S. HRG. 112–953 

OVERSIGHT HEARING ON THE ENVIRONMENTAL 

PROTECTION AGENCY’S IMPLEMENTATION OF 

THE SAFE DRINKING WATER ACT’S UNREGULATED DRINKING WATER CONTAMINANTS PROGRAM 

HEARING 

BEFORE THE

COMMITTEE ON 

ENVIRONMENT AND PUBLIC WORKS 

UNITED STATES SENATE 

ONE HUNDRED TWELFTH CONGRESS 

FIRST SESSION 

JULY 12, 2011 

Printed for the use of the Committee on Environment and Public Works 

( 

Available via the World Wide Web: http://www.fdsys.gov 

OVERSIGHT HEARING ON THE ENVIRONMENTAL PROTECTION AGENCY’S IMPLEMENTACONTAMINANTS PROGRAM TION OF THE SAFE DRINKING WATER ACT’S UNREGULATED DRINKING WATER 

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23–819 PDF 2017

S. HRG. 112–953 

OVERSIGHT HEARING ON THE ENVIRONMENTAL 

PROTECTION AGENCY’S IMPLEMENTATION OF 

THE SAFE DRINKING WATER ACT’S UNREGULATED DRINKING WATER CONTAMINANTS PROGRAM 

HEARING 

BEFORE THE

COMMITTEE ON 

ENVIRONMENT AND PUBLIC WORKS 

UNITED STATES SENATE 

ONE HUNDRED TWELFTH CONGRESS 

FIRST SESSION 

JULY 12, 2011 

Printed for the use of the Committee on Environment and Public Works 

( 

Available via the World Wide Web: http://www.fdsys.gov 

(II) 

COMMITTEE ON ENVIRONMENT AND PUBLIC WORKS 

ONE HUNDRED TWELFTH CONGRESS 

FIRST SESSION 

BARBARA BOXER, California, Chairman 

MAX BAUCUS, Montana 

THOMAS R. CARPER, Delaware 

FRANK R. LAUTENBERG, New Jersey 

BENJAMIN L. CARDIN, Maryland 

BERNARD SANDERS, Vermont 

SHELDON WHITEHOUSE, Rhode Island 

TOM UDALL, New Mexico 

JEFF MERKLEY, Oregon 

KIRSTEN GILLIBRAND, New York 

JAMES M. INHOFE, Oklahoma 

DAVID VITTER, Louisiana 

JOHN BARRASSO, Wyoming 

JEFF SESSIONS, Alabama 

MIKE CRAPO, Idaho 

LAMAR ALEXANDER, Tennessee 

MIKE JOHANNS, Nebraska 

JOHN BOOZMAN, Arkansas 

BETTINA POIRIER, Majority Staff Director and Chief Counsel 

RUTH VAN MARK, Minority Staff Director 

(III) 

C O N T E N T S 

Page 

JULY 12, 2011 

OPENING STATEMENTS 

Boxer, Hon. Barbara, U.S. Senator from the State of California ........................ 1 

Inhofe, James M., U.S. Senator from the State of Oklahoma .............................. 3 

Lautenberg, Frank, U.S. Senator from the State of New Jersey ......................... 5 

Boozman, Hon. John, U.S. Senator from the State of Arkansas ......................... 6 

Cardin Hon. Benjamin, U.S. Senator from the State of Maryland ..................... 7 

Sessions, Hon. Jeff, U.S. Senator from the State of Alabama, prepared statement ...................................................................................................................... 177 

WITNESSES 

Trimble, David C., Director, Natural Resources And Environment, Government Accountability Office .................................................................................. 8 

Prepared statement .......................................................................................... 11 

Responses to additional questions from: 

Senator Boxer ............................................................................................ 36 

Senator Inhofe ........................................................................................... 41 

Perciasepe, Hon. Robert, Deputy Administrator, Environmental Protection 

Agency ................................................................................................................... 43 

Prepared statement .......................................................................................... 45 

Responses to additional questions from: 

Senator Boxer ............................................................................................ 55 

Senator Whitehouse .................................................................................. 59 

Senator Inhofe ........................................................................................... 63 

Goldman, Lynn, Md, Mph, American Public Health Association, Dean of 

the School of Public Health and Health Services and Professor of Environmental and Occupational Health at the George Washington University ........ 88 

Prepared statement .......................................................................................... 90 

Responses to additional questions from: 

Senator Boxer ............................................................................................ 97 

Senator Inhofe ........................................................................................... 97 

Araiza, Anthony, General Manager, West Valley Water District ........................ 98 

Prepared statement .......................................................................................... 100 

Responses to additional questions from Senator Inhofe ............................... 103 

Joseph A. Cotruvo, Ph.D., President, Joseph Cotruvo and Associates ................ 106 

Prepared statement .......................................................................................... 108 

Responses to additional questions from Senator Inhofe ............................... 118 

Patierno, Steven R., Ph.D., Executive Director, George Washington University 

Cancer Center ....................................................................................................... 124 

Prepared statement .......................................................................................... 137 

Responses to additional questions from Senator Inhofe ............................... 161 

Griffiths, Jeffrey, K., Md, MPH and TM, Chair, Drinking Water Committee, 

Science Advisory Board of United States Environmental Protection Agency, 

Professor of Public Health and of Medicine, Tufts University School of 

Medicine ................................................................................................................ 165 

Prepared statement .......................................................................................... 167 

ADDITIONAL MATERIAL 

Peer Review Workshop for EPS’s Draft Toxicological Review of Hexavalent 

Chromium; Reviewer Post-Meeting Comments ................................................. 179 

(1) 

OVERSIGHT HEARING ON THE ENVIRONMENTAL PROTECTION AGENCY’S IMPLEMENTATION OF THE SAFE DRINKING 

WATER ACT’S UNREGULATED DRINKING 

WATER CONTAMINANTS PROGRAM 

WEDNESDAY, JULY 12, 2011 

U.S. SENATE, COMMITTEE ON ENVIRONMENT AND PUBLIC WORKS, Washington, DC. 

The full committee met, pursuant to notice, at 10 a.m. in room 

406, Dirksen Senate Office Building, Hon. Barbara Boxer (chairman of the full committee) presiding. 

Present: Senators Boxer, Inhofe, Lautenberg, Cardin, 

Whitehouse, Udall, Boozman. 

STATEMENT OF HON. BARBARA BOXER, 

U.S. SENATOR FROM THE STATE OF CALIFORNIA 

Senator BOXER. Welcome, everybody. I have called this hearing 

to examine one of the most important Federal public health safeguards in our Country, the Safe Drinking Water Act. Everyone has 

a right to clean and safe drinking water, and it is essential to the 

health of our children and our families that drinking water be free 

from harmful chemicals and pollutants. 

In order to ensure that enough is being done to protect our Nation from emerging contaminants, I, along with Representatives 

Waxman and Markey, asked the GAO to investigate the unregulated contaminant program. The report is being released today. I 

want to thank the GAO, the Government Accountability Office, for 

their work and welcome them here today, along with our Deputy 

Administrative. We welcome both. 

The GAO investigation addresses the stunning fact that the EPA 

has not made a determination to regulate any new drinking water 

contaminants, with one very recent exception. And that failure to 

move goes back to 1996 when the law was last amended. This law 

was signed by Gerald Ford, who made a very eloquent statement 

about it. I think it is very important to remember that our landmark laws are bipartisan in nature, and why I think it is so important that we work across party. 

This is what Gerald Ford said when he signed the Safe Drinking 

Water Act: ‘‘Nothing is more essential to the life of every single 

American than clean air and pure food and safe drinking water. 

There have been strong national programs to improve the quality 

of our air and the purity of our food.’’ He said, ‘‘This bill, the Safe 

2 

Drinking Water Act, will provide us with the protection we need 

for drinking water.’’ 

And since I have mentioned Gerald Ford, I want to note the 

passing of this incredible former First Lady, Betty Ford, who I had 

the pleasure of meeting and following her career. I know I speak 

for everybody when we send our deepest condolences. Her life was 

a life that really impacted others because she had courage to speak 

about things that a lot of people were fearful to speak about, 

whether it was breast cancer or the challenges facing society or addiction to drugs and alcohol. I just think her kind of leadership, 

which she really did not seek the spotlight, had a tremendous impact. I wanted to make that note. 

So when we talk about this report and why it is so important, 

I think the report is in the spirit of what Gerald Ford said, that 

we expect to have safe drinking water. In order to do that, we need 

to update our laws to make sure that we are not allowing certain 

contaminants in the water. And the GAO is very clear that EPA 

has to find what contaminants are dangerous and get them out of 

the water. 

In 1996, Congress amended the Safe Drinking Water Act and directed EPA to use the best available science to create drinking 

water safeguards that would protect the most vulnerable, including 

infants, children and pregnant women. As the GAO report shows, 

the development of new standards for unregulated drinking water 

contaminants, such as perchlorate, were derailed in a process that 

failed to use the best available science. 

We all say we want the best available science, Republicans, 

Democrats. We can just decide something is bad for you because it 

sound bad, has a scary name. We have to know the science behind 

it. And the fact is, we haven’t seen any action since 1996 with this 

recent exception of perchlorate, for which I am very grateful. Because I have a bill with Senator Feinstein to regulate perchlorate. 

But it shouldn’t be necessary to go to one contaminant at a time. 

The EPA has to act. 

Scientific information has shown that certain emerging contaminants in our drinking water, such as perchlorate and chromium– 

6 could be harmful to children and families. Perchlorate, which is 

used in rocket fuel, fireworks and road flares, poses a threat to 

human health when found in drinking water. It impairs the function of the thyroid gland, which harms children’s development and 

can result in decreased learning capacity. EPA needs to have a 

process that vigorously addresses these contaminants to help ensure the safety of our water. 

GAO’s report lays out a transparent and accountable framework 

that can help to ensure the EPA uses the best available science 

when creating drinking water protections for our communities and 

our families. Again, I am so glad that EPA is here today. And they 

are going to tell us about the implementation of the program. We 

are also going to hear GAO’s testimony on the agency’s need to use 

science to ensure that the Federal Government provides strong 

public health protections against drinking water contaminants. 

And with that, Senator Inhofe. 

3 

STATEMENT OF HON. JAMES M. INHOFE, 

U.S. SENATOR FROM THE STATE OF OKLAHOMA 

Senator INHOFE. Thank you, Madam Chairman. And let me tell 

you, I appreciate your remarks about Betty Ford. I happened to 

know her quite well. And you are right, she got into things, talking 

about alcoholism and she probably accomplished more than any 

other First Lady I can think of. They are celebrating her life today. 

I think everyone in this room agrees that providing safe, clean, 

affordable drinking water is essential and should be a national priority. At the cornerstone of the Safe Drinking Water Act is the idea 

that we should be controlling those substances that pose risks to 

public health. Unfortunately, the system that EPA uses to determine health risks, the IRIS system, the Integrated Risk Information System, has decades-long issues in crafting risk assessments. 

The National Academy of Sciences has recently pointed out that 

IRIS assessments have suffered from lack of transparency and consistency and problems with evaluating studies and the weight of 

evidence. These problems continue to persist in the face of the NAS 

and Congress repeatedly imploring EPA to correct these issues. 

Without having a good foundation and sound science, practices in 

all areas of EPA’s regulatory system are suffering, including drinking water. Unbiased, high quality scientific analyses are important 

foundations for making drinking water decisions. When the risk assessment that EPA is producing are unable to maintain the highest 

possible standards for scientific quality and integrity, every decision that follows is called into question. 

Senator Vitter and I are trying to get the EPA to explain how 

they plan to address the systematic concerns with IRIS that were 

raised by the recent NAS review of formaldehyde, a chemical we 

have decades of experience and information to draw upon. Now, if 

the IRIS review of a chemical that we have a great history on is 

so fundamentally flawed, it is hard to imagine how we will end up 

with good science on chemicals of emerging concern. I am looking 

forward to hearing from the EPA about how they plan to address 

these concerns. 

As analytical techniques continue to improve, we are able to detect constituents at increasingly lower levels. This ever-increasing 

ability to detect will allow the numbers of chemicals in our water 

to increase infinitely. However, it is important that we do not associate any detection with risk. In nearly every case, the extremely 

low levels that we are detecting are well below the dosage that 

would affect public health. To be perfectly clear, exposure does not 

mean there is risk. This is just one more example of the importance 

of getting robust science to guide our policy decisions to help correctly communicate to the public what the risk associated with a 

particular contaminant is. 

It is no surprise that nearly half of Americans are concerned 

about the quality of their drinking water when headlines and talking points are filled with alarmist stories of chemical detection with 

no information about what that means specifically to their health. 

Furthermore, it makes no sense to continue to tighten drinking 

water standards and send drinking water down an aging and failing infrastructure system. EPA estimates that just to keep up with 

the current drinking water requirements over the next 20 years, elVerDate Aug 31 2005 11:45 Apr 10, 2017 Jkt 000000 PO 00000 Frm 00009 Fmt 6633 Sfmt 6633 S:\_EPW\DOCS\23819.TXT VERN
4 

igible drinking water systems will need over $300 billion in infrastructure investment. We need to improve our Nation’s drinking 

water facilities by authorizing and reauthorizing the State Revolving Loan Fund programs, both for drinking water as well as for 

wastewater. 

I was extremely disappointed by EPA’s cuts of almost $950 million to the SRF program. By making these cuts, EPA is increasing 

funding mandates on water providers throughout the Country. We 

can’t expect our communities to continue to provide safe drinking 

water if they don’t have the resources to do it. 

This is something I have been talking about for such a long period of time. And I know my State of Oklahoma is not totally different than other States. But we have a lot of communities that 

just can’t afford these things. When I was mayor of Tulsa, I have 

to say that the biggest problems, and I know Bob Perciasepe has 

heard me say this before, the biggest problems facing our cities are 

really not prostitution and crime and all, it is unfunded mandates. 

And that is something that we have to be familiar with and have 

to address. 

And finally, Madam Chairman, I would like to request that the 

record of this hearing be kept open for two additional weeks, so 

that both outside groups and our witnesses here today have the opportunity to review the GAO report on unregulated contaminants, 

which has been unavailable to the public until now. So I think we 

may need to have the access to that, and then have a comment period. 

I am looking forward to hearing from the witnesses today, especially Dr. Patierno, who will share his expertise in chromium–6, 

something that is very interesting to me concerning the problems 

that exist in my city of Norman, Oklahoma, Dr. Cotruvo, who can 

share his lengthy public health and drinking water experience, and 

Deputy Administrator Bob Perciasepe, whom we have gotten to 

know very well. 

We are going to try to stay and maybe expedite this and get it 

all done throughout the second panel. Thank you, Madam Chairman. 

[The prepared statement of Senator Inhofe follows:] 

STATEMENT OF SENATOR JAMES M. INHOFE, U.S. SENATOR

FROM THE STATE OF OKLAHOMA

Thank you, Madam Chairman, for taking the time today to continue our discussions about Federal drinking water programs. I know that everyone in this room 

agrees that providing clean, safe, affordable drinking water is essential, and should 

be a national priority. 

At the cornerstone of the Safe Drinking Water Act is the idea that we should be 

controlling those substances that pose risks to public health. Unfortunately, the system that EPA uses to determine health risks, the Integrated Risk Information System (IRIS), has a decade’s long issues in crafting risk assessments. The National 

Academy of Sciences (NAS) has recently pointed out that IRIS assessments have 

suffered from a lack of transparency, inconsistency, and problems with evaluating 

studies and the weight of evidence. These problems continue to persist in the face 

of NAS and Congress repeatedly imploring EPA to correct these issues. Without 

having a good foundation and sound science practices, all areas of EPA’s regulatory 

system suffer, including drinking water. 

Unbiased, high-quality, scientific analyses are important foundations for making 

drinking water decisions. When the risk assessments that EPA is producing are unable to maintain the highest possible standards for scientific quality and integrity, 

every decision that follows is called into question. Sen. Vitter and I are trying to 

5 

get EPA to explain how they plan to address the systematic concerns with IRIS that 

were raised in the recent NAS review of Formaldehyde, a chemical we have decades 

of experience and information to draw upon. If the IRIS review for a chemical that 

we have great history on is so fundamentally flawed, it is hard to imagine how we 

will end up with good science on chemicals of emerging concern. I am looking forward to hearing from EPA about how they plan to address these concerns soon. 

As analytical techniques continue to improve, we are able to detect constituents 

at increasingly lower levels. This ever increasing ability to detect will allow the 

numbers of chemicals in our water to increase infinitely. However, it is important 

that we do not associate any detection with risk. In nearly every case, the extremely 

low levels we are detecting are well below the dosage that would affect public 

health. To be perfectly clear, exposure does not mean there is risk. This is just one 

more example of the importance of getting robust science to guide our policy decisions and to help correctly communicate to the public what the risk associated with 

a particular contaminant is. It is no surprise that nearly half of Americans are concerned about the quality of their drinking water when headlines and talking points 

are filled with alarmist stories of chemical detection with no information about what 

that means to their health. 

Furthermore, it makes no sense to continue to tighten drinking water standards 

and send drinking water down an aging and failing infrastructure system. EPA estimates that just to keep up with the current drinking water requirements over the 

next 20 years, eligible drinking water systems will need over $300 billion in infrastructure investments. We need to improve our nation’s drinking water facilities by 

reauthorizing the State Revolving Loan Fund programs, both for drinking water and 

waste water. 

I was extremely disappointed by EPA’s cuts of almost $950 million to the SRF 

program. By making these cuts, EPA is increasing unfunded mandates on water 

providers throughout the country. We cannot expect our communities to continue to 

provide safe drinking water if they do not have the resources to meet their infrastructure needs. We have the responsibility to ensure clean, safe, and affordable 

water for our country by providing the necessary resources to our states and local 

governments. 

Finally, Madam Chairman, I would like to request that the record for this hearing 

be kept open for 2 weeks, so that both outside groups and our witnesses here today, 

have the opportunity to review this GAO report on Unregulated Contaminants, 

which has been unavailable to the public until now, and provide a more robust understanding of the recommendations and suggestions for the committee record. 

I look forward to hearing from all of our witnesses today, especially Dr. Patierno, 

who will share his expertise in chromium–6, something of interest to me and to my 

constituents in Norman, OK, Dr. Cotruvo, who can share his lengthy public health 

and drinking water experience, and Deputy Administrator Perciasepe. Thank you. 

Senator BOXER. Thank you so much. And without objection, we 

will keep the record open for 2 weeks so everyone can avail themselves of the report and ask questions of the GAO. 

Senator Lautenberg, welcome. 

STATEMENT OF HON. FRANK LAUTENBERG, 

U.S. SENATOR FROM THE STATE OF NEW JERSEY 

Senator LAUTENBERG. Thank you very much, Madam Chairman, 

for holding this hearing. 

Clean water is as fundamental to our health as breathing clean 

air. And since the Safe Drinking Water Act came about 37 years 

ago, we have made significant progress making sure the Nation’s 

water supply is clean and safe. But we still have a lot of work to 

do. 

Research shows that there are more than 140 chemicals in our 

drinking water that EPA does not regulate. In some parts of the 

Country, these chemicals include gasoline additives and pesticides. 

Other States, natural gas drillers inject diesel fuel and chemicals 

into the ground as part of the now more popular fracking process, 

and EPA is powerless to do anything about it because of a loophole 

6 

in the Safe Drinking Water Act that I and others on this Committee have fought to repeal. 

In many ways, we are still being haunted by the neglect of the 

prior Administration, which repeatedly failed to set limits and 

strengthen regulations on pollution in the Nation’s water supply. 

During the Bush years, each and every time that the EPA was 

given an opportunity to improve water safety, the agency chose not 

to act. Today, we are going to hear the results of a new Government Accountability Office investigation that sheds additional light 

on this negligence and misplaced priorities. 

GAO’s investigation shows how much the Bush administration 

looked the other way, instead of regulating dangerous chemicals 

like perchlorate, a toxic substance found in rocket fuel. Instead of 

letting agency scientists and the law drive regulatory decisions, political appointees, acting and meeting in secret, pushed the agency 

away from putting limits on perchlorate. Fortunately, under President Obama, EPA is moving in the right direction once again working on the public’s behalf. 

The agency is taking steps to set new limits on chemicals in our 

drinking water and doing more to determine the impact of natural 

gas drilling on our Country’s water supply. The EPA is making 

good use of the tools under the Safe Drinking Water Act. But the 

law itself limits EPA’s ability to protect the public’s right to know. 

The Safe Drinking Water Act only allows EPA to regulate temporary monitoring of a small group of unregulated contaminants. 

So the public has no idea that they might be drinking water laden 

with unregulated contaminants like flame retardants, gasoline additives or other toxics. 

Twenty-five years ago, I authored the Right to Know law on toxic 

chemical releases to make sure that people know about potentially 

hazardous substances in their communities. The public also has a 

right to know what is in their water. That is why I introduced the 

Drinking Water Right to Know Act. My bill would require a targeted increase in monitoring for unregulated pollutants that could 

be hazardous. 

In addition, the bill would require EPA to make information on 

contaminants in drinking water more readily available online and 

in plain English. More information on contaminants will empower 

citizens and help government to make better decisions on pollutants in the water supply. 

So I look forward to hearing from our witnesses about how we 

can work together to make sure that every person has clean, safe 

water to drink when they turn on the tap. Thank you, Madam 

Chairman. 

Senator BOXER. Thank you very much. 

Senator Boozman. 

STATEMENT OF HON. JOHN BOOZMAN, 

U.S. SENATOR FROM THE STATE OF ARKANSAS 

Senator BOOZMAN. I will have a statement for the record. Thank 

you. 

[The prepared statement of Senator Boozman was not submitted 

at time of print.] 

Senator BOXER. Thank you. 

7 

Senator Cardin. 

STATEMENT OF HON. BENJAMIN CARDIN, 

U.S. SENATOR FROM THE STATE OF MARYLAND 

Senator CARDIN. Madam Chairman, I am going to put my full 

statement in the record, and I do want to welcome our witnesses. 

I just want to underscore a point that has been said, and that is 

Americans expect when they turn their taps on they are going to 

get clean water. And we have to make sure that in fact becomes, 

maintains reality. 

The way that unregulated contaminants are handled raises concern. We know we review them every 5 years, but to move it to regulation has been extremely cumbersome and difficult. Some of our 

States have acted because the Federal Government has not been 

able to act. I am not sure that is the substitute for the Federal 

Government acting. To me, this is one of our principal responsibilities, to make sure that we have safe and clean water for the people of this Nation. 

I look forward to this hearing. 

[The prepared statement of Senator Cardin follows:] 

STATEMENT OF HON. BENJAMIN L. CARDIN, U.S. SENATOR

FROM THE STATE OF MARYLAND

Madame Chairman, thank you. 

Readily available, safe drinking water is an expectation most people in our Nation 

have come to take for granted. 

The Safe Drinking Water Act Amendments of 1996 contain a provision that requires the EPA to evaluate chemicals in our drinking water and to regulate them 

as necessary. In its recent preliminary regulatory determination, however, the EPA 

has decided that it will NOT regulate 11 new chemicals found in our Nation’s tap 

water. 

One such contaminant is perchlorate—a chemical found in fireworks, explosives, 

and rocket motors. The chemical has been found in the drinking water of at least 

35 states and the District of Columbia. Perchlorate has been found in breast milk. 

It is known that perchlorate affects the ability of the thyroid gland to take up iodine. Iodine is needed to make thyroid hormones that regulate many body functions 

after they are released into the blood. Children are more likely to be affected by perchlorate than adults because thyroid hormones are essential for normal growth and 

development. 

Despite the known science on the hazards of perchlorate, EPA has failed to establish a safety standard for perchlorate in drinking water. 

Lacking Federal leadership, several states, including Maryland have adopted their 

own standards for perchlorate in drinking water. These standards range from 2 

parts-per-billion in Massachusetts to 6 parts-per-billion in California. In 2002, my 

State of Maryland established a 1 ppb advisory level for perchlorate. There are two 

communities in Maryland that exceeded this advisory level and have since installed 

treatment facilities: the city of Aberdeen in Harford County and the Sherwood Forest Mobile Home Park in Cecil County. 

Given the disparities in the advisory levels and standards for perchlorate from 

one State to another, our constituents may be confused and concerned that there 

is no National standard for perchlorate in their water given the risks, in particular 

to pregnant mothers and young children, of low-level exposure to perchlorate. 

My concerns about perchlorate, particularly the EPA’s failure to provide a regulation requiring monitoring of and notification for perchlorate in drinking water have 

led me to co-sponsor two bills that you, Madame Chairman have offered: S. 24, the 

‘‘Perchlorate Monitoring and Right-to-Know Act of 2007’’ and S. 150. the ‘‘Protecting 

Pregnant Women and Children From Perchlorate Act of 2007’’ 

A similar lack of action has existed for trichloroethylene or TCE: Even after a 

draft EPA Risk Assessment found that TCE was as much as 40 times more carcinogenic than previously thought, rather than setting a more protective standard for 

TCE in drinking water, the EPA called for more study. 

8 

Given the EPA’s recent history of not particularly timely development of new regulations, I am concerned that it may be several years before this new information 

might be incorporated into new regulatory standards. 

Existing scientific studies strongly suggest that the EPA should be acting expeditiously on developing regulations to limit exposure to perchlorate and TCE. It is unfortunate, that as with other environmental matters, the EPA has dragged its feet 

when the health of our Nation is at risk. 

I look forward to the testimony from today’s witnesses in helping to clarify this 

issue. There is scientific justification for the EPA to act quickly on these chemicals 

in our drinking water. If EPA cannot act, we must. Our country deserves better. 

Thank you Madame Chairman. 

Senator BOXER. Thank you very much. 

We will now turn to our panel. I think we are going to hear from 

the Government Accountability Office first, so that Hon. Deputy 

Administrator can respond to their findings. So Mr. Trimble, we 

welcome you and we thank you for all your heard work. We look 

forward to your presentation. You can summarize in 5 minutes, 

and if we have further questions, we will ask them. 

STATEMENT OF DAVID C. TRIMBLE, DIRECTOR, NATURAL RESOURCES AND ENVIRONMENT, GOVERNMENT ACCOUNTABILITY OFFICE 

Mr. TRIMBLE. I am pleased to be here today to discuss our report 

being released today on EPA’s program for regulating additional 

drinking water contaminants. We evaluated EPA’s implementation 

of the 1996 amendments to the Safe Drinking Water Act, and reviewed the process and findings EPA used in making its 2008 preliminary decision to not regulate perchlorate. 

We found that systemic limitations in EPA’s implementation of 

the requirements for determining whether additional drinking 

water contaminants warrant regulation have impeded the agency’s 

process in assuring the public of safe drinking water. These limitations start with the contaminants that EPA chooses to assess for 

regulation. Specifically, EPA’s selection of contaminants for regulatory determination in 2003 and 2008 was driven by data availability, not consideration of greatest public health concern, as specified in the statute. In fact, an EPA official described the agency’s 

regulatory determinations as addressing the low-hanging fruit. 

Moreover, EPA has not been effective in obtaining occurrence of 

health effects data on contaminants, both of which are essential for 

regulatory determination. This program to test drinking water for 

unregulated contaminants has not tested as many contaminants as 

it could have. Also, in many cases, the testing methods have not 

been sufficiently robust to detect the contaminants at the level of 

public health concern, reducing the credibility of EPA’s determinations. Information on the health effects of contaminants has also 

lagged due to longstanding challenges in the IRIS program and coordination issues. 

Importantly, EPA has not developed regulations, policies or guidance for interpreting the amendments’ broad statutory criteria for 

selecting contaminants and making decisions on them. In the absence of guidance, EPA has been informally applying a policy that 

contaminants need to occur in drinking water on a national scale 

to warrant regulation. 

However, a national occurrence threshold is not required by the 

Safe Drinking Water Act, which in fact provides relief to public 

9 

water systems for monitoring when a contaminant occurs in certain 

areas but not in others. We have concerns about EPA implementing a critical policy which interprets the Act and has important implications for the safety of public drinking water when this 

policy has neither been defined nor subjected to public review. 

Also, the credibility of some of the EPA’s regulatory determinations is limited by a lack of transparency, clarity and consistency. 

For example, EPA states unequivocally in its regulatory determination support document that there is no data to indicate children are 

more sensitive to manganese than adults, but in its health effects 

support document discusses studies that do associate manganese in 

drinking water with learning disabilities in children. Our report details numerous other limitations that reduce the credibility of 

EPA’s regulatory determinations, including making determinations 

on the basis of outdated and incomplete occurrence data, and the 

Office of Water’s lack of protocol and methods for assessing drinking water health risks for children and other sensitive sub-populations, 15 years after the 1996 amendments required EPA to consider effects on such populations. 

Regarding perchlorate, we found that the process and analyses 

EPA relied on to support its preliminary determination to not regulate were atypical, lacked transparency and limited the agency’s 

independence in developing and communicating its scientific findings. While EPA’s 2011 decision to regulate perchlorate included an 

analysis on the potential health impact on children, addressing key 

shortcomings in the 2008 decision, it is not clear at this time 

whether EPA has committed to do such analyses in the future. 

We made 17 recommendations to support the development of 

transparent, clear and consistent regulatory determinations. An 

overarching recommendation is that EPA develop policies and guidance clearly articulating the agency’s interpretation of the Act’s 

statutory criteria as well as eight additional recommendations 

identifying specific components of this guidance. 

EPA’s response to this recommendation was that it was not practical to establish policies or guidance, citing various complexities. 

However, the complexities cited would argue for rather than 

against the need to develop guidance for staff in applying the criteria. 

EPA also said it did not support developing policies or guidance 

because under the statutory criteria, it is the Administrator’s judgment as to whether regulation presents a meaningful opportunity 

for health risk reduction after considering information presented by 

agency staff. The statutory criteria are so broadly stated that they 

could potentially be interpreted to support regulating all the contaminants on the candidate list, some of them or none of them. 

It is precisely for these reasons that we believe it is essential for 

EPA staff to have sufficient guidance on applying the criteria consistently and transparently, so that the Administrator’s judgment 

can be based on sound and consistent information. Without such 

guidance, the basis for EPA’s determinations and the quality of 

their support can fluctuate over time as a result of, among other 

reasons, changes in agency leadership and staff. 

In large measure, EPA’s response to our recommendations essentially endorses conducting business as usual, a response that does 

10 

not acknowledge the scope and significance of the implementation 

limitations we identified. 

I would be happy to respond to any questions that you or other 

members of the Committee may have. 

[The prepared statement of Mr. Trimble follows:] 

11 


23819.001

GAO 

For Release on Delivery 

Expected at 10:00 a.m. EDT Tuesday, July 12,2011 

GA0-11-803T 

United States Government Accountability Office 

Testimony 

Before the Committee on Environment 

and Public Works, U.S. Senate 

SAFE DRINKING WATER 

ACT 

Improvements in 

Implementation Are Needed 

to Better Assure the Public 

of Safe Drinking Water 

Statement of David C. Trimble, Director 

Natural Resources and Environment 
12 


23819.002

Madam Chairman Boxer, Ranking Member lnhofe, and Members of the 

Committee: 

I am pleased to be here today to discuss highlights of GAO's report on 

the Environmental Protection Agency's (EPA) implementation of 

requirements for determining whether additional drinking water 

contaminants warrant regulation. As you know, the number of potential 

drinking water contaminants is vast-as many as tens of thousands of 

chemicals may be used across the country, and EPA has identified more 

than 6,000 chemicals that it considers to be the most likely source of 

human or environmental exposure. The potential health effects of 

exposure to most of these chemicals, and the extent of their occurrence 

in drinking water, are unknown. Under 1996 amendments to the Safe 

Drinking Water Act, every 5 years EPA is to detenmine for at least five 

contaminants whether regulation is warranted, considering those that 

present the greatest public health concern. EPA issued final regulatory 

determinations in 2003 and 2008 on a total of 20 contaminants, deciding 

in each case not to regulate. In fact, EPA did not recommend any new 

contaminants for regulation until February 2011, when it reversed its 

controversial 2008 preliminary decision to not regulate perchlorate, an 

ingredient in rocket fuel and other products. 

This statement summarizes our report being released today that (1) 

evaluates the extent to which EPA's implementation of the 1996 

amendments has helped assure the public of safe drinking water and (2) 

reviews the process and scientific analyses EPA used to develop the 

2008 preliminary decision to not regulate perchlorate.' In preparing this 

testimony, we relied on our work supporting the accompanying report, 

which contains a detailed description of our scope and methodology. All 

of the work for this report was performed in accordance with generally 

accepted government auditing standards. 

1

GAO, Safe Drinking Water Act EPA Should Improve Implementation of Requirements on 

Whether to Regulate Additional Contaminants, GA0-11·254 (Washington, D.C.: May 27, 

2011). 
13 


23819.003

Systemic Limitations 

in EPA's 

Implementation of 

Requirements for 

Determining Whether 

to Regulate Additional 

Contaminants Have 

Impeded Progress in 

Helping Assure the 

Public of Safe 

Drinking Water 

EPA Has Neither Identified 

the Drinking Water 

Contaminants of Greatest 

Public Health Concern Nor 

Fully Used Its Authority to 

Obtain Data for Making 

Regulatory Determinations 

As detailed in our report, we found the following concerning the extent to 

which EPA's implementation of the 1996 amendments has helped assure 

the public of safe drinking water. 

EPA has not effectively implemented the 1996 amendments' requirement 

to consider, for regulatory determinations, contaminants that present the 

greatest public health concern. The contaminant candidate list' that the 

amendments require EPA to develop every 5 years represents one level 

of prioritization as EPA selects from a larger universe those contaminants 

the agency believes warrant consideration for regulation. However, EPA 

officials told us that its Office of Water, which has primary responsibility 

for implementing the requirements of the Safe Drinking Water Act, has 

not ( 1) further ranked or otherwise prioritized the contaminants on the list 

on the basis of public health concern or (2) prioritized contaminants on the basis of public health concern when selecting them for regulatory 

determinations. In fact, for 16 of the 20 regulatory determinations made 

through January 2011, EPA based its decisions to not regulate on its 

assessment that public exposure to these drinking water contaminants 

was minimal-that is, there was limited or no occurrence of them in public 

drinking water systems. An EPA official described these determinations 

as addressing the "low hanging fruit"-rather than the contaminants of 

greatest public health concern. Overall, data availability-not 

2

The 1996 amendments require that EPA identify and publish a list every 5 years of 

unregulated contaminants that may require regulation; the list is called the contaminant 

candidate list. 
14 


23819.004

consideration of greatest public health concern-has been the primary 

driver of EPA's selection of contaminants for regulatory determinations. 

To assess unregulated contaminants against the statutory criteria, EPA 

needs sufficient information on both (1) the occurrence of these 

contaminants in drinking water--called occurrence data-to assess the 

population potentially being exposed and the levels of that exposure and 

(2) the human health effects that may result from exposure to the 

contaminants in drinking water. EPA has made some progress in 

developing the occurrence and health effects data it needs, but for many 

contaminants EPA lacks sufficient occurrence and health effects data to 

support regulatory determinations, which continues to limit its ability to 

make these decisions. Specifically, in finalizing its current candidate list 

comprising 116 contaminants, EPA indicated that the agency lacked 

sufficient occurrence or health effects data, or both, for making regulatory 

determinations for at least 100 of the contaminants. Moreover, in 2009 

EPA's Science Advisory Board recommended that the agency further 

prioritize among the contaminants on the candidate list because the list 

was too large, noting that prioritizing the contaminants on the list would 

help the agency meet its goal of selecting contaminants for regulatory 

determinations that "have the greatest opportunity to improve the safety 

of drinking water and protect public health." 

In addition, in its testing program for unregulated contaminants-which 

can provide key occurrence data to inform regulatory determinationsEPA has fallen short in both the number of contaminants tested and the 

utility of the data provided because of management decisions and 

program delays. For example, despite having the authority to require 

testing for up to 30 drinking water contaminants in each 5-year cycle, in 

implementing the first two cycles of the testing program, EPA required 

that only 51 contaminants be tested-thereby not availing itself of its 

authority to obtain occurrence data for 9 additional contaminants. 

Moreover, in some cases, the occurrence data EPA used to support its 

regulatory determinations were based on testing (analytic) methods that 

were not sufficiently sensitive to identify the presence of contaminants at 

EPA's health reference level-the level that EPA uses in assessing 

whether to regulate specific contaminants.' For 9 of the 20 contaminants 

health reference level is the estimated level of exposure to a contaminant in drinking 

water below which adverse health effects are not likely. 
15 


23819.005

for which EPA made regulatory determinations in 2003 and 2008, the 

minimum reporting level-the lowest level of a contaminant at which 

detections can be reported under testing protocols-exceeded EPA's 

health reference level. For example, for dieldrin-an insecticide banned 

by EPA for all uses in 1987 because of concerns about harm to human 

health and its ability to persist in the environment for decades-the 

agency relied on testing data obtained using minimum reporting levels 

ranging from 10 to 2,200 times higher than EPA's health reference level' 

EPA reported in its regulatory determination documents for dieldrin that it 

was detected in 0.06 percent of samples.' However, in subsequent 

testing of source water for drinking water wells using more sensitive tests 

with minimum reporting levels near and below EPA's health reference 

level, the U.S. Geological Survey (USGS) detected dieldrin in 3.1 percent 

of public well samples. Importantly, nearly all of USGS's detections were 

at levels above EPA's health reference level. USGS was able to detect 

dieldrin-and determine its presence above EPA's level of public health 

concern-in these groundwater well samples because it used a lower 

minimum reporting level for its testing than EPA used for its regulatory 

determinations. This is significant because, as USGS has reported, when 

a reporting level exceeds a health benchmark, a contaminant may be 

present at a concentration greater than the health benchmark but remain 

undetected, resulting in greater uncertainty in evaluating the contaminant 

concentration in the context of public health. EPA's testing program 

obtains data using minimum reporting levels that are often higher than 

those used by the USGS in its National Water Quality Assessment 

Program-ranging from 2 to more than 600 times higher. 

In addition, the lack of timely health assessment data on drinking water 

contaminants continues to limit EPA's ability to make regulatory 

determinations. As a result of long-standing productivity problems in 

EPA's Integrated Risk Information System (IRIS) program-managed by 

the Office of Research and Development-EPA has not been able to 

keep its existing chemical toxicity assessments current or to complete 

not disclose that the data presented were not sufficiently sensitive to detect 

occurrence at the agency's health reference level. 

5

1n this testimony, as in our report, we refer to Federal Register notices regarding EPA's 

regulatory determinations (notices) and EPA's regulatory determination support 

documents individually and collectively, as appropriate. When referring to these 

documents collectively, we use the term "regulatory determination documents." 
16 


23819.006

EPA Lacks Policies or 

Guidance on Applying the 

Broad Statutory Criteria 

for Selecting Contaminants 

for Regulatory 

Determinations and 

Making the Determinations 

assessments of the most important chemicals of concern.'· ' For 

example, from 1998 through 2008, the Office of Water lacked current IRIS 

assessments' or other sufficient health information for 24 chemical 

contaminants on the candidate lists, and the Office of Research and 

Development completed assessments for only 2 of the 24. Moreover, the 

Office of Water's current needs for health effects information for 

contaminants on the current candidate list have roughly doubled-when 

publishing the third candidate list in 2009, EPA identified health effects 

information gaps for 44 of the 104 chemicals on the list. Importantly, most 

of these contaminants with information gaps (1) are not on the IRIS 

agenda (i.e., assessments are neither under way nor planned) and (2) 

have not been identified by the Office of Water as priorities for IRIS 

assessments. 

The Safe Drinking Water Act requires EPA to select contaminants for 

regulatory detenninations that present the greatest public health concern. 

However, EPA has not defined the characteristics of contaminants of 

greatest public health concern or developed a process for prioritizing the 

contaminants on its candidate list for regulatory determination on this 

basis. As a result, EPA lacks criteria and a process for identifying those 

contaminants on its candidate list that pose the greatest public health 

concern. 

Moreover, under the act, in selecting contaminants that present the 

greatest public health concern, EPA is to consider the effect of these 

contaminants on subpopulations at greater risk of adverse health effects 

from exposure to drinking water contaminants. In addition, EPA has 

6

See GAO, Chemical Assessments: Low Productivity and New Interagency Review 

Process Limit the Usefulness and Credibility of EPA's Integrated Risk Information System, 

GAQ-08-440 (Washington, D.C.: Mar. 7, 2008). 

7

1n January 2009 we added transforming EPA's processes for assessing and controlling 

toxic chemicals as a highMrisk area in our report-updated in February 2011-on 

governmentwide high-risk areas requiring increased attention by executive agencies and 

Congress. See GAO, High-Risk Series: An Update, GA0-09-271 (Washington, D.C: 

January 2009); and High Risk Series: An Update, GA0-11-278 (Washington. D.C.: 

February 2011 ). 

8

IRIS assessments provide EPA's toxicity assessments of contaminants that may cause 

cancer and those that may cause neurological or other noncancer effects, or both. EPA 

uses IRIS or comparable toxicity assessments to develop health reference levels for the 

drinking water contaminants. 
17 


23819.007

stated that in making regulatory determinations, the act requires the 

agency to consider the effects the contaminants have on sensitive 

subpopulations, such as infants, children, those with kidney or liver 

diseases or weakened immune systems, and the elderly. Children, for 

example, represent a sensitive subpopulation because they may be more 

highly exposed to toxic substances in drinking water and at greater risk of 

adverse health effects than adults as a result of consuming more water 

per unit of body weight than adults. Children may also have increased 

susceptibility following exposure to drinking water contaminants because 

they continue to develop both behaviorally and physiologically throughout 

childhood. Furthermore, in 1995, EPA published its Policy on Evaluating 

Health Risks to Children, which states that the agency will "consider the 

risks to infants and children consistently and explicitly as a part of risk 

assessments generated during its decision making process," and to "the 

degree permitted by available data in each case, the Agency will develop 

a separate assessment of risks to infants and children or state clearly why 

this is not done: In 2006, EPA developed a general guidance document 

for all EPA program offices on implementing its 1995 children's health 

policy, as well as several technical guidance documents that could help 

the Office of Water develop its own guidance specific to assessing the 

sensitivity of children to drinking water contaminants. 

Notwithstanding the requirements of the Safe Drinking Water Act and 

EPA's 1995 children's health policy, the Office of Water did not implement 

a specific approach for considering children's health in developing its 

2003 and 2008 regulatory determinations. In addition, the Office of Water 

has not developed guidance for its staff on when and how to analyze the 

effects of drinking water contaminants on children-or other sensitive 

subpopulations-for the purposes of identifying the drinking water 

contaminants of greatest concern on which to make regulatory 

determinations and to ensure it consistently and explicitly considers risks 

to children in making these determinations, such as by developing 

separate health reference levels for children. While EPA identified 

children as a sensitive population in 11 of the 20 regulatory 

determinations it completed in 2003 and 2008, Office of Water officials 

confirmed that for these 20 determinations, EPA did not develop separate 

health reference levels for children or make adjustments to its health 

assessments. 

The 1996 amendments also provide three broad criteria for EPA to use in 

making regulatory determinations, all of which must be met for EPA to 

determine that regulation is warranted. Notably, two of the criteria are so 

broadly stated that they could potentially be interpreted so as to lead to 
18 


23819.008

regulating all of the contaminants on candidate lists, some of them, or 

none of them. Specifically, the second statutory criterion-that a 

contaminant is "known to occur or there is a substantial likelihood that the 

contaminant will occur in public water systems with a frequency and at 

levels of public health concern"-is susceptible to varying interpretations. 

For example, different people may reasonably have differing views on the 

frequency and levels of occurrence that represent a public health 

concern. The third criterion-that regulation of the contaminant presents 

"in the sole judgment of the Administrator ... a meaningful opportunity for 

health risk reduction"-is expressly discretionary, and similarly open to 

differing interpretations. 

Importantly, the Office of Water has not developed policies or guidance to 

help EPA staff apply these broad criteria. Guidance that might help EPA 

staff apply the criteria transparently and consistently could, among other 

things, (1) define or set thresholds or parameters for assessing whether a 

contaminant occurs, or is substantially likely to occur, in public water 

systems with a frequency and at levels of public health concern and (2) 

provide factors or characteristics of situations that would present 

meaningful opportunities for health risk reduction. We note that such 

guidance could also serve as the basis for an internal review mechanism 

to help EPA ensure consistent implementation of the statutory criteria. 

Office of Water officials could not describe examples of what would meet 

the three criteria beyond stating that "there are no bright lines" and that 

they would "know it when we see it." Without clarifying guidance, EPA's 

regulatory determinations lack transparency, and EPA is at risk of making 

inconsistent determinations, undermining the program's credibility and the 

agency's ability to assure the public of safe drinking water. 

In the absence of regulations or guidance for applying the broad statutory 

criteria, EPA appears to apply an informal policy that contaminants 

warranting regulation should occur in public water systems on a "national" 

scale. For example, documents supporting EPA's 2003 regulatory 

determinations state that the consideration of geographic distribution "is 

important because the agency is charged with developing national 

regulations, and it may not be appropriate to develop [national primary 

drinking water regulations] for regional or local contamination problems." 

In addition, some EPA officials serving on regulatory determination work 

groups told us that a contaminant must occur "nationally" to warrant a 

determination to regulate. Notably, however, the Safe Drinking Water Act 

does not require that contaminants be found in public water systems on a 

national basis for an Administrator to find a meaningful opportunity for 

health risk reduction. In fact, other parts of the statute provide for relief 
19 


23819.009

from monitoring and flexibilities for instances in which a contaminant 

occurs in certain areas but not in others. Moreover, there is nothing in the 

act's committee reports suggesting that a contaminant need occur 

nationally to support a decision to regulate. Without EPA guidance 

providing a definition or parameters, an informal "national occurrence" 

standard is open to shifting interpretations, potentially affecting the 

consistency and credibility of EPA's decision making. To the extent EPA 

is informally applying an unspecified national occurrence requirement for 

contaminants to be evaluated as occurring "with a frequency and at levels 

of public health concern," EPA is implementing a critical policy and 

interpretation of the Safe Drinking Water Act that has neither been 

defined nor subjected to public review. 

Further, regarding the third statutory criterion, EPA has not articulated 

guidelines or thresholds for how it is to assess whether regulating a 

specific contaminant would provide a meaningful opportunity for health 

risk reduction. The absence of guidelines on what scenario or scenarios 

might illustrate "a meaningful opportunity for health risk reduction" 

increases the potential for inconsistent decision making and reduces the 

decisions' transparency. 

In addition, EPA has not developed any guidance on the circumstances 

that would trigger a re-evaluation of a prior decision to not regulate or the 

process the agency would use in conducting a re-evaluation. In at least 

one instance-1, 1 ,2,2-tetrachloroethane-an updated IRIS assessment 

became available after EPA's determination to not regulate, but the 

agency has not announced whether it will reconsider the determination. In 

addition, as discussed in the following section, the credibility of some of 

EPA's regulatory determinations was limited. As we reported, we believe 

EPA should consider whether it needs to re-evaluate any of its past 

determinations to not regulate in light of the systemic and individual 

shortcomings we identified. In the absence of policies or guidance that 

identifies the circumstances that would trigger such re-evaluations and 

the process the agency would use in conducting them, it is not clear how 

and when such re-evaluations would occur. 
20 


23819.010

The Credibility of Some of 

EPA's Regulatory 

Determinations As 

Presented in Federal 

Register Notices and 

Support Documents Is 

Limited by a Lack of 

Transparency, Clarity, and 

Consistency 

The Safe Drinking Water Act requires EPA to ensure that, in its regulatory 

determinations, among other things, the presentation of information on 

public health effects is comprehensive, informative, and understandable. 

In addition, to the extent that EPA's regulatory determination notices and 

key support documents are transparent, clear, and consistent regarding 

the occurrence and health effects data the agency relied on, the credibility 

of the determinations is enhanced. However, for the regulatory 

determinations that EPA has made to date, some of the notices and 

support documents lack these key qualities. For example, as reflected in 

the following excerpts from EPA's regulatory determination documents for 

manganese and boron, EPA's presentation of health effects information 

on some contaminants lacked clarity, consistency, and transparency. 

EPA's 2003 regulatory determination support document for 

manganese states unequivocally that there are "no data to indicate 

children are more sensitive to manganese than adults." However, 

EPA's 2003 health effects support document for manganese 

discusses studies that identify an association between exposure to 

manganese in drinking water and learning disabilities in children and 

concludes that additional studies are needed to investigate the 

possibility that children are more sensitive than adults. 

EPA's regulatory determination support document for manganese 

notes that infants and newborns may be potentially susceptible to 

manganese toxicity, but this key document does not disclose that 

newborns may be exposed to high levels of manganese from infant 

formula or that these high levels of manganese in formula can be 

magnified when it is reconstituted with manganese-contaminated 

water. 

In its regulatory support document for boron, the Office of Water (1) 

identified the primary adverse effects identified from studies of 

animals after chronic exposure to low doses of boron as generally 

involving the testes and the developing fetus and (2) stated that 

animal studies identify the developing fetus as "potentially sensitive to 

boron" and concluded that boron concentrations greater than the 

health reference level "might" have an effect on prenatal development. 

In contrast, the Office of Water's May 2008 Drinking Water Health 

Advisory for Boron-developed in conjunction with the regulatory 

determination and published just 2 months before the regulatory 

determination was issued-states that there are "compelling lines of 

evidence to suggest that the testicular morphological effects" reported 

in studies of animals are applicable to children. Also, the Office of 
21 


23819.011

Water's health advisory was not limited to prenatal exposure as it 

concluded that exposure to boron between birth and puberty may 

result in adverse cellular effects that would "affect testicular function." 

EPA's Summary Document from the Health Advisory for Boron and 

Compounds provides an important warning regarding infants' 

exposure to boron in drinking water that is not included in either EPA's 

drinking water advisory for boron or its regulatory determination 

support document discussed above. Specifically, the summary 

document states that water containing boron "at levels above the HA 

[health advisory]" should not be used to prepare food or formula for 

infants. EPA does not identify which of the exposure duration health 

advisories it is referring to in this warning. 

In addition, EPA's regulatory determination documents lack transparency 

and clarity regarding how EPA determined its hea~h reference levels 

were protective of children. In addressing seven of the contaminants in its 

2003 regulatory determination notice for which EPA identified children as 

a sensitive subpopulation, EPA did not explain the potential effect of not 

developing separate health reference levels for children (or not making 

adjustments to its health assessments to reflect increased sensitivity) on 

its ability to ensure that the health reference levels used in the regulatory 

determinations were protective of children. Instead, EPA stated that the 

agency had not yet determined a protocol for making a regulatory 

determination for a chemical for which body weight and drinking water 

intake of infants or a particular childhood age group would be the basis of 

a regulatory action. As discussed earlier, health assessments based on 

adult weight and drinking water intake may not fully account for the risks 

to children of exposure to drinking water because they consurne more 

water per unit of body weight and may have other susceptibilities, as well. 

Regarding its 2008 notice that included four contaminants for which 

children were identified as a sensitive subpopulation, Office of Water 

officials told us they would have developed separate assessments for 

children if they had determined children were "particularly sensitive" to the 

adverse health effects of contaminants being considered for regulation. 

However, EPA did not explain in its regulatory determination notices or 

support documents the basis for its determinations that children were not 

particularly sensitive to the adverse health effects of the contaminants 

considered for regulation-even for those contaminants, such as 

manganese and boron, for which EPA had determined children were a 

sensitive subpopulation. EPA also did not explain how the sensitivity of 

children can be evaluated in the absence of a separate assessment 

based on the weight and drinking water intake of children. 
22 


23819.012

Also, EPA's regulatory determination notices lack transparency and clarity 

regarding the limitations of new or updated health advisories the agency 

issued in conjunction with 9 decisions to not regulate. According to EPA, 

the advisories are to provide, for example, "guidance to communities that 

might be exposed to elevated concentrations." However, the regulatory 

determination notices do not acknowledge that when EPA determines 

regulation is not warranted but a health advisory is needed, it will 

generally be up to states, localities, and consumers to determine whether 

such contaminant levels are found in public water systems in their 

jurisdiction. Importantly, because public water systems are not typically 

required to test for the presence of unregulated contaminants, information 

on the levels of the contaminants in individual public water systems may 

be outdated or unavailable. While some states-such as California and 

Massachusetts-can promulgate their own drinking water regulations, 

others are statutorily prohibited from, or otherwise constrained in, 

enacting more stringent regulations than EPA has promulgated or 

promulgating their own drinking water regulations for contaminants that 

EPA does not regulate. In addition, individuals may have to have their 

water tested by a laboratory to determine how much of these unregulated 

contaminants are present in their drinking water to heed, for example, 

EPA's warning in some cases to not use drinking water with contaminants 

in excess of certain levels to prepare infant food or formula. Moreover, 

EPA releases its drinking water advisories by posting them on its Web 

site and does not issue public notification of them, such as a press 

release, which potentially limits awareness of the health advisories. 

Our report provides information on the following limitations that also 

reduce the credibility of EPA's completed regulatory determinations: (1) 

EPA's explanations of the occurrence data EPA relied on to assess 

known and likely occurrence of contaminants in drinking water lack 

transparency, clarity, and consistency; (2) EPA's regulatory 

determinations lack clarity regarding its reliance on outdated and limited 

occurrence data to support some determinations; (3) the regulatory 

determination documents lack transparency and clarity regarding EPA's 

reliance on minimum reporting levels greater than its health reference 

levels; and (4) EPA lacked consistency and clarity in making 

determinations when IRIS assessments were either in process or needed 

to be updated. 
23 


23819.013

The Process and 

Analyses EPA Relied 

on to Support Its 

Preliminary 

Determination on 

Perchlorate Were 

Atypical, Lacked 

Transparency, and 

Limited the Agency's 

Independence in 

Developing and 

Communicating Its 

Scientific Findings 

EPA Used a Less Inclusive, 

Less Transparent, and 

More Directive Process in 

Developing Its Preliminary 

Regulatory Determination 

on Perchlorate Than Its 

Usual Process 

As detailed in our report, we found the following concerning the process 

and scientific analyses EPA used to develop its 2008 preliminary 

determination to not regulate perchlorate. 

In contrast to EPA's usual regulatory determination process, which is 

managed by a work group of professional staff with relevant expertise 

from across the agency, EPA officials decided that the agency's 

continuing deliberations on perchlorate would be managed by a less 

inclusive, small group of high-level officials, such as the Deputy 

Administrator and several Assistant Administrators. Notably, EPA did not 

include the Office of Children's Health Protection in its small group 

despite EPA's and the National Academies' conclusion that iodide uptake 

inhibition from perchlorate exposure had been identified as a concern in 

connection with increasing the risk of neurodevelopmental impairment in 

fetuses of pregnant women with iodine deficiency and to developmental 

delays and decreased learning capability in infants and children. This 

group of high-level officials managed the regulatory determination 

process for perchlorate both within EPA and externally with the 
24 


23819.014

Perchlorate Interagency Working Group,' whose work was coordinated by 

the Council on Environmental Quality." According to an EPA briefing 

document, the Perchlorate Interagency Working Group was established in 

2002 "to identify and help resolve perchlorate science and science policy 

issues." 

In contrast to the usual process EPA used for its regulatory 

determinations, in which EPA staff with relevant expertise develop and 

submit options to the Assistant Administrator for the Office of Water for 

review and selection, the Assistant Administrator directed the Office of 

Water staff in developing the preliminary determination for perchlorate to 

draft a preliminary determination that reflected the agency's decision to 

not regulate perchlorate and to support it with a detailed and specific 

rationale that EPA and other members of the Perchlorate Interagency 

Working Group had agreed to, under the leadership and coordination of 

the Council on Environmental Quality." EPA Office of Water officials told 

us that they believed this agreement-which is not part of the record for 

the preliminary regulatory determination-was developed by senior 

officials from the Council on Environmental Quality, the Department of 

Health and Human Services (HHS), EPA, the Office of Management and 

Budget (OMB), and the U.S. Department of Agriculture. The agreement 

focused on how EPA should address the key science issues concerning 

perchlorate in its preliminary regulatory determination and specified (1) a 

health reference level of 15 parts per billion of perchlorate in drinking 

water and (2) the rationale for EPA to support the conclusion that this 

health reference level would be protective of pregnant women and their 

fetuses as well as of infants and children. 

9

The Perchlorate Interagency Working Group includes officials from the Office of 

Management and Budget (OMS} and the Office of Science and Technology Policy, both 

part of the Executive Office of the President; Department of Defense; National Aeronautics 

and Space Administration; Department of Energy; the Department of Health and Human 

Services' Food and Drug Administration and the Agency for Toxic Substances and 

Disease Registry; Department of Agriculture; and Department of the Interior. 

10The Council on Environmental Quality, which is part of the Executive Office of the 

President, coordinates federal environmental efforts in the development of environmental 

policies and initiatives. 

11According to an EPA official, the agreement was documented in an unattributed two~ 

page white paper and faxed to EPA from the Council on Environmental Quality in early 

August 2008; EPA made some editorial changes to the document but did not alter the 

substance of the agreement 
25 


23819.015

EPA Established a 

Reference Dose for 

Perchlorate but Subjected 

It to a More Limited 

Review Than the Agency's 

Standard IRIS Assessment 

Review Process 

In developing an IRIS assessment of perchlorate, EPA established a 

reference dose" on the basis of the National Academies' 

recommendations, but subjected it to a more limited review than the 

agency's standard IRIS assessment review process." EPA's 2002 draft 

IRIS assessment of perchlorate-from which EPA derived a drinking 

water equivalent level of 1 part per billion"-drew significant attentionincluding from such federal agencies as the Department of Defense, the 

Department of Energy, and the National Aeronautics and Space 

Administration, because of the implications such a level could have on 

their operations if EPA were to develop a drinking water regulation for 

perchlorate. According to a senior EPA official, the controversy that arose 

over the draft IRIS assessment of perchlorate ''was like nothing I had ever 

seen or have seen since." As a result of the divergent views between 

EPA and the other federal agencies, the Administrator of OMB's Office of 

Information and Regulatory Affairs urged the four interested agencies to 

convene a National Academies panel to review the draft IRIS 

assessment. Convened in October 2003, the panel conducted this review 

and issued its report in January 2005." 

The National Academies 2005 perchlorate report made several key 

recommendations to EPA on the basis of a different study from those on 

which EPA had based its 2002 draft IRIS assessment on perchlorate. The 

National Academies' recommended reference dose was more than 20 

times higher than the one proposed in EPA's draft IRIS assessment. 

EPA's final internal review of the revised IRIS assessment for 

perchlorate-termed a consensus review-differed from the agency's 

12A reference dose is an estimate of the total daily oral exposure to a contaminant-for 

example, from food and water-that is not likely to cause "appreciable risk of deleterious 

effects during a lifetime.» A reference dose is a key component of the calculation EPA 

uses to derive a health reference level for drinking water contaminants. 

~he National Academies consists of four private, nonprofit organizations that advise the 

federal government on scientific and technical matters: the National Academy of Sciences, 

National Academy of Engineering, Institute of Medicine, and National Research Council. 

14A drinking water equivalent level represents the estimated exposure to a contaminant 

that is assumed to be protective for noncarcinogenic health effects during a lifetime of 

exposure. EPA calculated this drinking water equivalent level using the reference dose 

that EPA proposed in its 2002 draft IRIS assessment and the agency's default 

assumptions for adult weight and daily drinking water intake. 

15National Academies, Health Implications of Perchlorate Ingestion (Washington, D.C., 

2005). 
26 


23819.016

EPA Relied on an Estimate 

of the Relative Exposure 

to Perchlorate from 

Drinking Water and Food 

That It Derived from a 

Novel Analysis and Used a 

Nontraditional Method to 

Calculate the Relative 

Source Contribution 

usual consensus review process. For example, the scope of the internal 

review was limited in that the IRIS program did not seek input from 

consensus reviewers on the scientific basis for the assessment as it 

typically does; rather, it sought input only on the extent to which the 

science in the IRIS Summary was not inconsistent with the major 

conclusions of the National Academies' report. At least two EPA offices 

essentially opted out of the consensus review process because of this 

limitation, which was a significant departure from the usual IRIS 

consensus practice. 

In developing its regulatory determination on perchlorate, EPA conducted 

a novel analysis to develop estimates of exposure to perchlorate for 

various subpopulations, which the agency subsequently used to calculate 

the relative source contribution-the allocated exposure to perchlorate 

from drinking water alone." Independent scientists who reviewed EPA's 

analysis noted that it had several limitations-in particular, uncertainties 

specific to the exposure estimate for pregnant women. Nonetheless, EPA 

relied on the exposure estimate for pregnant women to calculate the 

relative source contribution, stating that the National Academies had 

identified pregnant women and their fetuses as the most sensitive 

subpopulation." 

In calculating the relative source contribution, EPA used a nontraditional 

method-called the subtraction method"-that was less conservative 

than the approach it had used for its other completed regulatory 

relative source contribution is an allocation of the estimated oral exposure to the 

contaminant from drinking water alone; it has a significant impact on the health reference 

level that the agency derives for contaminants with noncancer adverse health effects. 

17The 2005 National Academies report on perchlorate contained varying characterizations 

of sensitive subpopulations, sometimes referring to pregnant women and their fetuses 

alone as the most sensitive subpopulation and other times including infants in this 

designation. In addition, the National Academies identified developing children as a 

sensitive population and people with compromised thyroid function and people who are 

iodide-deficient as potentially sensitive populations. 

1

srhe subtraction method allocates the entire reference dose to the known sources of 

exposure by subtracting the known nontarget sources of exposure and allocating the 

remainder of the reference dose to the target-in this case, drinking water-even in cases 

where the total estimated exposure is less than the reference dose. This method has the 

effect of removing any cushion between the existing exposure levels and the reference 

dose. 
27 


23819.017

According to Key EPA 

Scientists, the Agency 

Mischaracterized 

Important Scientific 

Findings That Emerged 

from Its Novel Analysis of 

the Sensitivity of Various 

Age Groups to Perchlorate 

in Drinking Water 

determinations. While EPA identified some of the limitations of the 

exposure analysis in its preliminary regulatory determination notice for 

perchlorate, it did not discuss the effects of the limitations on EPA's 

exposure analysis. Although the agency's guidance for calculating the 

relative source contribution cautions against using the subtraction method 

in the absence of adequate data representative of at-risk populationsand EPA lacked data to estimate exposure to perchlorate for certain 

populations-the agency did not explain that the method it used to 

calculate the relative source contribution for perchlorate was the 

subtraction method or its reasoning for selecting this method. 

In early 2008, EPA used a physiologically based pharmacokinetic (PBPK) 

model" to (1) evaluate the relative sensitivity of sensitive subpopulations 

to the health reference level the agency had developed based on 

pregnant women and their fetuses and (2) address concerns that some 

sensitive subpopulations, such as infants, exposed at the health 

reference level may receive concentrations of perchlorate above the 

reference dose. For its preliminary regulatory determination, the agency 

used the model in a novel way and, according to some key EPA 

scientists, mischaracterized the findings of the modeling analyses by 

selecting and presenting information in such a way as to support the 

agreed-upon conclusion that a health reference level of 15 parts per 

billion was protective of all sensitive subpopulations, including infants. 

While EPA's Office of Research and Development conducted numerous 

sensitivity analyses with the PBPK model, EPA presented the results of a 

PBPK analysis in its October 2008 preliminary regulatory determination 

for perchlorate to support its conclusion that a health reference level of 15 

parts per billion was protective of all sensitive subpopulations, including 

infants, and stated that using the model in this way could reduce some of 

the uncertainty regarding the sensitivities of subpopulations other than 

pregnant women. However, Office of Research and Development officials 

disagreed with the way EPA presented the information in its preliminary 

regulatory determination notice, saying the agency did not sufficiently 

explain the uncertainties and limitations of the analysis, presenting the 

information more conclusively than was appropriate. 

19PBPK models are complex and involve numerous underlying assumptions that are 

imbedded in mathematical representations of the processes associated with how a 

contaminant behaves within, and is ellminated from, the body. 
28 


23819.018

Further, the table EPA published in the preliminary regulatory 

determination notice presenting the results of the PBPK analysis included 

data that may not be consistent with EPA's conclusion that a health 

reference level of 15 parts per billion was protective of all subpopulations. 

That is, the table provided sufficient data for informed readers of the 

preliminary determination to calculate that infants and young children 

could be exposed to doses of perchlorate at levels as high as 5.5 times 

greater than the reference dose, supporting the concern that infants and 

young children may, in fact, be more vulnerable to perchlorate exposure. 

While EPA's regulatory determination notice stated that the modeled 

exposure exceeds the reference dose for some subpopulations, the 

agency was not explicit about the extent to which the reference dose is 

exceeded-as calculated above-and did not explain the implications of 

this result on its conclusion that the health reference level of 15 parts per 

billion is protective of all subpopulations. In providing comments on the 

draft notice to the Office of Water, an Office of Research and 

Development scientist noted that the agency's failure to present a 

comparison of the estimated daily exposure with the reference dose 

constituted a "serious omission," and characterized the infants' estimated 

exposure as "substantially higher" than the reference dose. 

EPA's limited presentation of the PBPK analyses conducted by the Office 

of Research and Development in its preliminary regulatory determination 

notice validated the concern expressed at the time by Office of Research 

and Development scientists who conducted the analyses: that individual 

analyses could be used out of context in a way that could be misleading. 

Specifically, an Office of Research and Development official stated in 

September 2008 that while his office and the Office of Water had 

developed careful and sophisticated PBPK analyses to support the 

agency's preliminary regulatory determination, "the use of these science 

results in [the] draft regulatory determination is seriously flawed and 

misleading." As a result, Office of Research and Development officials 

and scientists that conducted the analyses concluded that the PBPK 

analysis done by the office did not support the draft preliminary regulatory 

determination's suggested health reference level of 15 parts per billion as 

being health protective for all sensitive subpopulations of concern to EPA. 
29 


23819.019

EPA's Independence in 

Developing and 

Communicating Its 

Scientific Findings Was 

Limited by Its Acceptance 

of External Input on the 

Preliminary Determination 

Notice 

Compounding scientists' concerns about the mischaracterization and lack 

of transparency regarding relevant scientific analyses, key language in 

EPA's preliminary regulatory determination notice appears to have been 

drafted by OMB rather than EPA. In working to finalize the preliminary 

regulatory determination notice, EPA's Office of Water worked with OMB, 

whose clearance of the notice was required per EPA's policy 

implementing Executive Order 12866 before the Office of Water could 

provide it to the EPA Administrator for review, approval, and publication in 

the Federal Register." According to the Office of Water, in four iterations 

of review, OMB sent EPA a substantial number of comments on the 

notice; in response, EPA "clarified its description of the supporting 

analysis and strengthened the rationale for the determination." The 

following example highlights OMS's role in reviewing and approving the 

specific wording of EPA's scientific analyses regarding perchlorate 

exposure in infants and children: 

Text EPA provided to OMB: "Because infants and children eat and drink more on 

a per body weight basis than adults, eating a normal diet and drinking water with 

15 (micrograms per liter} of perchlorate is likely to result in exposure that is 

greater than the reference dose in these groups." 

Revised text provided to EPA by OMS: «Because infants and children eat and 

drink more on a per body weight basis than adults, eating a normal diet and 

drinking water with 15 (micrograms per liter] of perchlorate may result in 

exposure that is greater than the reference dose in these groups." 

By changing three words, OMB downplayed EPA's characterization of the 

health risks of perchlorate exposure. Importantly, the EPA scientist who 

wrote the text provided to OMB noted to EPA reviewers-before it was 

sent to OMB in August 2008-that the PBPK model actually showed 

exposures at levels "much higher" than the reference dose, but also said 

that he believed describing the exposure scenario as "likely" was the 

strongest characterization that might be retained through OMB review. In 

addition, in September 2008, during its review of the draft preliminary 

determination notice and before clearing it for publication, OMB reminded 

20The objectives of this executive order are to enhance planning and coordination with 

respect to both new and existing regulations; to reaffirm the primacy of federal agencies in 

the regulatory decision-making process; to restore the integrity and legitimacy of 

regulatory review and oversight; and to make the process more accessible and open to 

the public. 
30 


23819.020

EPA's Response to 

Our 

Recommendations 

Does Not Appear to 

Acknowledge the 

Scope and 

Significance of the 

Implementation 

Limitations We 

Identified 

EPA that it expected the notice to "state a clear conclusion that the HRL 

[health reference level] is protective of all subpopulations, as agreed to in 

the August framework"-and accordingly, this conclusion appeared in the 

agency's October 2008 preliminary determination notice." 

We provided a draft of this report to the Administrator of EPA for review 

and comment. In commenting on the draft report, EPA agreed with 2 of 

the 17 recommendations we made to improve its implementation of the 

Safe Drinking Water Act. Specifically, EPA agreed with our 

recommendations regarding its drinking water health advisories, stating 

that it would evaluate their utility and determine whether and how to 

revise the advisories to better serve states, localities, public water 

systems, and the public. However, EPA did not agree to implement the 

remaining 15 recommendations we made, including an overarching 

recommendation that EPA develop policies or guidance that clearly 

articulate the agency's interpretation of the Safe Drinking Water Act's 

broad statutory criteria-as well as eight additional recommendations 

identifying specific components of this guidance and calling for review of 

the draft guidance by one of EPA's independent advisory committees and 

the establishment of an internal review mechanism to help ensure the 

determinations are consistent with the guidance. Another key 

recommendation with which EPA disagreed was for EPA to include in the 

public record OMS's and other federal agencies' comments on and 

revisions to regulatory determination notices and support documents to 

improve transparency and help EPA ensure that it maintains the fairness 

and openness of its operations. 

We made these recommendations to support the development of 

regulatory determinations that are transparent, clear, and consistent and 

that follow applicable agency policy. However, EPA said it believed that 

establishing policies or guidance for regulatory determinations was not 

"practicable" because of the many combinations of health effects factors 

and potential ranges of frequencies and levels of contaminants measured 

in drinking water. We do not believe that the existence of variables or 

complexities is a basis for not developing guidance for EPA staff to 

implement the statutory requirements for regulatory determinations. In 

21According to an EPA official, ~August frameworkn refers to the agreement that was faxed 

to EPA from the Council on Environmental Quality that included this conclusion as a key 

component of the rationale EPA and other federal agencies agreed to in August 2008. 
31 


23819.021

fact, the complexities cited would argue for, rather than against, the need 

to develop guidance for staff on applying the criteria. EPA also did not 

agree with these recommendations on the basis that policies or guidance 

could "inhibit its ability to continually improve its actions." This perspective 

suggests that guidance per se lacks flexibility. We do not agree that 

guidance and flexibility are incompatible or that developing guidance 

would inhibit EPA's ability to improve its actions. Rather, flexibility can 

and should be incorporated into guidance by establishing parameters or 

options for areas in which flexibility is deemed appropriate. 

Moreover, consistency and accountability are lacking in this important 

program because EPA has not developed guidance on the application of 

the broad statutory criteria, which are susceptible to varying 

interpretations. In its comments, EPA highlighted that, under these 

criteria, ultimately it is the Administrator's judgment as to whether 

regulation of a contaminant in drinking water presents a meaningful 

opportunity for health risk reduction, after considering the information 

presented by agency staff. As stated in our report, the statutory criteria 

are so broadly stated that they could potentially be interpreted so as to 

lead to regulating all the contaminants on the candidate list, some of 

them, or none of them. It is precisely for these reasons that we believe it 

is essential for the staff to have sufficient guidance on applying the broad 

criteria consistently and transparently so that the Administrator's 

judgment can be based on sound and consistent information. Without 

such guidance, the basis for EPA's determinations and the quality of the 

documentation the staff use to support them can fluctuate over time as a 

result of, among other reasons, changes in agency leadership and staff. 

In addition, regarding our recommendation that EPA provide in the public 

record OMB's and other federal agencies' comments on and revisions to 

regulatory determination documents, EPA's position is that unless 

required by law, it is not a good policy because, among other things, the 

documents may be confusing to the public and undermine the ultimate 

policy choice. We disagree and believe that to improve transparency of 

these determinations, which are by law committed to the Administrator's 

judgment, EPA should consistently make these documents available in 

the public record, regardless of whether there is a specific legal 

requirement for disclosure. 

In large measure, EPA's response to our recommendations essentially 

endorses conducting business as usual; a response that does not seem 

to acknowledge the scope and significance of the implementation 

limitations we identified. We are concerned that EPA's lack of 

responsiveness to our recommendations may reflect a misplaced reliance 
32 


23819.022

Contacts and 

Acknowledgements 

on the Office of Water to independently improve the management of this 

important program in the absence of (1) regulations, policies, or guidance 

that we believe are needed to provide a framework for current and future 

staff to apply in identifying and evaluating contaminants for regulation; (2) 

the identification of clear and specific actions needed to address our 

recommendations; and (3) an internal review mechanism to ensure 

identified actions are implemented effectively. We believe that EPA needs 

to adopt all of the recommendations in our report to better assure the 

public of safe drinking water. 

Madam Chairman Boxer, Ranking Member lnhofe, and Members of the 

Committee, this concludes our prepared statement. I would be happy to 

respond to any questions that you or other Members of the Committee 

may have. 

For further information about this testimony, please contact me at (202) 

512-3841. Christine Fishkin, Jamie Meuwissen, Elizabeth Beardsley, Kiki 

Theodoropoulos, and Michael Derr also made key contributions to this 

statement. 
33 


23819.023

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34 


23819.024

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1.) Please Print on Recycled Paper 
35 


23819.025

United States Government Accountability Office 

Washington, DC 20548 

November 3, 2011 

The Honorable Barbara Boxer 

Chairman 

The Honorable James M. lnhofe 

Ranking Member 

Committee on Environment 

and Public Works 

United States Senate 

Attention: Jonathan Aronchick 

We appreciate having had the opportunity to appear before the Senate Committee 

on Environment and Public Works on July 12, 2011, to speak about GAO's work on 

EPA's implementation of the Safe Drinking Water Act provisions related to 

unregulated drinking water contaminants. 

Enclosed are GAO's responses to the questions you submitted for the hearing 

record related to our testimony, Safe Drinking Water Act: Improvements in 

Implementation Are Needed to Better Assure the Public of Safe Drinking Water, 

GA0-11-803T, July 12, 2011. If you or your staff have any questions, please contact 

me at (202) 512-3841 or TrimbleD@gao.gov. 

David C. Trimble 

Director, Natural Resources 

and Environment 

Enclosure 

cc: Grant Cope 

Elizabeth Fox 
36 


23819.026

Enclosure 

GAO Response to Questions for the Record 

Senate Environment and Public Works Committee Hearing 

"Oversight Hearing on the Environmental Protection Agency's Implementation of the Safe 

Drinking Water Act's Unregulated Drinking Water Contaminants Program" 

July 12, 2011 

Response to Questions Submitted by Senator Barbara Boxer 

1. The GAO found that EPA lacks "criteria and a process for identifying those [drinking 

water] contaminants on its candidate list that pose the greatest public health concern ... " 

Could you please explain the practical importance of this type of criteria for ensuring 

transparency and accountability in EPA's decisions to regulate or not regulate drinking 

water contaminants? 

Establishing criteria and a process for identifying contaminants on the candidate lists that 

present the greatest public health concern is important if EPA is to target its limited resources 

on obtaining the data it needs to support regulatory determinations on priority contaminants. 

The Safe Drinking Water Act requires EPA to select contaminants for consideration for 

regulatory determinations that present the greatest public health concern. As we reported, 1 data 

availability-not consideration of greatest public health concern-has been the primary driver of 

EPA's selection of contaminants for regulatory determinations. Moreover, EPA has reported that 

it lacked sufficient occurrence or health effects data, or both, for making regulatory 

determinations for at least 100 of the contaminants on the current (2009) contaminant candidate 

list, which includes 17 contaminants that have been on all three candidate lists. In light of 

increased fiscal constraints and programmatic demands, it is increasingly important for EPA to 

establish criteria and a process so that it can focus its resources on those contaminants that 

may pose the greatest public health concern. The increased transparency provided by criteria 

and a defined process would support EPA's compliance with the statutory requirement for 

prioritization and provide a basis for EPA's accountability for its decisions, with the overarching 

goal of better assuring the public of safe drinking water. 

'GAO, Safe Drinking Water Act: EPA Should Improve Implementation of Requirements on Whether to Regulate 

Additional Contaminants, GA0-11-254 (Washington, D.C .. May 27, 2011). 
37 


23819.027

Enclosure 

2. The GAO recommended that EPA develop and allow the public to review guidance that 

describes the Agency's interpretation of the Safe Drinking Water Act's criteria for making 

regulatory determinations. GAO recommended that such guidance specify thresholds 

that must be met to support a regulatory finding; factors for determining when 

monitoring and health effects data are adequate to support such a finding; and the 

drafting of comprehensive, consistent and understandable documents. Given the GAO's 

review of EPA's current practices, how significant are the changes contemplated by 

these recommendations? 

The changes are potentially significant in that they seek to improve the consistency and 

accountability of this important program. Accountability and consistency has been lacking in the 

program, in part, because EPA has not developed guidance on the application of the broad 

statutory criteria, which are susceptible to varying interpretations. As we reported, the statutory 

criteria are so broadly stated that they could potentially be interpreted so as to lead to regulating 

all the contaminants on the candidate list, some of them, or none of them. It is precisely for 

these reasons that we believe it is essential for EPA staff to have sufficient guidance on 

applying the broad criteria consistently and transparently so that the Administrator's judgment 

can be based on sound and consistent information. Without clarifying guidance, EPA's 

regulatory determinations lack transparency, and EPA is at risk of making inconsistent 

determinations, undermining the program's credibility and the agency's ability to assure the 

public of safe drinking water. Based on the findings in our report, we recommended that EPA 

develop and make available to the public policies or guidance that clearly articulates the 

agency's interpretation of the Safe Drinking Water Act's broad statutory criteria for making 

regulatory determinations and that the guidance should, among other things: (1) specify any 

thresholds or parameters that the agency requires to be met to support a positive finding; (2) 

include factors for determining when the occurrence and health effects data the agency 

identifies are adequate to support a regulatory determination; and (3) establish a process to 

ensure that the presentation of health effects and occurrence information in regulatory 

determination notices and support documents is comprehensive, consistent, informative, and 

understandable. We believe that EPA needs to adopt all of the recommendations in our report 

to better assure the public of safe drinking water, including and especially, developing and 

implementing regulations, policies, or guidance to provide a framework for current and future 

staff to apply in identifying and evaluating contaminants for regulation. 
38 


23819.028

Enclosure 

3. The GAO found that the Office of Water "has not developed guidance for when and 

how to analyze the effects of drinking water contaminants on children ... " The GAO also 

recommended that EPA create "approaches ... to evaluate [contaminants] health effects 

on sensitive subpopulations, Including such groups as infants and children ... " If EPA 

develops this type of guidance, could it help to ensure that the Agency considered 

children's health issues in a more consistent and transparent fashion that it has in the 

past? Are there other potential benefits? 

Developing and implementing the guidance we recommended could help ensure that EPA 

considers children's health issues in a more consistent and transparent way than it has in the 

past. Moreover, if implemented effectively, the guidance could help ensure that EPA's 

regulatory determinations fully consider the subpopulations that are the most sensitive to 

adverse health effects from drinking water contaminants. rather than just the average adult. As 

we reported, EPA Office of Water's lack of guidance for when and how to analyze the effects of 

drinking water contaminants on children in its regulatory determinations hampers EPA's ability 

to consistently apply and document (1) the Safe Drinking Water Act requirement to consider 

sensitive subpopulations when selecting contaminants for regulatory determination and (2) 

EPA's children's health policy, which specifies how health risks to infants and children should be 

analyzed when the agency is developing a policy, regulatory action, or decision. For example, in 

the 2008 regulatory determinations, the Office of Water did not implement a specific approach to 

considering children's health. Further, it did not explain how or whether the agency determined 

that a separate assessment for children or an adjustment to the health reference level was not 

warranted. 

4. The GAO found that in 2008, EPA's "Assistant Administrator [for Water] directed the 

staff to develop a determination to not regulate perchlorate and to support a specific 

exposure level as protective of all populations ... [as] outlined in an agreement between 

high-level officials at EPA and other federal agencies ... " GAO also found that "key 

language" in the determination "notice appears to have been drafted by OMB rather than 

EPA." In your opinion, will requiring transparency with these types of activities help to 

ensure accountability in government decision-making and also help to ensure better use 

of science in making regulatory determinations? 

By implementing our recommendation to include in the public record OMB's and other federal 

agencies' comments on and revisions to regulatory determination notices and support 

documents, EPA could help ensure accountability in government-decision making and could 

help ensure better use of science in EPA's regulatory determinations. For example, as we 

reported, by changing three words in EPA's preliminary regulatory determination for perchlorate, 
39 


23819.029

Enclosure 

OMS downplayed EPA's characterization of the health risks of perchlorate exposure, and 

overall, the changes EPA made in response to OMS's comments presented EPA's conclusions 

with greater certainty than key EPA scientists stated they were comfortable supporting. Making 

public OMS's and other federal agencies' comments on and revisions to regulatory 

determination notices and support documents is key to enhancing the public's understanding of 

the degree to which EPA is maintaining its independence in developing and communicating the 

results of its scientific analyses. 

5. The GAO found that 9 of EPA's 20 determinations not to regulate a contaminant 

involved data from monitoring activities that could not detect these contaminants at 

levels that posed a health concern. The GAO recommended that EPA develop a process 

to "reconsider whether to regulate a contaminant" that it has determined not to regulate, 

and to "consider whether the agency needs to reevaluate any of its past determinations 

to not regulate." Could you please describe the importance of EPA developing this type 

of guidance for protecting public health? 

It is important that EPA develop guidance establishing a process for when and how it will reevaluate contaminants for which new or revised scientific information becomes available in 

order to (1) provide transparency and clarity regarding the circumstances under which EPA 

would undertake a re-evaluation of a completed regulatory determination and (2) help ensure 

consistency in the re-evaluations. Under the 1996 amendments of the Safe Drinking Water Act, 

EPA is to select for consideration those unregulated contaminants that present the greatest 

public health concern, evaluate their occurrence and the potential health risks associated with 

them, and decide whether a regulation is needed for at least five contaminants every 5 years. 

While EPA officials told us that EPA could reconsider a determination to not regulate a 

contaminant on the basis of new scientific data, such as a new IRIS health assessment, the 

agency has not developed any guidance on the circumstances that would trigger such a reevaluation. For example, in the case of 1,1 ,2,2-tetrachloroethane, EPA made a determination to 

not regulate the contaminant in 2008.2 In 2010, EPA's IRIS program finalized an updated health 

assessment, reclassifying 1,1 ,2,2-tetrachloroethane from a "possible" to a "likely" human 

carcinogen. However, the agency has not announced whether it will reconsider its determination 

for this contaminant. In addition, in 2003, EPA made a determination to not regulate dieldrin on 

the basis that its occurrence in drinking water at frequencies or concentrations significant for 

'Prior to the 1980s, 1,1 ,2,2-tetrachloroethane (a volatile organic compound, or VOC) was used in the production of 

other chemicals, such as trichloroethylene (TCE) and tetrachloroethylene (PCE). It was also used as a metal 

degreaser and a component of paint removers, varnishes and lacquers, and photographic films. While no longer 

generated as an end product, it is still produced as a byproduct in the manufacturing of other synthetic chemicals. 
40 


23819.030

Enclosure 

public health concern is low, according to EPA data.3 As we reported, however, using more 

sensitive tests than EPA used, the U.S. Geological Survey (USGS) conducted a study from 

1993 to 2007 in which it detected dieldrin in 3.1 percent of public well samples.' Importantly, 

essentially all of the detections were at levels that exceeded EPA's health benchmark and 

would not have been found if USGS had used the reporting levels on which EPA based its 

regulatory determination. As we reported, the decisions to not regulate these contaminants were 

made in the absence of policies or guidance on applying the broad statutory criteria in the Safe 

Drinking Water Act. We continue to believe it is important for EPA to develop the guidance we 

recommended-and consider whether the agency needs to re-evaluate any of its past 

determinations to not regulate in order to better assure the public of safe drinking water. 

6. The GAO found that 17 contaminants have been on all three of EPA's lists of priority 

drinking water contaminants since 1998, Including RDX or "Royal Demolition Explosive," 

which EPA has classified as a possible human carcinogen. The GAO also raised 

concerns with a potential lack of information that EPA has on some of these 

contaminants. What types of issues do these 17 contaminants raise concerning EPA's 

implementation of the unregulated contaminant program? 

The primary concern for which these 17 contaminants serve as examples is that a lack of 

coordinated occurrence and health effects data continues to limit EPA's ability to make timely 

regulatory determinations on contaminants of concern to EPA. EPA has identified these 17 

contaminants as candidates for drinking water regulation since 1998 and yet has been unable to 

make determinations on them because of insufficient occurrence or health effects data. While 

data on exposure and health effects from similar time frames are generally needed to develop 

credible regulatory determinations and drinking water regulations, EPA's current processes for 

conducting Integrated Risk Information System (IRIS) assessments and selecting contaminants 

for its unregulated contaminants testing program are not producing such data. EPA is collecting 

nationally representative occurrence data on some contaminants to identify exposure at levels 

of public health concern but is not at the same time actively pursuing IRIS assessments or other 

credible sources for health effects information. 

3

Dieldrin is an insecticide that was banned by EPA in 197 4 for most uses, except for the control of termites, and 

banned for all uses in 1987 because of concern about environmental damage, harm to human health, and its ability to 

persist in the environment for decades. 

4

USGS, Quality of Source Water from Public-Supply Wells in the United States, 1993-2007 (Reston, Va., May 2010). 
41 


23819.031

Enclosure 

Response to Questions Submitted by Senator James M. lnhofe 

1. Why did GAO not take into account the EPA Inspector General's review of perchlorate 

in both its 2010 report and this report? 

The EPA Inspector General's science review of perchlorate-which included an alternative 

health risk assessment developed by the Inspector General-was published in 2010, 2 years 

after EPA's preliminary regulatory determination on perchlorate and thus was not relevant to the 

objectives of our 2011 report. 5 The objectives of the 2011 report were to (1) evaluate the extent 

to which EPA's implementation of the 1996 amendments' requirement for determining whether 

to regulate potentially harmful contaminants has helped assure the public of safe drinking water 

and (2) review the process and scientific analyses EPA used to develop its 2008 preliminary 

regulatory determination on perchlorate. We do cite the EPA Inspector General's report on 

perchlorate in our 2010 report. 6 However, we did not include detailed information on the 

conclusions of the EPA Inspector General's report because the issues included in it were 

beyond the scope of our report. Our 2010 report7 examined (1) what is known about the extent 

to which perchlorate occurs in the nation's water and food supply and its likely sources; (2) what 

actions DOD, NASA, and DOE have taken to respond to or lessen perchlorate releases; and (3) 

what actions states, such as California and Massachusetts, have taken to regulate perchlorate. 

2. You highlighted a lack of transparency as a major issue in the Unregulated Drinking 

Water Contaminants Program. What benefits would the Agency and the Public obtain 

from consistent guidance on how EPA is interpreting and using its statutory authority? 

By developing guidance that specifies how EPA is interpreting and using its statutory authority, 

the agency will establish a framework for more credible decision making and increased 

accountability. For example, establishing criteria or parameters related to what level and 

frequency of contaminant occurrence may warrant regulation and its definitions of what 

characterizes a contaminant of "greatest public health concern" and "meaningful opportunity" for 

public health risk reduction will provide: ( 1) a framework to guide EPA program staff through the 

data gathering and analytical stages of the regulatory determinations and (2) benchmarks that 

5

GA0-11-254. 

6

EPA, Office of Inspector General Scientific Analysis of Perchlorate, Report No. 10-P-0101 (Washington, D.C., April 

2010). 

7

GAO, Perchlorate: Occurrence Is Widespread but at Varying Levels; Federal Agencies Have Taken Some Actions to 

Respond to and Lessen Releases, GA0-10-769 (Washington, D.C.: Aug. 12, 2010). 
42 


23819.032

Enclosure 

EPA can use to support and defend its science-based regulatory determinations to protect 

public health. The increased transparency that would result from EPA developing the guidance 

we recommended would support the agency's implementation of the statutory requirements and 

provide a basis on which EPA can be held accountable for its decisions, with the overarching 

purpose of better assuring the public of safe drinking water. 

3. Do deficiencies in IRIS affect EPA's ability to make regulatory decisions in the Drinking 

Water program? 

IRIS assessments provide certain scientific data on health effects that EPA needs to develop 

drinking water health reference levels-the benchmark the agency uses to evaluate whether 

contaminants occur in public drinking water at levels of public health concern. The lack of 

productivity of EPA's IRIS program has impeded effective implementation of EPA's regulatory 

determinations. As we reported, EPA identified health effects information gaps for 44 of the 104 

chemicals on its third candidate list published in 2009. However, the number of contaminants 

with health effects information gaps may be higher because there are another 24 contaminants 

on the candidate list for which EPA does not have an IRIS assessment or for which an IRIS 

assessment is either in progress or has not yet begun. As you know, because EPA had not 

developed sufficient chemical assessment information to limit public exposure to many 

chemicals that may pose substantial health risks, in January 2009 we added EPA's processes 

for assessing and controlling toxic chemicals to our list of areas at high risk for waste, fraud, 

abuse, and mismanagement or-as is pertinent to EPA's implementation of the IRIS program 

and the Toxic Substances Control Act-in need of enhanced oversight and transformation.• 

8

GAO, High-Risk Series: An Update, GA0-09-271 {Washington, D.C.: January 2009); and High Risk Series: An 

Update, GA0-11-278 {Washington, D.C.: February 2011). 
43 

Senator BOXER. Thank you very much for your hard work. 

And now it is time to hear from the EPA, and Mr. Perciasepe, 

welcome. 

STATEMENT OF HON. ROBERT PERCIASEPE, DEPUTY 

ADMINISTRATOR, ENVIRONMENTAL PROTECTION AGENCY 

Mr. PERCIASEPE. Thank you. Thank you for inviting me today, 

Chairman Boxer and Ranking Member Inhofe and members of the 

Committee here today to discuss EPA’s drinking water program. 

Making sure that all Americans have water that is safe to drink 

is one of the fundamental elements of EPA’s mission. I appreciate 

the Committee’s and the Government Accountability Office’s attention to the matter of the Safe Drinking Water Act. While EPA, 

under the leadership of Administrator Jackson, has made some key 

strides with the drinking water program, there is always room for 

improvement, and the GAO report makes some critical recommendations which we welcome. 

Strong science and the law are the foundation of our decisionmaking at EPA. We agree with GAO that our approach to drinking 

water regulation must be transparent, consistent, clear and driven 

by the need to protect public health. In 1996, Congress updated the 

Safe Drinking Water Act to improve the way we decide when a contaminant needs to be regulated. The new process was designed to 

assure that future standards focus on public health protection and 

are based on the best available science. 

Over the years, we have sought advice from a number of independent organizations on how to achieve this goal, including the 

National Academy of Sciences, the Science Advisory Board, and the 

National Drinking Water Advisory Council. Recommendations from 

these groups have contributed to a strong foundation for our program, and we continue to build on those past efforts. The draft 

GAO report makes it clear we have more work to do. 

Specifically, GAO provides recommendations about developing 

policies to interpret the broad statutory criteria that the Safe 

Drinking Water Act outline for making regulatory determinations. 

It is crucial that these criteria are transparent and protect vulnerable populations, particularly children. To achieve these goals, I 

have asked the Office of Water to consult with an independent 

panel of scientists on the process EPA uses. 

Specifically, they will consider how we evaluate contaminants 

against the criteria, how will we use the best available science to 

make a decision and how will we assess the greatest public health 

risk and how to consider vulnerable populations, especially children. We will make our regulatory determination process publicly 

available and we will review the process every 5 years as we conduct the regulatory determination cycle. 

Currently, we are in our third cycle of evaluating unregulated 

contaminants. In implementing this process, we are applying the 

lessons learned from the previous iterations. Our recent actions are 

consistent with many of GAO’s recommendations. To ensure that 

we are best protecting public health, GAO recommends that EPA 

develop criteria to identify contaminants that pose the greatest 

risk. In our most recent contaminant candidate list, EPA used a 

significantly improved process. We cast a wide net to identify posVerDate Aug 31 2005 11:45 Apr 10, 2017 Jkt 000000 PO 00000 Frm 00049 Fmt 6633 Sfmt 6633 S:\_EPW\DOCS\23819.TXT VERN
44 

sible contaminants. And from this universe of more than 7,000 candidates, we narrowed the list down to 116 that represent the greatest risk. The multi-step process was science-based and informed by 

the public and independent science experts. All of the supporting 

information is available to the public. From within this list, we 

have identified the contaminants with enough data to make a decision in this cycle, and we are focusing our decision on those with 

the greatest public health concern. 

In order to have the occurrence data we need to make good decisions, GAO recommends that we improve our unregulated contaminants monitoring program. We agree with the GAO’s recommendations . In March, we proposed our most recent list of contaminants 

for monitoring and we are taking full advantage of the statutory 

authority to test for 30 contaminants. We also proposed minimum 

reporting limits that are much lower than have been required in 

the past, making the data obtained more useful. 

This Administration also recognizes that we need to change the 

paradigm for determining and developing drinking water regulations to protect public health. We are implementing the drinking 

water strategy announced by the Administrator last year. In particular, we have established a focus on regulating multiple contaminants as a single group. By doing so, we can achieve public 

health protection more expeditiously and in a way that is more 

cost-effective for water utilities to implement. 

We are also focusing on fostering innovative drinking water technology, so that drinking water treatment is available and affordable. We are leveraging the authorities of multiple statutes to protect drinking water sources from contamination. 

In closing, Madam Chairman, protecting the resource of clean, 

safe water is one of EPA’s highest priorities. We are committed to 

continually improving our methods and I look forward to working 

with this Committee and the GAO in our efforts to best protect the 

American people. 

Thank you. 

[The prepared statement of Mr. Perciasepe follows:] 

45 


23819.033

Testimony of 

Robert Perciasepe, Deputy Administrator 

U.S. Environmental Protection Agency 

Before the 

Committee on Environment and Public Works 

United States Senate 

July 12, 2011 

Chairman Boxer, Ranking Member Inhofe, and Members of the Committee, thank 

you for inviting me to discuss how the EPA decides when to set new drinking water 

standards. The public relies on EPA to ensure the safety of the water they drink every 

day, and EPA takes this responsibility very seriously. 

Strong science and the law are the foundation of our decision-making at EPA. 

Under the Safe Drinking Water Act (SDWA), EPA identifies priority contaminants that 

are known or anticipated to occur in public water systems and then evaluates whether 

new drinking water standards are warranted for these contaminants. EPA appreciates the 

Government Accountability Office's (GAO) attention to the important matter of setting 

drinking water regulations and we welcome GAO's input about how to do this most 

effectively. EPA has reviewed GAO's draft report so my testimony reflects my 

consideration of the recommendations in that version. GAO's draft recommendations 

address three key areas for EPA to improve implementation of requirements on whether 

to regulate additional contaminants: 

1. Development of criteria to identify contaminants that pose the greatest health 

risk; 
46 


23819.034

2. Improvement of EPA's unregulated contaminants testing program, and 

3. Development of policies or guidance to interpret the broad statutory criteria. 

EPA agrees with GAO that consistency, transparency and clarity are essential in assuring 

the safety of public drinking water and our credibility with the public. While we have 

made substantial progress in achieving this goal, we agree that there is room for 

improvement. We are committed to actions to ensure that the public has confidence that 

the EPA's decisions are protective of their health and based on a thorough consideration 

of the best available science and information. 

EPA is in the third cycle of evaluating unregulated contaminants as required by 

the 1996 SD W A amendments. EPA has completed the third Contaminant Candidate List 

(CCL), proposed the third Unregulated Contaminant Monitoring Rule (UCMR3), is 

developing the third round of regulatory determinations, and also recently made the offcycle determination to regulate perchlorate. We are continually learning from each 

iteration of this process and are currently applying lessons learned from previous 

determinations. We believe the improvements we have made go a long way towards 

addressing GAO's concerns. 

Administrator Jackson also announced last year a new vision for better protecting 

drinking water including changing the paradigm of evaluating individual contaminants 

for regulation. Under the new drinking water strategy, EPA is committed to: 

1. Considering regulation of groups of contaminants to better protect public health 

by streamlining decision-making and in a way that is likely more cost-effective 

for water systems to implement 

2. Fostering development of new drinking water technologies 
47 


23819.035

3. Leveraging other regulatory programs such as the Toxic Substances Control Act 

and the Federal Insecticide, Fungicide, and Rodenticide Act as appropriate to 

protect sources or drinking water 

4. Working with states to share more complete drinking water monitoring data to 

support evaluation of drinking water protection nationwide and make information 

accessible to the public. 

We are implementing this strategy as we conduct the ongoing cycle of regulatory 

determinations. 

Identifying Contaminants of Concern 

The first step of the evaluation is to identify the contaminants of greatest public 

health concern, which EPA does through the Contaminant Candidate List. In the most 

recent CCL, published in October 2009, EPA used the advice from the National 

Academy of Sciences and the National Drinking Water Advisory Council to develop and 

usea significantly improved, more transparent and reproducible multi-step process to 

ensure more effective identification of public health threats. We cast a wide net in 

identifYing possible drinking water contaminants including those nominated by the 

public. From an initial universe of7,500 contaminants, EPA evaluated available 

occurrence and potential health effects data for this universe and incorporated public 

input and expert review. Through this review, we selected from this universe a list of 116 

priority contaminants that we found to be of the greatest public health concern based on 

both the severity of the health effect and the anticipated occurrence. EPA also improved 

transparency by making all data and criteria used to classifY contaminants publicly 

available on the EPA's website. 
48 


23819.036

GAO's report expresses concern that EPA's past decisions have been driven not 

by considering the greatest health concern but by considering available data. EPA agrees 

that we can improve our process to better focus on contaminants that may be of public 

health concern. The improved approach in the most recent CCL was a substantial step 

forward in achieving this by using a rigorous scientific process to better ensure that the 

contaminants on the list are the ones that should be of highest priority for public health 

protection. Because the new CCL selection process targeted candidates based on 

possible health effects or exposure rather than just on available data, the list includes 

emerging contaminants that are not currently well enough understood to discern whether 

regulation of drinking water could improve public health. EPA cannot make a credible 

decision driven by science without sufficient understanding of the potential for impacts to 

the health of the American people. Therefore, EPA has since narrowed the CCL down to 

a "short list" of 32 contaminants that have sufficient data to make a determination within 

the statutory timeline. This short list is being prioritized for regulatory decision making 

in this cycle based on the greatest public health concern. Those determinations will be 

announced by next summer for public comment. We believe this approach addresses 

GA 0' s concerns with previous cycles of our process. 

Collect and Evaluate Information 

For the remaining candidates, obtaining robust data and information regarding 

potential impacts is essential. For the current CCL, the evaluation of contaminants 

included a discussion of data gaps so that further information can be collected to support 
49 


23819.037

future decisions. The CCL classifies the contaminants based on the need for health 

effects data or occurrence data or analytical methods. 

To obtain occurrence data, EPA uses the Unregulated Contaminant Monitoring 

Rule (UCMR) and also looks to data collected by others such as the states and the U.S. 

Geological Survey. GAO had a number of recommendations to improve the UCMR 

process. We agree with the GAO recommendations regarding UCMR. They are 

consistent with the most recent UCMR proposal, published in March, in which EPA 

looked at health effects information to target the contaminants of greatest concern. We 

also proposed, as GAO has recommended, to use our full statutory authority to require 

testing for 30 contaminants and to conduct full assessment monitoring rather than more 

limited screening surveys. Additionally, the proposed UCMR generally requires much 

lower minimum reporting levels than have been required in the past, making the data 

obtained more useful in determining the likelihood of health impacts when contaminants 

are detected. 

Good data about health effects are also essential, and EPA's Office of Water 

identifies priority contaminants and health assessment information needs and coordinates 

with the EPA's Office of Research and Development and external organizations. The 

Agency searches the available literature and participates in scientific meetings to identify 

evolving science that may support evaluation of health effects. EPA has also made 

substantial revisions to the Integrated Risk Information System (IRIS) process to provide 

assessments in a timely fashion to best support regulatory decisions. EPA has reduced 

the IRIS backlog and shortened the risk assessment development time while ensuring 
50 


23819.038

rigorous peer review. Strengthening and streamlining the IRIS process is a continuing 

and ongoing priority for EPA. 

Regulatory Determination 

According to SDW A, EPA must make determinations for at least five 

contaminants from each CCL. SDW A defines three criteria to determine whether it is 

appropriate for EPA to regulate a potential drinking water contaminant: 

• The contaminant may have an adverse effect on the health of persons; 

The contaminant is known to occur or there is a substantial likelihood the 

contaminant will occur in public water systems with a frequency and at levels 

of public health concern; and 

• In the sole judgment of the Administrator, regulation of the contaminant 

presents a meaningful opportunity for health risk reductions for persons 

served by public water systems. 

EPA determines whether an adverse health effect may occur, identifies what levels of 

exposure may result in public health concern and then evaluates how extensive potential 

exposure at those levels might be. Finally, the Administrator must decide whether 

regulatory action taken by EPA would serve to reduce public health risk in a meaningful 

way. A decision by the Administrator to regulate a contaminant is the beginning of the 

SDWA regulatory development process. EPA has extensive further requirements 

regarding analyses of health benefits, costs, and treatment technologies that must be 

conducted before a National Primary Drinking Water Regulation is proposed and made 

final. 
51 


23819.039

In the first two cycles of regulatory determinations, EPA made negative 

determinations for 20 contaminants, in each case deciding that the occurrence of the 

contaminant was not at a frequency and level of public health concern to merit a new 

drinking water standard. This February, EPA made the Agency's first positive 

determination, when we announced that we will be developing a proposed drinking water 

standard for perchlorate by February 2013 at the latest. 

Strong science must be the foundation of decisions regarding the criteria defined 

by SDW A, but science alone cannot fully address the criteria. As the GAO describes in 

their report, there are a large number of factors that can impact our understanding of what 

levels would be of concern and how likely those are to occur, such as the severity of 

health effects, the potency of the contaminant, the geographic distribution and levels of 

drinking water detections, or other possible sources of exposure. In its regulatory 

determinations, EPA has sought out and evaluated available information on these factors 

and based our determinations on our best understanding of the existing information. 

Given the many possible combinations of factors and the constantly evolving 

science, it is essential that the bases for EPA's decisions be clearly presented so that the 

public can have confidence in our actions. For our regulatory determinations, our 

Federal Register notices and supporting documentation list the primary occurrence and 

health effects data, describe our evaluation of whether this information is sufficient, and 

explain our approach for deriving endpoints. 

The concerns that GAO raises indicate that we have not always done this 

effectively enough. We will do a better job in the future. EPA will work to improve the 
52 


23819.040

transparency of regulatory determination so that the public can better understand how 

EPA came to its conclusions and most effectively comment or review. EPA will make 

this information available when we publish our preliminary determination. 

In response to the GAO recommendations, EPA will also consult with an 

independent panel of scientists on the regulatory determinations, specifically on the 

evaluation of the contaminants against the first and second criteria defined by SDW A, the 

use of best available science to develop the determination, and whether the determination 

focuses on the greatest public health risk. We will post the regulatory determination 

process publicly and review the process every five years as we conduct the regulatory 

determination cycle. 

Regulating Contaminants as Groups 

As I stated earlier, in parallel to these improvements to the standard regulatory 

determination process, EPA is changing the regulatory approach that has primarily 

addressed contaminants one at a time. In February, Administrator Jackson announced 

that we are developing a single regulation to include up to 16 volatile organic compounds 

that may cause cancer. Several of these contaminants are on the current CCL and others 

are currently regulated and need to be revised. By considering them as part of a group 

rather than through individual regulatory determinations, we can address the public health 

concerns from a larger portion of our priority list at one time, achieving greater health 

protection more expeditiously and in a way that is likely to be more cost-effective for 

utilities to implement. 
53 


23819.041

In the current round of regulatory determinations, EPA is also evaluating whether 

to regulate nitrosamines (currently on the CCL) as a group. We have found these 

disinfection byproducts in a number of water systems and considering them as a group 

would allow us to take advantage of shared analytical methods and treatment or control 

processes, as well as making a greater impact on public health because nitrosamines 

sometimes co-occur, and because controlling nitrosamines also reduces exposure to 

related disinfection byproducts. 

Conclusion 

Clean and safe water is the foundation of healthy communities, healthy families, 

and a healthy economy. EPA is committed to continuing to improve our methods in 

using science and the law to best protect public health. I greatly appreciate the leadership 

of this Committee in helping to protect drinking water. We look forward to coordinating 

with this Committee as we work to achieve these important goals. 
54 


23819.042

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

The Honorable Barbara Boxer 

Chainnan 

MAR 1 2 2012 

Committee on Environment and Public Works 

United States Senate 

Washington, D.C. 20510-6175 

Dear Chainnan Boxer: 

OFFICE OF CONGRESSIONAL AND 

INTERGOVERNMENTAL RElATIONS 

Thank you for the opportunity to respond to the questions for the record following the July 12, 

2011 hearing on "The EPA's Implementation ofthe Safe Drinking Water Act's Unregulated 

Drinking Water Contaminants Program," The attached document has responses to the questions. 

I hope that this infonnation is useful to you and the members of the committee. 

If you have any further questions, please contact me or your staff may call Sven-Erik Kaiser in 

my office at (202) 566-2753. 

Attachment 

s;~ 

in Ganesan 

Associate Administrator 

lntemet Address (URL) • http:f/www.epa.gov 

Recycled/Racyclable • Pt1nted with Vegetable OM Based Inks on RecyCled Paper (Minimum 25% Postconsumet) 
55 


23819.043

Senate Committee on Environment and Public Works 

"Oversight Hearing on the EPA's Implementation of the Safe Drinking Water Act's Unregulated 

Drinking Water Contaminants Program" 

Questions for the Record 

July 12, 2011 

Senator Boxer 

Boxer 1. The GAO found that "EPA does not have criteria for identifying contaminants of 

greatest public health concern ... " and recommended that EPA develop criteria to identify 

contaminants that pose the greatest public health risk." 

