% of the dose was

recovered in the urine. With reference to this series of human dosing studies, O’Flaherty

et al. (2001) concluded that based on the reduction capacity estimated by De Flora et al.

(1997), “Even if all of the maximum single or multiple 5-mg doses had been ingested

instantaneously, the total reducing capacity of gastric juice should not have been

exceeded. Nonetheless, it is clear, based on total urinary chromium excretion, that a

consistently greater percentage of the Cr+6 than of the Cr+3 was absorbed. This

observation, consonant with other observations in humans (Donaldson and Barreras,

1966), implies that some Cr+6 escaped reduction in the stomach and entered portal venous

blood. The greater absorption of Cr+6 than Cr+3 does not imply that the reduction

capacity of gastric juice was exceeded, but rather that absorption from the gastrointestinal

tract is so rapid that it is able to compete effectively with reduction in the stomach.” This

implies that, regardless of the reductive capacity of human gastric fluid, the kinetics of

Cr+6 uptake from the gastrointestinal tract favor absorption of at least a portion of an

ingested dose. The rapid uptake of Cr+6 compared with Cr+3 appears to result from the

transport of anionic, Cr+6-containing, chromate or dichromate complexes across cell

membranes by the general anion transport system that is also responsible for transport of

SO4

-2 and PO4-3 (Cohen et al., 1993). Cr+3, on the other hand, crosses cell membranes

only by passive diffusion. Thus, whether Cr+6 is absorbed directly from the stomach as

suggested by the epidemiologic data of Beaumont et al. (2008), or from the small

29
intestine as observed in the mice in the NTP study, the evidence strongly suggests that, as

in the mouse, in exposed humans there can be significant exposure to unreduced Cr+6

even at low doses. This is consistent with the previously cited data on the kinetics of

gastric emptying in mice.

Kerger et al. (1996a) proposed an alternative explanation for the more rapid and complete

appearance of Cr in blood following Cr+6 ingestion compared to Cr+3 ingestion. They

proposed that Cr+6 is reduced in the stomach to an organic complex that is a particularly

absorbable form of Cr+3 . However, the literature cited in support of the existence of such

complexes (Ronai, 1969; Edel and Sabbioni, 1985; Gargas et al., 1994; Kortenkamp and

Beyersmann, 1987) does not address the formation of Cr+3 complexes in the

gastrointestinal tract and/or does not establish the existence of an organic Cr+3 complex

with absorption characteristics similar to Cr+6. One of the studies cited (Gargas et al.,

1994) examined the appearance of Cr+3 ingested as chromium picolinate, a synthetic

organic nutritional supplement designed to maximize the otherwise low bioavailability of

Cr+3. Gargas et al. (1994) report the bioavailability of Cr+3 from chromium picolinate as

2.8%. Even given the attempt to maximize Cr+3 gastrointestinal uptake through the use

of this organic complex, the bioavailability of Cr+3 is still considerably smaller than the

value of 6.9% reported by Kerger et al. (1996b) for ingested Cr+6. Another study

(Gonzalez-Vergara et al., 1981) also employed novel synthetic (pyridoxilidene and

nicotinic acid) complexes with Cr+3 with no indication that such complexes are produced

in the gastrointestinal tract. Levis et al. (1978), who are also cited in support of this

explanation, hypothesize absorption of Cr+3 due to stable “chelates and coordination

complexes” formed in cell culture environments. However, they also note that internal

cell concentrations of Cr resulting from incubation with soluble Cr+3 are 20 times lower

than those resulting from the same concentration of Cr+6. Mertz (1969, 1971) notes that

the formation of coordination complexes with small molecules in the intestine and intake

of Cr already bound to glucose tolerance factor make Cr available for absorption from the

intestines. However, these observations do not distinguish between Cr+3 and Cr+6 in this

regard, nor do they suggest that their absorption is comparable. Mertz (1969) also notes

that Cr binds selectively to siderophilin, which facilitates its transport to tissues. Here,

again, the valence of Cr is not specified and this binding is identified as a phenomenon in

serum rather than in the intestines. O’Flaherty et al. (2001) conclude that the explanation

of the formation of such Cr+3 complexes “ ... is considered implausible, because no

known complexes of CrIII are absorbed to the extent that CrVI is.”