Will EPA develop such criteria in order to determine which drinking water contaminants should 

be regulated and ensure that the criteria are used in a transparent and accountable process? 

Answer: As committed during the July 2011 hearing, the EPA has already consulted with an 

independent panel of scientists on its third set of regulatory determinations (RD3). We 

specifically requested our expert reviewers to provide input on: the Agency's evaluation of the 

contaminants against the first and second Safe Drinking Water Act (SDWA) criteria [ I) 

contaminant may have an adverse effect on the health of persons; 2) contaminant is known to 

occur or there is substantial likelihood that it will occur in public water systems with a frequency 

and at levels of public health concern]; whether the agency used the best available science to 

evaluate these criteria; and whether the process focuses oti the contaminants of greatest potential 

public health risk. 

Note that the third SDWA criterion (regulation of the contaminant presents a meaningful 

opportunity for health risk reduction for people served by public water systems) is subject to the 

sole judgment of the Administrator. The Administrator's decision of whether there is a 

meaningful opportunity to regulate a contaminant will take into consideration the input gathered 

from our expert panel and other relevant information. 

We plan to publish the third round of regulatory determinations in the Federal Register and allow 

the public to comment on the process and factors used to identify and evaluate contaminants for 

this round of regulatory determinations. We will also make available the support document that 

provides more detail about the approach for RD3 and the input and advice provided by our 

expert reviewers. 

Boxer 2. The Safe Drinking Water Act requires EPA to prioritize contaminants for regulation 

that present "the greatest public health concern," including for infants and children. The GAO 

found that the Office of Water "has not developed guidance for when and how to analyze the 

effects of drinking water contaminants on children ... " The GAO recommended that EPA create 

"approaches ... to evaluate [contaminants] health effects on sensitive subpopulations ... " 

Will EPA develop such approaches and ensure their use in a transparent and accountable 

process? 
56 


23819.044

Answer: The EPA agrees that it is important to evaluate health effects on sensitive populations 

such as infants and children, pregnant women, the elderly, and individuals with a history of 

serious illness. The EPA describes its consideration of the susceptibility of populations and life 

stages in the agency's health effects documents supporting each regulatory determination. To 

the extent that information is available in the literature defining the impacts on individuals with 

special susceptibilities, such as liver or kidney impairments, the information is provided to 

support the EPA's decision making. Immunocompetence is routinely considered in decisions 

concerning the occurrence of pathogenic organisms in source water and finished water. As 

described in the response to Boxer I above, the EPA is assuring a transparent and accountable 

consideration of effects to sensitive populations by requesting expert review and seeking public 

review and comment. 

Boxer 3. Your testimony today states that EPA will consult with an independent panel of 

scientists when the Agency's is making certain determinations about whether a contaminant 

should be regulated under the Safe Drinking Water Act. I think that this is very welcome news. 

Will EPA ensure that these scientific deliberations are transparent to the public? 

Answer: Yes, when the EPA publishes its third round of regulatory determinations, we will 

publish the supporting documentation, which will include a summary of the input and advice 

provided by the expert panel of scientists. 

Boxer 4. The GAO found that 9 of EPA's 20 determinations not to regulate contaminants 

involved data from monitoring activities that could not detect contaminants at levels that posed a 

health concem. 

The GAO recommended that EPA develop a process to "reconsider whether to regulate a 

contaminant" that EPA has determined it will not regulate, and to "consider whether the agency 

needs to reevaluate any of its past determinations to not regulate [contaminants)." 

Will EPA develop such a process, and is EPA considering whether to reevaluate any past 

decisions? 

Answer: Yes, the EPA will reevaluate new health effects and occurrence information for 

contaminants that the EPA previously issued determinations not to regulate (i.e., a negative 

regulatory determination) in future Contaminant Candidate List (CCL) processes. 

If new health or occurrence information becomes available on these contaminants, this 

information is considered to evaluate whether the contaminant should be listed on future CCLs 

and evaluated in future regulatory determinations processes. 

BoxerS. Deputy Administrator Perciasepe, the GAO found that in 2008, EPA's "Assistant 

Administrator [for Water] directed the staff to develop a determination to not regulate 

perchlorate and to support a specific exposure level as protective of all populations. This 

direction was outlined in an agreement between high-level officials at EPA and other federal 
57 


23819.045

agencies ... " GAO also found that "key language" in the "notice appears to have been drafted by 

OMB rather than EPA." GAO has recommended that EPA include such agency comments in the 

public record in the future. 

Will EPA agree to increase the level of public transparency in making these determinations? 

Answer: The EPA will work to ensure the transparency, clarity and consistency of its regulatory 

determinations so the public can understand how the EPA came to its conclusions and so that the 

public can most effectively review and provide comment. 

The EPA will identify for the public the substantive changes that occurred during the Office of 

Management and Budget (OMB) review and those changes made at the suggestion or 

recommendation ofOMB. Specifically, the EPA will make available, in the public docket, 

documents that show the specific changes made to any regulatory determination notice (or other 

regulatory action) in the course of interagency review under Executive Orders 12866 and 13563 

(either redline versions of the notice or before and after versions of the notice). 

Boxer 6. In February 2011, Administrator Jackson reversed a Bush Administration decision not 

to regulate perchlorate with an announcement that EPA would move forward and develop a 

drinking water safeguard for this dangerous chemical. Perchlorate is a toxic contaminant that 

can harm children's development. I strongly support Administrator Jackson's decision. 

Could you tell me if EPA is moving forward with the process of developing a drinking water 

standard for perchlorate? Also, can you assure me that the Agency will continue to use the best 

available science in crafting this standard? 

Answer: The EPA is moving forward with the process to develop a perchlorate standard. The 

EPA is continuing to evaluate the science on perchlorate health effects and occurrence to develop 

a proposed drinking water standard for perchlorate. The EPA will also evaluate the feasibility 

and affordability of treatment technologies to remove perchlorate and will examine costs and 

benefits of potential standard levels. The EPA intends to publish the proposed regulation for 

public review and comment in 2013 and expects to promulgate a final regulation within 18 

months of the proposal. 

The EPA is committed to using the best available science and peer reviewed data in developing a 

drinking water standard for perchlorate. The agency will consult with our Science Advisory 

Board and with the National Drinking Water Advisory Council in developing the standard. In 

addition, the EPA will provide an opportunity for public comment on the proposed regulation, 

and will carefully evaluate and consider any new studies and data submitted by public 

commenters in developing a final regulation. 

Boxer 7. In September 2010, EPA released a draft scientific assessment that found chromium-6 

in drinking water is "likely to be carcinogenic to humans." In February 2011, EPA proposed to 

require monitoring for 30 contaminants, but did not include chromium-6 on that list. However, 

EPA asked utilities to voluntarily monitor for this heavy metal and has also asked for comment 

on whether monitoring should be mandatory. 
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23819.046

Could you please tell me the status of EPA's consideration of whether to require monitoring for 

hexavalent chromium? 

Answer: The EPA is in the process of developing the final Unregulated Contaminant 

Monitoring Regulation (UCMR 3) rule for the next cycle of unregulated contaminant monitoring 

and anticipates publication soon. 

The EPA is giving serious consideration to the public comments we received on the question of 

including chromium 6 in the UCMR 3 rule. The EPA is also carefully considering what 

contaminant it would drop to stay within the 30 contaminant limit set by Congress if it does 

decide to require monitoring for hexavalent chromium. We will announce our decision when we 

publish the final rule. 

Boxer 8. In September 2010, EPA released a draft scientific assessment that found the 

chromium-6 in drinking water is "likely to be carcinogenic to humans." The Agency has said it 

expects to finish this assessment in 2011. 

Could you please tell me when in 20 II EPA will complete this assessment? 

Answer: The EPA received the Final External Peer Review Report on the draft assessment and 

it was posted on the Integrated Risk Information System (IRIS) website (www.epa.gov/iris) in 

July 2011. The agency is currently considering the recommendations of the peer review panel, 

along with the comments we have received from the public, in determining the next steps for this 

assessment. A revised timeline for completion of the assessment will be available on IRISTrack 

(http://cfpub.epa.gov/ncealiristrac/index.cfmin) in the near future. 
59 


23819.047

Senator Whitehouse 

Whitehouse 1. EPA's website links to a 2004 report entitled "PPCPs in the Environment: Future 

Research - Beginning with the End Always in Mind" (http://www.epa.gov/ppcp/pdf/daughtonneeds.pdf). The report identifies the following "Needs and Gaps" in research about and policy 

responses to Pharmaceuticals and Personal Care Products (PPCPs): 

• resource coordination; 

• the sources and origins of PPCPs; 

• detection of PPCPs in drinking water; 

• methods for standardizing the chemical monitoring of water; and 

• communication of risk and public outreach. 

What advances have been made with regard to pharmaceuticals in drinking water in the areas 

listed in this report in the 7 years since it was published? 

Answer: The EPA has been actively involved in efforts to identify compounds that are found in 

drinking water and surface water and determine appropriate responses to these issues. The 

agency continues to evaluate the potential risks to both human health and aquatic life that may be 

posed by these contaminants in water and is working with the relevant federal agencies. 

The most comprehensive assessment of pharmaceuticals in drinking water occurred during the 

third Contaminant Candidate List (CCL3) evaluation process in 2008 (draft) and 2009 (final). In 

the CCL3 process, the EPA evaluated the potential adverse health effects of pharmaceuticals and 

their occurrence in public drinking water systems to determine if pharmaceuticals should be 

added to the list. 

Since the publication of the draft CCL3 in 2008, several publications focused on the occurrence 

of pharmaceuticals in ambient water and drinking water were released. The EPA conducted 

additional data collection efforts to comprehensively review recent published information from 

the U.S. Geological Survey (USGS) and other sources to identify the best available occurrence 

information for pharmaceuticals. The EPA identified new occurrence data for 81 contaminants 

from 22 sources. The Agency also conducted additional literature reviews to identify the best 

available health effects information and identified data from sources including: the EPA Office 

of Pesticide Programs; the Food and Drug Administration (FDA) Center for Drug Evaluation and 

Research; the FDA Center for Veterinary Medicine; the Joint Food and Agriculture 

Organization/World Health Organization Expert Committee on Food Additives; and the 

European Medicines Agency. 

The EPA used the new data in the same process that was described in the draft CCL3 Federal 

Register notice to further evaluate pharmaceutical contaminants. Based upon this reevaluation 

with new data, the EPA concluded that one antibiotic (erythromycin) and nine hormones (17 

alpha-estradiol, 17 beta-estradiol, equilenin, equilin, estriol, estrone, ethinyl estradiol, mestranol, 

and norethindrone), should be included on the CCL3 because these contaminants are known or 

substantially likely to occur in public water systems and may be appropriate for regulation. 

In February, 2011, the EPA proposed the third Unregulated Contaminant Monitoring Regulation 

(UCMR 3). The EPA proposed to require monitoring for 30 contaminants (including 7 
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23819.048

hormones) using an analytical method developed by the EPA. The EPA expects to promulgate 

the UCMR 3 in 2012 and begin data collection in 2013. The EPA uses data collected through 

UCMR to evaluate the frequency and level of contaminant occurrence, to support the 

determination of whether to regulate a contaminant, and to inform future contaminant candidate 

lists. In addition, the EPA is collaborating with the USGS to monitor finished drinking water and 

source water at more than 20 drinking water treatment plants across the nation to evaluate both 

the occurrence of contaminants of emerging concern in water (including pharmaceuticals) as 

well as the effectiveness of treatment on reducing levels. Results of this study are scheduled to 

be released in 2013. 

Whitehouse 2. How does EPA plan to improve coordination internally with the many offices 

and laws (Resource Conservation and Recovery Act, Superfund, Clean Water Act, Safe Drinking 

Water Act) that regulate pharmaceuticals? How does the agency plan to improve coordination 

with other agencies such as the FDA? 

Answer: The EPA continues to coordinate its efforts related to pharmaceuticals internally and 

externally. Within the EPA, we form agency-wide workgroups designed to allow all the EPA 

offices and regions to participate. These workgroups serve as the agency's internal means for 

coordination of the many offices and laws, including the Resource Conservation and Recovery 

Act, Superfund, the Clean Water Act, and the Safe Drinking Water Act, that may relate to the 

regulation of pharmaceuticals. 

The agency also has ongoing collaborative efforts with the U.S. Geological Survey, the 

Department of Agriculture, the Office of National Drug Control Policy, and the Food and Drug 

Administration. In addition, the EPA has committed to establishing an interagency workgroup or 

other formal mechanism, as recommended by the Government Accountability Office (GAO) in 

its September 2011 report entitled "Action Needed to Sustain Agencies' Collaboration on 

Pharmaceuticals in Drinking Water," to collaborate and coordinate research on pharmaceuticals 

and, as appropriate, other contaminants in drinking water that present the greatest public health 

concern. To establish this mechanism, the EPA will build upon existing interagency 

collaborations with relevant federal partners and take steps identified by the GAO to: 1) define 

roles and responsibilities of existing and other relevant federal partners; 2) identify experts and 

other resources that each agency can bring to bear on the issue; and 3) develop a process for 

monitoring, evaluating, and reporting to the public the results of the collaborative research 

efforts. The EPA initiated this effort in late 2011. 

Whitehouse 3. Is EPA planning to assess the risks to human health associated with long-term 

exposure to low concentrations of pharmaceuticals and the possible combined effects of 

pharmaceuticals, particularly on the general population and/or sensitive subpopulations? 

Answer: The EPA assesses risks to the general population and/or sensitive subpopulations 

based on available occurrence and health effects data. As data becomes available for 

pharmaceuticals, the EPA plans to identify which, if any, of these contaminants require 

regulation or further research. The agency faces challenges in obtaining sufficient occurrence 

and health effects data on pharmaceuticals in drinking water to support these analyses and 

decisions. However, as mentioned in response to Whitehouse I above, the agency has proposed 

to·collect occurrence information on seven hormones under the UCMR 3, and the agency is 
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working with the Food and Drug Administration and the U.S. Geological Survey to obtain this 

data. 

Whitehouse 4. What preventative measures is EPA implementing or planning to implement, to 

address pharmaceuticals in drinking water • such as rational drug use and education of 

prescribers and/or the public to reduce disposal and discharges to the environment? 

Answer: The EPA developed a consumer information sheet about how to properly dispose of 

household medications and has given step by step demonstrations (upon request) at local events. 

The information sheet was designed to educate the public as well as prescribers on ways to 

reduce the introduction of pharmaceuticals into the environment by following steps to properly 

dispose of unwanted or unused pharmaceuticals. The information sheet is available online at: 

http://water.epa.gov/scitechlswguidance/ppcp/upload/ppcpflyer.pdf. 

However, disposal of household pharmaceuticals is a complex issue which requires collaboratio: 

among federal agencies such as the Drug Enforcement Administration (DEA). The EPA is 

working with the DEA to inform the public about the environmental benefits of reducing 

discharges of household pharmaceuticals to the environment. 

The DEA, in conjunction with state and local law enforcement agencies, conducts National 

Prescription Drug Take-Back Days. Nearly 4,000 state and local law enforcement agencies 

participated in these events, collecting more than 498 tons of pills. In October 20 I 0, the 

President signed the Safe and Secure Drug Disposal Act, amending the Controlled Substances 

Act to allow the DEA to develop a process for people to safely dispose of their prescription 

drugs. While that process is under development, DEA continues to conduct Take-Back Days. 

The next Take-Back Day is scheduled on April28, 2012. The EPA continues to work with the 

DEA to advertise these events. See http://www.deadiversion.usdoj.gov/drug disposal/takebacki 

for more information. 

Whitehouse S. Is EPA planning to undertake or fund any type of research to help waste water 

treatment facilities determine the practicalities of filtering out pharmaceuticals? 

Answer: In 20 I 0, the EPA released the results of an extensive literature review of published 

studies of the effectiveness of various treatment technologies for contaminants of emerging 

concern (CECs), including pharmaceuticals. The EPA also released a report that discusses som< 

of the results of the literature search, including removals of specific CECs across common 

wastewater treatment technologies. The report discusses 16 of the more than 200 CECs present 

in the database, and the average percent removals achieved by full scale treatment systems that 

employ six of the more than 20 reported treatment technologies. Wastewater treatment plant 

operators, designers, and others may find this information useful in understanding ways to 

remove CECs from wastewater. The extent of contaminant removal varies greatly with the 

specific CEC and type of water treatment. 

Whitehouse 6. Is EPA planning to or currently engaged in any monitoring of surface water, 

groundwater or wastewater effluent to assess possible occurrence levels and exposure to 
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particularly concerning pharmaceuticals, including estrogen compounds and cyotoxic 

chemotherapy drugs such as Mustargen? 

Answer: The agency is sampling surface water at 154 sites across the nation for a suite of 54 

pharmaceuticals, including estrogenic compounds, as part of the National Rivers and Streams 

Assessment survey. Data for this survey is scheduled to be released during spring 2012. 

Currently, the EPA is not sampling for cytotoxic chemotherapy drugs such as mustargen. 
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Senator Inhofe 

Inhofe 1. The GAO report "Safe Drinking Water Act: EPA Should Improve Implementation of 

Requirements on Whether to Regulate Additional Contaminants" was made public at the 

Hearing. EPA agreed with two of the recommendations GAO made, yet at the hearing you 

appeared to agree with the majority of the report rather than just two recommendations. 

Please describe how you will or will not be addressing each of the GAO's recommendations 

outlined in the report. 

Answer: Please see the enclosed letter from the EPA Chief Financial Officer Barbara Bennett to 

Congressman Issa dated December I 5, 20 II, describing how the EPA is addressing each of the 

Government Accountability Office's (GAO) recommendations. 

lnhofe 2. The National Academy of Sciences and the Agency for Toxic Substances and Disease 

Registry have concluded that the group of individuals most susceptible to harm from perchlorate 

exposure is fetuses of iodine-deficient pregnant women. In 2005, EPA accepted these 

conclusions. Does EPA continue to concur with those conclusions? If not, what is EPA's new 

position and what scientific support exists for EPA's new position? 

Answer: The EPA continues to concur with the National Academy of Sciences' statements on 

page 27 of the 2005 report. "The thyroid hormones are critical determinants of growth and 

development in fetuses, infants, and young children. Thus, fetuses and preterm newborns 

constitute the most sensitive populations although infants and developing children are also 

considered sensitive populations. People who have compromised thyroid function resulting from 

conditions that reduce thyroid hormone production and people who are iodide-deficient also 

constitute potentially sensitive populations." Therefore, the EPA believes that fetuses, preterm 

newborns, infants, developing children, people with compromised thyroid functions and people 

with iodide deficiencies are all sensitive populations. 

Inhofe 3. EPA has historically used a default body weight of 70 kg body weight and water intake 

oftwo liters per day to translate a reference dose to a drinking water concentration. These default 

values are already biased conservatively (low weighted adult in the numerator /large drinking 

volume in the denominator) and the RID already has safety factors embedded into its derivation. 

Does EPA intend to change the use of these default values for all chemicals it evaluates for 

MCLIMCLGs? Why? How will EPA ensure any changes in default values are adequately vetted 

with the scientific community and transparently explained to the public? 

Answer: The EPA has traditionally used a body weight estimate of 70 kg and daily 

consumption estimate of2 liters per day to derive Maximum Contaminant Level Goals (MCLGs) 

for noncarcinogenic contaminants. These values have corresponded to the mean body weight for 

the general population and the upper (90th percentile) consumption rate estimates. However 

when there are "subpopulations that are identified as likely to be at greater risk of adverse health 

effects due to exposure to contaminants in drinking water than the general population," section 
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l412(b)(3)(C)(i)(V) of the Safe Drinking Water Act requires that the EPA analyze the effects of 

contaminant on these groups. The EPA will utilize the best available, peer reviewed science and 

data collected in accordance with accepted methods when evaluating the effects of contaminants 

on subpopulations likely to be at greater risk than the general population. Because infants' and 

children's body weight and drinking water consumption differ substantially from the general 

population, the EPA will utilize the best available, peer reviewed exposure data and analyses 

specific to these subpopulations since these groups have been identified as being likely to be at 

greater risk of adverse health effects. EPA also continues to explore the use of peer reviewed 

physiologically based pharmacokinetic (PBPK) modeling to assess exposure in sensitive 

subpopulations. The EPA will assure transparency of these analyses by publishing them for 

public review and comment with the proposed MCLG and drinking water rule. 

Inhofe 4. The National Academy of Sciences has stated that inhibition of iodide uptake is the 

only effect of perchlorate exposure that had been consistently documented in humans from 

environmental levels of perchlorate exposure. ATSDR has stated that this effect has never been 

observed in humans. Can you point to any peer reviewed study in which inhibition of iodide 

uptake has been observed in human subjects from exposure to perchlorate in the environment? 

Answer: The National Academy of Sciences (NAS) identified a mode of action model or 

pathway that leads from perchlorate exposure to inhibition of iodide uptake in the thyroid which 

in tum leads to changes in thyroid hormones and thyroid stimulating hormone (TSH) levels. The 

NAS stated "that the most reasonable pathway of events after sustained changes in thyroid 

hormone and TSH secretion would be thyroid hypertrophy or hyperplasia, possibly followed by 

hypothyroidism in people unable to compensate with an increase in thyroid iodide uptake. At 

that point, the pathway would diverge to two potential outcomes-metabolic sequelae (such as 

decreased metabolic rate and slowing of the function of many organ systems) at any age and 

abnormal growth and development offetuses and children." The NAS clarified that "inhibition 

of thyroid iodide uptake is the only effect that has been consistently documented in humans 

exposed to perchlorate." Since the publication of the NAS report in 2005, a significant body of 

literature has been published that examines the association between perchlorate exposure and 

changes in thyroid hormones and TSH levels. 

Studies demonstrating an association between perchlorate exposure in the enviromnent and 

thyroid hormone or TSH perturbations include: 

• Blount et al., 2006. Urinary perchlorate and thyroid hormone levels in adolescent and 

adult men and women living in the United States. Environ. Health Perspect. 114:1865· 

1871; 

• Steimnaus et al., 2010. Perchlorate in drinking water during pregnancy and neonatal 

thyroid hormone levels in California. J. Occup. Environ. Med. 52:1217-1524; and 

• Cao et al., 2010. Goitrogenic anions, Thyroid-stimulating hormone, and thyroid hormone 

in infants. Environ. Health Perspect. 118:1332-1337. 

Inbofe 5. EPA's Office of Inspector General has stated that "the nature of the public health issue 

[inhibition of iodide uptake] not only meets the requirements to be addressed by a cumulative 

risk assessment approach, but ... requires the use of a cumulative risk assessment approach to 
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accurately characterize the nature of the problem and to identifY an effective and cost-efficient 

solution to the problem." 

Will EPA conduct a cumulative risk assessment on perchlorate and the other chemicals that have 

the same mechanism of action? 

Answer: Currently the EPA has no plans to conduct" a cumulative risk assessment for 

perchlorate and other chemicals that may have the same mechanism of action. As indicated by 

the Inspector General's report on perchlorate, there are significant gaps in the research and data 

that need to be filled in order to inform a cumulative risk assessment. However, the EPA will 

qualitatively consider the Inspector General's conclusions and recommendations as it develops 

the proposed perchlorate standard. 

The EPA is required to review and revise, as appropriate, its drinking water standards at least 

every six years. Any revision must maintain or improve public health protection. When there 

are sufficient scientific data to assess the cumulative risks of perchlorate and other contaminants, 

the EPA will review this information to evaluate whether any revisions of the National Primary 

Drinking Water Regulations are appropriate. 

lnbofe 6. EPA's Office of Inspector General has concluded that of the three common goitrogens 

(thiocyanate, nitrate and perchlorate), perchlorate accounts for less that l percent of iodide 

uptake inhibition, while the other two chemicals account for more than 99 percent of iodide 

uptake inhibition. What is the meaningful opportunity for public health risk reduction through 

the regulation of perchlorate, given that more than 99% of the effect is related to other 

chemicals? 

Answer: The EPA does not believe there are sufficient scientific data currently available to 

assess and characterize the combined and relative risk of these contaminants. The estimation 

technique the Office of the Inspector General used to estimate the amount of iodide uptake 

inhibition that would occur from perchlorate relative to thiocyanate, nitrate, and other substances 

is highly uncertain. The EPA concluded that perchlorate meets the SDWA's three criteria for 

regulating a contaminant: 

• the potential adverse effects of the contaminant on the health of humans; 

• the frequency and level of contaminant occurrence in public drinking water systems; and 

• whether regulation of the contaminant presents a meaningful opportunity for reducing 

public health risks. 

The science that led to this decision has been peer reviewed by independent scientists and public 

health experts, including the National Academy of Sciences. However, as noted above, the EPA 

will qualitatively consider the Inspector General's conclusions and recommendations as it 

develops the proposed perchlorate standard. 

As described in the response to Inhofe 5 above, the EPA is required to review and revise, as 

appropriate, its drinking water standards at least every six years. Any revision must maintain or 

improve public health protection. When there are sufficient scientific data to assess the 
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cumulative risks of perchlorate and other contaminants, the EPA will review this information to 

evaluate whether any revisions of the National Primary Drinking Water Regulations are 

appropriate. 

Inbofe 7(a). Is more recent water quality data available for perchlorate occurrence than the 

2001-2005 data that EPA stated it has been using? Please provide any new data. 

Answer: The EPA is continuing to collect and evaluate other drinking water occurrence data for 

perchlorate in addition to the data collected under the Unregulated Contaminant Monitoring Rule 

(UCMR) from 2001 to 2005 (see 

http://water.epa.gov/lawsregs/rulesregs/sdwalucrnr/data.cfrn#ucrnr2005). These additional data 

sources include: 

• The California Department of Public Health (see 

http://www.cdph.ca.gov/certlic/drinkingwater/Pages/Perchlorate.aspx; 

• The Massachusetts Department ofEnviromnental Protection (see 

http://www.mass.gov/dep/cleanup/sites/percsour,pdf); 

• The New Jersey Drinking Water Quality Institute (see 

http://www.state.nj.us/dep/watersupply/perchlorate mel 10 7 05.pdf; and 

• The American Water Works Association (see 

http:/ /www.awwa.orglfiles/ Advocacy/PerchlorateOccurrenceReportFinalb02092005.pdQ. 

Inbofe 7(b ). Additionally, in 2001-2005 the Colorado River contained up to about I 0 ppb of 

perchlorate. According to a 2008 report by the Nevada Division of Environmental Protection, 

perchlorate levels in the Colorado River have decreased to less than 2 ppb. How is EPA taking 

this into account? 

Answer: The EPA is evaluating the Nevada Division ofEnviromnental Protection's perchlorate 

monitoring in the Lower Colorado River. We believe this information and other ambient water 

monitoring data can be informative with regard to the trends of perchlorate contamination in 

some source waters. However, we believe that samples collected from treated or finished water 

are more representative of the level of perchlorate in tap water because many systems combine 

water from multiple sources before treating and distributing that water to consumers. The EPA 

will utilize the best available, peer reviewed science and data collected in accordance with 

accepted methods when developing estimates of the number of systems that have perchlorate 

contamination and the populations exposed at various health reference levels. 

Inhofe 8. The National Academy of Sciences, the Agency for Toxic Substances and Disease 

Registry, and EPA's Office oflnspector General have all concluded that a drinking water 

equivalent level of24.5 ppb would be conservative and health protective. What peer-reviewed 

studies does EPA have that purport to demonstrate that this level is not sufficiently protective of 

human health? 

Please provide a list of all peer-reviewed studies that EPA has that purport to demonstrate a 

causal link between exposure to perchlorate at drinking water levels of24.5 ppb and below and 

adverse health effects. 
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Answer: In their 2005 Report, the National Academy of Sciences (NAS) concluded "that an 

RID [Reference Dose] of 0.0007 mglkg per day should protect the health of even the most 

sensitive populations." The NAS report does not convert the perchlorate RID into a drinking 

water concentration. There are several factors that must be considered in identifYing a health 

protective drinking water level based on the RID. These include exposure through other sources 

(e.g., food) and selection of appropriate parameters to convert a dose per unit of body weight (the 

RID) into a drinking water concentration. The NAS did not address these issues. 

The EPA has not determined the level of perchlorate in drinking water at which no known or 

anticipated adverse effects on the health of persons occur and which allows an adequate margin 

of safety. In determining such a level, referred to in the Safe Drinking Water Act (SDW A) as a 

maximum contaminant level goal (MCLG), the agency will consider a variety of studies and 

factors. It is important to note that under SDWA, the agency must consider both effects to the 

general population and effects to sensitive subpopulations in identifYing the health protective 

level. Once the MCLG is identified, the EPA must identifY an enforceable regulatory standard, 

referred to as the maximum contaminant level (MCL). 

Under SDWA, there are several steps for determining the MCL. First, EPA identifies a 

candidate MCL that is as close as "feasible" to the MCLG. The EPA evaluates feasibility by 

examining the available technologies that have been evaluated in the field. The EPA takes cost 

into consideration as part of the feasibility determination. The EPA also conducts a health risk 

reduction cost analysis which includes an evaluation of costs and benefits (both quantified and 

unquantified). 

The EPA then makes a determination as to whether or not the benefits of the MCL justifY or do 

not justifY the costs based on this analysis. If EPA finds that benefits at the feasible level do not 

justifY costs, EPA may set a less stringent MCL that maximizes health benefits at a cost that is 

justified. The EPA will lay out the full rationale for its proposed MCLG and MCL when it 

publishes the proposed rulemaking for public comment. It will also identifY and include in the 

rule making docket all studies and research relied on for those determinations. 

Inhofe 9. How does EPA determine conflicts of interest when putting together peer review 

panels for IRIS Risk assessments? 

Answer: All draft human health assessments developed under the Integrated Risk Information 

System (IRIS) Program are subjected to rigorous, independent external peer review, most 

frequently by panels of scientific experts assembled by an external peer review service provider 

via a contractual agreement with the EPA. Panels assembled by the peer review service provider 

follow the procedures described on the IRIS website 

(http://www.epa.gov/iris/pdfs!Policy IRIS Peer Reviews. pdf), Procedures include requiring 

panel members to submit periodic notifications certifYing that no conflicts or potential conflicts 

of interest exist while reviews are ongoing. 

Sometimes a panel of experts is convened by either the EPA's Science Advisory Board (SAB) or 

the National Research Council (NRC). The established process for independent peer review by 
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both the SAB and NRC and their respective procedures for detennining conflicts of interest and 

any appearance ofbias or lack of impartiality, are available on the SAB's website 

(http://yosemite.epa.gov/sab/sabproduct.nsf/WebSABSO/NominationExperts?OpenDocument) 

and on the National Academies' website (http://www.nationalacademies.org/coi/index.html). 

lnhofe 10. Please provide the peer review comments in their entirety from the peer review panel 

for the Draft IRIS Risk Assessment for hexavalent chromium submitted to EPA. 

Answer: The peer review report for the draft Integrated Risk Infonnation System (IRIS) health 

assessment for hexavalent chromium is available on the EPA's website at: 

http:/ /cfpub.epa.gov /ncealiris drafts!recordisplay.cfm ?deid=2214 3 3 

Inhofe 11. In a recent House Committee on Science, Space, and Technology hearing, there was 

discussion of EPA's use of overly conservative toxicity values along with overly conservative 

exposure scenarios. Does EPA feel these conservative assumptions result in characterizations of 

risk that accurately allows decision makers to address real world situations? 

Answer: The EPA develops toxicity values and exposure scenarios specifically so that decision 

makers can address real world situations. They are not intended to be overly conservative; 

rather, they are based on the best available infonnation. Where reliable infonnation on specific 

toxicity and exposure parameters is not available, the EPA makes its best infonned estimates of 

values or ranges for these parameters. 

Sometimes, the choice of parameters in the absence of adequate data involves the use of default 

assumptions. The use of default assumptions was discussed and endorsed by the National 

Academy of Sciences (NAS) in "Science and Judgment in Risk Assessment" (1994), and the 

assumptions that the EPA makes follow the principles discussed by the NAS. Default 

assumptions do sometimes entail a degree of public health conservatism. The EPA is transparent 

about its default assumptions with decision makers and the public. The EPA believes that good 

risk management decisions should be based on a risk characterization which "integrates 

infonnation from ... the risk assessment and synthesizes an overall conclusion about risk that is 

complete, infonnative, and useful for decision makers." 

http://www.epa.gov/spc/pdfs/rchandbk.pdf). Further, the EPA subjects all of its risk assessments 

to external peer review and specifically asks peer reviewers to comment on the appropriateness 

of the major assumptions. This provides a check to ensure that decision makers have the best 

scientific infonnation available. 

Inhofe 12. Does EPA feel it is appropriate to set, based on application of EPA exposure 

assumptions, exposure levels below background concentration levels of ambient air, public 

drinking water sources, soil, or sediment? If so, how does EPA recommend State and local 

governments meet these levels? 

Answer: The EPA recognizes that contaminants which may cause adverse health effects even at 

background levels present special regulatory challenges. There is no one size fits all answer to 

these situations. As EPA develops standards and regulations, it evaluates each contaminant 
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carefully, based on statutory requirements to promulgate regulatory requirements that best meet 

the goals of the authorizing statute to protect public health and the environment. 

Inhofe 13. In May EPA announced reforms to the IRIS process as well as subsequent plans for 

further improvement as a direct response to the National Academy of Sciences Review of the 

Agency's Draft IRIS Assessment of Formaldehyde. 

Does EPA believe it is addressing all of the NAS recommendations? Does EPA intend to 

document/demonstrate what specific activities have been put into place in order to implement the 

recommendations? 

Answer: The EPA agrees with and is implementing all of the recommendations of the National 

Research Council (NRC), offered in their review of EPA's draft assessment of formaldehyde, 

that are related to the development of Integrated Risk Information System (IRIS) assessments. 

The EPA is addressing the NRC recommendations in a manner that is consistent with the NRC's 

"Roadmap for Revision" in Chapter 7 of the formaldehyde review report. Specifically, the NRC 

stated that "the committee recognizes that the changes suggested would involve a multiyear 

process and extensive effort by the staff of the National Center for Environmental Assessment 

and input and review by the EPA Science Advisory Board and others." 

The IRIS Progress Report (http://www.epa.gov/iris/pdts/irisprogressreport2011.pd!) outlines the 

EPA's plan to implement the recommendations and will be followed up with updates that 

document specific activities as they occur. 

Most recently, the EPA requested public nominations of scientific experts for appointment to the 

EPA's Science Advisory Board Chemical Assessment Advisory Committee. The EPA will 

request advice from the committee as the agency implements the NRC recommendations. 
70 

Senator BOXER. I want to thank you very much, and say I think 

your response to this report is refreshing, that you are looking at 

it and embrace the notion of transparency, which is critical, science 

is critical. 

In September 2010, EPA released a draft scientific assessment 

that found that chromium–6 in drinking water is ‘‘likely to be a 

carcinogenic to humans.’’ The agency said it expects to finish this 

assessment in 2011. Can you give us the date when you will complete this assessment? 

Mr. PERCIASEPE. That assessment we did last year was submitted for peer review. We just received a response from the peer 

reviewers and we are reviewing that. I would still think, depending 

on what depth we may need to go into, and I have to hedge the 

exact date here a little bit, based on the fact that we have not yet 

reviewed all the advice we got from the peer review. I think we are 

still on track to finish before the end of the year. 

Senator BOXER. Good. I am going to assume that unless I hear 

otherwise. We have had our problems with chromium–6. I am really glad that you moved forward on it. 

In February 2011, Administrator Jackson reversed a Bush administration decision not to regulate perchlorate with an announcement that EPA would move forward to develop a drinking water 

standard. I ask unanimous consent to place in the record comments 

from California urging you on in that regard. 

[The referenced information follows.] 

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American Water Works 

Association 

The Authoritative Resource on Sate Water SM 

July 26, 2011 

The Honorable Barbara Boxer, Chair 

The Honorable James M. lnhofe, Ranking Member 

Senate Committee on Environment and Public Works 

Washington, DC 20515 

Dear Senators, 

The American Water Works Association (AWWA) appreciates the committee's holding the 

hearing record open for two weeks following the July 12 hearing titled, "Oversight Hearing on 

the Environmental Protection Agency's Implementation of the Safe Drinking Water Act's 

Unregulated Drinking Water Contaminants Program." We request that this letter be made part of 

the hearing record. 

The American Water Works Association (AWWA) is an international, nonprofit, scientific and 

educational society dedicated to the improvement of drinking water quality and supply. 

Founded in 1881, the Association is the largest organization of water supply professionals in the 

world. Our 55,000-plus members represent the full spectrum of the drinking water community: 

treatment plant operators and managers, environmental advocates, engineers, scientists, 

academicians, and others who hold a genuine interest in water supply and public health. Our 

membership includes more than 4,000 water systems that supply roughly 80 percent of the 

nation's drinking water. AWWA and its member utilities are dedicated to safe water. 

With respect to the July 12 hearing, we would like to make the following key points: 

1. We believe the SDWA legislation of 1996 to be fundamentally sound. 

AWWA believes that Congress created a sound, science-based methodology for determining 

what new substances to regulate in the 1996 Amendments to the Safe Drinking Water Act 

(SDWA). In February 2011, we testified before this committee: 

"The SDWA was amended in 1996 to provide a scientifically sound and transparent method 

for selecting the appropriate substances for regulation and for selecting the appropriate 

maximum contaminant level for contaminants. We should allow the best available science, 

not the political process, to be the ultimate driver in regulatory decisions." 

2. With respect to the Unregulated Drinking Water Contaminants program provisions 

within the 1996 SDWA, there are success stories with respect to prior regulatory 

determinations about certain compounds. 

In many cases, substances of potential public health concern simply do not occur in drinking 

water sources and therefore, should be not be regulated. The following five substances in the 
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second regulatory determination in 2008 were found to have no occurrence in 3,479 systems 

across the U.S. under the first Unregulated Contaminant Monitoring Rule (UCMR1): 

1, 1-dichloro-2,2-bis(p-chlorophenyl)ethylene; 

1 ,3-dichloropropene; 

2,6-dinitrotoluene; 

s-ethyl-dipropylthiocarbamate; and 

Terbacil. 

One substance (2,4-dinitrotoluene) had a single detection. Regulating these non-occurring 

substances would have expended scarce resources for EPA and water utilities without providing 

any health benefits whatsoever. Deciding to not regulate these six substances in the second 

regulatory determination was appropriate, and shows that the CCL process is working. 

Additionally, EPA appropriately determined that regulating some other substances simply did 

not make sense as regulation would not provide a meaningful opportunity for health risk 

reduction. For example, the relative amount of sodium in drinking water is miniscule compared 

to food and sodium regulation in drinking water would not make a meaningful contribution to 

protecting public health. The same can be said for numerous other substances. 

3. While the Safe Drinking Water Act is fundamentally sound, we agree there is a 

need to improve upon its implementation with respect to its Unregulated Drinking 

Water Contaminant provisions. 

We acknowledge that the U.S. Environmental Protection Agency faces challenges in 

determining what substances to regulate in drinking water, particularly given the lack of health 

effects and occurrence data for many of the substances under consideration. But some of the 

challenges stem not so much from a lack of data as from problems with implementation. The 

GAO report "Safe Drinking Water Act: Improvements in Implementation Are Needed to Better 

Assure the Public of Safe Drinking Water" points to some systemic problems in the agency's 

implementation with which we largely concur. 

First, we agree with the GAO that EPA needs to systematically implement the SDWA to better 

address substances that present the greatest potential for threats to public health. The agency 

and the public would benefit from criteria and processes for prioritizing such substances in its 

Candidate Contaminant List (CCL) and regulatory determination processes. 

Second, as the comments above indicate, we concur with the GAO that greater transparency is 

needed in the regulatory determination process. 

Third, concerning the lack of data for potential substances of concern in drinking water, AWWA 

has for years urged EPA to set a research agenda more closely aligned with and designed to 

support its regulatory agenda. Congress has a responsibility here as well, not only in oversight, 

but in ensuring that the agency has the resources needed to utilize or develop occurrence and 

health effects research. 
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4. We remind Congress to consider any improvements in the Unregulated Drinking 

Water Program in the context of a comprehensive approach to protecting public 

health. 

While focusing on identifying and elaborating new standards for substances of emerging 

concern from the unregulated contaminants program, we remind Congress that treating water to 

the highest standards at a water treatment facility does not guarantee public health protection. It 

is vital that utilities also be able to convey drinking water to customers through a properly 

constructed and operated water infrastructure system. We appreciate that this committee has 

recognized the importance of federal support for investment in water infrastructure, including the 

State Revolving Loan Fund program. Another important issue is capacity development, as too 

many water providers struggle to meet increasingly complex regulatory requirements without 

adequate financial, technical, and managerial capacity. 

Given all the above, we believe it is imperative that any improvements considered in the 

Unregulated Drinking Water program ensure that public resources be committed to those 

elements of the water supply system which have the most meaningful effect upon public health. 

There is a significant relationship between the regulatory agenda, infrastructure, and 

management capacity in that every dollar spent on treating for substances that don't represent a 

genuine public health threat is a dollar that could instead be used to rehabilitate or replace aging 

infrastructure and to build managerial capacity. 

Once again, the Association appreciates the opportunity to comment on these critical matters 

and offers to work with the Committee further in advancing the treatment and provision of safe 

drinking water to the American public. 

Sincerely, 

Tom Curtis 

Deputy Executive Director 

Headquarters Office: 

6666 W. Quincy Avenue, Denver CO 80235 

T 303.794.771111 F 303.347.0804 

www.awwa.org 

Government Affairs Office: 

1300 Eye Street NW, Suite 701W 

Washington, DC 20005 

T 202.628.8303 II F 202. 628.2846 
74 

Senator BOXER. Perchlorate is a toxic contaminant that can harm 

children’s development. I strongly support Administrator Jackson’s 

decision. Could you tell me if EPA is moving forward with the process of developing a drinking water standard for perchlorate and 

can you assure me the agency will continue to use the best available science in crafting this standard? 

Mr. PERCIASEPE. Yes, I can assure you on both counts, Madam 

Chairman. We are moving ahead on the process that is initiated by 

making the regulatory determination. And of course, we are going 

to be looking at all the best available science, as we had it, to make 

the right determination and as we will continue to improve the 

science that is available as we go through the regulatory process. 

Senator BOXER. OK. Let me just say, given, I think, the criticisms that have come forward from the GAO, and the reality that 

so little has been done, the fact that you are moving on two contaminants out of so many, at least it is a start. So I am going to 

be watching very carefully to make sure that we don’t get caught 

up in politics or anything else. This is about safety for our people. 

And we need to know. So I am going to be following those very, 

very closely. 

Deputy Administrator, the GAO found that ‘‘EPA does not have 

criteria for identifying contaminants of greatest public health concern.’’ They recommended that EPA develop ‘‘criteria to identify 

contaminants that pose the greatest health risks.’’ Will EPA develop such criteria, in order to determine which drinking water 

contaminants should be regulated, and ensure that the criteria are 

used in a transparent and accountable process? 

Mr. PERCIASEPE. What I mentioned in my opening statement is 

that EPA, it is sort of, things are going on in parallel here. In addition to the review that GAO has done, which has provided us 

meaningful input and good recommendations on transparency, we 

have also been in the process of trying to make those improvements at the same time. It is sort of, if you play ice hockey, it is 

sort of like changing the line on the fly, as they call it. 

Senator BOXER. Oh, wait a minute, that is my husband’s domain. 

He loves ice hockey. 

Mr. PERCIASEPE. So you are familiar with that concept. 

Senator BOXER. I can barely follow the puck, but I think it is exciting. But could you say what you said again, so I can ask him 

when I get home? Say it again, I am serious. 

Mr. PERCIASEPE. Changing the line on the fly. 

Senator BOXER. Changing the line on the fly. Thank you. I will 

check it out with him when I get home. 

[Laughter.] 

Mr. PERCIASEPE. I apologize for bringing a sports analogy in 

here. 

But the point being that while GAO is looking at what we have 

done in the past, we are preparing the next round of the candidate 

contaminant list. And in doing that process, we have tremendously 

expanded this number of sources of science and data that we have 

looked at. And as I mentioned, we had almost 7,500 different contaminants we look at. I am doing this, because it is sort of like the 

top of a funnel. And as you go down the funnel to what you might 

recommend for a regulatory determination at the bottom, we are 

75 

building a very specific process that starts with the 7,000, looks at 

what we know about the severity of their health impacts, impacts 

on children or sensitive populations, occurrence information. 

And we are moving down that list. We have publicly produced a 

document. We have had public hearings on it, about how we went 

from the 7,500 down to a smaller number. And now what we are 

focusing on 116 of them, each step of the way we have laid out a 

deliberate process. 

Senator BOXER. OK, just to cut through, and I am going to give 

myself one more minute and give it to everybody else, the answer 

is yes? 

Mr. PERCIASEPE. Yes. 

Senator BOXER. The EPA is going to develop these criteria? 

Mr. PERCIASEPE. Yes. 

Senator BOXER. And you are going to make it public, and it will 

be transparent. 

Mr. PERCIASEPE. And we are building it off of this. 

Senator BOXER. OK. So my last question, and then I have a 

bunch I am going to ask you for in writing that are very important 

to me, the GAO found that 17 contaminants that have been on all 

three of EPA’s lists of priority drinking water contaminants since 

1998, and here we are, a long way from that, including RDX, or 

royal demolition explosive, which EPA has classified as a possible 

human carcinogen. The GAO raised concern with the potential lack 

of information that EPA has on some of these contaminants. We 

are talking about now a narrow list of 17 that have appeared on 

all these lists since 1998. 

Will you agree to have EPA brief my staff on the status of the 

agency’s information collection analysis activities for each of those 

17 contaminants? And also at that time describe when EPA expects 

to consider these contaminants for a regulatory determination? 

Mr. PERCIASEPE. Yes, of course. And I could add, I know that we 

are short on time, but a number of, a significant number of those 

are now on the final list that we are looking at in the current process. And a number of the other ones have been put on the unregulated contaminant monitoring list that David brought up in his testimony that we have expanded that list and will now get more data 

on them. So we are moving on pretty much all of those 17. 

Senator BOXER. Thank you, and I will get you all these other 

questions I have, and turn to Senator Inhofe for 6 minutes. 

Senator INHOFE. Thank you, Madam Chairman. And I would 

hope in the future, Mr. Administrator, if you are going to use a 

sports analogy, for my benefit, use either football or baseball, so we 

would understand. 

[Laughter.] 

Senator BOXER. Or soccer is good. 

Mr. PERCIASEPE. Well, we have a spiritual soccer event earlier in 

the week. 

Senator INHOFE. It could have been that the Chairman asked 

this question during her first minute, because I was distracted during that time. But I would like to know if there is anything further 

you could say on how the EPA is going to address the concerns that 

were raised by the NAS formaldehyde report, specifically the systemic concerns raised by the IRIS risk assessment process. 

76 

Mr. PERCIASEPE. In 2009, Administrator Jackson set in motion a 

number of improvements to the IRIS program to begin with. As you 

pointed out, maybe 10 years, where things had been allowed to perhaps lag for a while. So one of the things we did is improve some 

of the elements of the process. We have increased resources for it. 

And today our Assistant Administrator for the Office of Research 

and Development, Paul Anastas, is going to be announcing a next 

round of improvements to the IRIS process. And many of those improvements are on that same continuum we have been on now for 

a couple of years, and I think are very responsive to the advice we 

have gotten and that we appreciate also from the National Academy of Sciences. 

So I am optimistic, Senator, that we are moving on that proper 

continuum to make sure that very critical, and I think you said it 

as well as anybody could in your opening statement, how important 

that IRIS process is to our program. 

Senator INHOFE. OK, you are on that. As I said also in my opening statement, and you have heard me talk about this before, the 

concern that I have from my background as mayor of a major city 

in terms of the unfunded mandates. And again, I know other 

States have problems, too. I think Oklahoma certainly has great 

problems in our small communities. 

Now, it is my understanding that the EPA section 319 program 

does help States in their implementation. Is this correct, in some 

of these programs? 

Mr. PERCIASEPE. I am not completely familiar with that question. 

Senator INHOFE. All right. Well, it is the EPA section 319 program. It does provide funds for some States and local entities to 

comply with some of these mandates. Is that correct? 

Mr. PERCIASEPE. I think that—I am not familiar with 319. I 

probably know all the programs, but I don’t know them by their 

numbers. 

Senator INHOFE. OK, well, I was given the information that some $27 million was transferred from that program to the program that 

is sent to China and other countries. 

Mr. PERCIASEPE. I am in the wrong law. It is in the Clean Water 

Act. Section 319 is in the Clean Water Act. I am familiar with that. 

That allows non-point source programs and State efforts on nonpoint source programs that can be used to do both source water 

protection and perhaps even wellhead protection in the proper context. 

Senator INHOFE. Well, I think it is significant to bring up this 

point. Because you will remember, although you weren’t in your 

current position, when Republicans were the majority and I chaired 

this Committee, I was concerned that people didn’t have access to 

where a lot of the money was going from the EPA, from the Environmental Protection Agency. And so we set up a data base. And 

this has been pretty successful. And just the other day, Chairman 

Upton, of a comparable committee over there in the House, was 

talking about the $718,000 that went to China’s EPA to assist with 

control of air emissions. And it goes on to talk about some of the 

other countries. 

I just found this to be kind of mind-boggling that would take 

place. Is that incorrect? Because I know this would be the AssistVerDate Aug 31 2005 11:45 Apr 10, 2017 Jkt 000000 PO 00000 Frm 00082 Fmt 6633 Sfmt 6633 S:\_EPW\DOCS\23819.TXT VERN
77 

ant Administrator for International Affairs. But that person does 

answer to you. 

Mr. PERCIASEPE. I will have to look into that, Senator. I don’t 

have any direct information at this particular moment on financial—— 

Senator INHOFE. OK, rather than just take it for the record, if 

you don’t mind calling me back to see if I’m wrong in this. Because 

if I’m wrong and if Congressman Upton is wrong, we are doing a 

disservice by talking about this. 

Mr. PERCIASEPE. I will make sure we get back to your office 

today. 

Senator INHOFE. That is all I could ask for. 

And I would say to you, Mr. Trimble, in your analysis, does EPA 

have all the statutory authority that they need to regulate contaminants of concern? 

Mr. TRIMBLE. Sir, in our report and our recommendations, the 

deficiencies or the problems and the systemic problems we found 

were of a nature that we felt could be addressed strictly within the 

current authority. That is why our recommendations are to EPA, 

if we don’t have a consideration for changing the statute, we believe that the EPA can take action quickly under existing authorities to define its policies and establish guidance, to interpret the 

statutes and implement them in such a way that it would be clear, 

consistent and provide a better product at the end of the day. 

Senator INHOFE. All right. Thank you very much. 

Thank you, Madam Chairman. 

Senator BOXER. Thank you very much. 

Senator Lautenberg. 

Senator LAUTENBERG. Thanks, Madam Chairman. 

Mr. Perciasepe, the New Jersey State legislature recently passed 

a ban on hydraulic fracking in my home State. But Pennsylvania 

and New York are moving ahead with fracking activities that could 

affect New Jersey’s water supply. 

Now, there is currently a loophole we are all familiar with in 

Federal law that exempts fracking operations from much of the 

Safe Drinking Water Act requirements. How does this loophole affect EPA’s ability to prevent fracking contamination in one State 

from affecting the health of people in another, not unlike the winds 

that blow across our State from the west? 

Mr. PERCIASEPE. Let me just say that on the movement of water 

on the surface of the, when the water, when the fracking fluids are 

used or whether there is produced water out of a well, when the 

water comes to the surface, those are under the jurisdiction, if they 

are discharged to waters of the United States, those are under the 

jurisdiction of the Clean Water Act. And there are requirements for 

how it would be pre-treated, or there would be State requirements 

on how it is pre-treated if it goes into a sewage treatment plant, 

or if it is reinjected into the ground, that would be covered by the 

underground injection and control program. 

So on the surface, I think, Senator, the Clean Water Act would 

apply. The exemption is the regulation of the injection of the 

fracking fluids into the ground to conduct that operation. Those are 

not regulated, or that action is not regulated under the underVerDate Aug 31 2005 11:45 Apr 10, 2017 Jkt 000000 PO 00000 Frm 00083 Fmt 6633 Sfmt 6633 S:\_EPW\DOCS\23819.TXT VERN
78 

ground injection and control program, which is in the Safe Drinking Water Act. 

Senator LAUTENBERG. Well, a few months ago, I asked you about 

the air quality impacts of natural gas drilling. And in your testimony you said that natural gas drilling would lead to ‘‘an increase 

in emissions of volatile organic compounds and nitrogen oxide.’’ But 

there was no offer of any concrete steps that EPA is taking to reduce these emissions. 

Is EPA taking any action now to address emissions from natural 

gas drilling? 

Mr. PERCIASEPE. I will mention two briefly, Senator. First, just 

in the last several weeks, EPA, the U.S. Department of Agriculture 

and the Department of Interior signed an MOU on how we would 

conduct air quality analyses on leasing programs, either in the national forests or on the Bureau of Land Management’s land, which 

again, mostly is going to be out west. But we have now a multiagency agreement on how we will analyze the air impacts and 

what requirements might be placed on the leases that are put forward. 

We just recently conducted that analysis on a lease that the Department of Interior, through the Bureau of Land Management, 

made in the Grand National Buttes in Utah. And those requirements, and the company there was Anadarko, those requirements 

are very progressive and forward-leaning. And the company is moving forward in that. 

Senator LAUTENBERG. When will we get something specific there 

to move on this? The studies are being done. 

Mr. PERCIASEPE. Right. So those requirements would be placed 

on those leases, and they would have to meet those standards. The 

second thing, though, that I want to mention, is EPA is also working on what under the Clean Air Act is called new source performance standards. So as a new well is produced or developed, it would 

be required to reduce the amount of volatile organic compounds 

that would be emitted while the well development is being conducted. 

So those proposals will be out later this year. And they will be 

in many ways similar to what we are requiring companies to do 

when they lease on Federal land. 

Senator LAUTENBERG. The concerns are real. But the pace seems 

fairly slow in doing something about it. 

Mr. PERCIASEPE. I appreciate that very much. 

Senator LAUTENBERG. In addition to individual pollutants in 

drinking water, people are often exposed to multiple contaminants. 

The interaction with each other can cause some health effects. Mr. 

Trimble, how might EPA consider the health effects of the combination of chemicals when they set drinking water contaminant 

limits? 

Mr. TRIMBLE. I think that is an important issue. I think it goes 

to the heart of our recommendations, which is that the EPA needs 

to have guidance and policy and criteria to explain how such issues 

are considered in their decisionmaking. For example, when they 

make a decision, at the end of the day they need to explain was 

it considered, how was it considered. If they have a policy and it 

is an exception to that policy, they need to transparently and clearVerDate Aug 31 2005 11:45 Apr 10, 2017 Jkt 000000 PO 00000 Frm 00084 Fmt 6633 Sfmt 6633 S:\_EPW\DOCS\23819.TXT VERN
79 

ly explain all the factors that led them to the outcome that they 

arrived at. 

So I think we don’t, we are not a science agency, so we are not 

making recommendations on how they should or shouldn’t do these 

things. But from our standpoint, you need to establish the road 

map for the staff to follow in getting there. And then that road map 

not only guides your operations throughout your execution of this 

program, but at the end of the day, when you explain your decisions, it allows you to clearly articulate the basis for your decisions 

and credibly defend them. 

Senator LAUTENBERG. Chairman, there are so many questions 

that arise from this review, so the record is, I understand, will be 

kept open. 

Senator BOXER. Absolutely, the record is open. And Senator 

Inhofe wanted to make a request. 

Senator INHOFE. Yes, I want to make a unanimous consent request, I didn’t know that hydraulic fracturing was going to be coming up. I would like to make an observation that the first hydraulic 

frack job was done 60 years ago in my State of Oklahoma and 

there has never been a documented case of ground contamination. 

I have a statement from some, about 15 other States, all the other 

States where this is a concern. I would like to have this put into 

the record, that states the same thing about their States as I stated about Oklahoma. 

Senator BOXER. Absolutely. That will be put in the record. 

[The referenced information follows.] 

80 


23819.332

STATEMENTS ON HYDRAULIC FRACTURING SUBMITTED BY 

STATE REGULATORS 

ALABAMA: Nick Tew, Ph.D., P.G. Alabama State Geologist & Oil and Gas Supervisor 

President, Association of American State Geologists 

"There have been no documented cases of drinking water contamination that have resulted from 

hydraulic fracturing operations to stimulate oil and gas wells in the State of Alabama." 

ALASKA: Cathy Foerster Commissioner Alaska Oil and Gas Conservation Commission 

"There have been no verified cases of harm to ground water in the State of Alaska as a result of 

hydraulic fracturing." 

COLORADO: David Neslin Director Colorado Oil and Gas Conservation Commission 

"To the knowledge of the Colorado Oil and Gas Conservation Commission staff, there has been 

no verified instance of harm to groundwater caused by hydraulic fracturing in Colorado." 

INDIANA: Herschel McDivitt Director Indiana Department of Natural Resources 

"There have been no instances where the Division of Oil and Gas has verified that harm to 

groundwater has ever been found to be the result of hydraulic fracturing in Indiana." 

KENTUCKY: Kim Collings, EEC Director- Kentucky Division of Oil and Gas 

"In Kentucky, there have been alleged contaminations from citizen complaints h1,1t nothing that 

can be substantiated, in every case the well had surface casing cemented to surface and 

production casing cemented." 

LOUISIANA: 

James Welsh Commissioner of Conservation Louisiana Department of Natural Resources 

"The Louisiana Office of Conservation is unaware of any instance of harm to groundwater in the 

State of Louisiana caused by the practice of hydraulic fracturing." 

MICHIGAN: Harold Fitch Director, Office of Geological Survey (OGS) Department of 

Environmental Quality 

"There is no indication that hydraulic fracturing has ever caused damage to ground water or, 

other resources in Michigan. In fact, the OGS has never received a complaint or allegation that 

hydraulic fracturing has impacted groundwater in any way." 

OKLAHOMA: 

Lori Wrotenbery Director, Oil and Gas Conservation Division Oklaho.ma Corporation 

Commission 

"You asked whether there has been a verified instance of harm to groundwater in our state from 

the practice of hydraulic fracturing. The answer is no." 

TENNESSEE: Paul Schmierbach Manager Tennessee Department of Environmental 

Conservation 

"We have had no reports of well damage due to fracking." 
81 


23819.333

TEXAS: Victor G. Carrillo Chairman Railroad Commission of Texas 

"The practice of reservoir stimulation by hydraulic fracturing has been used safely in Texas for 

over six decades in tens of thousands of wells across the state." 

SOUTH DAKOTA: Fred Steece Oil and Gas Supervisor Department of Environment and 

Natural Resource 

"In the 41 years that I have supervised oil and gas exploration, production and development in 

South Dakota, no documented case of water well or aquifer damage by the fracking of oil or gas 

wells, has been brought to my attention. Nor am I aware of any such cases before my time." 

WYOMING: Rick Marvel Engineering Manager Wyoming Oil and Gas Conservation 

Commission & Tom Doll Oil and Gas Commission Supervisor Wyoming Oil and Gas 

Conservation Commission 

"There have been no documented cases of groundwater contamination from fracture 

stimulations in Wyoming." 

PENNSYLVANIA: John Hanger Department of Environmental Protection Secretary "It's 

our experience in Pennsylvania that we have not had one case in which the fluids used to break 

off the gas from 5,000 to 8,000 feet underground have returned to contaminate ground water." 

NEW YORK: Taury Smith, State Geologist "Those are exaggerated problems (of 

contamination); each incident wasn't the result ofhydro-fracking. There were incidents of 

groundwater contamination near frack sites, but they were unrelated." 
82 

Senator BOXER. And now we turn to Senator Boozman. 

Senator BOOZMAN. Thank you, Madam Chair. 

I think one of the things that is so important as we deal with 

the agencies and things, especially in dealing with issues that really do affect our health, economic matters in the sense that, trying 

to conform to some of the things that we come up with literally will 

cost billions of dollars in some instances, that you have to have 

confidence in the system. Mr. Trimble, you talked about the agency 

performing tests where they were doing testing and maybe, I will 

put it in my terms and you can correct me, maybe they were testing for one part and then the test itself was only good to two parts. 

Sometimes only good up to 2,200 parts, and then making decisions 

based on that. 

Can you comment further on that? 

Mr. TRIMBLE. Sure. In the decisions that they made in, I believe 

it was 2003, 2008, to not regulate about 20 contaminants, I believe 

in 9 of those the testing data that the program had developed was 

not at a level sufficient to detect the contaminant at the point of 

concern. So exactly to your point, it wasn’t robust enough to test 

it at the level of concern. 

That raises lots of issues. But what we talk about in the report, 

when we reviewed the determination in getting to the issue of the 

credibility, is that the impact of that limitation is either completely 

not addressed in the documentation explaining the decision, the determination decision, or it is buried. There could be other reasons 

why, maybe it is best available, maybe you have other data. But 

the issue and the basis for reaching a decision regarding sort of a 

big piece of this as well. So you have the problem with the testing 

program, but then you also have just the lack of explanation of, 

well, OK, you had this problem, how did you get to this point. 

Senator BOOZMAN. You also have the problem, too, in the sense 

that you can have a false sense of security, can’t you, in the sense 

that a test was supposedly performed and everything was OK? 

Mr. TRIMBLE. Absolutely. In four of the decisions, they did not 

acknowledge that there was this limitation or the impact of that 

limitation on their conclusion that there was no health issue. 

Senator BOOZMAN. So is that currently going on? Has that gone 

on recently? 

Mr. TRIMBLE. Our review looked at the 20 decisions since the 

1996 amendments. And in nine of them, we identified this issue. 

So that, I believe the last group we looked at was 2008. 

Senator BOOZMAN. Can you comment on that? Is that currently 

still a problem, or in the recent years been a problem? 

Mr. PERCIASEPE. We feel, and I mentioned in my opening comments that we have modified our approach to looking at the unregulated contaminant monitoring that we are allowed to do. We 

are making sure that the testing that is done there, and these are 

the suite of contaminants that we are concerned about but that are 

not currently regulated, and we are trying to determine if their occurrence, as David just mentioned, is prevalent enough that it 

would be of concern. But you have to measure for the right level. 

And we are confident that we have modified our processes to be 

able to do that. 

83 

Senator BOOZMAN. Mr. Trimble, you talked about the three criteria for acting and went on to say that some of the people in the 

EPA, when interviewed, said there are no bright lines, that we will 

know it when we see it. Was that from scientists, or were these political people? How does anybody that is familiar with the scientific 

process, how do you deal with statements like that? 

Mr. TRIMBLE. I would have to check who it was. But I think it 

was more the regulatory office. And the issue of the discussion was 

not seeing the need or not recognizing the need for guidance or a 

road map in terms of how you interpret this. It was sort of a we 

know it when we see it kind of decision. Again, the problem as you 

have pointed out is the credibility that leads to, where you can 

have, you may see it, I may see it, somebody else may see something different, you can have changes over time, changes between 

people. And that is the issue I was trying to get at. 

Senator BOOZMAN. Yes, sir? 

Mr. PERCIASEPE. And I also want to point out that we are equally 

concerned with this and are happy to get this advice. I think how 

to solve this problem is probably open to a little bit of interpretation. GAO has laid out, I think, a thoughtful way to do it. We are 

in the process of trying to do it differently this time with developing our current list of contaminants that we are concerned about. 

We have gone through a very deliberate process that dissects those 

three large categories that are in the Act, like is there an adverse 

health effect. Well, we are looking at potency, we are looking at 

sensitive populations, and we are screening down to that. We also 

have to look at the occurrence data. We are looking at both regional as well as national data. 

The last step, and as I testified, the last step in the judgment 

of the Administrator is there a meaningful opportunity for a health 

risk reduction, well, we are proposing now, and what I have asked 

the water program to look into is the staff working on those first 

two, developing a report and then having that report subject to a 

peer review which asks these questions. And then the Administrator would have in front of her both the staff report on the adverse health effects and the occurrence or substantial occurrence 

data across the Country or regionally, and that judgment would be 

able to be also enhanced, by that peer review of that work that was 

done. 

So in that final step which is in the judgment of the Administrator, they would have both of those pieces of information together. And I feel that if we get that peer review to ask some of 

these questions, because there are so many different kinds of contaminants, that they will be able to see if the staff properly did the 

integration. However, we are learning from doing it differently this 

time, and we definitely want to have the criteria, as the Chairwoman mentioned, be clearly laid out. 

Senator BOOZMAN. Thank you. 

Mr. TRIMBLE. I would just note, we have not obviously reviewed 

the latest thinking and proposals. I am encouraged by the direction. I note a couple of things. One, the triaging of the contaminants of concern into the candidate list down to now the current 

list has about 116 contaminants. That is, EPA has indicated that 

is a prioritization, and it is. It is a first step. 

84 

But 116 is not enough of a prioritization to guide your direction 

from there. In 2009, the Science Advisory Board also had a report 

encouraging EPA to prioritize. 

Senator BOOZMAN. Thank you, Madam Chair. 

Senator BOXER. Thank you very much. 

Senator Cardin? 

Senator CARDIN. Thank you, Madam Chair. 

Mr. Trimble, first, let me thank you for this work. I think it is 

extremely important. Mr. Perciasepe, I appreciate the attitude of 

EPA to make sure we get this right. 

It is very concerning when we know that we have had lists now 

of unregulated contaminants. And there has been inconsistency in 

the methods used to determine whether there should be regulation. 

Our bottom line is to protect the public health of the people of this 

Nation. They rely upon us to determine whether water is safe to 

drink. And we have a responsibility to make sure that in fact is 

being done. 

What was most disconcerting about the GAO report, you showed 

the listing of new contaminants for regulation, lacked a procedural 

safeguard, that there were different variations of assessment based 

on the chemical involved. So Mr. Perciasepe, let me just ask you 

first, to what extent do you think the variation of the assessment 

procedures from one chemical to another affects the reliability of 

the EPA determinations? 

Mr. PERCIASEPE. Well, Senator, there may be radionucleides, 

there could be viruses, there could be bacteria or other protozoans 

as well as chemicals. So all of them have different ways you have 

to analyze them. I think that there is a commonality of approach, 

though, when you start with a large list and you start looking at 

which one of those things on that large list has the most potential 

health effects, whether it be sensitive sub-populations or whether 

it be to the general population. And you keep narrowing that list 

down. David used triage, I would call it some deliberate screening. 

And I agree that you get that list down to 116, that is not 

enough. Because this is really the precursor to deciding what your 

priorities are to actually do a regulation on. So this is not itself a 

regulation, this is just deciding what to regulate. 

And I think it is important that we do the best job we can, publicly and with criteria and with disclosure on how we got that, including peer review. And then from that list, the Administrator has 

to decide. And we think the list will be smaller than 116. 

In this instance, we think it will be down to 30 or maybe less. 

And that is what we will ask the outside reviewers to say, do we 

have that list right, and did we ask these right questions. And then 

that is what would be in front of the Administrator, to make that 

determination. 

Senator CARDIN. Can we learn something from perchlorate, in 

that it was included in the first uncontaminated chemical monitoring rule, so at least many years ago, there was at least some 

concern about it? Perchlorate is a toxic component of rocket fuel 

that may disrupt the production of thyroid hormones which are 

particularly sensitive for fetuses in young children. I know that the 

Administration is moving forward on perchlorate. Can you at least 

give us the status of where we are on that, and whether there is 

85 

a lesson to be learned as to why we did not investigate this issue 

earlier? 

Mr. PERCIASEPE. Well, obviously it went through the process that 

I just described as it was designed in many respects in the detail 

that GAO looked at. But since that time, we have re-looked at the 

science there. We looked at not just modeling of these physiological 

effects, but also the actual exposures to children and pregnant 

women. That forms some of the key basis for the Administrator 

making that determination that it is required to go through the 

regulatory process. 

So we are starting that regulatory process now, once that determination is made. 

Senator CARDIN. What is the timeframe? 

Mr. PERCIASEPE. I have to say, with the proposal and then the 

final, it is probably going to be at least 2 years. 

Senator CARDIN. And one of the questions I would raise, first of 

all, I think that is too long. But I understand the procedures you 

have to go through. You used the funnel example as to how many 

contaminants are listed, and you need to make a priority judgment 

as to which are the greatest at risk and what can be regulated effectively for public health. 

Perchlorate to me was too high up in that funnel, it should have 

been at a lower part of that funnel at an earlier stage, which I 

think is what the GAO report really, this is an example of the inadequate testing that was done earlier dealing with perchlorate. So 

I hope we have learned that lesson to get it down to the level of 

regulation earlier. And I would hope that you would work with us 

to streamline the regulatory process, because if the risk factors are 

to a vulnerable group, this requires us to act in a more expedited 

way. 

Mr. PERCIASEPE. I agree, and I think, I mean, I have worked 

with some members here today on that Safe Drinking Water 

Amendment in 1996, when I was in the government back in the 

1990’s. We deliberately wrote into the law that the Administrator 

had to make a decision. They couldn’t just keep analyzing, there 

had to be a decision made. I think that what has happened is that 

the transparency of that decision and the criteria used to get to 

that point, and the peer review of the staff and science that was 

being used in that final step by the Administrator to decide yea or 

nay on a regulatory determination, it is that point that got, I think, 

bent out of shape in the last time the determination was made. 

And I believe that having a peer review at that particular moment 

will, you never solve every problem, but it will, I think provide 

more transparency to why and if the Administrator is making decisions based on the most current science. 

Senator CARDIN. Thank you very much. 

Senator BOXER. Thank you, Senator. 

Senator Udall, welcome. 

Senator UDALL. Thank you, Senator Boxer, and thank you for 

holding this important hearing. 

First, on behalf of the residents of northern New Mexico, I would 

like to say thank you to the EPA, represented here by Deputy Administrator Bob Perciasepe. The EPA provided really important 

emergency air monitoring resources, including their ASPECT plane 

86 

to monitor air quality during the largest forest fire in New Mexico’s 

history, which threatened Los Alamos National Laboratory and 

many other areas in New Mexico. Those air monitoring results, the 

independence of them, the State looked at things, EPA looked and 

the lab had their numbers. 

But these air monitoring results we have seen so far show no unusual pollution beyond what would be in a normal forest fire, and 

EPA’s independent presence on the ground and in the air was of 

great value, I think, in providing the public with credible information. So I thank you for that. 

On the topic of today’s hearing, several witnesses, including the 

GAO, observed that the EPA has not fully taken into account the 

impact of unregulated drinking water contaminants on children’s 

health, I know Senator Cardin mentioned this earlier, as required 

by the Safe Water Drinking Act and the agency’s policy on children’s health. And perchlorate, in particular, is one that concerns 

me in the decisionmaking on perchlorate. Perchlorate is an unregulated contaminant found in many drinking water systems in the 

west, which is home to many of our Nation’s major military facilities. New Mexico’s communities are proud to host our Nation’s 

armed forces, but all communities, I think, deserve clean drinking 

water. 

Just as with forest fires, we are counting on EPA to provide accurate, independent scientific information and take the appropriate 

action. 

So let me first start with Mr. Trimble on this issue of children’s 

health and the overview of the work you did. Could you please describe the safe Drinking Water Act and the EPA policy requirements on considering children’s health impacts in drinking water 

standards, and then two, describe your report’s findings on how 

EPA met these requirements, and three, describe how recommendations you made relate to those children’s health requirements. 

Mr. TRIMBLE. Sure. First, the requirement to prioritize contaminants of greatest concerns includes a provision that prioritization 

should include impacts on children and other sensitive sub-populations. So that is sort of square one, that is part of the 

prioritization. And again, it goes to the recommendation we have 

regarding how you establish that priority list. 

In observing how that has been implemented with EPA and our 

work in doing those determinations has not integrated specific risk 

assessments for children as part of its determinations. In 2003, in 

the determinations they made to not regulate additional contaminants, it acknowledged that it did not have the methodology to address the impact on children, but did not then make any other explanation as to how they were going to figure out whether this was 

protective of children’s health. 

In 2008, they made additional determinations where they acknowledge that these contaminants had an effect on children’s 

health, but they did not explain in their analysis how that potential risk was addressed in their analysis, and how their conclusion 

that these contaminants did not need regulation was protective of 

children’s health. 

87 

In perchlorate, similar concerns were raised about children’s 

health. In fact, that is one of the reasons why the decision was reconsidered, was concerns about uncertainty as well as the impact 

on children. What I would note in that regard is while the EPA, 

from my understanding, has decided to regulate perchlorate, it is 

not clear at this point whether the additional analysis it did in reconsidering its decision for perchlorate will be carried forward in 

future determinations. So in other words, are they going to integrate that analysis and the impact on children in future determinations. It is still a little unclear from what we have been given 

whether that would happen. 

Senator UDALL. Thank you. 

Mr. Perciasepe, could you please respond to what we have heard 

from the GAO on children’s health and what will EPA do to implement GAO recommendations in this area? 

Mr. PERCIASEPE. We feel that we are required by law under the 

Safe Drinking Water Act to consider the sensitive populations. I 

won’t read it, I have it sitting here in front of me, but I can guarantee you that it is in the law and it is clearly laid out, and the 

GAO has pointed this out clearly, that we should be making a clear 

analysis. It depends on what the health effect of the substance is. 

I would have to say some may not have an impact on children in 

the same way. 

But it doesn’t negate the need to do it. So we would be agreeing 

with that assessment that we need to be doing that. That is going 

to be also part of our overall screening process of even when it gets 

down to being looked at as a hard contaminant. For instance, we 

have a contaminant on our current list, a subset of that 116 we 

mentioned earlier, for instance, strontium, which has a very important negative effect on bone development, particularly in children. 

So that is one that is on the list particularly because of children. 

Senator UDALL. Thank you. Thank you, Madam Chair. 

Senator BOXER. Thank you. And Senator Udall, thank you for all 

your work on children’s health. This is so critical, because we have 

a responsibility to them. 

I just want to thank the panel very, very much, GAO for your 

clarity. I think this report was very good. And I think it should be 

embraced by everyone, because what you are basically saying is 

transparency in these decisions, move quicker on these decisions, 

and get it done. And I applaud the EPA for your response, which 

is, we hear you. I just want you to know, we are watching on chromium–6 and perchlorate. I am looking forward to that briefing. 

And by the way, anybody else who wants to hear the briefing on 

these 17 contaminants that have been looked at since 1998 and 

nothing has been done, we are going to have a briefing. We just 

might do it as an open briefing, but I will let you know about that. 

Mr. PERCIASEPE. OK, we are ready. 

Senator BOXER. Well, we appreciate it so much. We really do appreciate it. And even your sports analogies which drew our interest. 

[Laughter.] 

Senator BOXER. Thank you very much. 

Mr. PERCIASEPE. I will remember to use football the next time. 

88 

Senator BOXER. Football, baseball, soccer, that is fine. It is good. 

Women’s soccer in particular would be good. 

[Laughter.] 

Senator BOXER. OK, panel two. Dr. Lynn Goldman; A.W. ‘‘Butch’’ 

Araiza; Dr. Joseph Cotruvo; Dr. Steven Patierno; and Dr. Jeffrey 

Griffiths. And each of you will have an opening of 5 minutes. And 

at that point, I will ask you to put the remainder of your statement 

in the record. So if you could summarize. 

We are going to start with Lynn Goldman and we are going to 

move down the table, if that is OK. So Dr. Lynn Goldman, welcome, American Public Heath Association, Dean of the School of 

Public Health and Health Services, and Professor of Environmental 

and Occupational Health, George Washington University. We are 

very delighted that you are here, thank you. 

STATEMENT OF LYNN GOLDMAN, MD, MPH, AMERICAN PUBLIC Health ASSOCIATION, DEAN OF THE SCHOOL OF PUBLIC 

HEALTH AND HEALTH SERVICES AND PROFESSOR OF ENVIRONMENTAL AND OCCUPATIONAL Health AT THE GEORGE 

WASHINGTON UNIVERSITY 

Dr. GOLDMAN. And thank you very much. It is my pleasure to 

testify about the Environmental Protection Agency’s implementation of the Safe Drinking Water Act’s unregulated drinking water 

contaminants program on behalf of the American Public Health Association. 

I have been a member of the APHA for almost 20 years. I am 

pleased to represent them today. The APHA is dedicated to protecting all Americans and their communities from preventable, serious health threats and assuring community-based health promotion and disease prevention activities and preventive health 

services are universally accessible in the United States. APHA has 

long advocated for strong environmental health laws. 

Safe drinking water is essential to public health, and our Nation’s drinking water supply is one of the safest in the world. Along 

with the EPA, our Nation’s State and local health departments also 

play a critical role to ensure the safety of our drinking water. Additionally, there are more than 170,000 public water systems providing water. 

While most of our drinking water does not violate EPA standards 

for maximum contaminant levels, the APHA is concerned over a 

number of public health issues. First, we are concerned about the 

decline in Federal resources to State to improve drinking water infrastructure. In fact, the final 2011 continuing resolution, and I 

have heard possibly the 2012 budget as well, is cutting significant 

amounts of money, hundreds of millions of dollars, from the EPA’s 

Safe Drinking Water State Revolving Fund. 

Second, we are concerned about unregulated contaminants. Tens 

of thousands of chemicals are on the market, but the vast majority 

of these will never appear in finished drinking water. Those that 

are in drinking water should be regulated by EPA so the public can 

be assured that levels are safe. 

A number of specific chemicals have been of concern to the 

APHA, including perchlorate, chromium–6, trichloroethylene and 

other volatile organic chemicals, and a number of pesticides and 

89 

disinfection products found in drinking water across the Nation. 

APHA expected that the 1996 Safe Drinking Water Act amendments would have increased the number of chemicals that were 

regulated by EPA. Unfortunately, EPA did not adopt new drinking 

water safeguards for chemicals after that law was passed. And a 

recent study by the Environmental Working Group reported that 

hundreds of unregulated contaminants are found in our drinking 

water systems. 

Why do we need to have MCLs? Minus the establishment of 

MCLs and health-based MCL goals, how are we to know that the 

levels of chemicals in drinking water are safe? In response, several 

States have undertaken drinking water regulation of unregulated 

contaminants, for example, the State of California regulating perchlorate, chromium–6, TCE, several VOCs and pesticides. But only 

national standards can assure safe drinking water across our entire 

Country. 

Third, we are concerned that EPA standards not only protect the 

average adult but also adequately protect infants and children, the 

frail elderly and those with weakened immune systems. As a pediatrician, I am particularly concerned about infants less than 6 

months of age who receive drinking water in infant formula. These 

infants consume five times more drinking water per body weight 

than adults. They therefore have greater exposure to any substance 

in drinking water than adults would. And yet, we also know that 

at these life stages that they may be more sensitive, less able to 

metabolize, detoxify and excrete toxic substances, as well as more 

sensitive to developmental effects, effects on growth and development. 

The 1996 Safe Drinking Water Act amendments directed the 

EPA to protect the general population, as well as those significant 

sub-populations. This authority needs to be exercised by the EPA 

in the regulation of unregulated contaminants. 

In conclusion, APHA suggests a number of ways that EPA could 

strengthen the regulation of chemicals in drinking water. EPA does 

need to implement the 2009 National Academy of Sciences report, 

Science and Decisions Advancing Risk Assessment, especially the 

recommended changes in risk assessment design, dose response 

modeling, uncertainty analysis and analysis of cumulative risk. Cumulative risk is not only the risk of multiple chemicals that might 

act in the same way, but also the risks that people have in other 

exposures in daily life that create public health threats like nutrition, exposure to smoke, other carcinogens. 

EPA needs to develop MCLs for unregulated toxic chemicals and 

pesticides that are present in drinking water so that the public can 

be confident of safety. And third, EPA needs to strengthen its efforts to protect vulnerable populations such as infants and children 

and pregnant women from risks of drinking water contaminants. 

Thank you for the opportunity to testify this morning, and I am 

happy to answer any questions. 

[The prepared statement of Dr. Goldman follows:] 

90 


23819.058

11 American 

Public Health 

Association 

800 l Street. ~W. • Washington. DC 20001 1710 

Phtme: ('202) 777-APHA • Fax: (202) 7i7.:2534 

www.aph<Lorg • ._•nmln~nl<.@aph<Lorg 

Testimony of Lynn Goldman, MD, MS, MPH 

Representing the American Public Health Association 

Oversight Hearing on the Environmental Protection Agency's Implementation of the Safe 

Drinking Water Act's Unregulated Drinking Water Contaminants Program 

Senate Committee on Environment and Public Works 

July 12, 2011 

Chairman Boxer, Ranking member Inhofe and members of the Committee, it is my pleasure to 

testify about the Environmental Protection Agency's Implementation of the Safe Drinking Water 

Act's Unregulated Drinking Water Contaminants Program on behalf of the American Public 

Health Association. 

My name is Lynn Goldman. I am Dean of the School of Public Health and Health Services at 

The George Washington University School of Public Health and Health Services. I am a 

pediatrician and an epidemiologist. I have done research on health impact of environmental 

contaminants and am a member of the Institute of Medicine. From 1993-98, I served as 

Assistant Administrator for the Office of Chemical Safety and Pollution Prevention at the US 

Environmental Protection Agency (EPA). Prior to joining the EPA I worked for eight years in 

public health with the California Department of Health Services. I also am a member of the 

Board of Trustees of the Environmental Defense Fund. I have been a member the American 

Public Health Association (APHA) for almost 20 years, and I am pleased to represent APHA at 

today's hearing regarding the public health implications of the Safe Drinking Water Act 

(SDWA). APHA is the nation's oldest and most diverse organization of public health 
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professionals in the world, dedicated to protecting all Americans and their communities from 

preventable, serious health threats and assuring community-based health promotion and disease 

prevention activities and preventive health services are universally accessible in the United 

States. APHA has long advocated for strong environmental health laws that adequately protect 

the health of the public from environmental hazards and we appreciate the opportunity to testify 

today on the public health implications of our nation's safe drinking water laws. 

The SDWA, last amended in 1996, is the nation's primary law to protect tbe public's health from 

harmful contaminants in the nation's drinking water supply. While the EPA administers the law 

and sets standards, the states have been delegated primary authority for enforcing drinking water 

programs. Our nation's state and local health departments also play a critical role in working 

with state drinking water regulators to ensure the safety of our drinking water. 

Safe drinking water is essential to public health. According to the EPA, there are currently more 

than 170,000 public water systems that provide water to most Americans, and our nation's 

drinking water supply is one of the safest in the world. Since its enactment, tbe SDW A has made 

significant improvements to the quality of the nation's public water supplies. An overwhelming 

majority of Americans receive their drinking water from sources that do not violate EPA's 

standards for maximum contaminant levels. At the same time, the public health community 

remains concerned over a number of issues that continue to provide risks to the public's health, 

including unregulated contaminants and decreasing federal resources to states to improve 

drinking water infrastructure. In fact, in the final 20 II continuing resolution cut more than $400 

million (or 30 percent) from the FY 2010 level for EPA's Drinking Water State Revolving Fund. 
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In addition, APHA believes more must be done to protect vulnerable populations that are more 

susceptible to exposures, including infants/children, pregnant women, immune-suppressed 

individuals, and the elderly when setting drinking water standards. 

There are weaknesses in federal statutes and regulations governing the safety of drinking water, 

and a number of EPA standards are being currently being reviewed and revised. In some 

instances, contaminants are not regulated, such as perchlorate and a number of pesticides. EPA 

standards may protect the average adult but may not adequately protect infants and children, the 

frail elderly, and those with weakened immune systems. Moreover, EPA standards are 

established to protect health while considering the water treatment costs and availability of 

clean-up technology. 

Numbers of Contaminants Unregulated 

At the time the SDW A was amended, APHA expected that this would result in an uptick in the 

numbers of chemicals regulated since SD W A gave EPA more flexibility to establish priorities 

for regulation of contaminants based on those that present the greatest public health concern, 

including for pregnant women, infants and children. Unfortunately, this expectation was not 

fulfilled, and my review of EPA's drinking water regulations at CFR 40 part 141 finds that EPA 

has not adopted any new drinking water safeguards for chemicals since enactment of the 1996 

law- neither to assure the safety of unregulated contaminants nor to change regulations for 

previously-:egulated chemical contaminates. Tens of thousands of chemicals are on the market 

but the vast majority of these will never appear in finished drinking water. Those that are in 
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drinking water should be regulated by the EPA so that the public can be assured that levels are 

safe. According to a December 2009 report issued by the Environmental Working Group, 

hundreds of additional unregulated contaminants have been found in our drinking water systems. 

Minus the establishment of clear Maximum Contaminant Levels (MCLs) and health-based MCL 

Goals, how are we to know that these chemicals in their water are "safe"? A number of specific 

chemicals have been of concern to the APHA and the public health community in states, 

including: perchlorate, chromium VI, trichloroethylene (TCE) and other VOCs, and a number of 

pesticides and disinfection products that are found in drinking water across the nation. 

Unfortunately, this situation has caused several states to undertake drinking water standard 

setting on their own, ahead of EPA action, most notably, the State of California, which has 

established standards for perchlorate, chromium VI, TCE and several other VOCs, and, a number 

of pesticides. While APHA believes that states are responsible for taking such steps to protect 

the public's health, we would prefer to see national standards that can effectively assure safe 

drinking water for all of the country. 

SDWA and Children, Pregnant Women, and Other Vulnerable Populations 

In 1995, APHA developed a policy that recommended that all environmental policy, legislation, 

and regulation protect children and we have long advocated that environmental health standards 

need to protect vulnerable populations. It is important to recognize that, as we state in that 

policy, "children are uniquely vulnerable to environmental exposures because they are in a 

dynamic state of growth, with many vital systems such as the nervous, immune, and respiratory 

systems not fully developed upon birth" and to understand "that children can have greater 
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exposures to environmental toxins". In the case of drinking water, children drink more water 

and consume more water indirectly through food, per body weight, than adults. According to 

EPA estimates, considering all drinking water intake, infants less than 6 months of age 

consuming drinking water in infant formula, per body weight consume five times more drinking 

water than adults consume in both drinking water and food. This means that they have greater 

exposure to any substance, microbial or chemical, that might be present in that drinking water. 

At various life stages- during pregnancy, in utero, in childhood, in old age, and during serious 

illnesses -- people may have different abilities to metabolize, detoxifY, and excrete certain many 

toxic substances, thus rendering them more vulnerable to health effects. Moreover, rapid growth 

and development in utero and in early childhood puts children at particular risk for exposure to 

environmental toxins that may disrupt normal developmental processes, and result in permanent 

irreversible damage. Traditionally EPA's environmental health regulations have been based on 

data primarily from research on healthy adult humans or animals and do not take into 

consideration the unique exposure patterns and sensitivities of children, pregnant women, the 

elderly and those with chronic diseases. 

In the 1996 SDW A amendments, Congress specifically directed the EPA to regulated 

unregulated contaminants that are at greater risk of adverse health effects due to exposures than 

the general population, including infants, children, pregnant women, the elderly, and individuals 

with a history of serious illness, or other subpopulations recognized to be of greater risk. 

Likewise, in establishing MCL goals, EPA was to protect the general population as well as these 

significant subpopulations. This authority has been exercised by EPA to craft stronger 
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regulations for microbial contaminants like cryptosporidia in drinking water, but not for 

addressing unregulated chemical contaminants. 

Conclusions and Recommendations for Improvement 

APHA suggests a number of ways that EPA could strengthen the regulation of chemicals in 

drinking water: 

I. In all of its risk assessment efforts, EPA needs to implement the 2009 National Academy 

of Sciences report Science and Decisions: Advancing Risk Assessment. This report 

recommended changes in how EPA designs risk assessments, models dose response for 

hazardous substances, and considers uncertainty in risk assessment. In particular it 

concluded that EPA needs to address the issue of cumulative exposures. Exposures to 

drinking water contaminants occur within a context of multiple exposures to other 

contaminants in drinking water, foods, household environments, and air. They also occur 

within contexts such as significant subpopulations who have other health problems that 

may contribute such as poor nutrition and exposure to tobacco smoke. All of these issues 

need to be considered in an overhaul of EPA's risk assessment processes, including for 

drinking water. 

2. EPA needs to more strongly weigh evidence that chemicals and pesticides are present in 

drinking water. Given that there are no mechanisms for systematic collection of 

information about levels of unregulated chemicals in drinking water, those toxic 

chemicals that are occurring frequently need MCLs so that they will be monitored and the 

public can be assured that levels are safe. 
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3. Each and every one of us was, at one time, a fetus or a child; have been or may become 

(if we are female) pregnant; has suffered from or may have a chronic illness; and (if we 

are lucky) may become elderly. These aren't "subpopulations"; they are life stages. EPA 

needs to strengthen its efforts to assure that vulnerable populations are protected as 

required by law. 

Thank you for the opportunity to testify this morning and I am happy to answer any questions. 
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23819.065

Goldman Follow Up Question Responses 

Questions from Senator Barbara Boxer 

I. Without specific guidance, the EPA will not be able to develop a science-based 

approach that can be applied consistently to ensure that children are protected 

from contaminants in drinking water considering multiple developmental stages. 

Yes, this is the type of action that APHA has in mind. 

Yes. The agency should take a proactive approach to identification of drinking 

water contaminants that are most likely to be hazardous to pregnant women and 

children, review those and, where appropriate, establish protective standards. 

2. Yes, this effort is consistent. APHA is concerned that the Agency move forward 

quickly, especially in the case of chemicals with similar toxicity that are likely to 

co-occur (for example, pesticides in surface water supplies). 

3. First, a six kg, six-month-old baby is about one-tenth the size of a 70 kg adult 

male. If the baby consumes five times more water per body weight than the adult, 

that means the infant is receiving a 50 times higher dose (per body weight) of any 

contaminant in the water. 

This has obvious practical importance, especially in the case of contaminants that 

are particularly toxic during development. 

Questions from Senator James Inhofe 

I. I have testified as an expert witness on two occasions. One I was a witness for a 

plaintiff and once I was a witness for the defendant. In the first instance, 

compensation was received by my university; in the second instance, I received 

some compensation. 

2. I have never served on a peer review panel for any EPA risk assessments. 

3. No, I do not. I believe that the drinking water infrastructure in the US in many 

areas is aging, and that over the years, the EPA and Congress will need to 

continue to focus attention on assuring that the American people have safe, 

plentiful, and affordable supplies of drinking water. 
98 

Senator BOXER. Thank you, Dr. Goldman. I think that cumulative risk issue is key. I thank you for bringing it up. 

And it is my pleasure to introduce Mr. A.W. ‘‘Butch’’ Araiza, General Manager of the West Valley Water District in the Inland Empire, my State. It has been my pleasure to work with them. They 

face some tough issues and we look forward to hearing from him. 

STATEMENT OF ANTHONY ARAIZA, GENERAL MANAGER, WEST 

VALLEY WATER DISTRICT 

Mr. ARAIZA. Thank you, Chairman Boxer and Ranking Member 

Inhofe and members of the Committee. It is an honor to be asked 

to testify today about the West Valley Water District’s success in 

removing perchlorate from groundwater. 

I would first like to communicate my profound thanks to Senator 

Boxer. Over the years, you and your staff have been key in helping 

us secure Federal funding for our remediation efforts in helping encourage the Environmental Protection Agency and the Department 

of Defense to aid in treatment of contaminated water in our region. 

West Valley Water District’s success is very much your success as 

well. Thank you. 

By way of introduction, I have been employed by the District for 

the past 48 years. I worked my way up through the ranks to the 

position of General Manager nearly 16 years ago. In 2002, the District became the lead agency in Inland Empire Perchlorate Task 

Force, which is responsible for coordinating the investigation and 

response to the serious perchlorate contamination crisis in our 

area. 

West Valley Water District was founded almost 60 years ago. 

Today, we serve almost 20,000 homes and businesses. Our staff 

and board of directors are dedicated to protecting, safeguarding 

and delivering clean water to our customers at the lowest reasonable price. The necessity to innovate was presented clearly by our 

need to remediate the effects of widespread perchlorate pollution in 

the inland empire. The source of that pollution is a 160-acre industrial site in Northern Rialto that used to be a U.S. Army munitions 

depot. The State of California has set a limit of perchlorate of 6 

parts per billion. 

But the plume migrating from the site has led to levels as high 

as 800 parts per billion. This has forced West Valley Water District 

and other water providers in the area to shut down or otherwise 

restrict the use of at least 22 groundwater production wells, representing more than half the region’s water supply. Additional 

groundwater production wells may need to be shut down as pollution continues to migrate. We have no choice other than to remediate. Groundwater is the main source of drinking water in the region and alternative sources are not reliable. Estimated costs for 

cleaning up the whole problem would be in the hundreds of millions of dollars and could take up to 30 years. 

Meanwhile, remediation poses a significant burden to the ratepayers in one of the most economically disadvantaged regions in 

the Nation. Facing this crisis and lacking the vast resources required to solve it, West Valley Water District has looked to innovative solutions. We were fortunate to find that the Department of 

Defense’s Environmental Security Technology Certification ProVerDate Aug 31 2005 11:45 Apr 10, 2017 Jkt 000000 PO 00000 Frm 00104 Fmt 6633 Sfmt 6633 S:\_EPW\DOCS\23819.TXT VERN
99 

gram, ESTCP, and its extremely capable executive director, Dr. 

Jeffery Marqusee. Under the leadership of Dr. Marqusee, ESTCP 

is demonstrating the most innovative and cost effective technologies that address DOD’s high priority environmental requirements. Our work with ESTCP is a win-win partnership, providing 

our customers clean water while collecting data that the ESTCP 

uses to advance cost-effective technology. 

The District’s perchlorate remediation efforts start in 2003, utilizing technology known as ion exchange. We are currently running 

five ion exchange systems, one of which is cutting edge and was 

made possible by the ESTCP. Ion exchange systems work by trapping perchlorate and resin, and our experience has proven them to 

be safe and reliable. 

With the support of ESTCP and the State of California, we have 

recently broken ground on the first facility to use naturally occurring biological process to treat drinking water. Bioremediation allows natural process to break down perchlorate and other harmful 

chemicals in the water. Bioremediation has proven safe and effective, does not use harmful chemicals and requires less annual 

maintenance than other methods. Our project will provide clean 

tap water to our customers and opportunities for ESTCP to save 

the Department of Defense significant amounts of money at sites 

they may have liability for across the Country. 

We are proud to provide a venue for demonstrating this pioneering green technology. It should not surprise you that West Valley Water District is not going to stop working toward the future, 

now that the bioremediation project is underway. We anticipate 

this project will become a component of a much larger undertaking 

to fully restore the region’s groundwater supplies. The District is 

currently working with the United States Environmental Protection Agency, the State of California and parties responsible for the 

contamination to develop a plan for final phase of groundwater 

clean-up. This is a lot of work yet to be done, and we are looking 

for more innovative solutions, just as we have been for the past 60 

years. 

Thank you for this opportunity to testify to the Committee, and 

I would be happy to take any questions. 

[The prepared statement of Mr. Araiza follows:] 

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Testimony of Anthony Araiza 

General Manager 

West Valley Water District 

before the 

US Senate Committee on Environment and Public Works 

"Oversight Hearing on the Environmental Protection Agency's Implementation of the Safe 

Drinking Water Act's Unregulated Drinking Water Contaminants Program" 

July 12, 2011 

Chairman Boxer, Ranking Member Inhofe, and Members of this Committee, it is an honor to be 

asked to testify today about West Valley Water District's success in removing perchlorate from 

groundwater as part of our mission to deliver safe and affordable drinking water to residents of 

the Rialto-Colton area. I am pleased to be able to share our experience with the Committee 

because we believe we have been part of the solution. 

Before I introduce myself or explain what we've been doing, I would first like to communicate 

my profound thanks to Senator Boxer. Over the years, you and your staff have been key in 

helping secure federal funding for our remediation efforts and helping encourage the 

Environmental Protection Agency and the Department of Defense to aid in the treatment of 

contaminated water in the region. West Valley Water District's success is very much your 

success as well. Thank you. 

By way of introduction, I have been employed by the District for the past 48 years. I worked my 

way up through the ranks to the position of General Manager nearly 16 years ago. In 2002, I 

became the lead member of the Inland Empire Perchlorate Task Force, which is responsible for 

coordinating the investigation and response to the serious perchlorate water contamination crisis 

impacting approximately 22 drinking water wells and 500,000 people in the Inland Empire. 

Background on WVWD 

When the predecessor to West Valley Water District started almost 60 years ago, it served a very 

rural area and few water customers. Today we serve almost 20,000 homes and businesses, and 

our service area encompasses more than 29 square miles. Despite the many changes, our staff 

and Board of Directors have maintained a constant dedication to protect, safeguard, and deliver 

water to our customers at the lowest, reasonable price. We serve as fiduciaries, for the public 

trust. We do so not only by sticking to the tried-and-true, but also by looking for new 

innovations- for example, we recently re-vamped our web site, developed a Facebook page and 

a started a Twitter feed to help ensure the very best service to our customers. 

Perchlorate 

The necessity to innovate was presented clearly by our need to remediate the effects of 

widespread perchlorate pollution, which affects the groundwater aquifers on which the West 
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Valley Water District, the Cities of Colton and Rialto and the Fontana Water Company rely to 

provide drinking water to the citizens of the Inland Empire. The source of that pollution is a 

160-acre industrial site in northern Rialto that used to be a U.S. Army munitions depot. The 

State of California has set a limit for perchlorate of six parts per billion, but the plume migrating 

from this site has led to levels as high as 800 parts per billion. Perchlorate contamination has 

forced the West Valley Water District and other water providers in the region to shut down or 

otherwise restrict the use of at least 22 groundwater production wells in the area, representing 

more than half of the region's water supply. Additional groundwater production wells may need 

to be shut down in the near future as the pollution continues to migrate. 

We have no choice other than to remediate. Groundwater is the main source of drinking water in 

the region, and the basin's water levels are approaching an historic low. Alternative sources 

such as the State Water Project are not reliable. The estimated cost of cleaning up the whole 

problem could be in the hundreds of millions of dollars and take up to 30 years. The necessity 

for remediation in the region poses a significant burden for ratepayers in one of the most 

economically disadvantaged regions in the nation. 

ESTCP 

Facing this crisis, and lacking the vast resources required to solve it, West Valley Water District 

has looked for innovative solutions. We were extremely fortunate to find the Department of 

Defense's Environmental Security Technology Certification Program (ESTCP) and its extremely 

capable executive director, Dr. Jeffrey Marqusee. ESTCP is DoD's environmental technology 

demonstration and validation program, which promotes innovative technologies. Under the 

leadership of Dr. Marqusee, the ESTCP program has succeeded in achieving its goal "to identify 

and demonstrate the most promising innovative and cost-effective technologies and methods that 

address DoD's high-priority environmental requirements." We work with ESTCP's 

Environmental Restoration Program, which has been a win-win partnership. The District and 

our customers benefit through cost-effective clean water, and the data collected helps the 

ESTCP's goal of advancing science and technology to provide the Department of Defense (DoD) 

with effective management and restoration of its contaminated assets. Dr. Marqusee has been a 

problem solver who has helped significantly in moving forward with solutions. 

Remediation 

The first stage of the District's perchlorate remediation efforts have utilized a technology known 

as ion exchange. We are currently running 5 ion-exchange systems, one of which is a cuttingedge design that was made possible by the ESTCP. Ion exchange systems work by trapping 

perchlorate in resin, and our experience has proven them to be safe and reliable. 

With encouragement and support from ESTCP and the State of California, we have entered a 

second stage of remediation by breaking ground on the first bio treatment facility to utilize a 

naturally occurring biological treatment process to treat contaminated drinking water supplies. 

Bioremediation allows natural processes to breakdown perchlorate and other harmful chemicals 

in the water. After this natural process occurs, the water is sent through a traditional water 

treatment process before it enters the drinking water system. Bioremediation is proven safe and 
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effective, does not use harmful chemicals, and requires less annual maintenance than other 

methods. 

The system under construction will utilize a fluidized-bed reactor, and we hope to combine it 

with an additional treatment system which will feature a fixed-bed reactor. Because the two 

systems will treat water from the same underground source, side by side, our partnership with the 

ESTCP will provide a unique opportunity to test the effectiveness, sustainability, and costs of 

each system a tremendous benefit that will help future clean-up efforts throughout the country. 

In other words, our project will provide opportunities for the Department of Defense to save 

significant amounts of money at sites they may have liability for across the country. We're proud 

to be a part of the solution and to provide a venue for demonstrating this pioneering "green" 

technology. 

I would be remiss if I did not acknowledge that these projects would not have gotten off the 

ground with out considerable help from others in addition to Senator Boxer and Dr. Marqusee. 

Senator Feinstein and Congressman Baca have been tremendous allies. The State of California 

has provided $1 0 million in grants, and the State Water Resources Control Board has also 

contributed crucial resources. 

Future 

It should not surprise you that West Valley Water District is not going to stop working towards 

the future now that the fluidized bed reactor construction is underway. As I just mentioned, we 

anticipate that this project will become a component of a much larger undertaking to fully restore 

the region's groundwater supplies. The District is currently working with the United States 

Environmental Protection Agency, the State of California and parties responsible for the 

contamination to develop plans for the final phase of groundwater cleanup. There is a lot of 

work yet to be done, and we are looking for more innovative solutions-just as we have been 

doing for the last 60 years. 

Summary 

The staff and Directors of West Valley Water District are fiduciaries for the public trust, 

dedicated to serving our 20,000 households clean water through efficient service. We are 

focused on the future and we're looking for innovative solutions to the challenges that confront 

us. Although the perchlorate crisis has presented a significant challenge in our region, we are 

working tirelessly to be part of the solution, not only for our ratepayers, but also- thanks to the 

ESTCP- for everyone else who is or may be affected by perchlorate in the future. With the help 

of our elected representatives including Senator Boxer, we are providing new technologies that 

can remove perchlorate safely and cost efficiently. This is a win-win collaboration for West 

Valley Water District, our customers, the DoD, and all Americans who rely on the delivery of 

clean, safe and affordable drinking water. 

Thank you. 
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855 West Base Line, P.O. Box 920 -- Rialto, Galifornia 92377-0920 

Phone {909) 875-1804 

November 2, 2011 

Senate Committee on Environment and Public Works 

Attn: Jonathan Aronchick 

410 Dirksen Senate Office Building 

Washington, DC 20005 

Dear Mr. Aronchick: 

Soard of Directors 

Earl Tillman, Jr. Presldenl 

Betty J. Gosney Vil;l;!Pf!ffllde!tl 

Alan G. Dyer 

Donald D. Olinger 

Jackie Cox 

Administrative Staff 

Anthony W. Araiza GenaralManager·Se<:telary 

Thomas J. Crowley, P. E Aul!otan!GeneraiMal'lager 

Deborah L Sousa Treasuw 

Peggy S. Asche Admm•!ll<aliveSecrl!tary 

Fax {909) 875-7284 Administration 

Fax {909) 875-1361 Engineering 

Fax {909) 875-1849 Customer Service 

Enclosed please find my answers to the questions submitted by Senator lnhofe for the hearing record for 

the July 12, 2011, hearing entitled, "Oversight Hearing on the Environmental Protection Agency's 

Implementation of the Safe Drinking Water Act's Unregulated Drinking Water Contaminants Program." As 

requested, I have reproduced the questions with my responses. 

I hope my answers are helpful to the Committee as it continues its work on this important topic. Please 

contact me if you have any further questions or need any additional information. 

Sincerely, 

WEST VALLEY WATER DISTRICT 

Established as a public agency in 1952 

The District is an Equal Opporttmity Provider 
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West Valley 

Watel' Distl'ic:t 

1. The Committee hears a lot from the smaller drinking water utilities around the country 

about issues complying with unfunded drinking water mandates such as new rules or 

additional testing. How does West Valley Water District respond to these mandates and 

challenges? 

West Valley Water District is accustomed to managing the difficulties of providing a 

clean, safe water supply while keeping costs reasonable for our ratepayers. Our 

organization believes that we should work to make certain our water is as clean as 

possible for our users because it is the right thing to do, not simply because we are 

required to meet state or federal standards. The State of California has set a limit for 

perchlorate of six parts per billion. We have been able to manage this challenge by 

working to implement cutting-edge water treatment technologies, like the bioremediation 

project I mentioned in my written and oral testimonies. Chairman Barbara Boxer, 

Senator Dianne Feinstein, and others have been successful in helping us develop 

innovative partnerships with federal and state agencies to test and help fund new 

technologies. 

2. What are some of the challenges that utilities face in responding to media reports about 

unregulated contaminants? 

Media reports can easily lead to concerns about the safety and quality of water supplies. 

West Valley Water District believes that public outreach is vital to ensuring our users 

have accurate information about their water supply, and we have employed an aggressive 

outreach strategy. As mentioned in my written testimony, we maintain a very 

informative website, we are on Facebook, and we even have a Twitter feed. Through 

these and other more traditional mediums - informational mailings, public meeting, etc. -

we work to educate our customers about information that can come through traditional 

media, the internet, and other sources. We strive to ensure our users know exactly what 

is in their water and the steps West Valley Water District is taking to protect the water 

supply. 

3. Are you supportive of a drinking water regulatory process that relies on science to help 

guide decision making? Do you think the current SDWA provides a clear, transparent, 

science-driven process for making decisions regarding drinking water regulation? 

West Valley Water District supports a sound scientific process as it relates to the 

regulatory framework for groundwater supplies. Few federal statutes are perfect, and the 

Safe Drinking Water Act is not without room for improvement. As Ranking Minority 

Member Inhofe pointed out in his opening statement, the National Academy of Sciences 

has recently pointed out that the Environmental Protection Agency's (EPA) risk 
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assessment system is flawed. And, as Chairman Boxer pointed out in her statement, a 

recent Govermnent Accountability Office report indicated that the EPA's process for 

developing standards for unregulated drinking water contaminants failed to use the best 

available science. West Valley Water District would be happy to participate in 

discussions with the committee if our experience could be helpful in finding ways to 

address J;hose issues. 
106 

Senator BOXER. Thank you very much, and we welcome our next 

speaker, Dr. Joseph Cotruvo, it is Joseph Cotruvo and Associates, 

LLC, Water, Environmental and Public Health Institute. Welcome, 

sir. 

STATEMENT OF JOSEPH A. COTRUVO, PH.D., PRESIDENT, 

JOSEPH COTRUVO AND ASSOCIATES 

Mr. COTRUVO. Thank you, Chairman Boxer and Senator Inhofe. 

I appreciate the opportunity to come and talk to you today. 

My name is Joseph Cotruvo. I have spent more than 35 years in 

public health matters. The doctorate is in physical organic chemistry. I was the first Director of EPA’s Drinking Water Standards 

division back about 35 year ago when the law was passed, and 

spent a number of years there. In the course of that, I was involved 

in developing many of the existing regulations and in fact, many 

of the methodologies that are used for regulating. 

The regulations are very comprehensive. We will talk about more 

of them later. Currently I work internationally on water quality 

and health. I do some non-U.S. Government assistance on regulatory activity and water quality. I serve on a number of advisory 

committees, including World Health Organization’s guidelines for 

drinking water quality, which is actually what the rest of the 

world, outside the United States, uses for establishing its drinking 

water safety. 

I am also a member of the D.C. Water and Sewer Authority 

board of directors here. I just want you to know, I am not representing any organization or institution. These are my personal 

thoughts, conclusions, and I am not receiving any compensation for 

this presentation. 

On the topic of drinking water quality and safety, almost all community water systems in the United States produce safe drinking 

water, including Washington, DC, although some may not believe 

that. It is a daunting task, because we are dealing with 65,000 

water supplies, 25 persons up to millions. And it is a very complex 

job that is a multi-faceted job. EPA has a role, States have a role, 

and of course, it is all underpinned by those guys that are out there 

every day, 24–7, producing that water at the tap. 

Overall, I think EPA has done a generally good job of implementing the Safe Drinking Water Act. The record shows that reported waterborne infectious disease outbreaks have declined since 

the implementation of the law. And the portion of outbreaks due 

to distribution systems and infrastructure has increased. So overall, it has declined, but that part has gone up. That tells us very 

clearly that the problem is infrastructure, it means that water 

going in from the plant better than the water that it coming out 

at the tap. 

So fixing that and maintaining is, I think, really the greatest 

public health challenge that we have to face here, and the greatest 

public health concern. 

The current regulations are very comprehensive. They cover a 

number of substances, 81 MCLs, microbial contamination, which 

basically covers all microbial contaminants, radionucleides all are 

covered. There is corrosion control, there are some specific items 

also. But the numbers even belie the reality in that they actually 

107 

cover much, much more than just those numbers, because all those 

regulations, or many of them in fact have a cascade of other substances that are protected against by the regulation that is in 

place. 

Regulations have to be protected. They are designed to be protective. They have certain safety factors built in, and that is what we 

expect. But it is of course important that they have to be realistic, 

and they have to be appropriate for the kind of risk involved, and 

the significance, and in fact, the strength of evidence that there is 

really a risk involved. That is why part of the task that EPA has 

is so difficult. Because with all of these new analytic methods, as 

you heard from the GAO, methods keep getting lower and lower. 

We used to talk about parts per million, now parts per billion, now 

parts per trillion, and next year it will be parts per quadrillion, 

probably. 

So what all that means is we are finding more and more substances. It doesn’t mean that there are more there than were there 

5 years ago or 10 years ago or whatever. It is just we are finding 

them now. Invariably, when one is measuring something at levels 

that are that low, it is really hard to conceive that there is some 

significant risk from exposure at that minute level. 

Just to put some things in perspective a little bit, I have a couple 

of items here mentioning reports on perchlorate and pharmaceuticals. I will just read you the report that came this year from 

the UNFAO, WHO, JECFA Committee: ‘‘The estimated dietary exposures of 0.7 micrograms per kilogram per day, highest, and 0.1 

microgram per kilogram per day, mean, including both food and 

drinking water, are well below the provisional maximum tolerable 

daily intake. The committee considered that these estimated dietary exposures were not of health concern.’’ 

So there are differing points of view that one has to deal with 

in this business. And that makes all of our jobs and EPA’s very difficult. 

[The prepared statement of Mr. Cotruvo follows:] 

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Senate Committee on Environment and Public Works 

Chair, Senator Barbara Boxer 

Oversight Hearing on USEPA's Implementation of the Safe Drinking Water Act's 

Unregulated Contaminants Program 

July 12, 2011 

Joseph A. Cotruvo, Ph D 

President: Joseph Cotruvo and Associates 

Washington, DC 

Good morning Senator Boxer and members of the committee. 

My name is Joseph Cotruvo. I have spent more than 35 years engaged in public health and 

environmental matters. My doctorate is in Physical Organic Chemistry. I was the first 

Director of EPA's Office of Drinking Water Criteria and Standards Division after passage 

of the Safe Drinking Water Act, where I had a role in developing many of the existing 

drinking water regulations and regulatory methodologies, and later in the Risk Assessment 

Division in Pollution Prevention and Toxics. Those regulations included comprehensive 

microbial protection, trihalomethanes, radionuclides, Surface Water Filtration, corrosion 

control, Volatile Synthetic Organic chemicals (VOCs) and numerous other organic and 

inorganic chemicals. Currently, I work internationally in water quality, health and 

technology including helping to establish a drinking water regulatory program in 

Singapore, and on desalination and health related issues and epidemiology in the Middle 

East, and on development of small water treatment technologies for home and small 

systems applications, as well as on some basic toxicology studies. I serve on several 

independent advisory groups dealing with drinking water safety issues, including many 

years with the World Health Organization's Drinking Water Quality Committee, which 

last week issued the 4th edition of the Guidelines for Drinking Water Quality. These are the 

benchmarks that most of the world outside of the US try to apply to define drinking water 

safety. I am also a member alternate of the Washington DC Water and Sewer Authority. 
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I am not here representing any organization or institution; these are my personal thoughts 

and conclusions, and I am not receiving any compensation for this presentation. 

• Drinking Water Quality and Safety 

Almost all public community water systems in the United States provide safe drinking 

water --including Washington, DC-- contrary to what some believe, and that is no small 

accolade and accomplishment when dealing with the 60,000 entities providing drinking 

water, which range in population from as few as 25 persons to millions. The quality and 

safety of drinking water in the United States is very good and almost always more 

consistent and better than the limited number of nations that have substantial drinking 

water regulatory and enforcement programs in place. We always strive for improvement. 

US policies and processes for safe drinking water provision have clear leadership standing 

in the world and many if not most of the existing methodologies for assessing and 

regulating drinking water quality and safety were developed here first. 

The EPA's and the states and water suppliers' implementation of the Safe Drinking Water 

Act of 1974 and the later amendments has significantly improved the safety of drinking 

water in the United States. It is indeed a joint effort that is implemented 24/7 by the often 

underappreciated professionals in the national drinking water delivery community that we 

all rely on to actually produce and deliver safe water. The comprehensive protection 

program includes regulation, advice, monitoring and system assessments and operations, 

supported by an enforcement program, and significant financial support from the federal 

government. The underpinning of it all is that there is that cadre of dedicated water 

professionals who manage and operate those water systems. 

The record shows that reported waterborne infectious disease outbreaks have declined 

since implementation of the SDWA began, but the portion of outbreaks due to distribution 

system infrastructure deficiencies has been increasing. That is a clear indication that the 

water coming out at the tap isn't always as good as the treated water leaving the plant. It is 

a symptom of our ageing infrastructure. Fixing and maintaining infrastructure and 

reducing leaks and contamination in distribution is where our priorities now belong. 
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• Current Regulations 

The 1986 amendments to the SDW A specified a long list of chemicals to be regulated, and 

also a requirement to regulate 25 additional substances every 3 years, which was physically 

impossible. As a result several MCLs were produced for substances that were unlikely to 

be present in significant amounts or nationally widespread or of significant concern. The 

compliance monitoring over the years has aptly demonstrated that, so there are actually 

more current regulations than needed to assure drinking water quality. The 1996 

amendments required EPA to produce a Contaminant Candidate List (CCL) every 5 years 

(3 produced to date), make a determination every 3 years whether up to 5 meet the criteria 

to warrant possible regulation, and also to periodically generate lists of unregulated 

contaminants to be analyzed (UCMR) in a number of locations to determine occurrence 

and exposure from water. In my opinion the CCLs and UCMRs have generally not been 

critically prepared to isolate high probability substances of potential concern in drinking 

water. Although systematic processes have been utilized to produce them, there must be a 

serious flaw in the decision logic as has been demonstrated by their results. 

The current regulations contain Maximum Contaminant Levels or Treatment 

Requirements for a broad spectrum of contaminants: 

0 81 MCLs for Organics and Inorganics including: 

27 pesticides and 9 DBPs (indicators) 

21 Inorganics 

24 VOCs and other synthetic organics 

0 Microbial: 6 Treatment requirements, Total Coliforms, E. Coli= all pathogens 

0 5 ( 2 group) Radionclide MCLs +Uranium= All radionuclides 

0 Lead and Copper corrosion control: Treatment Requirements 

0 Acrylamide and Epichlorohydrin: Treatment Technique as product quality. 

The coverage is actually far greater than the numbers imply. Essentially all microbial 

pathogens are covered by the MCL and treatment requirements. All radionuclides are 

covered by those rules, and treatment requirements such as surface water filtration and 

disinfection address numerous contaminants simultaneously. Disinfection byproducts 
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(DBP) indicator MCLs are intended to trigger techniques that result in across the board 

reductions of many unmeasured DBPs. 

The existing regulations are very comprehensive as they cover almost all of the categories 

of potential drinking water contaminants including: microbial contamination in great 

detail, natural precursor products, inorganic chemicals, disinfection byproducts indicators, 

radionuclides, corrosion products, volatile and other synthetic organics, and pesticides. 

The potential for microbial contamination bas been and will always be the greatest threat 

to health from drinking water. Although some outbreaks still occur, public water suppliers 

with Safe Drinking Water Act mandates have very successfully dealt with microbial threats, 

and no regulatory action should ever jeopardize our continued control over that threat. 

As they should be, the regulations are designed to be protective, and they are produced 

with very conservative health assessments that are generally designed to overestimate 

potential risks. They utilize conservative default assumptions in the absence of detailed 

data, e.g. on margins of safety for toxicity, linear non threshold extrapolation models, and 

on occurrence and exposure and relative source contributions. I look at conservative risk 

assessments and regulations as a sort of insurance policy that we pay for to assure that 

there are not now and will not be meaningful risks from drinking water. However, 

conservative assumptions must be rational and carry mainstream scientific credibility, 

because, in a way they can convert risk assessors into pseudo regulators. The issue is 

ultimately how large should the safety margins be, and how much insurance cost is 

appropriate. There is a price, and over regulation can also have negative consequences, 

such as by unnecessarily increasing costs to consumers, and by limiting use of some 

beneficial technologies. Also, excessively dwelling on hypothetical and negligible risk 

concerns by regulators and the press also drives some people to mistrust their public 

drinking water supply and pay the extra (perhaps lOOOx) cost of bottled water. 

• Examples of Some Pending Issues 

When producing a national regulation, EPA must, by law as well as common sense, 

determine that the contaminant is of national significance, is a risk to health, and also 
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demonstrate that the regulation will achieve meaningful health risk reduction. Here are 

several examples of current issues that are being debated and considered for regulation by 

EPA, including perchlorate, pharmaceuticals and nitrosamines. It is useful to put them into 

perspective relative to the context of the SDWA mandates. 

• Perchlorate 

Perchlorate in the environment, diet, and drinking water at low parts per billion levels, is 

potentially both of natural and anthropogenic origin. Perchlorate (as well as many other 

common anions) competes with iodine uptake by the thyroid and under some condition and 

dose could cause adverse consequences especially in infants. There has been much debate 

in the US, and some states have set standards or action levels at very low parts per billion 

(ppb) levels. EPA produced a guideline at 15 Jtg /L (15 ppb) in drinking water, and is now 

engaging in a regulatory development process. On the other hand in 2011, JECFA, the 

Joint Expert Committee on Food Additives, an independent multinational external 

toxicology/health science advisory committee of the UN Food and Agriculture Organization 

and World Health Organization released their analysis of the level of concern for 

perchlorate in Report 959. After reviewing the toxicology, epidemiological and clinical 

evidence, the NAS report from which EPA derived its current value, and dietary and water 

concentrations including much US data, they calculated a provisional maximum tolerable 

daily intake (PMTDI) of 10 Jtglkg bw/day and drew the following conclusion: 

"The estimated dietary exposures of 0. 7 Jtglkg bw/day (highest) and the 0.1 Jtg/kg 

bw/day (mean), including both food and drinking water, are well below the PMTDI. 

The committee considered that these estimated dietary exposures were not of health 

concern." 

• Pharmaceuticals 

In recent years there have been reports of detections of several pharmaceuticals in some 

drinking waters mostly at parts per trillion levels. Pharmaceuticals can reach drinking 

water from upstream wastewater discharges and runoff from some animal feed lots. They 

are attenuated to some degree partly by the wastewater treatment process, environmental 

passage (biodegradation, decomposition and dilution), and drinking water treatment. Most 

of the entry of pharmaceuticals into wastewater comes from human excretion of 
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pharmaceuticals and metabolites after therapeutic uses; some fairly small portion derives 

from improper disposal of unused drugs. One week ago the World Health Organization 

issued a report that included contributions from a 10 member Working Group on 

Pharmaceuticals in Drinking Water, of which I was a member, and which reviewed much 

US and other data. WHO concluded: 

"Trace quantities of pharmaceuticals in drinking water are very unlikely to pose 

risks to human health because of the substantial margin of exposure or margin of 

safety between the concentrations detected and the concentrations likely to evoke a 

pharmacological effect." (i.e. the therapeutic dose) 

"The current levels of exposure to pharmaceuticals in drinking water also suggest 

that the development of formal guideline levels for pharmaceuticals in the WHO 

Guidelines for Drinking Water Quality is unwarranted." 

Furthermore, WHO went on to say that routine monitoring and installation of specialized 

drinking water treatment was not deemed necessary. 

• Nitrosamines 

Nitrosamines are formed in some industrial processes, ubiquitous in foods, generated 

during cooking of proteins, and produced by humans endogenously from ingestion of 

precursor substances and oxynitrogen compounds. They are also found in some drinking 

waters at parts per trillion levels, especially those with upstream wastewater discharges, 

and also in some cases from disinfection with chloramine and from some polymers used as 

part of water treatment. Almost all nitrosamines are considered to be carcinogens. EPA 

has listed several nitrosamines in its Contaminant Candidate List (CCL 3) and in 

unregulated contaminant monitoring lists. 

The regulatory challenge is that the portion of daily exposure to nitrosamines due to 

drinking water in those locations is likely less than 1% of all the other sources. The 

monitoring costs are substantial, and water treatment possibilities are not completely 

defined. Because the relative source contribution from drinking water sources is so 

minimal, a low Maximum Contaminant Level that would impact some number of those 

water supplies that contain some nitrosamine would have a negligible, if any, impact on 

risk and public health. 
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• Unregulated Contaminants 

In theory, addressing unregulated contaminants in a formal way is a reasonable concept to 

determine which substances may warrant further assessment and possible regulation 

because of exposure from water and potential health risk. However, my sense is that EPA's 

approach to date has not been very efficient or effective. The process has been operating 

for more than 10 years and a huge amount of preparative and assessment work has been 

done, but there have not been any new regulations developed to date. In fact, that points to 

the obvious conclusion that there are not very many contaminants that demand national 

regulation to protect public health beyond those already on the books. On the other hand, I 

think EPA could have arrived at the same conclusion and identified perhaps a few 

candidates for regulatory consideration by a much more direct and efficient process. It 

should be said in EPA's defense that they were partly driven to that process by advice from 

a National Academy of Sciences report and from their statutory National Drinking Water 

Advisory Council. 

• Recommendations 

Overall public drinking water quality in the US is very good and the drinking water is safe, 

and undoubtedly safer than it has ever been since the introduction of centralized water 

supplies more than 200 years ago, and introduction of collected wastewater discharges. The 

potential for microbial contamination has and will always be the greatest threat. Public 

water suppliers with Safe Drinking Water Act mandates have very successfully dealt with 

microbial threats, and no action should ever jeopardize our continued control over that 

threat. 

However, concerns are commonly raised because current and rapidly developing analytical 

technologies continually produce new detections at parts per billion, parts per trillion and 

even lower levels, and this trend will continue and expand, because analytical science 

becomes more sensitive. Those exposures are essentially always extremely unlikely, if not 

implausible, to have any meaningful risk. These amounts are many orders of magnitude 

below levels that can show a detectable effect from even high dose testing. 
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The number of substances in the water environment can be large, but the amounts are very 

small, and the likelihood for requiring national drinking water regulations to protect 

public health for many ofthem present at minute trace levels is not great. As a practical 

matter, it is simply physically and economically impossible, and also unnecessary to have a 

long list of regulated substances in drinking water. The resources and time required to 

produce regulations are great and the process is not responsive to the needs of water 

suppliers and public health officials to have access to a scientifically credible basis for rapid 

decision making when a substance is detected. That is precisely why the EPA Office of 

Drinking Water initiated the Health Advisory program in -1980, and about 200 Health 

Advisories currently exist. 

Risk assessments always utilize conservative default assumptions when detailed 

information is not available. Scientific knowledge progresses constantly and sometimes the 

risks may turn out to be less than previously estimated or assumed, so it is essential for 

credibility to always allow or require EPA to utilize the best mainstream scientific 

information when it is making regulatory decisions. It might be necessary to amend the 

"anti backsliding" provision in the SDWA to allow that to occur. 

• Suggested Drinking Water Act Implementation Strategy 

We have accumulated a significant history of drinking water composition and quality in 

the US and causes of contamination have decreased, so it would be logical and appropriate 

to utilize that knowledge to revise our approach to assuring the safety of public water 

supplies. Following is a suggested more efficient strategy to cope with the realities of safe 

drinking water provision and assurance of continued safe and safer drinking water. 

I. Eliminate regulations that do not meet significant risk, national significance and 

meaningful risk reduction tests. Many regulations on the books have negligible occurrence 

near levels of concerns demonstrated by many years of compliance monitoring. Convert 

them into Health Advisories (see # 7). This might require legislation. 

2. There needs to be application of rational and faster prescreening and prioritization 

methodologies such as the Threshold of Toxicological Concern (TTC) that has been used 

for years by the FDA and other organizations to screen food additives. 

3. Add probably a small number of regulations that would meet the 1996 SDW A tests 

(national prevalence, health risk and meaningful risk reduction), and periodically update 

as needed. 
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4. When regulating, utilize individual MCLs, Treatment Technologies, or groupings as 

justified and technically appropriate. These have been traditionally utilized in 

implementation of the Safe Drinking Water Act, to date. 

5. Always utilize the best available mainstream science when performing risk assessments 

and making regulatory decisions. 

6. Emphasize addressing ageing distribution system infrastructure, because it is the likely 

greatest current and future drinking water health risk concern that requires correction. 

7. Periodically carry out national surveys of source and finished drinking water 

composition so as to be up to date on what is present and might warrant regulation. 

8. Greatly expand the number of peer reviewed Drinking Water Health Advisories 

(DWHAs) on substances that have been or would probably be in drinking waters. This is 

very cost effective and it would provide a compendium of substances in drinking water 

with authoritative guidance on health significance. It is responsive to the needs of water 

suppliers and public health officials for making rapid judgments in the event of a detection 

of a new substance. 

e.g. DWHAs for most pesticides and pharmaceuticals can be readily produced from 

existing very robust registration and drug approval data bases.c 

9. EPA should provide guidance and assistance to states to help them develop local need 

regulations-health advisory assessments, technology performance and cost, and analytical 

methods. 

10. EPA should facilitate applications of needed and new technologies developed by others, 

e.g. provide technical assistance to states and water suppliers by technology demonstrations, 

and via Technology Verification programs. 

11. More aggressively use Clean Water Act, Toxic Substances Control Act, and Pesticide 

authorities to reduce contaminant introduction and burdens on Public Water Supplies. 

12. Focus on preventing environmental and drinking water contamination. Industrial 

discharge controls have been successful. Manage pharmaceuticals by improved sewage 

treatment and controls on disposal, and pesticides by water basin management controls on 

applications. 

13. Focus CCLs and UCMRs on most likely candidates rather than as shopping lists 

containing only a few candidates of likely concern. 

14. Partner with states for sharing validated water monitoring data in a usable common 

electronic format so that information on national circumstances and trends is always 

readily available. 
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This proposed strategy is: 

0 Comprehensive and forward looking, anticipatory and sustainable. 

0 Provides coverage of many more contaminants, essential benchmarks, and 

nationally consistent advice. 

0 Consistent with the SDW A mandate to protect public health. 

0 Much more efficient and cost effective for EPA, the states and water suppliers. 

0 Reduces wasteful compliance monitoring for public water suppliers 

0 Further reduces source contamination. 

0 Reduces uncertainties for water and state and local regulatory officials and the 

public. 

0 An opportunity to get ahead of the curve on interpretations of significance of trace 

contaminant detections using margins of safety and margins of exposure (MOE) to 

provide officials and the public a better perspective on DW quality and "risk" e.g. 

pharmaceuticals. 

0 It provides timely leadership and national consistency on water health related issues, 

(applications in Hazardous Waste Sites, Superfund ••• ) 

Downsides-not many. 

• States might not always regulate their local need contaminants-EPA has 

supervisory oversight authorities under the SDWA implementation. 

• State regulations will not always be uniform---they aren't always now. 

The bottom line is that this approach is responsive to the need, sustainable, credible, 

and efficient, and it provides more public health protection and much more bang for 

the buck! 

Thank you for the opportunity to present these comments and suggestions. 
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23819.082

Environment and Public Works Committee Hearing 

July 12, 2011 

Follow-up Questions from Senator James M. lnhofe and responses for written submission 

from Joseph Cotruvo 

1. Given the limited resources that utilities and EPA have when making investments in 

improving drinking water, where do you think the area of greatest need is? 

The greatest need is certainly not for new regulations that add costs with questionable or 

marginal benefits, if any. Currently, there is excellent regulatory coverage of significant 

contaminants and perhaps only a few additional might be appropriate in the near term, if they 

can be properly justified according to the requirements of the 1996 amendments to the Safe 

Drinking Water Act. The principal needs are for investment in infrastructure repair and 

replacement and especially in water distribution system infrastructure. Leaks, water loss or 

unaccounted-for water are major cost drains and sources of waste for water supplies and the 

environment, and of public resources. Recent information from the Singapore and Tokyo water 

systems indicates that due to their continued commitment, their unaccounted-for water is now 

on the order of 5% or less, which is much lower than in the recent past. By comparison 

Philadelphia estimates on the order of 30% unaccounted-for water and that is not unusual in 

the US. Pipe breaks in old systems also result in major disruptions and costly repairs. 

Contamination can enter leaking distribution piping during periods of low pressure. 

Infrastructure replacement needs to be accelerated, because it is cost effective to bring older 

water systems up to shorter average lifetimes. This would reduce water loss, pipe breaks and 

emergency repairs. Infrastructure upgrades are economically and environmentally beneficial 

and sustainable because they reduce waste of water and money. 

2. How would increased federal assistance in fixing and maintaining infrastructure provide 

meaningful opportunities for public health improvement? Would funding the SRF 

program be important in accomplishing this? 

Improvements in water treatment provide high quality water entering water distribution 

systems. The incidence of reported waterborne disease outbreaks has been reduced in the US, 

but the portion of distribution-related outbreaks and cases has been increasing. Much of this 

trend is due to legionellosis cases. Old and leaking distribution systems can result in 

recontamination or regrowth of some microorganisms, partly due to inability to maintain 

disinfectant residuals in the system. Old pipe is commonly cluttered with tubercle encrusted 

surfaces that harbor biofilms and microbes, and restrict the water flow. Better maintained and 

cleaner systems result in cleaner water reaching consumers. Federal assistance by revolving 

loan funds to be repaid at interest provide incentives for water suppliers to improve their 
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23819.083

systems. This is cost effective by improving water safety, reducing leaks, water losses and pipe 

breaks, so that the economics of water production would improve for most supplies. It also is a 

source of labor intensive shovel-ready projects for job creation for the long term, because it 

requires a continuous commitment from the water supplier. 

3. California and Massachusetts recently set MCLs for perchlorate at 6 ppb and 2 ppb, 

respectively, and EPA may set an even lower level for drinking water. Based upon your 

experience, does reducing perchlorate to levels like these have any additional health 

benefit to the public? 

Perchlorate is present in the environment from both natural and anthropogenic origins. It is 

among the several anions that compete with uptake of iodine by the thyroid. In its unregulated 

contaminant monitoring rule data from 2001-2005 EPA found that 4.1% of the more than 3800 

systems tested exceeded 4Jlg/L (4 ppb). So, it is not a very widespread issue in drinking water. 

Perchlorate is not unique to drinking water; it is also present at parts per billion levels in 

numerous foods. EPA's 2009 interim guideline of 15 jlg /l (15 parts per billion) in drinking water 

utilized a conservative 2005 recommendation from the National Academy of Sciences of 24 Jlg 

/day. This was based upon a non adverse NOEL (No Observed Effect level) of 7 micrograms per 

kilogram of body weight per day, and reference dose of 0. 7 micrograms per kilogram of body 

weight per day, that is "likely without appreciable risk of deleterious effects over a lifetime". 

The reference dose value is conservative because it was derived from a No Observed Effect 

level (NOEL) rather than a NOAEL (No Observed Adverse Effect level), and then an additional 

10 fold uncertainty (safety) factor was applied. USFDA did not recommend changes in diet, and 

it did not recommend taking dietary supplements with additional iodine. EPA is now engaging 

in a regulatory development process and the outcome is unknown, but the implication is that 

EPA thinking is that the guideline is too high and should be reduced. 

In 2004 the American Thyroid Association reported results from occupational studies indicating 

that "even long-term, intermittent high perchlorate exposure does not appear to alter thyroid 

function significantly, and it would also be expected that perchlorate in drinking water may not 

have a significant effect." A Chilian study (Tellez, 2004) of nonsmoking pregnant women 

determined that perchlorate in drinking water at levels as high as 1141lg/l (114 ppb) during 

pregnancy did not affect maternal thyroid status early in gestation or fetal thyroid stratus at 

birth. 

The 2011 analysis of the Joint Expert Committee on Food Additives (JECFA), an independent 

multinational external toxicology/health science advisory committee of the United Nations 

Food and Agriculture Organization and World Health Organization came to a very different 

conclusion than EPA, CA or MA. In their report, they stated that none of the ecological studies 
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23819.084

showed any relationship between perchlorate concentrations in drinking water and the 

incidence of thyroid diseases. After reviewing the toxicology, epidemiological and clinical 

evidence, including the NAS report from which EPA derived its current value, and dietary and 

water concentrations including much US data, JECFA calculated a provisional maximum 

tolerable daily intake (PMTDI) of 10 jlg/kg bw/day, with a 10 fold uncertainty (safety) factor to 

cover high risk subgroups. This value is equivalent to 700 jlg per day for a standard US 70 kg 

person, and 100 jlg per day for a standard 10 kg infant/child. JECFA concluded that current daily 

exposures from food and drinking water were well below that PMTDIIevel so they were not of 

health concern. 

Thus, the current EPA Guideline of 15 jlg /l (15 ppb) in drinking water is already conservative 

and protective of health, and the much lower California and Massachusetts standards would 

not be expected to result in any benefits. According to the JECFA conclusions all of those values 

(EPA, CA, MA) would be extremely conservative. Thus, a low drinking water standard would 

not reduce risk or contribute to greater benefits to public health. 

4. EPA's Office of Inspector General has concluded that of the three common goitrogens 

(thiocyanate, nitrate and perchlorate), perchlorate amounts for Jess than 1 percent iodide 

uptake inhibition, while the other two chemicals account for more than 99% of uptake 

inhibition. What is the meaningful opportunity for public health risk reduction through 

the regulation of perchlorate, given that more than 99% of the effect is related to other 

chemicals? 

Numerous anions have some competition with iodide for transport into the thyroid, and anions 

are ubiquitous. Perchorate has high binding affinity, but the other anions are present in much 

greater amounts. Nitrate is frequently present in drinking waters and also in the diet both 

naturally, e.g. in vegetables like spinach, and as an additive in some processed foods. 

Thiocyanate is primarily a natural dietary component from several vegetables like broccoli and 

cabbage, and endogenously produced from precursors in the natural diet. Smokers have 

particularly high levels of thiocyanate in their systems. Much experimental toxicology has been 

conducted with rats, however, rats are much more sensitive to disturbances in thyroid function 

than humans, so rat data must be carefully interpreted and probably deserves uncertainty 

factors of less than one rather than the usual multiples of 10 that are common for animal to 

human extrapolation. Thus, human data are the best types of information for human risk 

assessments for these types of chemicals and endpoints. A scientifically valid analysis would not 

consider the anions individually, but evaluate aggregate effects from all sources to determine 

whether there is a risk that needs to be reduced and, if so, which strategy would be most 

effective and sensible, especially for smokers. 
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23819.085

S. How do deficiencies in IRIS risk assessments affect EPA's ability to make regulatory 

decisions in the Drinking Water program? 

IRIS has the role of providing objective, timely, comprehensive and authoritative risk 

assessments to support EPA's regulatory programs. To accomplish this it must produce 

scientifically credible assessments and in a timely manner. The draft chromium VI IRIS 

document is a recent example of a non scientifically credible IRIS assessment as demonstrated 

by the comments from the recent external peer review that EPA conducted. In order to be 

credible IRIS must represent mainstream scientific thinking and not be an organ for either 

internal or external fringe positions that could unreasonably constrain policy choices. Its work 

must undergo very detailed external broad spectrum independent peer reviews by qualified 

credible scientists, and then its conclusions must be practical and represent the weight of 

scientific evidence along with the alternative options and their strengths and weaknesses. IRIS 

should not wander into hypothetical risks that are very small, low probability and de minimis, of 

dubious validity, and which can never be verified. It must include the peer review results in its 

recommendations to the regulatory programs. The EPA regulatory programs usually have 

scientific staff, and the responsibility to produce beneficial and defensible regulations, so they 

should have the opportunity to not accept the IRIS conclusions for good cause, if they are not 

defensible. 

With a few exceptions, drinking water is usually among the smallest contributors to human 

exposure to natural and anthropogenic environmental contaminants; diet is surely the greatest 

source of exposure and risk for most natural and synthetic substances. It could be argued that 

in some cases drinking water regulations may give excessive attention to drinking water and 

skew priorities when it is a negligible contributor to actual risks. 

6. Other comments 

A. The Safe Drinking Water Act as amended in 1996 has the elements needed to provide 

continuing protection of public health through safe drinking water. However, EPA's 

recent implementation strategy needs to be updated to become more efficient and 

provide the kind of comprehensive guidance that public water supplies need to cope 

with new potential concerns or questions that may arise. The existing regulatory list 

contains numerous superfluous standards that just add monitoring and administrative 

costs without commensurate benefits. The cost and time required to produce a 

regulation are very high, so only a few substances would warrant that expenditure. At 

the same time water suppliers and states and the public are left without benchmarks to 

address questions about significance or lack of significance of trace substances that are 

being detected by ever more sensitive analytical methods. 
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23819.086

National standards should only be developed within the three determinants of the 1996 

SDWA amendments: 1. National significance, 2. Risk to health, and 3. Meaningful risk 

reduction. Localized issues should be dealt with by states with technical assistance from 

EPA. The testimony contains a suggested 14 point comprehensive strategy that provides 

a starting point for EPA to update its approach to providing leadership for safe drinking 

water assurance and to be responsive to the current and future needs of all of the 

stakeholders. Its principal recommendation is a shift away from producing more costly 

and inefficient MCLs and standards to much greater emphasis on scientifically based 

and peer reviewed Health Advisory guidelines that can be much more comprehensive 

and timely and supportive of the roles of the states, and much less costly to produce. 

B. The current Safe Drinking Water Act has an "anti backsliding" provision that makes it 

very difficult for EPA to raise an MCL if more current scientific data demonstrates that 

the actual risk from the substance in drinking water is less than what was thought when 

the original MCL was written. This utterly rejects the essentiality of good science in 

making regulatory decisions and requires that water suppliers make continued 

expenditures to meet a discredited standard. Any water supplier or state has the right to 

meet a lower standard if they choose to do so, and can justify it to their constituents, but it 

should not be required by federal law. This provision is a candidate for reconsideration when 

the congress next reviews the Safe Drinking Water Act. 

C. wastewater reuse for beneficial purposes is also worthy of much greater 

encouragement by the federal government. Wastewater has finally been recognized as 

a beneficial resource that should be maximally exploited, rather than treated and 

discarded. It is a source of nitrogen and phosphorous nutrients, biosolids for beneficial 

land application, oils and grease as potential biofuels, methane for energy production, 

and water for irrigation, as well as highly treated water for direct or indirect human 

consumption. Treatment technology of recycled water for potable use is similar to 

desalination of seawater and only about half the cost. It has been demonstrated at full 

scale that the current advanced wastewater treatment processes can produce water 

that is at least as high as and probably higher quality than good quality natural waters. 

Recycle programs for commodities like paper, plastic, and glass are usually difficult to 

justify economically because of the high cost of collection and transport. On the other 

hand wastewater recycling already has the collection and transport system (sewers) in 

place, so it is an ideal application. Wastewater recycling has the additional benefit of 

reducing discharges to the water environment, so it helps to clean our waterways. 
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23819.087

Beneficial wastewater reuse is a phenomenon whose time has arrived. It is a win, win, 

win! 
124 

Senator BOXER. Your time has passed, and we need to move on. 

We thank you. 

Dr. Steven Patierno, we are glad that you are here. And let me 

give your background to the folks. Executive Director of the George 

Washington Cancer Institute. We are very happy you are here. And 

is also Vivian Gil here with you? 

Mr. PATIERNO. I am sorry? 

Senator BOXER. Is Vivian Gil here with you? 

Mr. PATIERNO. No. Vivian Gil is a historical donor to the George 

Washington University endowing a professorship, which I hold. 

Senator BOXER. Aha, that is what I was going to say. It says, this 

is so funny, it says Vivian Gil Distinguished Professor of Oncology, 

so I thought she was a distinguished professor. 

[Laughter.] 

Senator BOXER. But we have you, and you are the distinguished 

professor. So we are thrilled that you are here. Go ahead. 

STATEMENT OF STEVEN R. PATIERNO, PH.D., EXECUTIVE DIRECTOR, GEORGE WASHINGTON UNIVERSITY CANCER CENTER 

Mr. PATIERNO. Before I start, I would like to also quickly apologize for the length of my comments. I only found I was invited to 

attend this on Friday and just didn’t have time to hone this down 

to exactly 5 minutes, as some of my colleagues have. 

Senator BOXER. We don’t have a choice, because we need to go 

on. So please, go ahead. Do your best. 

Mr. PATIERNO. Madam Chairman, Ranking Member Inhofe, and 

members of the Committee, thank you for this opportunity to address you today. As you have heard, my name is Steve Patierno, 

I am the Director of the GW Cancer Institute. I am also a professor 

of pharmacology and physiology in the School of Medicine and a 

professor of environmental and occupational health in the School of 

Public Health. 

I have been conducting research for over 31 years on hexavalent 

chromium, 23 of those funded by the National Institute of Environmental Health sciences and the National Cancer Institute. I have 

published many papers, served on many review committees. I 

would also like to disclose that although I have never worked for, 

consulted directly for any company associated with any chromiumrelated industry, over the course of 30 years of working in chromium, I have on seven occasions served as an expert for the defense in chromium litigation. 

A recent release issued by the Environmental Working Group entitled Chromium–6 in U.S. Tap Water reported that very low levels 

of hexavalent chromium were found in drinking water in 31 U.S. 

cities. Most of the cities with the highest of these low levels of ambient chromium–6 have little or no proximity to any chromium-related industry, indicating that these levels likely constitute a natural background, results that are neither new nor unexpected. The 

report and the associated media coverage, which was purposeful in 

referring to chromium–6 as the carcinogenic Erin Brockovich chemical, has caused unnecessary fear and alarm, as these levels constitute no health risks to humans. 

125 

It should be stated that there is a vast literature on occupational 

and industrial exposures to high doses of chromium compounds for 

long periods of time, a large literature on animal studies and defined systems. Valid conclusions are only drawn when complementary data come together and fundamental principles and practices 

of pharmacology and toxicology must be factored into an accurate 

analysis. 

Taken together, the consistent message is that only long-term 

high dose exposures to chromate dusts, as encountered in the chromate production pigment and plating industry have been associated with human cancer, and only for cancers of the respiratory 

tract. The same studies also showed there was no consistent association with any increased risk of other cancers. This is attested to 

by every major government international agency review ever written. 

All three major areas of risk related study, epidemiology, animal 

studies and cell culture provide clear evidence for very high threshold levels for both toxicity and carcinogenicity. And in fact, epidemiologic studies can identify no effect levels despite long-term occupational exposures that are factored over a 45 year work history. 

Likewise, published meta-analyses, investigated gastrointestinal 

tract cancers, and all of this analysis shows no significant risk in 

malignancy for any GI site. 

An often overlooked fact is Dr. John Morgan, an epidemiologist 

working for the State of California Cancer Registry has been tracking cancer incidence in the town of Hinckley, California, the Erin 

Brockovich town, for the past 15 years. And he recently reported 

that not only is there no excess of total cancer or any specific type 

of cancer in Hinckley, there is actually fewer cancers than expected. 

Evidence for a very high threshold level for chromium carcinogenicity is found in the recent NTP animal studies where rodents 

were administered very high concentrations of chromium–6 in the 

drinking water continuously for 2 years. Tumors were only observed at the two highest doses, and they were so high the water 

was unpalatable to rodents. And they resulted in long-term chronic 

tissue damage. There is a figure in my comments. I would ask you 

to note the sharp high-dose threshold for toxicity and that no toxicological effects were observed, even at doses that are nearly five 

orders of magnitude, 100,000 times higher than the average tap 

water concentration reported in the EWP report. 

Despite Hollywood depictions, chromium–6 is not potent as either a toxic or a carcinogen. Enormous quantities of chromium are 

needed to evoke any kind of toxicity in humans or animals. This 

is because humans have a many-tiered, innate mechanism of protection against chemical toxicities of any sort, including chromium. 

It is critical to understand that chromium compounds exist in two 

forms, a trivalent form, which is an essential element required for 

human physiology, and a hexavalent form. Hexavalent chromium is 

very, very rapidly and completely reduced to trivalent chromium by 

a number of protective mechanisms and fluids in the human body. 

And recent studies have shown that this conversion takes place in 

less than a minute. 

126 

If it does reach a cell, there are intra-cellular mechanisms of protection that prevent it from directly interacting with DNA. So it is 

not correct to assume that if a vanishingly small amount of 

hexavalent chromium reaches a cell, it automatically becomes carcinogenic. 

[The prepared statement of Mr. Patierno follows:] 

127 


23819.088

Testimony of Dr. Steven R. Patierno, Ph.D. 

Executive Director, George Washington University Cancer Institute 

Before the U.S. Senate Subcommittee on Environment and Public Works: Full Committee Hearing 

Entitled "Oversight Hearing on the Environmental Protection Agency's Implementation of the 

Safe Drinking Water Act's Unregulated Drinking Water Contaminants Program". 

Tuesday, July 12, 2011; EPW Hearing Room- 406 Dirkson 

Chairman Boxer, Ranking Member lnhofe, and members of the committee, thank you for the 

opportunity to appear before you today to testify on EPA's work on the Safe Drinking Water Act's 

Unregulated Drinking Water Contaminants Program, specifically with respect to hexavalent 

chromium. 

My name is Steven Patierno and I am the Executive Director of The George Washington 

University Cancer Institute (GWCI) and the Vivian Gil Distinguished Professor of Oncology at the 

George Washington University. I am also a Professor of Pharmacology and Physiology, and 

Genetics at The George Washington University School of Medicine and Health Sciences; a 

Professor of Environmental and Occupational Health at The George Washington University School 

of Public Health and Health Services; and the Founding Director of the Molecular and Cellular 

Oncology Program. I am actively engaged in several areas of cancer research and intervention 

including drug discovery, cancer health disparities, patient navigation and cancer survivorship, but 

what is most relevant to today's discussion is that I have been conducting research on hexavalent 

chromium for 31 years and my basic science laboratory has been funded by the National Institute of 

Environmental Health Science and/or the National Cancer Institute, continuously for 23 years, to 

study cellular and molecular mechanisms of hexavalent chromium toxicology and carcinogenesis. I 

have published more than 120 peer-reviewed scientific papers and have served on numerous 

review panels for hexavalent chromium risk assessment including both the previous and the current 

EPA Toxicological Review of Hexavalent Chromium in support of the Summary Information on the 

Integrated Risk Information System (IRIS). I would also like to disclose that although I have never 

worked or consulted directly with or for any company associated with the chromium production or 

use industries, over the course of 30 years working in chromium toxicology I have, on seven 

occasions, served as an expert for the defense in chromium litigation. 

A recent press release issued by the Environmental Working Group, entitled "Chromium-6 in 

U.S. Tap Water" reported that very low levels of hexavalent chromium were found in drinking water 

from 31 U.S. cities. The average level reported was 0.18 ppb with a range of 0.03 to 13 ppb. 

Interestingly, most of the cities with the highest levels of ambient Cr(VI) have little or no proximity to 
128 


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any chromium-related industry, indicating that these low levels constitute a natural background. 

Given that chromium is a natural component of the earth's crust, these results were neither new nor 

unexpected. The EWG report and the associated media coverage, which was purposeful in 

referring to Cr(VI) as the "Carcinogenic Erin Brockovich Chemical", has caused unnecessary fear 

and alarm, as these levels constitute no health risk to humans. 

Before analyzing this low level exposure scenario it should be stated that there is a vast 

literature on occupational and industrial exposures to high doses of chromate compounds for long 

periods of time, as encountered in the chromate production and plating industries in the early to 

mid-1900s. There is also a very large literature on the effects of chromate compounds in animals 

and in defined systems such as cell culture. Valid conclusions are typically drawn when 

complementary data from different types of studies indicate that an observed effect is reproducible, 

dose-dependent, free from confounding variables and statistically significant. Concepts which are 

fundamental to the principles and practice of pharmacology and toxicology, including dose, duration 

of exposure, route of exposure, metabolism, toxicokinetics and detoxification, must also be factored 

into an accurate analysis. 

Taken together, the consistent message is that only long-term, high dose exposures to 

moderately or highly insoluble particulate forms of either chromate dusts or concentrated chromic 

acid mists, (or in the words of the International Agency for Research on Cancer [IACR]. "as 

encountered in the chromate production, pigment and plating industry") have been associated with 

human cancer and even then, only for cancers of the respiratory tract. These same studies, which 

consistently detected a positive correlation with respiratory cancer, also showed there is no 

consistent association with any increased risk for any other cancers. This is attested to by every 

major government or international agency-related review ever written including the International 

Agency for Research on Cancer (IARC), the previous US EPA Toxicological Review, and the 

Agency for Toxic Substances and Disease Registry (ATSDR). 

Furthermore, all three major areas of risk-related study, (epidemiology, animal and cell 

culture) provide clear evidence for a very high threshold levels for both toxicity and carcinogenesis. 

For example, in an analysis of occupational risk in a cohort of more than 2300 workers in the 

chromate production plant by Gibb et al, widely cited as a reference paper for risk assessment, no 

increased risk for lung cancer was observed in long-term occupationally exposed workers exposed 

to concentrated mixtures of chromic acid mist and/or dusts of chromate particulates at a mean 

exposure level of 450 ng/m3

-yr (Odds ratio of .96 factored over a 45 year work history). These 

exposures were high enough to cause severe nasal tissue damage including perforation of the 

nasal septa. At more than a 9-fold increase in mean exposure (4,200 ng/m3

-yr), the Odds ratio for 
129 


23819.090

risk was increased to 1.42 but the increase was still not statistically significant. Statistical 

significance must be achieved in order to validate that a given observation is real and not the result 

of chance. Only when exposure was 7-fold higher again (30,000 ng/m3-yr), did the odds ratio for 

risk achieve statistical significance at 1.57. It should also be noted that 116 of 122 workers who 

developed lung cancer after long-term, high-dose exposure, were also smokers. 

Likewise, a published Meta-Analysis investigated Gastrointestinal Tract cancers (oral, 

esophageal, stomach, small intestine, colon and rectal) in all epidemiology studies of Cr(VI) 

exposed workers published after 1950 (Gatto et al. 2010), including 32 studies from various 

industries wherein airborne concentrations were extremely high and resulted in oral exposure as 

evidenced by yellow stained teeth, tongues and Gl distress showed no significant increased risk for 

malignancy at any Gl site. 

An additional often-overlooked fact is that Dr. John Morgan. an excellent epidemiologist 

working for the State of California Cancer Registry, has been tracking cancer incidence in the town 

of Hinckley CA (the "Erin Brockovich" town) for the past 15 years. He recently reported that not 

only is there no excess of total cancer or any specific type of cancer in Hinckley, there are actually 

fewer cancers than expected. 

Further evidence for a very high threshold exposure level for Cr(VI) carcinogenesis is found 

in rodent carcinogenesis bioassays conducted by the National Toxicology Program (Stout et al, 

2009) and in a multi-center analysis of Cr(VI) Mechanism of Action by ToxStrategies (first paper just 

published by Thompson et al, 2011 ). In the NTP study mice and rats were exposed to 

extraordinarily high concentrations of Cr(VI) (0, 14, 57, 172, or 516 ppm of sodium dichromate 

dihydrafte) in the drinking water continuously for two years. In the NTP study, tumors were only 

observed in the small intestine of mice at the highest two dose groups, relative to concurrent 

controls, and at all the doses in the NTP study, the high concentration exposures resulted in longterm tissue damage in these tissues. Thompson et al. 2011 administered Cr(VI) in drinking water to 

mice at the same concentrations as NTP in the NTP study and at two lower concentrations, 

including the current Federal drinking water standard (the Maximum Contaminant Level or MCL). 

As shown below from Thompson et al. 2011, no toxicological effects were observed even at doses 

in the mouse small intestine that are nearly five orders of magnitude (100,000 times) higher than 

the average tap water concentration of Cr(VI} in that study, which was conduted in Birmingham, 

Alabama. The drinking water concentrations of Cr(VI) for the control animals ((not dosed with 

Cr(VI)) in the Thompson et al. 2011 study, and the NTP study which was conducted by the same 

laboratory, are consistent with the levels reported across the US in the EWP report. 
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23819.091

80 

E 

:::! 

55 60 

"0 

0 

:::! 

0 40 0) 

'i::: 

(..) 20 

TapHzO 

(control) 

1 

Drinking Water, mg/L Cr(VI) 

The following figure shows the NTP study doses relative to the current MCL and the 95'" 

percentile drinking water concentration in the US and in California for which extensive drinking 

water monitoring data exists. Note the break in scale for the highest dose. 

l' f, ! f ' . ' 1 ~ 

TolaiCrMCL 

It is important to understand that despite Hollywood depictions, Cr(VI) is not "potent" as 

either a toxin or a carcinogen and in fact if anything should be recognized as a very weak potential 

carcinogen. Enormous quantities of chromium(VI) are needed to evoke any kind of toxicity 

(demonstrated and documented by cases of accidental or suicidal poisonings) in humans or 

animals, and as mentioned above, respiratory carcinogenicity in humans is only associated with 

high-dose, long term occupational exposure to chromate dusts or concentrated chromic acid mists. 
131 


23819.092

As stated by Paracelsus in the 161

h century (who is widely recognized as the father of pharmacology 

and toxicology), "all substances are poisons and there is none which is not a poison, the right dose 

differentiates a poison from a remedy). For example, over-the-counter drugs from a local pharmacy 

(like acetaminophen) have either no effect or a therapeutic effect at low doses, but at high doses 

even such widely available drugs can be toxic or even lethal. In the same way humans have manytiered, innate mechanism of protection against chemical toxicities of any sort, including Cr(VI). 

To understand this, is it critical to understand that chromium compounds exist, for the most 

part, in two chemical forms called hexavalent and trivalent chromium. Trivalent chromium ((Cr(lll)) 

is chemically very stable and is actually an essential element that is required for normal human 

physiology. We get large amounts of Cr(lll) from our diet. After ingestion, very little of it is 

absorbed and the part that is absorbed into the body does not easily get into cells. Its essential 

activity as co-factor in insulin regulation seems to take place at the surface of cells but not inside. 

In contrast, hexavalent chromium is chemically similar to sulfate (another essential element) and it 

can slip inside of cells posing as sulfate. However, Cr(VI) is chemically unstable and easily 

converted to Cr(lll) by a large number of natural component of our saliva, gastric juice, lung fluids, 

and blood components. Once Cr(VI) is converted to Cr(lll) outside of our cells it behaves as an 

essential element and does not readily enter cells. 

Recent studies using human gastric juice show that even large quantities of concentrated 

solutions of Cr(VI) are completely converted to Cr(lll) in less than a minute. At low concentrations 

the conversion is almost instantaneous. This means that unless enormous concentrations of Cr(VI) 

employed, high enough to overwhelm the conversion capacity of our body fluids, little or no Cr(VI) 

will reach the surface of any cell. In addition, any tiny amount Cr(VI) that temporarily escapes 

instantaneous reduction would encounter the mucous lining of the respiratory tract and Gl tract and 

not have easy access to the actual surface of any cell. If by chance a massive dose is administered, 

high enough to saturate the conversion capacity of lung or gastric fluids, some Cr(VI) may be 

absorbed into the bloodstream. There it will encounter an even greater capacity of our blood 

plasma to convert Cr(VI) to Cr(lll) making it virtually impossible for Cr(VI) to arrive at other tissues 

as Cr(VI) except at massively toxic concentrations. This conversion capacity serves as a powerful 

barrier to Cr(VI) toxicity and its existence creates a very high threshold for Cr(VI) toxicity: it must be 

overwhelmed before toxicity can occur. The fact that all cells have a sulfate transport system that 

Cr(VI) can piggy-back on does mean that Cr(VI) can cause every type of cancer. One cannot 

ignore all the many-tiered protective barriers that prevent Cr(VI) from getting to surface of cells 

distal to the point of entry. Even at the massive doses administered in the NTP study, Cr(VI) did not 

cause cancer outside the Gl tract. 
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Once Cr(VI) finally enters a cell it has to be converted to other forms of chromium inside the 

cell in order to become toxic. It also encounters additional barriers to becoming toxic because 

these other forms of chromium are quickly bound up by protective molecules inside of cells and 

rendered inactive. Only when an intracellular concentration of Cr(VI} is reached that overwhelms 

these protective barriers can it begin to interact with important macromolecules like DNA and 

protein. The excess Cr(VI) may damage these molecules through a process of chemical oxidation, 

usually leading to the destruction of the cell. Cr(lll) formed inside of a cell by conversion from 

Cr(VI) is capable of binding to DNA and many studies, including from my own laboratory, have 

reported on what we thought was a mutagenic mechanism of action as result of DNA damage by 

Cr(lll). However, for many years we have been concerned about the fact that very high doses and 

highly contrived experimental conditions, high enough to kill most of the exposed cells, were 

necessary to detect mutagenesis. Many of us, including eight of the nine current reviewers of the 

Draft Toxicological Review, have come to understand that what we thought was mutagenesis was 

in fact more likely a process of selection for chance survivors of the toxic treatment. The ninth 

member abstained, but only out of concern that the EPA's linear default model is a historical 

precedent that is not likely to be overruled. Nevertheless, it more likely that the carcinogenic MOA 

of Cr(VI) under high dose, long term exposure conditions is due to chronic tissue damage, 

inflammation and chronic regenerative cell proliferation. At doses lower than the threshold there 

simply is no MOA because there is no toxicity or carcinogenesis. These important concepts need 

to be considered by the EPA. 

Before I conclude I need to address one other important issue. As one can quickly discern 

from the EWG report there are some who have taken to espousing the opinion that even 

vanishingly small. short term exposures to Cr(VI) are capable of causing a plethora of human 

diseases and virtual every type of cancer known to mankind. To support this premise reference is 

frequently made to two "opinion" articles in the literature, one in 1997 and one in 2006, published in 

the journal Critical Reviews in Toxicology. These articles were written and published at a time 

when the senior author was actively engaged as an expert witness for the plaintiffs in high-profile 

chromium lawsuits, but this involvement was not disclosed in the 1997 article. Production of the 

2006 article was paid for by the plaintiffs law firm but this was only partially disclosed. These 

papers were cited by the Draft 2010 EPA Toxicological Review of Hexavalent Chromium, but 

premise of these papers is not accepted by the general scientific community and it was 

unanimously rejected by the current nine-member review panel of the Draft Toxicological Review 

because the methodology applied is severely flawed. 

It is well established that when large scale epidemiological studies examining many 
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endpoints are conducted, random fluxuation due to the breadth of the study will result in number of 

false positives (random, usually small and nonstatistically significant increases in risk rates for 

specific cancer). Statistical significance is extremely critical in epidemiological studies because the 

number of comparisons in a typical epidemiological study make it inevitable that some increased 

SMRs or Odds Ratios would be arrived at by chance. One must look for consistency across 

multiple studies to determine whether it is real. In these articles whatever instances that could be 

found in any epidemiology study of chromium, of an elevated Standard Mortality Ratio (SMR) were 

picked and presented in tabular form with no consideration of the fact that most of these instances 

were small, non-statistically significant elevations that were ignored or discounted by the original 

authors because of confounding factors. The paper also failed to show that many of these random 

non-significant elevations in some cancers in one selected study were counter-balanced by either 

no elevation or decreased SMRs in other studies. This is incorrect scientific methodology but it 

illustrates the importance of critically evaluating epidemiological data from original journal articles 

and not relying on an opinion paper as the Draft EPA Toxicological Review apparently did. 

Thank you for the opportunity to address you today. 
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Further Background on Chromium and Chromium Carcinogenesis 

Examples of Additional Papers Incorrectly Cited to Suggest that Occupational Exposures to 

Cr(VI) can cause cancers other than Respiratory Cancer. 

Another published paper that is sometimes cited (including in the 2010 Draft EPA 

Toxicological Review of Chromium) in an attempt to link environmental chromium exposure to NonHodgkin's lymphoma is Sick et at, Int. J. Hematol. 64:257-262, 1996). This paper should be retracted 

from the scientific literature. Two of the authors were lead lawyers for the plaintiffs in several highprofile chromium lawsuits, now immortalized by the Hollywood movie "Erin Brockovich". They listed 

their "academic" credentials as the Department of Hematology at the University of Tasmania in 

Australia. The other three authors were paid expert witnesses for the plaintiffs in the same case, which 

was active at the time. None of this was disclosed in the paper. The two cases of Non-Hodgkin's 

lymphoma discussed in this case report were plaintiffs in the active lawsuit and the information was 

supplied by the lawyers. Moreover, at best this report is merely a case-report (not even a case-control 

study), merely reporting that two people in Hinckley CA, at that time, had been diagnosed with NonHodgkin's lymphoma. 

In contrast, for perspective it is important to look at historical occupational exposures that 

were associated with increased risk (summarized in the ATSDR Toxicological Profile for Chromium, by 

IARC, and by OSHA in the Federal Register). The history of the recognition of certain chromium 

compounds in lung and other respiratory cancers traces back to Scotland in the late nineteenth century 

and to Germany in the 1920's. In the work environment of the 1920's through 1950's, the levels of dust 

in factories were so high that is was said to be difficult to see across the factory floor (hundreds of 

micrograms to milligrams of chromium per cubic meter of air). Workers had no protective gear and they 

would leave work with chromate dust encrusted on their clothes and in their noses. Much of the dust 

would be inhaled and swallowed. Worker safety protocols and health monitoring were non-existent. 

The increases in lung cancer provoked the application of modern industrial hygiene practices in these 

industries, and by the 1960's most Western plants using chromium had instituted industrial hygiene 

practices that dramatically reduced exposure to airborne particulate chromates and virtually eliminated 

the adverse health outcomes associated with chromium exposure. 
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The consensus of scientific opinion, summarizing a very large number of epidemiological 

studies, animal studies and mechanistic studies, is that an increased risk of lung cancer can be 

associated with long-term, high dose exposure to either acid mists of soluble Cr(VI), or highly insoluble 

particulate dusts of calcium, lead or zinc chromate, as they were encountered in occupational settings 

such as mining or production industries. Even under such heavy exposure conditions, there was no 

association of exposure with increased risk for cancer of any other organ system other than lung 

cancer, and most of the chromium-exposed workers were also smokers. Among soluble chromates, 

only occupational inhalation exposure to concentrated mists of chromic acid in the chrome plating 

industry were associated with increased risk for respiratory cancer. 

For example, in risk-associated production industries (dichromate and chromium trioxide) 

and pigment industries, men were exposed to concentrated dusts of the low solubility particles of lead 

chromate and zinc chromate. In some plants, exposure levels were found to range from 10,000 to 

190,000 ng/m3 for an average of 18 years. In some plants, CrVI exposures averaged around 170,000 

ng/m3, often for more than 20 years. Some risk was noted after a two year exposure in a plant with 

exposure levels above 400,000 ng/m3. In other plants, multi-year exposures of 250,000-490,000 

ng/m3 were associated with lung cancer. Among pigment workers, an SMR of 190 was determined for 

workers exposed for more than 2 years at 500,000-2,000,000 ng/m3. Another plant yielded at risk 

workers with exposures of 500,000-1,500,000 ng/m3 for 6-9 years. 

In risk-associated chrome plating industries, men were exposed to mists of concentrated chromic acid. 

Risks were generally limited to men with greater than 15years work. A three month exposure to 

chromic acid mist found no excess. Only men working directly with or near the chromic acid baths were 

at risk. In an Italian plant, increased risk was found for men working at least 1 year near the baths with 

airborne levels of the acid mist at 60,000 ng/m3. In a Czech plant, air levels near the baths were above 

400,000 ng/m3. 

Basic Principles of Chromium Toxicology 

Chromium is used in many different industrial and commercial practices and products 

including stainless steel, chrome plating, leather tanning, as an anti-rust agent, and in various dyes, 

paints and alloys as a pigment Urban air concentrations from air pollution average 10-30 ng/m3 but 

can range up to 500 ng/m3

. Soil typically contains 40-400 ppm (ng/mg) of chromium. Chromium in 

foods is present in the range of 20-520 ppb (ng/g) but varies widely with food type. Chromium is also 

present in tobacco, and is found in cigarette smoke. 

There are two major "oxidation" states of chromium that are important for understanding the 

biology and chemistry of chromium, i.e., chromium(lll) (Cr(lll), Cr+3, trivalent chromium) and 
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chromium(VI) )Cr(VI), Cr+6, hexavalent chromium). Other oxidation states exist, and with the 

exception of chromium(O), which is neutral as chromium metal and is inert, these other oxidation forms 

are transient. Chromium(lll) is the form of chromium found in nature, usually complexed with several 

other elements in ores or soils. It is also the form that is an essential trace element in humans and is 

found in virtually all plants and animals. Chromium is ubiquitous in the environment (principally as 

chromium(lll), and is found in water, air, soil and rock. 

Chromium(VI) forms many different types of compounds, such as highly insoluble titanium 

and lead chromate, moderately soluble calcium chromate and zinc chromate, and the highly soluble 

sodium dichromate. Chromium( Ill) also forms many different types of compounds including "inorganic" 

forms such as chromium chloride, and "organic" or biological forms such as chromium picolinate and 

Low Molecular Weight Chromium complex (LMWCr). Each form exhibits different physico-chemical 

and biological properties. 

The word "Chromate" or "chromate ion" or "chromate oxyanion" refers to chromium(VI) 

bound to four oxygen atoms (Cr04

•2), and is the fully "dissolved" form of chromium(VI) which is able to 

cross cell membranes. The ability of chromate to dissolve is highly dependent on the initial form, i.e., 

the slightly soluble calcium, zinc and lead chromate releases chromate slowly, while the highly soluble 

sodium dichromate releases chromate readily in solution. 

A major discovery was made by Mertz and co-workers at the U.S. Department of Agriculture 

in the 1950s, which was later confirmed by Schroeder, indicating that Cr(lll) is an essential trace 

element in animals. Schroeder and colleagues showed that an absence of chromium in the diet led to 

glucose intolerance (a diabetic-like state) in animals. This was confirmed in a number of other 

laboratories, and chromium joined selenium, iron, zinc, calcium and other metals on the list of elements 

that are essential in the diet for normal health. 

Later studies of total parenteral nutrition (TPN), receiving all nutrition from the artificial liquid 

diet in an intravenous bag, further confirmed that chromium is an essential element. Lack of chromium 

produces progressive glucose intolerance, a diabetic-like state that was not responsive to the addition 

of insulin, but immediately reversed after adding Cr(lll) to the TPN. Cr(lll) is now included in all TPN 

solutions. Every major nutritional expert, society, organization, and government and international panel 

has concluded that chromium(lll) is an essential trace element in humans. Chromium(lll) is added to 

many over-the-counter multi-vitamin and mineral supplements. Only one or two papers have attempted 

to say otherwise, at least one of which was written under financial inducement by a law firm with a 

vested interest in characterizing all Cr, including Crill, as a potential hazard (see preceding comments). 

Except for those few citations it is almost universally accepted that Crill is an essential element. 

Chromium(VI) exists at low levels in nature, but is produced industrially for commercial 
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purposes by oxidizing the chromium(lll) to chromium(VI) using a process called roasting (strong 

oxidizing conditions and very high temperatures). Biological systems do not posses the oxidizing 

power needed to convert chromium(lll) to chromium(VI) but chromium(VI) is readily chemically reduced 

to chromium(lll) both in the environment and in humans and those of other animals and plants. The 

rapid and ready reduction of chromium(VI) to chromium(lll), and chromium(lll)'s essentiality in humans, 

is critical for understanding how chromium interacts with the human body. 

As described above there is broad consensus that long-term occupational inhalation 

exposure to dusts of the intermediate soluble forms, calcium chromate, zinc chromate and lead 

chromate, or to concentrated chromic acid mists is associated with an increased risk of lung and other 

respiratory cancers. These historic occupational exposures to chromium (VI) that were associated with 

respiratory cancer were also characterized by irritation of skin by direct contact with very high 

concentrations of chromium dusts or acidic solutions. Nasal irritation has also been observed with high 

concentrations of airborne chromium(VI) and is a hallmark of occupational exposures. These overt 

dermal effects have not been observed below the current occupational thresholds. It is welldocumented that these workers frequently had chromium-related skin lesions. Follow-up of these 

workers has demonstrated no increase in skin cancer or other cancers. 

Animal studies show that certain intermediate soluble forms of chromium(VI) (calcium in its 

sintered form, zinc and lead chromate) rather than the soluble forms (like sodium chromate) or the 

highly insoluble forms (such as barium chromate), are potentially carcinogenic at the site of exposure. 

These studies also indicate that these compounds are not carcinogenic at any sites distal to the route 

of exposure. Even the compounds of intermediate solubility are only weakly carcinogenic in these 

tests, and only if the animals are exposed in a way that circumvents a normal exposure route. These 

include directly injecting chromium(VI) compounds into muscles, lungs or trachea, or implanting caged 

cholesterol pellets of chromium compounds into the animals' lungs. Positive results were only seen 

with the highest, oftentimes overtly toxic doses, and even under such conditions the tumor incidence 

was low. 

The NTP toxicology studies on subchronic oral exposure are technically well done. The 

principle issue that needs to not be lost in the detail is that even the lowest dose was 14.3 mg/L (ppm) 

of sodium dichromate dehydrate) (5ppm of CrVI), a concentration sufficient to overwhelm oral and 

gastric reductive capacity. Despite these enormous doses most of the observations did not exhibit a 

consistent pattern of dose or duration dependence. It is also important to recognize that these 

enormous doses of CrVI actually serve to deliver an enormous amount of Crill to the organs and cells 

in question. Remembering that Crill is not without biological activity (acting as a co-factor in insulin 

action), it is entirely possible the some of the observed effects are due to the physiological effects of 
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massive Crill overload. 

Even at these high doses a consistent relationship between severity and dose was not 

observed. This implies the presence of effects caused by indirect mechanisms, likely chronic 

inflammation and/or tissue damage only observed at the highest doses. Urinalysis shows effects due 

to decreased water intake due to poor palatability of the yellow water. This dehydration alone is 

capable of rendering epithelial tissues more fragile. Changes in organ weights were only observed at 

doses above 500ppm (180 ppm Cr(VI). 

The results of the NTP assays are described repeatedly as ''without clear dose-response 

relationship". Indeed, minimal to mild histiocytic cellular infiltration was observed in all groups including 

the control animals. Even less toxicity was observed in mice compared to rats; in fact even at 1000 

ppm for 3 months there was no evidence of any hepatotoxicity, only mild changes in some 

hematological indices that were attributed to changes in body weight (probably caused by massive Crill 

overloading and its potential effects on insulin and glucose metabolism). What needs to be appreciated 

is that the lowest dose used in any of these studies is at or above saturation of gastric reductive 

capacity and yet still very little toxicity was observed except at the two highest doses (and often only at 

the one highest dose). At the lower end of these very high doses, only inconsistent observations were 

made and when "toxicity" was reported it was generally ranked minimal to mild. Only the index of Liver 

(fatty change) was ranked as moderate, but that was identical to the ranking of that same index in the 

Controls. The main point here is that these are massive doses and they are eliciting minimal effects. 

This important concept should not be lost in the mass of detailed results. 

The NTP carcinogenesis studies in rats and mice show that there is no carcinogenic 

response except at the two highest doses that also produce chronic tissue damage at the sites of 

carcinogenicity. The dose-response is definitively non-linear, as is the absorption data described 

above. Given that the lowest dose is already above the reductive capacity of the oral cavity and 

stomach, these data provide strong evidence of the protective effects of the reductive capacity of blood 

components. 

It should be noted that the NTP's published report by Stout et al [Hexavalent Chromium is 

Carcinogenic to F344/N Rats and B6C3F1 Mice after Chronic Oral Exposure, Environmental Health 

Perspectives 117: 716, 2009] presents an inaccurate discussion of potential mechanism of action, 

drawn heavily from the 2006 Costa article, especially in criticizing the work of DeFiora. In point of fact, 

the results of the NTP assay give nearly definitive proof that the work of DeFiora is correct. Even the 

lowest dose of the NTP assay exceeds the reductive capacity of the oral cavity and upper digestive 

tract. Yet little toxicity and no carcinogenicity is observed except at the two highest doses. 

The argument by Stout et al that the NTP doses were below gastric reduction-saturation, 
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based on a supra-linear (decreasing response with dose) rather than sub-linear (increasing response 

with dose) dose response is incorrect. If the doses were below saturation of reductive capacity, as the 

dose increased the ratio of unreduced CrVI to reduced CrVI (Crill) in the stomach would increase (due 

to depletion of reductive capacity), and absorption would show an increasing rate of response (opposite 

of what was observed) because of an increased percentage of the total Cr that would be in the 

unreduced hexavalent state. Yet both absorption and toxicity exhibit a decreasing rate of response with 

dose in the NTP assay. This would actually be expected at supra-saturation doses: once the reductive 

capacity of the oral, digestive and blood components is exceeded, the organs receiving the highest 

amount of CrVI will sustain inflammatory tissue damage provoking tissue regeneration. It is unlikely 

that such tissue damage would display dose dependence since it only occurred at the two highest 

doses of the assay and it is a complex, disseminated biological response. It is likely then that a 

combination of three factors contribute to the high dose carcinogenic response: (i) tissue damage with 

regenerative cell profieration, (ii) regenerative cell proliferation in the presence of macromolecular 

damage, and (iii) regenerative cell proliferation occurring in the presence of massive Crill loading, 

which may affect insulin-dependent proliferative signaling. Thus, the scientific evidence does not 

support the conclusion that low level environmental exposure to chromium(VI) is associated with health 

effects of any kind. 

This is explained by the complex chemico-biological interactions and pharmacodynamics of 

chromium and the ability of the body to rapidly and effectively reduce chromium(VI), the potentially toxic 

form of chromium, to chromium(lll), the biologically essential form of chromium. At the physiological 

level, a broad scientific and governmental consensus has embraced the Physiological Reduction model 

first put forward by Dr. Silvio De Flora. At the cellular level, a broad scientific and governmental 

consensus has embraced the Uptake-reduction model of Dr. Karen Wetterhahn. 

At the physiological level, De Flora and co-workers have produced a model of chromium 

reductive metabolism that explains the highly selective toxicity of chromium(VI) to the respiratory 

system at high doses. The extracellular components of tissues and body fluids possess tremendous 

capacity for reducing chromium(VI) to the essential element chromium(lll). This is true for all three 

routes of exposure that are relevant to humans, i.e., inhalation (breathing of dusts, mists, vapors, etc.), 

oral ingestion (swallowing of chromium from dusts, mists, etc. in mucous and saliva, food and water 

intake) and dermal exposure (dusts, solids, mists and liquids on the skin). Dermal exposure to 

chromium(VI) does not yield significant absorption of chromium because the dermis acts as a physical 

barrier and also has high reduction potential. Similarly, oral ingestion of chromium(VI), even at high 

doses, is expected to result in rapid and near-complete (depending on the dose) reduction of 

chromium(VI) to chromium( Ill) with little chance for absorption of chromium(VI). Total absorption rarely 
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exceeds 1 0% by this route. The reason for this is that the saliva, the gastric juices, and then the 

intestinal fluids all have enormous reductive capacity for chromium(VI). Recent studies have shown that 

even concentrated solutions of Cr(VI) (1mM) are almost completely reduced to Cr(lll) by gastric juice in 

less than one minute. Only very high doses of Cr(VI) will overwhelm this reductive capacity. Small 

amounts of Cr(VI) that temporarily escape reduction (because the rapid reductive rate constant is not 

perfectly instantaneous) will encounter the mucous barrier lining the respiratory and gastrointestinal 

tract and not get ready access to the cell surface. At extremely high doses some Cr(VI) may be 

absorbed as Cr(VI) (see below for further discussion), but will quickly be reduced to Cr(lll) by 

interaction with huge quantities of reductive agents in the blood. As shown in many studies, it is 

extremely difficult to deliver a genotoxic dose of Cr(VI) to a tissue distal to the point of injection: except 

under conditions of massive dose the administered Cr(VI) arrives at distal tissues as Cr(lll). 

At the cellular level, the Wetterhahn uptake-reduction model describes the intracellular 

metabolism of chromium. Chromium(lll) crosses cell membranes very poorly due to its structure and 

charge. Chromium(VI), on the other hand, is taken up by cells much better, since it has the same basic 

structure and charge as phosphate and sulfate. It should be noted that although it is often stated in 

review articles that chromium "readily crosses cell membranes", only a fraction of the available 

chromium(VI) outside the cell (2-10% in most cases, depending on the system) actually crosses into 

cells even under idealized cell culture conditions. Once inside the cell, chromium(VI) is rapidly 

reduced, ultimately to chromium(lll). In the process of this reduction, it goes through various 

intermediates, including chromium(V) and chromium(IV). It may also generate reactive oxygen species 

and other radical species but this is still under investigation, especially since it is now know that Cr(VI) 

is capable of direct oxidation of biological macromolecules. Following chromium(VI) uptake and 

reduction by cells, various forms of DNA damage can be measured, and chromium(VI) treatment of 

cells can increase mutations. Thus, the basic model for chromium carcinogenesis is that Cr(VI) 

reduction outside of cells is protective and Cr(VI) reduction inside the cell can lead to macromolecular 

binding, DNA damage and toxicity (explained in more detail below). 

Chromium(lll) crosses cell membranes only poorly due to its chemistry, and functions 

outside the cell by forming an amino acid complex, which is called either Low Molecular Weight Cr 

complex (LMWCr) or Chromodulin. This complex binds to the external surface of cells and enhances 

insulin signaling thereby helping to control glucose tolerance of our bodies and this mechanism is now 

well-established. A normal diet provides approximately 50-200 ug of Cr(lll) per day and daily ingestion 

is required since Cr(lll) is readily excreted from the blood into urine. Only 0.5-2% of available 

chromium(lll) is absorbed. 
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Summary of Principles Derived from My Own Chromium Research 

Not all chromium (VI) compounds are equally carcinogenic: 

Trivalent chromium (Crill) is nearly completely negative in virtually every animal bioassay 

and every in vitro assay it has been tested in. It is not recognized as a either a mutagen or a 

carcinogen and in fact there is strong evidence that it functions as an necessary dietary essential 

element. Thus, this report will focus primarily on hexavalent chromium (CrVI) but will address trivalent 

chromium when necessary. 

It is extremely important to understand that Cr(VI) exists in many different forms. There are 

completely soluble forms of Cr(VI), such as sodium and potassium chromate, which dissolve easily in 

water. Cr(VI) also exists in the form of solid particles which exhibit varying degrees of solubility. Some 

particulate forms are almost completely insoluble and can hardly dissolve in water at all (such as 

titanium chromate), some are mostly insoluble such that only a small amount dissolves in water (forms 

such as lead chromate), and there are moderately insoluble forms that dissolve to a moderate degree 

(forms such as calcium chromate and zinc chromate). 

Early on it was recognized that both the epidemiological studies on chromate-exposed 

workers, and the in vivo (in the living animal) carcinogenesis assays of Cr (VI)-exposed animals, 

revealed that not all Cr (VI) compounds could be implicated as carcinogens. The epidemiological 

studies revealed that the site of action was almost exclusively limited to the respiratory tract and that 

the exposures were primarily through inhalation of either large quantities of particulate chromium (VI) 

compounds of limited solubility (moderately to highly insoluble) for long periods of time (as in chromium 

mining and chromate production), or chronic inhalation of a chromic acid mist (chrome platers). This is 

why the International Agency for Research on Cancer (IARC) has classified Cr(VI) as a carcinogen, "as 

it is encountered in the chromate production, chromate pigment production and chromium plating 

industries". 

The early animal carcinogenesis data supported this concept. Less than half of the total 

animal experiments yielded a positive result, and the vast majority of animal experiments using soluble 

hexavalent chromium compounds were negative; only rarely and inconsistently was an extremely weak 

response observed using multiple repeated high dose exposure regimens. The early studies showed 

that only the particulate compounds of limited solubility were capable of tumor induction and only at the 

site of administration. This strongly suggested that there was something unique about the chromium 

particles of limited solubility such that long term, high dose exposure to them was weakly, but 

measurably, carcinogenic. It was this hypothesis that I first tested while still a postdoctoral fellow at 
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USC and then continued researching independently at GWUMC. These studies have helped us 

understand the differential carcinogenic potential of different Cr(VI) compounds. 

The concepts emerging from my laboratory are summarized below. Using lead chromate as 

a prototypical particulate of limited solubility, we found that these particles are negatively charged and 

approximately 1 micron in diameter in the shape of hexagonal rods. Upon inhalation they are capable 

of deposition on any impact surface and most will adhere to the mucous lining of the respiratory tract. 

Most of the inhaled particles will be engulfed by specialized particle-scavenging cells called 

macrophages and removed through the mucous escalator to the mouth where they are spit out or 

swallowed. Some of the particles may adhere to cells of respiratory tract, and although they exhibit 

only limited solubility in water, the particles in contact with the cell surface begin to dissolve in the 

immediate micro-environment of the cell. Some of the particles are also internalized into lung 

fibroblasts or epithelial cells by a process of engulfment called phagocy1osis. Only doses and durations 

of exposure high enough and long enough to evoke marked amounts of cell killing are capable of 

inducing mutagenesis and carcinogenesis. Most of the Cr(VI) oxyanions being dissolved from the 

particle outside the cell are quickly reduced to the trivalent form of chromium which is not readily 

absorbed by cells. The capacity of the extracellular milieu to reduce Cr(VI) to Cr(lll) is markedly 

increased by the presence of ascorbate (vitamin C). In fact, the ability of ionic Cr(VI) produced by 

particle dissolution to get into cells and cause DNA damage can be nearly completely obviated by 

supplementing the cell grow1h medium with physiological concentrations of ascorbate. Some of the 

Cr(VI) released from the particles onto the surface of the cells will enter the cells through the anion 

transport system and undergo reductive metabolism to form pentavalent, tetravalent and ultimately 

trivalent species inside the cell. The principle intracellular reductants are ascorbate (vitamin C) and 

glu1athione (GSH). Oxidative intermediates may also be generated in the process, but whether they 

are produced in normal cells at non-lethal doses, and what their role is in cellular responses to 

chromium, has not yet been fully established. 

Modeling Particle Effects with Soluble Cr(VI): Evidence for a threshold: We have modeled the 

release of Cr(VI) ions from particles of limited solubility, in vitro, using soluble Cr(VI) compounds such 

as sodium chromate. Even in a cell culture medium, a completely "closed" system, which has a limited 

capacity to reduce Cr(VI) to Cr(lll), we have found that continuous exposure for at least 18-24 hours 

was required to achieve intracellular levels of chromium comparable to what lead chromate particles 

could achieve. Comparable intracellular levels could also be achieved using much higher doses for 

shorter periods of time (the concentration x time ratio) but it was found that at any given dose, no 

additional uptake or toxicity could be achieved by treatment times exceeding 24 hours. Thus, no 
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cumulative uptake or damage would be expected for durations of exposure longer than 24 hours. The 

uptake of soluble Cr(VI) is extremely sensitive to the addition of reducing components in the culture 

medium and is nearly completely blocked by the addition of vitamin C due to the nearly instantaneous 

reduction of Cr(VI) to Cr(lll). The amount of uptake in the presence of vitamin C is nearly identical to 

the low levels of uptake which occur when dosing with Cr(lll). Thus, there is clear evidence of a "no 

effect level" and a threshold for chromium toxicity, simply based on the composition of the extracellular 

medium and prevention of Cr(VI) uptake. 

We have also found clear evidence for an intracellular threshold for toxicity. For example, 

treatment of human lung fibroblasts with less than 1 uM sodium chromate [approximately 50 ppb of 

Cr(VI), or 50,000 ng/L of culture solution), for 24 hours, had absolutely no effect on cell survival, 

whereas 2, 4, 6 and 8 uM for 24 hours dose-dependently decreased cell survival from 100% to <5%, in 

one of the steepest survival/dose curves that we have ever observed. Thus, at the level of 50 ppb of 

pure hexavalent chromium in a closed system with limited reductive capacity (i.e. a petri dish), the 

chromium which enters the cell is effectively dealt with and completely detoxified. Given the enormous 

reductive capacity of human body fluids, one would have to start with a massive dose of soluble Cr(VI) 

to deliver 50 ppb (1 uM) directly to a cell. 

We have proven that the DNA damaging, pseudo-mutagenic and neoplastic potential of 

Cr(VI) compounds occurs only at doses which overwhelm both the extracellular and intracellular 

protective mechanisms and cause forms of cell death known as apoptosis and terminal growth arrest 

Over the past ten or so years, through research funded by the NIH, my laboratory has established the 

understanding that chromium carcinogenesis at the cellular level is likely to be a chronic process of 

selection of rare cells exhibiting gradually increasing resistance to cell death in the presence of chronic 

tissue damage due to long-term chronic exposure to toxic agents. Most recently, we have begun to 

identify the genes and molecular changes responsible for this rare conversion of lung cells to death 

resistance and my laboratory is at the forefront of elucidating the role of the ATM, AKT, ATR, PLK, 

ERK, p53 and other genes in the evolution of Cr(VI)-induced cancer. 

We have also conducted and published studies on the role of DNA repair in chromium 

carcinogenesis. Contrary to the statements made by those who merely assume that inhibition of DNA 

will lead to increased mutagenesis, we and others have actually proven that the opposite is true of 

chromium. Under certain circumstances of exposure of cells to significant doses of Cr(VI), we do 

indeed find that the treatment can inhibit DNA repair, but we have also found that the loss of DNA 

repair leads to decreased mutagenesis. Chromium mutagenesis is actually suppressed in cell strains 

lacking specific DNA repair genes. This does not support the theory that Cr(VI) is carcinogenic by 

inhibition of DNA repair. Taken together, this indicates that at low dose exposures, no carcinogenic 
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response should be expected, and at high dose, long term exposure, at best only a weak carcinogenic 

response should be expected because the predominant effect of these doses is to induce cell death. 

This is entirely consistent with the epidemiological studies linking an increased incidence of lung cancer 

with high-dose, long-term exposure to highly insoluble chromate particulates or tissue-damaging 

chromic acid mists. 

Why then did occupational chromium(VI) exposure increase risk of lung and other 

respiratory cancers? Firstly, workers were breathing in large concentrations of chromium-laden dusts, 

particularly those that penetrate to the deep lung (PM2 s or less). Secondly, workers were chronically 

exposed to these dusts 8 hours per day, 5 days per week, 50 weeks per year, 15-40 years, such that 

there was a large daily and cumulative dose. Thirdly, exposure was to the intermediate soluble forms 

of chromium(VI) such that the particles allow slow dissolution of chromium(VI) to the cells surrounding 

the particle slowly over time. Studies of the lungs of chromium workers have shown massive 

accumulation of chromium(VI) dusts in these individuals, sometimes leading to chromium levels that 

could approach 10% of the weight of the lung. Because these intermediate soluble chromium(VI) 

particles dissolve slowly and are poorly cleared, they remain for very long periods of time. 

It should be also be noted that although several epidemiological studies have suggested 

that the chromium-related risk of lung cancer in these workers may be distinguished over and above 

the risk from smoking, the vast majority of lung cancer cases were in chromium workers who were also 

smokers. Obviously, smoking is an additional potential source of chronic tissue damage. 

Even particulate Cr(VI) compounds are weak transforming agents: The prediction made above is 

borne out in experiments showing that at least moderately toxic doses of Cr(VI) were required in order 

to cause a measurable mutagenic or neoplastic effect in several different types of cultured cells. These 

experiments further demonstrated the uniqueness of some of the particulate forms of Cr(VI) since only 

the particles of limited solubility were able to induce morphological or neoplastic transformation. These 

concepts have been further confirmed by other independent investigators as well. Even highly 

cytotoxic doses of completely soluble chromates (sodium chromate) or moderately soluble chromates 

(calcium chromate administered in its particulate form), were unable to induce morphological or 

neoplastic transformation. Thus, independent of dose or relative toxicity, the soluble chromium 

compounds were exceedingly inefficient as transforming agents. This is likely to be due, at least in 

part, to the extracellular and intracellular protective threshold mechanisms described above. However, 

it is also important to note that the relative potency of even the particulate chromates in causing cell 

transformation is extremely weak relative to a classic organic polycyclic aromatic hydrocarbon such as 

3-methylcholanthrene. In experiments where completely non-toxic doses of 3-methylcholanthrene 
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would cause multiple cell transforming events in every culture dish, the chromate particles would barely 

induce 1 or 2 transforming events total in 20 culture dishes, and only at highly toxic concentrations. 

Because a culture dish is a closed, non-dynamic system, it is relatively easy to overwhelm 

the extracellular reducing capacity of the culture medium and force the uptake of Cr(VI) by the cells. It 

is important to recognize, however, that cells also have a number of intracellular barriers to chromium 

toxicity as well. For example, during the reduction of Cr(VI), the newly formed Cr(lll) is bound 

extensively by and to free amino acids, glutathione and small peptides and in this "liganded" state is 

dramatically less available for binding to critical macromolecules such as DNA. Also, many of the 

oxidative effects of Cr(VI) are reversed or blocked by antioxidants such as Vitamin E. If the intracellular 

barriers are also overwhelmed, reductive intermediates of Cr(VI) can cause a spectrum of DNA 

damage including single strand breaks, chromium-DNA adducts, DNA-protein crosslinks, DNA-DNA 

crosslinks, and chromosomal aberrations. Much of this genotoxicity can be prevented by pretreatment 

of cells with anti-oxidant vitamins such as ascorbate and tocopherol (Vitamin E). My laboratory has 

identified the chromium-induced DNA-DNA crosslink as one of the most damaging genotoxic lesions. 

We published a study showing that the presence of glutathione (a key intracellular protectant found in 

mM concentrations inside of cells) prevents formation of DNA-DNA crosslinks during chromium 

exposure. Another important consideration is that much of the DNA damage induced by chromium is 

repaired rapidly (for example all of the DNA breaks induced by toxic doses are repaired within 6 hours 

after treatment). We and others have shown that DNA repair may itself be partially inhibited by toxic 

Cr(VI) exposures. However, we have also shown that the loss of certain types of DNA repair leads to 

decreased chromium mutagenesis, not increased mutagenesis. Thus, not only are there profound 

barriers to chromium toxicity outside of cells in the body fluids, there are also barriers to chromium 

toxicity inside of cells as well. 

Intersection of our work with Chromium Absorption and Distribution: The extracelluar fluids of 

the human body possess enormous capacity to quickly reduce Cr(VI) to Cr(lll), thus Cr(VI) is not 

considered a systemic toxicant except at extremely high doses administered as a single dose. There is 

no oxidizing environment in the human body capable of re-oxidizing Cr(lll) to Cr(VI), thus Cr(lll) is the 

final stable product. At doses which do not overwhelm the reductive capacity of a tissue or a system, 

the Cr(VI) is quickly reduced to Cr(lll) with a half-life measured in seconds. Cr(lll) is most likely 

absorbed by either passive or facilitated diffusion through the interstitial spaces surrounding the 

mucosal cells lining the tissue. After a single dose, absorbed Cr(lll) will enter the bloodstream and 

transient increases in tissue chromium are followed by rapid elimination in the urine and accumulation 

of chromium in tissues cannot be detected. Chronic intake of high doses of Cr(lll) will result in 

sustained increases in tissue levels of Cr(lll), which quickly decrease as soon as the ingestion is 
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ceased. Human infants are born with high tissue levels of Cr(lll) which decrease with aging, probably 

due to nutritional deficiencies. 

When Cr(VI) is administered, most of it will be quickly reduced to Cr(lll) by fluids associated 

with the treated tissue. Cr(VI) will apparently be absorbed better than Cr(lll), but even at high doses, 

on average, less than 10% of Cr(VI) is absorbed. Another barrier to absorption of any Cr(VI) that 

temporarily escapes reduction is the mucous lining of the respiratory and gastrointestinal track. The 

mechanism of the increased absorption of the excess Cr(VI) is not known, but all of the possibilities 

would result in further reduction to Cr(lll). One possibility is that it would be taken up by mucosal cells 

as Cr(VI), then reduced to Cr(lll) intracellularly (and probably bound to peptides), as it is being 

transported across the mucosal cells for release into the blood. A second possibility is that the "excess" 

Cr(VI} is not really Cr(VI) at all, but a newly-formed type of Cr(lll) which is absorbed better than free 

Cr(lll). Recent published reports add significant support to the growing understanding that newly 

formed Cr(lll) is chemically and reactively different than "aged" Cr(lll), thus it is possible that newly 

formed Cr(lll), produced by reduction from Cr(VI), may be better absorbed from the Gl tract than aged 

Cr(lll}. This would further explain why total chromium absorption from the Gl tract is greater with 

Cr(VI), but that the chromium arriving in the blood and distal tissues is Cr(lll). It is also possible that at 

high doses, very small amounts of the excess Cr(VI) could theoretically be carried through the mucosal 

lining with the passive diffusion (absorption) of water. In this case the Cr(VI) would be absorbed by 

passing through the interstitial spaces and not by being transported into and across the cells of the 

mucosal lining. Regardless of how or in what form it crosses the mucosal lining, the absorbed 

chromium will make it into the blood stream where it will immediately encounter the enormous reducing 

capacity of red blood cells and the enormous protein binding capacity of the blood plasma. These 

principles are well established and recognized, having been reviewed by E.J O'Fiaherty in 1995 in 

Toxicology of Metals: Biochemical Aspects. 

Absorption by inhalation is limited by particle size, solubility, and phagocytic elimination by 

the 23 billion pulmonary macrophages (particle scavenging cells). The vast majority of inhaled 

particulates are efficiently removed by the muco-ciliary escalator. Highly soluble particles and aqueous 

mists (droplets) are reduced to Cr(lll) by ascorbate, glutathione and other reducing equivalents present 

in lung lavage fluid in high concentrations. Highly insoluble particles, administered at high chonic 

exposure levels that overwhelm macrophage capacity, may persist in the respiratory tract and may 

contact the cells of the lung lining, leading to the events described above. 

It is clear that we are faced with a unique situation in assessing the MOA of Cr(VI) at it 

relates to low-dose risk assessment. It is abundantly clear from all the science that the effects of Cr(VI) 

at the massive doses necessary to produce tissue toxicity and carcinogenesis in rodents, have no 
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bearing on the effects of low-dose, environmentally-relevant exposures. This is consistently borne out 

by epidemiological, animal and cell experimentation. This is especially pertinent in relation to whether 

or not Cr(VI) should be considered with a mutagenic MOA. I have spent more than 25 years studying 

the genotoxic properties of Cr(VI) and I have frequently contributed to the plethora of studies showing 

DNA damage and what we thought was associated mutagenesis. There is no doubt that Cr(VI) can be 

forced to be genotoxic and "mutagenic" under experimentally contrived systems using high doses that 

evoke major amounts of cell death. The question is, is Cr(VI) mutagenic at environmentally-relevant 

exposure levels? The growing consensus is that it is not. 

In hindsight many of us "DNA damage and repair" scientists have come to appreciate 

several important factors: (i) DNA damage is only observed at very high dose that kill a lot of cells, (ii) 

Cr(VI) is at best a very weak "mutagen", requiring very high doses that kill most cells and experimental 

"backflips" to select for survivors, and (iii) what we thought was "mutagenesis" is actually selection for 

stochastic cell survivors of massive toxic insult. Dr. Rossman's group at NYU has shown that the base 

sequence of the genes used for mutation detection and selection is intact and that the changes in gene 

expression enabling selection are epigenetic, not mutagenic. Our group has shown that what we really 

selected for at toxic exposures are cells that are resistant to apoptosis, and Dr. Zhitkovich's group at 

Brown has shown that the "mutant" cells were actually surviving cells that were selected for changes in 

specific forms of DNA repair. Again, this only occurs at doses that kill a lot of cells, not dis-similar to the 

high-dose rodent assays wherein tumors were only observed at doses that produced chronic and fairly 

severe tissue damage. 

Regulatory agencies may be under certain historical precedents and pressures to deem 

Cr(VI) with a mutagenic mode of action simply because there are published studies that have "Cr(VI)" 

and "mutation" equated in the title (some of these papers are my own), but this decision would not be 

based on recent science. At high, tissue damaging doses, one can get tumors to form and those 

tumors will have mutations in specific genes because that is the molecular history of how that particular 

type cancer develops. It will not have any relation to chemically-specific mutations caused by Cr(VI) 

because Cr(VI) is an exceedingly poor mutagen. Even at the low end of the very high NTP doses there 

is no MOA because there is little or no toxicity, no mutagenecity, and no carcinogenesis. Extrapolating 

linearly from events observed at the two highest doses of the NTP assay, to anything close to reality for 

environmental exposure, is simply not scientific. 

Summary: The carcinogenicity of Cr(VI) is limited to certain forms of Cr(VI) (highly insoluble particulates 

and mists of concentrated chromic acid) and require long-term exposure to high doses: Taken 

together, the experimental observations provide a mechanistic basis for understanding why the 
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epidemiological data shows that the carcinogenicity of Cr(VI) is limited to occupational settings. Only in 

occupational settings, and especially certain occupational settings prior to 1970, did the inhalation 

exposure reach sufficiently high levels of the intermediate soluble particles to induce a carcinogenic 

response. Indeed, several studies have suggested that even the risks for occupational chromiumrelated respiratory cancer decreased after simple industrial hygiene measures, such as wearing a 

mask, were implemented. Animal carcinogenesis studies show that completely soluble Cr(VI) is not 

carcinogenic by inhalation or ingestion. 

It should be noted from the above discussion that Cr(VI) compounds are able to to induce 

genotoxic damage in experimental animals when administered through routes that bypass or 

overwhelm the natural defense mechanisms, such as through intra-peritoneal injection, intra-tracheal 

instillation, or intra-gastric injection . For example, the massive reductive capacity of blood, which 

normally prevents adverse effects of Cr(VI) at a distance from the portal of entry, can only be 

overwhelmed by intra-peritoneal doses that exceed 50 mg per kg body weight. These protective 

mechanisms were acknowledged by the U.S. EPA when setting a maximum contaminant level goal 

(MCLG) of 100ug chromium/liter. USEPA reported that "the reduction of chromium(VI) to chromium( ill) 

occurs in mammals". The saliva and gastric juice in the upper alimentary tract of mammals, including 

humans, have a varied capability to reduce chromium(VI), with the gastric juice having notably high 

capacity. Likewise, the tracheo-bronchial tract and lungs also display high reducing capacity capable of 

handling the inhalation of droplets of aqueous chromium(VI) as alleged in this case. To the extent that 

chromium(VI) might survive these reduction environments, the blood plasma and red blood cells, as 

well as other organs/tissues such as the liver, are also reducing environments. Thus, the body's 

normal physiology provides detoxification for chromium(VI), which provides protection from the oral 

toxicity of chromium(VI)." 

These conclusions are illustrative of the fact that Cr(VI) is poorly toxic and poses no 

carcinogenic risk by the oral route or by inhalation of droplets of water containing chromium(VI) [not 

including concentrated chromic acid mist]. Regarding human lethality, most humans survive even 10-

15 grams of acute ingestion with the lethal oral dose of chromates is estimated at 50-70mg 

chromium(VI)/kg body weight. Studies in mice, rats, dogs and rabbits, wherein Cr(VI) was administered 

in drinking water at doses far in excess of drinking water standards for long periods of time, revealed no 

adverse effects. The USEPA cites that no adverse health effect was observed in a family drinking 

Cr(VI)-contaminated well water for 3 years. Likewise, in a 24 year period of follow-up, there was no 

increase in cancer in residents of Southern Mexico drinking groundwater containing 0.9 mg/liter total 

chromium. No increase of cancer was found in residents of Glascow drinking water contaminated with 

chromium(VI) from chromate slag in soil containing 10,000mg total chromium, followed for 30 years. 
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Chromium contamination of drinking water in Woburn Massachussets was put forward as working 

hypothesis to explain a purported excess of mortality to leukemia in children, but this hypothesis was 

shown to be incorrect by the same research group. 

The WHO concluded that "there is insufficient evidence to implicate chromium as a 

causative agent of cancer in any organ other than the lung". Likewise IARC concluded that "for cancers 

other than of the nasal and sinonasal cavity, no consistent pattern of cancer risk has been shown 

among workers exposed to chromium compounds". The ATSDR report reached the same conclusion. 

The I ARC Working Group reviewed the animal and human studies that show the existence of threshold 

mechanisms of Cr(VI) toxicity and carcinogencity and "interpreted these findings as indicating 

mechanisms that limit the activity of chromium(VI) compound in vivo". Likewise, in its prior toxicological 

review the USEPA concluded that "the body's normal physiology provides detoxification for 

chromium(VI)" and the US Department of Health and Human Services indicated that these 

"mechanisms limit the bioavailability and attenuate the potential effects of chromium(VI) in vivo". 

Concluding Comments 

There is a massive body of literature documenting what is referred to as the Uptake-Reduction 

model of chromium toxicity. Simply stated, Cr(lll) (an essential element) is incapable of crossing 

cellular membranes to any significant extent. Its normal physiological function is to facilitate the 

interaction of insulin with insulin's receptor on cell surface. Thus, the Cr(lll) that we normally receive in 

large quantities from our diet, does not enter into cells. In contrast, the Cr(VI) oxyanion passes easily 

across cellular membranes because it is structurally similar to sulfate and phosphate and it piggybacks 

on the cell's anion transport system. Cr(VI) itself is relatively un-reactive with other cellular 

macromolecules (like DNA or proteins) but once inside of cells, Cr(VI) gets metabolically reduced (thus 

the Uptake-Reduction Model) by intracellular reductants (ascorbate, glutathione, cysteine, etc.) to form 

potentially reactive intermediates Cr(V), Cr(IV) and ultimately Cr(lll). Once it reaches its lowest energy 

state [Cr(lll)] it cannot leave the cell as Cr(lll) because it can't cross the cell membrane. If it gets 

completely liganded (bound) to small peptides or amino acids, it can presumably leave the cell by 

passive diffusion. Under no circumstances would it be feasible or possible for Cr(lll) inside of cells to 

be oxidized back to Cr(VI). Although this reaction can be forced to take place in a chemistry lab, the 

oxidizing power required to catalyze this reaction is completely incompatible with life and would destroy 

any cell near it. 

It would be hard to exaggerate the importance of these fundamental concepts, which are 

uniformly accepted and embraced by both the scientific and regulatory communities. They explain why 

Cr(lll)-piccolinate can be a $200 million/year dietary supplement industry, whereas Cr(VI), in certain 
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forms and doses, can potentially be an occupational hazard. These concepts also provide a 

foundation for the importance of the human body's physiological extracellular reducing systems that 

mitigate the toxicologic potential of even large quantities of Cr(VI). Once extracellular Cr(VI) is reduced 

to Cr(lll) outside of the cell, it becomes an essential nutrient and is incapable of causing any damage. 

It should be recognized that there is a vast literature describing the genotoxic and mutagenic effects of 

supra-threshold doses of Cr(VI) in experimental systems. There is also a vast literature on the effects 

of carcinogen doses of Cr(VI) on cell biology, gene expression and the process of malignant 

transformation. To the philosophical extent that scientists can "know" anything, much is known about 

Cr(VI) as an occupational carcinogen and therefore much can be ascertained about doses and 

exposures that represent true risks. It is not proper scientific methodology to ignore this knowledge and 

broadly state that the mechanism of chromium-induced lung cancer is either unknown or caused by 

oxidative stress or reactive oxygen species produced as a result of extracellular reduction of chromium. 

It should also be noted that compared to many organic mutagens (ie., certain specific PAH's), Cr(VI) is 

only weakly mutagenic, if mutagenic at all, and only at markedly toxic doses. It is inappropriate 

scientific methodology to simply state that Cr(VI) is mutagenic (without qualification) and therefore imply 

that any dose, no matter how small, will be a carcinogenic risk. Moreover, in several in vitro assays for 

neoplastic transformation, soluble Cr(VI) is actually unable to induce neoplastic transformation, even 

though the dose was high enough to damage DNA, presumably because its mechanism of mutation 

induction does contribute very well to transformation. There is no foundation for the belief, or the 

conclusions drawn from it, that any dose (concentration) of hexavalent chromium may meaningfully 

contribute to both risk and causation. This concept departs from accepted scientific methodology, 

which does not embrace a semantic or philosophic argument about whether a vanishingly small amount 

(down to a few atoms or molecules) of any substance can contribute to risk or causation. What is 

important to science and society is whether that risk or causation is meaningful, and this where 

experimental data provides appropriate information. The available data, much of which I have reviewed 

in this report, indicates that a very high dose is required for the carcinogenic effect of a limited number 

of forms of Cr(VI) as they can be encountered in the chromium industry. 

At a minimum, methodologies and conclusions regarding Cr(VI) risk and causation have to 

be qualified with the critical concept of dose and detoxification thresholds. Most body compartments 

have enormous reductive power and rapidly reduce Cr(VI) to Cr(lll), an essential nutritional element. 

Body fluids contain high concentrations of a number of reducing agents (including ascorbate), each of 

which will contribute independently and additively to Cr(VI) reduction to Cr(lll). Many of these biological 

reducing agents are present in great excess over the concentrations of chromium that could be 

delivered by environmental exposure. It is not acceptable to not give adequate (or any) weight to the 
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extensive documentation of the reducing power of extracellular body fluids. To overlook this is to have 

overlooked the most basic principles oftoxicology regarding detoxification. 

Even if one presumes that a high enough dose of Cr(VI) actually gets to the cells of a 

particular tissue, one must also consider that there are a number of intracellular protective mechanisms 

which generate an intracellular threshold which must be breached by a high enough dose, before 

genotoxic endpoints will be reached. It is virtually inconceivable that such a high dose could be 

delivered to a cell with an environmental exposure, as is claimed by plaintiffs in this case. First of all, 

the cell's cytoplasm contains high (millimolar) concentrations of reducing agents, just like the 

extracellular environment. Most of the Cr(VI) which is reduced inside of the cell is converted to Cr(lll) 

with its binding capacities quickly saturated with small molecules such as cysteine (and other amino 

acids) and small peptides such as the tri-peptide glutathione. In this liganded state, Cr(lll) is virtually 

unreactive with additional macromolecules because its binding coefficient to protein is much higher 

than molecules such as DNA. Indeed, it is well know that binding of Cr(lll) to peptides such as 

glutathione, prevents binding of the Cr(lll) to DNA and also prevents the formation of several other DNA 

lesions. 

Our current models for chromium genotoxicity require a dose of Cr(VI) high enough that 

some Cr(VI) can be reduced to its reactive intermediates in the immediate vicinity of the DNA, by a 

reductant which will not itself bind the intermediate and prevent it from interacting with the DNA. If such 

a dose is received, damage to DNA can occur. But that is not the end of the operative protective 

measures of the cell. My laboratory was the first to show that low levels of Cr(VI)-induced DNA 

damage trigger a classic DNA damage response (p53 induction) which stops the cell from dividing until 

it repairs the damage. This is now widely reproduced and accepted by the scientific community. Most 

types of Cr(VI)-induced DNA damage are effectively repaired within 8-24 hours after occurring. My 

laboratory was also the first to demonstrate that if the amount of DNA damage is too large to easily 

repair, the otherwise transient cell cycle arrest will convert to terminal growth arrest or apoptotic cell 

death. These are generally accepted mechanisms whereby a damaged cell will be eliminated and will 

no longer be a target for mutagenesis or neoplastic transformation. Thus, it is not appropriate for 

anyone to imply that if any dose, nor matter how small, of Cr(VI) reaches a cell, that cell is automatically 

a candidate for cancer initiation. This methodology is not supported by the peer-reviewed scientific 

literature and not accepted by the expert scientific community. It patently ignores the facts about the 

basic toxicology (physiological disposition and metabolism) of chromium and ventures into the realm of 

theoretical "biological plausibility". Likewise, it is also scientifically inappropriate to refer to 

mathematically-derived regulatory values as though they represent a biologically relevant threshold, 

above which genotoxic damage to a cell and development of cancer is nearly an inevitable outcome. 
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Selected Personal References 

PEER REVIEWED ARTICLES: MOLECULAR TOXICOLOGY AND CARCINOGENESIS 

1. Patierno, S.R., Costa, M., Lewis, V. and Peavy, D.: "Inhibition ofLPS toxicity for macrophagcs by 

metallothionein inducing agents". J. cl Immunology.JU: 1924-1929 (1983). 

2. Patiemo, S.R., Pellis, N.R., Evans, R.M. and Costa, M.: "Application of a modified >l3Hg binding assay 

for metallothionein". L.ili; Sciences, ,22:1629-1636 (1983). 

3. Evans, R.M., Patierno, S.R., \Vang, D.S., Cantoni, 0. and Costa, M.: "Growth inhibition and 

metallothionein induction in cadmium resistant cells by essential and non-essential metals". 

Molecular Pharmacology 2;!:77-83 (1983). 

4. Patierno, S.R. and Costa, M.: "DNA-protein crosslinks induced by nickel compounds in intact cultured 

mammalian cells". iJJJ;m.lli2l Interactions, 2![:75-91 (1985). 

5. Patierno, S.R., Sugiyama, M., Basilian,J.P. and Costa, M.: "Preferential DNA-protein crosslinking 

induced by NiCb in magnesium-insoluble regions of fractionated Chinese hamster ovary cell 

chromatin". Cancer Res., :!,;!:5784-5794 (1985). 

6. Sugiyama, M., Patierno, S.R. and Costa, :tvf.: "Characterization of DNA lesions induced by CaCr04 in 

synchronous and asynchronous cultured mammalian cells". l\1olecular Pharmacology, 2.2:606-613 

(1986). 

7. Patierno, S.R. and Costa, M.: "Characterization of nickel-induced binding of nuclear proteins to DNA in 

intact mammalian cells". ~ ~. Biophys., 2:113-126 (1987). 

8. Patierno, S.R., Sugiyama, M. and Costa, M.: "Effects of nickel (II) on DNA-protein binding, thymidine 

incorporation and sedimentation pattern of chromatin fractions from intact mammalian cells". 

Biochemical Toxicology, ~:13-23 (1987). 

9. Sunderman, F.W. Jr., Hopfer, S.M., Knight, J.A., McCully, K.S., Cecutti, AG., Thornhill, P.G., Conway, 

K., :Miller, C., Patierno, S.R and Costa, M.: "Physicochemical characteristics and biological effects of 

nickel oxides in relationship to nickel oxides carcinogenesis". Carcinqgenesis l\:305-313 (1987). 

10. Patierno, S.R. and Landolph,J.R.: "Transformation of C3H/IOT'f, mouse embryo cells to focus 

formation and anchorage independence by insoluble lead chromate but not soluble calcium 

chromate: Relationship to mutagenesis and internalization of lead chromate particlesn. Cancer Res., 

11!: 5280-5288 (1988). 

II. Patierno, S.R., Lehman, N. and Landolph, J.R.: "Study of the ability of aspirin, phenacetin and 

acetaminophen to induce cytoxicity, mutation and neoplastic transformation in C3I-I/10'1'1fz mouse 

embryo cells". Cancer ll&§.., :12: 1038-1044 (1989). 