Specific evidence against the hypothesis of intestinal uptake of Cr+3 as a readily absorbed

organic complex is provided by Donaldson and Barreras (1966) who incubated Cr+6 in

human gastric fluid (pH 1.4) for 30 minutes and then perfused the material into the

jejunum of human subjects. Whereas approximately 25% of Cr+6 perfused into the

jejunum without pre-incubation in gastric fluid was absorbed, the gastric fluid incubation

resulted in nearly complete inhibition of absorption from the jejunum. This provides

direct evidence that reduction of Cr+6 in the stomach does not result in readily absorbable

forms of Cr+3.

Evidence for low dose gastrointestinal absorption of Cr+6 by mice

30
The assumption that, based on evidence from human studies, Cr+6 is absorbed from the

gastrointestinal tract of mice more rapidly than it is reduced to Cr+3 is supported by

reports of systemic effects of low oral doses of Cr+6. Davidson et al. (2004) found that in

hairless mice exposed to UV light, significantly more skin tumors were produced in the

mice that also received 0.13 mg/L Cr+6 as potassium chromate in their drinking water.

This concentration is only 3% of the lowest drinking water concentration in the NTP

study. Murthy et al. (1996) found ultrastructural abnormalities in the ovaries of Swiss

albino mice given 5 mg/L Cr+6 in their drinking water, the same concentration as the

lowest concentration in the NTP study. Dana Devi et al. (2001) investigated the levels of

single strand DNA breaks in leukocytes as reflected in the comet assay in mice

administered a single oral dose of potassium dichromate. At the lowest dose, 0.59 mg/kg

as potassium dichromate (0.21 mg/kg as Cr+6), as well as at higher doses, there was a

statistically significant increase in DNA breaks compared to controls as measured by the

length of the comet tail. The lowest dose in that study (as Cr+6) is about half the lowest

daily dose received in the NTP study. These observations are consistent with the low

dose uptake of Cr+6 from the gastrointestinal tract of mice, but they are not consistent

with known systemic effects of Cr+3.

Conclusions regarding reduction capacity

In summary, there does not appear to be any clear evidence to support a hypothesis that

the tumors in the mouse small intestine resulted from the Cr+6 doses in the NTP chronic

bioassay overwhelming the reduction capacity of the gastrointestinal tract. In contrast,

both the pharmacokinetic data on Cr accumulation in the various organs and the

comparison of the mouse Cr+6 intake rate to the Cr+6 reduction rate in human gastric fluid

provide evidence that the observed tumor incidence in the mice cannot be explained by

exceedance of the reduction capacity.

31
Appendix B

Cancer Potency Derivation Based on the NTP Sodium Dichromate Chronic

Bioassay by USEPA Office of Prevention, Pesticides and Toxic Substances (OPPTS)

The USEPA OPPTS conducted a risk assessment and cancer potency derivation based on

the NTP’s sodium dichromate chronic bioassay in conjunction with its consideration of

the pesticide reauthorization of copper-chromate-arsenic (CCA) treated wood (USEPA,

2008a, b). The narrative portion of the assessment concluded that, with respect to the

2005 USEPA Cancer Guidelines, Cr(VI) is “Likely to be Carcinogenic to Humans” based

on the presence of oral mucosa and tongue tumors in male and female rats and tumors of

the small intestine in male and female mice at doses that were adequate, but not

excessive, to assess carcinogenicity. There is clear evidence that Cr(VI) is mutagenic and

convincing evidence supporting a mutagenic mode of action.” (USEPA, 2008a). The

quantitative derivation of a cancer potency estimate was based on combined intestinal

tumors (duodenum, jejunum, and ileum) in female mice using the linearized multistage

model (Q1*) and (body-weight)3/4 scaling of doses. Based on this approach, they derived

a human cancer potency estimate of 0.79 (mg/kg/day)-1.

The approach followed by the USEPA OPPTS differs from the one followed in this

document in several respects. The OPPTS chose a potency derived from female mice (as

opposed to rats of either sex or to male mice) because that species and sex was the most

sensitive. That is, it yielded the largest potency estimate. In contrast, the cancer potency

estimate derived in this document is based on male mice. The choice of male mice for

the assessment provided in this document was based on the observation that, although

female mice yielded a slightly larger estimate of potency with some benchmark dose

models, the overall fit of those models to the female mouse data were poor and would

generally be considered unacceptable.

The OPPTS also chose to use the linearized multistage model to calculate the cancer

potency slope directly from the fit of the data to that model. In contrast, this document

used the approach recommended in the current USEPA Cancer Guidelines (USEPA,

2005a) that calls for the slope to be calculated from a straight line extending from the

point-of-departure (POD) to the point corresponding to zero incremental dose-zero

incremental response. The two approaches are not equivalent and it is unclear why

OPPTS chose not to follow the current USEPA (2005) guidelines. Also, OPPTS

assumed a weight of 30 g for the female mice for use in the allometric dose conversion

from mouse to human. In this document, the body weight for males and female control

mice (50 and 53 g, respectively) was used for the allometric dose conversion. The basis

for the choice of 30 g by OPPTS is unclear given that the time-weighted average weights

for these mice varied from 42-53 g depending on the dose. Finally, the OPPTS

assessment identified a mutagenic mode of action for Cr+6 carcinogenicity by the oral

route of exposure. At the present time, the criteria for this determination are not clear and

the age dependent adjustment factor for mutagenic MOA was not used in the risk

assessment presented in this document.

32
Despite these differences in approach and interpretation, it is interesting to note that the

OPPTS cancer potency estimate based on female mice, 0.79 (mg/kg/day)-1, and its

potency estimate based on male mice, 0.65 (mg/kg/day)-1 are close to the estimate of 0.50

(mg/kg/day)-1 provided in this document. Both OPPTS and the risk assessment presented

herein conclude that under the current USEPA Cancer Guidelines, Cr+6 is “likely to be

carcinogenic to humans.”

33
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37
Fig. 1. Fig. 2.

Incidence of oral tumors in male rats Incidence of oral tumors in female

(not adjusted for number of animals rats (not adjusted for number of animals

at-risk – see text) at-risk – see text)

Male rats

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25 30

sodium dichromate dose (mg/kg/day)

Female Rats

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25 30

sodium dichromate dose (mg/kg/day)

Fig. 3. Fig. 4.

Incidence of intestinal tumors in male mice Incidence of intestinal tumors in

(not adjusted for number of animals female mice (not adjusted for

at-risk – see text) of animals at-risk – see text)

Female mice

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25 30

sodium dichromate dose (mg/kg/day)

Male mice

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25 30

sodium dichromate dose (mg/kg/day)

38
Fig. 5

Intestinal neoplasms in male mice benchmark dose modeling – Multistage cancer model

BMD (benchmark dose) – the dose corresponding to the 10% response rate after adjusting for response rate

in controls

BMDL (benchmark dose-low) – the dose corresponding to the lower confidence bound on the 10%

response rate after adjusting for response rate in controls

39
Fig. 6

Intestinal neoplasms in female mice benchmark dose modeling – Multistage cancer

model

BMD (benchmark dose) – the dose corresponding to the 10% response rate after adjusting for response rate

in controls

BMDL (benchmark dose-low) – the dose corresponding to the lower confidence bound on the 10%

response rate after adjusting for response rate in controls

40
Fig. 7

Intestinal neoplasms in combined male and female mice benchmark dose modeling –

Multistage cancer model

BMD (benchmark dose) – the dose corresponding to the 10% response rate after adjusting for response rate

in controls

BMDL (benchmark dose-low) – the dose corresponding to the lower confidence bound on the 10%