12. !\!iura, T., Pacierno, S.R. and Landolph, J.R.: "Morphological and neoplastic transformation in 

C3H/10T1/2 mouse embryo cells by insoluble carcinogenic nickel compounds 11 • Environmental and 

~Mutagenesis,l:!:: 65-78,1989. 

13. Wise, J.P., Leonard, J., and Patierno, S.R.: "Clastogenesis by particulate PbCrO, in hamster and human 

cells". Mutation Research, ill: 69-79, 1992. 

14. Xu,J., \X'ise,J.P., and Patierno, S.R: uDNA damage induced by lead chromate in cultured mammalian 

cells". Mutation Research 280: 129-136, 1992. 

15. Manning, F.C.R, Xu,J., and Patierno) S.R.: "Transcriptional inhibition by carcinogenic chromate: 

Relationship to DNA damage.:" Molecular Carcinogenesis,[;: 270-279, 1992. 

16. \Vise, J.P., Orenstein,J.M., and Patierno, S.R.: "Inhibition of Lead Chromate Clastogcnesis by Ascorbate: 

Relationship to Particle Dissolution and Uptake". Carcinqgenesis, 1.:!:: 429-434, 1993. 

17. Patierno, S.R., Dirscherl, L.) and Xu,J.: "Transformation of rat tracheal epithelial cells to "immortal 

growth variants' by carcinogenic particulate and soluble nickel compounds".l\futation Research .2QQ: 

179-193, 1993. 

18. Clawson, G.A., Norbeck, L.L., Isom, H.C., Wise, J.P., and Patiemo, S.R.: "Nuclear scaffold protease 

inhibitor blocks chemically induced transformation of embryonic fibroblasts". Cell Growth and 

Differentiation,;±: 589-594, 1993. 
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19. Bridgewater, L.C., Manning, F.C.R., Woo, E.S., and Patierno, S.R.: "DNA polymerase arrest by adducted 

trivalent chromium. Molecular Carcinogenesis, 9: 122-133, 1994. 

20. Blankenship, L.J., Manning, F.C.R., Orenstein, J., and Patierno, S.R.: "Apoptosis is the mode of cell death 

induced by carcinogenic chromate". Toxicology and Applied Pharmacology. 126: 75-83, 1994. 

21. Xu, J., Manning, F.C.R., and Patiemo, S.R.: "Preferential formation and repair of chromium (VI)-induced 

DNA adducts and DNA-protein crosslinks in the nuclear matrix". Carcinqgenesis 15:1443-1450, 

1994. 

22. Wise, J.P., Stearns, D., Wetterhahn, K., and Patierno, S.R.: "Cell-mediated dissolution of carcinogenic 

Lead Chromate particles: role of individual dissolution products in clastogenesis". Carcinogenesis, 

15:2249-2254, 1994. 

23. Bridgewater, LC., Manning, FCR., and Patierno, SR: "Base-specific arrest of in vitro DNA replication by 

carcinogenic chromium: relationship to DNA interstrand crosslinking". Carcinogenesis, Vol 

15:2421-2427, 1994. 

24. Stearns, D.M., Wise, J.P.Sr., Patierno, S.R., and Wetterhahn, K.E., "Chromium(Ill) Picolinate produces 

chromosome damage in Chinese hamster ovary cells". The FASEB Tournai, 9:16643-1648, 1995). 

25. Bub ley, G.J., Xu, J., Kupiec, N., Sanders, D., Foss, F., O'Brien, M., Emi, Y., Teicher, B.A., and Patierno, 

S.R., "Effect of DNA conformation on Cisplatin adduct formation.,_ Biochemical Pharmacology 

51:717-721,1996. 

26. Xu, J., Manning, F.C.R., Bubley, G., and Patierno, S.R.: "Chromium (VI) treatment of normal human lung 

cells results in guanine-specific DNA polymerase arrest, DNA-DNA crosslinks and S-phase blockade 

of cell cycle. Carcinogenesis 17:1511-1517, 1996. 

27. Costa, M. Zhitkovitch, A., Gargas, M., Paustenbach, D., Finley, B., Kuykendall,]., Billings, R., Carlson, 

T.J., \Vetterhahn, K., Xu, J., Patierno, S.R, Bogdanf£Y, M.: "Interlaboratory validation of a new assay 

for DNA-protein crosslinks. Mutation Research 369: 13-21, 1996. 

28. Mirsalis, J.C., Paustenbach, D.J., Kerger, B.D., and Patierno, S.R.: "Chromium (VI) at plausible drinking 

water concentrations is not genotoxic in vivo." Environmental and Molecular tvfutagenesis 28:60-63, 

1996. 

29. Blankenship, L.B., Carlisle, D.,L., Orenstein,J.M., Dye, L.E.Ill, Wise, J.P., and Patierno S.R.: "Induction 

of apoptotic cell death by particulate lead chromate: differential effects of vitamins C and E on 

genotoxicity and survival. Toxicology and Applied Pharmacology. 146: 270-280 1997. 

30. Singh, J.T., Bridgewater, LC., and Patierno, SR: "Differential sensitivity of Chromium mediated DNA 

interstrand crosslinks and DNA-protein crosslinks to disruption by alkali and EDTA" (Toxicological 

Sciences, 45:72-76, 1998. 

31. Singh,.J.T., McLean,J.A., Pritchard, D.E., Montaser, A., and Patierno, S.R.: "Sensitive quantitation of 

chromium-DNA adducts by Inductively Coupled Plama Mass Spectroscopy with a direct injection 

high efficiency nebulizer." T qxicological Sciences, 46:260-265, 1998. 

32. Bridgewater, LC., !vfanning, FCR., and Patierno, SR: "Arrest of processivity of mammalian DNA 

polymerases alpha and beta by carcinogenic chromiurn. 11 (Molecular Carcinogrnesis, 23:201-206, 

1998. 

33. Singh,J., Pritchard D., Carlilse, D.L, McLean,J.A, Montaser, A., Orenstein,J.M., and Patierno, S.R. 

Internalization of carcinogenic lead chromate particles by cultured normal human lung epithelial 

cells: formation of intracellular lead inclusion bodies and induction of apoptosis". Toxicology and 

Applied Pharmacology, 161,240-248, 1999). 

34. Carlisle, D.L., Blankenship, L.J., Orenstein, J., and Patierno, S.R.: "Apoptosis and p53 induction in 

human lung fibroblasts exposed to chromium(Vl): effect ascorbate and tocopherol" Toxicological 

Sciences 55:60-68, 2000. 

35. Carlisle, D.L., and Patierno, S.R. "Chromium (VI) induces p53-dependent apoptosis in diploid 

human lung and mouse dermal fibroblasts". Mqlecular Carcinogenesis 28:111-118, 2000. 

36. Pritchard, D., and Paticrno, S.R. "Cyclosporin A inhibits chromium (Vl)-induced apoptosis and 

mitochondrial cytochrome c release and restores clonogenic survival in CHO cells.'' Carcinogenesis, 

21:2027-2033,2000. 

37. O'Brien, T., Xu, J., and Patierno, S.R. "Effects of glutathione on chromium-induced DNA crosslinking and 
154 


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DNA polymerase arrest." Molecular and Cellular BiochemistJ;y 222:173-182, 2001. 

38. Pritchard, D.E., Ceryak, S., Ha, L., Fornsaglio,J., Hartman, S., O'Brien, T.J. and Patierno, S. 

"Mechanism of Apoptosis and determination of cellular fate in chromium (VI)-exposed populations 

oftelemerase-immortalized human fibroblasts." Cell Growth and Differentiation 12:487-496,2001. 

Featured on journal cover. 

39. Vicheck, S.K., O'Brien, T., Pritchard, D., Ha, L., Ceryak, Fornsaglio, J., and S., Patierno, S. "Fanconi 

anemia complementation group A cells are hypersensitive to chromium (VI)-induced toxicity." 

Environmental Health Perspectives 110:773-777,2002. 

40. O'Brien, T., Fornsaglio,J., Ceryak, S.,and Patierno, S. R."Effects of hexavalent chromium on the 

survival and cell cycle distribution of DNA repair-deficient S. Cerevisae . " DNA Repair, I :617-

627,2002. 

41. Xu,J., Manning, F.C.R., O'Brien, T.J., Ceryak, S., and Patierno, S.R.: "Mechanisms of chromiuminduced suppression of RNA synthesis in cellular and cell-free systems: relationship to RNA 

polymerase arrest" Mol. Cell. Biochem 255:151-160, 2004. 

42. Ceryak, S., Zingariello, C., O'Brien, T. an Patierno, S.R. (2003) Induction of pro-apoptotic and cellcycle-inhibiring genes io chromium (VI)-treated human lung fibroblasts: lack of effect of ERK. Mcl 

Cell. Biochem 255:139-149, 2004. 

43. O'Brien, T., Mandel, H., Pritchard, DE., and Patierno, S.R. "Critical Role of chromium (Cr)-

DNA) interations in the formation of Cr-induced polymerase arresting lesions; a quantitative 

PCR (Q-PCR) approach." Biochemistry, 41:12529-12537,2002. 

44. Ha, L., Ceryak, S., and Patiemo, S.R., "Ataxia telangiectasia mutated gene (A TM) is required for both 

apoptosis and recovery/ escape from terminal growth arrest after genotoxic insult with chromium 

(VI)". Journal of Biological ChemistJ;y: 278, 17,885-17,894, 2003. 

45. Ha, L., Ceryak, S., and Patierno, S.R., "Generation ofS phase-dependent DNA double strand breaks by 

Cr(VI) exposure: Involvement of A TM in Cr(VI) induction induction of gamma-H2AX". 

Carcinogenesis 25:2265-2274, 2004. 

46. O'Brien, T.J., Brooks, B and Patierno, S.R. Nucleotide excision repair functions in the removal of 

chromium-induced DNA damage in mammalian cells Mol. Cell. Biochem. 279:85-95, 2005. 

47. Fornsaglio, J.L., O'Brien, T.J. and Patierno, S.R. Differential impact of ionic and coordinate covalent 

chromium (Cr)-DNA binding on DNA replication Mol. Cell. Biochem. Molecular and Cellular 

Biochemistry: 279:149-155, 2005. 

48. Daryl E. Pritchard, Susan Ceryak, Keri P. Ramsey, Travis J. O'Brien, Linan Ha, Jamie L. Fornsaglio, 

Dietrich A. Stephan, and Steven R Patierno., Resistance to apoptosis, increased growth potential, 

and altered gene expression in cells that survived genotoxic hexavalent chromium {Cr(VI)] exposure 

Molecular and Cellular Biochemistry: 279:169-181, 2005. 

49. Vicheck, S.K., Ceryak, S., O'Brien, 1J., and Patierno, S.R.: FANCD2 monoubiquitination and activation 

by hexavalent chromium [Cr(VI)] exposure: activation is not required for repair of Cr-induced DSBs. 

Mutation Research 610:21-30: 2006. 

50. O'Brien, TJ.,Jiang, GH., Mandel, HG., Westphal, CS., Kahen, K., Montaser, A., States,JC., and Patiemo, 

S.R.: Incision of trivalent chromium ([Cr(III)]-induced DNA damage by Bacillus caldotenex UvrABC 

endonuclease. Mutation Research: 610: 85-92, 2006. 

51. O'Brien, TJ., Mandel, HG., Sugden, KD., Komarov, AM., and Patierno, S.R.: Hypoxia impedes 

the formation of chromium-DNA adducts in a cell-free system. Biochemical Pharmacology, 

70: 1814-22, 2005. 

52. Carlisle, D., and Patierno, SR. "Carcinogenesis and Molecular Genetics". In Cancer Risks and 

Biomarkers., Shields, P (ed). Marcel Dekker, Inc. New York 2005. 

53. Pritchard, D., Ceryak,S., Ramsey, K., O'Brien, T., Ha, L., Fornsaglio, J., Stephan, D., and Patierno, S. 

Resistance to apoptosis, increased growth potential and altered gene expression in cells that survived 

genotoxic exposure. Molecular and Cellular BiochemistJ;y 279: 169-181, 2005. 

54. O'Brien, T.J., Jiang, G.H., Chun, G, Mandel, H. G., Westphal, C.S., Kahen, K., Montaser, A., 
155 


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States,J.C., and Patierno, S.R. (2006) Incision of trivalent chromium [Cr(III)]-induced DNA 

damage by Bacillu.r mldotenax UvrABC endonuclease. Mutation. Resean'h. 610(1-2):85-92 

55. Bae, D.B., Camilli, T.C., Chun, G., Lal, M., Wright, K., O'Brien, T.J., Patierno, S.R. and Ceryak, 

S., Bypass of hexavalent chromium-induced growth arrest by a protein tyrosine phosphatase 

inhibitor: enhanced survival and mutagenesis. Mutation Research. 660:40-46, 2009. 

56. Brooks, B., O'Brien, T.J., Ceryak, S., and Patierno, S.R .. Excision Repair is required for genotoxininduced mutagensis in mammalian cells. Carcinog.enesis: 29:1064-1069, 2008. 

57. Beaver, L.M., Stemmy, E.J., Constant, S.L., Schwartz, A, Little, L.G., Gigley,J.P., Chun, G., 

Sugden, K.D., Ceryak, S., and Patierno, S.R. Lung injury, inflammation and AKT signaling 

following inhalation of particulate hexavalent chromium. Toxicology and Applied 

Pharmacology 235:47-56, 2009. 

58. O'Brien, T.J., Preston Witcher, Bradford Brooks and Steven R. Patierno. DNA Polymerase zeta 

is essential for hexavalent chromium-induced mutagenesis. Mutation Research, 26:663, 77-

83,2009. 

59. Lal, MA, BaeD, Camilli TC, Patierno SR, Ceryak S. AKT1 mediates bypass of the G 1-S 

checkpoint after genotoxic stress in normal human cells. Cell Cycle 8:10, 1589-1602,2009. 

60. Beaver, L.M., Stemmy, E.J., Constant, S.L., Schwartz, A, Damsker, JM., Chun, G., Sugden, K.D., 

Ceryak, S., and Patierno, S.R. Lung inflammation, injury and Proliferative Response 

follo\\~ng repetitive particulate hexavalent chromium exposure. Environmental Health 

Perspectives: 117 (12): 1896-1902, 2009. 

61. ChunG, BaeD, Nickens K, O'Brien TJ, Parierno SR, Ceryak S. Polo-like kinase 1 enhances survival and 

mutagenesis after genotoxic stress in normal cells through cell checkpoint bypass. 

Carcinogenesis. 2010 Jan 20. [Epub ahead ofprint)J>\l!D: [Puh\kd as suppli<'d by 

ptlblisherJRelated articles. 

62. Nickens KP, Patierno SR, Ceryak S. Chromium genotoxicity: A double-edged sword. Chern 

Bioi Interact. 2010 Apr 27. [Epub ahead of print]. 

MOLECULAR TUMOR CELL BIOLOGY/ PHARMACOLOGY 

63. Shuin, T., Billings, P.C., Roy-Burman, P., Lillehaug,J.R., Patierno, S.R. and Landolph. J.R.: 

"Enhanced expression of c-myc and decreased expression of c-fos protooncogenes in 

chemically and radiation transformed C3H/10T'/, C18 mouse embryo cell lines". Cancer 

Res., .:12:5302-5311 (1986). 

64. Patiemo, S.R., Tuscano,.J.M., Landolph,J.R. and Lee, AS.: "Increased expression of the glucoseregulated gene encoding GRP78 in chemically and radiation transformed C3H/10T'/' mouse embryo 

cell lines". Cancer Res., ±I:6220-6224 (1987). 

65. Clawson, G.A., Norbeck, L.L., Hatem, C.L., Isom, H. C., Rhodes, C., Amiri, P., McKerrow, 

6J.H., Patierno, S.R., and Fiskum, G.: Ca 2+ -regulated serine protease associated %th the 

nuclear scaffold". Cell Growth and Differentiation, J: 827-838, 1992. 

66. Leyton, J., Manyak, M.J., Mukherjee, A.B., and Patiemo, S.R.: "Recombinant Human Uteroglobin inhibits 

invasiveness of human metastatic prostate cells and the release of arachidonic acid stimulated by 

fibroblast-conditioned medium", Cancer Research, Advances in Brief, 54: 3696-3700, 1994. 

67. Widra, E., Weeraratna, A., Stepp, M.A., Stillman, R., and Patierno, S.R., "Modulation of 

implantation-associated integrin expression but not uteroglobin by steroid hormones in an 

endometrial cell line." Human Reproduction, 3:563-568,1997. 

68. Weeraratna, A., Cajigas,J.A., Schwartz, A., Manyak, M.J., and Patierno, S.R.: "Loss of 

uteroglobin expression in prostatic cancer:relationship to advancing grade." Clinical Cancer 

~. 3: 2295-2300, 1997. 

69. Lin, Z.P., Boller, Y.C., Amer, S.M., Russell, R.L., Pacelli, KA., Paticrno, S.R., and Kennedy, K.A. 
156 


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Prevention ofBrefeldin A-induced resistance to teniposide (V1v126) by the proteasome inhibitor MG132: Involvement of NF-kB activation in drug resistance. Cancer Research, 58:3059-3065, 1998. 

70. Fernandez, P.M.,Jacobs, L.K., A.T., Tsangaris, T.N., Tabbara, S., Manning, F.C.R, Schwartz, 

A., Kennedy, KA., and Patierno, S.R.: "Differential transcriptional and translational 

overexpression of the glucose-regulated stress protein (GRP78) in malignant but not benign 

human breast lesions. Breast Cancer Research and Tteatrnent, 59:15-26,000. 

71. Attiga, FA., Fernandez, PM., Weeraratna, AT, Manyak, MJ., and Patierno, SR. Inhibitors of 

prostaglandin synthesis inhibit prostate tumor cell invasiveness and reduce the release of 

matrix metalloproteinases. Cancer Research 60, 4629-4637,2000. 

72. Brandes, L.M., Lin, Z.P., Patierno, S.R., and Kennedy, KA. Reversal of physiologic stressinduced resistance to Topoisomerase II inhibitors using an inducible phosphorylation sitedeficient dominant negative mutant oflkB-alpha. Molecular Pharmacology 60:559-567, 

2001. 

73. Boller, YC, Brandes, L.M., Russell, RL., Lin, Z.P., Patierno, S.R., and Kennedy, KA. 

Prostaglandin Al inhibits stress-induced NF-kB activation and reverses resistance to Topol!. 

Inhibitors. Oncology Research 112:383-395, 2001. 

74. Lugassy, C., Haroun RL, Brem H., Tyler BM.,Jones RV., Fernandez, PM., Patierno, SR., 

Kleinman, HK, and Barnhill, RL., Pericytic-like angiotropism of glioma and melanoma cells. 

American J. Dermatopathology 24:473-478, 2002. 

75. Lugassy, C., Kleinman, H.K, Fernandez, P.M., Patierno, S.R., Webber, M.M., Ghanem, G., 

Spatz, A., Barnhill, R.L. Human melanoma cell migration along capillary-like structures in 

vitro: A new dynamic model for studying extravascular migratory metastasis. ]. Invest. 

Dermatol. 119(3):703-704, 2002 

76. Patiemo, SR., Manyak, MJ., Fernandez, PM., Baker, AB., Weeraratna, AT, Chou, D., Szlyk, G., Geib, 

KS., \'V'alsh, C., and Patteras.,J. Uteroglobin: A potential novel tumor suppressor and molecular 

therapeutic for prostate cancer. Clinical Prostate Cancer 1,2: 118-124, 2002. 

77. Fernandez, P.M., Patierno SR and Rickles FR. Tumor angiogenesis and blood coagulation. In: Lugassy 

G, Falanga A, Kakkar AJ, Rickles FR (eds): Thrombosis and Cancer, lvlartin Dunitz Ltd, London, pg 

69-99, 2004. 

78. Rickles, F.R, Patierno, S.R., and Fernandez, P.M. Targeting the endothelium in cancer- the importance 

of the interaction of hemostatic mechanisms and the vascular wall for tumor growth and 

angiogenesis. Pathophysiology of Hacmostasis and 11uombosis 33: 1-10, 2003. 

79. Lugassy, C., Kleinman, H.K, Engbring,JA, Welch, D.R, Harms, J.P., Rufner, R., Ghanem, G., Barnhill, 

R.L. and Patierno, Pericyte-like location of GFP-tagged melanoma cells. Ex vivo and in vivo studies 

of extravascular migratory metastasis. A Journal Pathology. 164(4)>1191-1198, 2004. 

80. Fernandez, P.M., Patierno, S.R. and Rickles, F.R Tissue factor and fibrin in tumor angiogenesis. Sem 

Thromb Hemost, 2004). 

81. Bianco FJ Jr, McHone BR, Wagner K, King A, Burgess J, Patiemo S,Jarrett T\XT Prevalence of Erectile 

Dysfunction in Men Screened for Prostate Cancer. Urology. Urology. 2009 Jul;7 4( 1 ):89·93. Epub 2009 

May 9.PMID 19428072{PubMed indexed for MEDLINE]Related articles. 

82. Wang BD, Kline CL, Pastor DM, Olson TL, Frank B, Luu T, Sharma AK, Robertson G, Weirauch MT, 

Patierno SR, Stuart JM, Irby RB, Lee NH. Prostate apoptosis response protein 4 sensitizes human 

colon cancer cells to chemotherapeutic 5-FU through mediation of an NF kappaB and microR.NA 

network. Mol Cancer. 2010 Apr 30;9>98. 

DISPARITIES, PATIENT NAVIGATION, OUTREACH, SURVIVORSHIP, POUCY 

83. Henson, D., and Patierno, S.R. Breast cancer aggressiveness and racial disparity. Breast Cancer Research 

and Treatment, Breast Cancer Research and Treatment, 87-291-296, 2004. 

84. Freund KM, Battaglia TA, Calhoun E, Dudley DJ, Piscella K, Paskett E, Raich PC, Roetzhein1 RG, 
157 


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Patient Navigation Research Program Group. National Cancer Institute Patient Navigation Research 

Program: methods, protocol, and measures. Cancer. 2008 Dec 15;113(12):3391~9. 

85. Wells KJ, Battaglia Ti\, Dudley OJ, Garcia R, Greene A, Calhoun E, MandelblattJS, Paskett ED, Raich 

PC, Patient Nav':igation Research Program Group. Patient navigation: state of the art or is it science? 

Cancer. 2008 Oct 15;113(8):1999-2010. 

86. Steven R Patierno, Nancy L. La Verda, Lisa M. Alexander, Paul H. Levine, Heather A. Young. 

Longitudinal Network Patient Navigation: Development of An Integrative Model to Reduce Breast 

Cancer Disparities in Washington, DC. Oncology Issues, March, 2010. 

87. Hossain, S., Barich, K, Henson, DE., Schwartz, AM, and Patierno, SR. Cancer Disparities bet\.veen 

African~ Americans and Caucasians: A Review of the Entire SEER Program. Submitted by editorial 

request: Cancer Epidemiology and Biomarkers, 2010. 

88. Ramsey S, Whitley E, Mears V\XI, McKoy JM, Everhart RM, Caswell RJ, Fiscella K, Hurd TC, Battaglia 

T, Mandelblatt, J; Patient Navigation Research Program Group. E\·aluating the cost-effectiveness of 

cancer patient navigation programs: and practical issues. Cancer. 2009 Dec 

1;115(23):5394-403. Review.l'\fll)- [Puh\lcd Hitkxcd ((,· \IFDJ ,l.'\E[Related rticles. 

89. \V'ang, B, Bianco, FJ, Tall,]. Andrawis, R, Frazier H, Patierno SR, Lee NH: Genomic studies on prostate 

cancer disparities associated with the African American population. Submitted, Mol Cancer 

Research, 2010. 

90. Kwabi-Addo B, Wang S, Chung W, Jelinek J, Patierno SR, Wang BD, Andrawis R, Lee, NH, Apprey V, 

Issa JP, lttmann M. Identification of differentially methylated genes in normal prostate tissues from 

African American and caucasian men. Clin Cancer Res. 2010 Jul15;16(14):3539-47. Epub 2010 Jul6. 

91. Jean-Pierre P, Fiscella K, Freund KM, ClarkJ, Darnell], Holden A, Post 0, Patiemo SR, Winters PC; the 

Patient Navigation Research Program Group. Structural and reliability analysis of a patient 

satisfaction with cancer-relatedcare measure: i\ Multisite patient navigation research program study. 

Cancer. 2011 Feb 15;117(4):854-861. doi: 10.1002/cncr.25501. Epub 2010 Oct 4. 

92. Rosenbaum,S, Leonard J., Chapman, M., and Patierno S. Cancer Survivorship and National Health Care 

Reform, In Press, Quality Health Care for Cancer Sut\'ivors, Feurerstein and Gatz Ed, Springer. 

Available February, 2011 

Chapters in Books and Invited Articles 

93. Costa, M., Kraker, A.J. and Patierno, S.R.: 11Toxicity and carcinogenicity of essential and non-essential 

metals". Progress in Clinical Biochemistt:y Vol. 1, pp.l-49 Springer-Verlag, 1984. 

94. Parierno, S.R: "Role of chemical delivery modes in toxicological studies11

• Hazard Assessment of 

chemicals - Current Developments. Vol.7~ J. Saxena, U.S., EPA (Ed.) Hemisphere Publishing 

Corporation, \Vashington, 1990. 

95. Manning, F.C.R., and Paticrno, S.R.: 11 Potential role of apoptosis in carcinogenesis". Current Topics in 

Molecular Pharmacology, J. Menon (Ed.), Council of Scientific Research Integration, Trivardrum, 

India (1994). 

96. Patiemo, S.R and Peavy, D.L.: "Induction of metallothionein in macrophages: A molecular mechanism 

for protection against LPS-mcdiated autolysis", Immunopharmacology of Endotoxicosis M.K. 

Agarwal, M. Yoshida (Eds.) Walter De Gruytcr and Co., Berlin- New York (1984). 

97. Patierno, S.R. and Landolph, J.R.: 11Soluble verses insoluble hexavalent chromate: Relationship of 

mutation to ill :ri.ttQ transfornution and particle uptake". Biological Trace Element Research, 

Humana Press. Volume 21, Number 1-3, 1989. 

98. Manning, F.C.R., Blankenship, L.J., Wise, J.P., Xu,J., Bridgewater, LC., and Patieroo, S.R: "Induction of 

apoptosis by carcinogenic chromate: Relationship to genotoxicity, cell cycle delay, and inhibition of 

macromolecular synthesis." Environmental Health Perspectives, 102 (Suppl3): 159-167, 1994. 

99. Patierno, S.R., Blankenship, LJ, Wise, J.P., Xu, J., Bridgewater, L.C., and I\:fanning, P.C.R.: 11Genotoxininduced apoptosis: Implications for Carcinogenesis". The Cell Cycle: Regulators Targets and 

Clinical AppliC<Ltions, Hu, V.W. (ed). Plenum Press, 1994, pp 331. 

100. Manning, F.CR, and Patiemo, S.R.: "Role of apoptosis in carcinogenesis". Cancer Investigation. 

Hirshaut (Ed.), Marcel Dekker, Inc. New York, 1996. 
158 


23819.119

10L Singh, J., Carlisle, D.L., Pritchard, D., and Patierno, S.R. "Chromium-induced genotoxicity and 

apoptosis: relationship to chromium carcinogenesis". Invited review, Oncology Reports, 5:1307-

1318, 1998. 

102. Carlisle, D., and Patierno, SR. "Carcinogenesis and Molecular Genetics". In Cancer Risks and 

Biomarkers., Shields, P (ed). Marcel Dekker, Inc. New York (2005) 

103. O'Brien, T., Ceryak, S., and Pat:ierno, SR., "Complexities of chromium carcinogenesis: role of cellular 

response, repair and recovery mechanisms". Special Metals issue of Mutation Research, 533:3-36, 

2003. 

104. Nickens KP, Patierno SR, Ceryak S. Chromium genotoxicity: A double-edged sword. Chern Biol 

Interact. 2010 Apr 27. [Epub ahead of print]. 

105. Rosenbaum,S, Leonard J ., Chapman, M., and Paticrno S. Cancer Survivorship and National Health Care 

Reform, In Press, Quality Health Care for Cancer Survivors, Feurerstein and Gatz Ed, Springer. 

Available February, 2011. 

Selected Additional References 

1. De Flora, S., Camoriano, A., Bagnasco, M., Bennicelli, C., Corbitt, G.E., and Kerger, B. Estimates of the 

chromiwn(VI) reducing capacity in human body compartments as a mechanism for attcntuating its 

potential toxicity and carcinogenicity. Carcinogenesis, 18:531-537, 1997. 

2. International Agency for Research on Cancer. Chromium, nickel and welding. IARC Monographs on the 

Evaluation of Carcinogenic Risks to Humans., Vo. 49: IARC, Lyon France, 1990. 

3. U.S.E.P.A Toxicological Review of Hexavalent Chromium (CAS No. 18540-29-9); In support of 

summary information on the Integrated Risk Information System (IRIS), 1997. 

4. U.S. E.P.A Draft Toxicological Review of Hexavalent Chromium, September 2010. 

5. Toxicological Proftle for Chromium. U.S. Department of Health and Human Services, Public Health 

Service, Agency for Toxic Substances and Disease Registry (ATSDR), 2000. 

6. National Toxicology Program. Technical report on the toxicology and carcinogenesis studies of sodium 

dichromate dehydrate in F344N rats and B6C3F1 mice (drinking water studies). NTP TR 546, NIH 

Publication No. 07-5887. US DHHS, 2008. 

7. Stout, MD, Hebert, RA, Kissling, GE et aL Hexavalent chromium is carcinogenic to F344/N rats and 

B6C3F1 mice after chronic exposure. Environmental Health Perspectives 117 (5):716-722, 2009. 

8. De Flora, S., Threshold mechanisms and site specificity in chromium (VI) carcinogenesis. Carcinogenesis 

21:533-541,2000. 

9. Borncff ct al., Mouse drinking study with 3,4~benzpyrene and potassium chromate. Arch. Hyg. 152:45-

65, 1968. 

10. Hansen, M., Johanson, ).and Menne, T., Chromium allergy: significance of both Cr(Ill) and Cr(Vl). 

Contact Dermatitis 49:206-212, 2003. 

11. Crump et al., "Dose~ response and risk assessement of airborne hexavalent chromium and lung cancer 

mortality" Risk Analysis 23:1147, 2003. 

12. Luippold, R.S., et al., «Lung cancer mortality among chromate production workers" Occup Environ 

Med 60: 451-457,2003 

13. Park et al., "Hevalcnt Chromium and lung cancer in the chromate industry: a quantitative risk 

assessment". Risk Analysis 24:1099, 2004. 

14. National Toxicology Program, Report on Carcinogens, 1Oth Edition, Department of Health and Human 

Sen~ces, 2002. 

15. Paustenbach, DJ, Sheehan, P.J., Paull, JM., Wisser, L.M., and Finley, B.L. Review of the allergic contact 

dermatitis hazard posed by chromium~contaminated soil: identifying a "safe" concentration. J. Toxicol. 

Environ. Health., 37:177-207,1992. 

16. Costa, M Toxicity and Carcinogenecity of Cr(VI) in animal models and humans. Critical Reviews in 

Toxicology (27(5): 431-442, 1997. 

17. Costa, M., Klein CB. Toxicity and Carcinogenecity of Chromium Compounds in Humans. Critical 

Reviews in Toxicology 36(2): 155-163,2006. 
159 


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18. Bick, R.L., Girardi TV., Lack, \V.J., Costa, M., Titelbawn, D. Hodgkin's disease in association with 

hexavalent chromium exposure. Int.J. Hematol. 64:257-262,1996. 

19. Mack, T. Cancers in the Urban Environment: Patterns of Malignant Disease in Los Angeles County and 

its Neighborhoods. Elsevier Inc. 200·'. 

20. \X!HO. Chromium. Environmental Health Criteria 61. Geneva: IPCS International Programme on 

Chemical Safety, World Health Organization; 1988. 

21. !ARC. Chromium, Nickel and Welding. !ARC Monograph on the Evaluation of Carcinogenic Risks to 

Humans, Volume 49. Lyon: International Agency for Research on Cancer, \X'JiO; 1990. 

22. ATSDR. Toxicological Proftle for Chromium (Update). Washington D.C.: Agency for Toxic Substances 

and Disease Registry, USDHHS, PHSSS; 1999. 

23. Hamilton JW, Wetterhahn KE. Chapter 19: Chromium In: Seiler HG, Sigel H, editors. Handbook on 

Toxicity of Inorganic Compounds. 1 ed. New York: Marcel Dekker, Inc.; 1987. P239-250. 

24. De FloraS, Wetterhahn KE. Mechanisms of chromium metabolism and genotoxicity. Life Chern Rep 

1989; 7:169-244. 

25. Mertz W. Chromium in human nurcition: a review. J Nutr 1993; 123:626-633. 

26. Anderson RA. Chromium as an essential nutrient for humans. RegToxicol Pharmacol1997; 26:S35-

S41. 

27. Lukaski HC. Chromium as a supplement. Annu Rev Nutr 1999; 19:179-302. 

28. Mertz W. Rlsk assessment of essential trace elements: new approaches to setting recommended dietary 

allowances and safety limits. Nutr Rev 1995; 53:179-185. 

29. Vincent JB. Mechanisms of chromium action: low-molecular-weight chromium-binding substance. 

Am College Nutr 1999; 18:6-12. 

30. EPA US. Toxicological review of trivalent chromium. In support of summary information on the 

Integrated Risk Information System (IRIS). U.S. Environmental Protection Agency, Washington D.C.; 

1998. 

31. EPAA US. Toxicological review of hexavcnt chromium. In support of summary information on the 

Integrated Risk Information System (IRIS). U.S. Environmental Protection Agency, Washington D.C.; 

1998. 

32. De Flora S, Camoirano A, Bagnasco M, Bennicelli C, Corbett GE, Kerger BD. Estimates of the 

chromium(VI) reducing capacity in human body compartments as a mechanism for attenuating its 

potential toxicity and carcinogenicity. Carcinogenesis 1997; 18:531-537. 

33. De Flora S. Threshold mechanisms and site specificity in chromium (VI) carcinogenesis. Carcinogenesis 

2000; 21:533-541. 

34. Jones RE. Hexavalent chrome: threshold concept for carcinogenicity. Biomed Environ Sci 1990; 3:20-

34. 

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November 3, 2011 

Jonathon Aronchick 

Senate Committee on Environment and Public Works 

410 Dirkson Senate Office Building, 

Washington, D.C. 20510 

Dear Mr. Aronchick, 

Below you will find my attempt to answer the questions posed to me by Senator Inhofe. As I 

did at my public testimony, and as I did both before and at the EPA Peer Review of the Draft 

IRIS Risk Assessment for Hexavalent Chromium, I disclose that over tbe course of a long 

career of research in chromium toxicology and carcinogenesis I have occasionally served, 

and am currently serving, as an expert witness in chromium-related litigation (<I 0 cases in 

30 years). This is normal activity for experts in the applied science of toxicology. 

Because one of the Senator's questions refers to an ongoing, multi-institutional research 

study (funded by the American Chemistry Council and coordinated by ToxStrategies) on the 

MOA and pharmacokinetics of hexavalent chromium exposure in rodents, I also disclose that 

the George Washington University (my employer) was awarded a small grant from 

ToxStrategies to conduct a small part of this very large multi-center study. I am not the 

Principle Investigator (PI) of that grant. Although the PI of that grant is a professional 

colleague of mine he is an independent faculty member and he does not work for me or 

report to me in any way. 

I. EPA has released a Draft IRIS Risk Assessment for hexavalent chromium. I understand 

that you were invited by EPA to participate on its peer review panel for this draft assessment 

and that the peer review comments were submitted to EPA. What was the gist of your 

comments to EPA on its Draft IRIS Assessment? In the draft Assessment, EPA uses a linear 

approach to develop its unit risk value. Did you agree with that approach? 

I was a member of the peer review panel for the draft assessment and my detailed comments 

are publically available. This gist of my comments to EPA is that an objective analysis of all 

lines of scientific inquiry into chromium carcinogenesis (occupational epidemiology, in vivo 

animal studies, and in vitro studies on cultured cells) point to a clear non-linear dose response 

curve for toxicity and tumor formation. There are clear no effect levels of exposure and even 

those no effect levels are very high exposure levels. Toxicity and tumor formation are only 

observed after very high levels of exposure for long periods of time and those high doses are 

associated with chronic tissue damage and subsequent chronic tissue regeneration. In rodent 

studies little or no tissue accumulation of total chromium can even be observed until mice are 

continuously exposed (with no option) to at least 14mg/L (ppm) of concentrated sodium 

dichromate in drinking water. Even under these chronic high dose conditions there is little or 

no change in even the most sensitive indices of exposure until the chronic exposure reaches 

at least 60 mg/L (ppm), and tumors are not observed unless tbe mice are exposed, 
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continuously for their lifetime, to 170-520 mg/L (ppm). At least one reason for this is that 

the vast majority of the hexavalent chromium taken in by these animals is reduced to trivalent 

chromium which is an essential nutrient for humans. In my opinion, and in the opinion of a 

strong majority of the Review Panel, it is inappropriate to linearly extrapolate from uniquely 

high dose effects, to speculative low dose potential, when there are clear no effect levels in 

between. 

2. We understand that recent mode of action and pharmacokinetic research findings were 

presented at the peer review panel meeting. What was your impression of this research? 

Should EPA include these new data in its revised Assessment? Are these data pivotal to our 

understanding about hexavalent chromium in drinking water? 

I assume that this question is referencing the large multi-institutional study that is funded by 

the ACC and coordinated by ToxStrategies. This study is being conducted at six or more 

independent laboratories including several unaffiliated academic institutions. Several papers 

from this study have already been published in the peer-reviewed scientific literature and 

apparently more are on the way. The stated goal of this research is to conduct MOA studies 

using the identical animal model and doses used in the NTP bioassay and to address gaps left 

unaddressed by the NTP assay. Given that these studies are being conducted in independent 

laboratories by independent investigators these data should be included in the EPA's revised 

Assessment. My recollection is that it was unanimously agreed upon by the Review Panel 

that the EPA should wait for and utilize this data. Judging from the published papers that I 

have seen so far, these data strongly reinforce the concept of the non-linear dose-dependence 

that I described above, so in that specific sense they are important but perhaps not pivotal (we 

already knew this from the NTP study. However, these studies are also providing very 

important insights into the MOA of hexavalent chromium under high dose exposure 

conditions. 

3. If the mode of action and pharmacokinetic research findings presented at the peer review 

panel meeting are not included in EPA's risk assessment, will the risk assessment provide an 

accurate picture of the current state of the science on hexavalent chromium oral exposure? 

As I conveyed in my public comments, the current Draft Assessment does not adequately 

consider even the existing data (prior to the current MOA study organized by ToxStrategies). 

All lines of scientific inquiry point to clear no effect levels, even at high levels of exposure. 

Independent of the ongoing MOA study, a strong majority of the Review Panel urged the 

EPA to reconsider its conclusion of a mutagenic mechanism of action. At least four of the 

members of the Panel, including myself, have spent many years studying the interactions 

between hexavalent chromium and DNA, and we each have contributed to the peer-reviewed 

literature on what we presumed was mutagenesis by hexavalent chromium. Most of us have 

gradually moved towards the opinion that although Cr(VI) can be forced to show what we 

presumed to be mutagenic activity in heavily contrived experimental systems, this 

"mutagenesis" only occurred at very high doses that killed the vast majority of exposed cells, 

suggesting that it was more likely that we were just selecting for chance survivors. Additional 

studies, wherein the presumably mutated genes were subjected to DNA sequencing, no 

consistent mutations or patterns of mutations in the base sequence of the genes could be 

found. Taking this exceedingly weak pseudo-mutagenesis together with the non-linear in 
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vivo dose response, and the fact that tumors were only observed at exceedingly high dose 

which also caused chronic tissue damage, most of us have shifted our thinking towards 

chronic tissue damage and its associated cell regeneration as a more probable MOA for high 

doses. At the low end of these high doses (which are still very high) there is no MOA 

because there are no measurable effects. 

4. Is the National Toxicology Program (NTP) study consistent with other scientific literature 

on the carcinogenic threshold exposure for hexavalent chromium? 

See answer to Question #3 above. 

5. In February, this Committee heard from EPA that they believed the NTP study argued for 

more stringent drinking water controls for hexavalent chromium. Based on your review of 

current literature, do you think this study calls into question the need for additional regulation 

of drinking water to protect people from getting cancer from hexavalent chromium? 

The NTP study merely confirmed what most experts already knew. Mammals, including 

humans, possess enormous capacity to convert hexavalent chromium to trivalent chromium 

which is non-toxic and non-carcinogenic and is actually an essential nutrient. Only a very 

small proportion of chromium exposure is ever actually absorbed into the body and even if a 

small amount hexavalent escapes conversion at the point of entry or in the stomach, the blood 

and the other tissues possess additional enormous capacity to convert Cr(Vl) to Cr(III) and 

bind it up in manner that prevents Cr(VI) from arriving at tissues or getting absorbed into 

cells as Cr(VI). At exceedingly high doses this protective capacity can be overwhelmed and 

both toxicity and carcinogenesis can ensue, but even then there are additional layers of 

intracellular protection against toxicity, mutagenesis and carcinogenesis. The NTP study 

merely confirmed that exceedingly high concentrations of hexavalent chromate, administered 

in rodents' drinking water with no option for a lifetime, can produce toxicity and 

carcinogenesis. At lower doses in the same study (which are still exceedingly high) there is a 

fairly dramatic threshold below which no toxicity or carcinogenesis can be observed. This 

strongly supports a non-linear dose response and the existence of a high-dose threshold, 

below which there is no effect. 

6. Did any member of the risk assessment review panel believe EPA should rely on the 

journal articles from Critical Reviews in Toxicology you mention in your testimony? Is a risk 

assessment that relies on such articles one that can stand up to scientific review? 

This was not discussed during the Review Panel so I have no way of knowing whether any 

panel member relied on those articles. My concerns regarding those articles can be found in 

my Comments on the draft Assessment. 

7. Is there anything else you would like to add for the record? 

Not at this time. 
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Steven R. Patierno, Ph.D. 

Director, The GW Cancer Institute 

Vivian Gill Distinguished Professor of Oncology 

Professor of Pharmacology and Physiology, Genetics and Urology 

The GW School of Medicine and Health Sciences 

Professor of Environmental and Occupational Health, and Global Health 

The GW School of Public Health and Health Services 

Founding Director, Molecular and Cellular Oncology Program 
165 

Senator BOXER. Thank you very much, sir. 

We are going to move to our final witness, Dr. Jeffrey Griffiths, 

Associate Professor, Department of Public Health and Community 

Medicine at Tufts. 

STATEMENT OF JEFFREY K. GRIFFITHS, MD, MPH AND TM, 

CHAIR, DRINKING WATER COMMITTEE, SCIENCE ADVISORY 

BOARD OF UNITED STATES ENVIRONMENTAL PROTECTION 

AGENCY, PROFESSOR OF PUBLIC HEALTH AND OF MEDICINE, TUFTS UNIVERSITY SCHOOL OF MEDICINE 

Dr. GRIFFITHS. Thank you, Chairman Boxer and Ranking Member Senator Inhofe, members of the Committee. Good morning. I 

really appreciate this opportunity to testify. 

My name is Jeff Griffiths, I am actually a professor now at Tufts 

University. And I am Chair of the Science Advisory Board’s Drinking Water Panel which advises the U.S. EPA. I am trained as a 

physician, internal medicine, pediatrics, infectious diseases, epidemiology. Relevant to this, I have been involved in regulation for 

about 15 years of drinking water contaminants. In fact, I was a 

member of both the National Academy of Sciences panel which recommended some changes in the way the EPA looks at unregulated 

contaminants, as well as the National Drinking Water Advisory 

Committee when it also then chewed on that report and then made 

some suggestions to the EPA. 

So I would just like to comment a bit on some of the ways in 

which I see there needs to be a sea change in the way these things 

are looked at. One of the things that is really important to acknowledge is there is a sea of new chemicals out there that we are 

finding. We are just finding more and more and more of them as 

detection capacity increases. Unfortunately, health effects data is 

really hard to get. It is expensive, it is time-consuming, et cetera. 

So we really only have health information on a small number of 

contaminants. 

So this, I think, means that the number of chemicals has essentially outstripped our capacity to get health information very easily. 

The second point that I would like to make along that line is of 

course that the health effects differ, depending on who you are. If 

you are a baby or pregnant mother or something like that, it is 

going to differ depending on what population you are part of. 

The next point that I would like to make is the need for transparency and a good public process. It forms a good, robust scientific 

basis for decisionmaking, because that means that things are out 

in the open. And also some confidence on the part of the public in 

terms of accepting the conclusions that are made about these compounds. And I would just comment that the 1998 contaminant list 

was considered quite murky and relied on expert opinion. You are 

going to hear a diversity of expert opinions about these contaminants. So I think the transparency is really important. 

I just wanted to go through the perchlorate story a little bit, because it seems to me that kind of illustrates these points. First off, 

we didn’t really know that perchlorate was in the water until detection capacity improved. That happened in the 1990’s. So then 

suddenly people were finding perchlorate everywhere. So that 

166 

means that monitoring and actually, good monitoring is really critical. 

The second point that I would make along that line of the perchlorate story is that it has been known for a while that perchlorate affects thyroid function. But because of the interest in perchlorate there were new studies done, there is new animal data. 

And some of that animal data, for example, suggests that perchlorate acts on thyroid function in a different way than we had 

thought and at lower levels. 

So that is consistent, in fact, with data that was accumulated by 

the CDC in doing a study of nearly 3,000 people where they found 

that women living in Atlanta and throughout the Country had less 

thyroid hormone in their system if they had more perchlorate in 

their system. So there seemed to be some kind of an inverse relationship. So if you are exposed to perchlorate, you may have lower 

thyroid levels. 

And then I think that illustrates the point that once something 

gets on a list, it means there is going to be more science done about 

this thing. So it has to make it onto the list. 

The last point that I would like to make in this story is that if 

we didn’t know anything about a specific sensitive population with 

perchlorate, we might have less concern. That is because perchlorate is concentrated in breast milk. So when a mother is nursing, she may be delivering high levels of perchlorate. And sort of 

perversely also less iodine than the baby needs. If you didn’t look 

at that particular population, you wouldn’t know this. 

And I have some data in here about that in my remarks. But the 

key thing is that by looking at a susceptible population, suddenly 

there is this new information that goes, ah, OK, so this is a group 

that is more susceptible not only inherently but because of the way 

mom delivers breast milk. So I think it is a really critical thing 

that there be data collected from susceptible populations. 

I want to comment that there were many people such as myself 

who were surprised that perchlorate was not identified for regulation in 2008. It was a controversial decision. And many people 

thought there was a lack of transparency around that. And that 

goes to the original point that I made, that transparency is really 

crucial in this. 

Drinking water contaminants are really high on the public discourse, and I think there are four things that have to be done. We 

have to look at groups of compounds for regulation. We need to devote sufficient resources, so that we can monitor these compounds. 

We have to look at health effects, both in the general population 

and in the susceptible populations. And last, the whole process has 

to be transparent, because this will maximize scientific credibility 

and public confidence. 

Thanks very much. 

[The prepared statement of Dr. Griffiths follows:] 

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Oral Testimony of Jeffrey K. Griffiths, MD MPH&TM 

Chair, Drinking Water Committee, Science Advisory Board of the US EPA 

Professor of Public Health and of Medicine, Tufts University School of Medicine 

Senate Committee on Environment and Public Works 

Oversight Hearing on the Environmental Protection Agency's Implementation of the Safe 

Drinking Water Act's Unregulated Drinking Water Contaminants Program 

July 12,2011 
168 


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Chairman Boxer, ranking Member Senator Inhofe, and Members of the Committee, good 

morning. Thank you for this opportunity to testify today before the Committee on Environment 

and Public Works regarding the implementation of the Safe Drinking Water Act's unregulated 

drinking water contaminants (UDWC) program. 

I am Jeffrey K. Griffiths, MD MPH&TM, a Professor of Public Health and of Medicine at Tufts 

University School of Medicine, and Chair of the Drinking Water Committee of the US EPA's 

Science Advisory Board. [My professional training has been as a pediatrician, internist, and 

infectious diseases epidemiologist. I was the Director of the public health program at Tufts 

University for five years, and have studied waterborne diseases for over twenty years. I have 

been involved in the national regulation of drinking water contaminants for approximately 15 

years, and served on the National Drinking Water Advisory Council for over a decade]. I was a 

member of both the 2001 National Academy of Sciences' National Research panel, and the 2004 

National Drinking Water Advisory Council committee, which recommended a significant sea 

change in the way the US EPA it assesses unregulated drinking water contaminants. We 

recommended that contaminants be selected for regulation using a systematic, scientifically 

sound, and transparent process. Please allow me to elaborate as this relates to today's hearing. 

An increasing number of contaminants and our capacitv to understand their health effects 

In order to implement the UDWC program, both water occurrence and health impact information 

is needed. Many challenges exist. Factually, ever more contaminants are being found in water as 

our detection capacities improve. However health effects information can be expensive and time 

consuming to acquire, and is currently available for only a small number of contaminants. The 
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number of known unregulated contaminants has now outstripped our capacity to analyze them all 

on an individual basis using traditional approaches. Furthermore, health effects differ for people 

at different life stages; what may be safe for one group may not be safe for another. These 

challenges must be acknowledged and overcome - but cannot serve as an excuse for inaction. 

Transparency, public participation, and confidence in the process 

A transparent and public process promotes a robust scientific basis for decision making, and 

confidence with acceptance by the public. The 1998 candidate contaminant list determination 

was quite murky and extensively relied on expert opinion. As well we know, experts can differ in 

their opinions. Implementation of the UDWC program should be transparent, welcome 

stakeholders and the public, and be mindful of social equity issues. 

The perchlorate story illustrates the importance of these points. 

Perchlorate was unexpectedly discovered in ground water when water testing improved in the 

1990s, and was found to be present in groundwater throughout the country. This demonstrates 

the benefits of continuously improving the monitoring of our water supply. 

In the 1990s, perchlorate was already known to interfere with the thyroid gland's uptake of 

iodine. Since then, new animal data1 suggests that perchlorate's adverse effects on the thyroid are 

found at unexpectedly low levels of exposure. Similarly, work by the Centers for Diseases 

Control (CDC) has shown that thyroid hormone levels were significantly lower in women who 

were exposed to low levels of perchlorate which had not been thought to affect thyroid function. 2 
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This data, and data from many other studies, suggests that perchlorate is of public health 

concern. It demonstrates the value of appropriately focusing resources so that new health 

information can be generated which may dispel, or increase, concerns about a contaminant. 

Perchlorate has now been found to be concentrated in breast milk. It may replace iodine which is 

essential for babies. Breast-feeding mothers may provide their infants with inadequate iodine and 

perversely feed them excessive perchlorate. A 2007 study 3 concluded that 90% of the babies of 

women who drank water containing perchlorate at the 2006 preliminary groundwater 

remediation goal set by the EPA of 24.5 micrograms per liter would receive a daily dose nearly 

three times the maximum amount established for infants. [This information was used by 

Massachusetts to set a drinking water standard of2 micrograms per liter,4 currently the most 

stringent standard in the United States]. The point is that breast fed babies get all of their 

nutrition from Mom. They are fed breast milk which concentrates perchlorate, and so their 

mothers have to drink low levels of perchlorate to protect their infants from perchlorate enriched 

milk. This information about a specific susceptible population provides a key insight into the 

potential health effects of perchlorate which would have been missed otherwise. 

It was surprising to many, including me, when in the face of this information, the EPA did not 

target perchlorate for regulation in 2008. This decision was controversial and used by many as an 

example of a lack of transparency in the UD WC evaluation process. Indeed, the fact that not one 

new chemical compound has been identified for regulation since the adoptions of the 1996 Safe 

Drinking Water Amendments suggests that implementation has been hampered by a lack of 
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scientific resources, a lack of data, or inadequate transparency. Many of the decisions to not 

regulate contaminants have been driven by a lack of information. 

In sum, 

Drinking water contaminants continue to be high on the burner of public discourse. In addition to 

perchlorate, unregulated compounds such as chromium VI, volatile organic compounds (VOCs) 

such as trichloroethylene and its chemical relatives, and a variety of agricultural and 

pharmaceutical compounds are of public health concern. The EPA has the opportunity to reinvigorate the implementation of the unregulated drinking water implementation process by: 

(1) analyzing groups of similar compounds for regulation when appropriate; 

(2) devoting sufficient resources so that worrisome compounds are adequately monitored on a 

national basis; 

(3) studying health effects in both the general population and in sensitive subpopulations; and 

(4) using an open and transparent process, which will maximize scientific credibility and build 

public confidence. 

Thank you for this opportunity to comment and I welcome any questions. 
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Cited publications 

1 McLanahan ED, Andersen ME, Campbell JL, Fisher JW. Competitive inhibition of thyroidal 

update of dietary iodide by perchlorate does not describe perturbations in rat serum total T4 and 

TSH. Environ Health Perspect 2009; 117:731-738. 

2 Blount BC, Pirkle JL, Osterloh JD, Valentin-Blasini L, Caldwell KL. Urinary perchlorate and 

thyroid hormone levels in adolescent and adult men and women living in the United States. 

Environ Health Perspect 2006 114:1865-1871. 

3 Ginsberg GL, Hattis DB, Zoeller RT, Rice DC. Evaluation of the US EPA/OSWER preliminary 

remediation goal for perchlorate in groundwater: focus on exposure to nursing infants. Environ 

Health Perspect 2997 115:361-369. 

4 Zewdie T, Smith CM, Hutcheson M, Rowan West C. 2010 Basis of the Massachusetts 

Reference Dose and Drinking Water Standard for Perchlorate. Environ Health Perspect 2010 

118:41-48. 
173 

Senator BOXER. Thank you. 

Senator Inhofe has asked if he can ask his questions first. 

Senator INHOFE. Mine will be very, very brief. I do have a lot of 

questions I want to ask for the record that I will be submitting. 

Dr. Patierno, I know that you have done extensive research into 

chromium–6, with particular emphasis on the carcinogenic and mutagenic efforts. As you know, my constituents in Norman, Oklahoma are very much concerned about this. I just wanted to get 

your expert opinion. First of all, is chromium–6 mutagenic? And 

second, are my constituents at health risk from drinking water at 

the low levels of chromium? I am talking about Norman, Oklahoma 

now, because I think you are aware of what is going on there. 

Mr. PATIERNO. Yes. Thank you for the question, Senator Inhofe. 

The question of whether hexavalent chromium is mutagenic has 

become a very interesting question. Those of us who work in DNA 

damage and carcinogenesis have known for years that in carefully 

contrived experimental conditions, we can force virtually any chemical to become carcinogenic, or excuse me, mutagenic. We have 

been concerned for a long time that in order to detect mutagenicity 

after exposure to chromium, one has to use enormous concentrations that actually kill the vast number of cells or tissue cells that 

get exposed. 

We have always been concerned about that. Recent studies have 

gone back in, actually sequenced the DNA sequence of the genes 

that we thought had been mutagenized, and found that the DNA 

sequence is still intact. And that has led to a current understanding, or an emerging understanding, that despite the fact that 

you can force chromium–6 to get into cells at very high doses and 

damage DNA, or bind to DNA, it appears that the mode of action 

is that we are killing cells and selecting for survivors. 

So that has raised very significant questions that came up 2 

months ago in the review panel for the toxicological review of chromium, and extensive discussion was made about that extensive 

question. 

Senator INHOFE. Are they at risk? 

Mr. PATIERNO. Are they at risk at the levels in tap water? 

Senator INHOFE. Yes. 

Mr. PATIERNO. Absolutely not. 

Senator INHOFE. OK, that is what I wanted them to hear. 

The last question I have is for Dr. Cotruvo. I appreciate your 

statement and your emphasis on fixing and maintaining infrastructure and reducing leaks and contamination. I was telling the 

Chairman here that I went through this experience when I became 

mayor of Tulsa. We had a deteriorated infrastructure. 

Do you think that the funding, the SRF program, the revolving 

fund program, would be important in accomplishing some of this 

infrastructure correction that we need? 

Mr. COTRUVO. Certainly, the financial demands are huge. Somehow we built all this without a lot of Federal support over the last 

200 years. But it has really come to the point where there is significant infusion that is needed to get these systems back up. And 

probably Federal funds are going to be necessary. 

Senator INHOFE. But revolving funds is what I was referring to. 

Mr. COTRUVO. Yes. 

174 

Senator INHOFE. OK, thank you very much. Thank you, Madam 

Chairman. 

Senator BOXER. Thank you. 

Dr. Patierno, I am struck by your confidence that chromium–6 is 

safe, and I am going to press you on that. First of all, you argue 

that too much was given to the animals. Isn’t this what we do in 

science? Because of their life span, isn’t that a normal test, that 

you give a lot to the animal so that you can see what is going on? 

Mr. PATIERNO. Yes, that is a standard procedure. I am not arguing that too much was given. What I am arguing is that so much 

was given that it was, it overwhelmed all possible protective mechanisms. And even at very, very, very high doses, not effect was observed until the two very highest doses, which went hand in glove 

with extensive toxicity to the tissue. 

Senator BOXER. But isn’t that being conservative, in your approach? Isn’t it the point? I understand that you have testified as 

a defense witness, isn’t that correct, on chromium–6 cases? 

Mr. PATIERNO. I have on occasion. 

Senator BOXER. Yes, you have. You made money doing that? 

Mr. PATIERNO. I have. 

Senator BOXER. Yes, you have. I don’t make money on any side 

of this thing. I am trying to protect the public, OK? So when you 

tell me too much was given, I want to be conservative about this. 

So Dr. Goldman, I think it gets to your point, and Dr. Griffiths 

as well, I would like to ask you to comment on the cumulative effect. You made the strong point, and I think Dr. Griffiths as well, 

what we are dealing with here, we are looking at something where 

if a little child is exposed, let’s say even in utero and continues to 

be exposed to these contaminants, it is a cumulative impact, is it 

not, Dr. Goldman? 

Dr. GOLDMAN. Yes, it is. It is a cumulative impact. It is also the 

impact of exposures over a lifetime. And I should say in the case 

of hexavalent chromium that this has been reviewed by expert bodies, like the International Agency for Research on Cancer. And they 

do conclude that it is a risky substance. They do not conclude that 

they can absolutely assure safety at the levels of exposure in the 

population. 

Senator BOXER. Dr. Griffiths? 

Dr. GRIFFITHS. Thank you. The issue of mixtures of chemicals is 

really a big one. Because we have a way in science of doing this 

kind of dancing on the head of a pin where we have the chemical 

or no chemical. But the truth is we live and we drink water with 

lots of different things in it. And we have exposures not only 

through water but air and food and so forth. And we really don’t 

have good tools for figuring that stuff out. 

So I think that we in fact do have to develop new tools. But also, 

what information we do have suggests that exposure to multiple 

chemicals, even at low levels, can be risky. So I think it is important to understand that sometimes it is not just one thing. It is like 

smoking and asbestos exposure. Your rates of lung cancer are really high if you both smoke and have exposure to asbestos. But they 

are much lower if you only do one. 

Mr. PATIERNO. May I comment? 

175 

Senator BOXER. Dr. Patierno, yes, I am going to let you have the 

floor in a minute. I am going to ask you this question. The National 

Toxicology Program, which is part of the U.S. Department of 

Health and Human Services, found that chromium–6 in drinking 

water caused cancer in the studies of animals. And you have taken 

issue with how much they got, I don’t know exactly what you said, 

you said, I don’t think they got too much, but it is how much—I 

was confused on what you were talking about. 

And then the EPA’s draft assessment on chromium–6 has found 

that this toxic heavy metal is ‘‘likely to be carcinogenic to humans 

when consumed in water.’’ So you take issue with this? 

Mr. PATIERNO. Madam Chairman, it is not just that I take issue. 

All nine members of the external review panel for the draft toxicological assessment of hexavalent chromium disagree with that assumption. It has to be qualified by dose. And the assessments that 

are done by international agencies on the carcinogenicity of chromium deal specifically with high level occupational exposures. And 

they even cite in the literature these are exposures as encountered 

in the chromate production pigment and plating industries. Because of the unique chemistry and biology of chromium, of 

hexavalent chromium, and the capability of our bodies to reduce 

hexavalent chromium very quickly to trivalent chromium, which is 

completely non-toxic, and in fact, is an essential element, make it 

unique among other chemicals and make it so that it is not scientifically sound to do a linear dose extrapolation at high doses. 

Senator BOXER. OK, OK, I get it. You don’t agree with it. I get 

it. Now, is there anything you agree with? We have standards for 

88 different chemicals, I am sure you are aware of this, over the 

years. Do you believe that these are safe and shouldn’t have had 

standards associated with them? 

Mr. PATIERNO. I have not looked into any of the others. I was 

only invited here today to speak on—— 

Senator BOXER. I know. But you are making a statement, you 

were a witness for the defense, I am questioning your objectivity. 

So I am trying to find out if you just feel that way on—I assume 

you think it is fine to limit the amount of chromium in the water, 

total chromium, is that correct? 

Mr. PATIERNO. Total chromium? 

Senator BOXER. Yes, total chromium, which is in fact regulated 

and has been for quite some time. 

Mr. PATIERNO. I am not sure I am following specifically your 

question. 

Senator BOXER. I am asking a question. There is 88—you are an 

expert—there are 88 chemicals that are regulated under the law 

where they have maximum contaminant levels. This piece of paper 

tells me that chromium is regulated. Are you aware of that? 

Mr. PATIERNO. Yes, I am. 

Senator BOXER. OK. Do you support that? 

Mr. PATIERNO. I do support that chromium needs to be regulated 

based on the occupational exposures that are so well characterized 

in the literature. 

Senator BOXER. This is drinking water. This isn’t occupational. 

This is the Safe Drinking Water law of the land. Do you agree that 

there should be a limit on chromium in drinking water? 

176 

Mr. PATIERNO. Madam Chairman, the question for me is not 

whether there should be. There of course should be. The question 

is at what level. 

Senator BOXER. OK, how about arsenic? Do you think we should 

regulate arsenic in drinking water? Yes or no? 

Mr. PATIERNO. Madam Chairman, I am here today to speak on 

hexavalent chromium. 

Senator BOXER. No, you are not really. You are not really. You 

are here because we are having an overview of the Safe Drinking 

Water program. And we are not asking you just on chromium. 

What about benzene? What about chlorine? Do you support that? 

Mr. PATIERNO. Any chemical that has potential toxicological impact needs to be evaluated and regulated. But each chemical needs 

to be evaluated based on the science supporting that specific chemical, the mechanisms of action of that chemical, the defense mechanisms that the body has against particular chemicals. All of that 

needs to be taken into consideration. 

One could argue using a different set of criteria that something 

as common and innocuous as acetaminophen shouldn’t be on the 

market because two might control a headache but 50 could dissolve 

your liver and create a lethal situation. So it is all about dose and 

form and duration. And then the basic principles of toxicology 

which argue in this case that human beings have very, very, very 

strong defense mechanisms, particularly against low levels of 

hexavalent chromium. 

Senator BOXER. Well, good luck with that theory, because you 

ought to talk to some people who had exposure who don’t feel that 

way. But you are a scientist, and I am saying to you, we have 88 

different chemicals that are regulated here. And if people came 

with your attitude and appeared in court in favor of the defense, 

we wouldn’t get anywhere. And we have a problem here, and that 

is why we called you together. Because since 1998, we haven’t regulated anything. 

And I think what you hear from the panel basically is, we may 

not have to regulate a lot of things. We agree that maybe, as Dr. 

Cotruvo said, existent in, and echoed by Dr. Griffith, they may be 

appearing in very small, minute quantities. And that is why we 

have to look at the risk factors, who is exposed to these, the cumulative impact, as Dr. Goldman suggested, and not just say, we appeared for the defense. That is just not going to get us anywhere, 

when money gets involved in the equation. 

We need to have pure science. And that is what I am for, pure 

science. 

Now, we will be very interested to see where the EPA takes it 

on perchlorate. And I could tell you that you just talked to my constituent here on perchlorate, when you have 800, what is it, 800 

parts per billion? 

Mr. ARAIZA. Yes. 

Senator BOXER. Instead of six? 

Mr. ARAIZA. Yes. 

Senator BOXER. And you smell that, as I have, and you see it, 

it is scary. It is scary. 

Mr. ARAIZA. Yes. 

177 

Senator BOXER. And we know the Safe Drinking Water Act is 

going to protect the most vulnerable. We are not talking about protecting a 250 pound man who is 6 foot 5 here. That is what we just 

to do. But guess what? A Boxer amendment a very long time ago 

did something your clients didn’t like, Dr. Patierno. We said, you 

have to protect the most vulnerable child. That was my amendment, God bless it, it passed. And so, no, we are going to be conservative when we set these standards, and we are going to look 

to see how we can protect the most vulnerable. 

But I want to thank the panel. You have all been very, very helpful. We are going to move on and see what happens with these 

rules on chromium–6, on perchlorate. We are going to see what 

happens when the EPA adopts many of the recommendations made 

by the GAO, because clearly, no one can tell me that we are any 

safer today when we haven’t even regulated one thing since 1998. 

It just doesn’t add up, when you look at the rates of cancer out 

there. Let’s just get real here. 

Especially I say to the doctors, we have cancer in people younger 

and younger. We have serious problems, we are spending billions 

of dollars, we don’t have a cure. And I will be darned if I am going 

to sit here and allow us to say that this isn’t important. This is 

very important and we are going to do our work. 

So we look forward to seeing the EPA’s response. I am very encouraged by today’s hearing, because I think the GAO’s very good 

work is going to lead us to more science, hopefully less politics, 

more transparency and more action. So thank you very much, all 

of you, for being here today. We stand adjourned. 

[Whereupon, at 12 p.m., the committee was adjourned.] 

[Additional material submitted for the record follows.] 

STATEMENT OF HON. JEFF SESSIONS, U.S. SENATOR

FROM THE STATE OF ALABAMA

Good morning Mr. Trimble and Dr. Perciasepe. Thank you for coming before our 

committee today. 

We are here today to consider EPA’s implementation of the Safe Drinking Water 

Act, which is designed to protect our nation’s drinking water supplies from harmful 

contamination. As the Ranking Member of the Subcommittee on Water & Wildlife, 

I am interested in learning more about this topic. 

Under this law, EPA currently regulates more than 90 drinking water contaminants—things like cryptosporidium, chlorine, E-coli, and arsenic, just to name a few. 

These are contaminants that we all want to ensure do not damage our drinking 

water. EPA is required by the Safe Drinking Water Act to periodically review existing standards as well as to consider whether new standards should be issued. 

I am aware that GAO has issued a report expressing an opinion that EPA should 

improve the way it decides whether to regulate additional contaminants. I am interested in learning more about that report. 

In addition, there are 4 issues, in particular, that I think we need to be sure to 

keep in mind. 

First, the Role of the States. The Safe Drinking Water Act, like many of our Federal environmental laws, is based on a concept known as ‘‘cooperative federalism.’’ 

EPA oversees the program, but the States have a critically important role in implementing and administering the program. 

In Alabama, our State environmental management agency works closely with 

more than 700 water systems in the State to ensure safe drinking water for our 

state’s 4 million citizens. The State enforces compliance with the drinking water 

standards. There are monthly, quarterly, and annual monitoring and reporting requirements to ensure compliance. By and large, our State has very good drinking 

water, and I believe we have an effective drinking water program. 

We cannot lose focus of the fact that what we do here, at the Federal level, results 

in mandates on the states. Those mandates are, in large measure, usually unfunded. 

178 

So, as we explore the possibility of greater regulation, we need to ensure there is 

close consultation with the states, as well as with the water systems themselves. 

Second, the Impact of New Standards on Water Systems and Water Users. When 

a new Federal MCL (maximum contaminant level) is issued, the water systems have 

to comply with that new standard. That becomes a new cost imposed on those who 

pay for the water. Rural water systems, in particular, often carry the heaviest burden when trying to comply with these standards. I think we need to be as flexible 

and reasonable as possible to ensure that excessive costs are not imposed on our 

rural communities. 

Third, the Role of Science—does the science justify new standards and the costs 

of those standards. If there are costs to be imposed, we need to make sure that the 

underlying decision to create any new standard is correct—based on the best available science. I think we need to know more about EPA’s methodology for establishing drinking water standards, and in particular, EPA’s ‘‘IRIS’’ system. 

Fourth, our Nation’s serious infrastructure needs. As Senator Inhofe has articulated, we have an aging infrastructure in this Nation. Our water infrastructure is, 

in many cases, being used well beyond intended lifespans. I am interested in learning more about the impact of our aging infrastructure on our ability to ensure safe 

drinking water. 

Thank you. 

179 


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Peer Review Workshop 

for EPA's Draft 

Toxicological Review of Hexavalent Chromium 

Reviewer Post-Meeting Comments 

July 6, 2011 
180 


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Contents 

Responses to Charge Questions .................................................................................................. 1 

General Charge Questions ................................................................................................................ 3 

GL Is the Toxicological Review logical, clear and concise? Has EPA clearly presented and 

synthesized the scientific evidence for noncancer and cancer hazard? .......................................... 3 

G2. Please identify any additional studies that would make a significant impact on the 

conclusions of the Toxicological Review ................................................................................... 41 

Chemical-Specific Charge Questions .............................................................................................. 49 

(A) Oral Reference Dose (RID) for Hexavalent Chromium ........................................................ 49 

AI. A two-year drinking water study of sodium dichromate dihydrate in rats and mice (NTP, 

2008) was selected as the basis for the derivation of the RID. Please comment on whether 

the selection of this study as the principal study is scientifically supported and clearly 

described. Please identify and provide the rationale for any other studies that should be 

selected as the principal study .................................................................................................... 49 

A2. Diffuse epithelial hyperplasia in the duodenum of female mice was selected as the critical 

effect for the RID. Please comment on whether the selection of this critical effect is 

scientifically supported and clearly described. Please identify and provide the rationale for 

any other endpoints that should be selected as the critical effect. ................................................ 51 

A3. Benchmark dose (BMD) modeling was applied to the incidence of diffuse epithelial 

hyperplasia in the duodenum of female mice to derive the point of departure (POD) for the 

RID. Has the BMD modeling been appropriately conducted and clearly described? Is the 

benchmark response (BMR) selected for use in deriving the POD (i.e., a 10% increase in 

the incidence of diffuse epithelial hyperplasia) scientifically supported and clearly 

described? ................................................................................................................................. 53 

A4. Please comment on the rationale for the selection of the uncertainty factors (UFs) applied 

to the POD for the derivation of the RID. Are the UFs scientifically supported and clearly 

described? If changes to the selected UFs are proposed, please identify and provide a 

rationale .................................................................................................................................... 54 

(B) Carcinogenicity of Hexavalent Chromium ............................................................................ 56 

B I. Under EPA's 2005Guidelines for Carcinogen Risk Assessment 

(www.epa.gov/iris/backgrd.html), hexavalent chromium is likely to be carcinogenic to 

humans by the oral route of exposure. Is the cancer weight of evidence characterization 

scientifically supported and clearly described? ........................................................................... 56 

B2. A mutagenic mode of carcinogenic action by all routes of exposure is proposed as the 

primary mode of action for hexavalent chromium. Please comment on whether this 

determination is scientifically supported and clearly described. Please comment on data 

available for hexavalent chromium that may support an alternative primary mode of 

action ..... , .................................................................................................................................. 59 
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B3. A two-year drinking water study in rats and mice (NTP, 2008) was selected for the 

derivation of an oral slope factor. Please comment on whether the selection of this study 

for quantification is scientifically supported and clearly described. Please identify and 

provide the rationale for any other studies that should be considered .......................................... 72 

B4. The incidence of adenomas and carcinomas combined in the small intestine of male mice 

from the NTP (2008) two-year drinking water study were selected to serve as the basis for 

the quantitative cancer assessment. Please comment on whether this selection is 

scientifically supported and clearly described. Please identify and provide tbe rationale for 

any other endpoints that should be selected to serve as tbe basis for the quantitative cancer 

assessment. ................................................................................................................................ 73 

B5. The oral slope factor was calculated by linear extrapolation from the POD (i.e., the lower 

95% confidence limit on the dose associated with I 0% extra risk of tumors of the small 

intestine in male mice). Has the modeling been appropriately conducted and clearly 

described? ................................................................................................................................. 75 

Additional Reviewer Comments ............................................................................................... 79 

Janusz Z. Byczkowski, DABT, Ph.D ................................................................................................. 81 

Konstantin Salnikow, Ph.D ................................................................................................................ 83 

Anatoly Zhitkovich, Ph.D .................................................................................................................. 87 

Appendix A: Individual Reviewer Comments ........................................................................ A·l 

Janusz Z. Byczkowski, DABT, Ph.D ............................................................................................... A-3 

Joshua W. Hamilton, Ph.D ............................................................................................................ A-11 

Monica Nordberg, Ph.D ................................................................................................................ A-29 

Steven R. Patierno, Ph.D ............................................................................................................... A-37 

Toby G. Rossman, Ph.D ................................................................................................. , .............. A-51 

Konstantin Salnikow, Ph.D ............................................................................................................ A-61 

John Pierce Wise, Sr.; Ph.D ........................................................................................................... A-73 

Anatoly Zhitkovich, Ph.D .............................................................................................................. A-99 

Yiliang Zhu, Ph.D ....................................................................................................................... A-113 

Appendix B: List of Reviewers ............................................................................................... B-1 

Appendix C: List of Observers .............................................................................................. C-1 

Appendix D: Agenda .............................................................................................................. D-1 
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Responses to Charge Questions 
183 


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Responses to Charge Questions 

General Charge Questions 

Gl. Is the Toxicological Review logical, clear and concise? Has EPA clearly presented and 

synthesized the scientific evidence for noncancer and cancer hazard? 

Reviewer 

Byczkowski 

Hamilton 

Comments 

Logical - yes; clear - mostly; concise - no, as there is a very large body of information 

covered by this Toxicological Review. 

While the scientific evidence of both noncancer and cancer hazards of oral exposures to 

Cr.,. has been appropriately reviewed and synthesized, the conclusions and numerical 

derivation of toxicity values are mostly based on strict (default) interpretation and literal 

application of U.S EPA guidelines, rather than on the current scientific understanding of 

the mode of action of Cr .... 

In general, the report is a concise presentation of the vast primary literature for chromium 

toxicology and carcinogenicity and the writing is generally clear. However, there are 

specific sections with deficiencies as noted below in detail, and many other sections and 

specific comments that are not logical. In many cases a statement in one section is either 

not a logical extension of the data presented, or is in opposition to a statement elsewhere. 

Overall, the greatest concern is with the logic regarding the choice of a mode of action 

(MOA), which is the basis for many of the subsequent assumptions that are made, the 

default values that are chosen, and risk assessment modeling that follows from these 

choices. In this reviewer's strong opinion and in the consensus opinion of the external 

reviewers who are experts in this area and who discussed this at the May 12, 20 II 

meeting Cr(VI) is highly unlikely to act via a mutagenic mode of action in vivo. Rather, 

a careful review of existing information, as well as emerging studies all strongly indicate 

that the likely MOA involves a threshold mechanism that supports both the physiological 

uptake-reduction model of DeFlora and the cellular uptake-reduction model of 

W etterhahn that were previously proposed. The current EPA draft document concludes 

that chromium(VI) acts via a mutagenic MOA by all routes of exposure, a conclusion that 

is illogical given the current state of knowledge of chromium biology and toxicology as 

already presented in this draft report, and also based on the recently emerging data from a 

series of 90-day rodent MOA studies sponsored by the American Chemistry Council 

(ACC). This is the most important and central point since the choice of a mutagenic 

MOA then drives all other considerations in this document. Specific areas of concern are 

outlined by chapter and section below in detail in a combined response to G l. and 02. 

Chapter 1 

EPA should include more definitive information about the literature it reviewed that 

contributed to this draft report. It currently states that the relevant literature was reviewed 

through September 2010 but the external reviewers all noted at the May 12 meeting a 

number of gaps in the literature being cited in the current draft. In addition, it would be 

helpful to know how many studies the EPA identified as being part of the chromium 

literature, how many it reviewed, how many it set aside or did not review, what criteria 
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Responses to Charge Questions 

were used to include or exclude a study, etc. For example, a statement such as: "The EPA 

identified 26,839 peer-reviewed scientific publications in PubMedfrom 1950 through 

September 2010 using the keyword 'chromium.' Of these, 9,456 were determined to be 

relevant to the current draft based on the criteria of covering aspects of chromium 

biology, toxicology, environmental chemistry and epidemiology." 

Cbapter2 

On page 14 the draft states that,"Natural occurrence of hexavalent chromium is rare ... " 

This statement should be qualified since geochemical surveys by the U.S. Geological 

Survey and others have indicated that there are background levels of naturally occurring 

Cr(VI) in most groundwater, and that these levels are typically in the range of 2-5 ppb but 

can be up to 3-5 times higher than that in certain areas. Likewise recent reports of Cr(VI) 

levels in soil and house dust indicate that there are natural sources, or at least sources that 

cannot be identified as being specifically anthropogenic, that contribute to the background 

levels and background exposures of people throughout the U.S. A more detailed literature 

review and discussion regarding background levels of Cr(VI) in soil, air and water, 

including more up-to-date information on such levels, should be included in this chapter 

to provide context for subsequent discussions regarding human exposure through drinking 

water. Keep in mind that, until recently, analytical methods to detect total chromium and 

to speciate chromium(VI) accurately were difficult, with higher detection levels than 

many natural sources contain. Further, speciation of chromium(VI) at very low levels has 

been very problematic until recently, so much of the older environmental data are 

inaccurate or had non-detect values where reanalysis has revealed widespread 

chromium(Vl) background levels in the environment. This is of importance not only for 

understanding the potential contribution of these background levels to total chromium 

exposure but also in setting practical regulatory levels. Clearly it is of little value for the 

EPA to calculate a maximum contaminant level (MCL) or public health goal (PHG) that 

is lower that typical background levels since this would be virtually impossible to achieve 

by most public drinking water systems with limited resources, and because such 

background levels are unlikely to represent a significant health risk (see discussion below). 

Chapter3 

This is an important chapter that would greatly benefit from reorganization and from 

synthesis of information. A number of studies are described here sequentially, and for 

several of these the draft document speculates on possible mechanisms. However, the 

order of presentation is not logical, and there is little in the way of more global synthesis 

of results and the conclusions that can be drawn or those areas that remain controversial 

or poorly understood- while there are several areas where a statement in one section is 

contradicted by a statement elsewhere. Because the toxicokinetics of chromium is so 

central to its biology and toxicology, this is a critical chapter for its overall evaluation and 

therefore this chapter should be revised. 

Regarding organization, Section 3.3 is extremely important for understanding the studies 

described throughout the remainder of the chapter, providing context for extracellular 

versus intracellular reductive metabolism. It is suggested that this section should be 

moved to just before or immediately after the current Section 3.1. Likewise, Section 3.6 

on Cr(III) and its nutritional benefit and essentiality is extremely important for 
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Responses to Charge Questions 

understanding how the body normally absorbs, distributes and excretes chromium. From 

the standpoint of human environmental exposures, Cr(VI) is primarily, but not exclusively 

an aothropogenically derived form of chromium that is principally encountered and used 

in occupational settings, but humans and all other life forms have been dealing, both 

physiologically and biochemically, with Cr(III) and reduction of background levels of 

Cr(VI) for the entire history of life on Earth. It is recommended that Section 3.6 should 

be moved to the beginning of this chapter and greatly expanded. The current section on 

this important topic is extremely short, superficial, and inaccurately presents this subject 

as an area of controversy in the field. 

Cr(III) nutritional biochemistry has been extensively studied over the past fifty-plus years 

with a large and robust literature. Only two toxicologists are cited as the sources of the 

"current debate" about this, whose individual views have been well aired in their review 

articles but which represent views that are generally considered to be well outside the 

mainstream of toxicology and nutritional biochemistry. Citing their views so prominently 

in this short section does not balance well with the wealth of studies and numerous other 

investigators over decades who have concluded that there is a beneficial role of Cr(III) in 

human and animal nutrition and have demonstrated an underlying biochemistry and 

physiology that supports this role. This section should be greatly expanded and treated in 

a more balanced fashion since it sets the stage for understanding how Cr(III) is treated by 

the body in all aspects of toxicokinetics, and therefore also provides valuable insight into 

Cr(VI) toxicokinetics that largely explains much of the experimental literature on 

chromium disposition in intact animals and humans as discussed in more detail below. 

See for example reviews by W Mertz (J Nutr 1993, 123:626-633; Nutr Rev 1995, 53:179-

185), RAAnderson (Reg Tox Pharm 1997, 26:S35-41), HC Lukaski (Ann Rev Nutr 1999, 

19:279-302) and JB Vincent (JAm College Nutr 1999, 18:6-12) for summaries of this 

earlier literature. 

Some of tbis information is alluded to- for example, the end of the primary paragraph on 

page 44 beginning with "Aitio et al. (1988) developed ... ", which represents an extremely 

important body of literature on Cr(III) toxicokinetics and which should be moved forward 

with section 3.6 and greatly expanded by citing other relevant literature. There are dozens 

of studies of Cr(III) uptake and kinetics. The current chapter leaves the impression that 

Cr(III) gastrointestinal (GI) uptake is uniformly low, and that chromium is not normally 

found ahove background levels in urine, therefore if one administers Cr(VI) and sees a 

dose-dependent increase in blood, urinary or tissue levels it is evidence of Cr(VI) uptake 

as CrCVIl. But there are a number of uptake studies of chromium picolinate and other 

natural and man-made Cr(III) complexes, as well as Cr(III) uptake in chromium-sufficient 

versus chromium-deficient diets, that defy this simple interpretation. For example, one 

study described on page 45 by Kerger et al. (1997) reported that "ingestion of chromium 

picolinate resulted in significantly elevated urine concentrations such that participants 

routinely exceeded background. " Similar elevations have been reported for chromium 

picolinate in the nutritional literature. Likewise, other studies have shown an inverse 

correlation between Cr(III) levels in diet and the uptake, distribution, tissue storage and 

excretion of Cr(III), indicating that the body tightly regulates Cr(III) kinetics to maintain a 

steady-state body burden and availability of the nutritionally active form of Cr(III), and 

can actively take up Cr(III) from the GI when the body senses that it is deficient, or 

decrease uptake and increase excretion when internal stores are sufficient or exceeded. 
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Responses to Charge Questions 

A related and key concept that is alluded to in this section but never addressed directly is 

the behavior of chromium in serum and red blood cells (RBC). It has been well 

established, beginning with RBC labeling studies going back to the 1950's in which RBC 

are incubated with Cr(VI) ex vivo, that Cr(VI) is readily taken up by RBC, rapidly 

reduced, and in the process forms highly stable chromium adducts on hemoglobin and 

other macromolecules which are very long lived, essentially remaining intact for the 

lifetime of the cell. In this way the half-life of RBC in humans and experimental animals 

was established (with human RBCs having a half-life of ca. 110-120 days) and this tool 

has also been used to look at RBC turnover. This is alluded to on page 27, where it states 

that "The partitioning of hexavalent chromium from plasma into erythrocytes is 

significant. It has been used as a biomonitoring endpoint ... and is responsible for the 

observed residence time of chromium in whole blood ... " Conversely, in the human and 

animal studies that were described in Chapter 3, it has been consistently shown that when 

Cr(VI) is administered orally or by gavage, there is a transient increase in both serum and 

RBC levels which rapidly return to baseline. Thus, it is highly unlikely that the chromium 

in the blood of these animals and humans was Cr(VI), or else the RBC would have been 

stably labeled. It was noted on page 45, for example, in describing the human studies of 

Paustenbach et al. (1996) where there was oral exposure to Cr(Vl) that "both plasma and 

RBC chromium concentrations returned rapidly to background levels within a few days, 

again suggesting that concentrations of 10 mg Cr(Vl)/L or less in drinking water of 

humans appears to be completely reduced to Cr( Ill) prior to systemic distribution. " This 

is an extremely important experimental observation in humans - and one of the most 

important statements in this draft document -- that directly addresses the issue of whether 

Cr(VI) is taken up by the human GI as Cr(VIl and whether it survives as Cr(Vl) in the 

circulation. 

Similar results were seen in the Sutherland et al. (2000) rat stndy in which the blood 

kinetics, and lack of chromium increases in brain or other distal tissues argued strongly 

that it was Cr(III) rather than Cr(VI) that was taken up by the gut. Were this not the case, 

they should have observed stable chromium labeling of the RBC and elevated chromium 

levels in distal tissues. But they reported the opposite, and this also strongly indicates in 

this key rodent study that Cr(VI) failed to survive as Cr(Vll in crossing into the 

bloodstream from a GI exposure. And elsewhere in Chapter 3, similar results are reported 

that would lead to the same conclusion, yet the text alludes to transient RBC uptake as 

possible evidence that Cr(VI) is being taken up from the GI tract as Cr(VI). This is a 

highly flawed, illogical argument that appears throughout this document. Likewise it is 

now well known that there are specific uptake, transport and storage mechanisms for 

nutritionally active Cr(III) that must be taken into account in any measurements of 

chromium in the blood or other tissues. In fact, none of the studies presented in Chapter 3 

provide any direct evidence that any Cr(VI escapes the GI tract as Cr(VI) except perhaps 

where normal gastric reduction is bypassed or the bolus doses are so large as to 

completely overwhelm the reductive capacity of the gut, which is likely what occurred in 

the NTP (2008) study. This should be more clearly described in this chapter, perhaps with 

a new section prior to current section 3.5 that presents the theoretical PBPK model since 

this model also critically relies on how one interprets the in vivo data. 
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Responses to Charge Questions 

Specific edits suggested below are based on these concerns (changes underlined): 

• Page 24, Line 24- " .. for the chromium administered as hexavalent ... " 

• Page 25, Line 7 " ... ofthe chromium administered as hexavalent ... " 

• Page 26, Line 3 - " .... Generally, absorbed chromium is ... " 

• Page 26, Line 9 - " ... toxicokinetics of chromium, .... " 

• Page 26, Line 16- " ... comparing administration o{Cr(III) ... " 

• Page 30, Line 2 - " ... bioavailability of chromium administered as ... " 

• Page 35, Lines 7-11- two sentences beginning with "The reason for the higher ... " 

This is highly speculative and should be deleted. This could all be Cr(III) rather than 

Cr(Vl) using the arguments above. 

• Page 36, Lines 25-28- end of sentence beginning with" ... indicating that a ponion of 

the Cr(Vl) escaped extracellular reduction ... " This is also highly speculative and is 

actually counter to the data presented, which clearly show transient blood levels that 

are indicative of Cr(lll) distribution, not Cr(Vl), as more appropriately alluded to in 

Lines 32-34 where is says "Brain, ovarian, and whole-blood concentrations were 

below detection limits in all exposed groups. The lack of concentrations in wholeblood was attributed to rapid delivery of Cr to tissues and clearance of plasma Cr." 

The lack of stable blood chromium clearly indicates that it could not have been 

absorbed as Cr(V!) or else the RBC would have shown significant and stable 

elevations. 

• Page 38, Line 13 - " ... did not alter GI uptake appreciably at these concentrations." 

• Page 41, Line I - "Chromium is capable of crossing the placenta." 

• Page 41, Line 15-17- "Absorption and elimination of chromium was evaluated .... 

Following ingestion by human volunteers of either trivalent or hexavalent chromium in 

single or multiple drinking water doses." 

• Page 42, Lines 23-26- This statement beginning with "Because the Cr(Vl) increases 

... " is incorrect. Increases in RBC chromium that are stable over time, with the same 

half-life as the RBC, would be indicative of uptake of chromium as Cr(V!) by the 

RBC, but if the increase in RBC chromium is transient, as in this case, then it cannot be 

due to Cr(VI) in the blood but is most likely a Cr(lll) complex that is carried by the 

RBC but not covalently bound. Evidence for this is cited elsewhere in this chapter (see 

middle of page 44 and top of page 46, for example) and in the broader Cr(lll) literature. 

The draft report is illogical since it currently argues this issue both ways depending on 

the study being discussed. 

• Page 42, Lines 30-35 -These two sentences beginning with "The higher 

bioavailability ... " are highly speculative and well outside the boundaries of the actual 

data presented and discussed above. If the report is going to speculate, it should pull 

all this into a separate section and synthesize it across all the studies that were cited. 
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Otherwise delete these speculations since they do not have a factual foundation. 

• Page 49, last line-" ... chromium administered as Cr!Vl) distributes ... " 

• Page 58, Lines 2-3 - " ... greater percentage of chromium administered as Cr(Vl) than 

Cr(Ill) is absorbed." Delete sentence reading "This implies that some Cr(Vf) escaped 

reduction ... " since this is not implied by the actual experimental data based on the 

above arguments. 

Taken together, these studies, as argued above, lead one to only one logical conclusion: 

Cr(VI) is not taken up by the gut as Cr(Vll, nor does it survive as Cr(Vl) in the systemic 

circulation in these studies. It is only by greatly exceeding the normal doses and reductive 

capacity of the gut that one can see any signs of Cr(Vl) surviving to reach other tissues. 

Therefore there is a clear biochemical barrier, a threshold, to Cr(VI) uptake and systemic 

exposure under normal physiological conditions. This must clearly be taken into account 

in any analysis of MOA and resulting risk assessment for this toxicant. 

Chapter4 

Liaoning Province, China studies- At the beginning of this chapter (Pages 68-76) the 

EPA presents an extensive review and discussion of the various reports related to the 

study of populations in China near a site of chromium-contaminated drinking water from 

a nearby industry. Depending on the authors and methods used- particularly certain 

assumptions regarding age adjustment, use of an urban area as a control population, and 

exposure estimates -- the reports of this data set either find no statistical association 

between stomach cancer incidence or a modest elevation. The other epidemiology studies 

cited in this chapter report no statistical correlation between drinking water chromium 

exposure and cancer incidence. It is of considerable concern to this reviewer that the EPA 

has chosen in some places to highlight this one positive report and elevate it to the level of 

their major recommendations, such as on Page 239 of Chapter 6 (Major Conclusions) 

where they state that there is "evidence of an association between oral exposure to 

hexavalent chromium and stomach cancer in humans." Yet elsewhere, they state, 

appropriately, that "this risk has not been established in other populations exposed to 

drinking water contaminated with hexavalent chromium. The epidemiology data are not 

sufficient to establish a causal association between exposure to hexavalent chromium by 

ingestion and cancer." (Page 201, Lines 20-23; and similar language on Page 205, Lines 

33-36) 

It is inappropriate to "cherry-pick" a single study -indeed, a single treatment among three 

treatments of the same study - if this is going to influence their major recommendations 

regarding the human carcinogenicity of Cr(Vl) via ingestion. Many of my other 

comments and edits relate to this concern, as well as previous concerns regarding the 

toxicokinetics of Cr(VI) via ingestion and the misinterpretation of those results with 

respect to mode of action and carcinogenic potential in animals and humans as detailed 

below. This reviewer feels strongly that the Liaoning Province study and its various 

treatments should be set aside, since the initial study (first reported in 1987) is highly 

flawed by today' s epidemiology standards, there is a lack of information that would allow 

others to reevaluate this study in the manner that would most directly address the potential 

correlation, and the three subsequent treatments by original authors Zhang and Li (1997), 
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by Beaumont et al. (2008) and by Kerger et al. (2009) -- each of which makes slightly 

different assumptions in order to fill critical data gaps -- reached different conclusions. It 

is also important to point out that this population was exposed not just to extremely high 

levels of Cr(VI) but also to industrial effluent which contained high levels of a number of 

other chemicals of concern including sulfates, acids and other toxicants. Thus, even if an 

association was found, it is not possible to attribute this to Cr(Vl) per se and could be the 

result of either other contaminants or a statistical anomaly based on the high rates of 

stomach cancer in China which are diet- and province-related. For these reasons this 

study and its series of treatments cannot be the basis for risk characterization or risk 

assessment, and should be set aside. 

Section 4.4 also requires extensive revision and expansion. The first paragraph reports 

only briefly on the extensive literature examining occupational exposures and cancer, 

principally lung cancer. This section, and this literature, should receive a much more 

extensive treatment since these studies are our best data regarding human exposures to 

Cr(VI). It should be noted that in addition to inhaling extremely high levels of Cr(VI) 

prior to the advent of industrial hygiene practices in the 1960's and 1970's (levels as high 

as several mg chromium(VI) per cubic meter) these workers had extensive dermal 

exposures, and also had extensive ingestion exposure since it is well known that 

individuals exposed to high levels of dust swallow a large fraction of what they inhale 

either through direct deposition in the mouth, nose and throat, and via mucocilliary 

clearance of the pulmonary system. Thus, these occupational epidemiology studies are 

extremely important in understanding the toxicology of chromium(VI). Several key 

points will be made below citing a few critical studies, but the EPA should more 

thoroughly review this entire literature. 

The U.S EPA's current risk assessment for chromium(VI) via inhalation is based 

principally on the studies by Mancuso of older worker populations through the 1960's 

prior to the advent of modern industrial hygiene practices. Similar risks were observed in 

other occupational studies of workers from the 1940's through 1970's who were exposed 

to much higher levels of chromium than are encountered in occupational settings today, 

such as those by RB Hayes et al. (Inti J Bpi 1979, 8:365-374; Am J lndust Med 1989, 

16:127-133), JM Davies (Br J Indust Med 1984, 41:158-168), T Sorahan et al. (Br J 

Indust Med 1987, 44:250-258), and R Kishi et al. (Am J Indust Med 1987, 11:67-74). 

Subsequent epidemiology studies of workers exposed to lower levels of chromium since 

the 1960's have not only provided estimates of risk at these high doses, but have also 

provided important information as to lower occupational levels at which no increases in 

lung cancer or other health effects were observed. These more modern studies have also 

taken into account other factors, such as accounting for cigarette smoking and other 

confounding variables, whereas many of the older studies did not control for these 

important confounders (see for example, KD Rosenman, Am J Indust Med 1996, 29:491-

500). It should be noted that, where it was reported, virtually all the lung cancer cases in 

these occupational studies occurred in smokers (see for example Pastides et al., Am J 

Indus! Med 1994, 25:663-675; T-C Aw, Reg Tox Pharrn 1997, 26:S8-12). These studies 

not ouly provide better estimates of the actual health risks attributable to occupational 

chromium exposure, but also an estimate of a practical threshold below which we would 

predict either no effects, or risk of effects that are so low that it cannot readily be detected 
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even in large populations of exposed people. 

Gibbet a!. performed a follow-up study (Am J Indust Med 2000, 38:115-126) of a worker 

population in Baltimore MD that had previously been studied by Hayes et a!. (Int J Epi 

1979, 8:365-374) in examining the relationship between chromium(VI) exposure and 

cancer incidence. In the Gibb study, the workers were stratified according to different 

levels of cumulative exposure to chromium, allowing a more detailed examination of the 

potential dose-response. Cumulative exposure was expressed as j.lg/m3

-years (1,000 

ng/m3

-years), integrating both the chromium level and the total time of exposure at that 

level. This is similar to smoking data that express cumulative dose as "pack-years" and is 

based on the observation that the risk of a 40 pack-year smoker who used I pack per day 

for 40 years is similar to that of a 40 pack-year smoker who used 2 packs per day for 20 

years. In the Gibb study, the lowest quartile of workers had exposure to chromium 

between 0 and 1.5j.lg/m3

-years (1,500 ng/m3-years). This group had an observed/expected 

lung cancer ratio of 0.96, i.e., it was slightly less than expected from the comparison 

population (the general population of Maryland) that had no occupational chromium 

exposure. 

Pastides et a!. examined a group of chromate production company workers in North 

Carolina (cited above), focusing on the possible differences in risk between cohorts of 

workers who were exposed to chromium under the older conditions and processes of the 

1940's through 1960's and those who began work after 1971 in a modernized factory in 

which both the chemical process and the exposure levels to chromium had been modified. 

They found a slightly increased risk of lung cancer, proportional to exposure, in the older 

cohort working under the higher dose exposure conditions, as had been reported 

previously in other studies. However, the workers in the modernized factory had no 

excess of lung cancer, all cancers, heart disease, or all causes of death over an 18 year 

period. Personal monitors for the workers indicated that the chromium(VI) levels were all 

below 50,000 ng!m3

, and most were below 25,000 ng!m3, with the majority in the range of 

500-10,000 ng/m3

• Average duration of employment was 9.5 years, such that cumulative 

dose would have averaged 4,750-95,000 ng/m3

-years. Dividing the workers into two 

groups of exposure, i.e., those working less than 10 years versus those working more than 

10 years, indicated no difference in mortality, further suggesting that these workers had no 

significant increase in cancer or other health risks from either the higher or lower 

chromium exposures. Similarly, Aw reported (cited above) that workers in the more 

modernized plants who were occupationally exposed to chromium since the 1960's 

showed no increase in disease risk, as was also noted by S Langard et al. (Br J lndust Med 

1990, 47:14-19). 

WJ Blot et a!. performed a large and comprehensive study (J Occup Environ Med 2000, 

42:194-199) of a group of 51,899 workers of the Pacific Gas & Electric (PG&E) 

Company. A sub-set of 3,796 these workers had been exposed occupationally to 

chromium(Vl), either as gas generator workers or trainees at the Kettleman CA station 

which used chromium as a rust inhibitor in cooling tower water at PG&E natural gas 

transfer stations from the 1950's through the 1980's. Examination of these workers for 

specific cancers, all cancers, specific non-cancer diseases, and all diseases indicated no 

increased incidence in any adverse health outcomes in relation to chromium exposure. In 

fact, the total cancer and lung cancer standardized mortality ratios (SMRs, 
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Responses to Charge Questions 

observed/expected ratios) were 0.64 and 0.81, and 0.55 and 0.57, respectively, for these 

two groups of chromium-exposed workers, which was less than those of the overall 

PG&E worker group and substantially less than those of the general California population 

against which they were compared. SMRs for all causes of death were also low (0. 79 and 

0.68, respectively). Likewise, JD Boice et al. performed a large and comprehensive study 

(Occup Environ Med 1999, 56:581-597) of a group of 77,965 workers at an aircraft 

manufacturing plant in California. A sub-set of 3,634 of these workers were exposed to 

chromates and other chemicals as part of airplane production for a total of 88,224 personyears of exposure and a mean of 24 working years per person of exposure. The SMR for 

total cancers was 0.93, and the SMR for lung cancer was 1.02. As with the Blot study, 

there was no association of any adverse cancer or non-cancer health outcome with 

chromium exposure in this group, nor did the overall worker population have an increase 

in overall or specific mortalities as compared to the general population despite exposure to 

a number of occupationally related chemicals. 

Taken together, these occupational studies indicate that, although previous historical 

exposure conditions were associated with a modest risk of lung and respiratory cancer 

(average of 2- to 4- fold increased lifetime risk, as compared, for example, to a 10- to 20-

fold increased risk for cigarette smokers), more recent occupational exposures at or below 

the current regulatory limits indicate that these represent levels that do not elevate cancer 

risk even for lifetime occupational exposures. Moreover, the previous exposures of 

concern in workers from the 1930's through the 1960's were at levels that typically 

exceeded I ,000,000 ng/m3 and also involved exposure to the most carcinogenic forms of 

chromates, i.e., the insoluble or slightly soluble forms such as lead chromate, zinc 

chromate and calcium chromate. The newer lifetime occupational exposure limits -- at 

which no increase in cancer risk or other health effects has been observed - represent 

daily exposures that are hundreds to thousands of times higher than would occur in an 

environmental setting or via U.S. drinking water. There are two other major conclusions 

that can be drawn from these occupational exposure studies. First, although dermal 

exposure to chromium(VI) was extensive- particularly prior to the advent of industrial 

hygiene practices in the 1970's- there is no evidence for increased risk of skin cancer, 

even in workers where the chromate levels were high enough to burn "chrome holes" in 

their skin or nasal septum. These chrome holes healed and were not associated with 

increased skin cancer risk in these workers. This is relevant to the very high doses of 

chromium(Vl) used in the NTP studies and a possible MOA. Second, taken together 

these occupational studies do not demonstrate an increased risk of GI cancers or other 

internal cancers, despite the fact that these workers swallowed a significant fraction of the 

dusts they were exposed to in the air. These data were recently summarized in a metaanalysis published in 2010 (NM Gatto eta!., Cancer Epi 2010, 34:388-399). 

Other specific edits and comments (changes underlined): 

• Page 200, Lines l-2 - Delete the phrase " ... and evidence of an association between 

oral exposure to hexavalent chromium and stomach cancer in humans" which is based 

on a single study and EPA's selective treatment of this result as discussed above. 

• Page 200, Lines 18-20- "This study found evidence of a modestly increased incidence 

ofstornach cancer mortalitv fOR 1.69. Cll.12-2.44!from 1970 to 1978 .... " 
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Responses to Charge Questions 

• Page 201, Lines 20-23- " ... was reported in a re-examination of a single study in 

JinZhou .. " 

• Page 209, last section, Bioavailability- This section is highly flawed in logic and 

presentation as discussed under Chapter 3. Specific edits are as follows: 

o Page, 209, last two Jines- "Quantitative studies of GI absorption of chromium 

administered as hexavalent chromium have estimated ... " 

o Page 210, first line- Please note that hexavalent chromium was not measured, 

and without exception has never been measured systemically as Cr(VI) following 

Gl absorption. It is an assumption that increased chromium uptake to the blood 

represents Cr(VI) uptake, but it could also represent other forms as discussed in 

Chapter 3 above. We know that certain forms of Cr(III) are much more readily 

taken up than others, and it is therefore possible, and perhaps quite likely, that 

reduction of Cr(VI) in the gut in the presence of organic molecules in the GI 

lumen leads to formation of complexes that are much more bioavailable than 

inorganic Cr(III) that is typically found in food, water and soil. You could also 

modify this phrase as a separate sentence to read "This may indicate that not all 

hexavalent chromium is reduced by the gastric juices of the stomach, or that 

reduction of Cr(Vl) in this environment leads to formation of organic chromium 

complexes that are more readily absorbed than inorganic Cr(lll)." 

o Page 210, Lines 7-12- Delete the sentence beginning "Thus, at oral doses within 

human exposure ranges ... " as per the argnment above or modify to include 

alternative interpretations as suggested above. 

o Page 210, Lines 28-30 - End of sentence beginning with " ... and uptake of 

hexavalent chromium into the tissues ... " delete this phrase or modify it as per 

the argument above. 

o Page 210, last line- Add a sentence after the last sentence on this page reading 

"However, none of those studies speciated the chromium that was absorbed 

systemically, and so the form( s) of chromium in the blood and other tissues is 

unknown following increased absorption of chromium following ingestion of 

hexavalent chromium. Therefore, it is not known whether chromium reaches the 

blood or distal tissues as chromium( VI) at doses relevant to human exposures. 

The lack of long-term labeling of RBCs by chromium in the animal and human 

studies argues that little, if any, chromium is absorbed as chromium( VI) under 

these exposure scenarios." 

o Page 213, Section 4.7.3.5. Lines 3-5- This statement is completely incorrect; 

there is no evidence for it as argued above. Delete this, or modify as follows 

"Chromium absorbed following ingestion exposure to chromium( VI) may be in 

forms that can reach the systemic circulation and distal tissues, thereby 

potentially affecting tissues beyond those at or near the site of entry. However, 

theform(s) of chromium following such uptake is not known." 
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Responses to Charge Questions 

ChapterS 

This reviewer strongly objects to use of a linear low-dose approach for Cr(VI) risk 

assessment given the clear evidence for a threshold mechanism due to extracellular 

reduction of chromium at doses of relevance to human exposure via drinking water. The 

conclusion by EPA of a mutagenic action of chromium - most of which is based on cell 

culture data where chromium exposures and other parameters were extreme and where 

metabolism and intracellular exposure are far different than in vivo exposures should 

not be the sole basis for use of this standard model which ignores the compelling 

toxicokinetic data sunnnarized in Chapter 3 of this draft. More importantly, as discussed 

above for Chapter 4, Cr(VI) is unlikely to act via a mutagenic MOA in vivo, and requires 

extraordinary experimental manipulation to be positive in cell culture and in vivo 

mutagenicity studies. While it is clear that Cr(VI) can cause certain forms of DNA and 

chromosomal damage or other changes, it is not clear whether any of these is premutagenic, and the in vivo data argue strongly against a mutagenic MOA under 

physiological conditions and normal routes of exposure. The document must more clearly 

differentiate between genotoxicity- i.e., damage to DNA or chromatin- and 

mutagenicity - or frank mutations that may result from DNA damage. Chromium(VI) can 

induce DNA damage but is a very weak mutagen at best, particularly in vivo. It is far 

more likely, and most consistent with all available data, that chromium(VI) acts via a nonmutagenic mechanism that involves a clear threshold- two threshold actually, one of 

which is extracellular and chemical involving reduction of chromium(VI) to 

chromium(III) and the other of which is biochemical and involves a threshold for cellular 

effects that lead to cell damage and cell death, resulting in turn in tissue proliferative 

responses that ultimately increase tumor risk via well known mechanisms of repeated 

tissue injury, compensatory cell proliferation and re-population. 

Given this most likely MOA based on a synthesis of several decades of chromium 

research, it is therefore inappropriate to use a linear low-dose extrapolation model for 

assessing risk via the ingestion route of exposure. It is clear from the animal and human 

studies that there is a threshold for in vivo effects that is based on the strong reducing 

capacity of the GI tract following oral exposures, and that at normal drinking water 

concentrations this will effectively protect from any in vivo exposure to Cr(Vl) as Cr(Vl). 

Thus, a more appropriate risk assessment method would be to do dose-response modeling 

from the 2008 NTP study and the more recent ACC-sponsored 90-day MOA studies, and 

then use an approach similar to that for the RID to calculate, with appropriate safety 

factors, a drinking water MCL that is protective based on threshold mechanisms. This 

should be done for Cr(VI) rather than the current MCL that is for total chromium, but it is 

likely that an MCLin the range of 50-100 ppb is going to be fully protective, including 

several uncertainty factors that separate it from the departure point of any likely human 

health effects for even the most sensitive individuals. 

Chromium is an excellent example of an opportunity to apply the concept of evidencebased risk assessment - which the EPA has claimed to be promoting for many years but 

has not, to date, actually applied in any meaningful risk assessment -- since there is a 

strong and compelling argument for use of a non-linear, threshold-based mechanism for 

chromium that logically leads to a real-world risk assessment that is based on that 

mechanism. Setting aside the 1987 Zhang and Li study and subsequent re-analyses as per 
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Responses to Charge Questions 

the arguments above, there is not a single credible epidemiology study linking exposure to 

chromium via ingestion with cancer risk or any other long-term health effects, including 

in the extensive occupational epidemiology literature which includes several decades of 

extremely high-dose exposure cohorts. And even taking into account the Zhang and Li 

study, there is not a single peer-reviewed report linking any health effects to 

chrornium(VI) at levels within a hundred-fold of likely environmental exposures via 

drinking water. The NTP 2008 animal data showed evidence of a cancer increase only at 

the highest doses, and the more recent ACC studies demonstrated hyperplasia consistent 

with a non-mutagenic MOA, which is also consistent with a threshold mechanism and 

which argues against developing a linear cancer slope factor from those data. 

Chapter6 

There is considerable disconnect between the conclusions provided in Chapter 6 and the 

more considered and detailed discussions of the primary data in the previous chapters. 

The language should be modified to reflect this understanding such that the conclusions 

and their application to risk assessment of chromium(VI) follow logically from the 

scientific evidence. Specific edits: 

• Page 235, Lines 18-19 - " ... resulting in substantial, and in some cases complete 

reduction of hexavalent chromium to trivalent chromium depending on the 

concentration, dose. and precise route and method of exposure." 

• Page 235, Lines 21-26- "The extent of absorption of. chromium from ingesting 

hexavalent chromium appears to be determined by both the solubility ..... in the Gl 

tract, but ingestion of both trivalent and hexavalent chromium results in systemic 

uptake of chromium. Trivalent chromium does not readily cross cell membranes 

except as part of certain organic complexes. Hexavalent chromium. if absorbed 

systemically, can easily .... " 

• Page 235, Lines 27-29 "Chromium absorbed systemically from the gut following 

ingestion of hexavalent chromium is distributed throughout the body . .... If hexavalent 

chromium is absorbed without extracellular reduction, it can cross cell membranes 

f!!1d, once inside the cell, .... " 

• Page 236, Line 3 - "Chromium absorbed systemically following hexavalent chromium 

ingestion is eliminated primarily in the urine as trivalent chromium" 

• Page 239, Lines 11-12 - delete the phrase " .. and evidence of an association between 

oral exposure to hexavalent chromium and stomach cancer in humans. " This is a 

significant statement to make, and as discussed above in reference to Chapter 4, the 

Zhang and Li studies ( 1987, 1997) should not be used to assess human cancer risk for 

oral exposure to chromium. 

• Page 240, Lines 12-14- As noted for Chapter 5 above, there is considerable concern 

with the default choice of a linear low-dose extrapolation model for cancer risk. 
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Nordberg 

Patierno 

Responses to Charge Questions 

EPA has presented and synthesized present knowledge about non-cancer and cancer 

hazards for hexavalent chromium. However, some further literature could be included in 

the document and paid attention to i.e, Langard and Costa Chapter 24 Chromium In: 

Nordberg OF, Fowler BA, Nordberg M and Friberg LT (Eds.) (2007) Handbook on the 

Toxicology of Metals, 3rd edition, Elsevier 487-510. 

Other chapters of interest e.g.,Chapter 10 Carcinogenicity by Ke, Costa and Kazantzis 

page 177-196. One chapter (14 by G. Nordberg and B A Fowler) deals with Risk 

assessment pages 281-301. 

A comparison between criteria for classification of carcinogenicity should be done 

between IARC, EU and USA. Hexavalent chromium is classified as a human carcinogen. 

This evaluation is also taken by USEPA for inhaled hexavalent chromium and related 

lung cancer. It should be highlighted that for some metals e.g., arsenic it has been reported 

in the scientific literature that oral intake i.e., via drinking water also can give rise to lung 

cancer though oral intake mostly is referred to cancer in the oral cavity or gastrointestinal 

system. 

My question is if the lung was studied in the NTP studies or any of the animal studies 

reported in the given report? Same question goes for the epidemiological studies that are 

cited? 

Table 3-7 page 30 reports levels of chromium in female controls both in kidney and in 

bone. It is not easy to find any comments on this in the document. Is there any analytical 

problem in this study? 

I will offer a response to this question in the form of general comments regarding specific 

sections of the Review in order of appearance in the text. In taking this approach my 

comments will also directly address questions (A)I-4 and (B)I-5. 

Page 7: The Environmental Protection Agency (EPA) should not be referencing a 2006 

review article by Costa and Klein to site background on environmental chemistry. This 

review article was not a critical review of the environmental chemistry of chromium. 

Even if the general background in that review article is accurate, The Toxicological 

Review of Hexavalent Chromium (TRHC) should cite primary references from chemical 

or environmental journals or compendiums. Also, this paper is mis-labeled in the 

reference list as a 2008 paper. [Correct reference is "Toxicity and Carcinogenicity of 

Chromium Compounds in Humans" Crit. Rev. Tox.: 36(2):155-163, 2006]. 

Moreover, the premise of this review article [in essence that even very low exposures to 

any form of CrVI, including in drinking water, can cause virtually any type of cancer in 

virtually every organ, as well as plethora of assorted other diseases], and the preceding 

review article on which it was largely based ["Toxicity and Carcinogenicity of Cr(VI) in 

Animal Models and Humans" Crit. Rev. Tox.: 27:431-442, 1997], should not be 

universally accepted by the EPA without critical evaluation. Much of the 

epidemiological methodology applied in these papers is flawed. In these papers, the 

author(s) repeatedly and selectively tabulated whatever instances could be found in any of 

the many epidemiologic studies of chromium, of an elevated Standard Mortality Ratio 

(SMR). These were presented with no mention of the fact that most of these instances 
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Responses to Charge Questions 

were small, non-statistically significant elevations (likely to be random fluctuations due to 

the large breadth of the studies), which were either ignored or discounted by the original 

authors because of confounding factors. The paper also failed to take into account that 

many of the small, non-statistically significant elevations in some cancers in one selected 

study, were counter-balanced by either no elevation or decreased SMRs in other studies. 

This "tabulation" approach does not constitute a true meta-analysis and is also statistically 

incorrect. 

There are also additional reasons that the EPA should be circumspect about citing either 

of these articles. The 2006 article, and its preceding counterpart published in 1997, were 

written and published at a time when the senior author was actively engaged as an expert 

witness for the plaintiffs in high-profile hexavalent chromium lawsuits. This involvement 

was not disclosed in the 1997 article, which was focused on attempting to implicate low 

dose exposure to CrVI in a broad array of human cancers. In the Acknowledgements 

section of the 2006 article there is partial disclosure that production of the paper was paid 

for in part by Baron and Budd. In fact, Baron and Budd is one the Jaw firms with whom 

the senior author was under contract with as an expert witness for the plaintiffs in an 

active lawsuit. This article specifically tried to implicate CrVI as a human drinking water 

carcinogen even at very low doses, as well as suggesting that exposure to CrVI causes a 

broad array of other diseases, including neuropsychiatric problems, for which there is no 

support. If the EPA is going to site these review articles it is critical that EPA conduct an 

independent critical review of every paper sited in these review articles. In the latter 

scenario it is certain that EPA will reach a different conclusion. 

I am not of the opinion that a scientist who serves as an expert witness should have to 

disclose all litigation-related work in all scientific publications, particularly not in reports 

of original laboratory research into mechanisms of action, or even in review articles that 

give an unbiased evaluation of the existing literature, especially as it relates to basic 

mechanisms of action. Indeed, in the world of chromium toxicology it is hard to find 

experts who have not participated in some sort of chromium-related litigation. However, 

these two articles do not merely describe original laboratory research or present an 

unbiased review of the literature (note that part of the 2006 review article is a 

recapitulation of an already published journal article on UV light and chromium 

exposure). These two particular articles are essentially position/opinion papers, with 

speculative declarations that even very low dose exposures to soluble CrVI can cause 

virtually any kind of cancer (and other disease) in virtually any organ, a theory of obvious 

benefit to any plaintiff's case in chromium-related litigation, but one that is not supported 

by either epidemiological studies or in vivo animal studies. 

Page 20-21: Although the draft TRHC frequently describes each specific study in this 

section and offers a conclusion/interpretation, the TRHC discussion of the Donaldson and 

Barrera paper ends with a reference to Table 3-1 with no summary. There is important 

information in Table 3-1 that strongly supports the capacity of gastric juice to rapidly 

reduce CrVI to Crill. Note that the uptake 'of Cr in intestinal rings was virtually identical, 

whether the starting material was Criii without gastric juice or CrVI plus gastric juice at 

pH 1.4. 

The text at the bottom of page 21 and the top of page 24 seems to be nuanced to cast 

doubt on the body of work of DeFlora, by using the word "suggested" (second line from 
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Responses to Charge Questions 

the bottom of pg 21), and then suggesting that the values of reducing capacity given by 

DeFlora "should be considered with some caution". This "caution" is based on 

speculation found in the paper cited (Zhitkovich, 2005), and reiterated in the 2006 Costa 

and Klein article (from where the draft TRHC apparently drew its language). This 

speculation is addressed in the supplementary materials under "Public Comments". The 

TRHC and the EPA should not cast doubt on the body of work by DeFlora. based on 

unsubstantiated speculation. 

Page 24-5: The TRHC should recognize and illustrate the main point of the absorption 

studies cited: no matter whether the original starting material is CrVI or Crill there is 

limited absorption and little retention of either: fecal recovery in rats was 98% for Crill 

and 97.7% for CrVI (pg 24) and in humans was 99.6% for Criii and 89.4% for CrVI. 

Pretreatment of CrVI with gastric juice completely inhibited absorption of CrVI after 

direct perfusion into the small intestine. On pg 27 another study indicates that 99% of 

CrVI is recovered in feces using rats gavaged with CrVI. On pg 28 another study 

indicates that maximal uptake after gavage of rats with CrVI occurred in liver and was 

only I%. Absorption in other organs was in the range of 0.1 to 0.2%. It is important to 

note that in all of these absorption studies, including drinking water studies, the increased 

tissue distribution was only observed after chronic administration of more than 5 ppm. 

Many of the studies used greater than l OOppm. The main point that there is very little 

absorption and retention of Cr even after administration of CrVI. 

Page 26: It is incorrect to state that absorbed "hexavalent" chromium is distributed 

throughout the body. Few studies actually speciated the Cr found in organs distal to the 

route of administration and even extremely large doses of CrVI, large enough to saturate 

reduction in the stomach and GI tract, do not deliver much more than trace amounts of 

CrVI to most distal organs because of the vast reducing capacity of blood components. 

The vast majority of Cr reaching distal organs arrives as Criii. The TRHC should make 

this absolutely clear. 

Page 34: The TRHC should provide an accurate summation of Table 3-8 which compares 

tissue chromium after ingestion of a very large, gastric reduction-saturating dose (12.9 

mg!L) of either Crill or CrVI. The only "organ" that showed consequential increased 

levels of Cr after CrVI compared to Criii was blood. Most of the other organs exhibited 

only trace amounts of Cr, even after this huge dose of CrVI, except for the intestine which 

showed significant and nearly identical increased Cr concentrations after both Criii and 

CrVI. This supports the conclusions of the supplementary data in the Public Comments 

showing that some sections of the intestine (jejunum for example) are sites of Cr 

accumulation, regardless of whether the source material is Criii or CrVI. 

Page 35: Note that there is no increased accumulation after 8 weeks of exposure compared 

to 4 weeks of exposure, even at the enormous dose of 130 ppm (mg!L). 

Page 36: The NTRC should not interject the commentary statement: "indicating that a 

portion of the CrVI escaped intracellular reduction in the GI tract and became bioavailable 

for systemic distribution". Like almost every other study this study measured total tissue 

Cr and did not speciate tissue Cr, and the NTRC therefore cannot speculate on what the 

form of Cr was that reached the tissues. Note in Table 3-8 the accumulation of Cr in the 

intestine and blood after CriiL 
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Page 37: Note the obvious threshold of increased bone and kidney concentrations after 10 

ppm compared to all lower doses, as well as estimated body Cr burdens. Note that in liver 

there is a significant Cr burden even after ZERO CrVI exposure. Note the strangely 

compressed scale of the Y axis: even the increase in females at I 0 ppm is only an increase 

from 0.3 to less than 0.5. These data demonstrate the exact opposite of the conjecture on 

page 36: there is a clear threshold of accumulation indicating saturation of reductive 

capacity. 

Pages 38-39: Virtually every study shows the same thing. The NTP study used a "low 

dose" that is already higher than the 10 ppm in the Sutherland study. What is being 

referred to here as a "dose-dependent" increase is already supra-saturation of gastric 

reductive capacity. What these studies really show is how little Cr is absorbed, even in 

tissues that are directly exposed (glandular stomach and forestomach), even after massive 

doses are administered. 

Page 41: It is inappropriate to make such an unqualified statement as found at the top of 

this page: "Hexavalent chromium is capable of crossing the placenta". This is only true 

in the highly contrived circumstances referenced below the statement wherein pregnant 

mice were given an IV injection of a massive dose of CrVI (!Omg/kg). 

Page 42: The TRHC does a good job describing the bioavailability and kinetics ofCr 

absorption in humans after Crill, CrVI in OJ and CrVI. It correctly acknowledges that the 

CrVJ-OJ was completely reduced and that even the full dose of CrVI was insufficient to 

overwhelm the reducing capacity of blood. The potential explanations offered are correct 

but need to add another possibility. Often overlooked is the fact that not all Crill is alike. 

Anyone who works with Criii in the laboratory knows that it undergoes aging in aqueous 

solution, even visibly changing color with time after solubilization. It is possible that 

Crill generated from newly reduced CrVI (as in the CrVI-OJ) may have some different 

biological parameters than straight Crill made up in water and allowed sit for a couple of 

days. In fact overall absorption of newly formed Criii may be higher than aged Crill, 

possibly as a function of its ability to form complexes with biological ligands that may 

alter its absorption potential. 

Page 49 bottom: It is inaccurate to state that "CrVI distributes to other tissues, notably the 

blood, kidney, and liver." Except for the cases of treatment with extreme doses, or use of 

pathways like intra-intestinal instillation or IP injection, the vast majority of Cr that 

arrives at distal tissues is Crill. Once again, it is critical that TRHC make that fact clear, 

otherwise it gives the appearance of non-objectivity. 

Page 50, last paragraph: The TRHC should not simply reiterate speculation that is found 

in the papers it cited (Zhitkovich 2005, Costa and Klein 2006), in suggesting that the 

mutagenicity of Cr may be underestimated in cultured cells because of lower levels of 

intracellular ascorbate when cells are cultured in absence of added ascorbate. Indeed, it is 

just as likely that the mutagenicity of CrVI in cultured cells is grossly overestimated, 

because the lack of ascorbate in the extracellular medium allows CrVI to persist in the 

extracellular medium thereby maximizing its uptake as the hexavalent oxyanion. At the 

very least the TRHC should discuss both possibilities and not give the appearance of bias. 

Page 58: It is inaccurate to state that model simulations "imply" that some CrVI escaped 

reduction in the stomach. This is circular reasoning. The "input" data that went into 
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formulating these models was based on experiments wherein massive doses of CrVI were 

administered, doses that would clearly exceed reductive capacity. It is not appropriate to 

then state that the model simulation "implies" that some CrVI escaped reduction, as 

though the model now supports a novel biological observation. It would be completely 

expected for the model to predict that scenario since it would logically emanate from the 

very data that was used to formulate the model. It is critical that the TRHC indicate that 

these models do not apply to, or accurately predict, the toxicokinetics of low, 

environmentally relevant doses of CrVL The discussion in the THRC does become more 

balanced on page 64 where the non-linear aspects of CrVI uptake, reduction and biodistribution are given some weight. 

Page 66: This section (3.6) needs to be completely rewritten as it lends undue weight to 

an opinion expressed in only one or two papers, at least one of which was written under 

financial inducement by a law firm with a vested interest in characterizing all Cr, 

including Criii, as a potential hazard (see preceding comments). The TRHC needs to not 

indiscriminately cite speculation found in review articles without more rigorous analysis. 

Except for those few biased citations it is almost universally accepted that Criii is an 

essential element. 

Page 68: Section 4.1.2,1ast sentence: This is nearly the ultimate example of how 

critically important it is for the TRHC to do its own critical analysis of the literature. The 

paper cited, (Bick et al, 1996) should not be cited under any circumstances and in fact it 

should be retracted from the scientific literature. Two of the authors, Walter Lack and 

Thomas Girardi, were two of the lead lawyers for the plaintiffs in several high-profile 

chromium lawsuits, now immortalized by the Hollywood movie "Erin Brockovich". 

They listed their "academic" credentials as the Department of Hematology at the 

University of Tasmania in Australia. The other three authors (Costa, Bick and 

Teitlebaum) were paid expert witnesses for the plaintiffs in the same case, which was 

active at the time. None of this was disclosed in the paper. The two cases of NonHodgkin' s lymphoma discussed in this case report were plaintiffs in the active lawsuit and 

the information was supplied by the lawyers. Moreover, at best this report is merely a 

case-report (not even a case-control study), merely reporting that two people in Hinckley 

CA, at that time, had been diagnosed with Non-Hodgkin's lymphoma. 

In contrast, the draft TRHC does not yet, but should reference the recent work of Dr. John 

Morgan, an epidemiologist for the California Cancer Registry. He has been tracking 

cancer incidence in the town of Hinckley CA (the "Brockovich" town) from 1996 to 

present. He recently published data showing that from 1996 to 2008, not only is there no 

excess of total cancer or any specific cancer in Hinckley, there are actually fewer cancers 

than expected. 

Pages 71-80: The draft TRHC conducts a very thorough depiction of the different 

interpretations of the Liaoning Province situation. What seems to get lost in the details is 

the larger picture. This is a Province of a country wherein the background rates of both 

stomach and lung cancer are high even in non-chromium exposed comparison groups, 

indicating the presence of other contributing risk factors. This is a situation where 

exposure is characterized in terms of high dose, long term "yellow water", yet despite this 

potential significant exposure, the question of whether there is an additional modest 

increase in risk for stomach cancer hangs on whether a particular industrial area is 
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included in the comparison group or not. There is much controversy surrounding the 

reports of cancer risk in this Province, but after discussing the controversy the draft THRC 

aligns itself with the method of re-analysis of Beamon! et a!. The THRC should then also 

cite the commentary by Allan Smith [Epidemiology 19:24, 2008] which accompanied the 

Beamont article: although Smith is sympathetic to Beamont's attempt to re-analyze the 

data, he also describes the extensive weaknesses of the approach. This is not the kind of 

data that a regulatory decision should be based on. 

Page 81: The NTP toxicology studies on subchronic oral exposure (Section 4.2.1) are 

technically well done. The principle issue that needs to not be lost in the detail is that the 

lowest dose was 62.5 ppm, an enormous concentration of little or no environmental 

relevance. This is a "yellow water" situation to the extreme. Despite these enormous 

doses most of the observations did not exhibit a consistent pattern of dose or duration 

dependence. It is also important to recognize that these enormous doses of CrVI actually 

serve to deliver an enormous amount of Crill to the organs and cells in question. 

Remembering that Crill is not without biological activity (acting as a co-factor in insulin 

action), it is entirely possible the some of the observed effects are due to the physiological 

effects of massive Crill overload. The extensive new data provided by ToxStrategies, 

described in the Public Comments, needs to be incorporated into the TRHC. 

Page 84: Again, a consistent relationship between severity and dose was not observed. 

This implies the presence of effects caused by indirect mechanisms, likely chronic 

inflammation and/or tissue damage only observed at the highest doses (see below). 

Urinalysis shows effects due to decreased water intake due to poor palatability of the 

yellow water. This dehydration alone is capable of rendering epithelial tissues more 

fragile. Changes in organ weights were only observed at doses above 500ppm (pg 86). 

Page 87-108: The results are described repeatedly as "without clear dose-response 

relationship". Indeed, minimal to mild histiocytic cellular infiltration was observed in all 

groups including the control animals. Even less toxicity was observed in mice compared 

to rats; in fact even at 1000 ppm for 3 months there was no evidence of any 

hepatotoxicity, only mild changes in some hematological indices that were attributed to 

changes in body weight (probably caused by massive Crill overloading and its potential 

effects on insulin and glucose metabolism). What needs to be emphasized here is that the 

lowest dose used in any of these studies is at or above saturation of gastric reductive 

capacity and yet still very little toxicity was observed except at the two highest doses (and 

often only at the one highest dose) (Tables 4-12. 4-13, 4-14). At the lower end of these 

very high doses, only inconsistent observations were made and when "toxicity" was 

reported it was generally ranked minimal to mild. Only the index of Liver (fatty change) 

was ranked as moderate, but that was identical to the ranking of that same index in the 

Controls. The main point here should be that these are massive doses and they are 

eliciting minimal effects. This important concept should not be lost in the mass of 

detailed results. 

Pages 109-120: The NTP carcinogenesis studies in rats and mice show that there is no 

carcinogenic response except at the two highest doses that also produce chronic tissue 

damage at the sites of carcinogenicity. The dose-response is definitively non-linear, as is 

the absorption data described above. Given that the lowest dose is already above the 
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reductive capacity of the oral cavity and stomach, these data provide strong evidence of 

the protective effects of the reductive capacity of blood components. 

It should be noted that the NTP's published report by Stout et al [Hexavalent Chromium is 

Carcinogenic to F344/N Rats and B6C3FI Mice after Chronic Oral Exposure, 

Environmental Health Perspectives 117: 716, 2009] presents an inaccurate Discussion of 

potential mechanism of action, drawn heavily from the 2006 Costa and Klein article, 

especially in criticizing the work of DeFlora. In point of fact, the results of the NTP 

assay, and the extensive additional data found in the Public Comments generated by a 

group of investigators around the country funded by ToxStrategies, give nearly definitive 

proof that the work of DeFiora is correct. Even the lowest dose of the NTP assay exceeds 

the reductive capacity of the oral cavity and upper digestive tract. Yet little toxicity and 

no carcinogenicity is observed except at the two highest doses. 

The argument by Stout et al that the NTP doses were below gastric reduction-saturation, 

based on a supra-linear (decreasing response with dose) rather than sub-linear (increasing 

response with dose) dose response is incorrect. If the doses were below saturation of 

reductive capacity, as the dose increased the ratio of unreduced CrVI to reduced CrVI 

(Crill) in the stomach would increase (due to depletion of reductive capacity), and 

absorption would show an increasing rate of response (opposite of what was observed) 

because of an increased percentage of the total Cr that would be in the unreduced 

hexavalent state. Yet both absorption and toxicity exhibit a decreasing rate of response 

with dose in the NTP assay. This would actually be expected at supra-saturation doses: 

once the reductive capacity of the oral, digestive and blood components is exceeded, the 

organs receiving the highest amount of CrVI will sustain inflammatory tissue damage 

provoking tissue regeneration. It is unlikely that such tissue damage would display dose 

dependence since it only occurred at the two highest doses of the assay and it is a 

complex, disseminated biological response. It is likely then that a combination of three 

factors contribute to the high dose carcinogenic response: (i) tissue damage with 

regenerative cell profieration, (ii) regenerative cell proliferation in the presence of 

macromolecular damage, and (iii) regenerative cell proliferation occurring in the presence 

of massive Criii loading, which may affect insulin-dependent proliferative signaling. 

Pages 122-149: For these studies on the potential reproductive toxicities caused by CrVI 

one can only hope that the TRHC and EPA will remember the 16"' century adage of 

Paracelcus "all substances are poisons, the right dose differentiates a poison from a 

remedy". These studies show reproductive toxicity at huge doses ofCrVI, often given 

using invasive administration procedure (IP or IV injection), with little relevance to 

environmental exposure levels. 

Pages 176-178: The in vivo studies showing DNA damage or mutagenicity in cells 

peripheral to the point of administration of CrVI were only positive when massive doses 

of CrVI were administered by gavage, direct instillation, or intravenous injection. 

Although some studies claim to find mutations in the absence of cytotoxicity these are 

highly contrived systems: for example eye spots in offspring of pregnant female mice 

given huge doses (62.5 ppm) of CrVI in drinking water. All studies of mutagenesis in 

cultured mammalian cells, including human cells, demonstrate that mutagenesis is only 

observed at doses that produce some degree of cytotoxicity and replication arrest. It 

should also be clarified in the TRHC that DNA damage and mutagenicity should not be 
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equated: while mutagenicity may result from DNA damage, the relationship is not simple 

or linear and is further complicated by DNA repair. Also, it is unclear whether all forms 

of DNA or chromatin alterations (collectively termed DNA damage) are pre-mutagenic. 

For example, in silico studies on DNA-protein crosslinks suggest that under certain 

circumstances Cr!II can serve as binary crosslinking agent between small peptides and 

DNA. However, in in vitro studies in cultured cells and in in vivo studies, it is not clear 

what is actually being measured by assays for DNA-protein crosslinks. This phenomenon 

may in fact only indicate that chromatin isolated from certain cells exhibits a higher 

degree of condensation during isolation, rendering chromatin proteins more difficult to 

extract. What appear to be DNA-protein crosslinks can be actually be observed in cells 

treated with agents that do not participate in or catalyze formation of an actual binary 

crosslink. 

Pages 202-214 (section4.7.3.2): Many of the preceding comments directly address and 

provide major qualifications to the MOA discussed in this section, including the 

interpretation of reductive capacity found in the Stout et al report of the NTP assay. 

It is clear that the EPA is faced with a unique situation in assessing the MOA of Cr(VI) at 

it relates to low-dose risk assessment. It is abundantly clear from all the science that the 

effects of Cr(VI) at the massive doses necessary to produce tissue toxicity and 

carcinogenesis in rodents, have no bearing on the effects of low-dose, environmentallyrelevent exposures. This is consistently borne out by epidemiological, animal and cell 

experimentation. 

This is especially pertinent in relation to whether or not Cr(VI) should be considered with 

a mutagenic MOA. I have spent more than 25 years studying the genotoxic properties of 

Cr(VI) and I have frequently contributed to the plethora of studies showing DNA damage 

and what we thought was associated mutagenesis. There is no doubt that Cr(VI) can be 

forced to be genotoxic and "mutagenic" under experimentally contrived systems and at 

high doses that evoke major amounts of cell death. However, in hindsight many of us 

"DNA damage and repair" scientists have come to appreciate several important factors: (i) 

DNA damage is only observed at very high dose that kill a lot of cells, (ii) Cr(VI) is at 

best a very weak "mutagen", requiring very high doses that kill most cells and 

experimental "backflips" to select for survivors, and (iii) what we thought was 

"mutagenesis" is actually selection for stochastic cell survivors of massive toxic insult. 

Dr. Rossman's group has shown that the base sequence of the genes used for mutation 

detection and selection is intact and that the changes in gene expression enabling selection 

are epigenetic, not mutagenic. Our group has shown that what we really selected for at 

toxic exposures are cells that are resistant to apoptosis, and Dr. Zhitkovich's group has 

shown that the "mutant" cells were actually surviving cells that were selected for changes 

in specific forms of DNA repair. Again, this only occurs at doses that kill a Jot of cells, 

not dis-similar to the high-dose rodent assays wherein tumors were only observed at doses 

that produced chronic and fairly severe tissue damage. This harkens to what is sometimes 

viewed as a landmark study of lung cancer and occupational exposure to high doses of 

Cr(VI) by Gibb et al. Occupational exposure to in the chromate production industry was 

categorized into 4 quartiles. The lowest two quartiles are huge levels of exposure by 

"environmental" standards, orders of magnitude beyond the even the highest known 

environmental exposures. The lowest quartile of exposure was essentially a No Effect 
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Level (no elevated risk) and the slightly elevated risk ratio in the second quartile was not 

statistically significant. Interestingly, of the total of 120 lung cancer cases found in 

chromium-exposed workers, 116 were also smokers. 

The EPA may be under certain historical regulatory precedents and pressures to deem 

Cr(VI) with a mutagenic mode of action simply because there are published studies that 

have "Cr(VI)" and "mutation" equated in the title (some of these papers are my own), but 

this decision would not be based on science. At high, tissue damaging doses one can get 

tumors to form and those tumors will have mutations in specific genes because that is the 

molecular etiology of how that particular cancer develops. It will have no relation to any 

chemically-specific mutations caused by Cr(VI) because Cr(VI) is an exceedingly poor 

mutagen. Even at the low end of very high doses there is NO MOA because there is NO 

toxicity, no mutagenecity, and no carcinogenesis. Extrapolating linearly from events 

observed at the two highest doses of the NTP assay, to anything close to reality for 

environmental exposure, is simply not scientific. If ever there was a textbook case to be 

made for a "threshold carcinogen", it is Cr(VI). 

In general, this was a clearly written document. However, my area of expertise is in the 

mutagenic and epigenetic mechanisms of action of carcinogens, and I found many more 

problems in those areas than in the rest of the document. I will discuss these in section 

B2. 

Here I will mention some errors in the rest of the text. 

p. 38, end of I" paragraph reads, "Uptake in guinea pigs did not appear to generally differ 

from that of rodents". The guinea pig (Cavia porcellus) is a rodent. 

p. 46, section 3.3, refers to transport of the hexavalent chromium oxyanion (for clarity, 

should this read chromate/dichromate?) by sulfate and phosphate transport system~ 

(should be pleural). It is claimed that this allows accumulation in cells at higher 

concentrations than the extracellaular concentration. Neither the transport systems nor the 

evidence for higher intracellular accumulation are referenced. Actually, what allows 

higher accumulations is the fact that Cr(6) is reduced in the cell to Cr(3), which cannot get 

out, so what accumulates is Cr(3). 

p. 201, 1" full paragraph: It is claimed that the key precursor events leading to chromiuminduced mutagenicity have been identified in animals. This is not so. It is not even true 

for mammalian cells in culture. Some ideas have been derived from cell culture studies 

(but with Cr-damaged shuttle vectors). Almost nothing is known about mutagenicity in 

animals, and nothing at all is known about the genetic changes occurring in animal tumors 

or in the target tissues. 

p. 202. Section 4. 7 .3. For reasons that will become clearer in my response to B2, this 

section is very flawed. I will mention just one point here: The confusion between 

"mutagenic" and "genotoxic" must be cleared up throughout this section (as well as 

throughout Section 4.5.). The thinking on this issue is very sloppy. A mutagenic mode of 

action is just that: it requires mutagenesis. 
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Salnikow In September 2010, the U.S. Environmental Protection Agency prepared the 

"Toxicological Review of Hexavalent Chromium" to assess health risks associated with 

hexavalent chromium exposure. This document will appear on the Agency's online 

database, the Integrated Risk Information System (IRIS). The existing IRIS file for 

hexavalent chromium, prepared in 1998, does not consider hexavalent chromium to be 

carcinogenic by the oral route of exposure. The purpose of the new document is to update 

the IRIS regarding noncancer and cancer health effects associated with oral exposure to 

hexavalent chromium after considering the latest scientific evidences. 

The prepared draft of the "Toxicological Review of Hexavalent Chromium" provides a 

detailed analysis of the data obtained in several studies carried out in 2008, 2009 by the 

National Toxicology Program along with other studies, formulates a mutagenic mode of 

carcinogenic action, and suggests that the reduction of orally administered hexavalent 

chromium in guts, even in low doses, is incomplete. The draft also calculates a cancer 

slope factor for humans. Recognizing the importance and relevance of this document, this 

reviewer needs to address some shortcomings of the document. In general this is a dense 

document cataloguing many diverse and sometimes controversial studies in the area of 

hexavalent chromium toxicology and carcinogenesis. Of course the limitations in 

available experimental data obtained from existing animal models, ongoing investigations 

regarding the mode of action (MOA) of hexavalent chromium, and uncertainties in 

epidemiological data make it difficult to prepare a comprehensive document that will fully 

address public health concerns. 

Chapter 3 should be reorganized and more emphasis should be given to the role of 

fhromium III in toxico- and pharmacokinetics as well as in biological effects produced by 

hexavalent chromium. Hexavalent chromium is generally considered a much more 

potent mutagen and carcinogen than trivalent chromium. Lack of carcinogenic effects 

observed with trivalent chromium compounds can be explained by poor permeability of 

cell membrane for this ion. However, considering that the end llroduct of intracellular 

reduction of hexavalent chromium is trivalent chromium, which rna~ accumulate in 

tissues, it is im!lortant to consider what role intracellular!:; dellosited trivalent chromium 

rna:; Qla~ in chromium toxicit:; and carcinogenesis. The majority of studies indicate that 

after intracellular reduction of hexavalent chromium to trivalent chromium, it can form 

various damaging DNA adducts. These adducts can inhibit the enzymatic activity of DNA 

polymerases, simultaneously increasing the rate of replication and the processivity of the 

DNA polymerase, and thereby decreasing its fidelity and causing more frequent errors. 

The frequency of errors increases with a dose-dependent increase in mutation frequency 

in vitro (Snow, 1991; Salnikow and Zhitkovich, 2008). Unfortunately, it is not clear how 

applicable these studies are to understanding the effects of trivalent chromium on DNA 

synthesis and cellular metabolism in vivo because the experiments were done in either in 

test tubes or in artificial model systems with concentrations far exceeding those obtained 

through environmental exposure (Snow, 1991; Dai et al., 2009). Numerous attempts have 

been done to study the distribution and retention of chromium (III) in vivo. Onkelinx 

studied tissue retention of 51CrC13 in groups of female Wistar rats of various ages (35, 60, 

and 120 days) after a single intravenous injection of trace amounts of 51CrCh (Onkelinx, 

1977). The study showed that total excretory clearance is the sum of three components: 

urinary clearance (fu), fecal clearance (fd), and a residual clearance (fs), corresponding to 

an apparently irreversible deoosition of chromium into lonrr term bodv reservoirs. 
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Consistent with the model, 51Cr was found to accumulate with time in several organs such 

as bone, kidney, spleen, and liver after a single intravenous injection of 51CrCJ,. These 

data are supported hy those obtained by O'Flaherty (O'Flaherty, 1996) and others 

indicating that the retention of chromium III by bone, liver, kidney, and spleen is 

prolonged. 

Also, to make the toxicological review concise, I suggest eliminating Table 4-2l"In vitro 

genotoxicity studies of hexavalent chromium in nonmammalian cells" and Table 4-24 "In 

vivo genotoxicity studies of hexavalent chromium in D. melanogaster" because these are 

irrelevant to the MOA of hexavalent chromium in mammalian systems. 

There are numerous errors throughout the text of the Draft. Some of them are shown at the 

end of these comments as Errata. 

Wise The Toxicological Review is logical, clear and concise. However, overall the document is 

inconsistent and thus, EPA has not presented and synthesized the scientific evidence for 

noncancer and cancer hazard in a clear manner. Some sections, primarily the ones focused 

on animal data are clearly presented and synthesized. These sections present the primary 

literature and discuss the merits of each study with balance and insight. 

Other sections, however, particularly those involving in vitro cell culture data and 

underpinning the mode of action are much less appropriately considered, are not wellpresented, and do not synthesize the underlying data very well. Determining a mode of 

action is a key part of the risk assessment. The Toxicological Review would be a stronger 

document if it fully analyzed and synthesized the primary literature to ascertain the 

possible modes of action for Cr(VI). The strengths, weaknesses and data gaps for each 

could have been highlighted and discussed, and then a rationale for the chosen mode of 

action presented. However, as presented this approach is not apparent. 

Instead, as presented, the document gives the impression that the mode of action was predetermined from a select set of review articles and the best case for that mode of action 

presented. Decisions appear to have been made to agglomerate all of the genotoxicity data 

into positive or negative proof of mutagenesis rather than more careful consideration of 

individual lesions. Confounding factors, such as ascorbate levels. are cautioned against, 

but inadequately and inaccurately presented and unevenly applied, which undermines 

confidence that the primary data were adequately considered and contributes to a 

perception that decisions were predetermined. This perception is strongly reinforced by 

poor management and citation of the underlying literature and a heavy reliance on a few 

select review articles that unfortunately miscite the primary literature. As a result, many 

sections of the Toxicological Review lack clarity, accuracy, synthesis and rigor and the 

rationale for the choice of mode of action seems predetermined and forced. Each of these 

factors is elaborated on in more detail below. 

I. Unnecessar:,: agglomeration of genotoxicit:,: data 

The Toxicological Review essentially combines all of the lesions related to genotoxicity 

(stated on page 212 as " ... including DNA adduct formation, DNA damage, gene 

mutations, chromosomal aberrations, and micronuclei/ormation") into one bundle and 

refers to them as "mutagenicity". This decision is typically based on the presumption that 
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the various genotoxic lesions will ultimately manifest as mutations in the primary DNA 

sequence. Hence, it is generally recognized that a crosslink or a strand break is not 

inherently a mutation, but that it may eventually manifest as one and thus, it is a 

mutagenic event. Based on the aggregation of all of these data, the Toxicological Profile 

declares Cr(VI) to be a mutagen and proposed a mutagenic mode of action. 

This approach is consistent with older practice and perception of these genotoxicity assays 

and it may be a useful approach for a chemical with a limited data set. However, the 

genotoxicity data for Cr(VI) is a rich data set and deserves a more sophisticated 

consideration. The Cr(VI) literature often distinguishes in its presentation between 

mutagenic and genotoxic lesions. The Toxicological Review does not carry forward that 

distinction and does not explain the rationale for ignoring it. However, it is an important 

distinction because not all of these lesions are likely to be mutagenic after Cr(VI) 

exposure. Aggregating these lesions oversimplifies the interpretation of the data and 

masks the fact that much of the primary data suggest that in actuality, Cr(VI) is a very 

weak mutagen. Discussing each class of lesion on its own merit with a more careful 

consideration of the primary literature would have better framed the strengths and 

limitations of the genotoxicity studies and brought this discussion into a clearer light. 

The fundamental problem underlying this section is a failure to clearly consider the 

primary literature to see that Cr(VI) is a weak mutagen when defined as an agent that can 

directly change the primary DNA sequence. Cr(VI)-induced mutations have indeed been 

observed in bacteria, cultured cells and animal studies. However, in most cases, one has 

to experimentally force the mutations to occur by using a high dose, a forced experimental 

system or a non-physiological exposure route. 

There is no real synthesis of this literature beyond listing outcomes in a table. The 

Toxicological Review also considers the results as simply positive or negative. That 

certainly is one approach; however, it misses the opportunity to consider the data more 

thoroughly. Some consideration should be given to potency and its potential impact and 

the robustness of the underlying assays. If the experimental data show that Cr(VI) induces 

a 2-fold increase in mutations, then the Toxicological Review would call that outcome 

positive. However, if in that same assay an established mutagen induced a 50-fold 

increase in mutations, then does Cr(VI) still appear to be a mutagen? Or does it suggest a 

different mode of action, particularly when the frequency of mutations have not been 

reported to increase in Cr(VI)-induced human tumors? Does the fact that ascorbate is 

higher in rodents make these mutations a rodent-specific event? A more careful and 

thoughtful presentation of the underlying data would have better informed the 

consideration of this mode of action. 

In addition, before lumping all of these genotoxic endpoints together as all mutagenic 

outcomes, a careful review and discussion of the primary mutagenesis literature is needed. 

That review needs to determine, for Cr(VI), which of the various lesions (e.g. DNA 

adducts, DNA crosslinks, DNA strand breaks, gene mutations, chromosome damage, etc.) 

actually occur in cells (for example as discussed below the adducts may not actually form 

in cells) and to what extent they occur. Then, the review needs to determine which, if 

any, of these lesions actually lead to gene mutations. The discussion below illustrates that 

there are reasons to doubt that the various lesions are all mutagenic outcomes. After this 
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analysis, those lesions that do form in cells and that do produce mutations could more 

reasonably be combined into a category of "mutagenicity". 

Perhaps, the data will indicate Cr(VI) is a mutagen, but, perhaps, the data indicate that one 

only gets mutations in the DNA sequence when systems are forced experimentally to do 

so at very high concentrations, due to species specific factors or by non-physiological 

exposure routes. If the latter were true, this possibility would suggest that mutations are 

not likely to occur in humans, raising direct implications for the mode of action decision. 

A more thorough treatment of the primary mutation data is needed to clarify these 

important points. 

There is concern that the discussion of some lesions is overstated while others are 

mentioned but not discussed. The section explaining DNA adducts is greatly overstated 

and also does not fully consider the primary literature. The section presents a case that 

implies the status and impact of the various potential adducts are known in cells and in 

vivo. The Toxicological Review even provides a structure of a Cr-DNA adduct. The 

major concern is that when the primary literature is fully considered, it becomes apparent 

that these adducts are all based on cell-free systems and no one has been able to clearly 

identify any specific adducts in cells, whole animals or humans beyond observing tangles 

of DNA, protein, and Cr that are considered to be DNA-DNA or DNA-protein crosslinks. 

The primary literature has only measured adduct levels in cells by isolating DNA and then 

measuring the amount of Cr associated with it or by nonspecific P32 postlabelling. These 

measures cannot ascertain how or if Cr is bound to the DNA, only that it is associated 

with it in some way. Some studies have synthesized adducts in cell free systems and 

applied them to cells, but that does not mean those specific adducts form in the cell. 

Thus, it is unknown if specific DNA adduct events occur in cells, whole animals or 

humans. Nonspecific adducts have been detected by postlabelling, but specific adducts 

remain elusive. To lump these studies together as clear evidence of mutagenicity gives 

them a weight of evidence that seems premature and inaccurate. Overall, this section is 

very misleading in its portrayal of the status of adducts as more understood than they 

actually are. 

Oxidative damage is also included as evidence of mutagenicity. However, discussion is 

missing to establish whether this oxidative damage is a direct effect of Cr(Vl) causing 

oxidative damage to DNA and thus, potentially a mutagenic event, or if this damage is 

actually indirect, resulting from overall oxidative stress to cells caused by high doses of 

Cr(Vl) depleting intracellular antioxidants. 

Cr(Vl)-induced strand breaks are cited as another type of mutagenic event. These lesions 

are discussed as post-replication-induced breaks. However, the discussion fails to 

question and discuss whether or not these are actually frank DNA breaks. As the 

Toxicological Review indicates, the studies have focused on gamma-H2A.X focus 

production as the measure of breaks. Data indicate that chromatin remodeling may also 

induce the production of gamma-H2A.X so they may not be frank DNA breaks after all. 

This possible explanation is missing from the section. 

DNA-protein crosslinks are included as a type of mutagenicity in the tables and 

description despite the fact that the document state.~ on page 186 that it is unknown if thev 
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are mutagenic: "Tests for the mutagenicity of these cross/inks have proved inconclusive 

(reviewed in Macfie et al., 2010), but the bulkiness of these lesions indicates the potential 

for genotoxicity ... " 

The most consistent genotoxic outcome is the production of damage to metaphase 

chromosomes manifested as aberrations, sister chromatid exchanges, and micronuclei. 

The fact that Cr(VI) induces these events was presented but not discussed. This lesion 

may be the key lesion as it is the most consistent and yet the mechanisms that may 

underlie it are ignored and not discussed. Chromosome damage could be a mutagenic 

lesion as assumed. Alternatively, it could be the consequence of epigenetic changes in the 

cell resulting from Cr binding to centrosomes in the mitotic spindle assembly apparatus or 

from bypass of the spindle assembly checkpoint. Cr(VI) has been shown to affect 

centrosomes and the spindle assembly checkpoint and perhaps it causes uneven pulling 

leading to breaks and errors in chromosome number. Cells with broken chromosomes 

may undergo apoptosis, while those with increased chromosome number may go on and 

survive as highly aneuploid cells. Cr(VI) has been shown to induce highly aneuploid cells 

that can clonally expand and survive. This outcome would not be consistent with a 

mutagenic mode of action. 

These concerns above are only magnified by the problems with uneven consideration of 

confounders and in poor management of the underlying literature described below. 

Together these factors give the impression of excluding or avoiding different syntheses of 

the data. More care and balance are needed to discuss and consider the genotoxicity data 

separately and evaluate if they are mutagenic markers. 

2. Uneven application of experimental confounders 

The perception of bias caused by bundling all of the genotoxicity endpoints together is 

magnified by an apparent uneven consideration of experimental confounders in the 

document. It appears that the Toxicological Review does not fully consider and present all 

of the relevant in vitro cell culture data that inform possible modes of action. Instead, 

selected examples of primary literature that reinforce one point of view are presented. 

This approach undermines the synthesis of the literature and because of a marked 

unevenness in presentation creates a perception of bias that should not be part of the 

analysis. 

For example, on page 184-185, the Toxicological Review gives the reader the impression 

that ascorbate-trivalent chromium-DNA adducts have been found to be highly mutagenic. 

However, the section did not describe the experimental detail that indicate the cells used 

in the study were abnormal and genetically modified so that they could not carry out 

proficient DNA repair or apoptosis, or that the cells were not actually treated directly with 

Cr(VI). These omissions stand in stark contrast to the experimental criticisms the 

Toxicological Review applies to other cell culture studies. 

For example, on page 4 7, the document states: 

"Caution should be used in interpreting cell culture data, as the cell culture medium 

could play a role in hexavalent chromium reduction, confounding the extent of 

intracellular hexavalent chromium reduction." 
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It then cites a couple of examples where Cr(V) was detected in extracellular cell culture 

medium. The use of the word "caution" leads the reader to conclude that many in vitro 

studies may be flawed due to this reduction. No explanation is offered as to why this 

outcome is a problem. No discussion is provided that points out whether this same type of 

reduction might be expected to occur outside of cells in the body and thus actually be 

normal. The practical reality is that reducing agents are present in the extracellular fluid 

and thus, some extracellular reduction probably occurs in the extracellular fluid. This 

factor is probably not appropriate as a cautionary one and may actually reflect 

physiological conditions. But, no balanced discussion is provided for the reader to decide 

if this factor is indeed a concern. 

A similar unevenness occurs during the presentation of the relative importance of 

ascorbate. The Toxicological Review states on page 50: 

"An additional important note on these biotransformations regards the 

interpretation and reliability of data from in vitro assays. In vivo, the intracellular 

levels of ascorbate are quite high (about I mM). In contrast, the levels of ascorbate 

in tissue culture media are quite low since generally it is not added to the media so 

that the only source is supplemented fetal bovine serum (FBS). With 10% FBS, the 

level of ascorbate in tissue cultured cells is only about 50 11M which is 20 times 

lower than that which is found in vivo (Zhitkovich, 2005 ). Therefore, experiments on 

mutagenesis and other toxic effects of hexavalent chromium in tissue culture may 

underestimate its mutagenic, genotoxic, and cell-transforming activities (Zhitkovich, 

2005; Costa and Klein, 2006)." 

No further discussion is presented. There is no presentation of the primary literature in 

cell culture showing the impact or lack of impact of this difference, just speculation that it 

might cause some underestimation. There is no presentation of the primary literature to 

establish what the ascorbate levels are in the relevant tissues of concern. There is just this 

one review article (Zhitkovich 2005) with some comment from a secondary review article 

(Costa and Klein 2006) cited. Closer inspection shows that the Costa and Klein review is 

actually citing the same Zhitkovich review so in the final consideration, this entire section 

relies only on Zhitkovich 2005 which, as discussed below in section 3.D., miscites the 

primary and secondary literature and draws a conclusion the primary literature does not 

closely support. 

Thus, the Toxicological Review draws attention to the possible presence of extracellular 

metabolism and lack of intracellular ascorbate as confounding factors, which it may have 

used to exclude some cell culture studies. But, by contrast, it makes no mention and 

expresses no concern about studies done in abnormal compromised cells treated only 

indirectly with Cr(Vl). This discrepancy makes the document and its treatment of the 

underlying literature seem uneven. 

Moreover, in its discussion of the impact of ascorbate, the Toxicological Review does not 

discuss the potential impact of ascorbate differences on the bacterial mutation studies or 

the possible impact of ascorbate differences on the animal genotoxicity data. For example, 

in Table 4-23 on page 172, the Toxicological Review indicates a positive effect for 

mutagenicity in mice after intratracheal instillation. Mice, however, have more ascorbate 

in their lung tissue. If one accepts the speculation in the Toxicological Review that 
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ascorbate-trivalent chromium-DNA adducts form and are highly mutagenic, then the 

elevated mutations in this study might simply be due to the elevated ascorbate levels in 

this species suggesting a species specific effect. Such a possibility would explain why 

there are mutations in rodents but not in human tumors. Regardless of which conclusion is 

correct, the point is that the Toxicological Review does not appear to apply this 

confounder it stresses in the in vitro work evenly to all studies reinforcing a perception of 

selective bias. 

Similarly, the Toxicological Review presents the primary research studies by Quievryn et 

aL, 2003; Voitkun eta!., 1998 as showing adduct effects, but both studies used the cell 

culture medium the Toxicological Review expresses concerns about and neither study 

addressed the ascorbate concern, but these aspects are not mentioned in the document. 

The absence of discussion of these confounders in these experiments give the impression 

that the Toxicological Review does not seem to apply its confounding criticisms evenly. 

This inconsistent presentation of experimental expectations and application of 

confounding factors creates a perception of uneven evaluation of the primary literature. 

Considered together, the language and approach suggest a strong bias against in vitro 

studies and the cautionary language should be removed to eliminate that bias. 

The discussion about ascorbate needs to be more balanced and thorough and the 

information better synthesized. The ascorbate section could be removed or if the EPA 

feels the issue needs to be considered, it should be fully vetted with a discussion of how 

differences in ascorbate might affect the interpretation of the bacterial mutagenesis studies 

and the rodent data. The discussion would need to also include the strengths and 

limitations of the primary literature. The relative merits of data from a primary normal 

human cell line without vitamin C supplementation versus data from a tumor-derived cell 

line with ascorbate supplementation would need to be presented and discussed. The 

various underlying phenotypic issues in cell lines would also need to be considered as a 

mitigating factor. Similarly, the technical limitations of ascorbate supplementation in 

culture would need to be considered including how long it is retained by the cell and the 

impact of its diffusion out of the cell and into the extracellular medium. 

This discussion would need to include a full evaluation of ascorbate levels in tissues of 

interest in humans and animals and whether those levels are intracellular or extracellular 

or both. It should include a full discussion of any data that show whether or not there is an 

actual impact of different ascorbate levels on outcomes inside the cell from cell culture 

studies. It should also include a discussion about the fact that ascorbate in the cell 

becomes depleted over time after Cr(Vl) exposure and whether the relevant exposure is 

when ascorbate levels are normal or depleted. 

3. Poor management and citation of the literature 

The above two concerns are further magnified by the presentation and management of the 

literature in the sections involving in vitro cell culture data and underpinning the mode of 

action. There is a tendency in the document to overstate the fipdings of the selected 

primary literature included in the document, raising questions about whether the primary 

literature was properly evaluated and weighed. There is inconsistent application of the 

phrases "in vitro" and "in vivo" resulting in substantial confusion regarding the underlying 
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literature and reinforcing a perception of inaccuracy in the document. There are flaws in 

citations, extensive direct quoting and long stretches of general paraphrasing of a small 

number of review articles raising questions about the heavy reliance on those articles and 

points of view, Finally, there is often a failure to check the underlying primary research 

studies cited in these review articles reinforcing the perception that the document relies on 

the review and not the underlying primary research data. Considered together, these 

aspects raise questions about the process of how the conclusions were drawn, create 

confusion about whether the primary data were fully reviewed or whether the view of the 

authors of those few review articles was simply adopted, and raise significant questions 

about the credibility of the overall evaluation. Each concern is explained in more detail 

below. 

A Overstating the findings of the selected primary literature 

There are concerns that the Toxicological Review over-generalizes its presentation of the 

primary literature, particularly with respect to in vitro cell culture studies. One example of 

this problem is seen in its discussion of the literature concerning DNA adducts where the 

Toxicological Review states on pages 184-185 that: 

"Although the ascorbate-trivalent chromium-DNA adducts are recovered less 

frequently in vitro due to the low concentrations of vitamin C present in commonly 

used tissue culture media (Zhitkovich, 2005}, these adducts have been shown to be 

the most mutagenic of all the ternary adducts (Quievryn et at., 2003)." ... "They 

have been detected in vitro in Chinese hamvter ovary cells following exposure to 

hexavalent chromium, and account for up to 50% of all chromium-DNA adducts. 

The ternary adducts have been found to cause mutagenic and replication-blocking 

lesions in human fibroblasts in vitro (Quievryn eta!., 2003; Voitkun eta!., 1998}." 

Thus, the reader is led to believe that ascorbate-trivalent chromium-DNA adducts have 

been found in cells and these adducts have been shown to be highly mutagenic. Careful 

examination of the two cited references reveals that the statement in the Toxicological 

Review quote listed above that states: 

"They have been detected in vitro in Chinese hamster ovary cells following exposure 

to hexavalent chromium ... " 

is incorrect. Detection of adducts in Chinese hamster ovary cells was not actually 

presented as data in either paper or mentioned in the text of either paper. The claim is 

unsubstantiated as presented, which makes it potentially misleading. 

Furthermore, when oue considers the experimental detail in Quievryn et aL, 2003 and 

Voitkun et al, 1998, one learns that the adducts were synthesized in a cell free system. A 

sequence of DNA was treated with Cr(VI) and ascorbate in a cell free system. Then, the 

damaged DNA sequence was administered to cells. The cells then converted the damaged 

DNA sequence to a mutation that was revealed when the sequence was recovered and 

sequenced. 

The detail also shows that the cells used were not normal human cells, but rather a SV40 

immortalized cell line. SV40 is known to silence p53 activity, among other cellular and 
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molecular changes, thus these cells were unable to carry out proficient DNA repair or 

apoptosis, as these are normally p53-dependent events. 

Thus, the studies did not show that these adducts were normally present or able to form 

inside the cell. They could not account for the fate of the adduct structure after the 

transfection process and are only assuming it remained intact. The studies did not show 

that mutations would have occurred normally inside the cell as a consequence of Cr(VI) 

exposure or as a consequence of these lesions. Moreover, they do not show that these 

events would have happened in a repair proficient or apoptosis-proficient cell. It could be 

that the only reason mutations were seen is that the cells' ability to repair or eliminate 

them through apoptosis was artificially turned off beforehand. 

An alternative interpretation of the studies could be that one can experimentally force a 

cell to generate a mutation in response to a Cr adduct if repair and apoptosis are silenced. 

Indeed, a step forward, but not one that establishes that adducts form or are mutagenic in 

cells. 

It is unclear why these two studies were chosen to show that Cr induces adducts in cells. It 

is remarkable that given the emphasis the Toxicological Review places on the importance 

of physiologically relevant cell cultures, that it would fail to mention or discuss the 

integrity of the cell line itself, which in these studies were not robust cells. The document 

seems to be saying that there is a problem with cell culture studies that have some 

extracellular metabolism of Cr(VI) or that might not have enough ascorbate, but there are 

no problems with studies in cells with compromised DNA repair and cell death pathways. 

There are more examples of this type of exaggeration of the implications of the primary 

literature in the Toxicological Review. These exaggerations obscure the mea,ning and 

applicability of the data and should be corrected. These exaggerations also undermine the 

integrity of the document and raise questions about its accuracy and process. Other studies 

in the primary literature may reemerge as more relevant if treated more evenly and these 

studies should be reconsidered and possibly presented. 

B. Flaws in citations, extensive direct quoting and long stretches of general paraphrasing 

One concern is that the Toxicological Review actually appears to directly quote sources 

without indicating the comments are quotes of the original source. For example, in the 

Toxicological Review on page 46, lines 13-15, the writing states: 

"Studies on the reduction of Cr(Vl) by extracts of rat liver, lung, or kidney have 

found that ascorbate accounted for at least 80% of Cr(Vl) metabolism in these 

tissues (Standeven et al., 1991,1992)." 

which is the exact same sentence that occurs in the Toxicological Review's Zhitkovich 

2005 reference. That reference states on its page 5: 

"Studies on the reduction of Cr-(Vl) by extracts from rat lung, liver, or kidney have 

found that ascorbate accounted for at least 80% of Cr(VI) metabolism in these 

target tissues (45, 46)." 
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Then on the same page, lines 20-22, the Toxicological Review states: 

"Depending on the nature of the reducing agent and its concentralion, this process 

can generate various amounts of unstable Cr(V) and Cr(IV) intermediates (Stearns 

et al., /994)." 

which is the exact same sentence that is in Zhitkovich 2005. That reference states on its 

pages 5-6: 

"Depending on the nature of the reducing agent and its concentration, this process 

can generate various amounts of unstable Cr(V) and Cr(N) intermediates ( 14-16)." 

Neither of these sentences are indicated as being exact quotes of the original source and 

neither one is attributed to the original source. This omission is a concern as it is 

important to know when the document is choosing to quote from a source directly. These 

examples are not the only occurrences of this type of error and the entire document needs 

to be checked to identify other such problems. 

In other instances, the Toxicological Review only changes a couple of words in a direct 

quote and fails to indicate it is a direct quote, which is also unacceptable. For example, the 

Toxicological Review states on page 50: 

"Therefore, experiments on mutagenesis and other toxic effects of hexavalent 

chromium in tissue culture may underestimate its mutagenic, genotoxic, and celltransforming activities (Zhitkovich, 2005; Costa and Klein, 2006)." 

The underlying reference by Costa and Klein 2006 states on its page 157: 

"Thus, experiments on mutagenesis and other toxic effects of hexavalent Cr in tissue 

culture may underestimate its mutagenic, genotoxic, and cell-transforming activity 

(Zhitkovich, 2005)." 

The quote in the Toxicological Review and the Costa and Klein review differ by only 

substituting a "Therefore" for a "Thus" at the beginning and "activities" for "activity" near 

the end. Changing two words does not avoid the need to offset this sentence as a direct 

quote. As written, it is sufficiently in the original authors' words that it is considered a 

direct quote. 

Similarly, in the Toxicological Review on page 46, the writing states: 

"Ascorbate is also the fastest reducer in the in vitro reactions, and its rate of 

reduction at I mM exceeds that of cysteine and glutathione by approximately 13 and 

61 times, respectively (Zhitkovich, 2005; Quivryn eta/., 2001 ). " 

which is the exact same sentence that is in the Toxicological Review's Zhitkovich 2005 

reference that states on its page 5: 

"Ascorbate is also the fastest reducer of Cr(Vl) in the in vitro reactions, and its rate 

of reduction at I mM concentration exceeds that of cysteine and glutathione 

approximately 13 and 61 times, respectively (48)." 
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Again, this language is a direct quote and needs to be offset in quotation to make that 

clear. 

This type of error also occurs with some frequency in the document and needs to be 

addressed. 

Next, there are numerous instances when the Toxicological Review extensively 

paraphrases a review articie and the meaning of the original passage is altered to another 

meaning that was not originally intended resulting in some overstatements and 

inaccuracies. For example, the Toxicological Review states on page 185: 

"Reduction of hexavalent chromium in vitro produces a large proportion of binary 

trivalent chromium-DNA adducts, but these have not been detected in vivo. It has 

been theorized that the formation of the ternary ad ducts described above occurs far 

more frequently due to the high concentration of ligands capable of complexing with 

trivalent chromium before it can bind to DNA. (Zhitkovich, 2005 ). " 

Given its general use of "in vitro" to mean "in cell culture", the Toxicological Review 

appears to be stating that binary trivalent chromium-DNA adducts occur in cell culture but 

not in in vivo studies. However, the underlying Zhitkovich review reference actually 

states that the binary adducts have been detected in a test tube and not in cell culture. 

Specifically, it states (bold added here for emphasis): 

"Reductive metabolism of Cr(Vl) in vitro usually generates a large number of 

binary Cr(Ill}-DNA adducts (22, 37, 53), but the presence of these DNA 

modifications in cells has not yet been established. The formation of binary Cr-DNA 

complexes in cells is expected to be strongly inhibited due to the abundance of 

intracellular ligands capable of rapid coordination to Cr( Ill) prior to its binding to 

DNA." 

This error occurs quite often and creates confusion about what the underlying literature is 

indicating. 

There is a reference to "Salnikov and Zhitkovich, 2009" in a couple of places and no 

citation for this reference is provided. In the citation list, there is one reference listed as 

"Salnikow, K; Zhitkovich, A. (2008)" and another just below it as "Salnikov, K; 

Zhitkovich, A. (2008)" that looks to be exactly the same reference. These details should 

be straightened out and the entire reference section rechecked. 

The occurrence of these various errors in citations undermines confidence in the 

Toxicological Review and raises significant concerns about process. It gives the 

impression that review articles formed the basis for the evaluation. rather than primary 

sources and, with the extensive quoting and paraphrasing, that some articles were simply 

integrated into the document. The use of these review articles in the document needs to be 

revised and addressed. 

C. Confusion due to inconsistent application of the phrases "in vitro" and "in vivo". 

The data in the Toxicological Review essentially fall into four groups. There are cell-free 

system studies, cell culture studies, whole animal studies and human studies. To describe 

these data, the phrases "in vitro" and "in vivo" are used. To most readers "in vivo" is 
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thought to refer to studies in the body and so include whole animal and in some instances 

human studies. By contrast, "in vitro" is thought to refer to cell culture studies. There are 

inconsistencies in the use of these terms in the Toxicological Review as some of the 

underlying references use "in vivo" to mean in cell culture and "in vitro" to mean in cell 

free systems. The Toxicological Review has often failed to clarify the underlying studies 

and carried the underlying language forward into the review. 

Two examples of this problem are presented in the preceding criticism, where the 

Toxicological Review elevated the celL~ in culture to an "in vivo" status, but there are 

many more occurrences in the document. There are two explanations for this outcome. 

One possibility is that in some places the Toxicological Review uses "in vitro" to mean in 

cell culture and in others to mean in cell free systems and in some places it uses "in vivo" 

to mean in whole animals and in others to mean in cell culture. The second is that the 

authors of the Toxicological Review did not realize that the underlying literature meant 

for "in vivo" to mean in cell culture and "in vitro" to mean in cell free systems. Regardless 

of which reason applies, as written the use of the terms is confusing and in some cases, 

such as the one explained above, misleading. The EPA needs to decide on a definition for 

these terms, present it, review the underlying literature to be sure they reflect what is 

meant and then apply them consistently in the document. 

D. Failure to check the underlying primary research studies in review articles. 

There was a failure to fully consider the underlying primary research articles in the review 

articles that are extensively cited. This failure creates a perception that the authors did not 

read beyond that review article, raising questions about process and whether primary data 

was evaluated at all. Where the document depends on a review article for its source, the 

underlying primary literature should be checked to confirm the integrity of the statements. 

One example of this problem is seen in the passage below from page 50 of the 

Toxicological Review: 

"An additional important note on these biotransformations regards the 

interpretation and reliability of data from in vitro assays. In vivo, the intracellular 

levels of ascorbate are quite high (about 1 mM). In contrast, the levels of ascorbate 

in tissue culture media are quite low since generally it is not added to the media so 

that the only source is supplemented fetal bovine serum (FBS). With 10% FBS, the 

level of ascorbate in tissue cultured cells is only about 50 f1M which is 20 times 

lower than that which is.found in vivo (Zhitkovich, 2005). Therefore, experiments on 

mutagenesis and other toxic effects of hexavalent chromium in tissue culture may 

underestimate its mutagenic, genotoxic, and cell-transforming activities (Zhitkovich, 

2005; Costa and Klein, 2006)." 

Thus, the Toxicological Review wants the reader to question cell culture studies if they do 

not contain intracellular levels of ascorbate in the mM range. However, the Zhitkovich 

review article that this section is entirely based upon miscites the primary literature on this 

matter and it appears the Toxicological Review did not check it. Specifically, as seen in 

the passage below from the Zhitkovich 2005 review, the claims about physiological levels 

of ascorbate being in the millimolar range rely on primary research papers that are its 

references 53 and 54. The Zhitkovich review states: 
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"Under standard tissue culture conditions, A549 and many other human and rodent 

cells either lack detectable ascorbate or contain it only at micromolar levels 

(physiological levels are in millimolar range) (53, 54) due to low concentrations of 

this vitamin in fetal bovine serum and its absence in the most commonly used types 

of growth media (DMEM, RPM! 1640, F/0, Fl2)." 

These two references are: 

"(53) Quievryn, G., Messer, J., andZhitkovich, A. (2002) Carcinogenic 

chromium( VI) induces cross-linking of vitamin C to DNA in vitro and in human lung 

A549 cells. Biochemistry 41, 3156-3167. 

(54) Salnikow, K., Donald, S. P., Bruick, R. K., Zhitkovich, A., Phang, J. M., and 

Kasprzak, K. S. (2004) Depletion of intracellular ascorbate by the carcinogenic 

metals nickel and cobalt results in the induction of hypoxic stress. J. Bioi. Chern. 

279, 40337-40344." 

Examination of these two references shows, however, that neither one offers any data or 

evidence of physiological ascorbate levels being in the mM range. Salnikow et al. 

measures the amount of ascorbate loss in cultured cells treated with nickel and cobalt. 

They do measure ascorbate levels in untreated control cells to determine the background 

level of their experimental system. But their study does not measure any levels of vitamin 

C in any physiological setting. Nor does the citation make any reference at all to any study 

that does. 

Quievryn et al., treats the human carcinoma cell line A549 with ascorbate and 

dihydroascorbate and then measures the amount of vitamin C inside the cell under these 

experimental conditions. But the study does not measure any levels of vitamin C in any 

physiological setting. The discussion section does make a comment that: "Human cells in 

vivo contain a millimolar concentration of Asc (43) ... ".However, that reference 43 is "43. 

Meister, A. ( 1994) J. Bioi. Chem. 269, 9397-9400", which is a review article concerning 

glutathione and ascorbate in rodents. It contains no mention of ascorbate in human cells. 

Thus, the EPA expresses a significant concern about in vitro cell culture studies based on 

a single review article that miscites the primary and secondary literature. This oversight 

implies that in the preparation of this Toxicological Review, the EPA did not access the 

primary literature and confirm the secondary and tertiary review articles. 

If one were to look at primary literature for ascorbate levels, one would find that these 

claims are overstated. Ascorbate can reach mM levels in the body, but they are not 

universally mM levels. For example, Slade et al., report lung ascorbate levels of 2.91 to 

62.35 mg/100 g (Slade, R., Stead, A. G., Graham, J.A., and Hatch, G.E. (1985) 

Comparison of lung antioxidant levels in humans and laboratory animals. Am. Rev. 

Respir. Dis. 131(5), 742-6). This measure can be converted to a range of 165 uM to 3.5 

mM. However, these are not intracellular levels, but rather the product of tissue 

homogenization and so a mixture of extracellular and intracellular sources. Thus, there is 

clearly variability in levels that span the uM and mM range, indicating that this factor may 

not be so essentiaL 
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E. Some exaggerations about Cr trans11ort in cells. 

Of less concern, but certainly in need of being addressed is some of the inaccurate 

language concerning Cr transport into the cell. In several places, cells are described as 

being impermeable to Cr(III). This characterization is too strong and inaccurate. Cr(III) 

will enter cells. It is just a slower uptake process than Cr(VI) uptake as Cr(UI) moves by 

simple diffusion and it requires a higher dose to create the concentration gradient to get in 

the cell. This language should be adjusted. 

Also, there is language implying Cr(VI) is actively transported into cells. Cr(VI) does 

enter rapidly by facilitated diffusion, but it is not an active transport process. These 

comments should be adjusted. 

F. Some typographical errors. 

There is also a mention on page !50 that states: 'As discussed in detail in Section 4.4.2 

(Intracellular Reduction) ... " It is actually section 4.5.2. 

In sum, these factors all combine to give the appearance that the mode of action was not 

fully and consistently considered. To make the document and its conclusions much 

stronger and more accurate and the rationale behind its decisions more transparent, the 

following steps should be taken: 1) The EPA needs to separate the genotoxicity literature 

into discrete endpoints and consider them individually. This consideration should be 

based on the primary literature, which should be presented in a more careful and coherent 

fashion so that the reader can understand the strengths, weaknesses and data gaps. 2) 

Based on that analysis the EPA should choose which lesions are the key lesions and 

explain the rationale for that choice. 3) Once the key lesions are chosen, the EPA should 

consider the possible mechanisms of action that may cause those lesions and determine if 

there are data to support those mechanisms of action. 4) Once the key lesions are 

identified and the likely mechanisms described, the EPA should explain its rationale for 

the one chosen to be the mode of action. Of course, non-genotoxic modes of action should 

also receive similar analysis and presentation. This approach would help the EPA 

determine the most robust mode of action based on the primary literature. In addition, the 

EPA should decide what factors are truly confounders of concern and then apply them 

evenly to all of the literature, reduce its use of secondary and tertiary review articles and 

improve its management and citation of the literature. 

Zhitkovich In general, I found the Draft to be well prepared and balanced in its presentation of 

various aspects of chromium-6 toxicology and carcinogenesis. It has a logical structure, 

leading a reader from the basics of redox chemistry of chromium-3 and chromium-6 and 

their interactions with biosystems to the detailed description of in vivo studies on 

bioavailability, tissue disposition and finally, toxic and carcinogenic effects. Weaknesses 

and strengths of the key in vivo studies along with the reasons for the inclusion or 

exclusion of specific findings were also clearly presented. Different sections vary 

somewhat in their degree of emphasis on the importance of one or another mechanistic 

aspect of Cr(Vl) toxicology, which is also reflective of divergent opinions in the field. As 

typical for any large document covering a complex topic, the Draft contains some 

information that is not up-to-date and would benefit from additional editorial work. 
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Suggested modifications and corrections: 

I) Section 2.1 "Environmental Sources and Occurrence" appears to draw a large amount 

of information from decades-old literature. The analytical approaches for the detection of 

both total Cr and Cr-6 underwent major improvements in 1980s and the older references 

to the amount of Cr-6 in various environmental media and biological samples should be 

looked at with a healthy degree of skepticism and scrutinized for potential 

overestimations. My specific concerns are related to the included values for Cr-61evels in 

soil, freshwater and seawater. All three sets of values are too high for the typical samples 

from the noncontaminated areas/sites. 

2) Table 2-1 "Industrial uses of hexavalent chromium compounds" is missing uses of 

sodium/potassium chromate and dichromate. The addition of information on sodium 

dichromate is particularly important in light of its testing for carcinogenicity by the NTP. 

3) p.l4, last para: Cr(III) oxidation to Cr(VI) by atmospheric oxygen can also occur in the 

presence of calcium oxide (Pillay et a!. 2003). 

4) Table 2-4 "Detection limits for methods ... " reports outdated values. The EPA's Method 

218.6 for Cr(VI) in water has a detection limit which is -100 times lower than detection 

limits listed in Table 2-4. A recent modification of this method affords detection of Cr(VI) 

at the 0.003 ppb level (Application Update 179 from Dionex). The detection sensitivity of 

flame AAS is also underestimated. Based on the discussion of work by Levine (2007) on 

p.l6 and other available literature, the detection limit for total Cr in water samples by 

ICP-MS reported in Table 2-4 is probably lower by a couple orders of magnitude. 

5) p.25, lines 5-6: in Donaldson and Barreras (1966), urinary excretion for orally 

administered Cr(VI) and Cr(III) were 2.1 and 0.5%, respectively (not 2.1 versus 1.5%). 

6) Table 3-5 and the discussion of these results appear contradictory. 

7) Table on p.39 is confusing: it reports daily doses of sodium dichromate dihydrate in the 

NTP-2008 study but the ratio mice:rats looks incorrect based on the data in the top two 

rows. It is also unclear why Cr(VI) consumption was compared between male rats and 

female mice and not between animals of the same sex. 

8) Finley et at. (1996) delivered a Cr-6 dose of0.005 mglkglday, not 0.005 mg (p.45). 

9) Section 3.3 describes Cr(VI) reduction by microsomal enzymes in detail on three 

pages. This degree of attention may create an erroneous impression about the importance 

of the specialized enzymatic processes in Cr-6 metabolism. There is a strong consensus in 

the field that Cr(Vl) reduction in mammalian cells is primarily accomplished 

nonenzymatically by ascorbate and small thiols such as glutathione and cysteine. As 

acknowledged in other sections of the draft, ascorbate alone accounts for reduction of 80-

95% Cr(Vl) depending on the tissue (Standeven and Wetterhahn, 1991, 1992). A 

combined contribution of ascorbate and thiols is responsible for more than 95% Cr(VI) 

reduction. These estimates from tissue preparations were confirmed by the measurements 

of individual reduction rates (Quievryn et at. 2003). It is clearly important to present 

mechanistic aspects of Cr(VI) reduction but the detailed focus should be on ascorbate and 

non-protein thiols, not enzymatic systems with a minimal contribution to the overall 
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Cr(VI) metabolism in vivo. The absence of Cr(V) intermediate during Cr(VI) reduction by 

ascorbate is especially important. 

1 0) Last sentence on p. 50: The description of vitamin C accumulation by cells is not 

entirely correct. Cellular accumulation of vitamin C via uptake of its oxidized form 

dehydroascorbic acid is a physiological mechanism that functions in all mammalian cells. 

It is particularly active in human cells, which leads to very efficient recycling and much 

lower daily requirements for vitamin C in humans compared to rodents (Nualart et al. 

2003, Mantel-Hagen et al. 2008). These differences between humans and rodents are 

relevant for the interspecies extrapolation. 

11) Figure 3-6 needs to be modified: 

a) Depiction of the cation channel with the comment "No effect" could be interpreted 

as indicating some nontoxic delivery route for chromium. Unlike some other toxic 

metals, cation channels play no role in uptake of Cr ions and the cation route should 

be deleted from this Figure. 

b) Although some Cr(III)-ligand complexes can exhibit a limited ability to enter cells, 

there is no evidence that they can react with DNA and cause mutagenic/genotoxic 

ternary Cr-DNA adducts, as shown in the Figure. The Figure should be modified by 

removing this nonexistent route of DNA damage. 

c) The route for the formation of DSB by mismatch repair needs to be revised. As 

demonstrated in a recent study by Reynolds MF eta! (2009), ternary Cr-DNA adducts 

are directly bound by mismatch repair proteins followed by DSB formation in 02 

phase without stalling replication forks in the preceding S-phase. 

12) Summary Table 4-20 should add the +(M) designation for mutagenesis of sodium 

dichromate in laboratory animal, as demonstrated by Cheng et al. 2000 (this study was 

later cited in Table 4-23). 

13) Table 4-23 "In vivo genotoxicity studies ... Mutations section" is missing references 

to two positive mutagenesis studies in vivo by ltoh and Shimada (1997, 1998). 

14) Section 4.7.3.1. Hypothesized Mode of Action: 

a) Ref. to Salnikow et al. (1992) in support of ternary complexes is inappropriate and 

could be replaced by Voitkun et al. (1998). 

b) Neither Zhitkovich (2005) nor Voitkun et al. (1998) dealt with "intrastrand DNADNA crosslinks". A study by Lloyd et al. (1998) is the only original report describing 

putative intrastrand crosslinks, which were generated in a buffer solution with massive 

concentrations of hydrogen peroxide. Tested under the same reaction conditions, 

essential metals copper and cobalt were even more potent inducers of these presumed 

crosslinks. There is no evidence for the formation of these cross links by chromium-6 

in cells or in acellular systems containing its main biological reducers. 

15) Tables 4-22 and 4-23 failed to include any references to studies reporting the 

formation of chromium-DNA adducts in cultured mammalian cells and in vivo. This is a 

critical omission as the presence of chromium-DNA adducts demonstrates a direct DNA-
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damaging mechanism for Cr(Vl) genotoxicity. The formation of DNA adducts was briefly 

discussed in other sections of the Draft. 

16) Discussion of a negative report on DNA damage by DeF!ora eta!. (2008) on 

pp.206-207: 

This study found no evidence of DNA damage in forestomach, glandular stomach and 

duodenum of female SKH-1 mice after a 9-month long exposure to 5 and 20 mg!L 

Cr(Vl) in drinking water. Based on the study by DeF!ora et a!. (2008), a high safety 

threshold argument was also made in some of the submitted public comments. The 

Draft argued that a shorter duration of exposure (9 months vs. 2 years in the NTP 

study) made the DeF!ora 2008 study "infeasible" for the comparison. With the 

exception of mutations and a potential accumulation of unrepaired damage in a 

population of the long-lived crypt stem cells, there are no other obvious factors 

suggesting that the formation of DNA damage by Cr(VI) in the entire duodenum 

during the ftrst half of the 2-year exposure would be significantly different from 

damage occurring at the end of the 2 years. 

However, the study by DeF!ora eta!. (1998) is uninformative for other reasons. The 

authors assayed tissues for two forms of DNA damage: DNA-protein crosslinks and 

8-oxo-dG (the Draft incorrectly described 8-oxo-dG as a DNA adduct; it is actually a 

base oxidation product). Both types of damage showed no increases above 

background in tissues of exposed animals; however, these negative results were 

predictable based on the technical !imitations of their analytical methodologies. Since 

Cr(VI) tumorigenesis occurred in the duodenum, I will limit my discussion to this 

tissue. 

1) DNA-protein cross !inks: 

A positive control consisting of mouse duodenal cells treated ex vivo with 1.6 mM Cr(VI) 

(83.2 mg!L) generated a 2.5-fold response. Such a low responsiveness was clearly 

insufficient to detect DNA damage for exposures with 4.2- and 16.6-times lower Cr(VI) 

levels in the 20 mg!L and 5 mg!L test groups, respectively, even in the unlikely scenario 

of no reduction and no dilution of Cr(VI) with stomach juices before reaching the 

duodenum. Although chronic exposures frequently leads to the accumulation of 

unrepairable damage, a dramatic increase in DNA-protein crosslinks during chronic 

Cr(VI) exposures would be not very likely. DNA-protein crosslinks are repairable lesions 

in mammalian cells in vivo and culture (Tsapakos et a!. 1983, Sugiyama et al. 1986, 

Zecevic et al. 2010) with the possible exception of human peripheral blood lymphocytes 

(Quievryn and Zhitkovich 2000). Furthermore, ongoing proliferation and shedding of 

cells in the duodenal villi would result in continuous dilution of damage and loss of 

previously exposed cells. 

2) 8-oxo-dG measurements: 

A positive control generated by exposure of mouse duodenal cells to 1.6 mM Cr(VI) (83.2 

mg!L) produced a 3.8-fold increase in the levels of 8-oxo-dG. It is doubtful that this assay 

sensitivity was sufficient to detect significant increases in 8-oxo-dG levels for even 

undiluted/unreduced 20mg!L and 5mg!L Cr(VI) concentrations that were used in the 

treatment groups. 8-oxo-dG as a biomarker of DNA damage has one critical limitation- a 
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short lifetime due to its rapid removal by base excision repair. Repair of 50% 8-oxo-dG 

occur within 30 min and is complete within 2 hr (Lan et al. 2004). Not only would this 

short lifetime prevent any accumulation of 8-oxo-dG during chronic exposures, but it 

would also make it very difficult to detect this lesion even after recently ingested water 

with a sufficiently high dose producing positive responses under ex vivo conditions. 

Zhu This EPA's Review is well organized overall and for most part well presented. The 

literature review is extensive and thorough. However, !he Review does not contain a set of 

clearly-stated criteria under which the literature was searched, critiqued, and synthesized. 

Specifically, was each published study judged with respect to design (including sample 

size), exposure assessment, choice of dose metrics, choice of endpoints, adequate doseresponse data, dose-response modeling, and positive findings? Whereas some of these 

criteria may have been used in the Review, the lack of a systematic approach may have 

compromised the consistency and transparency of this review process. In its independent 

review of the EPA's IRIS Documents on Formaldehyde and Dioxins, for example, the 

National Academies of Science and National Research Council have strongly advocated 

the adoption of a systematic review approach to EPA's IRIS risk assessment process 

(NAS 2006, 2011). The present Review of Hexavalent Chromium once again 

demonstrates the need for adopting a systematic review approach. 

G2. Please identify any additional studies that would make a significant impact on the 

conclusions of the Toxicological Review. 

Reviewer Comments 

Byczkowski It seems that up to date (around the year 2009), all important studies (and some 

unimportant too) have been already covered by !his Toxicological Review. However, I 

strongly suggest !hat the results of study presented at the workshop by ToxStrategies, Inc., 

and especially the physiologically based pharmacokinetic (PBPK) modeling by Summit 

Toxicology, LLP., should be included in the revised Toxicological Review of Hexavalent 

Chromium document. 

Hamilton See G 1. above. 

Nordberg To my understanding there are new experimental studies with more for this purpose fitted 

doses. It could be worthwhile to await the outcome of these studies to find out whether 

more appropriate values for both NOAEL and LOAEL will be reported and include these 

in an appendix if feasible. 

Any further data obtained in the cited NTP studies should be included and presented to the 

reader. It would be of interest to know if there are any data on lung cancer or other effects 

from the NTP studies. 

During the workshop a number of ongoing studies were presented and it was suggested 

that they be paid attention to. It is always an advantage to get more and more information 

and research is always going on. 
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Patierno 

Rossman 

In my opinion it is however important to set recommendations for exposure to toxic 

agents in order to protect humans from developing adverse health effects. It is a human 

right to be protected from unwanted exposure which also will cause unnecessary worry 

during the time from alert to protection. People expect regulatory agencies to make 

evaluations and set exposure limits. Studies underway even if published in peer review 

scientific journals should be carefully evaluated and scrutinized by EPS 's working group 

to determine if presented data is reliable e.g., based on a number of factors such as, just to 

mention a few, how large are the studies and what is the power o the study, analytical 

procedures that include quality control so data is validated and to be trusted. Based on 

experience it takes time before data will be available even for ongoing studies. I 

recommend that IRIS, EPA sets a recommendation based on information presented in the 

draft document. In case important information which can change any evaluation shows up 

in time, such data can be included in the final document as an appendix or addendum. It is 

important in Risk Assessment to keep in mind that any recommendation set for exposure 

levels values needs to be reevaluated over time because by new techniques e.g., rapid 

development of usage of "omics" has to be considered. In view of said it is important to 

draw conclusions now and on data available now and not to wait. 

The TRHC should absolutely consider the extensive new data being provided by 

ToxStrategies and presented in part in the supplementary section under Public Comments. 

Some of these will be presented in section B2, as they pertain to mode of action. 

It is extremely important that the new information supported by American Chemical 

Council (performed by ToxStudies and others) should be considered before the final 

document is completed. They address a number of missing data sets. It is already clear 

that proliferative increases occur in the mouse duodenum at doses of Cr(6) lower than 

those that cause tumors. Also, there is evidence for cytotoxicity at these lower 

concentrations that may be driving the proloferative responses. 

The fact that Cr is an essential element needs to be addressed. What are the implications 

for a threshold? 

It is possible that dietary Cr(6) is significant and should be evaluated. All parts of grain 

contain Cr(6) and 10% of the Cr in bread is Cr(6) (Mishra et al., Food Chern. Toxicol. 

33:393-397, 1995; Soares et al., J. Agric. Food Chern. 58:1366-1370). 

River waters have a median Cr value [which is probably Cr(6)] of 10 ppb (range <1-30), 

and even rainwater has a range from 0.14-0.9 (ATSDR, Chromium, Draft for Public 

Comment, online). 

A recent meta-analysis of cancers of the G.I. tract among those occupationally exposed to 

Cr(6), concludes that these workers are not at greater risk than the general population 

(Gatto et al., Cancer Epidemiol. 34:388-399, 2010). Inhalation exposure usually leads to 

G.I. exposure, suggesting a possible threshold if the ingested dose can be estimated. 
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Salnikow 

Responses to Charge Questions 

Key issues that should have an impact on the conclusions of the draft of the Toxicological 

Review of Hexavalent Chromium are: I) the use of appropriate animal models, 2) an 

understanding of the chromium carcinogenic MOA, including genotoxic (mutagenic) and 

non-genotoxic (epigenetic) mechanisms and their interrelations, 3) the co-carcinogenic 

effects of hexavalent chromium, and 4) the role of iron metabolism in chromium 

carcinogenesis. Unfortunately, studies to address these issues are either not done or are in 

the early stages of research. Thus, it is too early to draw any conclusive decisions on risk 

assessment of hexavalent chromium in drinking water. 

Are used animal models appropriate for risk assessment? 

Although the NTP studies provide evidence that oral exposure to hexavalent chromium 

induced tumors in rodents, the main argument against these studies is that the toxic and 

carcinogenic effects could be achieved/observed only at high chromium concentrations, 

which significantly exceed human exposure levels. Also, it is noted that biological effects 

were seen only at chromium concentrations that overwhelmed the cellular defense 

systems (reducing capabilities). Ascorbate is a major reducing agent of hexavalent 

chromium in biological fluids and tissues (see review (Zhitkovich, 2005; Salnikow and 

Zhitkovich, 2008)). Humans cannot synthesize ascorbate in the body because of a 

mutation in the L-gulono-y-lactone oxidase gene coding for the final enzyme in ascorbate 

metabolism and thus ascorbate is supplemented through the diet. Unlike humans, 

laboratory mice and rats, used for carcinogenicity assays, are capable of synthesizing 

ascorbate endogenously. Because ascorbate regulates many cell and tissue functions that 

are critical for cancer development, the changes in the level of ascorbate should be 

considered in animal models of choice (Salnikow and Kasprzak, 2005). It is impossible to 

deplete tissue ascorbate levels by metal exposure in wild-type rodents because the enzyme 

producing ascorbate will be up-regulated when the level of ascorbate drops below a 

critical point. To avoid this problem for in vivo testing of the toxic and carcinogenic 

effects of heavy metals, which efficiently destroy ascorbate, an appropriate model is the 

use of mice or rats that like humans cannot synthesize ascorbate (Kasprzak eta/., 2011). 

Two rodent model systems unable to synthesize ascorbate are available: Gulo-/- mice 

(Maeda et at., 2000), aud a similar rat strain (Mizushima et al., 1984). Our preliminary 

data show that when Gulo-/- mice were supplemented with ascorbate in drinking water 

their blood and tissue ascorbate levels were undistinguishable from that in wild type mice. 

However, ascorbate levels were significantly decreased by metal exposure in Gulo-/- mice 

but not in wild-type mice, in which the enzyme responsible for ascorbate production was 

activated in response to metal exposure (ascorbate depletion) (Kasprzak et al., 2011). In 

this model system we found that the reduction in ascorbate levels increased acute toxicity 

induced by Ni3S2 in Gulo-/- mice and that Gulo-/- mice were more susceptible than wildtype mice to nickel-induced carcinogenesis. Additionally, in tumor transplantation assays, 

Gulo-/- mice had shorter tumor latency than wild-type mice. After the lag period 

established tumor growth rates were comparable in Gulo-/- and wild-type mice. Although 

cancer initiation and development is a very complicated process our results indicate that 

ascorbate is a potentially important part of the molecular mechanisms of metal 

carcinogenesis and acute toxicity. 

Ascorbate is involved in diverse biological activities. Ascorbate is essential for the 

function of numerous 2-oxoglutarate-dependent hydroxvlases. This group of hydroxylases 
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Responses to Charge Questions 

includes the asparaginyl and prolyl hydroxylases, FIH-1 and PHD!, PHD2, PHD3, which 

are responsible for H!Fa hydroxylation (Epstein et al., 2001; Mahon et al., 2001; 

Hewitson et al., 2002; Lando et al., 2002); the collagen prolyl-4-hydroxylases 

(Myllyharju, 2003), which is critical for extracellular matrix formation; and a new class 

of histone and DNA demethylases that remove methyl group through hydroxylation (Shi, 

2007). We already pointed out that the level of ascorbate is critical for metal 

carcinogenesis mainly by affecting epigenetic pathway (Salnikow and Zhitkovich, 2008). 

More recently a link between changes in ascorbate concentration and DNA demethylation 

of human embryonic stem cells (hESCs) has been identified (Chung et al., 2010). Thus, 

ascorbate levels have the potential to directly impact the differentiation of hESCs and the 

reprogramming of somatic cells. 

Given that ascorbate has diverse cellular functions and ascorbate levels are critical to 

interpreting carcinogenic effects of heavy metals, the animal models described in the 

Toxicological Review are not the most appropriate. The results obtained in NTP 2007 

studies are consistent with the idea that ascorbate is an important factor to consider. In 

preliminary toxicokinetic studies in which animals were exposed to chromium in drinking 

water for 21 days chromium concentrations in the blood of the guinea pigs (which are 

unable to synthesis ascorbate) was greater than chromium concentrations in the blood of 

the rats or mice suggesting greater absorption (less reduction) of chromium in guinea pigs. 

httn://ntg.niehs.nih.gov/ntglhtdocs/ST rgts/tox072.gdf 

I suggest that new studies similar to the NTP studies with several dietary concentrations of 

ascorbate and lower chromium does (i.e., those more relevant to environmental exposures), 

be done in ascorbate-deficient rats or mice or both animal models. Additionally, because 

of more efficient depletion of ascorbate in tissues of Gulo-/- animals by chromium these 

animal models will show whether Cr(III) and Cr(VI) kinetic that were developed using the 

wild type animals (O'Flaherty, 1996; O'Flaherty et al., 2001) will be applicable to Gulo-/-

animals. This will allow for adjustment of kinetic models, if needed, and identification of 

new or confirming known compartments of chromium retention. 

Wise Although this review is focused on oral exposures, some insight may be gleaned from the 

inhalation exposure data. Specifically, the data on mutations in lung tumors for Cr(VI)-

exposed workers should be considered. These data show a lack of mutations in those 

tumors suggesting that mutagenicity as considered as a primary change in the DNA 

sequence is not a key event in the mechanism of action. They are consistent with the fact 

that one only sees these types of mutations in mammalian experimental models when one 

forces them by applying very high doses. These studies are: 

Katabami M, Dosaka-Akita H, Mishina T, Honma K, Kimura K, Uchida Y, et al. Frequent 

cyclin Dl expression in chromate induced lung cancers. Hum Pathol 2000; 31 : 973-9. 

Kondo K, Hino N, Sasa M, Kamamura Y, Sakiyama S,Tsuyuguchi M, et al. Mutations of 

the p53 gene in human lung cancer from chromate-exposed workers. Biochem 

Biophy Res Commun 1997; 239 : 95-100. 

Ewis AA, Kondo K, Lee J, Tsuyuguchi M, Hashimoto M, Yokose T, et al. Occupational 

cancer genetics: Infrequent ras oncogenes point mutation in lung cancer samples 

from chromate workers. AmJ Ind Med 2001; 40:92-7. 
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Responses to Charge Questions 

Hirose T, Kondo K, Takahashi Y, Ishikura H, Fujino H, Tsuyuguchi M, eta!. Frequent 

microsatellite instability in lung cancer from chromate-exposed workers. Mol 

Carcinog 2002; 33 : 172-80. 

Takahashi Y, Kondo K, Hirose T, Nakagawa H, Tsuyuguchi M, Hashimoto M, et al. 

Microsatellite instability and protein expression of the DNA mismatch repair gene, 

hMLHI, of lung cancer in chromate-exposed workers. Mol Carinog 2005; 42: 150-8. 

Kondo K, Takahashi Y, Hirose Y, Nagao T, Tsuyuguchi M, Hashimoto M, eta!. The 

reduced expression and aberrant methylation ofpl6INK4a in chromate workers 

with lung cancer. Lung Cancer 2006; 53 : 295-302. 

Ewis AA, Kondo K, Dang F, Nakahori Y, Shinohara Y, Ishikawa M, et al. Surfactant 

protein B gene variations and susceptibility to lung cancer in chromate workers. Am 

J Ind Med 2006; 49: 267-73. 

There are also studies of Cr(Vl)-induced neoplastic transformation of cells in culture. 

These need to be considered and included. In particular, reports by Xie et. al., show that 

cells must acquire a DNA double strand break repair phenotype to undergo transformation 

indicating escape from repair may be a key event in the mode of action. These studies are: 

Patierno SR, Banh D, Landolph JR. Transformation of C3H/10Tl/2 mouse embryo cells 

to focus formation and anchorage independence by insoluble lead chromate but not 

soluble calcium chromate: relationship to mutagenesis and internalization of lead 

chromate particles. Cancer Res 1988; 47 : 3815-23. 

Xie H, Holmes AL, Wise SS, Huang S, Peng C, Wise Sr JP. Neoplastic transformation of 

human bronchial cells by lead chromate particles. Am J Respir Cell Mol Bioi 2007; 

37:544-52. 

Xie H, Wise SS, Wise Sr. JP. Deficient repair of particulate chromate-induced DNA 

double strand breaks leads to neoplastic transformation. Mutat Res 2008; 649 : 230-8. 

The document only considered mismatch repair, but there are important data showing that 

other DNA repair pathways must be overcome to induce genotoxicity and carcinogenesis. 

Double strand breaks and their repair, in particular, are important. The following studies 

should be added to the repair/DNA double strand break discussion: 

Xie, H., Holmes, A.L., Young, J.L., Qin, Q., Joyce, K, Pelsue, S.C., Peng, C., Wise, S.S., 

Jeevarajan, A., Wallace, W.T., Hammond, D. and Wise, Sr., J.P. Zinc Chromate 

Induces Chromosome Instability and DNA Double Strand Breaks in Human Lung 

Cells. Toxicology and Applied Pharmacology, 234: 293-299, 2009. 

Xie H, Wise SS, Holmes AL, Xu B, Wakeman T, Pelsue SC, eta!. Carcinogenic lead 

chromate induces DNA double-strand breaks and activates ATM kinase in human 

lung cells. Mutat Res 2005; 586: 160-72. 

Xie H, Wise SS, Wise Sr. JP. Deficient repair of particulate chromate-induced DNA 

double strand breaks leads to neoplastic transformation. Mutat Res 2008; 649 : 230-8. 

Stackpole MM, Wise SS, Goodale BC, Duzevik EG, Munroe RC, Thompson WD, et a!. 

Homologous recombination protects against particulate chromate-induced genomic 

instability in Chinese hamster cells. Mutat Res 2007; 625:145-54. 
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Responses to Charge Questions 

Camrye E, Wise SS, Milligan P, Gordon N, Goodale B, Stackpole M, eta!. Ku80 

deficiency does not affect particulate chromate-induced chromosome damage and 

cytotoxicity in Chinese hamster ovary cells. Toxicol Sci 2007; 97: 348-54. 

Bryant HE, Ying S, Helleday T. Homologous recombination is involved in repair of 

chromium-induced DNA damage in mammalian cells. Mutat Res 2006;599:116-23. 

Grlickova-Duzevik EG, Wise SS, Munroe RC, Thompson WD, Wise Sr. JP XRCC1 

protects cells against particulate chromate-induced chromosome damage and 

cytotoxicity in Chinese hamster ovary cells. Tox Sci 2006a;92(2):409-15. 

Grlickova-Duzevik E, Wise SS, Munroe RC, Thompson WD, Wise Sr JP. XRCC I 

protects cells from chromate-induced chromosome damage, but does not affect 

cytotoxicity. Mutat Res 2006; 610(1-2):31-7. 

Vilcheck SK, Ceryak S, O'Brien TJ, Patierno SR. FANCD2 monoubiquitination and 

activation by hexavalent chromium [Cr(Vl)] exposure: Activation is not required for 

repair of chromium(Vl)-induced DSBs. Mutat Res 2006;6!0:21-30. 

Savery LC, Grlickova-Duzevik E, Wise SS, Thompson WD. Hinz JM, Thompson LH, 

Wise Sr. JP. Role of the Fancg gene in protecting cells from particulate chromateinduced chromosome instability. Mutat Res 2007, 626(1-2):120-127. 

There needs to be a stronger and clearer discussion about aneuploidy as a potential 

mechanism. These studies should be added to that discussion (some are in the document 

already): 

Holmes, A.L., Wise, S.S., Pelsue, S.C., Aboueissa, A., Lingle, W., Salisbury, S., Gallaher, 

J. and Wise, Sr., J.P. Chronic exposure to zinc chromate induces centrosome 

amplification and spindle assembly checkpoint bypass in human lung fibroblasts. 

Chemical Research in Toxicology, 23(2): 386-395, 2010. 

Guerci A, Seoane A, Dulout FN. Aneugenic effects of some metal compounds assessed by 

chromosome counting in MRC-5 human cells. Mutat Res 2000; 469 : 35-40. 

Seoane AL, Guerci AM, Dulout FN. Malsegregation as a possible mechanism of 

aneuploidy induction by metal salts in MRC-5 human cells. Environ Mol Mutagen 

2002; 40 : 200-6. 

Holmes AL, Wise SS, Sandwick SJ, Lingle WL, Negron VC, Thompson WD, et al. 

Chronic exposure to lead chromate causes centrosome abnormalities and aneuploidy 

in human lung cells. Cancer Res 2006; 66: 4041-8. 

Wise SS, Holmes AL, Xie H, Thompson WD, Wise Sr JP. Chronic exposure to particulate 

chromate induces spindle assembly checkpoint bypass in human lung cells. Chern 

Res Toxico12006; 19: 1492-8. 

The following two studies should be added to the clastogenicity results, particularly in 

light of one reviewer's comments that telomerase may be important as the second paper 

suggests telomerase does not affect Cr genotoxicity: 
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Zhitkovich 

Zhu 

Responses to Charge Questions 

Xie, H., Holmes, A.L., Wise, S.S., Gordon, N. and Wise, Sr., J.P. Lead chromate-induced 

chromosome damage requires extracellular dissolution to liberate chromium ions 

but does not require particle internalization or intracellular dissolution. Chemical 

Research in Toxicology, 17(10): 1362-1367,2004. 

Wise SS, Elmore LW, Holt SE, Little JE, Antonucci PG, Bryant BH, et al. Telomerasemediated lifespan extension of human bronchial cells does not affect hexavalent 

chromium-induced cytotoxicity or genotoxicity. Mol Cell Biochem 2004; 255: 

103-11. 

Finally, there are misleading comments about DNA-DNA crosslinks. The Toxicological 

Review states they are unlikely to form in vivo. When one studies the underlying review 

cited as evidence by Salnikow and Zhitkovich, it becomes apparent that by in vivo they 

mean cells in culture or whole animals. Thus, the review implies that Cr -DNA-DNA 

crosslinks would not be predicted to occur in cells or whole animals, however, data from 

Josh Hamilton and Karen Wetterhahn show Cr DNA-DNA crosslinks do form in vivo. 

The inclusion of Josh Hamilton's study showing DNA-DNA crosslinks in vivo would 

correct the inaccurate conclusion in the Toxicological Review that these lesions do not 

occur in vivo. I cannot locate that paper in the time frame available, but Josh Hamilton is a 

reviewer and should be able to provide it. 

The Draft included information from all major studies that have a significant impact on 

the main conclusions. It does not list or discuss every study published on Cr(VI) but there 

was also no systematic exclusion. In Section 4.4, I would recommend adding an important 

report by Gibb et al. (2000), which is the largest epidemiological study of cancer risk due 

to inhalation exposure to Cr(VI). While the omission or inclusion of this study does not 

change the overall conclusion about Cr(VI) carcinogenicity to humans via inhalation, 

Gibb et al. (2000) provided strong evidence of chromate dose-dependence for lung cancer 

risk and its independence of the common confounder, tobacco smoking. 

NA 
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Chemical-Specific Charge Questions 

(A) Oral Reference Dose (RID) for Hexavalent Chromium 

At. A two-year drinking water study of sodium dichromate dihydrate in rats and mice (NTP, 

2008) was selected as the basis for the derivation of the RID. Please comment on whether 

the selection of this study as the principal study is scientifically supported and clearly 

described. Please identify and provide the rationale for any other studies that should be 

selected as the principal study. 

Reviewer Comments 

Byczkowski The two-year drinking water study by NTP (2008) seems to be the most comprehensive 

from all available chronic bioassays, and thus, it is suitable as the basis for derivation of 

the RID. 

However, there is still some concern regarding the selection of the NTP study for 

developing of RID (this document states -C. f. P. 85, L# I: " ... Urinalysis showed doserelated decreased volume and increased specific gravity, consistent with decreased water 

intake. NTP (2007) suggested that decreased water intake was due to decreased 

palatability ... " and then, P.89. L# 34: " ... Drinking water consumption was reduced ... ") 

Thus, the reduced drinking water consumption, and consequently at least partial 

dehydration, may have increased the osmolality of gastrointestinal fluid, which could be a 

significant confounder in the chronic toxicity study, even though (according to the quotation 

on P. 120) the Technical Report by NTP (2008) attempted to clismiss such a concern. 

Hamilton No concerns regarding AI. And A2. This reviewer would point out that the hyperplasia 

that was chosen as this endpoint, while appropriate for this RID, is also appropriate for 

considering the carcinogenic MOA as well, as argued above and below and taking into 

considering the recently reported ACC studies that should be considered in this regard. 

Nordberg EPA suggests an oral RID of 9x 1

Q.4 mglkg-day. 

The epidemiology studies in Liaoning province, China (p 68-76 in draft report) reported 

increased incidence of cancer after intake of drinking water contaminated with hexavalent 

chromium. It is stated in the document to be the only reported human data. That study 

supports the statement of hexavalent chromium in drinking water to be carcinogenic. 

It should be explained to the reader why sodium dichromate dehydrate was chosen for oral 

exposure study. Data in the literature indicates that bioavailability and bioaccesibility 

depends on species of the compound and also exposure media. Are there any data on 

hexavalent chromium species in the drinking water in the general environment? The NTP 

studies that are reported have used doses that are much higher than reported present 

concentration in drinking water in the general environment. Thus values for LOAEL is 

identical to lowest administered dose. It has not been possible because of applied doses to 
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set a NOAEL. 

Patierno The two-year drinking water study of sodium dichromate dyhydrate in rats and mice 

(NTP, 2008) is the most thorough and technically well-conducted study available. It is 

likely the best study available for selection. However, the interpretation of and 

conclusions drawn from that study need to be re-evaluated in light of the issues raised in 

my preceding comments and the additional data shown in the Pubic Comments and 

coming available from a multi-institutional study sponsored by ToxStrategies. 

Rossman This does seem like the best and most complete study to use. 

Salnikow Outside of my area of expertise. 

Wise This study is the proper study based on the available data. The study is flawed because 

only very high doses were considered in the study, thus, there is concern that it may not 

reflect events at lower doses. The EPA is in the unique position that a study that repeats 

the one above and extends it to lower doses is almost completed. The EPA should wait for 

the final results of that study to make the most informed analysis. 

Zhitkovich The NTP-2008 is the best available study of chromium-6 toxicity via oral exposure and its 

choice for the calculation of the RID is scientifically sound and was clearly explained in 

the Draft. 

Zhu The Review offers EPA's rational for selecting the NTP's two-year drinking water study 

of sodium dichromate dihydrate in rats and mice (NTP, 2008). EPA's justification 

includes the lack of reliable epidemiological data, solid design of the NTP' s experiment, 

its controlled exposure regimens, the sensitivity of the endpoint, the availability of doseresponse data, and consistency with hypothesized genotoxicity MOA. These justifications 

are acceptable. There are still merits to include other studies, particularly the 3-month 

sodium dichromate dehydrate drinking water exposure of rats and mice (NTP 2007) in 

calculating RIDs. Inclusion of additional and all qualified studies is especially beneficial 

for better quantifying uncertainties and variations arising from different studies due to 

different study designs, strain/species of animals, and exposure regimens. As in a 

systematic review, studies meeting selection criteria should all be included for review and 

for analysis. Selecting a final RID then becomes a risk management decision. 
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A2. Diffuse epithelial hyperplasia in the duodenum of female mice was selected as the critical 

effect for the RID. Please comment on whether the selection of this critical effect is 

scientifically supported and clearly described. Please identify and provide the rationale 

for any other endpoints that should be selected as the critical effect. 

Reviewer Comments 

Byczkowski Diffuse hyperplasia, in itself, is considered to be a physiological (normal) response to 

several stimuli, as the cells of a hyperplastic growth remain sensitive to normal regulatory 

control mechanisms. Such a physiological proliferation of celts may be secondary to 

several pathological factors (e.g., the increased osmolality, changed pH, infection by 

Helicobacter, etc). Still, the proliferation in "diffuse hyperplasia" is a normal processalthough, it may be generated in response to abnormal condition (in contrast to neoplasia, 

where the proliferation in itself becomes abnormal). On the other hand, the hyperplasia is 

a common early preneoplastic response to potentially carcinogenic stimuli. 

Considering its rather ubiquitous and nonspecific character, it is remarkable that even no 

single case of epithelial hyperplasia was found during the two-year study by NTP (2008) 

in duodena of as many as I 00 mate and female control mice. Taking this result at a face 

value, it may be assumed that the diffuse epithelial hyperplasia in duodena could be an 

early biomarker of oral exposure to Cr+6, at least in the two-year NTP (2008) bioassay 

study. 

However, another end point- the pathological changes in the liver were noted in the same 

study with significantly increased frequency at the lowest dose employed. Even though 

they may be considered somewhat tess "specific" than the epithelial hyperplasia (because 

they were observed at tow frequency also in controls), but perhaps they could be more 

relevant to the systemic toxicodynamics of Cr+6- appearing further away from the portal 

of entry then the hyperplasia and apparently being less sensitive to the potentially 

confounding effects of the reduced drinking water consumption. 

Hamilton See AI. above. 