response rate after adjusting for response rate in controls

41
Fig. 8

Intestinal neoplasms in combined male and reduced female mice (female high-dose

excluded) benchmark dose modeling – Gamma multi-hit model

BMD (benchmark dose) – the dose corresponding to the 10% response rate after adjusting for response rate

in controls

BMDL (benchmark dose-low) – the dose corresponding to the lower confidence bound on the 10%

response rate after adjusting for response rate in controls

42
Fig. A-1a

Concentration of Cr in female mouse kidney tissue at selected times in conjunction with

drinking water exposure to sodium dichromate

Female mouse kidney

Cr concentration

Mean; Whisker: Mean-.95 Conf. Interval, Mean+.95 Conf. Interval

conc d6

conc d13

conc d182

conc d371 -100 0 100 200 300 400 500 600

Sodium dichromate water concentration mg/L

-5

0

5

10

15

20

25

30

35

43
Fig. A-1b

Concentration of Cr in female mouse liver tissue at selected times in conjunction with

drinking water exposure to sodium dichromate

Female mouse liver

Cr concentration

Mean; Whisker: Mean-.95 Conf. Interval, Mean+.95 Conf. Interval

conc d6

conc d13

conc d182

conc d371 -100 0 100 200 300 400 500 600

Sodium dichromate water concentration (mg/L)

-10

0

10

20

30

40

50

60

70

44
Fig. A-1c

Concentration of Cr in female mouse non-glandular stomach tissue at selected times in

conjunction with drinking water exposure to sodium dichromate

Female mouse non-glandular stomach

Cr concentration

Mean; Whisker: Mean-.95 Conf. Interval, Mean+.95 Conf. Interval

conc d6

conc d13

conc d182

conc d371 -100 0 100 200 300 400 500 600

Sodium dichromate water concentration (mg/L)

-10

-5

0

5

10

15

20

25

30

45
Fig. A-1d

Concentration of Cr in female mouse glandular stomach tissue at selected times in

conjunction with drinking water exposure to sodium dichromate

Female mouse glandular stomach

Cr concentration

Mean; Whisker: Mean-.95 Conf. Interval, Mean+.95 Conf. Interval

conc d6

conc d13

conc d182

conc d371 -100 0 100 200 300 400 500 600

Sodium dichromate water concentration (mg/L)

-20

0

20

40

60

80

100

120

46
Fig. A-1e

Concentration of Cr in female mouse plasma at selected times in conjunction with

drinking water exposure to sodium dichromate

Female mouse plasma

Cr concentration

Mean; Whisker: Mean-.95 Conf. Interval, Mean+.95 Conf. Interval

conc d6

conc d13

conc d182

conc d371 -100 0 100 200 300 400 500 600

Sodium dichromate water concentration (mg/L)

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

47
Fig. A-1f

Concentration of Cr in female mouse erythrocytes at selected times in conjunction with

drinking water exposure to sodium dichromate

Female mouse erythrocytes

Cr concentration

Mean; Whisker: Mean-.95 Conf. Interval, Mean+.95 Conf. Interval

conc d6

conc d13

conc d182

conc d371 -100 0 100 200 300 400 500 600

Sodium dichromate water concentration (mg/L)

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

48
Fig. A-1g

Concentration of Cr in female mouse urine at selected times in conjunction with drinking

water exposure to sodium dichromate

Female mouse urine

Cr concentration

Mean; Whisker: Mean-.95 Conf. Interval, Mean+.95 Conf. Interval

conc d371

conc d5

conc d12

conc d181 -100 0 100 200 300 400 500 600

Sodium dichromate water concentration (mg/L)

-60

-40

-20

0

20

40

60

80

49
Fig. A-2

Concentration of Cr in male mouse blood and kidney after 21 days of exposure in

conjunction with drinking water exposure to sodium dichromate

Male mouse - liver and kidney Cr concentration - 21 day study

-50 0 50 100 150 200 250 300 350

Sodium dichromate water conc (mg/L)

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Cr blood conc

Cr kidney conc

50