Nordberg Perhaps some information on possible effects on the lung should be comment on. The 

document should give information about solubility of different chromium (VI) species 

should be given and specifically for the chromium species that have been used in the 

quoted studies. Soluble salts are mentioned on page 54 under 7 but soluble in what media 

is not mentioned. The reference WHO/IPCS (2006) Environmental Health Criteria 234, 

Elemental speciation in Human Health Risk Assessment, WHO, Geneva is recommended 

to be included. 

Patierno The selection of Diffuse epithelial hyperplasia in the duodenum of female mice as the 

critical effect for the RID should be re-evaluated in light of the issues raised in my 

preceding comments. It must be considered in the context of the non-linear, dose-related 

issues discussed above regarding saturation of reductive capacity and definitive threshold 

data for toxicity and carcinogenicity. 
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Rossman This seems like an appropriate choice, but it's outside my area of expertise. 

Salnikow Outside of my area of expertise. 

Wise This endpoint is a proper endpoint based on the available data, but is not necessarily a 

toxic outcome. To allow for better understanding, RID's for other endpoints should he done 

and presented including some continuous endpoints. The EPA may conclude this endpoint 

is the critical effect, but this approach makes the analysis more transparent, open and clear. 

Zhitkovich The incidence of diffuse epithelial hyperplasia in the duodenum of female mice was the 

most sensitive histological response observed in Cr(Vl)-exposed groups and therefore, it 

was appropriately selected as the critical effect for the RID. 

Zhu EPA considered seven non-cancer endpoints for deriving RIDs (Table 5-l ): chronic liver 

inflammation in female rats, histiocytic cellular infdtration in the liver of female mice, 

diffuse epithelial hyperplasia in the duodenum of the male and female mice, histiocytic 

cellular infdtration in the mesenteric lymph nodes of male and female mice, and 

cytoplasmic cellular alteration of acinar epithelial cells in the pancreas of female mice. 

All seven are quanta! response from the NTP's 2-year chronic exposure study. The 

selections were largely driven by the dose-response data these effects exhibited. After 

dose-response modeling and the estimation of benchmark dose (BMD) for each of these 

select effects, diffuse epithelial hyperplasia in the duodenum of the female mice was 

chosen as the critical endpoint simply because it yielded the smallest BMD and its 

corresponding lower confidence limit (BMDL). It must be noted that the dose-response 

model for this critical effect was done only after deleting the two highest doses. As a 

result, the dose-response modeling relied on only three dose level (including the control), 

leaving little room for any flexible dose-response forms other than the "linear" multistage model with a polynomial of I degree of freedom. 

Instead of relying on a select "critical" effect, EPA could report a range of RIDs based on 

a set of qualified and select effects. As a result, EPA will be able to a range of RIDs, 

projecting the uncertainty and variation of RIDs arising from man y sources and affording 

risk management the opportunity to make an informed choice of a final RID (NAS, 20 I 0). 

This is important as EPA is moving towards enhancing analysis of uncertainty and 

variation in risk assessment. 

To this end, EPA could have benefited greatly by including additional endpoints from this 

principle study as well as other qualified studies. Potential candidates include histiocytic 

cellular infiltration in the duodenum of female rats and male mice, histiocytic cellular 

inflammation in pancreatic lymph nodes of male rats, and histiocytic cellular infiltration 

in the liver of female rats. EPS considered only the quanta] responses in this Review for 

the purpose of computing RIDs. It is unclear why effects of continuous measurement scale 

were not considered. Many of these effects show unequivocal dose-response (e.g. Tables 

4-12 and 4-13) and seem to be relevant to the hypothesized MOA of hexavalent 

chromium. The availability of EPA's software BMDS for dose-response modeling and 

benchmark dose computation makes it practical and useful to consider continuous effects 

for RID derivation as well. 
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A3. Benchmark dose (BMD) modeling was applied to the incidence of diffuse epithelial 

hyperplasia in the duodenum of female mice to derive the point of departure (POD) for 

the RID. Has the BMD modeling been appropriately conducted and clearly described? Is 

the benchmark response (BMR) selected for use in deriving the POD (i.e., a 10% 

increase in the incidence of diffuse epithelial hyperplasia) scientifically supported and 

clearly described? 

Reviewer Comments 

Byczkowski The benchmark dose modeling was applied in accordance with the U.S. EPA Benchmark 

Dose Technical Guidance Document (2000) in the prescribed manner. However, the 

modeled BMD, derived from the diffuse epithelial hyperplasia, was supported only by the 

three dose-points (including zero dose). In contrast, the pathological changes in the liver 

were consistently fitted, for both female rats and mice with Log-logistic model which 

included all the five data points (including controls), and thus, they could be considered to 

be robust dose-response adverse effects for derivation of BMD. So, the pathological 

changes in the liver may be used as alternative end points for derivation of BMD. 

Hamilton This reviewer is not an expert on modeling and cannot comment on A3 in detail. 

Nordberg Yes. 

Patierno See answer to A2 above. 

Rossman This does seem like the best and most complete study to use (but it's a bit outside my area 

of expertise). 

Salnikow Outside of my area of expertise. 

Wise The BMD modeling has been appropriately conducted and clearly described. To allow for 

better understanding, BMR modeling at 5% and 1% should be done and presented. This 

approach makes the analysis more transparent, open and clear. 

Zhitkovich BMD modeling and the calculations of the POD both appear to be appropriately 

performed. 

Zbu EPA should be commended for conducting BMD modeling for multiple effects with 

different model forms. For the modeling of the incidence of diffuse epithelial hyperplasia 

in the duodenum of female mice, EPA should provide a more detailed discussion on the 

limitation of the dose-response modeling (See A2). Uncertainties due to model choice, 

variation in the shape of seemingly equally well-fit models also can be quantified to a 

degree by considering multiple benchmark response levels (BMR) for each model. EPA 

used only BMR~lO%. It makes perfect sense to also consider BMR~5% or even 

BMR~ I% when such a choice is supported by the data. This is the general 

recommendation of EPA's own guideline (EPA, 2000). By doing so EPA would be able to 

quantitatively demonstrate uncertainty and variations due to the choice of different models 
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Responses to Charge Questions 

and different BMR levels. Additionally, EPA should briefly but clearly define the BMD 

concept and methodology in an appendix to improve the readability for readers unfamiliar 

with the process. 

A4. Please comment on the rationale for the selection of the uncertainty factors (UFs) applied 

to the POD for the derivation of the RID. Are the UFs scientifically supported and 

clearly described? If changes to the selected UFs are proposed, please identify and 

provide a rationale. 

Reviewer Comments 

Byczkowski While the uncertainty factors (UFs) were applied in accordance with the U.S. EPA 

guidelines, the use of the UF = 10 for extrapolating the toxicity value based on effects in 

the portal of entry of Cr"' in animals to predict GI effects in humans, seems to be 

problematic. Thus, both the reductive capacity of human gastrointestinal fluids and the 

antioxidant protection of human tissues exceed those in mice. So, adequately nourished 

and hydrated humans should be less vulnerable than mice to the adverse GI effects of oral 

exposure to Cr+6

, rather then 10 times more sensitive, as the UA=IO may suggest. 

Hamilton No concerns regarding A4. Uncertainty factors are policy decisions, not scientific ones, 

and we can neither prove nor disprove any of the assumptions on which they are based nor 

can we accurately determine when and how such factors might be applied. Based on 

previous EPA doctrine, these seem to be consistent with previous applications. 

Nordberg The reason for chosen UFs is clearly and properly described. 

It is known that the reduction of hexavalent chromium to chromium three is influenced by 

vitamin C. This can perhaps be used in setting UFs and thus not only choose the standard 

UFs of 10 between species and I 0 for interindividual differences. Humans can not 

synthesize vitamin C and are thus depending on vitamin C supplementation. The tested 

animals i.e., the mouse and the rat both produce vitamin C themselves. In this context a 

laboratory animal that resembles the human by being depended on vitamin C 

supplementation might be used in future studies. The concentration of vitamin C in tissues 

and organs are very important in evaluation of carcinogenic metals. It is likely to be 

involved in the mechanism in causing cancer and plays a role in the MOA. 

Patierno The Uncertainty Factors must be re-evaluated in the context of the non-linear doseresponse data, the clear evidence of thresholds for toxicity and carcinogenicity and the 

fact that these high-dose, supra-saturation experiments cannot be extrapolated linearly to 

low or vanishingly small doses. 

Rossman This is outside my area of expertise. 

Salnikow Outside of my area of expertise. 
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Wise The rationale for the selection of the uncertainty factors (UFs) applied to the POD for the 

derivation of the RID are appropriate. It was suggested that an UF for children and those 

with different conditions be used. People with different conditions (e.g. antacid use) are 

already contemplated in the UF applied for interindividual variation. Currently, such a 

factor for children is not included in the EPA guidelines. It could be that the same 

interindividual variation may apply in that case as well. 

Zhitkovich Two UFs were applied: UF= I 0 for interspecies extrapolation to humans and UF= I 0 for 

interindividual variability in the human population. The interspecies UF was used because 

there is no available information to quantitatively assess the true differences in chromium6 toxicokinetics and toxicodynamics between humans and laboratory rodents. There are, 

however, two biological factors that point to a potentially greater sensitivity of humans 

relative to mice. The first is related to the fact that telomerase was shown to suppress 

genetic damage by chromium-6 (Glaviano eta!. 2006). All mouse cells express telomerase 

while only stem cells retain telomerase expression in human tissues. The second factor is 

the difference in ascorbate metabolism. Human cells actively recycle ascorbate (Nualart et 

a!. 2003, Mantel-Hagen eta!. 2008), resulting in -100 times lower requirements for this 

vitamin by humans relative to rodents. A more economical use of vitamin C by humans 

also results in lower ascorbate concentrations in the extracellular fluid (for example, as 

reported for bronchoalveolar lavage fluid by Slade eta!. 1993), which would more rapidly 

detoxify chromium-6 via extracellular reduction. However, No specific information about 

ascorbate concentrations in the extracellular environment of the duodenum and jejunum of 

mice and humans is currently available. 

The interindividual variability in sensitivity to chromium-6 was not studied and the 

application of the safety coefficient (UF) is definitely appropriate in this case. However, 

the proposed UF=IO likely underestimates the range of the interindividual variability. The 

Draft has a brief discussion on the common presence of genetic polymorphism in DNA 

repair genes as one source of interindividual differences. Four major DNA repair 

pathways (mismatch repair, nucleotide excision repair, base excision repair and 

homologous recombination) are known to impact the extent of genetic damage and 

cytotoxicity by Cr(VI), and the use of UF= I 0 to account for interindividual differences in 

the overall DNA repair would assume a quite low degree of variability for each repair 

process (overall I 0-fold variation would result from a very narrow 1.8-fold variation in 

each process: 1.84 = 10.5). 

Chromium-6 toxicity can be affected on three levels: I) differences in extracellular 

detoxification, 2) differences in cellular uptake and 3) differences in cellular/genomic 

defense mechanisms. A 5-fold variation at each stage would give a potential 125-fold 

variation in the general population. A study by Donaldson and Barreras (1966) showed 

that individuals with pernicious anemia had 4-times higher systemic uptake of chromium6 due to its lower detoxification in the stomach. Widespread use of antacid medications 

has a clear potential to diminish reduction rates of chromium-6. No systematic studies on 

potential variations in chromium-6 uptake have been performed yet, but two human lung 

carcinoma lines, H460 and A549, displayed a 5-fold difference in chromium-6 

accumulation (Macfie eta!. 2010). A caveat of using information from these two cell lines 

is that they are malignant and therefore it is not possible to determine whether their 
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differences were present in the initial normal cells or whether it is a side effect of different 

transformation processes. 

The Draft correctly stated on p.214 that there is no information about susceptibility of 

children to chromium-6 toxicity. In this case, it would be clearly appropriate to use 

additional UF=IO to account for a potential early life susceptibility. If EPA considers it 

unnecessary, then the exclusion of this UF should be justified in Section 5.1.3. My 

recommendation would be to use UF=IOO to account for the combined effects of the 

interindividual variability in susceptibility and early life exposures. 

Zhu The use of uncertainty factors (UFs) in this review is well described and is consistent with 

EPA's guidance documents for RIDs. Exposure in earlier stage of life was discussed. 

(B) Carcinogenicity of Hexavalent Chromium 

Bl. Under EPA's 2005Guidelines for Carcinogen Risk Assessment (www.epa.gov/iris/backgrd.html), 

hexavalent chromium is likely to be carcinogenic to humans by the oral route of exposure. 

Is the cancer weight of evidence characterization scientifically supported and clearly 

described? 

Reviewer Comments 

Byczkowski Even though the U.S. EPA (2005) guidelines were applied appropriately, there is no direct 

evidence of dose-dependent carcinogenicity by orally administered Cr+6 in humans (C.f. 

P. 236, L# 33: " ... EPA concluded that the exposure-response analyses presented by Zhang 

and Li ( 1997), Beaumont et al. (2008), and Kerger et al. (2009) are not based on the 

quality of data that is needed to support a conclusion regarding the presence or absence 

of a dose-response among the observed cancer rates in these villages. The other 

epidemiologic studies did not find a significant correlation between hexavalent chromium 

concentrations in drinking water (or proximity to the source of hexavalent chromium sail 

contamination) and cancer ... ") 

The classification of hexavalent chromium as "likely to be carcinogenic to humans by the 

oral route of exposure" is based on carcinogeniesis observed only at the highest dose 

levels employed in animal studies. Therefore, it may, or may not be relevant to humans at 

environmentally relevant exposure concentrations. 

Hamilton Regarding B I. and as outlined in the detailed comments under G I. and G2., this reviewer 

is concerned that the evidence for carcinogenicity is not strong in animals and is not 

supported by human epidemiology. As also noted in comments under Gl. and G2., there 

is considerable concern with statements by EPA in sections of this draft, particularly 

under Major Conclusions in Chapter 6, regarding evidence for human carcinogenesis 

based on a single human epidemiology study that in turn is a re-analysis of another study 

that lacks critical information that would be useful in fully assessing relative cancer risk. 

However, based on the current criteria for selection of this designation, it appears to be 
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Nordberg 

Patierno 

Rossman 

Salnikow 

Wise 

Responses to Charge Questions 

consistent with EPA doctrine since there is evidence of increased tumors in animals under 

certain exposure conditions. 

Though it is an American document prepared for US I would recommend also to consult 

and cite documents published by the United Nations Organizations such as International 

Agency for Research on Cancer (IARC) and World Health Organization (WHO). 

Recommended literature is IARC Monographs on the Evaluation of Carcinogenic Risks to 

Humans Volume49 (1990), Lyon, France and Chromium, Nickel and Welding 1-677 

pages and !ARC Monographs on the Evaluation of Carcinogenic Risks to Humans 

Volume 100 which is in preparation and further information on www.iarc.fr 

Hexavalent chromium is classified as a human carcinogen. It is not clear to the reader why 

classification can be different upon different route of exposure. 

The cancer weight of evidence characterization is not scientifically supported. The 

conclusion that hexavalent chromium is "likely to be carcinogenic to humans by the oral 

route of exposure" is not scientifically supportable given the issues raised in my 

comments above. The non-linear dose data in both the NTP studies and the data 

preliminarily discussed in the Public Comments clearly demonstrate that the toxicities and 

carcinogenesis observed at these extremely high, obviously supra-saturating doses, cannot 

and should not be extrapolated to lower doses. See detailed comments above. 

Given the fact that there is not enough human data to firmly establish carcinogenicity, but 

there is animal data, "likely to be" is reasonable but "possibly carcinogenic at high dose" 

would be more accurate. 

Hexavalent chromium has been classified by IARC as carcinogenic to humans (group I) 

via inhalation route of exposure based on results obtained in human and animal studies. 

However, when animals were exposed to hexavalent chromium in drinking water the 

carcinogenic effects were observed only at very high doses, which are irrelevant to human 

exposure. These results seems to cast doubt on carcinogenicity of hexavalent chromium 

via oral route of exposure and yet as I already pointed out in G2 the reason for only high 

chromium doses producing carcinogenic effect may be stemming from the inappropriate 

animal models which have higher protective ascorbate levels as compared to humans. 

Although, more human and animal studies are required to make an informed conclusion 

the ability of hexavalent chromium to produce tumors makes it likely to be carcinogenic 

by oral exposure. 

Another important consideration is that hexavalent chromium is a co-carcinogen and 

consumption of water with other toxic or carcinogenic compounds will result in 

unraveling chromium carcinogenic effects at much lower doses. Additionally, people with 

chronic inflammation of digestive tract could be more susceptible to chromium-induced 

carcinogenesis. 

The general lack of accuracy in the document in its handling of citing, paraphrasing and 

considering the underlying literature is of some concern in this presentation. There is a lot 

of cell culture and animal data showing genotoxicity and clastogenicity, however, the 
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Zbitkovich 

motivation for these studies were largely inhalational exposure-induced cancer. It seems if 

they can support one route, they could support the other, but it functionally means the data 

underpinning the oral route of exposure is the one NTP study. 

That NTP study is flawed because only very high doses were considered in the study, 

thus, there is significant concern that it may not reflect events at lower doses. The EPA 

requirement for a "likely to be carcinogenic to humans" classification defined as 

" ... appropriate when the weight of the evidence is adequate to demonstrate carcinogenic 

potential to humans but does not reach the weight of evidence for the descriptor 

"Carcinogenic to Humans." It is understandable why an initial assessment of "likely to be 

carcinogenic" was chosen as the guidance states: " Supporting data for this descriptor may 

include: ... 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". 

The next possible descriptor is "suggestive evidence of carcinogenic potential". It is 

indicated as " ... appropriate when the weight of evidence is suggestive of carcinogenicity; 

a concern for potential carcinogenic effects in humans is raised, but the data are judged 

not sufficient for a stronger conclusion." It is also said to cover " ... evidence associated 

with varying levels of concern for carcinogenicity, ranging from a positive cancer result in 

the only study on an agent...". 

Thus, there is conflicting guidance for Cr(VI) on these descriptors. On the one hand, there 

is a study of multiple species and genders possibly qualifying it for a descriptor of "likely 

to be carcinogenic to humans". On the other hand there is only one study showing this 

outcome making it suitable for a descriptor of "suggestive evidence of carcinogenic 

potential". 

In studying the data and descriptors, it appears to be premature to conclude that the weight 

of the evidence is nearly adequate for demonstrating carcinogenic potential to humans. 

Moreover, in considering the spirit of the guidelines for the two descriptors, it is clear that 

"likely to be carcinogenic" descriptor is contemplating that the data concerning multiple 

species, genders, strains etc. will come from multiple studies not just one. It is also clear 

that the "suggestive evidence of carcinogenic potential'' descriptor is intended for a 

database with flaws. This database is flawed by the lack of multiple studies and the fact 

that the one study available relied on very high doses. Accordingly, a descriptor of 

"suggestive evidence of carcinogenic potential" is more appropriate at this time. 

However, the EPA is in the unique position to soon have another study that repeats the 

NTP study is almost completed. The EPA should wait for the final results of that study to 

make the most informed analysis. If it too shows tumors at all doses, then the stronger 

descriptor would be justified. 

The classification of hexavalent clrromium as "likely to be carcinogenic to humans" via 

the oral route of exposure is supported by evidence of its tumorigenicity in the oral cavity 

of female and male rats and in the small intestine of female and male mice. An increased 

incidence of stomach cancers in the JinZhou area (China), which was contaminated with 

high concentrations of clrromium-6 in drinking water, is supportive of the selected 

classification. Even if the ecological study from China is excluded, the weight of evidence 
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from animal studies is adequate to designate hexavalent chromium as "likely to be 

carcinogenic to humans" via the oral exposure. 

Zhu The descriptor of "likely to be carcinogenic to humans" for hexavalent chromium is 

consistent with EPA's Guidelines for Carcinogen Risk Assessment. EPA gave a clear 

description of the hypothesized MOA. 

82. A mutagenic mode of carcinogenic action by all routes of exposure is proposed as the 

primary mode of action for hexavalent chromium. Please comment on whether this 

determination is scientifically supported and clearly described. Please comment on data 

available for hexavalent chromium that may support an alternative primary mode of 

action. 

Reviewer Conunents 

Byczkowski The positive laboratory results of mutagenicity tests do not prove genotoxicity and are not 

necessarily biologically relevant to humans exposed in vivo to the environmentally 

relevant concentrations of Cr'6

.The mutagenicity results from humans occupationally 

exposed to Cr., (Table 4-25) were inconsistent (this document states- C.f. P. 178, L# 14: 

" .. .In general, associations between hexavalent chromium exposure and mutagenicity in 

workers are uncertain ... ") and actually, the results presented in the document did not 

prove the direct genotoxic mode of action of Cr., in vivo. 

An alternative, indirect mode of action seems plausible to this reviewer. As discussed in 

Section 4.5.2., the intracellular products of one-electron reduction of Cr., in vivo, causing 

oxidative stress and free-radical damage to cellular macromolecules, seems to be 

responsible for initiating carcinogenicity at relatively low dose levels. Erosion of GI tract 

mucosa with inflammation that followed at high Cr.., dosage (discussed in section 4.2.1) 

apparently caused promotion in the lesions, which eventually progressed to benign and 

malignant tumors. Importantly, such a mode of action implies a threshold phenomenon, 

i.a. due to antioxidant protection of cells. 

Hamilton There is considerable evidence that Cr(Vl) is genotoxic in cell culture and in vitro, and 

under certain extreme conditions it can also be shown to be mutagenic. However, there is 

far less support that Cr(VI) is genotoxic or mutagenic in vivo by the oral route of exposure 

at doses of relevance to humans; conversely, there is considerable evidence that there are 

protective threshold mechanisms that significantly impact the ability of Cr(VI) to reach 

target tissues and cause DNA damage under physiological conditions. In addition, while 

alternative mechanisms are briefly discussed, these are essentially dismissed without 

extensive treatment. As noted in the document, there is not a large literature on 

alternative mechanisms, but this is largely because, since the discovery of the genotoxic 

potential of Cr(VI) some forty years ago, most of the field has only focused on this one 

aspect, and I suspect it would be very difficult to get peer-reviewed funding to study nongenotoxic mechanisms for this toxicant. It is important to note, however, that there are no 

reports of increased skin cancer under occupational exposure settings, despite the fact that 

workers until the past few decades were directly exposed to Cr(VI) on the skin to the 
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extent that they formed "chrome holes" that eventually healed. Yet this direct application 

and clear signs of chromium reduction and toxicity directly on the skin produced no 

increased skin cancer risk. Likewise, the occupational exposure literature only recently 

investigated the role of smoking status in chromium-related lung cancer risk. The 

increased risk of lung cancer associated with Cr(VI) exposure is modest considering the 

exposure levels and duration of exposures that span decades, and virtually all of the 

cancer cases in the epidemiology studies were seen in smokers, suggesting an interaction 

but one that is very modest. This in turn suggests alternative mechanisms such as 

inflammation, oxidative damage, damage-induced proliferation, and other mechanisms 

not directly tied to the ability of Cr(VI) to enter cells and damage DNA. These should 

considered and explored in more detail, since they are the basis for many of the 

assumptions regarding the risk of cancer from oral exposure to Cr(Vl). 

Nordberg Again this might be linked to differences among hexavalent chromium species regarding 

for example solubility in body fluids. 

Somewhere in the document it should be pointed that iron can reduce hexavalent 

chromium to chromium three. This has been done in some products. This is touched by on 

page 48 line 18. 

The document describes in detail the possible MOA. !ARC 1990 stated" ... chromium 

(VI) compounds on the basis of the combined results of epidemiological studies, 

carcinogenicity studies in experimental animals, and several types of other relevant data 

which support the underlying concept that chromium(VI) ions generated at critical sites in 

the target cells are responsible for the carcinogenic action observed" 

However, also alternatives like DNA- methylation and other epigenetic mechanisms 

should be considered, because for many metals DNA-methylation is recognized as a 

possible mode of action also addressed as mechanism for carcinogenicity. Effects on cell 

signalling and gene expression may also serve as mechanisms involved in carcinogenesis 

of metaHic compounds. See further discussion in review by Davidsson et al, Chapter 5 in 

Nordberg eta! (eds) Handbook on the Toxicology of Metals, p79-IOO 

Patierno The determination of a mutagenic mode of action by all routes of exposure should be reevaluated before proposing it as the primary mode of action. Nearly all indices (NTP 

studies, inhalation studies, mammalian cell mutagenesis studies etc) indicate that 

carcinogenicity of CrVI is only observed under exposure conditions that evoke cellular 

toxicity, inflammatory tissue damage, and tissue regeneration. The DNA damage and 

presumed mutagenicity (actually epigenetic or stochastic selection of cells that survived 

toxicity) of CrVI is only observed at doses that also cause cell death and tissue damage. 

In vivo, these effects are only achieved at very large doses that clearly overwhelm the 

reductive capacity of the oral cavity, stomach and blood components resulting in a sharp 

threshold of carcinogenesis only at the two highest NTP doses. I have spent more than 25 

years studying the molecular mechanisms of CrVI genotoxicity and mutagenesis and I 

have a deep appreciation for its capacity to interact with cells and alter DNA and DNA 

replication and transcription. However, just because CrVI is capable of causing DNA 

damage and what we thought was "mutagenicity" (see above) in carefully contrived 

experimental systems, does not mean that it does so under physiologically and 
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environmentally relevant conditions. It is much more likely that the chronic tissue 

damage, with accompanying inflammation and subsequent proliferative regeneration, 

possibly in the presence of unrepaired DNA damage, all of which are only observed at the 

highest doses, is the principle mode of action. 

By definition, the "mode of action" (MOA) of a carcinogen is "a sequence of key events 

and processes, starting with interaction of the agent with a cell, proceeding through 

operational and anatomical changes, and resulting in cancer formation" (USEP A, 2005). 

For mutagenesis to be a carcinogenic MOA, the agent must at the very least cause 

heritable mutations in mammalian cells. The mutations should be induced in a 

concentration range with low toxicity (preferably similar to concentrations seen in human 

exposures), and the mutations should be induced in the target tissues in animal 

experiments and in humans. Human and animal tumors should also show genetic 

alterations consistent with the types of mutations induced by the agents, and these should 

be early events. 

The information about Cr(6) is lacking for much of these criteria. In fact, the human 

tumor data support an epigenetic mechanism more than a mutagenic one. 

Genotoxic is not the same as mutagenic, and sections 4.5 and 4. 73 must be completely 

rewritten, as they consistently confuse these terms. Standard genotoxicity assays were not 

designed to inform specific modes of tumor induction. With the exception of 

mutagenesis, these other assays (non-mutagenic assays) do not measure heritable events, 

but rather measure evidence of DNA damage or its repair. Non-mutagenic assays include 

chromosome aberrations, micronuclei, comet assays, DNA lesion measurements, and 

DNA repair assays. These assays are useful for hazard identification or as biomarkers of 

exposure. They provide only supportive evidence that mutagenesis might be a MOA. 

DNA damage per se does not inform us about eventual heritable change, which is the true 

issue. Most (but not all) mutagens cause heritable changes in DNA sequences by causing 

damage to DNA (pre-mutagenic lesions) that is converted to mutation after cell division. 

Table 4-21 should be deleted, as results in bacteria are not relevant to tumorigenic MOA. 

A simple statement that Cr(6) is mutagenic in bacteria should suffice (referencing a 

review such as Klein CB. "Carcinogenicity and genotoxicity of chromium" In: 

Toxicology of Metals (Chang LW, ed). Boca Raton, FL:CRC Press, 1996, pp.205-219. 

Table 4-22 represents positive results in a group of assays that measure both mutagenic 

and non-mutagenic endpoints (including a paper on epigenesis, Klein et al., 2002, which 

should be removed, as this is not a genotoxic event; neither is disruption of mitosis, which 

can have many causes). Table 4.22 has neither information on the concentrations 

inducing positive results, nor on the toxicity of those treatments. 

This table is also deficient in the most important results in mammalian cells, i.e. 

mutagenesis. All of the studies reported are mouse lymphoma cell studies, yet later in the 

document (page 203), reference is made to mutations at the HGPRT locus in "Chinese 

Hamster ovary cells (V79 and AT3-2)" V79 is not a Chinese hamster ovary cell, it is a 

Chinese hamster lung fibroblast. In fact, one such study using V79 cells (Sugiyama et al., 

Mutat. Res. 260:19-23, 1991) shows a modest positive effect only in a narrow 

concentration range. A CHO line (AA8) showed a small increase (3.4 fold) at a dose 
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giving 75% survival (Brooks et al., 2008). There is a fuller discussion of chromate 

mutagenicity (with references) in: Klein CB. "Carcinogenicity and genotoxicity of 

chromium" In: Toxicology of Metals (Chang LW, ed). Boca Raton, FL:CRC Press, 1996. 

pp.205-219. This article points out that chromium compounds are mutagenic in a narrow 

dose range, possible because of persistent toxicity after treatment (e.g. residual toxicity 

was seen a week after treatment of V79-derived G 12 cells). 

Chromosome aberrations and DNA strand breaks can occur as a result of cytotoxicity. 

Dead cells do not become tumors. Unless assays for cytotoxicity are performed, it is not 

possible to know whether DNA damage occurs in cells that can replicate to form clones. 

Traditional cytogenetic assays rely on short-term cell survival to generate the mitotic 

figures necessary for analyses; the long-term viability of these treated cells cannot be 

determined. Thus, the relevance of this kind of data for carcinogenic MOA is 

questionable. To measure cytotoxicty, the gold standard is clonal survival, a method that 

is common in gene mutation assays, but not in other genotoxicity assays. Short-term 

survival assays, such as MTT, neutral red, and trypan blue, as well as measurements of 

mitotic index that are commonly used in cytogenetic assays, fail to detect early or delayed 

apoptotic events. Trypan blue detects only necrosis. MTT and neutral red assays can be 

delayed to allow time for apoptosis to develop, at which point the results approach clonal 

survival (Komissarova eta!., Toxicol. Appl. Pharmacal. 202:99-107, 2005). As 

mentioned above, Cr(6) causes delayed residual toxicity (Klein eta!. Environ. Health 

Perspect. 102 (suppl3):63-67, 1994), and thus clonal survival or at least apoptosis at later 

times after exposure, are essential in establishing cytotoxicity levels. Normal human 

fibroblasts show -80% loss of clonality after a 25h exposure to 2 11M sodium chromate 

[Vilcheck et al., Environ. Health Perspect. 110 (Sup5):773-777, 2002]. 

Micronuclei can result from DNA damage or from malsegregation of chromosomes. It 

has been recommended that this assay should be performed under conditions of high 

survival (an increase of >90% in number of viable cells) and that markers for apoptosis 

and necrosis be included [Kirsch-Voiders, et a!. (2003) Report from the in vitro 

micronucleus assay working group. Mutat. Res. 540:153-163]. In the case ofCr(6), at 

lower concentrations, most of lhe micronuclei are kinetecore-positive, meaning that they 

arise from malsegregation and not DNA strand breaks (Seoane and Delout, Mutat. Res. 

490:99-106, 2001; Figgitt eta!., Mutat. Res. 688:53-61, 2010). Those that are kinetecorenegative (arising from chromosome breaks) occurred only at the highest concentrations. 

Thus, Cr(6) induces aneuploidy rather than DNA damage at lower concentrations (Holmes 

et al., 2010; Figgitt eta!., Mutat. Res. 688(1-2):53-61, 2010). Aneugenesis is caused by 

alterations in proteins, not DNA, and has thresholds. 

It should also be noted that the mouse lymphoma assay (MLA), chromosome aberration 

assay (CA) and micronucleus assay (MN) give a large number of false positives, even 

compared with the Ames test. Chemicals that are non-carcinogenic after thorough testing 

in both male and female rats and mice are often positive in these assays (Kirkland eta!., 

Mutation Research 584 (2005) 1-256). 

The Comet assay detects single and double strand DNA breaks as well as alkali-labile 

sites. Nucleotide excision repair (NER) and base excision repair (BER) of adducts can 

create breaks as intermediates. Single strand breaks are quickly repaired and are not 

regarded as significant premutagenic lesions. During apoptosis, DNA fragmentation into 
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segments of 180 base pairs occurs, whether or not the apoptosis was induced by a 

genotoxic event (Choucroun et al. 2001, Mutat. Res. 478:89-96; Henderson et al.,.l998, 

Mutagenesis 13:89-94.) Necrotic cells also display DNA damage (Fairbairn et al., 1996 

Scanning 18:407-416.). In order to avoid false positive responses, Henderson et al. (1998) 

suggests that the concentration of test substance should produce> 75% viability. 

In summary, standard genotoxicity assays from hazard identification exercises cannot be 

used to establish a mutagenic MOA, because these assays do not measure heritable events 

and because the doses used in such assays are usually too high. 

Other MOA's have not been adequately considered. These include, for example, selection 

for Cr-resistance, resistance to apoptosis, and aneuploidy. The evidence for a mutagenic 

MOl is weak. Mutations can result from DNA damage, but can also be a secondary effect 

of the loss of mismatch repair, aneuploidy, and other types of genomic instability (in other 

words, it is a later effect). With the exception of the mouse lymphoma system, Cr(6) is 

only weakly mutagenic in mammalian cells, rarely giving more than a 3-fold increase in 

mutant fraction over background levels (in endogenous genes), and in a very narrow (and 

toxic) dose-range with a strong threshold (reviewed in Nickens et al., 2010). 

In some cases the "mutations" have been shown to be epimutations resulting from altered 

DNA methylation (Klein eta!., 2002). Since none of the other studies on mammalian 

cells looked for epigenetic inactivation, this calls into question whether the "mutants" 

seen are really mutants. These are important considerations for the MOA of Cr(6), since 

cells grown in the presence of Cr(6) show selection for cells with inactivated mismatch 

repair (MMR) genes. These cells are Cr(6)-resistant and could be the result of either 

mutation or epigenetic inactivation (reviewed in Salnikow and Zhitkovich, 2008). Cells 

with epigenetically inactivated MLHl (a MMR gene) were seen in human lung A549 cells 

exposed to Cr(6) (Sun eta!., Toxicol. Appl. Pharmacol. 237:258-266, 2009). MMRdeficient cells are mutators (having a high spontaneous mutation rate) and show 

microsatellite instability. An important consideration for MOA is the fact that chromiuminduced lung cancer cells also show epigenetically-inactivated MMR genes (Takahashi et 

al.. 2005). Also against the idea of a mutagenic MOI is the fact, discussed in Salnikow 

and Zhitkovich (2008), that Cr-induced lung tumors in humans lack p53 mutations, in 

contrast to lung tumors associated with other agents such as tobacco smoke, and the fact 

that the few mutations found do not correspond to the types of mutations caused by Cr in 

in vitro systems. The fact that Cr is essential also implies that oral Cr(Vl) could supply the 

necessary Cr, again implying a threshold at nontoxic doses. Also, experiments from the 

Costa laboratory (Davidson et al.,Toxicol. Appl. Pharmacol. 196:431-437, 2004; Uddin et 

a!., Toxicol. Appl. Pharmacol. 221:329-338, 2007) showing that chromate in drinking 

water is a cocarcinogen with solar UV, and the implications of this finding, are not 

discussed. 

Other problems (by page): 

Page 176, top of the page, is a good example of the confusion between mutagenicity and 

other endpoints. The statement "Hexavalent chromium-induced mutagenicity has been 

demonstrated following oral exposure" is misleading. There is only one mutagenicity 

assay showing positive effects on eyespots (presumed deletions) in offspring of female 

rats given drinking water with 62.5 mg Cr(6)/L. The deletions were not confirmed, so the 
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eyespots might be epigenetic events (as Klein found with so-called mutants). All of the 

other assays are for non-mutagenic endpoints, and tend to be negative for drinking water 

exposure, but positive for gavage (a more toxic type of exposure). 

Page 178, 4.5.!.2, it is claimed that mutagenicity has been evaluated in humans 

experimentally exposed to hexavalent chromium. No such studies appear in Table 4-25. 

The paragraph mistakenly refers to mutagenicity many times. 

Page 186: It is not ER (excision repair) that is responsible for removal of bulky lesions, 

butNER (nucleotide excision repair) that is. Reynolds et al. 2004 is missing in 

references. 

Page 187, bottom. It doesn't make sense that mice given 0 mglkg Cr(6) should have a 

significant level of apoptosis (compared to what?). 

Page 188: The Dai et al 2009 paper does not measure mutation frequency in human cells. 

Page 190: 3'd paragraph: The mutational spectrum of chromate is not clear. See the 

review by Klein referenced above. 

Page 202: Key Events, #3: The authors skip from discussing mechanisms of DNA 

damage by Cr to "overall genomic instability which can lead to mutations if not 

adequately repaired". Genomic instability can occur as a result of other factors besides 

DNA damage, and genomic instability is not repaired (DNA damage can be, but the repair 

often leads to apoptosis). As discussed above, toxic exposure would play a role in the 

selection of Cr-resistant and/or apoptosis-resistant cells. There is no obvious tie-in here 

between DNA damage and mutagenesis as a key event, since cells resistant to Cr could 

have arisen by epigenetic silencing (and this may also be a mechanism in resistance to 

apoptosis). In a sense, this point is recognized in #4, but in postulating apoptosis as a key 

event, the authors do not seem to realize the implications, i.e. that a toxic dose is needed 

for carcinogenicity. They suggest that selection for resistance to apoptosis is due to 

mutations (either pre-existing or Cr-induced) but there is no evidence for tllis. Besides 

altered DNA methylation, other mechanisms for the appearance of Cr(VI)-resistance in 

exposed cells include epigenetic effects via altered histone modification (Sun et al., 

Toxicol. Appl. Pharmacal. 237:258-266, 2009) as well as reduction of Cr(VI) transport 

via down-regulation of sulfate ion transporter activity, and resistance to apoptosis via 

altered gene expression (upregulation of survival pathways and down-regulation of 

apoptotic pathways) (discussed in Nickens et al., 2010) 

p. 204-210: What is described as "mutagenicity" is not in all cases. 

p. 206, end of paragraph 1: It is claimed that there is evidence that Cr(6) induces 

mutagenicity in tissues at the site of entry and systemically at doses relevant to human 

exposure. Where is the evidence for this? 

p. 206, bottom: Again, it is important to note that DNA damage can lead to apoptosis as 

well as mutation, so it does not necessarily support a mutagenic MOA. 

p. 207: De Flora et al., 2008, did not look for mutagenesis, they looked for DNA damage. 
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p. 208: Neither O'Brien eta!., 2005 nor Eastmond eta!., 2008 is in references. 

p. 209: Low (non-toxic) concentrations would not provide selective pressure for Crresistant, mismatch repair deficient cells. Especially in vivo, toxicity would be the driving 

force to stimulate the outgrowth of such cells. 

p. 209, z•d. paragraph: This is a lot of speculation (should be, may be). 

p. 211, bottom: It is claimed that the study by NTP found no evidence of tissue damage or 

necrosis. Did they look for apoptosis? It is also claimed that most available studies found 

Cr-induced genetic damage at doses below those that inducing cytotoxicity. This has not 

been demonstrated, since clonal survival (the only assay that will detect delayed toxicity) 

was not performed in these studies. 

p. 212, end: " ... giving rise to mutagenicity (including DNA adduct formation, DNA 

damage, gene mutations, chromosomal aberrations and micronuclei formation" This is 

nonsense, as is the next sentence. There is no evidence for mutagenicity at the target 

tissue at all. 

p. 213: More confusion between mutagenicity and other endpoints. It is not true that other 

hypothesized MOA's have not been demonstrated. There is actually more tumor evidence 

for an epigenetic MOA. Thus, the weight of evidence favors the alternative. This is not a 

numbers game. Epigenetic studies are relatively new, compared with DNA damage and 

other "genotoxic" studies, so there are fewer studies. 

p. 214 top: Has EPA concluded this? Then what is the purpose of reviewing this 

document? 

p. 238, bottom: More confounding of mutagenesis and other endpoints. 

Additionaijla(lers showing e(ligenetic effects of Cr 

Schnekenburger et al., (2007) Chromium cross-links histone deacetylasel-DNA 

methyltransferasel complexes to chromatin, inhibiting histone remodeling marks 

critical for transcriptional activation. Mol. Cell Bioi. 27(20): 7089-101. 

Sun et al. (2011) Comparison of gene expression profiles in chromate-transformed BEAS2B cells. PloS ONE 6(3): e17982. Doi:10.1371/journal.pone.OOI7982. 

Ali et al. (2011) Aberrant DNA methylation of some tumor suppressor genes in lung 

cancers from workers with chromate exposure. Mol. Carcinogenesis 50(2):88-99. 

Salnikow MOA. Genotoxic effect of hexavalent chromium. 

The draft of the Toxicological review provides a substantial body of information 

regarding the mutagenic potential of hexavalent chromium and concludes that hexavalent 

chromium is carcinogenic by a mutagenic MOA. Indeed this topic has been studied 

extensively. The results of in vitro and in vivo studies provide substantial evidence for the 

mutagenic activity of hexavalent chromium, which is mediated through the generation of 

the highly reactive chromium intermediates penta- and tetravalent chromium, reactive 

oxygen species, and trivalent chromium formed during the intracellular reduction of 
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hexavalent chromium. These chromium and oxygen species can react with DNA, leading 

to oxidative DNA damage, chromium-DNA adducts, DNA strand breaks, and 

chromosomal aberrations. Despite these studies, the significance of chromium-induced 

DNA damage in the mechanisms of chromium carcinogenicity is not clear. If DNA 

damage/mutations are important and a causative factor in chromium-induced 

carcinogenesis, then these mutations should be frequent in chromium-induced tumors. 

However, very few publications exist in this respect. In animal studies, De Flora et al. (De 

Flora et al., 2008) found no evidence of DNA-protein crosslinks and DNA adducts in the 

duodenum following drinking water chromium exposures. Other available hexavalent 

chromium drinking water exposure studies that measured mutagenicity in mice also could 

not detect evidence of micronucleus induction in the blood or bone marrow (Mirsalis et 

al., 1996; De Flora et al., 2006; De Flora et al., 2008). Analysis of lung cancers from 

chromate-exposed workers revealed that p53 mutations are not very frequent, with only 

six missense mutations identified in 4 (20%) of the 20 chromate lung cancer samples 

(Kondo et al., 1997). There were fewer mutations in the patients with lung cancers who 

had been exposed to chromate than in those who had not (20% vs about 50%). This study 

also revealed that there was no association between p53 mutations and the period spent 

working in chromate factories. It is conceivable that chromium causes genotoxic effects, 

damage DNA, but this damage is efficiently repaired and do not play any role in 

carcinogenic effects of chromium. 

Thus, in order to confirm or refute a possible role of chromium genotoxic/mutagenic 

effects in chromium-induced carcinogenesis, comprehensive analyses of mutations in 

oncogenes and tumor suppressor genes in experimentally induced tumors in animals 

should be done first, followed by more detailed sequence analyses of chromium-exposed 

human tumors. A feasible study that could be done in a short period of time (assuming 

that tumor samples collected in the NTP studies are stored and frozen) is exon only global 

sequencing of DNA from chromium-induced rat tumors versus spontaneous tumors 

(several spontaneous tumors of different origin were observed in NTP 2008 study). These 

studies will allow comparison of tumor driving mutations versus passenger mutations. 

MOA, Epigenetic effects of hexavalent chromium. 

Global changes in the epigenetic landscape are a hallmark of cancer. The initiation and 

progression of cancer, traditionally seen as a genetic disease, is now realized to involve 

epigenetic abnormalities along with genetic alterations. Recent advancements in the 

rapidly evolving field of cancer epigenetics have shown extensive reprogramming of 

every component of the epigenetic machinery in cancer including DNA methylation, 

histone modifications, nucleosome positioning and non-coding RNAs, specifically 

microRNA expression (Sharma et al., 2010). Epigenetic effects have also been observed 

following hexavalent chromium exposure (Salnikow and Zhitkovich, 2008; Arita and 

Costa, 2009). Increased DNA methylation was observed in the promoter region of the 

tumor suppressor gene pl6 and the MMR gene hMLHI, indicating that chromium can 

induce epigenetic effects (Takahashi et at., 2005; Kondo eta!., 2006). Gene transcription 

has also been shown to be affected by exposure to hexavalent chromium in vitro via 

epigenetic mechanisms. Sun et al. (Sun et aL, 2009) found alterations in the levels of 

histone methylation in human lung A549 cells exposed to hexavalent chromium, 

indicating the capability of these exposures to lead directly to chanoes in gene expression. 
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Taken together, these studies suggest that epigenetic mechanisms may contribute to the 

carcinogenicity of hexavalent chromium. However, it is not clear whether epigenetic 

changes produced by chromium exposure are acting alone or linked to chromium 

genotoxic effects and that both genetic and epigenetic changes are essential for tumor 

appearance and evolution. More research is needed in this area including the identification 

of changes in DNA methylation (analyses of frequency of inherited silencing of tumor 

suppressors in tumors and in miRNA expression patterns following chromium exposure in 

animal models and humans before any conclusions can be drawn regarding the role of 

epigenetics in the carcinogenic effects of hexavalent chromium. 

MOA, Co-carcinogenic effects of chromium. 

Co-carcinogenic effects of hexavalent chromium were reviewed recently (Salnikow and 

Zhitkovich, 2008). The majority of occupational and probably all environmental 

exposures to hexavalent chromium occur as co-exposures with other carcinogens. The 

most common examples of co-exposures occur among stainless steel welders, and among 

hexavalent chromium-exposed workers who are also smokers. Two reports from the Costa 

Lab (Davidson et al., 2004; Uddin et al., 2007) provided strong experimental data 

demonstrating that hexavalent chromium can act as a potent co-carcinogen for UVinduced skin tumors. In both studies, the presence of hexavalent chromium in drinking 

water caused dose-dependent increases in the frequency of skin tumors in UV-irradiated 

hairless mice. Hexavalent chromium alone produced no tumors, indicating that it acted a 

strong enhancer of UV-initiated tumorigenesis. Supplementation with vitamin E or 

selenomethionine had no effect on hexavalent chromium-mediated enhancement of skin 

carcinogenesis suggesting that co-carcinogenic effects were not oxidant-mediated. It is 

noteworthy that the level of chromium in skin directly exposed to UV had significantly 

higher levels of chromium than underbelly skin that was not directly exposed to UV in 

mice exposed to UV and 5 ppm K2Cr04 (P < 0.05) (Davidson et al., 2004). This raises an 

interesting question, does inflammation, whatever the source, facilitate chromium 

accumulation or delay chromium clearance? This is an important and understudied area. 

The identified co-carcinogenic effects of hexavalent chromium raise an intriguing 

possibility that much lower doses of chromium could be hazardous under certain 

circumstances when exposure to chromium in drinking water is combined with other 

harmful exposures. 

Another important area of research is an understanding of the role of Inflammation/colitis 

in hexavalent chromium carcinogenesis. It is well know that at least 20% of all cancers 

arise in association with infection and chronic inflammation and even those cancers that 

do not develop as a consequence of chronic inflammation, exhibit extensive inflammatory 

infiltrates with high levels of cytokine expression in the tumor microenvironment. 

Aberrant activation ofNF-KB and/or STAT3 is found in over 50% of all cancers and 

renders premalignant and fully transformed cells resistant to apoptosis and speeds up their 

rate of proliferation, thereby increasing tumor growth. It is extremely important to test 

whether hexavalent chromium will be more carcinogenic and at lower doses in animals in 

which colitis was induced, for example by sodium dextran sulfate. 

MOA. Interference with iron metabolism. 

In rats neoplastic changes were found at sites of tissue contacts with the highest 

concentrations of hexavalent chromium, i.e. oral cavity. This may be explained by the 
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immediate damaging effects of chromium on DNA and other cellular components. At the 

same time frequent nonneoplastic changes were observed in duodenum of male and 

female rats and neoplastic changes were observed in duodenum of male and female mice. 

These data cannot be explained by the direct effect of hexavalent chromium, which should 

be mostly reduced by the time it reaches small intestine. Considering that in other organs 

such as liver and kidney which accumulate significant amount of chromium no tumors 

were observed, it is important to do more research on the mechanism of tumor 

development in small intestine. Specifically, duodenum is a place where iron absorption 

takes place. An analysis of ferroreductase expression and iron metabolism will help to 

shed light on whether an alteration in iron metabolism in this tissue may be responsible 

for chromium carcinogenic effects. 

Wise This document is supposed to be limited to an oral drinking water exposure. lt is 

inappropriate to extend any finding to "all routes of exposure" in this document and such 

evidence has not been considered or presented for dermal or inhalation routes. 

In its defense of a mutagenic mode of action, the Toxicological Review states on page 213 

that: 

"In addition to the evidence supporting a mutagenic mode of action in test animals, 

alternative or additional hypothesized modes of action for hexavalent chromium 

carcinogenicity have not been demonstrated." 

There are three concerns with this statement. First, it seems to imply that other modes of 

action need to be "demonstrated" not simply supported. The frank reality is that no mode 

of action for Cr(Vl) has been demonstrated, even the mutagenic mode of action is only 

supported and not demonstrated. There should not be a double standard here where the 

mutagenic mode of action needs to only be supported, while other modes must be 

demonstrated. The word "demonstrated" should be changed to "supported" to be 

consistent with the beginning of the passage. 

The second concern is that the statement is inaccurate. We supported an alternative mode 

of action to the induction of mutations in our paper that is cited in the Toxicological 

Review. Specifically, in Holmes, A.; Wise, SS; Wise, Sr., JP (2008) Carcinogenicity of 

hexavalent chromium. Indian J Med Res 128:353-372, we argue that the mechanism for 

Cr(Vl) does not involve mutations in the primary sequence of the DNA as it is a weak 

mutagen. Instead, we argue for a genotoxic mechanism leading not to mutations but 

changes in chromosome number and structure. This point of view is not considered much 

in the Toxicological Review, but is well-supported in the review and does offer an 

alternative mode of action that should have been discussed. It is as well-supported as the 

mutagenic mode of action and so it is inaccurate to state other views have not been 

demonstrated to the extent a mutagenic mode of action has been. 

The third concern with the statement is that it seems to imply that the approach taken in 

determining the mode of action was to consider those possibilities suggested in reviews of 

the literature. Therefore, because there is no review article synthesizing the literature to 

suggest a mode of action, there are no other modes of actions to consider. A better 

approach would have been to consider the primary literature and consider some possible 

modes of action that emerge from the data, but that have not yet emerged as a review article. 
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There are other modes of action that emerge and two possibilities are presented below. 

There are data to support these modes that should be synthesized, evaluated and considered. 

The mutagenic mode of action as the primary mode of action is not sufficiently 

scientifically supported or described in the Toxicological Review. Many concerns in the 

presentation with respect to proper citation of results, bias against cell culture studies, and 

an incomplete consideration of the primary literature are discussed above. The only 

approach taken was to consider all of these lesions in bulk as simply all representative of 

mutagenic events and not consider the possible confounding factors for each that may 

indicate they are not mutagenic events. 

A more careful consideration of the primary literature, considering each endpoint on its 

own merit could argue against a mutagenic mode of action that involves changes to the 

primary sequence of the DNA strand resulting in mutations. Cr(VI)-induced human 

tumors rarely contain such mutations and Cr(VI)-induced mutations are most often 

generated in experimental systems when one artificially forces them to occur by using 

extraordinarily high doses or systems with compromised repair and cell death pathways or 

by non-physiological exposure routes. It is unlikely that Cr(VI) is a mutagen at low doses. 

The most consistent outcome in the primary literature appears to be impacts on metaphase 

chromosomes. These outcomes occur at relatively low doses, in intact healthy human cells 

and across species in cell culture, whole animal and human worker studies. The question 

remains and this document does not address the underlying mechanism for this outcome. 

Induction of aneuploidy is another promising mode of action. 

One mode of action could involve direct damage to the DNA strand resulting in an 

alteration in chromosome structure or number. This mode would have key events that 

include: I) Uptake of Cr(Vl), 2) intracellular reduction of Cr(Vl), 3) interaction of 

reductant products with DNA strands, 4) production of chromosomal changes, 5) escape 

of DNA repair and apoptosis and 6) expansion of damaged cells. 

Alternatively, the mode of action might not involve direct damage to the DNA. Instead, it 

could involve direct interactions with the mitotic spindle apparatus and be more of an 

epigenetic event. This mode would have key events that include: I) Uptake of Cr(VI), 2) 

intracellular reduction of Cr(VI), 3) accumulation of intracellular reductant products, 4) 

interaction of reductant products with mitotic spindle apparatus, perhaps binding to the 

centrosomes, 5) production of chromosomal changes, 6) bypass of the spindle assembly 

checkpoint, 7) escape of apoptosis and 8) expansion of damaged cells. 

Zhitkovich Mutagenic mode of action: Hexavalent chromium was overwhelmingly positive for 

genotoxicity in a large variety of cells and organisms. It was also consistently mutagenic 

in bacterial and mammalian test systems. The mutagenicity and genotoxicity of Cr-6 

result from a direct DNA-damaging mechanism, as evidenced by the induction of 

mutagenic chromium-DNA adducts and other forms of DNA damage. Formation of 

chromium-specific DNA lesions at environmentally relevant Cr-6 concentrations and 

sensitivity of genotoxic responses to manipulations of cellular DNA repair further support 

the role of direct DNA damage as a primary cause of genotoxicity. Since Cr-6 is taken up 

via ubiquitously expressed sulfate transporters and its metabolism in cells occur via 

ubiquitously present ascorbate, glutathione and cysteine, there is no reason to believe that 
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the formation of DNA damage in the small intestinal cells and in more extensively studied 

cell types would be significantly different. Thus, diverse lines of evidence are fully 

consistent with a mutagenic mode of carcinogenic action for hexavalent chromium. The 

Draft clearly presented the main arguments for this designation. However, as pointed out 

above, Tables 4-22 and 4-23 need to be supplemented with information on Cr-DNA 

adducts. 

Supporting the importance of Cr-DNA adducts in chromate tumorigenicity are findings 

from the MOA study by the ACC in which levels of adducts were dramatically higher in 

the duodenum and jejunum of mice vs. rats. This result mirrors species differences in the 

intestinal carcinogenesis by chromate and it could not be explained by differences in 

tissue accumulation of chromium. 

In contrast to clear positive mutagenicity and genotoxicity data from in vivo studies and 

ascorbate-restored mammalian cell cultures, aneuploidy and epigenetic responses have not 

yet been tested in animal models and so far have been observed only in ascorbatedeficient cells. In fact, Sun et el. (2009) have found that the induction of epigenetic 

changes by chromate in human cultured cells occurs only under ascorbate-depleted 

conditions. 

The measurements of mutations in KRAS and p53 genes as part of the MOA study 

sponsored by the American Chemistry Council would not necessarily provide a clear 

answer about the mutagenic mode of action. A short 3-months duration of this study vs. 2 

years for the NTP bioassay certainly diminishes its ability to detect mutations. Among the 

proposed mutation readouts, three KRAS codons represent a very small and consequently, 

insensitive mutagenic target. This gene was only rarely mutated in chromate-associated 

human lung cancers (Ewis et al. 2001). The p53 gene is also uncommonly mutated in 

cancers among chromate workers (Kondo et al. 1 997). The presence or absence of KRAS 

or p53 mutations do not serve as a strong test for the validity of the mutagenic mode of 

carcinogenic action, as the frequency of cells with mutated KRAS or p53 can increase 

through selection of the pre-existing mutant clones whereas transformation process can 

result from mutagenic events in other components of KRAS and p53 pathways. For 

example, a large wave of early thyroid cancers among Chernobyl radioiodine-exposed 

children was caused by translocations in growth factor receptors with almost no RAS and 

p53 mutations (Nikiforov et al. 1996, Suchy et a!. 1998, Williams 2002). 

Potential alternative modes of action: Two lines of in vivo evidence have been presented 

to point to a potentially nonmutagenic mode of carcinogenic action. One is based on the 

drinking water study by DeFlora et al. 2008, which found negative results for DNA 

damage in the duodenum of mice. However, as discussed in detail above, this study used 

assays that were insensitive for detection of DNA damage by the employed doses of 

Cr(VI)in drinking water. Therefore, the negative results of this work were expected and 

therefore, uninformative. 

The other observation leading to the discussion of nonmutagenic or indirectly mutagenic 

mechanisms of carcinogenicity was the presence of diffuse epithelial hyperplasia in the 

NTP bioassay. Although the NTP study has not found significant necrosis in the small 

intestine of exposed mice, it is quite possible that the observed hyperplasia was a typical 

manifestation of regenerative responses. A combination of increased proliferation and 
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inflammation could be presented as an alternative mechanism for indirect induction of 

mutations due to higher rates of cell division and by reactive oxygen species released by 

the recruited inflammatory cells. This carcinogenic pathway would exhibit a strongly 

sublinear, threshold-type dose dependence, as it relies on the induction of cell death and 

small doses would not kill cells. Inflammatory events could also be linked to cell death of 

chromium-damaged cells, which release pro-inflammatory molecules. The extent of 

hyperproliferation in chromium-exposed groups was modest, and considering the overall 

very high rate of cell division in the small intestine, it is hard to see how somewhat faster 

replication would provide dramatically more spontaneous mutations required for cancer 

development. At best, the cytotoxicity-induced compensatory proliferation mechanism 

and the mutagenic mode should co-exist at high tumorigenic doses. 

The results from the MOA study sponsored by the ACC argue against significant 

inflammatory responses in the duodenum of chromate-exposed mice, as no increases in 

the levels of 8-oxodG and in the panel of 22 cytokines have been observed. A statistically 

significant drop in the ratio of GSH/GSSG was small in its magnitude, further 

demonstrating that tumorigenic doses were not associated with the state of strongly 

elevated oxidative stress and inflammation. 

The presence of chromium-induced hyperplasia could also be viewed as a manifestation 

of cancer-protective responses by the small intestine. Elimination of genetically damaged 

cells by apoptosis or another form of cell death is a firmly established protective 

mechanism against cancer. Thus, there are two opposing interpretations for the 

toxicological significance of the observed hyperplasia: one is pro-tumorigenic and another 

is anti-tumorigenic. The supralinear shape of dose-tumor incidence responses in the NTP2008 studies for female mice is consistent with the engagement of cancer-protective 

mechanisms. Tumor incidence vs. dose in male mice visually displayed a linear dosedependence (as shown in Stern 2010). Thus, a hypothetical cytotoxicity-based mechanism 

with the expected dose-response sublinearity is contradicted by the available evidence. 

Zhu A mutagenic mode of action was proposed as the primary mode of action. On the one 

hand, EPA discussed data gap and uncertainties about the mutagenic MOA and other 

possible MOAs. On the other hand, EPA defended the mutagenic MOA despite the lack of 

data evidence. For example, the only animal study that investigated target tissue 

genotoxicity (De Flora et al. 2008) reported negative results for DNA-protein crosslinks 

and DNA adducts in forestomach, glandular stomach, and duodenum of mice exposed to 

hexavalent chromium in drinking water for 9 month. EPA dismisses the negative finding 

on the basis of a shorter duration of the study compared with the 2-year NTP study. 
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B3. A two-year drinking water study in rats and mice (NTP, 2008) was selected for the 

derivation of an oral slope factor. Please comment on whether the selection of this study 

for quantification is scientifically supported and clearly described. Please identify and 

provide the rationale for any other studies that should be considered. 

Reviewer Comments 

Byczkowski Apparently, the two-year drinking water study by NTP (2008) is the only, up to date, 

appropriate cancer bioassay of Cr"'" by oral route (this document states - C.f. P. 224, L# 

24: " ... No other adequate studies of hexavalent chromium carcinogenicity by ingestion are 

available ... ") 

Hamilton There are no significant concerns about selection of this study, it is clearly the best 

available study for this type of risk assessment, with the caveats about how these data are 

interpreted and modeled as discussed elsewhere. 

Nordberg In this document it is not explained to the reader what decided the selection of chromium 

species and selection of doses and how the chosen doses relate to exposure in drinking 

water in the general population. Lethal doses should also be given for the chromium 

species that have been used in the quoted studies. 

To evaluate and compare the outcome of studies and concentration levels in tissues, a 

ratio of concentration in dry weight to concentration in wet weight would make it possible 

and easier to compare reported results and also of possible intake of hexavalent chromium 

in drinking water. It is told that the animals by increasing exposure to hexavalent 

chromium in drinking water showed a decrease in intake of drinking water. Influence on 

different tissues will be found in doing this. On page 112 NTP 2008 decreased body 

weights could be explained by reduced drinking water consumption. 

Patierno See previous comments above. The NTP study is the best study available but the 

interpretation of the data and conclusions drawn from it are incorrect. Important 

supplementary data is preliminarily discussed in the Public Comments. 

Rossman This seems to be the only choice. 

Salnikow Outside of my area of expertise. 

Wise This study is the proper study based on the available data. The study is flawed because 

only very high doses were considered in the study, thus, there is concern that it may not 

reflect events at lower doses. The EPA is in the unique position that a study that repeats 

the one above and extends it to lower doses is almost completed. The EPA should wait for 

the final results of that study to make the most informed analysis. 

Zhitkovicb The selection of a two-year drinking water study in rats and mice by the NTP (2008) for 

the calculation of an oral slope factor is appropriate. The NTP study was well designed 

and well executed. No other multiple-dose chronic oral carcinogenicity study in animals is 
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available and the dose-dependence from the single ecological study linking chromium-6 

in drinking water to human stomach cancers cannot be reliably estimated. 

Zhu The selection of the NTP's 2-year drinking water study in rats and mice (NTP, 2008) is 

justified. Reasons for why existing epidemiological data were not used for estimating 

cancer slope factor are acceptable. 

84. The incidence of adenomas and carcinomas combined in the small intestine of male mice 

from the NTP (2008) two-year drinking water study were selected to serve as the basis 

for the quantitative cancer assessment. Please comment on whether this selection is 

scientifically supported and clearly described. Please identify and provide the rationale 

for any other endpoints that should be selected to serve as the basis for the quantitative 

cancer assessment. 

Reviewer Conunents 

Byczkowski Even though in the NTP (2008) bioassays, only the exposures to the two highest doses of 

Cr" in mice and to the highest one dose in rats produced statistically significant 

carcinogenicity, the selection of this study for derivation of cancer slopes seems to be 

justified by the description provided in the document. 

Regarding the (common) practice of combining adenomas and carcinomas in modeling 

cancer risk, an explanation should be provided that the adenoma is a benign tumor of 

epithelial tissue with the tendency to become malignant, thus it may - or may not - lead to 

cancer. 

Also, the results of NTP (2008) cancer bioassay could be interpreted with the assumption 

of a threshold. 

Hamilton There is concern about selection of these endpoints to represent cancer risk in these 

animals as the basis for a human risk assessment. The high doses required to induce these 

lesions are well above a threshold level that would be of concern in humans under normal 

exposure scenarios, and these almost certainly represent a scenario where natural 

reductive defense mechanisms were overwhelmed by the doses of chromium used. Takeu 

together with the more recent 90-day ACC sponsored studies, the MOA for chromium(VI) 

is most likely a non-mutagenic one involving tissue damage and reproliferation, and 

would only be seen at doses that are unlikely to ever occur in a human exposure setting, 

particularly via drinking water. Thus, a threshold based risk assessment is most 

appropriate, similar to the treatment used for the non-cancer endpoints which are likely to 

be directly related to the cancer MOA. 

Nordberg See comments above. It should be noted that pH is different in different parts of the 

gastrointestinal system. 
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Responses to Charge Questions 

Patierno See previous comments above. The NTP study is the best study available but the 

interpretation of the data and conclusions drawn from it are incorrect. Important 

supplementary data preliminarily discussed in the Public Comments. 

Rossman This seems to be the only choice. 

Salnikow It seems that combining the incidence of adenomas and carcinomas in small intestine was 

the proper choice for modeling cancer risk. This is supported by the available data and 

clearly described. The fact that only highest doses produced a carcinogenic effect may 

indicate high reducing capacity in tested model systems (wild type mice and rats). As 

suggested in A2 exposing Gulo-/- mice or rats to hexavalent chromium may result in 

tumor appearance at lower doses. If this will be the case the extrapolation to 

environmentally relevant doses of chromium exposure will be more feasible. 

Wise If one is going to rely on this NTP study, the selection of the incidence of adenomas and 

carcinomas in the small intestine of male mice from the NTP (2008) two-year drinking 

water study to serve as the basis for the quantitative cancer assessment is appropriate. 

However, scientifically these lesions are not the same and are not necessarily linked. 

Thus, one or the other should be used. 

Zhitkovich The choice of the combined incidence of adenomas and carcinomas in the small intestine 

of male mice from the NTP-2008 study for the quantitative cancer assessment was based 

on a better fit of the multistage model for the male mouse data than for the female mouse 

data. However, it was unclear why a combination of male and female mouse data sets was 

not used. 

Zhu For dose-response assessment EPA considered the incidences of adenoma and carcinoma 

combined in the small intestine of male and female B6C3F mice (Tables 5.3 and 5.4), in 

the oral cavity (mucosa and tongue) (Tables 5.3 and 5.4) in rats (NTP 2008). (Note the 

denominators that determine the tumor incidence in small intestine are not consistent with 

those in Table 4.19). EPA did not consider the incidence of other neoplasm because the 

incidence is not dose-dependent. 

The incidence of adenoma or carcinoma in the oral cavity in both male and female rats 

elevated only at the highest dose, but not at the three lower doses (up to 2.1 and 2.4 

mg/kg-d for male and female respectively). To fit a dose-response model to these 

incidence data that exhibited hockey-sticker shape of dose-response requires a nonlinear 

(curve-linear) functional form or even a threshold model. Such curve-linear pattern seems 

inconsistent with the hypothesized genotoxic MOA. The Jack of dose-response in these 

two endpoints was cited as reason for not advancing these two endpoints for final doseresponse analysis. Better justification is needed. 
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Responses to Charge Questions 

85. The oral slope factor was calculated by linear extrapolation from the POD (i.e., the lower 

95% confidence limit on the dose associated with 10% extra risk of tumors of the small 

intestine in male mice). Has the modeling been appropriately conducted and clearly 

described? 

Reviewer Comments 

Byczkowski The oral cancer slope factor was estimated in accordance with the U.S. EPA (2005) 

guidelines, but to this reviewer tbe linear cancer extrapolation seems inconsistent with the 

prior noncancer RID modeling. While the early preneoplastic epithelial hyperplastic 

lesions have been modeled as a threshold phenomenon in derivation of RID, the resultant 

neoplastic lesions were modeled as a no-threshold linear phenomenon in derivation of 

cancer slope factors. There seems to be some contradiction, as a threshold-bearing 

precursor cannot result in the no-threshold adverse effect. 

In addition, tbe allometric scaling of cancer slopes, extrapolated from animals to humans 

based on body weight (to the 3/4 power), seems to be inconsistent with the portal of entry 

site of action in the GI tract. Typically, this is the local concentration of a chemical at the 

target site/target tissue that drives an adverse response. Therefore, more appropriate could 

be a physiologically based pharmacokinetic (PBPK) scaling based on animal-to-human 

ratio of differences between the rate of absorption and the rate of reduction of Cr.,. to cr•' 

in gastrointestinal tract contents, as well as the rate of loss of Cr.,. from intestinal tract 

contents to the feces. 

It seems that appropriately developed PBPK model which recognizes physiological 

specificity of the different segments of gastrointestinal tract in animals and humans, as 

presented at the workshop by Summit Toxicology, LLP., may be critical in understanding 

the mode of action of orally administered Cr +6 and in quantitative evaluation of its effects. 

Hamilton As discussed in detail under G 1. and 02. responses, this reviewer has considerable 

concerns about tbe use of a linear low-dose extrapolation model for assessing chromium 

cancer risk. The evidence, when objectively assessed, strongly argues for threshold 

mechanisms botb in the gut and systemically, and there is little or no evidence that 

chromium reaches the systemic circulation as chromium(Vl) under exposure scenarios of 

relevance to human exposures. There is no better candidate for departure from these 

default EPA assumptions than chromium if EPA is serious about evidence-based risk 

assessment. Many of the public comments that were available to the reviewers just before 

and after our May 2011 meeting also raise these issues, and the EPA should, in particular, 

wait until the recently reported 90-day MOA and PK studies are published and available 

to tbem, at which point they should give serious consideration to how these new data 

inform the likely MOA. Given that most toxicology profiles are only revised every 10-15 

years, it is worth waiting for these studies, and taking them as well as the external 

reviewer comments in mind toward a revised document that will be more accurate and 

will better stand the test of time. The EPA might also consider asking for a National 

Research Council Special Emphasis Panel to review all these materials and make a 

recommendation to EPA regarding chromium(Vl) as has been done for other several other 

key toxicants of concern. In any event the current draft's risk assessment treatment of 

chromium(VI) is highly flawed and grossly mischaracterizes the likely risk of human 
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Responses to Charge Questions 

health effects of chromium(VI) in drinking water based on a careful and thorough 

assessment of all the available evidence. 

Nordberg The model is clearly described. However I feel very uneasy about extrapolation to lower 

exposure levels because of chosen exposure doses where LOAEL is identical to the lowest 

exposure dose administered. 

Other organizations like IARC do not perform any quantitative evaluation of carcinogenic 

agents/substances. Threshold concentrations are problematic because of lack of 

knowledge of how the carcinogenicity develops. Once an organism has been exposed to a 

substance that can give rise to cancer there is a possibility to such an effect to occur. 

Patierno See previous comments above. The linear extrapolation from the POD is not appropriate. 

CrVI toxicity and carcinogenicity demonstrates distinct non-linearity and there is little or 

no relation between what is observed at the highest doses in the NTP study and any 

physiologically-appropriate or environmentally-relevant exposure. 

Rossman This is outside my field of expertise. 

Salnikow Outside of my area of expertise. 

Wise The modeling has been appropriately conducted and clearly described. More methods are 

needed to make it clearer. It is possible that Cr(VI) acts via a threshold. The EPA is in the 

unique position that a study that repeats the NTP study and extends it to lower doses is 

almost completed. This study will help clarify if there is a threshold. The EPA should wait 

for the final results of that study to make the most informed analysis. 

Zhitkovich The calculation of the oral slope factor from the POD was appropriately performed. As 

per US-EPA 2005 Guidelines for Carcinogen Risk Assessment, a linear extrapolation to 

low doses was used based on the selection of the mutagenic mode of carcinogenic action 

for chromium-6. 

The ability of ingested chromium-6 to cause adverse effects at both environmentally 

relevant and much higher doses has been questioned. given the reported high chromate 

reducing capacity of the gastric juice and a limited systemic penetration of chromium after 

oral exposure (as extensively reviewed by the Draft). These considerations led to the 

formulation of the threshold model of chromium-6 carcinogenesis, which postulates that 

only doses that exceed the reducing capacity of the tissue (stomach for ingestion 

exposures) would be carcinogenic (DeFlora 2000). This model would argue that despite 

the selection of a mutagenic mode of action with the resulting recommendation for default 

linear extrapolation, the complete detoxification of low-to-moderate chromium-6 doses in 

the stomach makes it inappropriate to perform linear extrapolation from the POD. 

The ability of gastric juices to reduce/detoxify chromium-6 is generally accepted in the 

field; however, studies with human volunteers and other considerations argue against the 

completeness of the detoxification process. For example, the bioavailability for Cr(Vl) 

was -1 0-times higher than for Cr(Vl) reduced with orange juice prior to ingestion 
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(Kuykendall et al. 1996). The extent of chromium-6 reduction in the stomach is 

influenced by three factors: its reduction capacity, reduction rate and stomach emptying 

time. Based on the reported high reduction capacity of the stomach (>80 mg!day, DeFlora 

et al. 1997), the rate of reduction by gastric juice under fasting conditions could exhibit 

pseudo-first order kinetics for a broad range of chromium-6 concentrations. This means 

that the percentage of reduced chromium-6 could be the same for both very small amounts 

and much larger amounts. Reduction of chromium-6 by artificial gastric juice has been 

found to follow first order reaction kinetics (Gammelgaard et al. 1999). Consistent with 

the first-order reaction kinetics, Donaldson and Barreras (1966) found that human subjects 

excreted in the 24-hr urine 2.1% of ingested 20 ng radioactive chromium-6 whereas 

Kerger et al. (1997) found that ingestion of 5 mg chromium-6 by human volunteers led to 

about 1.43% excretion during the first 24 hr (1.43% excretion is a conservative 114"' 

estimate from the average 4-day excretion value of 5. 7% ). Thus, the bioavailability of 20 

ng and 5 mg chromium-6 (250,000-fold range) looks quite similar. 

Donaldson and Barreras (1966) also performed a very important experiment on the 

bioavailability of 20 ng radioactive chromium-6 that was directly delivered into the 

duodenum of human subjects. In this case, they found that 10.6% of chromium was 

excreted in the urine. Since the duodenal delivery represent 100% nonreduced chromium6, then the amount of nonreduced chromium-6 in their oral route experiment can be 

estimated from the urinary excretion of 2.1% divided by 0.106 = 19.8%. For the study by 

Kerger et al (2007), the same type of calculations gives an estimate of 14.3% nonreduced 

chromium-6 reaching the duodenum. Gammelgaard et al. (1999) calculated a half-life of 

23 min for reduction ofO.lmg/L chromium-6 (current MCL for total chromium) by 

artificial gastric juice. After 1 hr, this reduction rate would leave 16.5% chromium-6. 

Zhu EPA carried out dose-response modeling and BMD estimation for the incidences of 

adenoma and carcinoma of small intestine in male and female mice separately. EPA stated 

that it relied on the multi-stage model because the model is preferred by the agency, but 

gave no justification or explanation. It went on to report an estimated slope of 0.09 

(mg!kg-day) and 0.10 (mg!kg-day) for male and female mice respectively. In section 5.3.4 

of the Review, EPA reported the CSFs derived on the basis of the cancer incidence of 

small intestine in male and female mice, and chooses that of male mice because of "the 

poor fit of the multistage model to the female mouse data". EPA did not provided 

adequate detail on the modeling efforts, or a discussion and justifications for its final 

selection (section 5.3.3 and 5.3.4). EPA did provide some detail in Appendix B2, which is 

essentially the direct output from running the BMDS software, but again no discussion or 

explanation of the output. It would be helpful and necessary that EPA substantially 

expand sections 5.3.3 and 5.3.4 to report in greater detail the modeling process, the issues 

encountered, and justify the decision and choice therein. 

On a more technical side, examination of Appendix B2 reveals that (1) no standard error 

is reported for the estimate of model coefficient, and (2) the coefficient of the second 

order in the polynomial was set to be zero, not estimated. The model did not fit the data at 

all despite a non-significant p-value for goodness-of-fit test. EPA did not give any 

explanation or discussion in this regard. The significance of a goodness-fit-test depends 

on sample size, and a non-significant result does not imply a correct model, especially 

within a range where there are no data. EPA should explore different options: trying 
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Responses to Charge Questions 

different models, considering omitting the highest dose, or considering combine male and 

female mice. 

Inclusion of multiple studies, multiple endpoints, multiple model choices, and various 

BMR levels for deriving a POD is increasingly desirable towards a more systematic and 

quantitative risk assessment paradigm. It will afford an opportunity to quantify the 

underlying uncertainties and variations associated with the choices and options made each 

many stages of the risk assessment process. Within the context of CSF for hexavalent 

chromium, EPA is in a position to conduct a more thorough and comprehensive 

assessmeut by including multiple endpoints, different model forms that allow for 

nonlinear dose-response, various BMR levels. The outcome will then demonstrate a range 

for POD and CSF to permit a better quantification and better understanding of 

uncertainties and variations. 
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Additional Reviewer Comments 
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Janusz Z Byczkowski, DABT, Ph.D. 

Additional Comments Submitted by Dr. Janusz Z. Byczkowski 

Typos and errors that should be corrected in the revised version: 

P. 24, L# 4: " ... direct measurement of residual Cr(VI) in the calorimetric reaction ... " 

Please change to: " ... colorimetric ... " 

P.26, L# 26: " ... Michaelis-Menten uptake kinetics ... " 

Please change to: " ... Michaelis-Menten-type uptake kinetics ... " 

P. 51, Figure 3-6, it would help the reader if non-standard abbreviations were explained in the figure legend -

e.g., DSB, SSB, etc. 

P. 52, L# 38: " ... to describe kinetics ofhumans ... " 

Please change to: " ... kinetics in humans ... " 

P. 84, L# 36: " ... colestasis, as other markers of colestasis ... " 

Please change to: " ... cholestasis ... " 

P. 229, L# 33: " ... (see Section 4.6.3.4) ... " 

Such a section does not exist in the reviewed document. 

P. 244, L# 7: " ... Bukowksi, JA. .. " 

Please change to: " ... Bukowski ... " 
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Konstantin Salnikow, Ph.D. 

Additional Comments Submitted by Dr. Konstantin Salnikow 

Errata: 

Regarding the form of the draft it should be noted that, although this is not a manuscript, it is necessary to 

correct errors and mistakes in the draft content. 

Below are several examples: 

I. K2Cr20 4 - does not exist (page 30, table 3-7; page 45), should it be K2Cr04• 

2. Table 2-1, page 6- Cr20, is chromium (Ill), not hexavalent chromium. BaCrO. is barium chromate, 

not barium oxide. 

3. Table 2-5, page 18- "accumulates Cr(V)", the intent of this statement is not clear because this form 

is short living and unlikely that is can accumulate. 

4. Page 35 and Table 3-9, KzCrO,- does not exist. 

5. None existing or wrong citations: 

Kumulainen, 1991 (page 7), should be Kumpulainen, 1992 (page 251 ). 

Salnikov and Zhitkovicb, 2009 (page 50); Salnikov and Zhitkovich, 2008 (page 257), should be 

Salnikow and Zhitkovich, 2008. 

Costa and Klein, 2004 (page 66); Costa, M.; Klein, C.B. 2008 (page 245), should be Costa and 

Klein, 2006. 

LeVina eta!., 2003, 2007, (page 50) should be Levina et al., 2003, 2007. 

Kasprzak, 1996 (page 189), no reference provided. 

Campbell J.L.; Tan,Y.; Clewell, H.J. (2009) Development of a PBPK model for hexavalent 

chromium in rats and mice to estimate exposure to oral mucosa and small intestine. Toxicologist 

108(1):98 (Abstract) Poster ID # 108. This is a questionable citation. 

Sun et a!. (2009), (page 188), no reference provided. 

Davies, JM. (1979) Lung cancer mortality of workers in chromate pigment manufacture: An 

epidemiological survey. J Oil Chern Assoc 62:157-163, (page 245), should be: J Oil Colour 

Chern Assoc 62:157-163. 

References: 

Arita, A., and Costa, M. (2009). Epigenetics in metal carcinogenesis: nickel, arsenic, chromium and 

cadmium. Metallomics 1, 222-228. 

Chung, T. L., Brena, R. M., Kolle, G., Grimmond, S.M., Berman, B. P., Laird, P. W., Pera, M. F., and 

Wolvetang, E. J. (2010). Vitamin C promotes widespread yet specific DNA demethylation of the 

epigenome in human embryonic stem cells. Stem Cells 28, 1848-1855. 
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Konstantin Salnikow, Ph.D. 

Dai, H., Liu, J., Malkas, L. H., Catalano, J., Alagharu, S., and Hickey, R. J. (2009). Chromium reduces the in 

vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome. Toxicol 

Appl Pharmaco/236, 154-165. 

Davidson, T., Kluz, T., Burns, F., Rossman, T., Zhang, Q., Uddin, A., Nadas, A., and Costa, M. (2004). 

Exposure to chromium (VI) in the drinking water increases susceptibility to UV -induced skin tumors in 

hairless mice. Toxicol Appl Pharmaco/196, 431-437. 

De Flora, S., D'Agostini, F., Balansky, R., Micale, R., Baluce, B., and Izzotti, A. (2008). Lack of genotoxic 

effects in hematopoietic and gastrointestinal cells of mice receiving chromium(VI) with the drinking 

water. Mutat Res 659, 60-67. 

De Flora, S., Iltcheva, M., and Balansky, R. M. (2006). Oral chromium(VI) does not affect the frequency of 

micronuclei in hematopoietic cells of adult mice and of transplacentally exposed fetuses. Mutat Res 

610, 38-47. 

Epstein, A. C., Gleadle, J. M., McNeill, L.A., Hewitson, K. S., O'Rourke, J., Mole, D. R., Mukherji, M., 

Metzen, E., Wilson, M. !., Dhanda, A., Tian, Y. M., Masson, N., Hamilton, D. L., Jaakkola, P., 

Barstead, R., Hodgkin, J., Maxwell, P. H., Pugh, C. W., Schofield, C. J., and Ratcliffe, P. J. (2001). C. 

elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl 

hydroxylation. Ce/1107, 43-54. 

Hewitson, K. S., McNeill, L.A., Riordan, M. V., Tian, Y. M., Bullock, A. N., Welford, R. W., Elkins, J. M., 

Oldham, N.J., Bhattacharya, S., Gleadle, J. M., Ratcliffe, P. J., Pugh, C. W., and Schofield, C. J. 

(2002). Hypoxia-inducible factor (HIP) asparagine hydroxylase is identical to factor inhibiting HIP 

(FIH) and is related to the cupin structural family. J Bioi Chern 277,26351-26355. 

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Effects of Ascorbic Acid on Carcinogenicity and Acute Toxicity of Nickel Subsulfide, and on Tumor 

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Kondo, K., Hino, N., Sasa, M., Kamarnura, Y., Sakiyama, S., Tsuyuguchi, M., Hashimoto, M., Uyama, T., 

and Monden, Y. ( 1997). Mutations of the p53 gene in human lung cancer from chromate-exposed 

workers. Biochem Biophys Res Commun 239, 95-100. 

Kondo, K., Takahashi, Y., Hirose, Y., Nagao, T., Tsuyuguchi, M., Hashimoto, M., Ochiai, A., Monden, Y., 

and Tangoku, A. (2006). The reduced expression and aberrant methylation of p 16(1NK4a) in chromate 

workers with lung cancer. Lung Cancer 53, 295-302. 

Lando, D., Peet, D. J., Gorman, J. J., Whelan, D. A., Whitelaw, M. L., and Bruick, R. K. (2002). FIH-1 is an 

asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. 

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Mirsalis, J. C., Hamilton, C. M., O'Loughlin, K. G., Paustenbach, D. J., Kerger, B. D., and Patierno, S. 

(1996). Chromium (VI) at plausible drinking water concentrations is not genotoxic in the in vivo bone 

marrow micronucleus or liver unscheduled DNA synthesis assays. Environ. Mol. Mutagen. 28, 60-63. 
262 


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Konstantin Salnikow, Ph.D. 

Mizushima, Y., Harauchi, T., Yoshizaki, T., and Makino, S. (1984). A rat mutant unable to synthesize 

vitamin C. Experientia 40, 359-361. 

Myllyharju, J. (2003). Prolyl4-hydroxylases, the key enzymes of collagen biosynthesis. Matrix Bioi. 22, 15-

24. 

O'Flaherty, E. J. (1996). A physiologically based model of chromium kinetics in the rat. Toxicol Appl 

Pharmaco/138, 54-64. 

O'Flaherty, E. J., Kerger, B. D., Hays, S.M., and Paustenbach, D. J. (2001). A physiologically based model 

for the ingestion of chromium(III) and chromium(VI) by humans. Toxicol. Sci. 60, 196-213. 

Onkelinx, C. (1977). Compartment analysis of metabolism of chromium(III) in rats of various ages. Am J 

Physiol 232, E478-484. 

Salnikow, K., and Kasprzak, K. S. (2005). Ascorbate depletion: a critical step in nickel carcinogenesis? 

Environ Health Perspect 113, 577-584. 

Salnikow, K., and Zhitkovich, A. (2008). Genetic and epigenetic mechanisms in metal carcinogenesis and 

cocarcinogenesis: nickel, arsenic, and chromium. Chem. Res. Toxicol. 21, 28-44. 

Sharma, S., Kelly, T. K., and Jones, P. A. (2010). Epigenetics in cancer. Carcinogenesis 31, 27-36. 

Shi, Y. (2007). Histone lysine demethylases: emerging roles in development, physiology and disease. Nat 

Rev Genet 8, 829-833. 

Snow, E. T. (1991). A possible role for chromium(Ill) in genotoxicity. Environ Health Perspect 92, 75-81. 

Sun, H., Zhou, X., Chen, H., Li, Q., and Costa, M. (2009). Modulation of histone methylation and MLHl 

gene silencing by hexavalent chromium. Toxicol Appl Pharmacal 237, 258-266. 

Takahashi, Y., Kondo, K., Hirose, T., Nakagawa, H., Tsuyuguchi, M., Hashimoto, M., Sano, T., Ochiai, A., 

and Monden, Y. (2005). Microsatellite instability and protein expression of the DNA mismatch repair 

gene, hMLH I, of lung cancer in chromate-exposed workers. Mol. Carcinog. 42, 150-158. 

Uddin, A. N., Burns, F. J., Rossman, T. G., Chen, H., Kluz, T., and Costa, M. (2007). Dietary chromium and 

nickel enhance UV-carcinogenesis in skin of hairless mice. Toxicol Appl Phannacol 221, 329-338. 

Zhitkovich, A. (2005). Importance of chromium-DNA adducts in mutagenicity and toxicity of 

chromium(VI). Chem. Res. Toxicol. 18, 3-11. 
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Anatoly Zhitkovich, Ph.D. 

Additional Comments Submitted by Dr. Anatoly Zhitkovich 

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Kuykendall JR, Kerger BD, Jarvi EJ, Corbett GE, Paustenbach DJ. Measurement of DNA-protein cross-links 

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1062-1070 (2003). 

Reynolds MF, Peterson-Roth EC, Bespalov IA, Johnston T, Gure1 VM, Menard HL, Zhitkovich A. Rapid 

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kidney ultrafiltrates. Carcinogenesis 12: 1733-1737 (1991 ). 

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Sun H, Zhou X, Chen H, Li Q, Costa M. Modulation of histone methylation and MLHI gene silencing by 

hexavalent chromium. Toxicol. Appl. Pharmacal. 237(3): 258-266 (2009). 
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Anatoly Zhitkovich, Ph.D. 

Tsapakos M. J., Hampton T. H. and Wetterhahn K. E. Chromium(VI)-induced DNA lesions and chromium 

distribution in rat kidney, liver, and lung. Cancer Res. 43: 5662-5667 (1983). 

Williams D. Cancer after nuclear fallout: lessons from the Chernobyl accident. Nat Rev Cancer. 2(7): 

543-549 (2002). 

Zecevic A., Hagan E., Reynolds M., Poage G., Johnston T. and Zhitkovich A. XPA impacts formation but 

not proteasome-sensitive repair of DNA-protein crosslinks induced by chromate. Mutagenesis 25: 

381-388 (20 10). 
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Appendix A: Individual Reviewer Comments 
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POST-MEETING COMMENTS SUBMITTED BY 

Janusz Z. Byczkowski, DABT, Ph.D. 
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Gl. 

Contract No. EP-C-07-024 

Task Order No. 92 

March 3, 2011 

Janusz Z Byczkowski, DABT, Ph.D. 

Toxicological Review of Hexavalent Chromium 

Post-meeting 

Answers to General Questions 

Logical - yes; clear - mostly; concise - no, as there is a very large body of information covered by this 

Toxicological Review. 

While the scientific evidence of both noncancer and cancer hazards of oral exposures to Cr'" has been 

appropriately reviewed and synthesized, the conclusions and numerical derivation of toxicity values are 

mostly based on strict (default) interpretation and literal application of U.S EPA guidelines, rather than on the 

current scientific understanding of the mode of action of Cr'". 

G2. 

It seems that up to date (around the year 2009), all important studies (and some unimportant too) have been 

already covered by this Toxicological Review. However, I strongly suggest that the results of study presented 

at the workshop by ToxStrategies, Inc., and especially the physiologically based pharmacokinetic (PBPK) 

modeling by Summit Toxicology, LLP., should be included in the revised Toxicological Review of 

Hexavalent Chromium document. 

Answers to Chemical-Specific Charge Questions: 

(A) Oral Reference Dose (RID) for Hexavalent Chromium 

Al. 

The two-year drinking water study by NTP (2008) seems to be the most comprehensive from all available 

chronic bioassays, and thus, it is suitable as the basis for derivation of the RID. 

However, there is still some concern regarding the selection of the NTP study for developing of RID (this 

document states- C. f. P. 85, L# 1: " ... Urinalysis showed dose-related decreased volume and increased 

specific gravity, consistent with decreased water intake. NTP (2007) suggested that decreased water intake 

was due to decreased palatability ... " and then, P.89. L# 34: " ... Drinking water consumption was reduced ... ") 

Thus, the reduced drinking water consumption, and consequently at least partial dehydration, may have 

increased the osmolality of gastrointestinal fluid, which could be a significant confounder in the chronic 
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Janusz Z. Byczkowski, DABT, Ph.D. 

toxicity study, even though (according to the quotation on P. 120) the Technical Report by NTP (2008) 

attempted to dismiss such a concern. 

A2. 

Diffuse hyperplasia, in itself, is considered to be a physiological (normal) response to several stimuli, as the 

cells of a hyperplastic growth remain sensitive to normal regulatory control mechanisms. Such a 

physiological proliferation of cells may be secondary to several pathological factors (e.g., the increased 

osmolality, changed pH, infection by Helicobacter, etc). Still, the proliferation in "diffuse hyperplasia" is a 

normal process although, it may be generated in response to abnormal condition (in contrast to neoplasia, 

where the proliferation in itself becomes abnormal). On tbe other hand, the hyperplasia is a common early 

preneoplastic response to potentially carcinogenic stimuli. 

Considering its rather ubiquitous and nonspecific character, it is remarkable tbat even no single case of 

epithelial hyperplasia was found during the two-year study by NTP (2008) in duodena of as many as 100 

male and female control mice. Taking this result at a face value, it may be assumed that the diffuse epithelial 

hyperplasia in duodena could be an early biomarker of oral exposure to Cr+6, at least in tbe two-year NTP 

(2008) bioassay study. 

However, another end point- the pathological changes in the liver were noted in the same study with 

significantly increased frequency at the lowest dose employed. Even though they may be considered 

somewhat less "specific" than the epithelial hyperplasia (because they were observed at low frequency also in 

controls), but perhaps they could be more relevant to the systemic toxicodynamics of cr+6 - appearing further 

away from tbe portal of entry then the hyperplasia and apparently being less sensitive to tbe potentially 

confounding effects of the reduced drinking water consumption. 

A3. 

The benchmark dose modeling was applied in accordance with the U.S. EPA Benchmark Dose 

Technical Guidance Document (2000) in the prescribed manner. However, the modeled BMD, derived from 

the diffuse epithelial hyperplasia, was supported only by the three dose-points (including zero dose). In 

contrast, the pathological changes in the liver were consistently fitted, for both female rats and mice with 

Log-logistic model which included all the five data points (including controls), and thus, they could be 

considered to be robust dose-response adverse effects for derivation of BMD. So, the pathological changes in 

the liver may be used as alternative end points for derivation of BMD. 

A4. 

While the uncertainty factors (UFs) were applied in accordance with the U.S. EPA guidelines, the use of the 

UF: 10 for extrapolating the toxicity value based on effects in the portal of entry of Cr+6 in animals to 
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Janusz Z. Byczkowski, DABT, Ph.D. 

predict GI effects in humans, seems to be problematic. Thus, both the reductive capacity of human 

gastrointestinal fluids and the antioxidant protection of human tissues exceed those in mice. So, adequately 

nourished and hydrated humans should be less vulnerable than mice to the adverse GI effects of oral 

exposure to Cr+6, rather then 10 times more sensitive, as the UA=lO may suggest. 

(B) Carcinogenicity of Hexavalent Chromium 

Bl. 

Even though the U.S. EPA (2005) guidelines were applied appropriately, there is no direct evidence of dosedependent carcinogenicity by orally administered Cr+<> in humans (C. f. P. 236, L# 33: " ... EPA concluded that 

the exposure-response analyses presented by Zhang and Li ( 1997), Beaumont et al. (2008), and Kerger et al. 

(2009) are not based on the quality of data that is needed to support a conclusion regarding the presence or 

absence of a dose-response among the observed cancer rates in these villages. The other epidemiologic 

studies did not find a significant correlation between hexavalent chromium concentrations in drinking water 

(or proximity to the source of hexavalent chromium soil contamination) and cancer ... ") 

The classification of hexavalent chromium as "likely to be carcinogenic to humans by the oral route of 

exposure" is based on carcinogeniesis observed only at the highest dose levels employed in animal studies. 

Therefore, it may, or may not be relevant to humans at environmentally relevant exposure concentrations. 

B2. 

The positive laboratory results of mutagenicity tests do not prove genotoxicity and are not necessarily 

biologically relevant to humans exposed in vivo to the environmentally relevant concentrations of Cr .... The 

mutagenicity results from humans occupationally exposed to Cr+6 (Table 4-25) were inconsistent (this 

document states- C. f. P. 178, L# 14: " .. .In general, associations between hexavalent chromium exposure and 

mutagenicity in workers are uncertain ... ") and actually, the results presented in the document did not prove 

the direct genotoxic mode of action of Cr+6 in vivo. 

An alternative, indirect mode of action seems plausible to this reviewer. As discussed in Section 4.5.2., the 

intracellular products of one-electron reduction of Cr+6 in vivo, causing oxidative stress and free-radical 

damage to cellular macromolecules, seems to be responsible for initiating carcinogenicity at relatively low 

dose levels. Erosion ofGI tract mucosa with inflammation that followed at high Cr+6 dosage (discussed in 

section 4.2.1) apparently caused promotion in the lesions, which eventually progressed to benign and 

malignant tumors. Importantly, such a mode of action implies a threshold phenomenon, i.a. due to 

antioxidant protection of cells. 
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Janusz Z. Byczkowski, DABT, Ph.D. 

B3. 

Apparently, the two-year drinking water study by NTP (2008) is the only, up to date, appropriate cancer 

bioassay of Cr+6 by oral route (this document states- C.f. P. 224, L# 24: " .. .No other adequate studies of 

hexavalent chromium carcinogenicity by ingestion are available ... ") 

B4. 

Even though in the NTP (2008) bioassays, only the exposures to the two highest doses of Cr+6 in mice and to 

the highest one dose in rats produced statistically significant carcinogenicity, the selection of this study for 

derivation of cancer slopes seems to be justified by the description provided in the document. 

Regarding the (common) practice of combining adenomas and carcinomas in modeling cancer risk, an 

explanation should be provided that the adenoma is a benign tumor of epithelial tissue with the tendency to 

become malignant, thus it may- or may not- lead to cancer. 

Also, the results of NTP (2008) cancer bioassay could be interpreted with the assumption of a threshold. 

BS. 

The oral cancer slope factor was estimated in accordance with the U.S. EPA (2005) guidelines, but to this 

reviewer the linear cancer extrapolation seems inconsistent with the prior noncancer RID modeling. While 

the early preneoplastic epithelial hyperplastic lesions have been modeled as a threshold phenomenon in 

derivation of RID, the resultant neoplastic lesions were modeled as a no-threshold linear phenomenon in 

derivation of cancer slope factors. There seems to be some contradiction, as a threshold-bearing precursor 

cannot result in the no-threshold adverse effect. 

In addition, the allometric scaling of cancer slopes, extrapolated from animals to humans based on body 

weight (to the 3/4 pcwer), seems to be inconsistent with the portal of entry site of action in the GI tract. 

Typically, this is the local concentration of a chemical at the target site/target tissue that drives an adverse 

response. Therefore, more appropriate could be a physiologically based pharmacokinetic (PBPK) scaling 

based on animal-to-human ratio of differences between the rate of absorption and the rate of reduction of cr•6 

to cr+3 in gastrointestinal tract contents, as well as the rate of loss of cr+6 from intestinal tract contents to the 

feces. 

It seems that appropriately developed PBPK model which recognizes physiological specificity of the 

different segments of gastrointestinal tract in animals and humans, as presented at the workshop by Summit 

Toxicology, LLP., may be critical in understanding the mode of action of orally administered Cr+6 and in 

quantitative evaluation of its effects. 
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Janusz Z. Byczkowski, DABT, Ph.D. 

Additional Comments: 

Typos and errors that should be corrected in the revised version: 

P. 24, L# 4: " ... direct measurement of residual Cr(VI) in the calorimetric reaction ... " 

Please change to: " ... colorimetric ... " 

P.26, L# 26: " ... Michaelis-Menten uptake kinetics ... " 

Please change to: " ... Michaelis-Menten-type uptake kinetics ... " 

P. 51, Figure 3-6, it would help the reader if non-standard abbreviations were explained in the figure legend -

e.g., DSB, SSB, etc. 

P. 52, L# 38: " ... to describe kinetics <if humans ... " 

Please change to: " ... kinetics in humans ... " 

P. 84, L# 36: " ... colestasis, as other markers of colestasis ... " 

Please change to: " ... cholestasis ... " 

P. 229, L# 33: " ... (see Section 4.6.3.4) ... " 

Such a section does not exist in the reviewed document. 

P. 244, L# 7: " ... Bukowksi, JA ... " 

Please change to: " ... Bukowski ... " 
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POST-MEETING COMMENTS SUBMITTED BY 

Joshua W. Hamilton, Ph.D. 
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Joshua W. Hamilton, Ph.D. 

Post-Meeting Reviewer Comments on 

EPA Draft Toxicological Review of Hexavalent Chromium 

(dated September 2010) 

Joshua W. Hamilton Ph.D. 61!2/2011 

Senior Scientist, Bay Paul Center, Marine Biological Laboratory 

Professor (MBL), Pathology and Laboratory Medicine, Brown University 

General Charge Questions: 

G l. Is the Toxicological Review logical, clear and concise? Has EPA clearly presented and synthesized 

the scientific evidence for non-cancer and cancer hazard? 

G2. Please identify any additional studies that would make a significant impact on the conclusions of 

the Toxicological Review. 

Reviewer Response: 

In general, the report is a concise presentation of the vast primary literature for chromium toxicology and 

carcinogenicity and the writing is generally clear. However, there are specific sections with deficiencies as 

noted below in detail, and many other sections and specific comments that are not logical. In many cases a 

statement in one section is either not a logical extension of the data presented, or is in opposition to a 

statement elsewhere. Overall, the greatest concern is with the logic regarding the choice of a mode of action 

(MOA), which is the basis for many of the subsequent assumptions that are made, the default values that are 

chosen, and risk assessment modeling that follows from these choices. In this reviewer's strong opinion -

and in the consensus opinion of the external reviewers who are experts in this area and who discussed this at 

the May 12, 20 II meeting - Cr(VI) is highly unlikely to act via a mutagenic mode of action in vivo. Rather, 

a careful review of existing information, as well as emerging studies all strongly indicate that the likely MOA 

involves a threshold mechanism that supports both the physiological uptake-reduction model of DeFlora and 

the cellular uptake-reduction model of Wetterhahn that were previously proposed. The current EPA draft 

document concludes that chromium(VI) acts via a mutagenic MOA by all routes of exposure, a conclusion 

that is illogical given the current state of knowledge of chromium biology and toxicology as already 

presented in this draft report, and also based on the recently emerging data from a series of 90-day rodent 

MOA studies sponsored by the American Chemistry Council (ACC). This is the most important and central 

point since the choice of a mutagenic MOA then drives all other considerations in this document. Specific 

areas of concern are outlined by chapter and section below in detail in a combined response to G I. and G2. 
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Joshua W. Hamilton, Ph.D. 

Chapter 1 

EPA should include more definitive information about the literature it reviewed that contributed to this 

draft report. It currently states that the relevant literature was reviewed through September 2010 but the 

external reviewers all noted at the May 12 meeting a number of gaps in the literature being cited in the 

current draft. In addition, it would be helpful to know how many studies the EPA identified as being part of 

the chromium literature, how many it reviewed, how many it set aside or did not review, what criteria were 

used to include or exclude a study, etc. For example, a statement such as: "The EPA identified 26,839 peerreviewed scientific publications in PubMed from 1950 through September 2010 using the keyword 

'chromium.' Of these, 9,456 were determined to be relevant to the current draft based on the criteria of 

covering aspects of chromium biology, toxicology, environmental chemistry and epidemiology." 

Chapter2 

On page 14 the draft states that, "Natural occurrence of hexavalent chromium is rare ... " This statement 

should be qualified since geochemical surveys by the U.S. Geological Survey and others have indicated that 

there are background levels of naturally occurring Cr(Vl) in most groundwater, and that these levels are 

typically in the range of 2-5 ppb but can be up to 3-5 times higher than that in certain areas. Likewise recent 

reports of Cr(VI) levels in soil and house dust indicate that there are natural sources, or at least sources that 

cannot be identified as being specifically anthropogenic, that contribute to the background levels and 

background exposures of people throughout the U.S. A more detailed literature review and discussion 

regarding background levels of Cr(VI) in soil, air and water, including more up-to-date information on such 

levels, should be included in this chapter to provide context for subsequent discussions regarding human 

exposure through drinking water. Keep in mind that, until recently, analytical methods to detect total 

chromium and to speciate chromium(VI) accurately were difficult, with higher detection levels than many 

natural sources contain. Further, speciation of chromium(Vl) at very low levels has been very problematic 

until recently, so much of the older environmental data are inaccurate or had non-detect values where 

reanalysis has revealed widespread chromium(VI) background levels in the environment. This is of 

importance not only for understanding the potential contribution of these background levels to total 

chromium exposure but also in setting practical regulatory levels. Clearly it is of little value for the EPA to 

calculate a maximum contaminant level (MCL) or public health goal (PHG) that is lower that typical 

background levels since this would be virtually impossible to achieve by most public drinking water systems 

with limited resources, and because such background levels are unlikely to represent a significant health risk 

(see discussion below). 
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Joshua W. Hamilton, Ph.D. 

Chapter 3 

This is an important chapter that would greatly benefit from reorganization and from synthesis of 

information. A number of studies are described here sequentially, and for several of these tbe draft document 

speculates on possible mechanisms. However, the order of presentation is not logical, and there is little in the 

way of more global synthesis of results and the conclusions that can be drawn - or those areas that remain 

controversial or poorly understood while there are several areas where a statement in one section is 

contradicted by a statement elsewhere. Because the toxicokinetics of chromium is so central to its biology 

and toxicology, this is a critical chapter for its overall evaluation and therefore this chapter should be revised. 

Regarding organization, Section 3.3 is extremely important for understanding the studies described 

throughout the remainder of the chapter, providing context for extracellular versus intracellular reductive 

metabolism. It is suggested that this section should be moved to just before or immediately after the current 

Section 3.1. Likewise, Section 3.6 on Cr(III) and its nutritional benefit and essentiality is extremely 

important for understanding how the body normally absorbs, distributes and excretes chromium. Prom the 

standpoint of human environmental exposures, Cr(VI) is primarily, but not exclusively an anthropogenically 

derived form of chromium that is principally encountered and used in occupational settings, but humans and 

all other life forms have been dealing, both physiologically and biochemically, with Cr(III) and reduction of 

background levels of Cr(VI) for the entire history of life on Earth. It is recommended that Section 3.6 should 

be moved to the beginning of this chapter and greatly expanded. The current section on this important topic 

is extremely short, superficial, and inaccurately presents this subject as an area of controversy in the field. 

Cr(III) nutritional biochemistry has been extensively studied over the past fifty-plus years with a large 

and robust literature. Only two toxicologists are cited as the sources of the "current debate" about this, 

whose individual views have been well aired in their review articles but which represent views that are 

generally considered to be well outside the mainstream of toxicology and nutritional biochemistry. Citing 

their views so prominently in this short section does not balance well with the wealth of studies and 

numerous other investigators over decades who have concluded that there is a beneficial role of Cr(III) in 

human and animal nutrition and have demonstrated an underlying biochemistry and physiology that supports 

this role. This section should be greatly expanded and treated in a more balanced fashion since it sets the 

stage for understanding how Cr(III) is treated by the body in all aspects of toxicokinetics, and therefore also 

provides valuable insight into Cr(VI) toxicokinetics that largely explains much of the experimental literature 

on chromium disposition in intact animals and humans as discussed in more detail below. See for example 

reviews by W Mertz (J Nutr 1993, 123:626-633; Nutr Rev 1995, 53:179-185), RA Anderson (Reg Tox Pharm 

1997, 26:835-41 ), HC Lukaski (Ann Rev Nutr 1999, 19:279-302) and JB Vincent (J Am College Nutr 1999, 

18:6-12) for summaries of this earlier literature. 

Some of this information is alluded to - for example, the end of the primary paragraph on page 44 

beginning with "Aitio et al. ( 1988) developed ... ", which represents an extremely important body of literature 
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on Cr(III) toxicokinetics and which should be moved forward with section 3.6 and greatly expanded by citing 

other relevant literature. There are dozens of studies of Cr(III) uptake and kinetics. The current chapter 

leaves the impression that Cr(III) gastrointestinal (Gl) uptake is uniformly low, and that chromium is not 

normally found above background levels in urine, therefore if one administers Cr(VI) and sees a dosedependent increase in blood, urinary or tissue levels it is evidence of Cr(VI) uptake as Cr(VIl. But there are a 

number of uptake studies of chromium picolinate and other natural and man-made Cr(III) complexes, as well 

as Cr(III) uptake in chromium-sufficient versus chromium-deficient diets, that defy this simple interpretation. 

For example, one study described on page 45 by Kerger et al. (1997) reported that "ingestion of chromium 

picolinate resulted in significantly elevated urine concentrations such that participants routinely exceeded 

background." Similar elevations have been reported for chromium picolinate in the nutritional literature. 

Likewise, other studies have shown an inverse correlation between Cr(III) levels in diet and the uptake, 

distribution, tissue storage and excretion of Cr(III), indicating that the body tightly regulates Cr(III) kinetics 

to maintain a steady-state body burden and availability of the nutritionally active form of Cr(III), and can 

actively take up Cr(III) from the GI when the body senses that it is deficient, or decrease uptake and increase 

excretion when internal stores are sufficient or exceeded. 

A related and key concept that is alluded to in this section but never addressed directly is the behavior of 

chromium in serum and red blood cells (RBC). It has been well established, beginning with RBC labeling 

studies going back to the 1950's in which RBC are incubated with Cr(VI) ex vivo, that Cr(VI) is readily 

taken up by RBC, rapidly reduced, and in the process forms highly stable chromium adducts on hemoglobin 

and other macromolecules which are very long lived, essentially remaining intact for the lifetime of the cell. 

In this way the half-life of RBC in humans and experimental animals was established (with human RBCs 

having a half-life of ca. 110-120 days) and this tool has also been used to look at RBC turnover. This is 

alluded to on page 27, where it states that "The partitioning of hexavalent chromium from plasma into 

erythrocytes is significant. It has been used as a biomonitoring endpoint .. . and is responsible for the 

observed residence time of chromium in whole blood ... " Conversely, in the human and animal studies that 

were described in Chapter 3, it has been consistently shown that when Cr(VI) is administered orally or by 

gavage, there is a transient increase in both serum and RBC levels which rapidly return to baseline. Thus, it 

is highly unlikely that the chromium in the blood of these animals and humans was Cr(VI), or else the RBC 

would have been stably labeled. It was noted on page 45, for example, in describing the human studies of 

Paustenbach et al. (1996) where there was oral exposure to Cr(Vl) that "both plasma and RBC chromium 

concentrations returned rapidly to background levels within a few days, again suggesting that concentrations 

of 10 mg Cr(Vl)IL or less in drinking water of humans appears to be completely reduced to Cr(IIJ) prior to 

systemic distribution. " This is an extremely important experimental observation in humans and one of the 

most important statements in this draft document -- that directly addresses the issue of whether Cr(VI) is 

taken up by the human GI as Cr(VI) and whether it survives as Cr(VI) in the circulation. 
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Similar results were seen in the Sutherland et al. (2000) rat study in which the blood kinetics, and lack of 

chromium increases in brain or other distal tissues argued strongly that it was Cr(III) rather than Cr(Vl) that 

was taken up by the gut. Were this not the case, they should have observed stable chromium labeling of the 

RBC and elevated chromium levels in distal tissues. But they reported the opposite, and this also strongly 

indicates in this key rodent study that Cr(Vl) failed to survive as Cr(VI) in crossing into the bloodstream 

from a GI exposure. And elsewhere in Chapter 3, similar results are reported that would lead to the same 

conclusion, yet the text alludes to transient RBC uptake as possible evidence that Cr(Vl) is being taken up 

from the GI tract as Cr(Vfl. This is a highly flawed, illogical argument that appears throughout this 

document. Likewise it is now well known that there are specific uptake, transport and storage mechanisms 

for nutritionally active Cr(III) that must be taken into account in any measurements of chromium in the blood 

or other tissues. In fact, none of the studies presented in Chapter 3 provide any direct evidence that any 

Cr(Vl escapes the GI tract as Cr(Vl) except perhaps where normal gastric reduction is bypassed or the bolus 

doses are so large as to completely overwhelm the reductive capacity of the gut, which is likely what 

occurred in the NTP (2008) study. This should be more clearly described in this chapter, perhaps with a new 

section prior to current section 3.5 that presents the theoretical PBPK model since this model also critically 

relies on how one interprets the in vivo data. 

Specific edits suggested below are based on these concerns (changes underlined): 

Page 24, Line 24- " ... for the chromium administered as hexavalent ... " 

• Page 25, Line 7- " ... ofthe chromium administered as hexavalent ... " 

Page 26, Line 3 - " .... Generally, absorbed chromium is ... " 

Page 26, Line 9 - " ... toxicokinetics of chromium, .... " 

Page 26, Line 16 - " ... comparing administration of Cr( Ill) ... " 

Page 30, Line 2 - " ... bioavailability of chromium administered as ... " 

• Page 35, Lines 7-11 -two sentences beginning with "The reason for the higher ... " This is highly 

speculative and should be deleted. This could all be Cr(lll) rather than Cr(VI) using the arguments 

above. 

Page 36, Lines 25-28 -end of sentence beginning with " ... indicating that a portion of the Cr(Vl) 

escaped extracellular reduction ... " This is also highly speculative and is actually counter to the data 

presented, which clearly show transient blood levels that are indicative of Cr(III) distribution, not 

Cr(VI), as more appropriately alluded to in Lines 32-34 where is says "Brain, ovarian, and wholeblood concentrations were below detection limits in all exposed groups. The lack of concentrations 

in whole-blood was attributed to rapid delivery of Cr to tissues and clearance of plasma Cr." The 

lack of stable blood chromium clearly indicates that it could not have been absorbed as Cr(VI) or else 

the RBC would have shown significant and stable elevations. 

Page 38, Line 13 " ... did not alter GI uptake appreciably at these concentrations." 
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Page 41, Line I "Chromium is capable of crossing the placenta." 

Page 41, Line 15-17 - "Absorption and elimination of chromium was evaluated .... Following 

ingestion by human volunteers of either trivalent or hexavalent chromium in single or multiple 

drinking water doses." 

• Page 42, Lines 23-26 This statement beginning with "Because the Cr(Vl) increases ... " is 

incorrect. Increases in RBC chromium that are stable over time, with the same half-life as the RBC, 

would be indicative of uptake of chromium as Cr(VI) by the RBC, but if the increase in RBC 

chromium is transient, as in this case, then it cannot be due to Cr(VI) in the blood but is most likely a 

Cr(III) complex that is carried by the RBC but not covalently bound. Evidence for this is cited 

elsewhere in this chapter (see middle of page 44 and top of page 46, for example) and in the broader 

Cr(I!I) literature. The draft report is illogical since it currently argues this issue both ways depending 

on the study being discussed. 

• Page 42, Lines 30-35 These two sentences beginning with "The higher bioavailability ... " are 

highly speculative and well outside the boundaries of the actual data presented and discussed above. 

If the report is going to speculate, it should pull all this into a separate section and synthesize it across 

all the studies that were cited. Otherwise delete these speculations since they do not have a factual 

foundation. 

Page 49, last line- " ... chromium administered as CriVIJ distributes ... " 

Page 58, Lines 2-3 - " ... greater percentage of chromium administered as Cr!Vl/ than Cr(lll) is 

absorbed." Delete sentence reading "This implies that some Cr(Vl) escaped reduction ... " since this 

is not implied by the actual experimental data based on the above arguments. 

Taken together, these studies, as argued above, lead one to only one logical conclusion: Cr(VI) is not 

taken up by the gut as Cr(VI), nor does it survive as Cr(Vl) in the systemic circulation in these studies. It is 

only by greatly exceeding the normal doses and reductive capacity of the gut that one can see any signs of 

Cr(VI) surviving to reach other tissues. Therefore there is a clear biochemical barrier, a threshold, to Cr(VI) 

uptake and systemic exposure under normal physiological conditions. This must clearly be taken into 

account in any analysis of MOA and resulting risk assessment for this toxicant. 

Chapter4 

Liaoning Province, China studies At the beginning of this chapter (Pages 68-76) the EPA presents an 

extensive review and discussion of the various reports related to the study of populations in China near a site 

of chromium-contaminated drinking water from a nearby industry. Depending on the authors and methods 

used particularly certain assumptions regarding age adjustment, use of an urban area as a control 

population, and exposure estimates -- the reports of this data set either find no statistical association between 

stomach cancer incidence or a modest elevation. The other epidemiology studies cited in this chapter report 
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no statistical correlation between drinking water cbromium exposure and cancer incidence. It is of 

considerable concern to this reviewer that the EPA has chosen in some places to highlight this one positive 

report and elevate it to the level of their major recommendations, such as on Page 239 of Chapter 6 (Major 

Conclusions) where they state that there is "evidence of an association between oral exposure to hexavalent 

chromium and stomach cancer in humans." Yet elsewhere, they state, appropriately, that "this risk has not 

been established in other populations exposed to drinking water contaminated with hexavalent chromium. 

The epidemiology data are not sufficient to establish a causal association between exposure to hexavalent 

chromium by ingestion and cancer." (Page 201, Lines 20-23; and similar language on Page 205, Lines 

33-36) 

It is inappropriate to "cherry-pick" a single study - indeed, a single treatment among tbree treatments of 

the same study - if this is going to influence their major recommendations regarding the human 

carcinogenicity of Cr(VI) via ingestion. Many of my other comments and edits relate to this concern, as well 

as previous concerns regarding the toxicokinetics of Cr(VI) via ingestion and the misinterpretation of those 

results with respect to mode of action and carcinogenic potential in animals and humans as detailed below. 

This reviewer feels strongly that the Liaoning Province study and its various treatments should be set aside, 

since the initial study (first reported in 1987) is highly flawed by today's epidemiology standards, there is a 

Jack of information that would allow others to reevaluate this study in the manner that would most directly 

address the potential correlation, and the tbree subsequent treatments by original authors Zhang and Li 

( 1997), by Beaumont et al. (2008) and by Kerger et al. (2009) -- each of which makes slightly different 

assumptions in order to fill critical data gaps -- reached different conclusions. It is also important to point out 

that this population was exposed not just to extremely high levels of Cr(VI) but also to industrial effluent 

which contained high levels of a number of other chemicals of concern including sulfates, acids and other 

toxicants. Thus, even if an association was found, it is not possible to attribute this to Cr(VI) per se and 

could be the result of either other contaminants or a statistical anomaly based on the high rates of stomach 

cancer in China which are diet- and province-related. For these reasons this study and its series of treatments 

cannot be the basis for risk characterization or risk asse%ment, and should be set aside. 

Section 4.4 also requires extensive revision and expansion. The first paragraph reports only briefly on 

the extensive literature examining occupational exposures and cancer, principally lung cancer. This section, 

and this literature, should receive a much more extensive treatment since these studies are our best data 

regarding human exposures to Cr(VI). It should be noted that in addition to inhaling extremely high levels of 

Cr(VI) prior to the advent of industrial hygiene practices in the 1960's and 1970's (levels as high as several 

mg chromium(VI) per cubic meter) these workers had extensive dermal exposures, and also had extensive 

ingestion exposure since it is well known that individuals exposed to high levels of dust swallow a large 

fraction of what they inhale either tbrough direct deposition in the mouth, nose and tbroat, and via 

mucocilliary clearance of the pulmonary system. Thus, these occupational epidemiology studies are 
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extremely important in understanding the toxicology of chromium(VI). Several key points will be made 

below citing a few critical studies, but the EPA should more thoroughly review this entire literature. 

The U.S EPA's current risk assessment for chromium(VI) via inhalation is based principally on the 

studies by Mancuso of older worker populations through the 1960's prior to the advent of modern industrial 

hygiene practices. Similar risks were observed in other occupational studies of workers from the 1940's 

through 1970's who were exposed to much higher levels of chromium than are encountered in occupational 

settings today, such as those by RB Hayes et al. (lntl J Epi 1979, 8:365-374; Am J Indust Med 1989, 16:127-

133), JM Davies (Br J Indust Med 1984, 41:158-168), T Sorahan et al. (Br J lndust Med 1987, 44:250-258), 

and R Kishi et al. (Am J Indust Med 1987, 11:67-74). Subsequent epidemiology studies of workers exposed 

to lower levels of chromium since the I 960' s have not only provided estimates of risk at these high doses, but 

have also provided important information as to lower occupational levels at which no increases in lung cancer 

or other health effects were observed. These more modern studies have also taken into account other factors, 

such as accounting for cigarette smoking and other confounding variables, whereas many of the older studies 

did not control for these important confounders (see for example, KD Rosenman, Am J Indust Med 1996, 

29:491-500). It should be noted that, where it was reported, virtually all the lung cancer cases in these 

occupational studies occurred in smokers (see for example Pastides et al., Am J Indust Med 1994, 25:663-

675; T-C Aw, Reg Tox Pharm 1997, 26:S8-12). These studies not only provide better estimates of the actual 

health risks attributable to occupational chromium exposure, but also an estimate of a practical threshold 

below which we would predict either no effects, or risk of effects that are so low that it cannot readily be 

detected even in large populations of exposed people. 

Gibbet al. performed a follow-up study (Am J lndust Med 2000, 38:1 15-126) of a worker population in 

Baltimore MD that had previously been studied by Hayes et al. (lnt J Epi 1979, 8:365-374) in examining the 

relationship between chromium(VI) exposure and cancer incidence. In the Gibb study, the workers were 

stratified according to different levels of cumulative exposure to chromium, allowing a more detailed 

examination of the potential dose-response. Cumulative exposure was expressed as J.lg/m3

-years (1,000 

ng/m3

-years), integrating both the chromium level and the total time of exposure at that level. This is similar 

to smoking data that express cumulative dose as "pack-years" and is based on the observation that the risk of 

a 40 pack-year smoker who used I pack per day for 40 years is similar to that of a 40 pack-year smoker who 

used 2 packs per day for 20 years. In the Gibb study, the lowest quartile of workers had exposure to 

chromium between 0 and 1.5 J.lg/m3

-years (1,500 ng/m3-years). This group had an observed/expected lung 

cancer ratio of 0.96, i.e., it was slightly less than expected from the comparison population (the general 

population of Maryland) that had no occupational chromium exposure. 

Pastides et al. examined a group of chromate production company workers in North Carolina (cited 

above), focusing on the possible differences in risk between cohorts of workers who were exposed to 

chromium under the older conditions and processes of the 1940's through 1960's and those who began work 
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Joshua W. Hamilton, Ph.D. 

after 1971 in a modernized factory in which both the chemical process and the exposure levels to chromium 

had been modified. They found a slightly increased risk of lung cancer, proportional to exposure, in the older 

cohort working under the higher dose exposure conditions, as had been reported previously in other studies. 

However, the workers in the modernized factory had no excess of lung cancer, all cancers, heart disease, or 

all causes of death over an 18 year period. Personal monitors for the workers indicated that the chromium(VI) 

levels were all below 50,000 ng/m3

, and most were below 25,000 ng/m3, with the majority in the range of 

500-10,000 ng/m3

• Average duration of employment was 9.5 years, such that cumulative dose would have 

averaged 4, 750-95,000 ng/m3 -years. Dividing the workers into two groups of exposure, i.e., those working 

less than I 0 years versus those working more than I 0 years, indicated no difference in mortality, further 

suggesting that these workers had no significant increase in cancer or other health risks from either the higher 

or lower chromium exposures. Similarly, Aw reported (cited above) that workers in the more modernized 

plants who were occupationally exposed to chromium since the 1960's showed no increase in disease risk, as 

was also noted by S Langard et al. (Br J Indust Med 1990, 47:14-19). 

WJ Blot et al. performed a large and comprehensive study (J Occup Environ Med 2000, 42: 194-199) of a 

group of 51,899 workers of the Pacific Gas & Electric (PG&E) Company. A sub-set of 3,796 these workers 

had been exposed occupationally to chromium(VI), either as gas generator workers or trainees at the 

Kettleman CA station which used chromium as a rust inhibitor in cooling tower water at PG&E natural gas 

transfer stations from the 1950's through the 1980's. Examination of these workers for specific cancers, all 

cancers, specific non-cancer diseases, and all diseases indicated no increased incidence in any adverse health 

outcomes in relation to chromium exposure. In fact, the total cancer and lung cancer standardized mortality 

ratios (SMRs, observed/expected ratios) were 0.64 and 0.81, and 0.55 and 0.57, respectively, for these two 

groups of chromium-exposed workers, which was less than those of the overall PG&E worker group and 

substantially less than those of the general California population against which they were compared. SMRs 

for all causes of death were also low (0.79 and 0.68, respectively). Likewise, JD Boice et al. performed a 

large and comprehensive study (Occup Environ Med 1999, 56:581-597) of a group of 77,965 workers at an 

aircraft manufacturing plant in California. A sub-set of 3,634 of these workers were exposed to chromates 

and other chemicals as part of airplane production for a total of 88,224 person-years of exposure and a mean 

of 24 working years per person of exposure. The SMR for total cancers was 0.93, and the SMR for lung 

cancer was 1.02. As with the Blot study, there was no association of any adverse cancer or non-cancer health 

outcome with chromium exposure in this group, nor did the overall worker population have an increase in 

overall or specific mortalities as compared to the general population despite exposure to a number of 

occupationally related chemicals. 

Taken together, these occupational studies indicate that, although previous historical exposure conditions 

were associated with a modest risk of lung and respiratory cancer (average of 2- to 4- fold increased lifetime 

risk, as compared, for example, to a 10- to 20-fold increased risk for cigarette smokers), more recent 
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Joshua W. Hamilton, Ph.D. 

occupational exposures at or below the current regulatory limits indicate that these represent levels that do 

not elevate cancer risk even for lifetime occupational exposures. Moreover, the previous exposures of 

concern in workers from the 1930's through the 1960's were at levels that typically exceeded 1,000,000 

ng/m3 and also involved exposure to the most carcinogenic forms of chromates, i.e., the insoluble or slightly 

soluble forms such as lead chromate, zinc chromate and calcium chromate. The newer lifetime occupational 

exposure limits -- at which no increase in cancer risk or other health effects has been observed represent 

daily exposures that are hundreds to thousands of times higher than would occur in an environmental setting 

or via U.S. drinking water. There are two other major conclusions that can be drawn from these occupational 

exposure studies. First, although dermal exposure to chromium(VI) was extensive - particularly prior to the 

advent of industrial hygiene practices in the 1970's- there is no evidence for increased risk of skin cancer, 

even in workers where the chromate levels were high enough to burn "chrome holes" in their skin or nasal 

septum. These chrome holes healed and were not associated with increased skin cancer risk in these workers. 

This is relevant to the very high doses of chromium(Vl) used in the NTP studies and a possible MOA. 

Second, taken together these occupational studies do not demonstrate an increased risk of GI cancers or other 

internal cancers, despite the fact that these workers swallowed a significant fraction of the dusts they were 

exposed to in the air. These data were recently summarized in a meta-analysis published in 2010 (NM Gatto 

et al., Cancer Epi 2010, 34:388-399). 

Other specific edits and comments (changes underlined): 

Page 200, Lines 1-2- Delete the phrase " ... and evidence of an association between oral exposure to 

hexavalent chromium and stomach cancer in humans" which is based on a single study and EPA's 

selective treatment of this result as discussed above. 

Page 200, Lines 18-20- "This study found evidence of a modestly increased incidence of stomach cancer 

mortality (OR 1.69, Cll./2-2.44/from 1970 to 1978 .... " 

Page 201, Lines 20-23 " ... was reported in a re-examination of a single study in JinZhou .. " 

Page 209, last section, Bioavailability This section is highly flawed in logic and presentation as 

discussed under Chapter 3. Specific edits are as follows: 

o Page, 209, last two lines- "Quantita!We studies ofG1 absorption of chromium administered as 

hexavalent chromium have estimated ... " 

o Page 210, frrst line Please note that hexavalent chromium was not measured, and without 

exception has never been measured systemically as Cr(Vl) following GI absorption. It is an 

assumption that increased chromium uptake to the blood represents Cr(Vl) uptake, but it could 

also represent other forms as discussed in Chapter 3 above. We know that certain forms of 

Cr(III) are much more readily taken up than others, and it is therefore possible, and perhaps quite 

likely, that reduction of Cr(Vl) in the gut in the presence of organic molecules in the GI lumen 

leads to formation of complexes that are much more bioavailable than inorganic Cr(III) that is 
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Joshua W. Hamilton, Ph.D. 

typically found in food, water and soil. You could also modify this phrase as a separate sentence 

to read "This may indicate that not all hexavalent chromium is reduced by the gastric juices of 

the stomach, or that reduction of Cr(Vl) in this environment leads to formation of organic 

chromium complexes that are more readily absorbed than inorganic Cr(lll)." 

o Page 210, Lines 7-12 - Delete the sentence beginning "Thus, at oral doses within human 

exposure ranges ... " as per the argument above or modify to include alternative interpretations as 

suggested above. 

o Page 210, Lines 28-30- End of sentence beginning with " ... and uptake of hexavalent chromium 

into the tissues ... " delete this phrase or modify it as per the argument above. 

o Page 210, last line Add a sentence after the last sentence on this page reading "However, none 

of those studies speciated the chromium that was absorbed systemically, and so the form(s) of 

chromium in the blood and other tissues is unknown following increased absorption of chromium 

following ingestion of hexavalent chromium. Therefore, it is not knmm whether chromium 

reaches the blood or distal tissues as chromium( VI) at doses relevant to human exposures. The 

lack of long-term labeling of RBCs by chromium in the animal and human studies argues that 

little, if any, chromium is absorbed as chromium(Vl) under these exposure scenarios. " 

o Page 213, Section 4.7.3.5. Lines 3-5 - This statement is completely incorrect; there is no 

evidence for it as argued above. Delete this, or modify as follows "Chromium absorbed 

following ingestion exposure to chromium( VI) may be in forms that can reach the systemic 

circulation and distal tissues, thereby potentially affecting tissues beyond those at or near the 

site of entry. However, the form(s) of chromium following such uptake is not known." 

Chapter 5 

This reviewer strongly objects to use of a linear low-dose approach for Cr(VI) risk assessment given the 

clear evidence for a threshold mechanism due to extracellular reduction of chromium at doses of relevance to 

human exposure via drinking water. The conclusion by EPA of a mutagenic action of chromium - most of 

which is based on cell culture data where chromium exposures and other parameters were extreme and where 

metabolism and intracellular exposure are far different than in vivo exposures - should not be the sole basis 

for use of this standard model which ignores the compelling toxicokinetic data summarized in Chapter 3 of 

this draft. More importantly, as discussed above for Chapter 4, Cr(VI) is unlikely to act via a mutagenic 

MOA in vivo, and requires extraordinary experimental manipulation to be positive in cell culture and in vivo 

mutagenicity studies. While it is clear that Cr(VI) can cause certain forms of DNA and chromosomal damage 

or other changes, it is not clear whether any of these is pre-mutagenic, and the in vivo data argue strongly 

against a mutagenic MOA under physiological conditions and normal routes of exposure. The document 

must more clearly differentiate between genotoxicity i.e., damage to DNA or chromatin- and mutagenicity 
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Joshua W. Hamilton, Ph.D. 

- or frank mutations that may result from DNA damage. Chromium(VI) can induce DNA damage but is a 

very weak mutagen at best, particularly in vivo. It is far more likely, and most consistent with all available 

data, that chromium(VI) acts via a non-mutagenic mechanism that involves a clear threshold - two threshold 

actually, one of which is extracellular and chemical involving reduction of chromium(VI) to chromium(III) 

and the other of which is biochemical and involves a threshold for cellular effects that lead to cell damage 

and cell death, resulting in turn in tissue proliferative responses that ultimately increase tumor risk via well 

known mechanisms of repeated tissue injury, compensatory cell proliferation and re-population. 

Given this most likely MOA based on a synthesis of several decades of chromium research, it is therefore 

inappropriate to use a linear low-dose extrapolation model for assessing risk via the ingestion route of 

exposure. It is clear from the animal and human studies that there is a threshold for in vivo effects that is 

based on the strong reducing capacity of the GI tract following oral exposures, and that at normal drinking 

water concentrations this will effectively protect from any in vivo exposure to Cr(VI) as Cr(Vl). Thus, a 

more appropriate risk assessment method would be to do dose-response modeling from the 2008 NTP study 

and the more recent ACC-sponsored 90-day MOA studies, and then use an approach similar to that for the 

RID to calculate, with appropriate safety factors, a drinking water MCL that is protective based on threshold 

mechanisms. This should be done for Cr(VI) rather than the current MCL that is for total chromium, but it is 

likely that an MCLin the range of 50-100 ppb is going to be fully protective, including several uncertainty 

factors that separate it from the departure point of any likely human health effects for even the most sensitive 

individuals. 

Chromium is an excellent example of an opportunity to apply the concept of evidence-based risk 

assessment - which the EPA has claimed to be promoting for many years but has not, to date, actually 

applied in any meaningful risk assessment -- since there is a strong and compelling argument for use of a 

non-linear, threshold-based mechanism for chromium that logically leads to a real-world risk assessment that 

is based on that mechanism. Setting aside the 1987 Zhang and Li study and subsequent re-analyses as per the 

arguments above, there is not a single credible epidemiology study linking exposure to chromium via 

ingestion with cancer risk or any other long-term health effects, including in the extensive occupational 

epidemiology literature which includes several decades of extremely high-dose exposure cohorts. And even 

taking into account the Zhang and Li study, there is not a single peer-reviewed report linking any health 

effects to chromium(VI) at levels within a hundred-fold of likely environmental exposures via drinking 

water. The NTP 2008 animal data showed evidence of a cancer increase only at the highest doses, and the 

more recent ACC studies demonstrated hyperplasia consistent with a non-mutagenic MOA, which is also 

consistent with a threshold mechanism and which argues against developing a linear cancer slope factor from 

those data. 
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Joshua W. Hamilton, Ph.D. 

Chapter 6 

There is considerable disconnect between the conclusions provided in Chapter 6 and the more considered and 

detailed discussions of the primary data in the previous chapters. The language should be modified to reflect 

this understanding such that the conclusions and their application to risk assessment of chromium(VI) follow 

logically from the scientific evidence. Specific edits: 

Page 235, Lines 18-19 - " . .. resulting in substantial. and in some cases complete reduction of 

hexavalent chromium to trivalent chromium depending on the concentration, dose. and precise route 

and method of exposure." 

Page 235, Lines 21-26 "The extent of absorption of chromium from ingesting hexavalent chromium 

appears to be determined by both the solubility ..... in the GI tract, but ingestion of both trivalent and 

hexavalent chromium results in systemic uptake of chromium. Trivalent chromium does not readily 

cross cell membranes except as part of certain organic complexes. Hexavalent chromium. if 

absorbed systemically, can easily .... " 

Page 235, Lines 27-29 - "Chromium absorbed systemically (rom the gut following ingestion of 

hexavalent chromium is distributed throughout the body. .. .. If hexavalent chromium is absorbed 

without extracellular reduction, it can cross cell membranes and, once inside the cell, .... " 

• Page 236, Line 3 - "Chromium absorbed systemically following hexavalent chromium ingestion is 

eliminated primarily in the urine as trivalent chromium. " 

Page 239, Lines 11-12- delete the phrase " .. and evidence of an association between oral exposure 

to hexavalent chromium and stomach cancer in humans." This is a significant statement to make, 

and as discussed above in reference to Chapter 4, the Zhang and Li studies (1987, 1997) should not 

be used to assess human cancer risk for oral exposure to chromium. 

Page 240, Lines 12-14 - As noted for Chapter 5 above, there is considerable concern with the default 

choice of a linear low-dose extrapolation model for cancer risk. 

Chemical-Specific Charge Questions: 

(A) Oral Reference Dose (RID) for Hexavalent Chromium 

AI. A two-year drinking water study of sodium dichromate dihydrate in rats and mice (NTP, 2008) 

was selected as the basis for the derivation of the RID. Please comment on whether the selection 

of this study as the principal study is scientifically supported and clearly described. Please identify 

and provide the rationale for any other studies that should be selected as the principal study. 

A2. Diffuse epithelial hyperplasia in the duodenum of female mice was selected as the critical effect for 

the RID. Please comment on whether the selection of this critical effect is scientifically supported 

and clearly described. Please identify and provide the rationale for any other endpoints that 

should be selected as the critical effect. 
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A3. Benchmark dose (BMD) modeling was applied to the incidence of diffuse epithelial hyperplasia in 

the duodenum of female mice to derive the point of departure (POD) for the RID. Has the BMD 

modeling been appropriately conducted and clearly described? Is the benchmark response 

(BMR) selected for use in deriving the POD (i.e., a 10% increase in the incidence of diffuse 

epithelial hyperplasia) scientifically supported and clearly described? 

A4. Please comment on the rationale for the selection of the uncertainty factors (UFs) applied to the 

POD for the derivation of the RID. Are the UFs scientifically supported and clearly described? If 

changes to the selected UFs are proposed, please identify and provide a rationale. 

Reviewer Response: 

No concerns regarding AI. And A2. This reviewer would point out that the hyperplasia that was chosen as 

this endpoint, while appropriate for this RID, is also appropriate for considering the carcinogenic MOA as 

well, as argued above and below and taking into considering the recently reported ACC studies that should be 

considered in this regard. This reviewer is not an expert on modeling and cannot comment on A3 in detail. 

No concerns regarding A4. Uncertainty factors are policy decisions, not scientific ones, and we can neither 

prove nor disprove any of the assumptions on which they are based nor can we accurately determine when 

and how such factors might be applied. Based on previous EPA doctrine, these seem to be consistent with 

previous applications. 

(B) Carcinogenicity of Hexavalent Chromium 

BI. Under EPA's 2005Guidelines for Carcinogen Risk Assessment (www.epa.gov/iris!backgrd.html), 

hexavalent chromium is likely to be carcinogenic to humans by the oral route of exposure. Is the 

cancer weight of evidence characterization scientifically supported and clearly described? 

B2. A mutagenic mode of carcinogenic action by all routes of exposure is proposed as the primary 

mode of action for hexavalent chromium. Please comment on whether this determination is 

scientifically supported and clearly described. Please comment on data available for hexavalent 

chromium that may support an alternative primary mode of action. 

B3. A two-year drinking water study in rats and mice (NTP, 2008) was selected for the derivation of 

an oral slope factor. Please comment on whether the selection of this study for quantification is 

scientifically supported and clearly described. Please identify and provide the rationale for any 

other studies that should be considered. 

B4. The incidence of adenomas and carcinomas combined in the small intestine of male mice from the 

NTP (2008) two-year drinking water study were selected to serve as the basis for the quantitative 

cancer assessment. Please comment on whether this selection is scientifically supported and 

clearly described. Please identify and provide the rationale for any other endpoints that should be 

selected to serve as the basis for the quantitative cancer assessment. 

BS. The oral slope factor was calculated by linear extrapolation from the POD (i.e., the lower 95% 

confidence limit on the dose associated with 10% extra risk of tumors of the small intestine in 

male mice). Has the modeling been appropriately conducted and clearly described? 
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Reviewer Response: 

Regarding B 1. and as outlined in the detailed comments under G l. and G2., this reviewer is concerned 

that the evidence for carcinogenicity is not strong in animals and is not supported by human epidemiology. 

As also noted in comments under G 1. and G2., there is considerable concern with statements by EPA in 

sections of this draft, particularly under Major Conclusions in Chapter 6, regarding evidence for human 

carcinogenesis based on a single human epidemiology study that in turn is a re-analysis of another study that 

lacks critical information that would be useful in fully assessing relative cancer risk. However, based on the 

current criteria for selection of this designation, it appears to be consistent with EPA doctrine since there is 

evidence of increased tumors in animals under certain exposure conditions. 

B2. There is considerable evidence that Cr(VI) is genotoxic in cell culture and in vitro, and under certain 

extreme conditions it can also be shown to be mutagenic. However, there is far less support that Cr(VI) is 

genotoxic or mutagenic in vivo by the oral route of exposure at doses of relevance to humans; conversely, 

there is considerable evidence that there are protective threshold mechanisms that significantly impact the 

ability of Cr(Vl) to reach target tissues and cause DNA damage under physiological conditions. In addition, 

while alternative mechanisms are briefly discussed, these are essentially dismissed without extensive 

treatment. As noted in the document, there is not a large literature on alternative mechanisms, but this is 

largely because, since the discovery of the genotoxic potential of Cr(VI) some forty years ago, most of the 

field has only focused on this one aspect, and I suspect it would be very difficult to get peer-reviewed 

funding to study non-genotoxic mechanisms for this toxicant. It is important to note, however, that there are 

no reports of increased skin cancer under occupational exposure settings, despite the fact that workers until 

the past few decades were directly exposed to Cr(VI) on the skin to the extent that they formed "chrome 

holes" that eventually healed. Yet this direct application and clear signs of chromium reduction and toxicity 

directly on the skin produced no increased skin cancer risk. Likewise, the occupational exposure literature 

only recently investigated the role of smoking status in chromium-related lung cancer risk. The increased 

risk of lung cancer associated with Cr(VI) exposure is modest considering the exposure levels and duration of 

exposures that span decades, and virtually all of the cancer cases in the epidemiology studies were seen in 

smokers, suggesting an interaction but one that is very modest. This in turn suggests alternative mechanisms 

such as inflammation, oxidative damage, damage-induced proliferation, and other mechanisms not directly 

tied to the ability of Cr(VI) to enter cells and damage DNA. These should considered and explored in more 

detail, since they are the basis for many of the assumptions regarding the risk of cancer from oral exposure to 

Cr(VI). 
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B3. There are no significant concerns about selection of this study, it is clearly the best available study for 

this type of risk assessment, with the caveats about bow these data are interpreted and modeled as discussed 

elsewhere. 

B4. There is concern about selection of these endpoints to represent cancer risk in these animals as the basis 

for a human risk assessment. The high doses required to induce these lesions are well above a threshold level 

that would be of concern in humans under normal exposure scenarios, and these almost certainly represent a 

scenario where natural reductive defense mechanisms were overwhelmed by the doses of chromium used. 

Taken together with the more recent 90-day ACC sponsored studies, the MOA for chromium(VI) is most 

likely a non-mutagenic one involving tissue damage and reproliferation, and would only be seen at doses that 

are unlikely to ever occur in a human exposure setting, particularly via drinking water. Thus, a threshold 

based risk assessment is most appropriate, similar to the treatment used for the non-cancer endpoints which 

are likely to be directly related to the cancer MOA. 

B5. As discussed in detail under G I. and G2. responses, this reviewer has considerable concerns about the 

use of a linear low-dose extrapolation model for assessing chromium cancer risk. The evidence, when 

objectively assessed, strongly argues for threshold mechanisms both in the gut and systemically, and there is 

little or no evidence that chromium reaches the systemic circulation as chromium(Vl) under exposure 

scenarios of relevance to human exposures. There is no better candidate for departure from these default 

EPA assumptions than chromium if EPA is serious about evidence-based risk assessment. Many of the 

public comments that were available to the reviewers just before and after our May 2011 meeting also raise 

these issues, and the EPA should, in particular, wait until the recently reported 90-day MOA and PK studies 

are published and available to them, at which point they should give serious consideration to how these new 

data inform the likely MOA. Given that most toxicology profiles are only revised every 10-15 years, it is 

worth waiting for these studies, and taking them as well as the external reviewer comments in mind toward a 

revised document that will be more accurate and will better stand the test of time. The EPA might also 

consider asking for a National Research Council Special Emphasis Panel to review all these materials and 

make a recommendation to EPA regarding chromium( VI) as bas been done for other several other key 

toxicants of concern. In any event the current draft's risk assessment treatment of chromium(VI) is highly 

flawed and grossly mischaracterizes the likely risk of human health effects of chromium(VI) in drinking 

water based on a careful and thorough assessment of all the available evidence. 
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POST-MEETING COMMENTS SUBMITTED BY 

Monica Nordberg, Ph.D. 
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Charge Questions 

General Charge Questions: 

Gl. Is the Toxicological Review logical, clear and concise? Has EPA clearly presented and 

synthesized the scientific evidence for noncancer and cancer hazard? 

EPA has presented and synthesized present knowledge about non-cancer and cancer 

hazards for hexavalent chromium. However, some further literature could be included in 

the document and paid attention to i.e, Langiird aud Costa Chapter 24 Chromium In: Nordberg GF, Fowler 

BA, Nordberg M and Friberg LT (Eds.) (2007) Handbook on the Toxicology of Metals, 3rd edition, Elsevier 

487-510. 

Other chapters of interest e.g.,Chapter 10 Carcinogenicity by Ke, Costa and Kazantzis page 177-196. One 

chapter (14 by G. Nordberg and B A Fowler) deals with Risk assessment pages 281-301. 

A comparison between criteria for classification of carcinogenicity should be done between 

IARC, EU and USA. Hexavalent chromium is classified as a human carcinogen. This evaluation is also taken 

by USEPA for inhaled hexavalent chromium and related lung cancer. It should be highlighted that for some 

metals e.g., arsenic it has been reported in the scientific literature that oral intake i.e., via drinking water also 

can give rise to lung cancer though oral intake mostly is referred to cancer in the oral cavity or 

gastrointestinal system. 

My question is if the lung was studied in the NTP studies or any of the animal studies reported in the given 

report? Same question goes for the epidemiological studies that are cited? 

Table 3-7 page 30 reports levels of chromium in female controls both in kidney and in 

bone. It is not easy to find any comments on this in the document. Is there any analytical 

problem in this study? 

G2. Please identify any additional studies that would make a significant impact on the conclusions 

of the Toxicological Review. 

To my understanding there are new experimental studies with more for this purpose fitted doses. It could be 

worthwhile to await the outcome of these studies to find out whether more appropriate values for both 

NOAEL and LOAEL will be reported and include these in an appendix if feasible. 
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Any further data obtained in the cited NTP studies should be included and presented to the reader. It would 

be of interest to know if there are any data on lung cancer or other effects from the NTP studies. 

During the workshop a number of ongoing studies were presented and it was suggested that they be paid 

attention to. It is always an advantage to get more and more information and research is always going on. 

In my opinion it is however important to set recommendations for exposure to toxic agents in order to protect 

humans from developing adverse health effects. It is a human right to be protected from unwanted exposure 

which also will cause unnecessary worry during the time from alert to protection. People expect regulatory 

agencies to make evaluations and set exposure limits. Studies underway even if published in peer review 

scientific journals should be carefully evaluated and scrutinized by EPS 's working group to determine if 

presented data is reliable e.g., based on a number of factors such as, just to mention a few, how large are the 

studies and what is the power o the study, analytical procedures that include quality control so data is 

validated and to be trusted. Based on experience it takes time before data will be available even for ongoing 

studies. I recommend that IRIS, EPA sets a recommendation based on information presented in the draft 

document In case important information which can change any evaluation shows up in time, such data can 

be included in the final document as an appendix or addendum. It is important in Risk Assessment to keep in 

mind that any recommendation set for exposure levels values needs to be reevaluated over time because by 

new techniques e.g., rapid development of usage of "omics" has to be considered. In view of said it is 

important to draw conclusions now and on data available now and not to wait. 

Chemical-Specific Charge Questions: 

(A) Oral Reference Dose (RID) for Hexavalent Chromium 

Al. A two-year drinking water study of sodium dichromate dihydrate in rats and mice (NTP, 

2008) was selected as the basis for the derivation of the RID. Please comment on whether the 

selection of this study as the principal study is scientifically supported and clearly described. 

Please identify and provide the rationale for any other studies that should be selected as the 

principal study. 

EPA suggests an oral RID of9x104 mg/kg-day. 

The epidemiology studies in Liaoning province, China (p 68-76 in draft report) reported increased incidence 

of cancer after intake of drinking water contaminated with hexavalent chromium. It is stated in the document 

to be the only reported human data. That study supports the statement of hexavalent chromium in drinking 

water to be carcinogenic. 
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It should be explained to the reader why sodium dichromate dehydrate was chosen for oral exposure study. 

Data in the literature indicates that bioavailability and bioaccesibility depends on species of the compound 

and also exposure media. Are there any data on hexavalent chromium species in the drinking water in the 

general environment? The NTP studies that are reported have used doses that are much higher than reported 

present concentration in drinking water in the general environment. Thus values for LOAEL is identical to 

lowest administered dose. It has not been possible because of applied doses to set a NOAEL. 

A2. Diffuse epithelial hyperplasia in the duodenum of female mice was selected as the critical 

effect for the RID. Please comment on whether the selection of this critical effect is scientifically 

supported and clearly described. Please identify and provide the rationale for any other endpoints 

that should be selected as the critical effect. 

Perhaps some information on possible effects on the lung should be comment on. The document should give 

information about solubility of different chromium (VI) species should be given and specifically for the 

chromium species that have been used in the quoted studies. Soluble salts are mentioned on page 54 under 7 

but soluble in what media is not mentioned. The reference WHO/IPCS (2006) Environmental Health Criteria 

234, Elemental speciation in Human Health Risk Assessment, WHO, Geneva is recommended to be 

included. 

A3. Benchmark dose (BMD) modeling was applied to the incidence of diffuse epithelial 

hyperplasia in the duodenum of female mice to derive the point of departure (POD) for the RID. 

Has the BMD modeling been appropriately conducted and clearly described? Is the benchmark 

response (BMR) selected for use in deriving the POD (i.e., a 10% increase in the incidence of 

diffuse epithelial hyperplasia) scientifically supported and clearly described? 

Yes 

A4. Please comment on the rationale for the selection of the uncertainty factors (UFs) applied to 

the POD for the derivation of the RID. Are the UFs scientifically supported and clearly described? 

If changes to the selected UFs are proposed, please identify and provide a rationale. 

The reason for chosen UFs is clearly and properly described. 

It is known that the reduction of hexavalent chromium to chromium three is influenced by vitamin C. This 

can perhaps be used in setting UFs and thus not only choose the standard UFs of I 0 between species and I 0 

for interindividual differences. Humans can not synthesize vitamin C and are thus depending on vitamin C 

supplementation. The tested animals i.e., the mouse and the rat both produce vitamin C themselves. In this 

context a laboratory animal that resembles the human by being depended on vitamin C supplementation 

might be used in future studies. The concentration of vitamin C in tissues and organs are very important in 
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evaluation of carcinogenic metals. It is likely to be involved in the mechanism in causing cancer and plays a 

role in the MOA. 

(B) Carcinogenicity of Hexavalent Chromium 

Bl. Under EPA's 2005 Guidelines for Carcinogen Risk Assessment 

(www.epa.gov/irislbackgrd.html), hexavalent chromium is likely to be carcinogenic to humans by 

the oral route of exposure. Is the cancer weight of evidence characterization scientifically 

supported and clearly described? 

Though it is an American document prepared for US I would recommend also to consult and cite documents 

published by the United Nations Organizations such as International Agency for Research on Cancer (!ARC) 

and World Health Organization (WHO). Recommended literature is IARC Monographs on the Evaluation of 

Carcinogenic Risks to Humans Volume 49 (1990), Lyon, France and Chromium, Nickel and Welding 1-677 

pages and !ARC Monographs on the Evaluation of Carcinogenic Risks to Humans Volume 100 which is in 

preparation and further information on www.iarc.fr 

Hexavalent chromium is classified as a human carcinogen. It is not clear to the reader why 

classification can be different upon different route of exposure. 

B2. A mutagenic mode of carcinogenic action by all routes of exposure is proposed as the primary 

mode of action for hexavalent chromium. Please comment on whether this determination is 

scientifically supported and clearly described. Please comment on data available for hexavalent 

chromium that may support an alternative primary mode of action. 

Again this might be linked to differences among hexavalent chromium species regarding for example 

solubility in body fluids. 

Somewhere in the document it should be pointed that iron can reduce hexavalent chromium to chromium 

three. This has been done in some products. This is touched by on page 48 line 18. 

The document describes in detail the possible MOA. IARC 1990 stated " ... chromium (VI) compounds nn 

the basis of the combined results of epidemiological studies, carcinogenicity studies in experimental animals, 

and several types of other relevant data which support the underlying concept that chromium(VI) ions 

generated at critical sites in the target cells are responsible for the carcinogenic action observed" 

However, also alternatives like DNA- methylation and other epigenetic mechanisms should be considered, 

because for many metals DNA-methylation is recognized as a possible mode of action also addressed as 

mechanism for carcinogenicity. Effects on cell signalling and gene expression may also serve as mechanisms 
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involved in carcinogenesis of metallic compounds. See further discussion in review by Davidsson et al, 

Chapter 5 in Nordberg et al (eds) Handbook on the Toxicology of Metals, p79-IOO 

B3. A two-year drinking water study in rats and mice (NTP, 2008) was selected for the derivation 

of an oral slope factor. Please comment on whether the selection of this study for quantification is 

scientifically supported and clearly described. Please identify and provide the rationale for any 

other studies that should be considered. 

In this document it is not explained to the reader what decided the selection of chromium species and 

selection of doses and how the chosen doses relate to exposure in drinking water in the general population. 

Lethal doses should also be given for the chromium species that have been used in the quoted studies. 

To evaluate and compare the outcome of studies and concentration levels in tissues, a ratio of 

concentration in dry weight to concentration in wet weight would make it possible and easier to compare 

reported results and also of possible intake of hexavalent chromium in drinking water. It is told that the 

animals by increasing exposure to hexavalent chromium in drinking water showed a decrease in intake of 

drinking water. Influence on different tissues will be found in doing this. On page 112 NTP 2008 decreased 

body weights could be explained by reduced drinking water consumption. 

B4. The incidence of adenomas and carcinomas combined in the small intestine of male mice from 

the NTP (2008) two-year drinking water study were selected to serve as the basis for the 

quantitative cancer assessment. Please comment on whether this selection is scientifically 

supported and clearly described. Please identify and provide the rationale for any other endpoints 

that should be selected to serve as the basis for the quantitative cancer assessment. 

See comments above. It should be noted that pH is different in different parts of the gastrointestinal system. 

BS. The oral slope factor was calculated by linear extrapolation from the POD (i.e., the lower 95% 

confidence limit on the dose associated with 10% extra risk of tumors of the small intestine in male 

mice). Has the modeling been appropriately conducted and clearly described? 

The model is clearly described. However I feel very uneasy about extrapolation to lower exposure levels 

because of chosen exposure doses where LOAEL is identical to the lowest exposure dose administered. 

Other organizations like IARC do not perform any quantitative evaluation of carcinogenic 

agents/substances. Threshold concentrations are problematic because of lack of knowledge of how the 

carcinogenicity develops. Once an organism has been exposed to a substance that can give rise to cancer 

there is a possibility to such an effect to occur. 
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POST-MEETING COMMENTS SUBMITTED BY 

Steven R. Patierno, Ph.D. 
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Toxicological Review of Chromium 

Charge Questions 

General Charge Questions 

I. Is the Toxicological Review logical, clear and concise? Has EPA clearly presented and synthesized the 

scientific evidence for non-cancer and cancer hazard? 

I will offer a response to this question in the form of general comments regarding specific sections of the 

Review in order of appearance in the text. In taking this approach my comments will also directly address 

questions (A)l-4 and (B)l-5. 

Page 7: The Environmental Protection Agency (EPA) should not be referencing a 2006 review article by 

Costa and Klein to site background on environmental chemistry. This review article was not a critical review 

of the environmental chemistry of chromium. Even if the general background in that review article is 

accurate, The Toxicological Review of Hexavalent Chromium (TRHC) should cite primary references from 

chemical or environmental journals or compendiums. Also, this paper is mis-labeled in the reference list as a 

2008 paper. [Correct reference is "Toxicity and Carcinogenicity of Chromium Compounds in Humans" Crit. 

Rev. Tox.: 36(2):155-163, 2006]. 

Moreover, the premise of this review article [in essence that even very low exposures to any form of CrVI, 

including in drinking water, can cause virtually any type of cancer in virtually every organ, as well as 

plethora of assorted other diseases], and the preceding review article on which it was largely based ["Toxicity 

and Carcinogenicity of Cr(VI) in Animal Models and Humans" Crit. Rev. Tox.: 27:431-442, 1997], should 

not be universally accepted by the EPA without critical evaluation. Much of the epidemiological 

methodology applied in these papers is flawed. In these papers, the author(s) repeatedly and selectively 

tabulated whatever instances could be found in any of the many epidemiologic studies of chromium, of an 

elevated Standard Mortality Ratio (SMR). These were presented with no mention of the fact that most of 

these instances were small, non-statistically significant elevations (likely to be random fluctuations due to the 

large breadth of the studies), which were either ignored or discounted by the original authors because of 

confounding factors. The paper also failed to take into account that many of the small, non-statistically 

significant elevations in some cancers in one selected study, were counter-balanced by either no elevation or 

decreased SMRs in other studies. This "tabulation" approach does not constitute a true meta-analysis and is 

also statistically incorrect. 
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There are also additional reasons that the EPA should be circumspect about citing either of these articles. 

The 2006 article, and its preceding counterpart published in 1997, were written and published at a time when 

the senior author was actively engaged as an expert witness for the plaintiffs in high-profile hexavalent 

chromium lawsuits. This involvement was not disclosed in the 1997 article, which was focused on 

attempting to implicate low dose exposure to CrVI in a broad array of human cancers. In the 

Acknowledgements section of the 2006 article there is partial disclosure that production of the paper was paid 

for in part by Baron and Budd. In fact, Baron and Budd is one the law firms with whom the senior author 

was under contract with as an expert witness for the plaintiffs in an active lawsuit. This article specifically 

tried to implicate CrVI as a human drinking water carcinogen even at very low doses, as well as suggesting 

that exposure to CrVI causes a broad array of other diseases, including neuropsychiatric problems, for which 

there is no support. If the EPA is going to site these review articles it is critical that EPA conduct an 

independent critical review of every paper sited in these review articles. In the latter scenario it is certain that 

EPA will reach a different conclusion. 

I am not of the opinion that a scientist who serves as an expert witness should have to disclose all litigationrelated work in all scientific publications, particularly not in reports of original laboratory research into 

mechanisms of action, or even in review articles that give an unbiased evaluation of the existing literature, 

especially as it relates to basic mechanisms of action. Indeed, in the world of chromium toxicology it is hard 

to find experts who have not participated in some sort of chromium-related litigation. However, these two 

articles do not merely describe original laboratory research or present an unbiased review of the literature 

(note that part of the 2006 review article is a recapitulation of an already published journal article on UV light 

and chromium exposure). These two particular articles are essentially position/opinion papers, with 

speculative declarations that even very low dose exposures to soluble CrVI can cause virtually any kind of 

cancer (and other disease) in virtually any organ, a theory of obvious benefit to any plaintiffs case in 

chromium-related litigation, but one that is not supported by either epidemiological studies or in vivo animal 

studies. 

Page 20-21: Although the draft TRHC frequently describes each specific study in this section and offers a 

conclusion/interpretation, the TRHC discussion of the Donaldson and Barrera paper ends with a reference to 

Table 3-1 with no summary. There is important information in Table 3-1 that strongly supports the capacity 

of gastric juice to rapidly reduce CrVI to CrilL Note that the uptake of Cr in intestinal rings was virtually 

identical, whether the starting material was Crill without gastric juice or CrVI plus gastric juice at pH 1.4. 

The text at the bottom of page 21 and the top of page 24 seems to be nuanced to cast doubt on the body of 

work of DeFlora, by using the word "suggested" (second line from the bottom ofpg 21), and then suggesting 
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Steven R. Patierno, Ph.D. 

that the values of reducing capacity given by DeFlora "should be considered with some caution". This 

"caution" is based on speculation found in the paper cited (Zhitkovich, 2005), and reiterated in the 2006 

Costa and Klein article (from where the draft TRHC apparently drew its language). This speculation is 

addressed in the supplementary materials under "Public Comments". The TRHC and the EPA should not 

cast doubt on the body of work by DeFlora, based on unsubstantiated speculation. 

Page 24-5: The TRHC should recognize and illustrate the main point of the absorption studies cited: no 

matter whether the original starting material is CrVI or Cr!II there is limited absorption and little retention of 

either: fecal recovery in rats was 98% for Cr!II and 97.7% for CrVI (pg 24) and in humans was 99.6% for 

Cr!II and 89.4% for CrVL Pretreatment of CrVI with gastric juice completely inhibited absorption of CrVI 

after direct perfusion into the small intestine. On pg 27 another study indicates that 99% of CrVI is 

recovered in feces using rats gavaged with CrVI. On pg 28 another study indicates that maximal uptake after 

gavage of rats with CrVI occurred in liver and was only 1%. Absorption in other organs was in the range of 

0.1 to 0.2%. It is important to note that in all of these absorption studies, including drinking water studies, 

the increased tissue distribution was only observed after chronic administration of more than 5 ppm. Many 

of the studies used greater than I OOppm. The main point that there is very little absorption and retention of 

Cr even after administration of CrVI. 

Page 26: It is incorrect to state that absorbed "hexavalent" chromium is distributed throughout the body. 

Few studies actually speciated the Cr found in organs distal to the route of administration and even extremely 

large doses of CrVI, large enough to saturate reduction in the stomach and GI tract, do not deliver much more 

than trace amounts of CrVI to most distal organs because of the vast reducing capacity of blood components. 

The vast majority of Cr reaching distal organs arrives as Criii. The TRHC should make this absolutely clear. 

Page 34: The TRHC should provide an accurate summation of Table 3-8 which compares tissue chromium 

after ingestion of a very large, gastric reduction-saturating dose (12.9 mg!L) of either Cr!II or CrVI. The 

only "organ" that showed consequential increased levels of Crafter CrVI compared to Crill was blood. Most 

of the other organs exhibited only trace amounts of Cr, even after this huge dose of CrVI, except for the 

intestine which showed significant and nearly identical increased Cr concentrations after both Crill and CrVI. 

This supports the conclusions of the supplementary data in the Public Comments showing that some sections 

of the intestine (jejunum for example) are sites of Cr accumulation, regardless of whether the source material 

is Cr III or Cr VI. 

Page 35: Note that there is no increased accumulation after 8 weeks of exposure compared to 4 weeks of 

exposure, even at the enormous dose of 130 ppm (mg/L). 
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Page 36: The NTRC should not interject the commentary statement: "indicating that a portion of the CrVI 

escaped intracellular reduction in the GI tract and became bioavailable for systemic distribution". Like 

almost every other study this study measured total tissue Cr and did not speciate tissue Cr, and the NTRC 

therefore cannot speculate on what the form of Cr was that reached the tissues. Note in Table 3-8 the 

accumulation of Cr in the intestine and blood after Crill. 

Page 37: Note the obvious threshold of increased bone and kidney concentrations after 10 ppm compared to 

all lower doses, as well as estimated body Cr burdens. Note that in liver there is a significant Cr burden even 

after ZERO CrVI exposure. Note the strangely compressed scale of theY axis: even the increase in females 

at I 0 ppm is only an increase from 0.3 to Jess than 0.5. These data demonstrate the exact opposite of the 

conjecture on page 36: there is a clear threshold of accumulation indicating saturation of reductive capacity. 

Pages 38-39: Virtually every study shows the same thing. The NTP study used a "low dose" that is already 

higher than the 10 ppm in the Sutherland study. What is being referred to here as a "dose-dependent" 

increase is already supra-saturation of gastric reductive capacity. What these studies really show is how little 

Cr is absorbed, even in tissues that are directly exposed (glandular stomach and forestomach), even after 

massive doses are administered. 

Page 41: It is inappropriate to make such an unqualified statement as found at the top of this page: 

"Hexavalent chromium is capable of crossing the placenta". This is only true in the highly contrived 

circumstances referenced below the statement wherein pregnant mice were given an IV injection of a massive 

dose of CrVI (!Omg/kg). 

Page 42: The TRHC does a good job describing the bioavailability and kinetics of Cr absorption in humans 

after Crill, CrVI in OJ and CrVL It correctly acknowledges that the CrVI-OJ was completely reduced and 

that even the full dose of CrVI was insufficient to overwhelm the reducing capacity of blood. The potential 

explanations offered are correct but need to add another possibility. Often overlooked is the fact that not all 

Crill is alike. Anyone who works with Crill in the laboratory knows that it undergoes aging in aqueous 

solution, even visibly changing color with time after solubilization. It is possible that Crill generated from 

newly reduced CrVI (as in the CrVI-OJ) may have some different biological parameters than straight Crill 

made up in water and allowed sit for a couple of days. In fact overall absorption of newly formed Crill may 

be higher than aged Crill, possibly as a function of its ability to form complexes with biological ligands that 

may alter its absorption potential. 
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Page 49 bottom: It is inaccurate to state that "CrVI distributes to other tissues, notably the blood, kidney, and 

liver." Except for the cases of treatment with extreme doses, or use of pathways like intra-intestinal 

instillation or IP injection, the vast majority of Cr that arrives at distal tissues is CrilL Once again, it is 

critical that TRHC make that fact clear, otherwise it gives the appearance of non-objectivity. 

Page 50, last paragraph: The TRHC should not simply reiterate speculation that is found in the papers it cited 

(Zhitkovich 2005, Costa and Klein 2006), in suggesting that the mutagenicity of Cr may be underestimated in 

cultured cells because of lower levels of intracellular ascorbate when cells are cultured in absence of added 

ascorbate. Indeed, it is just as likely that the mutagenicity of CrVI in cultured cells is grossly overestimated, 

because the lack of ascorbate in the extracellular medium allows CrVI to persist in the extracellular medium 

thereby maximizing its uptake as the hexavalent oxyanion. At the very least the TRHC should discuss both 

possibilities and not give the appearance of bias. 

Page 58: It is inaccurate to state that model simulations "imply" that some CrVI escaped reduction in the 

stomach. This is circular reasoning. The "input" data that went into formulating these models was based on 

experiments wherein massive doses of CrVI were administered, doses that would clearly exceed reductive 

capacity. It is not appropriate to then state that the model simulation "implies" that some CrVI escaped 

reduction, as though the model now supports a novel biological observation. It would be completely 

expected for the model to predict that scenario since it would logically emanate from the very data that was 

used to formulate the model. It is critical that the TRHC indicate that these models do not apply to, or 

accurately predict, the toxicokinetics of low, environmentally relevant doses of CrVI. The discussion in the 

THRC does become more balanced on page 64 where the non-linear aspects of CrVI uptake, reduction and 

bio-distribution are given some weight. 

Page 66: This section (3.6) needs to be completely rewritten as it lends undue weight to an opinion expressed 

in only one or two papers, at least one of which was written under financial inducement by a law ftrm with a 

vested interest in characterizing all Cr, including Crill, as a potential hazard (see preceding comments). The 

TRHC needs to not indiscriminately cite speculation found in review articles without more rigorous analysis. 

Except for those few biased citations it is almost universally accepted that Crill is an essential element. 

Page 68: Section 4.1.2, last sentence: This is nearly the ultimate example of how critically important it is for 

the TRHC to do its own critical analysis of the literature. The paper cited, (Bick et al, 1996) should not be 

cited under any circumstances and in fact it should be retracted from the scientific literature. Two of the 

authors, Walter Lack and Thomas Girardi, were two of the lead lawyers for the plaintiffs in several highprofile chromium lawsuits, now immortalized by the Hollywood movie "Erin Brockovich". They listed their 
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"academic" credentials as the Department of Hematology at the University of Tasmania in Australia. The 

other three authors (Costa, Bick and Teitlebaum) were paid expert witnesses for the plaintiffs in the same 

case, which was active at the time. None of this was disclosed in the paper. The two cases of NonHodgkin's lymphoma discussed in this case report were plaintiffs in the active lawsuit and the information 

was supplied by the lawyers. Moreover, at best this report is merely a case-report (not even a case-control 

study), merely reporting that two people in Hinckley CA, at that time, had been diagnosed with NonHodgkin's lymphoma. 

In contrast, the draft TRHC does not yet, but should reference the recent work of Dr. John Morgan, an 

epidemiologist for the California Cancer Registry. He has been tracking cancer incidence in the town of 

Hinckley CA (the "Brockovich" town) from 1996 to present. He recently published data showing that from 

1996 to 2008, not only is there no excess of total cancer or any specific cancer in Hinckley, there are actually 

fewer cancers than expected. 

Pages 71-80: The draft TRHC conducts a very thorough depiction of the different interpretations of the 

Liaoning Province situation. What seems to get lost in the details is the larger picture. This is a Province of 

a country wherein the background rates of both stomach and lung cancer are high even in non-chromium 

exposed comparison groups, indicating the presence of other contributing risk factors. This is a situation 

where exposure is characterized in terms of high dose, long term "yellow water", yet despite this potential 

significant exposure, the question of whether there is an additional modest increase in risk for stomach cancer 

hangs on whether a particular industrial area is included in the comparison group or not. There is much 

controversy surrounding the reports of cancer risk in this Province, but after discussing the controversy the 

draft THRC aligns itself with the method of re-analysis of Beamon! et al. The THRC should then also cite 

the commentary by Allan Smith [Epidemiology 19:24, 2008) which accompanied the Beamont article: 

although Smith is sympathetic to Beamont's attempt to re-analyze the data, he also describes the extensive 

weaknesses of the approach. This is not the kind of data that a regulatory decision should be based on. 

Page 81: The NTP toxicology studies on subchronic oral exposure (Section 4.2.1) are technically well done. 

The principle issue that needs to not be lost in the detail is that the lowest dose was 62.5 ppm, an enormous 

concentration of little or no environmental relevance. This is a "yellow water" situation to the extreme. 

Despite these enormous doses most of the observations did not exhibit a consistent pattern of dose or duration 

dependence. It is also important to recognize that these enormous doses of CrVI actually serve to deliver an 

enormous amount of Crill to the organs and cells in question. Remembering that Crill is not without 

biological activity (acting as a co-factor in insulin action), it is entirely possible the some of the observed 
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effects are due to the physiological effects of massive Criii overload. The extensive new data provided by 

ToxStrategies, described in the Public Comments, needs to be incorporated into the TRHC. 

Page 84: Again, a consistent relationship between severity and dose was not observed. This implies the 

presence of effects caused by indirect mechanisms, likely chronic inflammation and/or tissue damage only 

observed at the highest doses (see below). Urinalysis shows effects due to decreased water intake due to poor 

palatability of the yellow water. This dehydration alone is capable of rendering epithelial tissues more 

fragile. Changes in organ weights were only observed at doses above 500ppm (pg 86). 

Page 87-108: The results are described repeatedly as "without clear dose-response relationship". Indeed, 

minimal to mild histiocytic cellular infiltration was observed in all groups including the control animals. 

Even less toxicity was observed in mice compared to rats; in fact even at 1000 ppm for 3 months there was 

no evidence of any hepatotoxicity, only mild changes in some hematological indices that were attributed to 

changes in body weight (probably caused by massive Criii overloading and its potential effects on insulin 

and glucose metabolism). What needs to be emphasized here is that the lowest dose used in any of these 

studies is at or above saturation of gastric reductive capacity and yet still very little toxicity was observed 

except at the two highest doses (and often only at the one highest dose) (Tables 4-12,4-13, 4-14). At the 

lower end of these very high doses, only inconsistent observations were made and when "toxicity" was 

reported it was generally ranked minimal to mild. Only the index of Liver (fatty change) was ranked as 

moderate, but that was identical to the ranking of that same index in the Controls. The main point here 

should be that these are massive doses and they are eliciting minimal effects. This important concept should 

not be lost in the mass of detailed results. 

Pages 109-120: The NTP carcinogenesis studies in rats and mice show that there is no carcinogenic response 

except at the two highest doses that also produce chronic tissue damage at the sites of carcinogenicity. The 

dose-response is definitively non-linear, as is the absorption data described above. Given that the lowest 

dose is already above the reductive capacity of the oral cavity and stomach, these data provide strong 

evidence of the protective effects of the reductive capacity of blood components. 

It should be noted that the NTP's published report by Stout et al [Hexavalent Chromium is Carcinogenic to 

F344/N Rats and B6C3Fl Mice after Chronic Oral Exposure, Environmental Health Perspectives 117: 716, 

2009] presents an inaccurate Discussion of potential mechanism of action, drawn heavily from the 2006 

Costa and Klein article, especially in criticizing the work of DeFlora. In point of fact, the results of the NTP 

assay, and the extensive additional data found in the Public Comments generated by a group of investigators 

around the country funded by ToxStrategies, give nearly definitive proof that the work of DeFlora is correct. 
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Steven R. Patierno, Ph.D. 

Even the lowest dose of the NTP assay exceeds the reductive capacity of the oral cavity and upper digestive 

tract. Yet little toxicity and no carcinogenicity is observed except at the two highest doses. 

The argument by Stout et al that the NTP doses were below gastric reduction-saturation, based on a supralinear (decreasing response with dose) rather than sub-linear (increasing response with dose) dose response is 

incorrect. If the doses were below saturation of reductive capacity, as the dose increased the ratio of 

unreduced CrVI to reduced CrVI (Crill) in the stomach would increase (due to depletion of reductive 

capacity), and absorption would show an increasing rate of response (opposite of what was observed) because 

of an increased percentage of the total Cr that would be in the unreduced hexavalent state. Yet both 

absorption and toxicity exhibit a decreasing rate of response with dose in the NTP assay. This would actually 

be expected at supra-saturation doses: once the reductive capacity of the oral, digestive and blood 

components is exceeded, the organs receiving the highest amount of CrVI will sustain inflammatory tissue 

damage provoking tissue regeneration. It is unlikely that such tissue damage would display dose dependence 

since it only occurred at the two highest doses of the assay and it is a complex, disseminated biological 

response. It is likely then that a combination of three factors contribute to the high dose carcinogenic 

response: (i) tissue damage with regenerative cell profieration, (ii) regenerative cell proliferation in the 

presence of macromolecular damage, and (iii) regenerative cell proliferation occurring in the presence of 

massive Crill loading, which may affect insulin-dependent proliferative signaling. 

Pages 122-149: For these studies on the potential reproductive toxicities caused by CrVI one can only hope 

that the TRHC and EPA will remember the 16'" century adage of Paracelcus "all substances are poisons, the 

right dose differentiates a poison from a remedy". These studies show reproductive toxicity at huge doses of 

CrVI, often given using invasive administration procedure (IP or IV injection), with little relevance to 

environmental exposure levels. 

Pages 176-178: The in vivo studies showing DNA damage or mutagenicity in cells peripheral to the point of 

administration of CrY! were only positive when massive doses of CrVI were administered by gavage, direct 

instillation, or intravenous injection. Although some studies claim to find mutations in the absence of 

cytotoxicity these are highly contrived systems: for example eye spots in offspring of pregnant female mice 

given huge doses (62.5 ppm) of CrVI in drinking water. All studies of mutagenesis in cultured mammalian 

cells, including human cells, demonstrate that mutagenesis is only observed at doses that produce some 

degree of cytotoxicity and replication arrest. It should also be clarified in the TRHC that DNA damage and 

mutagenicity should not be equated: while mutagenicity may result from DNA damage, the relationship is 

not simple or linear and is further complicated by DNA repair. Also, it is unclear whether all forms of DNA 

or chromatin alterations (collectively termed DNA damage) are pre-mutagenic. For example, in silico studies 
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Steven R. Patiemo, Ph.D. 

on DNA-protein crosslinks suggest that under certain circumstances Criii can serve as binary crosslinking 

agent between small peptides and DNA. However, in in vitro studies in cultured cells and in in vivo studies, 

it is not clear what is actually being measured by assays for DNA-protein crosslinks. This phenomenon may 

in fact only indicate that chromatin isolated from certain cells exhibits a higher degree of condensation during 

isolation, rendering chromatin proteins more difficult to extract. What appear to be DNA-protein crosslinks 

can be actually be observed in cells treated with agents that do not participate in or catalyze formation of an 

actual binary crosslink. 

Pages 202-214 (section 4.7.3.2): Many of the preceding comments directly address and provide major 

qualifications to the MOA discussed in this section, including the interpretation of reductive capacity found 

in the Stout et al report of the NTP assay. 

It is clear that the EPA is faced with a unique situation in assessing the MOA of Cr(VI) at it relates to lowdose risk assessment. It is abundantly clear from all the science that the effects of Cr(VI) at the massive 

doses necessary to produce tissue toxicity and carcinogenesis in rodents, have no bearing on the effects of 

low-dose, environmentally-relevent exposures. This is consistently borne out by epidemiological, animal and 

cell experimentation. 

This is especially pertinent in relation to whether or not Cr(VI) should be considered with a mutagenic MOA. 

I have spent more than 25 years studying the genotoxic properties of Cr(VI) and I have frequently contributed 

to the plethora of studies showing DNA damage and what we thought was associated mutagenesis. There is 

no doubt that Cr(VI) can be forced to be genotoxic and "mutagenic" under experimentally contrived systems 

and at high doses that evoke major amounts of cell death. However, in hindsight many of us "DNA damage 

and repair" scientists have come to appreciate several important factors: (i) DNA damage is only observed at 

very high dose that kill a lot of cells, (ii) Cr(VI) is at best a very weak "mutagen", requiring very high doses 

that kill most cells and experimental "backflips" to select for survivors, and (iii) what we thought was 

"mutagenesis" is actually selection for stochastic cell survivors of massive toxic insult. Dr. Rossman's group 

has shown that the base sequence of the genes used for mutation detection and selection is intact and that the 

changes in gene expression enabling selection are epigenetic, not mutagenic. Our group has shown that what 

we really selected for at toxic exposures are cells that are resistant to apoptosis, and Dr. Zhitkovich's group 

has shown that the "mutant" cells were actually surviving cells that were selected for changes in specific 

forms of DNA repair. Again, this only occurs at doses that kill a lot of cells, not dis-similar to the high-dose 

rodent assays wherein tumors were only observed at doses that produced chronic and fairly severe tissue 

damage. This harkens to what is sometimes viewed as a landmark study of lung cancer and occupational 

exposure to high doses of Cr(VI) by Gibb et al. Occupational exposure to in the chromate production 
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Steven R. Patierno, Ph.D. 

industry was categorized into 4 quartiles. The lowest two quartiles are huge levels of exposure by 

"environmental" standards, orders of magnitude beyond the even the highest known environmental 

exposures. The lowest quartile of exposure was essentially a No Effect Level (no elevated risk) and the 

slightly elevated risk ratio in the second quartile was not statistically significant. Interestingly, of the total of 

120 lung cancer cases found in chromium-exposed workers, 116 were also smokers. 

The EPA may be under certain historical regulatory precedents and pressures to deem Cr(VI) with a 

mutagenic mode of action simply because there are published studies that have "Cr(VI)" and "mutation" 

equated in the title (some of these papers are my own), but this decision would not be based on science. At 

high, tissue damaging doses one can get tumors to form and those tumors will have mutations in specific 

genes because that is the molecular etiology of how that particular cancer develops. It will haven? relation to 

any chemically-specific mutations caused by Cr(VI) because Cr(Vl) is an exceedingly poor mutagen. Even 

at the low end of very high doses there is NO MOA because there is NO toxicity, no mutagenecity, and no 

carcinogenesis. Extrapolating linearly from events observed at the two highest doses of the NTP assay, to 

anything close to reality for environmental exposure, is simply not scientific. If ever there was a textbook 

case to be made for a "threshold carcinogen", it is Cr(Vl). 

General Charge Questions: 

G2. The TRHC should absolutely consider the extensive 9ew data being provided by ToxStrategies and 

presented in part in the supplementary section under Public Comments. 

Chemical-Specific Charge Questions: 

A!. The two-year drinking water study of sodium dichromate dyhydrate in rats and mice (NTP, 2008) is the 

most thorough and technically well-conducted study available. It is likely the best study available for 

selection. However, the interpretation of and conclusions drawn from that study need to be re-evaluated in 

light of the issues raised in my preceding comments and the additional data shown in the Pubic Comments 

and coming available from a multi-institutional study sponsored by ToxStrategies. 

A2. The selection of Diffuse epithelial hyperplasia in the duodenum of female mice as the critical effect for 

the RID should be re-evaluated in light of the issues raised in my preceding comments. It must be considered 

in the context of the non-linear, dose-related issues discussed above regarding saturation of reductive 

capacity and definitive threshold data for toxicity and carcinogenicity. 
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Steven R. Patierno, Ph.D. 

A3. See answer to A2 above. 

A4. The Uncertainty Factors must be re-evaluated in the context of the non-linear dose-response data, the 

clear evidence of thresholds for toxicity and carcinogenicity and the fact that these high-dose, suprasaturation experiments cannot be extrapolated linearly to low or vanishingly small doses. 

(B) Carcinogenicity of Hexavalent Chromium 

B 1. The cancer weight of evidence characterization is not scientifically supported. The conclusion that 

hexavalent chromium is "likely to be carcinogenic to humans by the oral route of exposure" is not 

scientifically supportable given the issues raised in my comments above. The non-linear dose data in both 

the NTP studies and the data preliminarily discussed in the Public Comments clearly demonstrate tbat the 

toxicities and carcinogenesis observed at these extremely high, obviously supra-saturating doses, cannot and 

should not be extrapolated to lower doses. See detailed comments above. 

B2. The determination of a mutagenic mode of action by all routes of exposure should be re-evaluated before 

proposing it as the primary mode of action. Nearly all indices (NTP studies, inhalation studies, mammalian 

cell mutagenesis studies etc) indicate that carcinogenicity of CrVI is only observed under exposure 

conditions that evoke cellular toxicity, inflammatory tissue damage, and tissue regeneration. The DNA 

damage and presumed mutagenicity (actually epigenetic or stochastic selection of cells that survived toxicity) 

of CrVI is only observed at doses that also cause cell death and tissue damage. In vivo, these effects are only 

achieved at very large doses that clearly overwhelm the reductive capacity of the oral cavity, stomach and 

blood components resulting in a sharp threshold of carcinogenesis only at the two highest NTP doses. I have 

spent more than 25 years studying the molecular mechanisms of CrVI genotoxicity and mutagenesis and I 

have a deep appreciation for its capacity to interact with cells and alter DNA and DNA replication and 

transcription. However, just because CrVI is capable of causing DNA damage and what we thought was 

"mutagenicity" (see above) in carefully contrived experimental systems, does not mean that it does so under 

physiologically and environmentally relevant conditions. It is much more likely that the chronic tissue 

damage, with accompanying inflammation and subsequent proliferative regeneration, possibly in the 

presence of unrepaired DNA damage, all of which are only observed at the highest doses, is the principle 

mode of action. 

B3. See previous comments above. The NTP study is the best study available but the interpretation of the 

data and conclusions drawn from it are incorrect. Important supplementary data is preliminarily discussed in 

the Public Comments. 
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Steven R. Patierno, Ph.D. 

B4. See previous comments above. The NTP study is the best study available but the interpretation of the 

data and conclusions drawn from it are incorrect. Important supplementary data preliminarily discussed in 

the Public Comments. 

B5. See previous comments above. The linear extrapolation from the POD is not appropriate. CrVI 

toxicity and carcinogenicity demonstrates distinct non-linearity and there is little or no relation between what 

is observed at the highest doses in the NTP study and any physiologically-appropriate or environmentallyrelevant exposure. 
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POST-MEETING COMMENTS SUBMITTED BY 

Toby G. Rossman, Ph.D. 
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Toby G. Rossman, Ph.D. 

Toxicological Review of Hexavalent Chromate 

General Charge Questions 

G l. Is the Toxicological Review logical, clear and precise? Has EPA clearly presented and synthesized 

the scientific evidence for noncancer and cancer hazard? 

In general, this was a clearly written document. However, my area of expertise is in the mutagenic and 

epigenetic mechanisms of action of carcinogens, and I found many more problems in those areas than in the 

rest of the document. I will discuss these in section B2. 

Here I will mention some errors in the rest of the text. 

p. 38, end of I" paragraph reads, "Uptake in guinea pigs did not appear to generally differ from that of 

rodents". The guinea pig (Cavia porce/lus) is a rodent. 

p. 46, section 3.3, refers to transport of the hexavalent chromium oxyanion (for clarity, should this read 

chromate/dichromate?) by sulfate and phosphate transport system§. (should be pleural). It is claimed that this 

allows accumulation in cells at higher concentrations than the extracellaular concentration. Neither the 

transport systems nor the evidence for higher intracellular accumulation are referenced. Actually, what 

allows higher accumulations is the fact that Cr(6) is reduced in the cell to Cr(3), which cannot get out, so 

what accumulates is Cr(3). 

p. 20 I, 1" full paragraph: It is claimed that the key precursor events leading to chromium-induced 

mutagenicity have been identified in animals. This is not so. It is not even true for mammalian cells in 

culture. Some ideas have been derived from cell culture studies (but with Cr-damaged shuttle vectors). 

Almost nothing is known about mutagenicity in animals, and nothing at all is known about the genetic 

changes occurring in animal tumors or in the target tissues. 

p. 202. Section 4.7.3. For reasons that will become clearer in my response to B2, this section is very flawed. 

I will mention just one point here: The confusion between "mutagenic" and "genotoxic" must be cleared up 

throughout this section (as well as throughout Section 4.5.). The thinking on this issue is very sloppy. A 

mutagenic mode of action is just that: it requires mutagenesis. 

G2. Additional studies that would make a significant impact on the conclusions of the Toxicological 

Review. 

Some of these will be presented in section B2, as they pertain to mode of action. 

It is extremely important that the new information supported by American Chemical Council (performed by 

ToxStudies and others) should be considered before the final document is completed. They address a number 

of missing data sets. It is already clear that proliferative increases occur in the mouse duodenum at doses of 

Cr(6) lower than those that cause tumors. Also, there is evidence for cytotoxicity at these lower 

concentrations that may be driving the proloferative responses. 

The fact that Cr is an essential element needs to be addressed. What are the implications for a threshold? 
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Toby G. Rossman, Ph.D. 

It is possible that dietary Cr(6) is significant and should be evaluated. All parts of grain contain Cr(6) and 

10% of the Cr in bread is Cr(6) (Mishra eta!., Food Chern. Toxicol. 33:393-397, 1995; Soares et a!., J. Agric. 

Food Chern. 58:1366-1370). 

River waters have a median Cr value [which is probably Cr(6)] of 10 ppb (range <1-30), and even rainwater 

has a range from 0.14-0.9 (ATSDR, Chromium, Draft for Public Comment, online). 

A recent meta-analysis of cancers of the G.!. tract among those occupationally exposed to Cr(6), concludes 

that these workers are not at greater risk than the general population (Gatto eta!., Cancer Epidemiol. 34:388-

399, 2010). Inhalation exposure usually leads to G. I. exposure, suggesting a possible threshold if the ingested 

dose can be estimated. 

(A) Oral Reference Dose (RID) for Hexavalent Chromium 

Al. A two-year drinking water study of sodium dichromate dihydrate in rats and mice (NTP, 2008) 

was selected as the basis for the derivation of the RID. Please comment on whether the selection 

of this study as the principal study is scientifically supported and clearly described. Please identify 

and provide the rationale for any other studies that should be selected as the principal study. 

This does seem like the best and most complete study to use. 

A2. Diffuse epithelial hyperplasia in the duodenum of female mice was selected as the critical effect for 

the RID. Please comment on whether the selection of this critical effect is scientifically supported 

and clearly described. Please identify and provide the rationale for any other endpoints that 

should be selected as the critical effect. 

This seems like an appropriate choice, but it's outside my area of expertise. 

A3. Benchmark dose (BMD) modeling was applied to the incidence of diffuse epithelial hyperplasia in 

the duodenum of female mice to derive the point of departure (POD) for the RID. Has the BMD 

modeling been appropriately conducted and clearly described? Is the benchmark response 

(BMR) selected for use in deriving the POD (i.e., a 10% increase in the incidence of diffuse 

epithelial hyperplasia) scientifically supported and clearly described? 

This does seem like the best and most complete study to use (but it's a bit outside my area of expertise). 

A4. Please comment on the rationale for the selection of the uncertainty factors (UFs) applied to the 

POD for the derivation of the RID. Are the UFs scientifically supported and clearly described? If 

changes to the selected UFs are proposed, please identify and provide a rationale. 

This is outside my area of expertise. 

(B) Carcinogenicity of Hexavalent Chromium 

Bl. Under EPA's 2005Guidelines for Carcinogen Risk Assessment (www.epa.gov/iris/backgrd.html), 

hexavalent chromium is likely to be carcinogenic to humans by the oral route of exposure. Is tbe 

cancer weight of evidence characterization scientifically supported and clearly described? 
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Toby G. Rossman, Ph.D. 

Given the fact that there is not enough human data to firmly establish carcinogenicity, but there is animal 

data, "likely to be" is reasonable but "possibly carcinogenic at high dose" would be more accurate. 

B2. A mutagenic mode of carcinogenic action by all routes of exposure is proposed as the primary 

mode of action for hexavalent chromium. Please comment on whether this determination is 

scientifically supported and clearly described. Please comment on data available for hexavalent 

chromium that may support an alternative primary mode of action. 

By definition, the "mode of action" (MOA) of a carcinogen is "a sequence of key events and processes, 

starting with interaction of the agent with a cell, proceeding through operational and anatomical changes, and 

resulting in cancer formation" (USEPA, 2005). For mutagenesis to be a carcinogenic MOA, the agent must 

at the very least cause heritable mutations in mammalian cells. The mutations should be induced in a 

concentration range with low toxicity (preferably similar to concentrations seen in human exposures), and the 

mutations should be induced in the target tissues in animal experiments and in humans. Human and animal 

tumors should also show genetic alterations consistent with the types of mutations induced by the agents, and 

these should be early events. 

The information about Cr(6) is Jacking for much of these criteria. In fact, the human tumor data support an 

epigenetic mechanism more than a mutagenic one. 

Genotoxic is not the same as mutagenic, and sections 4.5 and 4.73 must be completely rewritten, as they 

consistently confuse these terms. Standard genotoxicity assays were not designed to inform specific modes 

of tumor induction. With the exception of mutagenesis, these other assays (non-mutagenic assays) do not 

measure heritable events, but rather measure evidence of DNA damage or its repair. Non-mutagenic assays 

include chromosome aberrations, micronuclei, comet assays, DNA lesion measurements, and DNA repair 

assays. These assays are useful for hazard identification or as biomarkers of exposure. They provide only 

supportive evidence that mutagenesis might be a MOA. DNA damage per se does not inform us about 

eventual heritable change, which is the true issue. Most (but not all) mutagens cause heritable changes in 

DNA sequences by causing damage to DNA (pre-mutagenic lesions) that is converted to mutation after cell 

division. 

Table 4-21 should be deleted, as results in bacteria are not relevant to tumorigenic MOA. A simple statement 

that Cr(6) is mutagenic in bacteria should suffice (referencing a review such as Klein CB. "Carcinogenicity 

and genotoxicity of chromium" In: Toxicology of Metals (Chang LW, ed). Boca Raton, FL:CRC Press, 1996, 

pp.205-219. 

Table 4-22 represents positive results in a group of assays that measure both mutagenic and non-mutagenic 

endpoints (including a paper on epigenesis, Klein et al., 2002, which should be removed, as this is not a 

genotoxic event; neither is disruption of mitosis, which can have many causes). Table 4.22 has neither 

information on the concentrations inducing positive results, nor on the toxicity of those treatments. 

This table is also deficient in the most important results in mammalian cells, i.e. mutagenesis. All of the 

studies reported are mouse lymphoma cell studies, yet later in the document (page 203), reference is made to 

mutations at the HGPRT locus in "Chinese Hamster ovary cells (V79 and AT3-2)" V79 is not a Chinese 

hamster ovary cell, it is a Chinese hamster lung fibroblast. In fact, one such study using V79 cells (Sugiyama 

et al., Mutat. Res. 260:19-23, 1991) shows a modest positive effect only in a narrow concentration range. A 

CHOline (AA8) showed a small increase (3.4 fold) at a dose giving 75% survival (Brooks et al., 2008). 

There is a fuller discussion of chromate mutagenicity (with references) in: Klein CB. "Carcinogenicity and 

genotoxicity of chromium'' In: Toxicology of Metals (Chang LW, ed). Boca Raton, FL:CRC Press, 1996, 

pp.205-219. This article points out that chromium compounds are mutagenic in a nanow dose range, 
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Toby G. Rossman, Ph.D. 

possible because of persistent toxicity after treatment (e.g. residual toxicity was seen a week after treatment 

of V79-derived Gl2 cells). 

Chromosome aberrations and DNA strand breaks can occur as a result of cytotoxicity. Dead cells do not 

become tumors. Unless assays for cytotoxicity are performed, it is not possible to know whether DNA 

damage occurs in cells that can replicate to form clones. Traditional cytogenetic assays rely on short-term 

cell survival to generate the mitotic figures necessary for analyses; the long-term viability of these treated 

cells cannot be determined. Thus, the relevance of this kind of data for carcinogenic MOA is questionable. To 

measure cytotoxicty, the gold standard is clonal survival, a method that is common in gene mutation assays, 

but not in other genotoxicity assays. Short-term survival assays, such as MTT, neutral red, and trypan blue, 

as well as measurements of mitotic index that are commonly used in cytogenetic assays, fail to detect early or 

delayed apoptotic events. Trypan blue detects only necrosis. MTT and neutral red assays can be delayed to 

allow time for apoptosis to develop, at which point the results approach clonal survival (Komissarova et al., 

Toxicol. Appl. Pharmacol. 202:99-107, 2005). As mentioned above, Cr(6) causes delayed residual toxicity 

(Klein et al. Environ. Health Perspect. 102 (suppl3):63-67, 1994), and thus clonal survival or at least 

apoptosis at later times after exposure, are essential in establishing cytotoxicity levels. Normal human 

fibroblasts show -80% loss of clonality after a 25h exposure to 211M sodium chromate [Vilcheck et al., 

Environ. Health Perspect. 110 (Sup5):773-777, 2002]. 

Micronuclei can result from DNA damage or from mal segregation of chromosomes. It has been 

recommended that this assay should be performed under conditions of high survival (an increase of >90% in 

number of viable cells) and that markers for apoptosis and necrosis be included [Kirsch-Voiders, et al. (2003) 

Report from the in vitro micronucleus assay working group. Mutat. Res. 540: 153-163]. In the case of Cr(6), 

at lower concentrations, most of the micronuclei are kinetecore-positive, meaning that they arise from 

malsegregation and not DNA strand breaks (Seoane and Delout, Mutat. Res. 490:99-106, 2001; Figgitt et al., 

Mutat. Res. 688:53-61, 2010). Those that are kinetecore-negative (arising from chromosome breaks) 

occurred only at the highest concentrations. Thus, Cr(6) induces aneuploidy rather than DNA damage at 

lower concentrations (Holmes et al., 2010; Figgitt et al., Mutat. Res. 688(1-2):53-61, 2010). Aneugenesis is 

caused by alterations in proteins, not DNA, and has thresholds. 

It should also be noted that the mouse lymphoma assay (MLA), chromosome aberration assay (CA) and 

micronucleus assay (MN) give a large number of false positives, even compared with the Ames test. 

Chemicals that are non-carcinogenic after thorough testing in both male and female rats and mice are often 

positive in these assays (Kirkland eta!., Mutation Research 584 (2005) 1-256). 

The Comet assay detects single and double strand DNA breaks as well as alkali-labile sites. Nucleotide 

excision repair (NER) and base excision repair (BER) of adducts can create breaks as intermediates. Single 

strand breaks are quickly repaired and are not regarded as significant premutagenic lesions. During 

apoptosis, DNA fragmentation into segments of 180 base pairs occurs, whether or not the apoptosis was 

induced by a genotoxic event (Choucroun et al. 2001, Mutat. Res. 478:89-96; Henderson et al.,.l998, 

Mutagenesis 13:89-94.) Necrotic cells also display DNA damage (Fairbairn eta!., 1996 Scanning 18:407-

416.). In order to avoid false positive responses, Henderson et al. (1998) suggests that the concentration of 

test substance should produce> 75% viability. 

In summary, standard genotoxicity assays from hazard identification exercises cannot be used to establish a 

mutagenic MOA, because these assays do not measure heritable events and because the doses used in such 

assays are usually too high. 
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Toby G. Rossman, Ph.D. 

Other MOA's have not been adeauately considered. These include, for example, selection for Cr-resistance, 

resistance to apoptosis, and aneuploidy. The evidence for a mutagenic MOl is weak. Mutations can result 

from DNA damage, but can also be a secondary effect of the loss of mismatch repair, aneuploidy, and other 

types of genomic instability (in other words, it is a later effect). With the exception of the mouse lymphoma 

system, Cr(6) is only weakly mutagenic in mammalian cells, rarely giving more than a 3-fold increase in 

mutant fraction over background levels (in endogenous genes), and in a very narrow (and toxic) dose-range 

with a strong threshold (reviewed in Nickens et al., 20 10). 

In some cases the "mutations" have been shown to be epimutations resulting from altered DNA methylation 

(Klein et al., 2002). Since none of the other studies on mammalian cells looked for epigenetic inactivation, 

this calls into question whether the "mutants" seen are really mutants. These are important considerations for 

the MOA of Cr(6), since cells grown in the presence of Cr(6) show selection for cells with inactivated 

mismatch repair (MMR) genes. These cells are Cr(6)-resistant and could be the result of either mutation or 

epigenetic inactivation (reviewed in Salnikow and Zhitkovich, 2008). Cells with epigenetically inactivated 

MLHl (a MMR gene) were seen in human lung A549 cells exposed to Cr(6) (Sun et al., Toxicol. Appl. 

Pharmacal. 237:258-266, 2009). MMR-deficient cells are mutators (having a high spontaneous mutation 

rate) and show microsatellite instability. An important consideration for MOA is the fact that chromiuminduced lung cancer cells also show epigenetically-inactivated MMR genes (Takahashi et.al., 2005). Also 

against the idea of a mutagenic MOI is the fact, discussed in Salnikow and Zhitkovich (2008), that Crinduced lung tumors in humans lack p53 mutations, in contrast to lung tumors associated with other agents 

such as tobacco smoke, and the fact that the few mutations found do not correspond to the types of mutations 

caused by Cr in in vitro systems. The fact that Cr is essential also implies that oral Cr(VI) could supply the 

necessary Cr, again implying a threshold at nontoxic doses. Also, experiments from the Costa laboratory 

(Davidson et al.,Toxicol. Appl. Pharmacal. 196:431-437, 2004; Uddin et al., Toxicol. Appl. Pharmacol. 

221:329-338, 2007) showing that chromate in drinking water is a cocarcinogen with solar UV, and the 

implications of this finding, are not discussed. 

Other problems (by page): 

Page 176, top of the page, is a good example of the confusion between mutagenicity and other endpoints. 

The statement "Hexavalent chromium-induced mutagenicity has been demonstrated following oral exposure" 

is misleading. There is only one mutagenicity assay showing positive effects on eyespots (presumed 

deletions) in offspring of female rats given drinking water with 62.5 mg Cr(6)/L. The deletions were not 

confirmed, so the eyespots might be epigenetic events (as Klein found with so-called mutants). All of the 

other assays are for non-mutagenic endpoints, and tend to be negative for drinking water exposure, but 

positive for gavage (a more toxic type of exposure). 

Page 178, 4.5.1.2, it is claimed that mutagenicity has been evaluated in humans experimentally exposed to 

hexavalent chromium. No such studies appear in Table 4-25. The paragraph mistakenly refers to 

mutagenicity many times. 

Page 186: It is not ER (excision repair) that is responsible for removal of bulky lesions, butNER (nucleotide 

excision repair) that is. Reynolds et al. 2004 is missing in references. 

Page 187, bottom. It doesn't make sense that mice given 0 mg/kg Cr(6) should have a significant level of 

apoptosis (compared to what?). 

Page 188: The Dai et al 2009 paper does not measure mutation frequency in human cells. 
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Toby G. Rossman, Ph.D. 

Page 190: 3'd paragraph: The mutational spectrum of chromate is not clear. See the review by Klein 

referenced above. 

Page 202: Key Events, #3: The authors skip from discussing mechanisms of DNA damage by Cr to "overall 

genomic instability which can lead to mutations if not adequately repaired". Genomic instability can occur as 

a result of other factors besides DNA damage, and genomic instability is not repaired (DNA damage can be, 

but the repair often leads to apoptosis). As discussed above, toxic exposure would play a role in the selection 

of Cr-resistant and/or apoptosis-resistant cells. There is no obvious tie-in here between DNA damage and 

mutagenesis as a key event, since cells resistant to Cr could have arisen by epigenetic silencing (and this may 

also be a mechanism in resistance to apoptosis ). In a sense, this point is recognized in #4, but in postulating 

apoptosis as a key event, the authors do not seem to realize the implications, i.e. that a toxic dose is needed 

for carcinogenicity. They suggest that selection for resistance to apoptosis is due to mutations (either preexisting or Cr-induced) but there is no evidence for this. Besides altered DNA methylation, other 

mechanisms for the appearance of Cr(VI)-resistance in exposed cells include epigenetic effects via altered 

histone modification (Sun et al., Toxicol. Appl. Pharmacal. 237:258-266, 2009) as well as reduction of 

Cr(VI) transport via down-regulation of sulfate ion transporter activity, and resistance to apoptosis via altered 

gene expression (upregulation of survival pathways and down-regulation of apoptotic pathways) (discussed 

in Nickens eta!., 201 0) 

p. 204-210: What is described as "mutagenicity" is not in all cases. 

p. 206, end of paragraph 1: It is claimed that there is evidence that Cr(6) induces mutagenicity in tissues at 

the site of entry and systemically at doses relevant to human exposure. Where is the evidence for this? 

p. 206, bottom: Again, it is important to note that DNA damage can lead to apoptosis as well as mutation, so 

it does not necessarily support a mutagenic MOA. 

p. 207: De Flora et al., 2008, did not look for mutagenesis, they looked for DNA damage. 

p. 208: Neither O'Brien et al., 2005 nor Eastmond et al., 2008 is in references. 

p. 209: Low (non-toxic) concentrations would not provide selective pressure for Cr-resistant, mismatch repair 

deficient cells. Especially in vivo, toxicity would be the driving force to stimulate the outgrowth of such 

cells. 

p. 209, 2"". paragraph: This is a lot of speculation (should be, may be). 

p. 211, bottom: It is claimed that the study by NTP found no evidence of tissue damage or necrosis. Did they 

look for apoptosis? It is also claimed that most available studies found Cr-induced genetic damage at doses 

below those that inducing cytotoxicity. This has not been demonstrated, since clonal survival (the only assay 

that will detect delayed toxicity) was not performed in these studies. 

p. 212, end: " ... giving rise to mutagenicity (including DNA adduct formation, DNA damage, gene mutations, 

chromosomal aberrations and micronuclei formation" This is nonsense, as is the next sentence. There is no 

evidence for mutagenicity at the target tissue at all. 

p. 213: More confusion between mutagenicity and other endpoints. It is not true that other hypothesized 

MOA's have not been demonstrated. There is actually more tumor evidence for an epigenetic MOA. Thus, 

the weight of evidence favors the alternative. This is not a numbers game. Epigenetic studies are relatively 

new, compared with DNA damage and other "genotoxic" studies, so there are fewer studies. 
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Toby G. Rossman, Ph.D. 

p. 214 top: Has EPA concluded this? Then what is the purpose of reviewing this document? 

p. 238, bottom: More confounding of mutagenesis and other endpoints. 

Additional papers showing epigenetic effects of Cr 

Schnekenburger et al., (2007) Chromium cross-links histone deacetylase !-DNA methyltransferase 1 

complexes to chromatin, inhibiting histone remodeling marks critical for transcriptional activation. Mol. Cell 

Bioi. 27(20): 7089-101. 

Sun et al. (2011) Comparison of gene expression profiles in chromate-transformed BEAS-2B cells. PloS 

ONE 6(3): el7982. Doi:l0.1371/journal.pone.0017982 

Ali et al. (2011) Aberrant DNA methylation of some tumor suppressor genes in lung cancers from workers 

with chromate exposure. Mol. Carcinogenesis 50(2):88-99 

83. A two-year drinking water study in rats and mice (NTP, 2008) was selected for the derivation of 

an oral slope factor. Please comment on whether the selection of this study for quantification is 

scientificaUy supported and clearly described. Please identify and provide the rationale for any 

other studies that should be considered. 

I do not think that a linear no threshold approach is valid. 

84. The incidence of adenomas and carcinomas combined in the small intestine of male mice from the 

NTP (2008) two-year drinking water study were selected to serve as the basis for the quantitative 

cancer assessment. Please comment on whether this selection is scientifically supported and 

clearly described. Please identify and provide the rationale for any other endpoints that should be 

selected to serve as the basis for the quantitative cancer assessment. 

This seems to be the only choice. 

85. The oral slope factor was calculated by linear extrapolation from the POD (i.e., the lower 95% 

confidence limit on the dose associated with 10% extra risk of tumors of the small intestine in 

male mice). Has the modeling been appropriately conducted and clearly described? 

This is outside my field of expertise. 
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POST-MEETING COMMENTS SUBMITTED BY 

Konstantin Salnikow, Ph.D. 
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Konstantin Salnikow, Ph.D. 

Revised comments on the Integrated Risk Information System (IRIS) assessment of 

hexavalent chromium carcinogenicity. 

These comments are part of an external peer-review of the scientific basis supporting the human health 

assessment of hexavalent chromium that will appear on the U.S. Environmental Protection Agency online 

database IRIS. 

Comments are prepared by Dr. Konstantin Salnikow, Ph.D. Program Director, Division of Cancer Biology, 

Cancer Cell Biology Branch, National Cancer Institute, NIH. 

G 1. Is the Toxicological Review logical, clear and concise? Has EPA clearly presented and synthesized 

the scientific evidence for noncancer and cancer hazard? 

In September 2010, the U.S. Environmental Protection Agency prepared the "Toxicological Review of 

Hexavalent Chromium" to assess health risks associated with hexavalent chromium exposure. This document 

will appear on the Agency's online database, the Integrated Risk Information System (IRIS). The existing 

IRIS file for hexavalent chromium, prepared in 1998, does not consider hexavalent chromium to be 

carcinogenic by the oral route of exposure. The purpose of the new document is to update the IRIS regarding 

noncancer and cancer health effects associated with oral exposure to hexavalent chromium after considering 

the latest scientific evidences. 

The prepared draft of the "Toxicological Review of Hexavalent Chromium" provides a detailed analysis of 

the data obtained in several studies carried out in 2008, 2009 by the National Toxicology Program along with 

other studies, formulates a mutagenic mode of carcinogenic action, and suggests that the reduction of orally 

administered hexavalent chromium in guts, even in low doses, is incomplete. The draft also calculates a 

cancer slope factor for humans. Recognizing the importance and relevance of this document, this reviewer 

needs to address some shortcomings of the document. In general this is a dense document cataloguing many 

diverse and sometimes controversial studies in the area of hexavalent chromium toxicology and 

carcinogenesis. Of course the limitations in available experimental data obtained from existing animal 

models, ongoing investigations regarding the mode of action (MOA) of hexavalent chromium, and 

uncertainties in epidemiological data make it difficult to prepare a comprehensive document that will fully 

address public health concerns. 

Chapter 3 should be reorganized and more emphasis should be given to the role of chromium III in texicoand pharmacokinetics as well as in biological effects produced by hexavalent chromium. Hexavalent 

chromium is generally considered a much more potent mutagen and carcinogen than trivalent chromium. 

Lack of carcinogenic effects observed with trivalent chromium compounds can be explained by poor 

permeability of cell membrane for this ion. However, considering that the end product of intracellular 

reduction of hex a valent chromium is trivalent chromium. which may accumulate in tissues. it is important to 

consider what role intracellularly deposited trivalent chromium may play in chromium toxicity and 

carcinogenesis. The majority of studies indicate that after intracellular reduction of hexavalent chromium to 

trivalent chromium, it can form various damaging DNA adducts. These adducts can inhibit the enzymatic 

activity of DNA polymerases, simultaneously increasing the rate of replication and the processivity of the 

DNA polymerase, and thereby decreasing its fidelity and causing more frequent errors. The frequency of 

errors increases with a dose-dependent increase in mutation frequency in vitro (Snow, 1991; Salnikow and 
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Konstantin Salnikow, Ph.D. 

Zhitkovich, 2008). Unfortunately, it is not clear how applicable these studies are to understanding the effects 

of trivalent chromium on DNA synthesis and cellular metabolism in vivo because the experiments were done 

in either in test tubes or in artificial model systems with concentrations far exceeding those obtained through 

environmental exposure (Snow, 1991; Dai et al., 2009). Numerous attempts have been done to study the 

distribution and retention of chromium (III) in vivo. Onkelinx studied tissue retention of 51CrCI, in groups of 

female Wistar rats of various ages (35, 60, and 120 days) after a single intravenous injection of trace amounts 

of 51CrCJ, (Onkelinx, 1977). The study showed that total excretory clearance is the sum of three components: 

urinary clearance (fu), fecal clearance (fd), and a residual clearance (fs), corresponding to an apparently 

irreversible deposition of chromium into long term body reservoirs. Consistent with the model, 51Cr was 

found to accumulate with time in several organs such as bone, kidney, spleen, and liver after a single 

intravenous injection of 51CrC13• These data are supported by those obtained by O'Flaherty (O'Flaherty, 1996) 

and others indicating that the retention of chromium III by bone, liver, kidney, and spleen is prolonged. 

Also, to make the toxicological review concise, I suggest eliminating Table 4-21 "In vitro genotoxicity studies 

of hexavalent chromium in nonmammalian cells" and Table 4-24 "In vivo genotoxicity studies of hexavalent 

chromium in D. melanogaster" because these are irrelevant to the MOA of hexavalent chromium in 

mammalian~ystems. 

There are numerous errors throughout the text of the Draft. Some of them are shown at the end of these 

comments as Errata. 

G2. Please identify any additional studies that would make a significant impact on the conclusions of 

the Toxicological Review. 

Key issues that should have an impact on the conclusions of the draft of the Toxicological Review of 

Hexavalent Chromium are: l) the use of appropriate animal models, 2) an understanding of the chromium 

carcinogenic MOA, including genotoxic (mutagenic) and non-genotoxic (epigenetic) mechanisms and their 

interrelations, 3) the co-carcinogenic effects of hexavalent chromium, and 4) the role of iron metabolism in 

chromium carcinogenesis. Unfortunately, studies to address these issues are either not done or are in the early 

stages of research. Thus, it is too early to draw any conclusive decisions on risk assessment of hexavalent 

chromium in drinking water. 

Are used animal models appropriate for risk assessment? 

Although the NTP studies provide evidence that oral exposure to hexavalent chromium induced tumors in 

rodents, the main argument against these studies is that the toxic and carcinogenic effects could be 

achieved/observed only at high chromium concentrations, which significantly exceed human exposure levels. 

Also, it is noted that biological effects were seen only at chromium concentrations that overwhelmed the 

cellular defense systems (reducing capabilities). Ascorbate is a major reducing agent of hexavalent chromium 

in biological fluids and tissues (see review (Zhitkovich, 2005; Salnikow and Zhitkovich, 2008)). Humans 

cannot synthesize ascorbate in the body because of a mutation in the L-gulono-y-lactone oxidase gene coding 

for the final enzyme in ascorbate metabolism and thus ascorbate is supplemented through the diet. Unlike 

humans, laboratory mice and rats, used for carcinogenicity assays, are capable of synthesizing ascorbate 

endogenously. Because ascorbate regulates many cell and tissue functions that are critical for cancer 

development, the changes in the level of ascorbate should be considered in animal models of choice 

(Salnikow and Kasprzak, 2005). It is impossible to deplete tissue ascorbate levels by metal exposure in wildtype rodents because the enzyme producing ascorbate will be up-regulated when the level of ascorbate drops 

below a critical point. To avoid this problem for in vivo testing of the toxic and carcinogenic effects of heavy 

metals, which efficiently destroy ascorbate, an appropriate model is the use of mice or rats that like humans 
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Konstantin Salnikow, Ph.D. 

cannot synthesize ascorbate (Kasprzak et al., 2011). Two rodent model systems unable to synthesize 

ascorbate are available: Gulo-/- mice (Maeda et al., 2000), and a similar rat strain (Mizushima et al., 1984). 

Our preliminary data show that when Gulo-/- mice were supplemented with ascorbate in drinking water their 

blood and tissue ascorbate levels were undistinguishable from that in wild type mice. However, ascorbate 

levels were significantly decreased by metal exposure in Gulo-/- mice but not in wild-type mice, in which the 

enzyme responsible for ascorbate production was activated in response to metal exposure (ascorbate 

depletion) (Kasprzak et al., 2011). In this model system we found that the reduction in ascorbate levels 

increased acute toxicity induced by Ni3S2 in Gulo-/- mice and that Gulo-/- mice were more susceptible than 

wild-type mice to nickel-induced carcinogenesis. Additionally, in tumor transplantation assays, Gulo-/- mice 

had shorter tumor latency than wild-type mice. After the lag period established tumor growth rates were 

comparable in Gulo-/- and wild-type mice. Although cancer initiation and development is a very complicated 

process our results indicate that ascorbate is a potentially important part of the molecular mechanisms of 

metal carcinogenesis and acute toxicity. 

Ascorbate is involved in diverse biological activities. Ascorbate is essential for the function of numerous 2-

oxoglutarate-dependent hydroxylases. This group of hydroxy lases includes the asparaginyl and prolyl 

hydroxylases, FIH-1 and PHD I, PHD2, PHD3, which are responsible for HIFa hydroxylation (Epstein et al., 

2001; Mahon et al., 2001; Hewitson et al., 2002; Lando et al., 2002); the collagen prolyl-4-hydroxylases 

(Myllyharju, 2003), which is critical for extracellular matrix formation; and a new class of histone and DNA 

demethylases that remove methyl group through hydroxylation (Shi, 2007). We already pointed out that the 

level of ascorbate is critical for metal carcinogenesis mainly by affecting epigenetic pathway (Salnikow and 

Zhitkovich, 2008). More recently a link between changes in ascorbate concentration and DNA demethylation 

of human embryonic stem cells (hESCs) has been identified (Chung eta/., 20 10). Thus, ascorbate levels 

have the potential to directly impact the differentiation of hESCs and the reprogramming of somatic cells. 

Given that ascorbate has diverse cellular functions and ascorbate levels are critical to interpreting 

carcinogenic effects of heavy metals, the animal models described in the Toxicological Review are not the 

most appropriate. The results obtained in NTP 2007 studies are consistent with the idea that ascorbate is an 

important factor to consider. In preliminary toxicokinetic studies in which animals were exposed to 

chromium in drinking water for 21 days chromium concentrations in the blood of the guinea pigs (which are 

unable to synthesis ascorbate) was greater than chromium concentrations in the blood of the rats or mice 

suggesting greater absorption (less reduction) of chromium in guinea pigs. 

http://nto.niehs.nih.gov/ntp/htdocs/ST rpts/tox072.pdf 

I suggest that new studies similar to the NTP studies with several dietary concentrations of ascorbate and 

lower chromium does (i.e., those more relevant to environmental exposures), be done in ascorbate-deficient 

rats or mice or both animal models. Additionally, because of more efficient depletion of ascorbate in tissues 

of Gulo-/- animals by chromium these animal models will show whether Cr(ill) and Cr(VI) kinetic that were 

developed using the wild type animals (O'Flaherty, 1996; O'Flaberty et al., 2001) will be applicable to Gulo-

/- animals. This will allow for adjustment of kinetic models, if needed, and identification of new or 

confirming known compartments of chromium retention. 

Al-A4 Oral Reference Dose (Rffi) for Hexavalent Chromium 

Outside of my area of expertise 
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Konstantin Salnikow, Ph.D. 

Carcinogenicity ofHexav.!tlent Chromium 

Bl. 

Hexavalent chromium has been classified by IARC as carcinogenic to humans (group I) via inhalation route 

of exposure based on results obtained in human and animal studies. However, when animals were exposed to 

hexavalent chromium in drinking water the carcinogenic effects were observed only at very high doses, 

which are irrelevant to human exposure. These results seems to cast doubt on carcinogenicity of hexavalent 

chromium via oral route of exposure and yet as I already pointed out in G2 the reason for only high 

chromium doses producing carcinogenic effect may be stemming from the inappropriate animal models 

which have higher protective ascorbate levels as compared to humans. Although, more human and animal 

studies are required to make an informed conclusion the ability of hexavalent chromium to produce tumors 

makes it likely to be carcinogenic by oral exposure. 

Another important consideration is that hexavalent chromium is a co-carcinogen and consumption of water 

with other toxic or carcinogenic compounds will result in unraveling chromium carcinogenic effects at much 

lower doses. Additionally, people with chronic inflammation of digestive tract could be more susceptible to 

chromium-induced carcinogenesis. 

B2. 

MOA. Genotoxic effect of hexavalent chromium. 

The draft of the Toxicological review provides a substantial body of information regarding the mutagenic 

potential of hexavalent chromium and concludes that hexavalent chromium is carcinogenic by a mutagenic 

MOA. Indeed this topic has been studied extensively. The results of in vitro and in vivo studies provide 

substantial evidence for the mutagenic activity of hexavalent chromium, which is mediated through the 

generation of the highly reactive chromium intermediates penta- and tetravalent chromium, reactive oxygen 

species, and trivalent chromium formed during the intracellular reduction of hexavalent chromium. These 

chromium and oxygen species can react with DNA, leading to oxidative DNA damage, chromium-DNA 

adducts, DNA strand breaks, and chromosomal aberrations. Despite these studies, the significance of 

chromium-induced DNA damage in the mechanisms of chromium carcinogenicity is not clear. If DNA 

damage/mutations are important and a causative factor in chromium-induced carcinogenesis, then these 

mutations should be frequent in chromium-induced tumors. However, very few publications exist in this 

respect. In animal studies, De Flora eta!. (De Flora et al., 2008) found no evidence of DNA-protein 

crosslinks and DNA adducts in the duodenum following drinking water chromium exposures. Other available 

hexavalent chromium drinking water exposure studies that measured mutagenicity in mice also could not 

detect evidence of micronucleus induction in the blood or bone marrow (Mirsalis et al., 1996; De Flora et al., 

2006; De Flora et al., 2008). Analysis of lung cancers from chromate-exposed workers revealed that p53 

mutations are not very frequent, with only six missense mutations identified in 4 (20%) of the 20 chromate 

lung cancer samples (Kondo et al., 1997). There were fewer mutations in the patients with lung cancers who 

had been exposed to chromate than in those who had not (20% vs about 50%). This study also revealed that 

there was no association between p53 mutations and the period spent working in chromate factories. It is 

conceivable that chromium causes genotoxic effects, damage DNA, but this damage is efficiently repaired 

and do not play any role in carcinogenic effects of chromium. 

Thus, in order to confirm or refute a possible role of chromium genotoxic/mutagenic effects in chromiuminduced carcinogenesis, comprehensive analyses of mutations in oncogenes and tumor suppressor genes in 

experimentally induced tumors in animals should be done first, followed by more detailed sequence analyses 
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Konstantin Salnikow, Ph.D. 

of chromium-exposed human tumors. A feasible study that could be done in a short period of time (assuming 

that tumor samples collected in the NTP studies are stored and frozen) is exon only global sequencing of 

DNA from chromium-induced rat tumors versus spontaneous tumors (several spontaneous tumors of 

different origin were observed in NTP 2008 study). These studies will allow comparison of tumor driving 

mutations versus passenger mutations. 

MOA, Epigenetic effects of hexavalent chromium. 

Global changes in the epigenetic landscape are a hallmark of cancer. The initiation and progression of cancer, 

traditionally seen as a genetic disease, is now realized to involve epigenetic abnormalities along with genetic 

alterations. Recent advancements in the rapidly evolving field of cancer epigenetics have shown extensive 

reprogramming of every component of the epigenetic machinery in cancer including DNA methylation, 

histone modifications, nucleosome positioning and non-coding RNAs, specifically microRNA expression 

(Sharma eta!., 2010). Epigenetic effects have also been observed following hexavalent chromium exposure 

(Salnikow and Zhitkovich, 2008; Arita and Costa, 2009). Increased DNA methylation was observed in the 

promoter region of the tumor suppressor gene p 16 and the MMR gene hMLH!, indicating that chromium can 

induce epigenetic effects (Takahashi eta!., 2005; Kondo eta!., 2006). Gene transcription has also been 

shown to be affected by exposure to hexavalent chromium in vitro via epigenetic mechanisms. Sun eta!. (Sun 

eta!., 2009) found alterations in the levels of histone methylation in human lung A549 cells exposed to 

hexavalent chromium, indicating the capability of these exposures to lead directly to changes in gene 

expression. Taken together, these studies suggest that epigenetic mechanisms may contribute to the 

carcinogenicity of hexavalent chromium. However, it is not clear whether epigenetic changes produced by 

chromium exposure are acting alone or linked to chromium genotoxic effects and that both genetic and 

epigenetic changes are essential for tumor appearance and evolution. More research is needed in this area 

including the identification of changes in DNA methylation (analyses of frequency of inherited silencing of 

tumor suppressors in tumors and in miRNA expression patterns following chromium exposure in animal 

models and humans before any conclusions can be drawn regarding the role of epigenetics in the 

carcinogenic effects of hexavalent chromium. 

MOA. Co-carcinogenic effects of chromium. 

Co-carcinogenic effects of hexavalent chromium were reviewed recently (Salnikow and Zhitkovich, 2008). 

The majority of occupational and probably all environmental exposures to hexavalent chromium occur as coexposures with other carcinogens. The most common examples of co-exposures occur among stainless steel 

welders, and among hexavalent chromium-exposed workers who are also smokers. Two reports from the 

Costa Lab (Davidson eta!., 2004; Uddin et al., 2007) provided strong experimental data demonstrating that 

hexavalent chromium can act as a potent co-carcinogen for UV-induced skin tumors. In both studies, the 

presence of hexavalent chromium in drinking water caused dose-dependent increases in the frequency of skin 

tumors in UV-irradiated hairless mice. Hexavalent chromium alone produced no tumors, indicating that it 

acted a strong enhancer ofUV-initiated tumorigenesis. Supplementation with vitamin E or selenomethionine 

had no effect on hexavalent chromium-mediated enhancement of skin carcinogenesis suggesting that cocarcinogenic effects were not oxidant-mediated. It is noteworthy that the level of chromium in skin directly 

exposed to UV had significantly higher levels of chromium than underbelly skin that was not directly 

exposed to UV in mice exposed to UV and 5 ppm K,Cro. (P < 0.05) (Davidson eta!., 2004). This raises an 

interesting question, does inflammation, whatever the source, facilitate chromium accumulation or delay 

chromium clearance? This is an important and understudied area. The identified co-carcinogenic effects of 

hexavalent chromium raise an intriguing possibility that much lower doses of chromium could be hazardous 
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Konstantin Salnikow, Ph.D. 

under certain circumstances when exposure to chromium in drinking water is combined with other harmful 

exposures. 

Another important area of research is an understanding of the role of Inflammation/colitis in hexavalent 

chromium carcinogenesis. It is well know that at least 20% of all cancers arise in association with infection 

and chronic inflammation and even those cancers that do not develop as a consequence of chronic 

inflammation, exhibit extensive inflammatory infiltrates with high levels of cytokine expression in the tumor 

microenvironment. Aberrant activation of NF-KB and/or STAT3 is found in over 50% of all cancers and 

renders premalignant and fully transformed cells resistant to apoptosis and speeds up their rate of 

proliferation, thereby increasing tumor growth. It is extremely important to test whether hexavalent 

chromium will be more carcinogenic and at lower doses in animals in which colitis was induced, for example 

by sodium dextran sulfate. 

MOA. Interference with iron metabolism. 

In rats neoplastic changes were found at sites of tissue contacts with the highest concentrations of hexavalent 

chromium, i.e. oral cavity. This may be explained by the immediate damaging effects of chromium on DNA 

and other cellular components. At the same time frequent nonneoplastic changes were observed in duodenum 

of male and female rats and neoplastic changes were observed in duodenum of male and female mice. These 

data cannot be explained by the direct effect of hexavalent chromium, which should be mostly reduced by the 

time it reaches small intestine. Considering that in other organs such as liver and kidney which accumulate 

significant amount of chromium no tumors were observed, it is important to do more research on the 

mechanism of tumor development in small intestine. Specifically, duodenum is a place where iron absorption 

takes place. An analysis of ferroreductase expression and iron metabolism will help to shed light on whether 

an alteration in iron metabolism in this tissue may be responsible for chromium carcinogenic effects. 

B3. 

Outside of my area of expertise. 

B4. 

It seems that combining the incidence of adenomas and carcinomas in small intestine was the proper choice 

for modeling cancer risk. This is supported by the available data and clearly described. The fact that only 

highest doses produced a carcinogenic effect may indicate high reducing capacity in tested model systems 

(wild type mice and rats). As suggested in A2 exposing Gulo-/- mice or rats to hexavalent chromium may 

result in tumor appearance at lower doses. If this will be the case the extrapolation to environmentally 

relevant doses of chromium exposure will be more feasible. 

B5. 

Outside of my area of expertise. 
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Konstantin Salnikow, Ph.D. 

Errata: 

Regarding the form of the draft it should be noted that, although this is not a manuscript, it is necessary to 

correct errors and mistakes in the draft content. 

Below are several examples: 

l. KzCrz04 - does not exist (page 30, table 3-7; page 45), should it be KzCr04 

2. Table 2-1, page 6 - Cr20 3 is chromium (III), not hexavalent chromium. BaCr04 is barium chromate, 

not barium oxide. 

3. Table 2-5, page 18- "accumulates Cr(V)", the intent of this statement is not clear because this form 

is short living and unlikely that is can accumulate. 

4. Page 35 and Table 3-9, K2Cr07 - does not exist 

5. None existing or wrong citations: 

Kumulainen, 1991 (page 7), should be Kumpulainen, 1992 (page 251). 

Salnikov and Zhitkovich, 2009 (page 50); Salnikov and Zhitkovich, 2008 (page 257), should be 

Salnikow and Zhitkovich, 2008. 

Costa and Klein, 2004 (page 66); Costa, M.; Klein, C.B. 2008 (page 245), should be Costa and 

Klein, 2006. 

LeVina et al., 2003, 2007, (page 50) should be Levina et al., 2003, 2007. 

Kasprzak, 1996 (page 189), no reference provided. 

Campbell J.L.; Tan,Y.; Clewell, H.J. (2009) Development of a PBPK model for hexavalent 

chromium in rats and mice to estimate exposure to oral mucosa and small intestine. Toxicologist 

108(1):98 (Abstract) Poster ID # 108. This is a questionable citation. 

Sun et al. (2009), (page 188), no reference provided. 

Davies, JM. (1979) Lung cancer mortality of workers in chromate pigment manufacture: An 

epidemiological survey. J Oil Chern Assoc 62:157-163, (page 245), should be: J Oil Colour Chern 

Assoc 62:157-163 

References: 

Arita, A., and Costa, M. (2009). Epigenetics in metal carcinogenesis: nickel, arsenic, chromium and 

cadmium. Metallomics 1, 222-228. 

Chung, T. L., Brena, R. M., Kolle, G., Grimmond, S. M., Berman, B. P., Laird, P. W., Pera, M. F., and 

Wolvetang, E. J. (2010). Vitamin C promotes widespread yet specific DNA demethylation of the 

epigenome in human embryonic stem cells. Stem Cells 28, 1848-1855. 

Dai, H., Liu, J., Malkas, L. H., Catalano, J., Alagharu, S., and Hickey, R. J. (2009). Chromium reduces the in 

vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome. Toxicol 

Appl Pharmaco/236, 154-165. 

Davidson, T., K1uz, T., Burns, F., Rossman, T., Zhang, Q., Uddin, A., Nadas, A., and Costa, M. (2004). 

Exposure to chromium (VI) in the drinking water increases susceptibility to UV-induced skin tumors in 

hairless mice. Toxicol Appl Pharmaco/196, 431-437. 

De Flora, S., D'Agostini, F., Balansky, R., Micale, R., Baluce, B., and Izzotti, A. (2008). Lack of genotoxic 

effects in hematopoietic and gastrointestinal cells of mice receiving chromium(VI) with the drinking 

water. Mutat Res 659, 60-67. 

De Flora, S., Iltcheva, M., and Balansky, R. M. (2006). Oral chromium(VI) does not affect the frequency of 

micronuclei in hematopoietic cells of adult mice and of transplacentally exposed fetuses. Mutat Res 

610,38-47. 
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Konstantin Salnikow, Ph.D. 

Epstein, A. C., Gleadle, J. M., McNeill, L.A., Hewitson, K. S., O'Rourke, J., Mole, D. R., Mukherji, M., 

Metzen, E., Wilson, M. I., Dhanda, A., Tian, Y. M., Masson, N., Hamilton, D. L., Jaakkola, P., 

Barstead, R., Hodgkin, J., Maxwell, P. H., Pugh, C. W., Schofield, C. J., and Ratcliffe, P. J. (2001). C. 

elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl 

hydroxylation. Cell107, 43-54. 

Hewitson, K. S., McNeill, L.A., Riordan, M. V., Tian, Y. M., Bullock, A. N., Welford, R. W., Elkins, J. M., 

Oldham, N.J., Bhattacharya, S., Gleadle, J. M., Ratcliffe, P. J., Pugh, C. W., and Schofield, C. J. 

(2002). Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF 

(FIH) and is related to the cupin structural family. J Bioi Chern 277, 26351-26355. 

Kasprzak, K. S., Diwan, B. A., Kaczmarek, M., Logsdon, D., Fivash, M. J., Jr., and Salnikow, K. (2011). 

Effects of Ascorbic Acid on Carcinogenicity and Acute Toxicity of Nickel Subsulfide, and on Tumor 

Transplants Growth in Gulonolactone Oxidase Knock-Out Mice and Wild-type C57BL Mice Toxicol 

Appl Phannacol submitted. 

Kondo, K., Hino, N., Sasa, M., Kamarnura, Y., Sakiyama, S., Tsuyuguchi, M., Hashimoto, M., Uyama, T., 

and Monden, Y. (1997). Mutations of the p53 gene in human lung cancer from chromate-exposed 

workers. Biochem Biophys Res Commun 239, 95-100. 

Kondo, K., Takahashi, Y., Hirose, Y., Nagao, T., Tsuyuguchi, M., Hashimoto, M., Ochiai, A., Monden, Y., 

and Tangoku, A. (2006). The reduced expression and aberrant methylation ofpi6(INK4a) in chromate 

workers with lung cancer. Lung Cancer 53, 295-302. 

Lando, D., Peet, D. J., Gorman, J. J., Whelan, D. A., Whitelaw, M. L., and Bruick, R. K. (2002). FIH-1 is an 

asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. 

Genes Dev 16, 1466-1471. 

Maeda, N., Hagihara, H., Nakata, Y., Hiller, S., Wilder, J., and Reddick, R. (2000). Aortic wall damage in 

mice unable to synthesize ascorbic acid. Proc. Nat!. Acad. Sci. U.S. A. 97, 841-846. 

Mahon, P. C., Hirota, K., and Semenza, G. L. (2001 ). FIH-1: a novel protein that interacts with HIF-lalpha 

and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev 15, 2675-2686. 

Mirsalis, J. C., Hamilton, C. M., O'Loughlin, K. G., Paustenbach, D. J., Kerger, B. D., and Patierno, S. 

(1996). Chromium (VI) at plausible drinking water concentrations is not genotoxic in the in vivo bone 

marrow micronucleus or liver unscheduled DNA synthesis assays. Environ. Mol. Mutagen. 28, 60-63. 

Mizushima, Y., Harauchi, T., Yoshizaki, T., and Makino, S. (1984). A rat mutant unable to synthesize 

vitamin C. Experientia 40, 359-361. 

Myllyharju, J. (2003). Prolyl4-hydroxylases, the key enzymes of collagen biosynthesis. Matrix Bioi. 22, 15-

24. 

O'Fiaherty, E. J. (1996). A physiologically based model of chromium kinetics in the rat. Toxicol Appl 

Pharmaco/138, 54-64. 

O'Flaherty, E. J., Kerger, B. D., Hays, S.M., andPaustenbach, D. J. (2001). A physiologically based model 

for the ingestion of chromium(III) and chromium(VI) by humans. Toxicol. Sci. 60, 196-213. 

Onkelinx, C. (1 977). Compartment analysis of metabolism of chromium(Ill) in rats of various ages. Am J 

Physio/232, E478-484. 

Salnikow, K., and Kasprzak, K. S. (2005). Ascorbate depletion: a critical step in nickel carcinogenesis? 

Environ Health Perspect 113, 577-584. 
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Konstantin Salnikow, Ph.D. 

Salnikow, K., and Zhitkovich, A. (2008). Genetic and epigenetic mechanisms in metal carcinogenesis and 

cocarcinogenesis: nickel, arsenic, and chromium. Chern. Res. Toxicol. 21, 28-44. 

Sharma, S., Kelly, T. K., and Jones, P. A. (2010). Epigenetics in cancer. Carcinogenesis 31, 27-36. 

Shi, Y. (2007). Histone lysine demethylases: emerging roles in development, physiology and disease. Nat 

Rev Genet 8, 829-833. 

Snow, E. T. (1991). A possible role for chromium(Hl) in genotoxicity. Environ Health Perspect 92,75-81. 

Sun, H., Zhou, X., Chen, H., Li, Q., and Costa, M. (2009). Modulation of histone methylation and MLHl 

gene silencing by hexavalent chromium. Toxicol Appl Pharmaco/237, 258-266. 

Takahashi, Y., Kondo, K., Hirose, T., Nakagawa, H., Tsuyuguchi, M., Hashimoto, M., Sano, T., Ochiai, A., 

and Monden, Y. (2005). Microsatellite instability and protein expression of the DNA mismatch repair 

gene, hMLH1, of lung cancer in chromate-exposed workers. Mol. Carcinog. 42, 150-158. 

Uddin, A. N., Burns, F. J., Rossman, T. G., Chen, H., Kluz, T., and Costa, M. (2007). Dietary chromium and 

nickel enhance UV-carcinogenesis in skin of hairless mice. Toxicol Appl Phannacol22l, 329-338. 

Zhitkovich, A. (2005). Importance of chromium-DNA adducts in mutagenicity and toxicity of 

chromium(VI). Chern. Res. Toxicol. 18, 3-11. 
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POST-MEETING COMMENTS SUBMITTED BY 

John Pierce Wise Sr., Ph.D. 
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John Pierce Wise Sr., Ph.D. 

Toxicological Review of Hexavalent Chromium 

PRE-MEETING WRITTEN COMMENTS 

CHARGE QUESTIONS 

General Charge Questions: 

Gl. Is the Toxicological Review logical, clear and concise? Has EPA clearly presented and 

synthesized the scientific evidence for noncancer and cancer hazard? 

The Toxicological Review is logical, clear and concise. However, overall the document is inconsistent and 

thus, EPA has not presented and synthesized the scientific evidence for noncancer and cancer hazard in a 

clear manner. Some sections, primarily the ones focused on animal data are clearly presented and 

synthesized. These sections present the primary literature and discuss the merits of each study with balance 

and insight. 

Other sections, however, particularly those involving in vitro cell culture data and underpinning the mode of 

action are much less appropriately considered, are not well-presented, and do not synthesize the underlying 

data very well. Determining a mode of action is a key part of the risk assessment. The Toxicological Review 

would be a stronger document if it fully analyzed and synthesized the primary literature to ascertain the 

possible modes of action for Cr(VI). The strengths, weaknesses and data gaps for each could have been 

highlighted and discussed, and then a rationale for the chosen mode of action presented. However, as 

presented this approach is not apparent. 

Instead, as presented, the document gives the impression that the mode of action was pre-determined from a 

select set of review articles and the best case for that mode of action presented. Decisions appear to have been 

made to agglomerate all of the genotoxicity data into positive or negative proof of mutagenesis rather than 

more careful consideration of individual lesions. Confounding factors, such as ascorbate levels, are 

cautioned against, but inadequately and inaccurately presented and unevenly applied, which undermines 

confidence that the primary data were adequately considered and contributes to a perception that decisions 

were predetermined. This perception is strongly reinforced by poor management and citation of the 

underlying literature and a heavy reliance on a few select review articles that unfortunately miscite the 

primary literature. As a result, many sections of the Toxicological Review lack clarity, accuracy, synthesis 

and rigor and the rationale for the choice of mode of action seems predetermined and forced. Each of these 

factors is elaborated on in more detail below. 
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John Pierce Wise Sr., Ph.D. 

I. Unnecessary agglomeration of genotoxicity data 

The Toxicological Review essentially combines all of the lesions related to genotoxicity (stated on page 212 

as " ... including DNA adduct formation, DNA damage, gene mutations, chromosomal aberrations, and 

micronuclei formation") into one bundle and refers to them as "mutagenicity". This decision is typically 

based on the presumption that the various genotoxic lesions will ultimately manifest as mutations in the 

primary DNA sequence. Hence, it is generally recognized that a crosslink or a su-and break is not inherently a 

mutation, but that it may eventually manifest as one and thus, it is a mutagenic event. Based on the 

aggregation of all of these data, the Toxicological Profile declares Cr(Vl) to be a mutagen and proposed a 

mutagenic mode of action. 

This approach is consistent with older practice and perception of these genotoxicity assays and it may be a 

useful approach for a chemical with a limited data set. However, the genotoxicity data for Cr(Vl) is a rich 

data set and deserves a more sophisticated consideration. The Cr(Vl) literature often distinguishes in its 

presentation between mutagenic and genotoxic lesions. The Toxicological Review does not carry forward 

that distinction and does not explain the rationale for ignoring it. However, it is an important distinction 

because not all of these lesions are likely to be mutagenic after Cr(Vl) exposure. Aggregating these lesions 

oversimplifies the interpretation of the data and masks the fact that much of the primary data suggest that in 

actuality, Cr(VI) is a very weak mutagen. Discussing each class of lesion on its own merit with a more 

careful consideration of the primary literature would have better framed the strengths and limitations of the 

genotoxicity studies and brought this discussion into a clearer light. 

The fundamental problem underlying this section is a failure to clearly consider the primary literature to see 

that Cr(VI) is a weak mutagen when defined as an agent that can directly change the primary DNA sequence. 

Cr(VI)-induced mutations have indeed been observed in bacteria, cultured cells and animal studies. 

However, in most cases, one bas to experimentally force the mutations to occur by using a high dose, a 

forced experimental system or a non-physiological exposure route. 

There is no real synthesis of this literature beyond listing outcomes in a table. The Toxicological Review also 

considers the results as simply positive or negative. That certainly is one approach; however, it misses the 

opportunity to consider the data more thoroughly. Some consideration should be given to potency and its 

potential impact and the robustness of the underlying assays. If the experimental data show that Cr(VI) 

induces a 2-fold increase in mutations, then the Toxicological Review would call that outcome positive. 

However, if in that same assay an established mutagen induced a 50-fold increase in mutations, then does 

Cr(VI) still appear to be a mutagen? Or does it suggest a different mode of action, particularly when the 

frequency of mutations have not been reported to increase in Cr(VI)-induced human tumors? Does the fact 
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John Pierce Wise Sr., Ph.D. 

that ascorbate is higher in rodents make these mutations a rodent-specific event? A more careful and 

thoughtful presentation of the underlying data would have better informed the consideration of this mode of 

action. 

In addition, before lumping all of these genotoxic endpoints together as all mutagenic outcomes, a careful 

review and discussion of the primary mutagenesis literature is needed. That review needs to determine, for 

Cr(VI), which of the various lesions (e.g. DNA adducts, DNA crosslinks, DNA strand breaks, gene 

mutations, chromosome damage, etc.) actually occur in cells (for example as discussed below the adducts 

may not actually form in cells) and to what extent they occur. Then, the review needs to determine which, if 

any, of these lesions actually lead to gene mutations. The discussion below illustrates that there are reasons 

to doubt that the various lesions are all mutagenic outcomes. After this analysis, those lesions that do form in 

cells and that do produce mutations could more reasonably be combined into a category of "mutagenicity". 

Perhaps, the data will indicate Cr(VI) is a mutagen, but, perhaps, the data indicate that one only gets 

mutations in the DNA sequence when systems are forced experimentally to do so at very high concentrations, 

due to species specific factors or by non-physiological exposure routes. If the latter were true, this possibility 

would suggest that mutations are not likely to occur in humans, raising direct implications for the mode of 

action decision. A more thorough treatment of the primary mutation data is needed to clarify these important 

points. 

There is concern that the discussion of some lesions is overstated while others are mentioned but not 

discussed. The section explaining DNA adducts is greatly overstated and also does not fully consider the 

primary literature. The section presents a case that implies the status and impact of the various potential 

adducts are known in cells and in vivo. The Toxicological Review even provides a structure of a Cr-DNA 

adduct. The major concern is that when the primary literature is fully considered, it becomes apparent that 

these adducts are all based on cell-free systems and no one has been able to clearly identify any specific 

adducts in cells, whole animals or humans beyond observing tangles of DNA, protein, and Cr that are 

considered to be DNA-DNA or DNA-protein crosslinks. The primary literature has only measured adduct 

levels in cells by isolating DNA and then measuring the amount of Cr associated with it or by nonspecific 

P32 postlabelling. These measures cannot ascertain how or if Cr is bound to the DNA, only that it is 

associated with it in some way. Some studies have synthesized adducts in cell free systems and applied them 

to cells, but that does not mean those specific adducts form in the cell. 
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John Pierce Wise Sr., Ph.D. 

Thus, it is unknown if specific DNA adduct events occur in cells, whole animals or humans. Nonspecific 

adducts have been detected by postlabelling, but specific adducts remain elusive. To lump these studies 

together as clear evidence of mutagenicity gives them a weight of evidence that seems premature and 

inaccurate. Overall, this section is very misleading in its portrayal of the status of adducts as more understood 

than they actually are. 

Oxidative damage is also included as evidence of mutagenicity. However, discussion is missing to establish 

whether this oxidative damage is a direct effect of Cr(VI) causing oxidative damage to DNA and thus, 

potentially a mutagenic event, or if this damage is actually indirect, resulting from overall oxidative stress to 

cells caused by high doses of Cr(VI) depleting intracellular antioxidants. 

Cr(VI)-induced strand breaks are cited as another type of mutagenic event. These lesions are discussed as 

post-replication-induced breaks. However, the discussion fails to question and discuss whether or not these 

are actually frank DNA breaks. As the Toxicological Review indicates, the studies have focused on gammaH2A.X focus production as the measure of breaks. Data indicate that chromatin remodeling may also induce 

the production of gamma-H2A.X so they may not be frank DNA breaks after all. This possible explanation is 

missing from the section. 

DNA-protein crosslinks are included as a type of mutagenicity in the tables and description despite the fact 

that the document states on page 186 that it is unknown if they are mutagenic: "Tests for the mutagenicity of 

these cross/inks have proved inconclusive (reviewed in Macfie et al., 2010), but the bulkiness of these lesions 

indicates the potential for genotoxicity ... " 

The most consistent genotoxic outcome is the production of damage to metaphase chromosomes manifested 

as aberrations, sister chromatid exchanges, and micronuclei. The fact that Cr(VI) induces these events was 

presented but not discussed. This lesion may be the key lesion as it is the most consistent and yet the 

mechanisms that may underlie it are ignored and not discussed. Chromosome damage could be a mutagenic 

lesion as assumed. Alternatively, it could be the consequence of epigenetic changes in the cell resulting from 

Cr binding to centrosomes in the mitotic spindle assembly apparatus or from bypass of the spindle assembly 

checkpoint. Cr(VI) has been shown to affect centrosomes and the spindle assembly checkpoint and perhaps it 

causes uneven pulling leading to breaks and errors in chromosome number. Cells with broken chromosomes 

may undergo apoptosis, while those with increased chromosome number may go on and survive as highly 

aneuploid cells. Cr(Vl) has been shown to induce highly aneuploid cells that can clonally expand and 

survive. This outcome would not be consistent with a mutagenic mode of action. 
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John Pierce Wise Sr., Ph.D. 

These concerns above are only magnified by the problems with uneven consideration of confounders and in 

poor management of the underlying literature described below. Together these factors give the impression of 

excluding or avoiding different syntheses of the data. More care and balance are needed to discuss and 

consider the genotoxicity data separately and evaluate if they are mutagenic markers. 

2. Uneven application of experimental confounders 

The perception of bias caused by bundling all of the genotoxicity endpoints together is magnified by an 

apparent uneven consideration of experimental confounders in the document. It appears that the 

Toxicological Review does not fully consider and present all of the relevant in vitro cell culture data that 

inform possible modes of action. Instead, selected examples of primary literature that reinforce one point of 

view are presented. This approach undermines the synthesis of the literature and because of a marked 

unevenness in presentation creates a perception of bias that should not be part of the analysis. 

For example, on page 184-185, the Toxicological Review gives the reader the impression that ascorbatetrivalent chromium-DNA adducts have been found to be highly mutagenic. However, the section did not 

describe the experimental detail that indicate the cells used in the study were abnormal and genetically 

modified so that they could not carry out proficient DNA repair or apoptosis, or that the cells were not 

actually treated directly with Cr(VI). These omissions stand in stark contrast to the experimental criticisms 

the Toxicological Review applies to other cell culture studies. 

For example, on page 47, the document states: 

"Caution should be used in interpreting cell culture data, as the cell culture medium could 

play a role in hexavalent chromium reduction, confounding the extent of intracellular 

hexavalent chromium reduction." 

It then cites a couple of examples where Cr(V) was detected in extracellular cell culture medium. The use of 

the word "caution" leads the reader to conclude that many in vitro studies may be flawed due to this 

reduction. No explanation is offered as to why this outcome is a problem. No discussion is provided that 

points out whether this same type of reduction might be expected to occur outside of cells in the body and 

thus actually be normal. The practical reality is that reducing agents are present in the extracellular fluid and 

thus, some extracellular reduction probably occurs in the extracellular fluid. This factor is probably not 

appropriate as a cautionary one and may actually reflect physiological conditions. But, no balanced 

discussion is provided for the reader to decide if this factor is indeed a concern. 
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John Pierce Wise Sr., Ph.D. 

A similar unevenness occurs during the presentation of the relative importance of ascorbate. The 

Toxicological Review states on page 50: 

"An additional important note on these biotransformations regards the interpretation and 

reliability of data from in vitro assays. In vivo, the intracellular levels of ascorbate are quite 

high (about I mM). In contrast, the levels of ascorbate in tissue culture media are quite low 

since generally it is not added to the media so that the only source is supplemented fetal 

bovine serum (FBS). With !Oo/o FBS, the level of ascorbate in tissue cultured cells is only 

about 50 pM which is 20 times lower than that which is found in vivo (Zhitkovich, 2005). 

Therefore, experiments on mutagenesis and other toxic effects of hexavalent chromium in 

tissue culture may underestimate its mutagenic, genotoxic, and cell-transforming activities 

(Zhitkovich, 2005; Costa and Klein, 2006)." 

No further discussion is presented. There is no presentation of the primary literature in cell culture showing 

the impact or lack of impact of this difference, just speculation that it might cause some underestimation. 

There is no presentation of the primary literature to establish what the ascorbate levels are in the relevant 

tissues of concern. There is just this one review article (Zhitkovich 2005) with some comment from a 

secondary review article (Costa and Klein 2006) cited. Closer inspection shows that the Costa and Klein 

review is actually citing the same Zhitkovich review so in the final consideration, this entire section relies 

only on Zhitkovich 2005 which, as discussed below in section 3.D., miscites the primary and secondary 

literature and draws a conclusion the primary literature does not closely support. 

Thus, the Toxicological Review draws attention to the possible presence of extracellular metabolism and lack 

of intracellular ascorbate as confounding factors, which it may have used to exclude some cell culture studies. 

But, by contrast, it makes no mention and expresses no concern about studies done in abnormal compromised 

cells treated only indirectly with Cr(Vl). This discrepancy makes the document and its treatment of the 

underlying literature seem uneven. 

Moreover, in its discussion of the impact of ascorbate, the Toxicological Review does not discuss the 

potential impact of ascorbate differences on the bacterial mutation studies or the possible impact of ascorbate 

differences on the animal genotoxicity data. For example, in Table 4-23 on page 172, the Toxicological 

Review indicates a positive effect for mutagenicity in mice after intratracheal instillation. Mice, however, 

have more ascorbate in their lung tissue. If one accepts the speculation in the Toxicological Review that 

ascorbate-trivalent chromium-DNA adducts form and are highly mutagenic, then the elevated mutations in 

this study might simply be due to the elevated ascorbate levels in this species suggesting a species specific 

effect. Such a possibility would explain why there are mutations in rodents but not in human tumors. 
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John Pierce Wise Sr., Ph.D. 

Regardless of which conclusion is correct, the point is that the Toxicological Review does not appear to 

apply this confounder it stresses in the in vitro work evenly to all studies reinforcing a perception of selective 

bias. 

Similarly, the Toxicological Review presents the primary research studies by Quievryn et a!., 2003; Voitkun 

et al., 1998 as showing adduct effects, but both studies used the cell culture medium the Toxicological 

Review expresses concerns about and neither study addressed the ascorbate concern, but these aspects are not 

mentioned in the document. The absence of discussion of these confounders in these experiments give the 

impression that the Toxicological Review does not seem to apply its confounding criticisms evenly. 

This inconsistent presentation of experimental expectations and application of confounding factors creates a 

perception of uneven evaluation of the primary literature. Considered together, the language and approach 

suggest a strong bias against in vitro studies and the cautionary language should be removed to eliminate that 

bias. 

The discussion about ascorbate needs to be more balanced and thorough and the information better 

synthesized. The ascorbate section could be removed or if the EPA feels the issue needs to be considered, it 

should be fully vetted with a discussion of how differences in ascorbate might affect the interpretation of the 

bacterial mutagenesis studies and the rodent data. The discussion would need to also include the strengths 

and limitations of the primary literature. The relative merits of data from a primary normal human cell line 

without vitamin C supplementation versus data from a tumor-derived cell line with ascorbate 

supplementation would need to be presented and discussed. The various underlying phenotypic issues in cell 

lines would also need to be considered as a mitigating factor. Similarly, the technical limitations of ascorbate 

supplementation in culture would need to be considered including how long it is retained by the cell and the 

impact of its diffusion out of the cell and into the extracellular medium. 

This discussion would need to include a full evaluation of ascorbate levels in tissues of interest in humans 

and animals and whether those levels are intracellular or extracellular or both. It should include a full 

discussion of any data that show whether or not there is an actual impact of different ascorbate levels on 

outcomes inside the cell from cell culture studies. It should also include a discussion about the fact that 

ascorbate in the cell becomes depleted over time after Cr(VI) exposure and whether the relevant exposure is 

when ascorbate levels are normal or depleted. 
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John Pierce Wise Sr., Ph.D. 

3. Poor management and citation of the literature 

The above two concerns are further magnified by the presentation and management of the literature in the 

sections involving in vitro cell culture data and underpinning the mode of action. There is a tendency in the 

document to overstate the findings of the selected primary literature included in the document, raising 

questions about whether the primary literature was properly evaluated and weighed. There is inconsistent 

application of the phrases "in vitro" and "in vivo" resulting in substantial confusion regarding the underlying 

literature and reinforcing a perception of inaccuracy in the document. There are flaws in citations, extensive 

direct quoting and long stretches of general paraphrasing of a small number of review articles raising 

questions about the heavy reliance on those articles and points of view. Finally, there is often a failure to 

check the underlying primary research studies cited in these review articles reinforcing the perception that the 

document relies on the review and not the underlying primary research data. Considered together, these 

aspects raise questions about the process of how the conclusions were drawn, create confusion about whether 

the primary data were fully reviewed or whether the view of the authors of those few review articles was 

simply adopted, and raise significant questions about the credibility of the overall evaluation. Each concern is 

explained in more detail below. 

A. Overstating the findings of the selected primary literature. 

There are concerns that the Toxicological Review over-generalizes its presentation of the primary literature, 

particularly with respect to in vitro cell culture studies. One example of this problem is seen in its discussion 

of the literature concerning DNA adducts where the Toxicological Review states on pages 184-185 that: 

"Although the ascorbate-trivalent chromium-DNA adducts are recovered less frequently in 

vitro due to the low concentrations of vitamin C present in commonly used tissue culture 

media (Zhitkovich, 2005 ), these adducts have been shown to be the most mutagenic of all the 

ternary adducts (Quievryn et al., 2003)." ... "They have been detected in vitro in Chinese 

hamster ovary cells following exposure to hexavalent chromium, and account .for up to 50% 

of all chromium-DNA adducts. The ternary adducrs have been .found to cause mutagenic 

and replication-blocking lesions in human fibroblasts in vitro (Quievryn et al., 2003; Voitkun 

et al., 1998)." 

Thus, the reader is led to believe that ascorbate-trivalent chromium-DNA adducts have been found in cells 

and these adducts have been shown to be highly mutagenic. Careful examination of the two cited references 

reveals that the statement in the Toxicological Review quote listed above that states: 

"They have been detected in vitro in Chinese hamster ovary cells following exposure to 

hexavalent chromium ... n 
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John Pierce Wise Sr., Ph.D. 

is incorrect. Detection of adducts in Chinese hamster ovary cells was not actually presented as data in either 

paper or mentioned in the text of either paper. The claim is unsubstantiated as presented, which makes it 

potentially misleading. 

Furthermore, when one considers the experimental detail in Quievryn et a!., 2003 and Voitkun et a!, 1998, 

one learns that the adducts were synthesized in a cell free system. A sequence of DNA was treated 

with Cr(VI) and ascorbate in a cell free system. Then, the damaged DNA sequence was administered to cells. 

The cells then converted the damaged DNA sequence to a mutation that was revealed when the sequence was 

recovered and sequenced. 

The detail also shows that the cells used were not normal human cells, but rather a SV 40 immortalized cell 

line. SV40 is known to silence p53 activity, among other cellular and molecular changes, thus these cells 

were unable to carry out proficient DNA repair or apoptosis, as these are normally p53-dependent events. 

Thus, the studies did not show that these adducts were normally present or able to form inside the cell. They 

could not account for the fate of the adduct structure after the transfection process and are only assuming it 

remained intact. The studies did not show that mutations would have occurred normally inside the cell as a 

consequence of Cr(VI) exposure or as a consequence of these lesions. Moreover, they do not show that these 

events would have happened in a repair proficient or apoptosis-proficient cell. It could be that the only reason 

mutations were seen is that the cells' ability to repair or eliminate them through apoptosis was artificially 

turned off beforehand. 

An alternative interpretation of the studies could be that one can experimentally force a cell to generate a 

mutation in response to a Cr adduct if repair and apoptosis are silenced. Indeed, a step forward, but not one 

that establishes that adducts form or are mutagenic in cells. 

It is unclear why these two studies were chosen to show that Cr induces adducts in cells. It is remarkable that 

given the emphasis the Toxicological Review places on the importance of physiologically relevant cell 

cultures, that it would fail to mention or discuss the integrity of the cell line itself, which in these studies were 

not robust cells. The document seems to be saying that there is a problem with cell culture studies that have 

some extracellular metabolism of Cr(VI) or that might not have enough ascorbate, but there are no problems 

with studies in cells with compromised DNA repair and cell death pathways. 

There are more examples of this type of exaggeration of the implications of the primary literature in the 

Toxicological Review. These exaggerations obscure the meaning and applicability of the data and should be 
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John Pierce Wise Sr., Ph.D. 

corrected. These exaggerations also undermine the integrity of the document and raise questions about its 

accuracy and process. Other studies in the primary literature may reemerge as more relevant if treated more 

evenly and these studies should be reconsidered and possibly presented. 

B. Flaws in citations. extensive direct quoting and long stretches of general paraphrasing 

One concern is that the Toxicological Review actually appears to directly quote sources without indicating 

the comments are quotes of the original source. For example, in the Toxicological Review on page 46, lines 

13-15, the writing states: 

"Studies on the reduction of Cr(Vl) by extracts of rat liver, lung, or kidney have found that 

ascorbate accounted for at least 80% of Cr(Vl) metabolism in these tissues ( Standeven et al., 

1991,1992)." 

which is the exact same sentence that occurs in the Toxicological Review's Zhitkovich 2005 reference. That 

reference states on its page 5: 

"Studies on the reduction of Cr-(Vl) by extracts from rat lung, liver, or kidney have found 

that ascorbate accounted for at least 80% of Cr(Vl) metabolism in these target tissues (45, 

46)." 

Then on the same page, lines 20-22, the Toxicological Review states: 

"Depending on the nature of the reducing agent and its concentration, this process can 

generate various amounts of unstable Cr(V) and Cr(1V) intermediates (Stearns et al., 1994)." 

which is the exact same sentence that is in Zhitkovich 2005. That reference states on its pages 5-6: 

"Depending on the nature of the reducing agent and its concentration, this process can 

generate various amounts of unstable Cr(V) and Cr(IV) intermediates (14-16)." 

Neither of these sentences are indicated as being exact quotes of the original source and neither one is 

attributed to the original source. This omission is a concern as it is important to know when the document is 

choosing to quote from a source directly. These examples are not the only occurrences of this type of error 

and the entire document needs to be checked to identify other such problems. 

In other instances, the Toxicological Review only changes a couple of words in a direct quote and fails to 

indicate it is a direct quote, which is also unacceptable. For example, the Toxicological Review states on page 

50: 
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John Pierce Wise Sr., Ph.D. 

"Therefore, experiments on mutagenesis and other toxic effects of hexavalent chromium in 

tissue culture may underestimate its mutagenic, genotoxic, and cell-transforming activities 

(Zhitkovich, 2005; Costa and Klein, 2006)." 

The underlying reference by Costa and Klein 2006 states on its page 157: 

"Thus, experiments on mutagenesis and other toxic effects of hexavalent Cr in tissue culture 

may underestimate its mutagenic, genotoxic, and cell-transforming activity (Zhitkovich, 

2005)." 

The quote in the Toxicological Review and the Costa and Klein review differ by only substituting a 

"Therefore" for a "Thus" at the beginning and "activities" for "activity" near the end. Changing two words 

does not avoid the need to offset this sentence as a direct quote. As written, it is sufficiently in the original 

authors' words that it is considered a direct quote. 

Similarly, in the Toxicological Review on page 46, the writing states: 

"Ascorbate is also the fastest reducer in the in vitro reactions, and its rate of reduction at 1 

mM exceeds that of cysteine and glutathione by approximately 13 and 61 times, respectively 

(Zhitkovich, 2005; Quivryn et at., 2001)." 

which is the exact same sentence that is in the Toxicological Review's Zhitkovich 2005 reference that states 

on its page 5: 

"Ascorbate is also the fastest reducer of Cr(Vl) in the in vitro reactions, and its rate of 

reduction at 1 mM concentration exceeds that of cysteine and glutathione approximately 13 

and 61 times, respectively (48)." 

Again, this language is a direct quote and needs to be offset in quotation to make that clear. 

This type of error also occurs with some frequency in the document and needs to be addressed. 

Next, there are numerous instances when the Toxicological Review extensively paraphrases a review article 

and the meaning of the original passage is altered to another meaning that was not originally intended 

resulting in some overstatements and inaccuracies. For example, the Toxicological Review states on page 

185: 

"Reduction of hexavalent chromium in vitro produces a large proportion of binary trivalent 

chromium-DNA adducts, but these have not been detected in vivo. It has been theorized that 
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John Pierce Wise Sr., Ph.D. 

the fonnation of the ternary adducts described above occurs far more frequently due to the 

high concentration of ligands capable of complexing with trivalent chromium before it can 

bind to DNA. (Zhitkovich, 2005)." 

Given its general use of "in vitro" to mean "in cell culture", the Toxicological Review appears to be stating 

that binary trivalent chromium-DNA adducts occur in cell culture but not in in vivo studies. However, the 

underlying Zhitkovich review reference actually states that the binary adducts have been detected in a test 

tube and not in cell culture. Specifically, it states (bold added here for emphasis): 

"Reductive metabolism of Cr(Vl) in vitro usually generates a large number of binary 

Cr( lll)-DNA adducts (22, 37, 53), but the presence of these DNA modifications in cells has 

not yet been established. The fonnation of binary Cr-DNA complexes in cells is expected to 

be strongly inhibited due to the abundance of intracellular ligands capable of rapid 

coordination to Cr(lll) prior to its binding to DNA." 

This error occurs quite often and creates confusion about what the underlying literature is indicating. 

There is a reference to "Salnikov and Zhitkovich, 2009" in a couple of places and no citation for this 

reference is provided. In the citation list, there is one reference listed as "Salnikow, K; Zhitkovich, A. 

(2008)" and another just below it as "Salnikov, K; Zhitkovich, A. (2008)" that looks to be exactly the same 

reference. These details should be straightened out and the entire reference section rechecked. 

The occurrence of these various errors in citations undermines confidence in the Toxicological 

Review and raises significant concerns about process. It gives the impression that review articles 

formed the basis for the evaluation, rather than primary sources and, with the extensive quoting and 

paraphrasing, that some articles were simply integrated into the document. The use of these review 

articles in the document needs to be revised and addressed. 

C. Confusion due to inconsistent application of the phrases "in vitro" and "in vivo". 

The data in the Toxicological Review essentially fall into four groups. There are cell-free system studies, cell 

culture studies, whole animal studies and human studies. To describe these data, the phrases "in vitro" and "in 

vivo" are used. To most readers "in vivo" is thought to refer to studies in the body and so include whole 

animal and in some instances human studies. By contrast, "in vitro" is thought to refer to cell culture studies. 

There are inconsistencies in the use of these terms in the Toxicological Review as some of the underlying 

references use "in vivo" to mean in cell culture and "in vitro" to mean in cell free systems. The Toxicological 

Review has often failed to clarify the underlying studies and carried the underlying language forward into the 

review. 
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John Pierce Wise Sr., Ph.D. 

Two examples of this problem are presented in the preceding criticism, where the Toxicological Review 

elevated the cells in culture to an "in vivo" status, but there are many more occurrences in the document. 

There are two explanations for this outcome. One possibility is that in some places the Toxicological Review 

uses "in vitro" to mean in cell culture and in others to mean in cell free systems and in some places it uses "in 

vivo" to mean in whole animals and in others to mean in cell culture. The second is that the authors of the 

Toxicological Review did not realize that the underlying literature meant for "in vivo" to mean in cell culture 

and "in vitro" to mean in cell free systems. Regardless of which reason applies, as written the use of the terms 

is confusing and in some cases, such as the one explained above, misleading. The EPA needs to decide on a 

definition for these terms, present it, review the underlying literature to be sure they reflect what is meant and 

then apply them consistently in the document. 

D. Failure to check the underlying primary research studies in review articles. 

There was a failure to fully consider the underlying primary research articles in the review articles that are 

extensively cited. This failure creates a perception that the authors did not read beyond that review article, 

raising questions about process and whether primary data was evaluated at all. Where the document depends 

on a review article for its source, the underlying primary literature should be checked to confirm the integrity 

of the statements. One example of this problem is seen in the passage below from page 50 of the 

Toxicological Review: 

"An additional important note on these biotransformations regards the interpretation and 

reliability of data from in vitro assays. In vivo, the intracellular levels of ascorbate are quite 

high (about I mM). In contrast, the levels of ascorbate in tissue culture media are quite low 

since generally it is not added to the media so that the only source is supplemented fetal 

bovine serum (FBS). With IO% FBS, the level of ascorbate in tissue cultured cells is only 

about 50 pM which is 20 times lower than that which is found in vivo (Zhitkovich, 2005). 

Therefore, experiments on mutagenesis and other toxic effects of hexavalent chromium in 

tissue culture may underestimate its mutagenic, genotoxic, and cell-transforming activities 

(Zhitkovich, 2005; Costa and Klein, 2006)." 

Thus, the Toxicological Review wants the reader to question cell culture studies if they do not contain 

intracellular levels of ascorbate in the mM range. However, the Zhitkovich review article that this section is 

entirely based upon miscites the primary literature on this matter and it appears the Toxicological Review did 

not check it. Specifically, as seen in the passage below from the Zhitkovich 2005 review, the claims about 

physiological levels of ascorbate being in the millimolar range rely on primary research papers that are its 

references 53 and 54. The Zhitkovich review states: 
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John Pierce Wise Sr., Ph.D. 

"Under standard tissue culture conditions, A549 and many other human and rodent cells 

either lack detectable ascorbate or contain it only at micromolar levels (physiological levels 

are in millimolar range) (53, 54) due to low concentrations of this vitamin in fetal bovine 

serum and its absence in the most commonly used types of growth media (DMEM, RPM! 

!640, FlO, Fl2)." 

These two references are: 

"(53) Quievryn, G., Messer, J., and Zhitkovich, A. (2002) Carcinogenic chromium(Vl) 

induces cross-linking of vitamin C to DNA in vitro and in human lung A549 cells. 

Biochemistry 41, 3156-3167. 

(54) Salnikow, K., Donald, S. P., Bruick, R. K., Zhitkovich, A., Phang, J. M., and Kasprzak, 

K. S. (2004) Depletion of intracellular ascorbate by the carcinogenic metals nickel and 

cobalt results in the induction of hypoxic stress. J. Bioi. Chern. 279, 40337-40344." 

Examination of these two references shows, however, that neither one offers any data or evidence of 

physiological ascorbate levels being in the mM range. Salnikow et a!. measures the amount of ascorbate loss 

in cultured cells treated with nickel and cobalt. They do measure ascorbate levels in untreated control cells to 

determine the background level of their experimental system. But their study does not measure any levels of 

vitamin C in any physiological setting. Nor does the citation make any reference at all to any study that does. 

Quievryn et al., treats the human carcinoma cell line A549 with ascorbate and dihydroascorbate and then 

measures the amount of vitamin C inside the cell under these experimental conditions. But the study does not 

measure any levels of vitamin C in any physiological setting. The discussion section does make a comment 

that: "Human cells in vivo contain a millimolar concentration of Asc (43) ... ". However, that reference 43 is 

"43. Meister, A. (1994) J. Bioi. Chern. 269, 9397-9400", which is a review article concerning glutathione and 

ascorbate in rodents. It contains no mention of ascorbate in human cells. 

Thus, the EPA expresses a significant concern about in vitro cell culture studies based on a single review 

article that miscites the primary and secondary literature. This oversight implies that in the preparation of this 

Toxicological Review, the EPA did not access the primary literature and confirm the secondary and tertiary 

review articles. 

If one were to look at primary literature for ascorbate levels, one would find that these claims are overstated. 

Ascorbate can reach mM levels in the body, but they are not universally mM levels. For example, Slade et 
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John Pierce Wise Sr., Ph.D. 

al., report lung ascorbate levels of 2.91 to 62.35 mg/100 g (Slade, R., Stead, A. G., Graham, J.A., and Hatch, 

G.E. (1985) Comparison of lung antioxidant levels in humans and laboratory animals. Am. Rev. Respir. Dis. 

131(5), 742-6). This measure can be converted to a range of 165 uM to 3.5 mM. However, these are not 

intracellular levels, but rather the product of tissue homogenization and so a mixture of extracellular and 

intracellular sources. Thus, there is clearly variability in levels that span the uM and mM range, indicating 

that this factor may not be so essential. 

E. Some exaggerations about Cr transport in cells. 

Of less concern, but certainly in need of being addressed is some of the inaccurate language concerning Cr 

transport into the cell. In several places, cells are described as being impermeable to Cr(III). This 

characterization is too strong and inaccurate. Cr(III) will enter cells. It is just a slower uptake process than 

Cr(VI) uptake as Cr(III) moves by simple diffusion and it requires a higher dose to create the concentration 

gradient to get in the cell. This language should be adjusted. 

Also, there is language implying Cr(VI) is actively transported into cells. Cr(Vl) does enter rapidly by 

facilitated diffusion, but it is not an active transport process. These comments should be adjusted. 

F. Some typographical errors. 

There is also a mention on page !50 that states: 'As discussed in detail in Section 4.4.2 (Intracellular 

Reduction) ... " It is actually section 4.5.2. 

In sum, these factors all combine to give the appearance that the mode of action was not fully and 

consistently considered. To make the document and its conclusions much stronger and more accurate and the 

rationale behind its decisions more transparent, the following steps should be taken: l) The EPA needs to 

separate the genotoxicity literature into discrete endpoints and consider them individually. This consideration 

should be based on the primary literature, which should be presented in a more careful and coherent fashion 

so that the reader can understand the strengths, weaknesses and data gaps. 2) Based on that analysis the EPA 

should choose which lesions are the key lesions and explain the rationale for that choice. 3) Once the key 

lesions are chosen, the EPA should consider the possible mechanisms of action that may cause those lesions 

and determine if there are data to support those mechanisms of action. 4) Once the key lesions are identified 

and the likely mechanisms described, the EPA should explain its rationale for the one chosen to be the mode 

of action. Of course, non-genotoxic modes of action should also receive similar analysis and presentation. 

This approach would help the EPA determine the most robust mode of action based on the primary literature. 

In addition, the EPA should decide what factors are truly confounders of concern and then apply them evenly 

to all of the literature, reduce its use of secondary and tertiary review articles and improve its management 

and citation of the literature. 
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John Pierce Wise Sr., Ph.D. 

G2. Please identifY any additional studies that would make a significant impact on the conclusions of 

the Toxicological Review. 

Although this review is focused on oral exposures, some insight may be gleaned from the inhalation exposure 

data. Specifically, the data on mutations in lung tumors for Cr(VI)-exposed workers should be considered. 

These data show a lack of mutations in those tumors suggesting that mutagenicity as considered as a primary 

change in the DNA sequence is not a key event in the mechanism of action. They are consistent with the fact 

that one only sees these types of mutations in mammalian experimental models when one forces them by 

applying very high doses. These studies are: 

Katabami M. Dosaka-Akita H, Mishina T, Honma K, Kimura K, Uchida Y, et a!. Frequent cyclin Dl 

expression in chromate induced lung cancers. Hum Pathol2000; 31 :973-9. 

Kondo K, Hino N, Sasa M, Kamamura Y, Sakiyama S,Tsuyuguchi M, et al. Mutations of the p53 gene in 

human lung cancer from chromate-exposed workers. Biochem Biophy Res Commun 1997; 239:95-100. 

Ewis AA, Kondo K, Lee J, Tsuyuguchi M, Hashimoto M, Yokose T, et al. Occupational cancer genetics: 

Infrequent ras oncogenes point mutation in lung cancer samples from chromate workers. Am J Ind Med 

2001; 40: 92-7. 

Hirose T, Kondo K, Takahashi Y, Ishikura H, Fujino H, Tsuyuguchi M, et a!. Frequent microsatellite 

instability in lung cancer from chromate-exposed workers. Mol Carcinog 2002; 33 : 172-80. 

Takahashi Y, Kondo K, Hirose T, Nakagawa H, Tsuyuguchi M, Hashimoto M, eta!. Microsatellite instability 

and protein expression of the DNA mismatch repair gene, hMLHI, of lung cancer in chromate-exposed 

workers. Mol Carinog 2005: 42: 150-8. 

Kondo K, Takahashi Y, Hirose Y, Nagao T, Tsuyuguchi M, Hashimoto M, et al. The reduced expression and 

aberrant methylation of p 16INK4a in chromate workers with lung cancer. Lung Cancer 2006; 53 : 295-302. 

Ewis AA, Kondo K, Dang F, Nakahori Y, Shinohara Y, Ishikawa M, et al. Surfactant protein B gene 

variations and susceptibility to lung cancer in chromate workers. Am J Ind Med 2006; 49 : 267-73. 

There are also studies of Cr(Vl)-induced neoplastic transformation of cells in culture. These need to be 

considered and included. In particular, reports by Xie et. al., show that cells must acquire a DNA double 

strand break repair phenotype to undergo transformation indicating escape from repair may be a key event in 

the mode of action. These studies are: 
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John Pierce Wise Sr., Ph.D. 

Patierno SR, Banh D, Landolph JR. Transformation of C3HI!OTI/2 mouse embryo cells to focus formation 

and anchorage independence by insoluble lead chromate but not soluble calcium chromate: relationship to 

mutagenesis and internalization of lead chromate particles. Cancer Res 1988; 47 : 3815-23. 

Xie H, Holmes AL, Wise SS, Huang S, Peng C, Wise Sr JP. Neoplastic transformation of human bronchial 

cells by lead chromate particles. Am J Respir Cell Mol Bioi 2007; 37: 544- 52. 

Xie H, Wise SS, Wise Sr. JP. Deficient repair of particulate chromate-induced DNA double strand breaks 

leads to neoplastic transformation. Mutat Res 2008; 649 : 230-8. 

The document only considered mismatch repair, but there are important data showing that other DNA repair 

pathways must be overcome to induce genotoxicity and carcinogenesis. Double strand breaks and their repair, 

in particular, are important. The following studies should be added to the repair/DNA double strand break 

discussion: 

Xie, H., Holmes, A.L., Young, J.L., Qin, Q., Joyce, K, Pelsue, S.C., Peng, C., Wise, S.S., Jeevarajan, A., 

Wallace, W.T., Hammond, D. and Wise, Sr., J.P. Zinc Chromate Induces Chromosome Instability and DNA 

Double Strand Breaks in Human Lung Cells. Toxicology and Applied Pharmacology, 234: 293-299, 2009. 

Xie H, Wise SS, Holmes AL, Xu B, Wakeman T, Pelsue SC, eta!. Carcinogenic lead chromate induces DNA 

double-strand breaks and activates ATM kinase in human lung cells. Mutat Res 2005; 586: 160-72. 

Xie H, Wise SS, Wise Sr. JP. Deficient repair of particulate chromate-induced DNA double strand breaks 

leads to neoplastic transformation. Mutat Res 2008; 649 : 230-8. 

Stackpole MM, Wise SS, Goodale BC, Duzevik EG, Munroe RC, Thompson WD, et a!. Homologous 

recombination protects against particulate chromate-induced genomic instability in Chinese hamster cells. 

Mutat Res 2007; 625:145-54. 

Camrye E, Wise SS, Milligan P, Gordon N, Goodale B, Stackpole M, et al. Ku80 deficiency does not affect 

particulate chromate-induced chromosome damage and cytotoxicity in Chinese hamster ovary cells. Toxicol 

Sci 2007; 97: 348-54. 

Bryant HE, Ying S, Helleday T. Homologous recombination is involved in repair of chromium-induced DNA 

damage in mammalian cells. Mutat Res 2006;599: 116-23. 
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John Pierce Wise Sr., Ph.D. 

Grlickova-Duzevik EG, Wise SS, Munroe RC, Thompson WD, Wise Sr. JP XRCCI protects cells against 

particulate chromate-induced chromosome damage and cytotoxicity in Chinese hamster ovary 

cells. Tox Sci 2006a;92(2):409-15. 

Grlickova-Duzevik E, Wise SS, Munroe RC, Thompson WD, Wise Sr JP. XRCCI protects cells from 

chromate-induced chromosome damage, but does not affect cytotoxicity. Mutat Res 2006; 61 0(1-2):31-7, 

Vilcheck SK, Ceryak S, O'Brien TJ, Patierno SR. FANCD2 monoubiquitination and activation by hexavalent 

chromium [Cr(VI)] exposure: Activation is not required for repair of chromium(Vl)-induced DSBs. Mutat 

Res 2006;610:21-30. 

Savery LC, Grlickova-Duzevik E, Wise SS, Thompson WD, Hinz JM, Thompson LH, Wise Sr. JP. Role of 

the Fancg gene in protecting cells from particulate chromate-induced chromosome instability. Mutat Res 

2007, 626(1-2):120-127. 

There needs to be a stronger and clearer discussion about aneuploidy as a potential mechanism. These studies 

should be added to that discussion (some are in the document already): 

Holmes, A.L, Wise, S.S., Pelsue, S.C., Aboueissa, A., Lingle, W., Salisbury, S., Gallaher, J. and Wise, Sr., 

J.P. Chronic exposure to zinc chromate induces centrosome amplification and spindle assembly checkpoint 

bypass in human lung fibroblasts. Chemical Research in Toxicology, 23(2): 386-395, 2010. 

Guerci A, Seoane A, Dulout FN. Aneugenic effects of some metal compounds assessed by chromosome 

counting in MRC-5 human cells. Mutat Res 2000; 469 : 35-40. 

Seoane AL, Guerci AM, Dulout FN. Malsegregation as a possible mechanism of aneuploidy induction by 

metal salts in MRC-5 human cells. Environ Mol Mutagen 2002; 40 : 200-6. 

Holmes AL, Wise SS, Sandwick SJ, Lingle WL, Negron VC, Thompson WD, et aL Chronic exposure to lead 

chromate causes centrosome abnormalities and aneuploidy in human lung cells. Cancer Res 2006; 66: 

4041-8. 

Wise SS, Holmes AL, Xie H, Thompson WD, Wise Sr JP. Chronic exposure to particulate chromate induces 

spindle assembly checkpoint bypass in human lung cells. Chern Res Toxicol 2006; 19 : 1492-8. 

The following two studies should be added to the clastogenicity results, particularly in light of one reviewer's 

comments that telomerase may be important as the second paper suggests !elomerase does not affect Cr 

genotoxicity: 
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John Pierce Wise Sr., Ph.D. 

Xie, H., Holmes, A.L., Wise, S.S., Gordon, N. and Wise, Sr., J.P. Lead chromate-induced chromosome 

damage requires extracellular dissolution to liberate chromium ions but does not require particle 

internalization or intracellular dissolution. Chemical Research in Toxicology, 17(10): 1362-1367, 2004. 

Wise SS, Elmore LW, Holt SE, Little JE, Antonucci PG, Bryant BH, et al. Telomerase-mediated lifespan 

extension of human bronchial cells does not affect hexavalent chromium-induced cytotoxicity or 

genotoxicity. Mol Cell Biochem 2004; 255: 103-11. 

Finally, there are misleading comments about DNA-DNA crosslinks. The Toxicological Review states they 

are unlikely to form in vivo. When one studies the underlying review cited as evidence by Salnikow and 

Zhitkovich, it becomes apparent that by in vivo they mean cells in culture or whole animals. Thus, the review 

implies that Cr -DNA-DNA crosslinks would not be predicted to occur in cells or whole animals, however, 

data from Josh Hamilton and Karen Wetterhahn show Cr DNA-DNA crosslinks do form in vivo. The 

inclusion of Josh Hamilton's study showing DNA-DNA crosslinks in vivo would correct the inaccurate 

conclusion in the Toxicological Review that these lesions do not occur in vivo. I cannot locate that paper in 

the time frame available, but Josh Hamilton is a reviewer and should be able to provide it. 

Chemical-Specific Charge Questions: 

(A) Oral Reference Dose (RfD) for Hexavalent Chromium 

Al. A two-year drinking water study of sodium dichromate dihydrate in rats and mice (NTP, 2008) was 

selected as the basis for the derivation of the RfD. Please comment on whether the selection of 

this study as the principal study is scientifically supported and clearly described. Please identify 

and provide the rationale for any other studies that should be selected as the principal study. 

This study is the proper study based on the available data. The study is flawed because only very 

high doses were considered in the study, thus, there is concern that it may not reflect events at lower 

doses. The EPA is in the unique position that a study that repeats the one above and extends it to 

lower doses is almost completed. The EPA should wait for the final results of that study to make the 

most informed analysis. 

A2. Diffuse epithelial hyperplasia in the duodenum of female mice was selected as the critical effect 

for the RfD. Please comment on whether the selection of this critical effect is scienti.[tcally 

supported and clearly described. Please identify and provide the rationale for any other endpoints 

that should be selected as the critical effect. 

This endpoint is a proper endpoint based on the available data, but is not necessarily a toxic outcome. 

To allow for better understanding, RID's for other endpoints should be done and presented including 
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John Pierce Wise Sr., Ph.D. 

some continuous endpoints. The EPA may conclude this endpoint is the critical effect, but this 

approach makes the analysis more transparent, open and clear. 

A3. Benchmark dose (BMD) modeling was applied to the incidence of diffuse epithelial hyperplasia in 

the duodenum of female mice to derive the point of departure (POD) for the RjD. Has the BMD 

modeling been appropriately conducted and clearly described? Is the benchmark response (BMR) 

selected for use in deriving the POD (i.e., a 10% increase in the incidence of diffuse epithelial 

hyperplasia) scientifically supported and clearly described? 

The BMD modeling has been appropriately conducted and clearly described. To allow for better 

understanding, BMR modeling at 5% and I% should be done and presented. This approach makes 

the analysis more transparent, open and clear. 

A4. Please comment on the rationale for the selection of the uncertainty factors (UFs) applied to the 

POD for the derivation of the RfD. Are the UFs scientifically supported and clearly described? If 

changes to the selected UFs are proposed, please identify and provide a rationale. 

The rationale for the selection of the uncertainty factors (UFs) applied to the POD for the derivation 

of the RID are appropriate. It was suggested that an UF for children and those with different 

conditions be used. People with different conditions (e.g. antacid use) are already contemplated in the 

UF applied for interindividual variation. Currently, such a factor for children is not included in the 

EPA guidelines. It could be that the same interindividual variation may apply in that case as well. 

(B) Carcinogenicity of Hexavalent Chromium 

Bl. Under EPA's 2005 Guidelines for Carcinogen Risk Assessment (www.epa.gov/iris/backgrd.html), 

hexavalent chromium is likely to be carcinogenic to humans by the oral route of exposure. Is the 

cancer weight of evidence characterization scientifically supported and clearly described? 

The general lack of accuracy in the document in its handling of citing, paraphrasing and considering 

the underlying literature is of some concern in this presentation. There is a lot of cell culture and 

animal data showing genotoxicity and clastogenicity, however, the motivation for these studies were 

largely inhalational exposure-induced cancer. It seems if they can support one route, they could 

support the other, but it functionally means the data underpinning the oral route of exposure is the 

one NTP study. 

That NTP study is flawed because only very high doses were considered in the study, thus, there is 

significant concern that it may not reflect events at lower doses. The EPA requirement for a "likely to 
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John Pierce Wise Sr., Ph.D. 

be carcinogenic to humans" classification defined as " ... appropriate when the weight of the evidence 

is adequate to demonstrate carcinogenic potential to humans hut does not reach the weight of 

evidence for the descriptor "Carcinogenic to Humans." It is understandable why an initial assessment 

of "likely to be carcinogenic" was chosen as the guidance states: "Supporting data for this descriptor 

may include: ... 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". 

The next possible descriptor is "suggestive evidence of carcinogenic potential". It is indicated as 

" ... appropriate when the weight of evidence is suggestive of carcinogenicity; a concern for potential 

carcinogenic effects in humans is raised, but the data are judged not sufficient for a stronger 

conclusion." It is also said to cover " ... evidence associated with varying levels of concern for 

carcinogenicity, ranging from a positive cancer result in the only study on an agent...". 

Thus, there is cont1icting guidance for Cr(VI) on these descriptors. On the one hand, there is a study 

of multiple species and genders possibly qualifying it for a descriptor of "likely to be carcinogenic to 

humans". On the other hand there is only one study showing this outcome making it suitable for a 

descriptor of "suggestive evidence of carcinogenic potential". 

In studying the data and descriptors, it appears to be premature to conclude that the weight of the 

evidence is nearly adequate for demonstrating carcinogenic potential to humans. Moreover, in 

considering the spirit of the guidelines for the two descriptors, it is clear that "likely to be 

carcinogenic" descriptor is contemplating that the data concerning multiple species, genders, strains 

etc. will come from multiple studies not just one. It is also clear that the "suggestive evidence of 

carcinogenic potential" descriptor is intended for a database with flaws. This database is flawed by 

the lack of multiple studies and the fact that the one study available relied on very high doses. 

Accordingly, a descriptor of "suggestive evidence of carcinogenic potential" is more appropriate at 

this time. 

However, the EPA is in the unique position to soon have another study that repeats the NTP study is 

almost completed. The EPA should wait for the final results of that study to make the most informed 

aualysis. If it too shows tumors at all doses, then the stronger descriptor would be justified. 
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B2. A mutagenic mode of carcinogenic action by all routes of exposure is proposed as the primary 

mode of action for hexavalent chromium. Please comment on whether this determination is 

scientifically supported and clearly described. Please comment on data available for hexavalent 

chromium that may support an alternative primary mode of action. 

This document is supposed to be limited to an oral drinking water exposure. It is inappropriate to 

extend any finding to "all routes of exposure" in this document and such evidence has not been 

considered or presented for dermal or inhalation routes. 

In its defense of a mutagenic mode of action, the Toxicological Review states on page 213 that: 

"In addition to the evidence supporting a mutagenic mode of action in test 

animals, alternative or additional hypothesized modes of action for 

hexavalent chromium carcinogenicity have not been demonstrated." 

There are three concerns with this statement. First, it seems to imply that other modes of action need 

to be "demonstrated" not simply supported. The frank reality is that no mode of action for Cr(V1) has 

been demonstrated, even the mutagenic mode of action is only supported and not demonstrated. 

There should not be a double standard here where the mutagenic mode of action needs to only be 

supported, while other modes must be demonstrated. The word "demonstrated" should be changed to 

"supported" to be consistent with the beginning of the passage. 

The second concern is that the statement is inaccurate. We supported an alternative mode of action to 

the induction of mutations in our paper that is cited in the Toxicological Review. Specifically, in 

Holmes, A.; Wise, SS; Wise, Sr., JP (2008) Carcinogenicity of hexavalent chromium. Indian J Med 

Res 128:353 - 372, we argue that the mechanism for Cr(VI) does not involve mutations in the 

primary sequence of the DNA as it is a weak mutagen. Instead, we argue for a genotoxic mechanism 

leading not to mutations but changes in chromosome number and structure. This point of view is not 

considered much in the Toxicological Review, but is well-supported in the review and does offer an 

alternative mode of action that should have been discussed. It is as well-supported as the mutagenic 

mode of action and so it is inaccurate to state other views have not been demonstrated to the extent a 

mutagenic mode of action has been. 

The third concern with the statement is that it seems to imply that the approach taken in determining 

the mode of action was to consider those possibilities suggested in reviews of the literature. 

Therefore, because there is no review article synthesizing the literature to suggest a mode of action, 

there are no other modes of actions to consider. A better approach would have been to consider the 
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primary literature and consider some possible modes of action that emerge from the data, but that 

have not yet emerged as a review article. There are other modes of action that emerge and two 

possibilities are presented below. There are data to support these modes that should be synthesized, 

evaluated and considered. 

The mutagenic mode of action as the primary mode of action is not sufficiently scientifically 

supported or described in the Toxicological Review. Many concerns in the presentation with respect 

to proper citation of results, bias against cell culture studies, and an incomplete consideration of the 

primary literature are discussed above. The only approach taken was to consider all of these lesions 

in bulk as simply all representative of mutagenic events and not consider the possible confounding 

factors for each that may indicate they are not mutagenic events. 

A more careful consideration of the primary literature, considering each endpoint on its own merit 

could argue against a mutagenic mode of action that involves changes to the primary sequence of the 

DNA strand resulting in mutations. Cr(Vl)-induced human tumors rarely contain such mutations and 

Cr(Vl)-induced mutations are most often generated in experimental systems when one artificially 

forces them to occur by using extraordinarily high doses or systems with compromised repair and 

cell death pathways or by non-physiological exposure routes. It is unlikely that Cr(VI) is a mutagen 

at low doses. 

The most consistent outcome in the primary literatnre appears to be impacts on metaphase 

chromosomes. These outcomes occur at relatively low doses, in intact healthy human cells and across 

species in cell culture, whole animal and human worker studies. The question remains and this 

document does not address the underlying mechanism for this outcome. Induction of aneuploidy is 

another promising mode of action. 

One mode of action could involve direct damage to the DNA strand resulting in an alteration in 

chromosome structure or number. This mode would have key events that include: I) Uptake of 

Cr(VI), 2) intracellular reduction ofCr(VI), 3) interaction of reductant products with DNA strands, 4) 

production of chromosomal changes, 5) escape of DNA repair and apoptosis and 6) expansion of 

damaged cells. 

Alternatively, the mode of action might not involve direct damage to the DNA. Instead, it could 

involve direct interactions with the mitotic spindle apparatus and be more of an epigenetic event. 

This mode would have key events that include: 1) Uptake of Cr(Vl), 2) intracellular reduction of 
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John Pierce Wise Sr., Ph.D. 

Cr(VI), 3) accumulation of intracellular reductant products, 4) interaction of reductant products with 

mitotic spindle apparatus, perhaps binding to the centrosomes, 5) production of chromosomal 

changes, 6) bypass of the spindle assembly checkpoint, 7) escape of apoptosis and 8) expansion of 

damaged cells. 

B3. A two-year drinking water study in rats and mice (NTP, 2008) was selected for the derivation of 

an oral slope factor. Please comment on whether the selection of this study for quantification is 

scientifically supported and clearly described. Please identify and provide the rationale for any 

other studies that should be considered. 

This study is the proper study based on the available data. The study is flawed because only very 

high doses were considered in the study, thus, there is concern tbat it may not reflect events at 

lower doses. The EPA is in the unique position that a study that repeats the one above and 

extends it to lower doses is almost completed. The EPA should wait for the final results of that 

study to make the most informed analysis. 

B4. The incidence of adenomas and carcinomas combined in the small intestine of male mice from the 

NTP (2008) two-year drinking water study were selected to serve as the basis for the quantitative 

cancer assessment. Please comment on whether this selection is scientifically supported and 

clearly described. Please identify and provide the rationale for any other endpoints that should be 

selected to serve as the basis for the quantitative cancer assessment. 

If one is going to rely on this NTP study, the selection of the incidence of adenomas and carcinomas 

in the small intestine of male mice from the NTP (2008) two-year drinking water study to serve as 

the basis for the quantitative cancer assessment is appropriate. However, scientifically these lesions 

are not the same and are not necessarily linked. Thus, one or the other should be used. 

BS. The oral slope factor was calculated by linear extrapolation from the POD (i.e., the lower 95% 

confidence limit on the dose associated with 10% extra risk of tumors of the small intestine in 

male mice). Has the modeling been appropriately conducted and clearly described? 

The modeling has been appropriately conducted and clearly described. More methods are needed to 

make it clearer. It is possible that Cr(VI) acts via a threshold. The EPA is in the unique position 

that a study that repeats the NTP study and extends it to lower doses is almost completed. This 

study will help clarify if there is a threshold. The EPA should wait for the final results of that 

study to make the most informed analysis. 
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POST-MEETING COMMENTS SUBMITTED BY 

Anatoly Zhltkovich, Ph.D. 
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Anatoly Zhitkovich, Ph.D. 

Review of a draft of Toxicological Review of Hexavalent Chromium prepared by the US-EPA 

in Support of Summary Information on the Integrated Risk Information System (IRIS) 

By Anatoly Zhitkovich, Ph.D. 

General Charge Questions: 

Brown University 

Providence RI 02912 

G 1. Is the Toxicological Review logical, clear and concise? Has EPA clearly presented and synthesized 

the scientific evidence for noncancer and cancer hazard? 

In general, I found the Draft to be well prepared and balanced in its presentation of various aspects of 

chromium-6 toxicology and carcinogenesis. It has a logical structure, leading a reader from the basics of 

redox chemistry of chromium-3 and chromium-6 and their interactions with biosystems to the detailed 

description of in vivo studies on bioavailability, tissue disposition and finally, toxic and carcinogenic effects. 

Weaknesses and strengths of the key in vivo studies along with the reasons for the inclusion or exclusion of 

specific findings were also clearly presented. Different sections vary somewhat in their degree of emphasis 

on the importance of one or another mechanistic aspect of Cr(VI) toxicology, which is also reflective of 

divergent opinions in the field. As typical for any large document covering a complex topic, the Draft 

contains some information that is not up-to-date and would benefit from additional editorial work. 

Suggested modifications and corrections: 

1) Section 2.1 "Environmental Sources and Occurrence" appears to draw a large amount of information 

from decades-old literature. The analytical approaches for the detection of both total Cr and Cr-6 

underwent major improvements in 1980s and the older references to the amount of Cr-6 in various 

environmental media and biological samples should be looked at with a healthy degree of skepticism and 

scrutinized for potential overestimations. My specific concerns are related to the included values for Cr-6 

levels in soil, freshwater and seawater. All three sets of values are too high for the typical samples from 

the noncontaminated areas/sites. 

2) Table 2-1 'Industrial uses of hexavalent chromium compounds" is missing uses of sodium/potassium 

chromate and dichromate. The addition of information on sodium dichromate is particularly important in 

light of its testing for carcinogenicity by the NTP. 

3) p.l4, last para: Cr(III) oxidation to Cr(Vl) by atmospheric oxygen can also occur in the presence of 

calcium oxide (Pillay et at. 2003). 

4) Table 2-4 'Detection limits for methods ... " reports outdated values. The EPA's Method 218.6 for Cr(Vl) 

in water has a detection limit which is -100 times lower than detection limits listed in Table 2-4. A recent 

modification of this method affords detection of Cr(VI) at the 0.003 ppb level (Application Update 179 

from Dionex). The detection sensitivity of flame AAS is also underestimated. Based on the discussion of 

work by Levine (2007) on p.l6 and other available literature, the detection limit for total Cr in water 

samples by ICP-MS reported in Table 2-4 is probably lower by a couple orders of magnitude. 

5) p.25, lines 5-6: in Donaldson and Barreras (1966), urinary excretion for orally administered Cr(Vl) and 

Cr(III) were 2.1 and 0.5%, respectively (not 2.1 versus 1.5% ). 
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Anatoly Zhitkovich, Ph.D. 

6) Table 3-5 and the discussion of these results appear contradictory. 

7) Table on p.39 is confusing: it reports daily doses of sodium dichromate dihydrate in the NTP-2008 study 

but the ratio mice:rats looks incorrect based on the data in the top two rows. It is also unclear why Cr(VI) 

consumption was compared between male rats and female mice and not between animals of the same sex. 

8) Finley et al. (1996) delivered a Cr-6 dose of 0.005 mg/kg/day, not 0.005 mg (p.45). 

9) Section 3.3 describes Cr(VI) reduction by microsomal enzymes in detail on three pages. This degree of 

attention may create an erroneous impression about the importance of the specialized enzymatic 

processes in Cr-6 metabolism. There is a strong consensus in the field that Cr(VI) reduction in 

mammalian cells is primarily accomplished nonenzymatically by ascorbate and small thiols such as 

glutathione and cysteine. As acknowledged in other sections of the draft, ascorbate alone accounts for 

reduction of 80-95% Cr(VI) depending on the tissue (Standeven and Wetterhahn, 1991, 1992). A 

combined contribution of ascorbate and thiols is responsible for more than 95% Cr(VI) reduction. These 

estimates from tissue preparations were confirmed by the measurements of individual reduction rates 

(Quievryn et al. 2003). It is clearly important to present mechanistic aspects of Cr(VI) reduction but the 

detailed focus should be on ascorbate and non-protein thiols, not enzymatic systems with a minimal 

contribution to the overall Cr(VI) metabolism in vivo. The absence of Cr(V) intermediate during Cr(VI) 

reduction by ascorbate is especially important. 

10) Last sentence on p. 50: The description of vitamin C accumulation by cells is not entirely correct. 

Cellular accumulation of vitamin C via uptake of its oxidized form dehydroascorbic acid is a 

physiological mechanism that functions in all mammalian cells. It is particularly active in human cells, 

which leads to very efficient recycling and much lower daily requirements for vitamin C in humans 

compared to rodents (Nualart et al. 2003', Montel-Hagen et al. 2008). These differences between humans 

and rodents are relevant for the interspecies extrapolation. 

II) Figure 3-6 needs to be modified: 

a) Depiction of the cation channel with the comment "No effect" could be interpreted as indicating 

some nontoxic delivery route for chromium. Unlike some other toxic metals, cation channels play no 

role in uptake of Cr ions and the cation route should be deleted from this Figure. 

b) Although some Cr(III)-ligand complexes can exhibit a limited ability to enter cells, there is no 

evidence that they can react with DNA and cause mutagenic/genotoxic ternary Cr-DNA adducts, as 

shown in the Figure. The Figure should be modified by removing this nonexistent route of DNA 

damage. 

c) The route for the formation of DSB by mismatch repair needs to be revised. As demonstrated in a 

recent study by Reynolds MF et al (2009), ternary Cr-DNA adducts are directly bound by mismatch 

repair proteins followed by DSB formation in G2 phase without stalling replication forks in the 

preceding S-phase. 

12) Summary Table 4-20 should add the +(M) designation for mutagenesis of sodium dichromate in 

laboratory animal, as demonstrated by Cheng et al. 2000 (this study was later cited in Table 4-23). 

13) Table 4-23 "In vivo genotoxicity studies ... Mutations section" is missing references to two positive 

mutagenesis studies in vivo by Itoh and Shimada (1997, 1998). 
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Anatoly Zhitkovich, Ph.D. 

14) Section 4.7.3.1. Hypothesized Mode of Action: 

a) Ref. to Salnikow et a!. ( 1992) in support of ternary complexes is inappropriate and could be replaced 

by Voitkun eta!. (1998). 

b) Neither Zhitkovich (2005) nor Voitkun eta!. (1998) dealt with "inttasttand DNA-DNA crosslinks". 

A study by Lloyd et a!. ( 1998) is the only original report describing putative intrasttand crosslinks, 

which were generated in a buffer solution with massive concenttations of hydrogen peroxide. Tested 

under the same reaction conditions, essential metals copper and cobalt were even more potent 

inducers of these presumed crosslinks. There is no evidence for the formation of these cross links by 

chromium-6 in cells or in acellular systems containing its main biological reducers. 

15) Tables 4-22 and 4-23 failed to include any references to studies reporting the formation of chromiumDNA adducts in cultured mammalian cells and in vivo. This is a critical omission as the presence of 

chromium-DNA adducts demonstrates a direct DNA-damaging mechanism for Cr(VI) genotoxicity. The 

formation of DNA adducts was briefly discussed in other sections of the Draft. 

16) Discussion of a negative report on DNA damage by DeFlora eta!. (2008) on pp.206-207: 

This study found no evidence of DNA damage in forestomach, glandular stomach and duodenum of female 

SKH-1 mice after a 9-month long exposure to 5 and 20 mg/L Cr(VI) in drinking water. Based on the study by 

DeFlora eta!. (2008), a high safety threshold argument was also made in some of the submitted public 

comments. The Draft argued that a shorter duration of exposure (9 months vs. 2 years in the NTP study) 

made the DeFlora 2008 study "infeasible" for the comparison. With the exception of mutations and a 

potential accumulation of unrepaired damage in a population of the long-lived crypt stem cells, there are no 

other obvious factors suggesting that the formation of DNA damage by Cr(VI) in the entire duodenum during 

the first half of the 2-year exposure would he significantly different from damage occurring at the end of the 

2 years. 

However, the study by DeFlora eta!. (1998) is uninformative for other reasons. The authors assayed tissues 

for two forms of DNA damage: DNA-protein crosslinks and 8-oxo-dG (the Draft incorrectly described 8-

oxo-dG as a DNA adduct; it is actually a base oxidation product). Both types of damage showed no increases 

above background in tissues of exposed animals; however, these negative results were predictable based on 

the technical limitations of their analytical methodologies. Since Cr(VI) tumorigenesis occurred in the 

duodenum, I will limit my discussion to this tissue. 

I) DNA-protein cross links: 

A positive control consisting of mouse duodenal cells treated ex vivo with 1.6 mM Cr(VI) (83.2 mg/L) 

generated a 2.5-fold response. Such a low responsiveness was clearly insufficient to detect DNA damage for 

exposures with 4.2- and 16.6-times lower Cr(VI) levels in the 20 mg/L and 5 mg/L test groups, respectively, 

even in the unlikely scenario of no reduction and no dilution of Cr(VI) with stomach juices before reaching 

the duodenum. Although chronic exposures frequently leads to the accumulation of unrepairable damage, a 

dramatic increase in DNA-protein crosslinks during chronic Cr(VI) exposures would be not very likely. 

DNA-protein crosslinks are repairable lesions in mammalian cells in vivo and culture (Tsapakos eta!. 1983, 

Sugiyama et al. 1986, Zecevic eta!. 2010) with the possible exception of human peripheral blood 

lymphocytes (Quievryn and Zhitkovich 2000). Furthermore, ongoing proliferation and shedding of cells in 

the duodenal villi would result in continuous dilution of damage and loss of. previously exposed cells. 
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Anatoly Zhitkovich, Ph.D. 

2) 8-oxo-dG measurements: 

A positive control generated by exposure of mouse duodenal cells to 1.6 mM Cr(Vl) (83.2 mg!L) produced a 

3.8-fold increase in the levels of 8-oxo-dG. It is doubtful that this assay sensitivity was sufficient to detect 

significant increases in 8-oxo-dG levels for even undiluted/unreduced 20mg/L and 5mg/L Cr(Vl) 

concentrations that were used in the treatment groups. 8-oxo-dG as a biomarker of DNA damage has one 

critical limitation- a short lifetime due to its rapid removal by base excision repair. Repair of 50% 8-oxo-dG 

occur within 30 min and is complete within 2 hr (Lan et al. 2004). Not only would this short lifetime prevent 

any accumulation of 8-oxo-dG during chronic exposures, but it would also make it very difficult to detect this 

lesion even after recently ingested water with a sufficiently high dose producing positive responses under ex 

vivo conditions. 

G2. Please identifY any additional studies that would make a significant impact on the conclusions of 

the Toxicological Review. 

The Draft included information from all major studies that have a significant impact on the main conclusions. 

It does not list or discuss every study published on Cr(VI) but there was also no systematic exclusion. In 

Section 4.4, I would recommend adding an important report by Gibb et a!. (2000), which is the largest 

epidemiological study of cancer risk due to inhalation exposure to Cr(Vl). While the omission or inclusion of 

this study does not change the overall conclusion about Cr(VI) carcinogenicity to humans via inhalation, 

Gibb et al. (2000) provided strong evidence of chromate dose-dependence for lung cancer risk and its 

independence of the common confounder, tobacco smoking. 

Chemical-Specific Charge Questions: 

(A) Oral Reference Dose (RID) for Hexavalent Chromium 

Al. A two-year drinking water study of sodium dichromate in rats and mice (NTP, 2008) was selected 

as the basis for the derivation of the RID. Please comment on whether the selection of this study as 

the principal study is scientifically supported and clearly described. Please identify and provide 

the rationale for any other studies that should he selected as the principal study. 

The NTP-2008 is the best available study of chromium-6 toxicity via oral exposure and its choice for the 

calculation of the RID is scientifically sound and was clearly explained in the Draft. 

A2. Diffuse epithelial hyperplasia in the duodenum of female mice was selected as the critical effect for 

the RID. Please comment on whether the selection of this critical effect is scientifically supported 

and clearly described. Please identify and provide the rationale for any other endpoints that 

should be selected as the critical effect. 

The incidence of diffuse epithelial hyperplasia in the duodenum of female mice was the most sensitive 

histological response observed in Cr(VI)-exposed groups and therefore, it was appropriately selected as the 

critical effect for the RID. 

A3. Benchmark dose (BMD) modeling was applied to the incidence of diffuse epithelial hyperplasia in 

the duodenum of female mice to derive the point of departure (POD) for the RID. Has the BMD 

modeling been appropriately conducted and clearly described. Is the benchmark response (BMR) 

selected for use in deriving the POD (i.e., a 10% increase in the incidence of diffuse epithelial 

hyperplasia) scientifically supported and clearly described? 

BMD modeling and the calculations of the POD both appear to be appropriately performed. 
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Anatoly Zhitkovich, Ph.D. 

A4. Please comment on the rationale for the selection of the uncertainty factors (UFs) applied to the 

POD for the derivation of the RID. Are the UFs scientifically supported and clearly described? If 

changes to the selected UFs are proposed, please identify and provide the rationale. 

Two UFs were applied: UF=IO for interspecies extrapolation to humans and UF=!O for interindividual 

variability in the human population. The interspecies UF was used because there is no available information 

to quantitatively assess the true differences in chromium-6 toxicokinetics and toxicodynamics between 

humans and laboratory rodents. There are, however, two biological factors that point to a potentially greater 

sensitivity of humans relative to mice. The first is related to the fact that telomerase was shown to suppress 

genetic damage by chromium-6 (Giaviano et a!. 2006). All mouse cells express telomerase while only stem 

cells retain telomerase expression in human tissues. The second factor is the difference in ascorbate 

metabolism. Human cells actively recycle ascorbate (Nualart eta!. 2003, Mantel-Hagen et al. 2008), 

resulting in -100 times lower requirements for this vitamin by humans relative to rodents. A more 

economical use of vitamin C by humans also results in lower ascorbate concentrations in the extracellular 

fluid (for example, as reported for bronchoalveolar lavage fluid by Slade et al. 1993), which would more 

rapidly detoxify chromium-6 via extracellular reduction. However, No specific information about ascorbate 

concentrations in the extracellular environment of the duodenum and jejunum of mice and humans is 

currently available. 

The interindividual variability in sensitivity to chromium-6 was not studied and the application of the safety 

coefficient (UF) is definitely appropriate in this case. However, the proposed UF=!O likely underestimates 

the range of the interindividual variability. The Draft has a brief discussion on the common presence of 

genetic polymorphism in DNA repair genes as one source of interindividual differences. Four major DNA 

repair pathways (mismatch repair, nucleotide excision repair, base excision repair and homologous 

recombination) are known to impact the extent of genetic damage and cytotoxicity by Cr(VI), and the use of 

UF=lO to account for interindividual differences in the overall DNA repair would assume a quite low degree 

of variability for each repair process (overall 1 O-f old variation would result from a very narrow 1.8-fold 

variation in each process: 1.84 = 1 0.5). 

Chromium-6 toxicity can be affected on three levels: 1) differences in extracellular detoxification, 2) 

differences in cellular uptake and 3) differences in cellular/genomic defense mechanisms. A 5-fold variation 

at each stage would give a potential 125-fold variation in the general population. A study by Donaldson and 

Barreras (1966) showed that individuals with pernicious anemia had 4-times higher systemic uptake of 

chromium-6 due to its lower detoxification in the stomach. Widespread use of antacid medications has a clear 

potential to diminish reduction rates of chromium-6. No systematic studies on potential variations in 

chromium-6 uptake have been performed yet, but two human lung carcinoma lines, H460 and A549, 

displayed a 5-fold difference in chromium-6 accumulation (Macfie et al. 201 0). A caveat of using 

information from these two cell lines is that they are malignant and therefore it is not possible to determine 

whether their differences were present in the initial normal cells or whether it is a side effect of different 

transformation processes. 

The Draft correctly stated on p.214 that there is no information about susceptibility of children to chromium6 toxicity. In this case, it would be clearly appropriate to use additional UF=!O to account for a potential 

early life susceptibility. If EPA considers it unnecessary, then the exclusion of this UF should be justified in 

Section 5.1.3. My recommendation would be to use UF=l 00 to account for the combined effects of the 

interindividual variability in susceptibility and early life exposures. 
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Anatoly Zhitkovich, Ph.D. 

(B) Carcinogenicity of Hexavalent Chromium 

Bl. Under EPA's 2005 Guidelines for Carcinogen Risk Assessment, hexavalent chromium is likely to 

be carcinogenic to humans by the oral route of exposure. Is the cancer weight of evidence 

characterization scientifically supported and clearly described. 

The classification of hexavalent chromium as "likely to be carcinogenic to humans" via the oral route of 

exposure is supported by evidence of its tumorigenicity in the oral cavity of female and male rats and in the 

small intestine of female and male mice. An increased incidence of stomach cancers in the JinZhou area 

(China), which was contaminated with high concentrations of chromium-6 in drinking water, is supportive of 

the selected classification. Even if the ecological study from China is excluded, the weight of evidence from 

animal studies is adequate to designate hexavalent chromium as "likely to be carcinogenic to humans" via the 

oral exposure. 

B2. A mutagenic mode of carcinogenic action by all routes of exposure is proposed as the primary 

mode of action for hexavalent chromium. Please comment whether this determination is 

scientifically supported and clearly described. Please comment on data available for hexavalent 

chromium that may support an alternative primary mode of action. 

Mutagenic mode of action: Hexavalent chromium was overwhelmingly positive for genotoxicity in a large 

variety of cells and organisms. It was also consistently mutagenic in bacterial and mammalian test systems. 

The mutagenicity and genotoxicity of Cr-6 result from a direct DNA-damaging mechanism, as evidenced by 

the induction of mutagenic chromium-DNA adducts and other forms of DNA damage. Formation of 

chromium-specific DNA lesions at environmentally relevant Cr-6 concentrations and sensitivity of genotoxic 

responses to manipulations of cellular DNA repair further support the role of direct DNA damage as a 

primary cause of genotoxicity. Since Cr-6 is taken up via ubiquitously expressed sulfate transporters and its 

metabolism in cells occur via ubiquitously present ascorbate, glutathione and cysteine, there is no reason to 

believe that the formation of DNA damage in the small intestinal cells and in more extensively studied cell 

types would be significantly different. Thus, diverse lines of evidence are fully consistent with a mutagenic 

mode of carcinogenic action for hexavalent chromium. The Draft clearly presented the main arguments for 

this designation. However, as pointed out above, Tables 4-22 and 4-23 need to be supplemented with 

information on Cr-DNA adducts. 

Supporting the importance of Cr-DNA adducts in chromate tumorigenicity are findings from the MOA study 

by the ACC in which levels of adducts were dramatically higher in the duodenum and jejunum of mice vs. 

rats. This result mirrors species differences in the intestinal carcinogenesis by chromate and it could not be 

explained by differences in tissue accumulation of chromium. 

In contrast to clear positive mutagenicity and genotoxicity data from in vivo studies and ascorbate-restored 

mammalian cell cultures, aneuploidy and epigenetic responses have not yet been tested in animal models and 

so far have been observed only in ascorbate-deficient cells. In fact, Sun et el. (2009) have found that the 

induction of epigenetic changes by chromate in human cultured cells occurs only under ascorbate-depleted 

conditions. 

The measurements of mutations in KRAS and p53 genes as part of the MOA study sponsored by the 

American Chemistry Council would not necessarily provide a clear answer about the mutagenic mode of 

action. A short 3-months duration of this study vs. 2 years for the NTP bioassay certainly diminishes its 

ability to detect mutations. Among the proposed mutation readouts, three KRAS codons represent a very 

small and consequently, insensitive mutagenic target. This gene was only rarely mutated in chromateassociated human lung cancers (Ewis et al. 200 I). The p53 gene is also uncommonly mutated in cancers 
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Anatoly Zhitkovich, Ph.D. 

among chromate workers (Kondo et al. 1997). The presence or absence of KRAS or p53 mutations do not 

serve as a strong test for the validity of the mutagenic mode of carcinogenic action, as the frequency of cells 

with mutated KRAS or p53 can increase through selection of the pre-existing mutant clones whereas 

transformation process can result from mutagenic events in other components of KRAS and p53 pathways. 

For example, a large wave of early thyroid cancers among Chernobyl radioiodine-exposed children was 

caused by translocations in growth factor receptors with almost no RAS and p53 mutations (Nikiforov et al. 

1996, Suchy eta!. 1998, Williams 2002). 

Potential alternative modes of action: Two lines of in vivo evidence have been presented to point to a 

potentially nonmutagenic mode of carcinogenic action. One is based on the drinking water study by DeF!ora 

eta!. 2008, which found negative results for DNA damage in the duodennm of mice. However, as discussed 

in detail above, this study used assays that were insensitive for detection of DNA damage by the employed 

doses of Cr(VI)in drinking water. Therefore, the negative results of this work were expected and therefore, 

uninformative. 

The other observation leading to the discussion of nonmutagenic or indirectly mutagenic mechanisms of 

carcinogenicity was the presence of diffuse epithelial hyperplasia in the NTP bioassay. Although the NTP 

study has not found significant necrosis in the small intestine of exposed mice, it is quite possible that the 

observed hyperplasia was a typical manifestation of regenerative responses. A combination of increased 

proliferation and inflammation could be presented as an alternative mechanism for indirect induction of 

mutations due to higher rates of cell division and by reactive oxygen species released by the recruited 

inflammatory cells. This carcinogenic pathway would exhibit a strongly sublinear, threshold-type dose 

dependence, as it relies on the induction of cell death and small doses would not kill cells. Inflammatory 

events could also be linked to cell death of chromium-damaged cells, which release pro-inflammatory 

molecules. The extent of hyperproliferation in chromium-exposed groups was modest, and considering the 

overall very high rate of cell division in the small intestine, it is hard to see how somewhat faster replication 

would provide dramatically more spontaneous mutations required for cancer development. At best, the 

cytotoxicity-induced compensatory proliferation mechanism and the mutagenic mode should co-exist at high 

tumorigenic doses. 

The results from the MOA study sponsored by the ACC argue against significant inflammatory responses in 

the duodenum of chromate-exposed mice, as no increases in the levels of 8-oxodG and in the panel of 22 

cytokines have been observed. A statistically significant drop in the ratio of GSHJGSSG was small in its 

magnitude, further demonstrating that tumorigenic doses were not associated with the state of strongly 

elevated oxidative stress and inflammation. 

The presence of chromium-induced hyperplasia could also be viewed as a manifestation of cancer-protective 

responses by the small intestine. Elimination of genetically damaged cells by apoptosis or another form of 

cell death is a firmly established protective mechanism against cancer. Thus, there are two opposing 

interpretations for the toxicological significance of the observed hyperplasia: one is pro-tumorigenic and 

another is anti-tumorigenic. The supralinear shape of dose-tumor incidence responses in the NTP-2008 

studies for female mice is consistent with the engagement of cancer-protective mechanisms. Tumor incidence 

vs. dose in male mice visually displayed a linear dose-dependence (as shown in Stern 2010). Thus, a 

hypothetical cytotoxicity-based mechanism with the expected dose-response sublinearity is contradicted by 

the available evidence. 
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Anatoly Zhitkovich, Ph.D. 

B3. A two-year drinking water study in rats and mice (NTP, 2008) was selected for the derivation of 

an oral slope factor. Please comment on whether the selection of this study for quantitation is 

scientifically supported and clearly described. Please identify and provide the rationale for any 

other studies that should be considered. 

The selection of a two-year drinking water study in rats and mice by the NTP (2008) for the calculation of an 

oral slope factor is appropriate. The NTP study was well designed and well executed. No other multiple-dose 

chronic oral carcinogenicity study in animals is available and the dose-dependence from the single ecological 

study linking chromium-6 in drinking water to human stomach cancers cannot be reliably estimated. 

B4. The incidence of adenomas and carcinomas combined in the small intestine of male mice from the 

NTP (2008) two-year drinking water study were selected to serve as the basis for the quantitative 

cancer assessment. Please comment on whether the selection is scientifically supported and clearly 

described. Please identify and provide the rationale for any other endpoints that should be 

selected to serve as the basis for the quantitative cancer assessment. 

The choice of the combined incidence of adenomas and carcinomas in the small intestine of male mice from 

the NTP-2008 study for the quantitative cancer assessment was based on a better fit of the multistage model 

for the male mouse data than for the female mouse data. However, it was unclear why a combination of male 

and female mouse data sets was not used. 

BS. The oral slope factor was calculated by linear extrapolation from the POD. Has the modeling 

been appropriately conducted and clearly described. 

The calculation of the oral slope factor from the POD was appropriately performed. As per US-EPA 2005 

Guidelines for Carcinogen Risk Assessment, a linear extrapolation to low doses was used based on the 

selection of the mutagenic mode of carcinogenic action for chromium-6. 

The ability of ingested chromium-6 to cause adverse effects at both environmentally relevant and much 

higher doses has been questioned, given the reported high chromate reducing capacity of the gastric juice and 

a limited systemic penetration of chromium after oral exposure (as extensively reviewed by the Draft). These 

considerations led to the formulation of the threshold model of chromium-6 carcinogenesis, which postulates 

that only doses that exceed the reducing capacity of the tissue (stomach for ingestion exposures) would be 

carcinogenic (DeF!ora 2000). This model would argue that despite the selection of a mutagenic mode of 

action with the resulting recommendation for default linear extrapolation, the complete detoxification of lowto-moderate chromium-6 doses in the stomach makes it inappropriate to perform linear extrapolation from the 

POD. 

The ability of gastric juices to reduce/detoxify chromium-6 is generally accepted in the field; however, 

studies with human volunteers and other considerations argue against the completeness of the detoxification 

process. For example, the bioavailability for Cr(VI) was -I 0-times higher than for Cr(VI) reduced with 

orange juice prior to ingestion (Kuykendall et al. 1996). The extent of chromium-6 reduction in the stomach 

is influenced by three factors: its reduction capacity, reduction rate and stomach emptying time. Based on the 

reported high reduction capacity of the stomach (>80 mglday, DeFiora et al. 1997), the rate of reduction by 

gastric juice under fasting conditions could exhibit pseudo-first order kinetics for a broad range of chromium6 concentrations. This means that the percentage of reduced chromium-6 could be the same for both very 

small amounts and much larger amounts. Reduction of chromium-6 by artificial gastric juice has been found 

to follow first order reaction kinetics (Gammelgaard eta!. 1999). Consistent with the first-order reaction 

kinetics, Donaldson and Barreras (1966) found that human subjects excreted in the 24-hr urine 2.1% of 

ingested 20 ng radioactive chromium-6 whereas Kerger et al. (1997) found that ingestion of 5 mg chromium-
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Anatoly Zhitkovich, Ph.D. 

6 by human volunteers led to about 1.43% excretion during the first 24 hr (1.43% excretion is a conservative 

114'• estimate from the average 4-day excretion value of 5.7%). Thus, the bioavailability of 20 ng and 5 mg 

chromium-6 (250,000-fold range) looks quite similar. 

Donaldson and Barreras (1966) also performed a very important experiment on tbe bioavailability of 20 ng 

radioactive chromium-6 that was directly delivered into the duodenum of human subjects. In this case, they 

found that 10.6% of chromium was excreted in the urine. Since the duodenal delivery represent 100% 

nonreduced chromium-6, then the amount of nonreduced chromium-6 in their oral route experiment can be 

estimated from the urinary excretion of 2.1% divided by 0.106 = 19.8%. For the study by Kerger eta! (2007), 

the same type of calculations gives an estimate of 14.3% nonreduced chromium-6 reaching the duodenum. 

Gammelgaard et a!. ( 1999) calculated a half-life of 23 min for reduction of O.lmg/L chromium-6 (current 

MCL for total chromium) by artificial gastric juice. After I hr, this reduction rate would leave 16.5% 

chromium-6. 

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De Flora S. Threshold mechanisms and site specificity in chromium(VI) carcinogenesis. Carcinogenesis 

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De FloraS, Camoirano A, Bagnasco M, Bennicelli C, Corbett GE, Kerger BD. Estimates of tbe 

chromium(VI) reducing capacity in human body compartments as a mechanism for attenuating its 

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De FloraS, D'Agostini F, Balansky R, Micale R, Baluce B, Izzotti A. Lack of genotoxic effects in 

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Donaldson RM Jr, Barreras RF. Intestinal absorption of trace quantities of chromium. J Lab Clin Med. 68(3): 

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Ewis AA, Kondo K, Lee J, Tsuyuguchi M, Hashimoto M, Yokose T, Mukai K, Kodama T, Shinka T, 

Monden Y, Nakahori Y. Occupational cancer genetics: infrequent ras oncogenes point mutations in 

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Gammelgaard B, Jensen K, Steffansen B. In vitro metabolism and permeation studies in rat jejunum: organic 

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Gibb H.J., Lees P.S.J., Pinsky P.F. and Rooney B.C. Lung cancer among workers in chromium chemical 

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Itoh S, Shimada H. Clastogenicity and mutagenicity of hexavalent chromium in lacZ transgenic mice. 

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Kerger BD, Finley BL, Corbett GE, Dodge DG, Paustenbach DJ. Ingestion of chromium(VI) in drinking 

water by human volunteers: absorption, distribution, and excretion of single and repeated doses. J 

Toxicol Environ Health 50(1): 67-95 (1997). 

Kondo, K., Hino, N., Sasa, M., Kamamura, Y., Sakiyama, S., Tsuyuguchi, M., Hashimoto, M., Uyama, T., 

and Monden, Y. Mutations of the p53 gene in human lung cancer from chromate-exposed workers. 

Biochem. Biophys. Res. Commun. 239: 95-100 (1997). 

Kuykendall JR, Kerger BD, Jarvi EJ, Corbett GE, Paustenbach DJ. Measurement of DNA-protein cross-links 

in human leukocytes following acute ingestion of chromium in drinking water. Carcinogenesis 17: 

1971-1977 (1996). 

Lan L, Nakajima S, Oohata Y, Takao M, Okano S, Masutani M, Wilson SH, Yasui A. In situ analysis of 

repair processes for oxidative DNA damage in mammalian cells. Proc Nat! Acad Sci USA 101(38): 

13738-13743 (2004). 

Macfie A., Hagan E. and Zhitkovich A. Mechanism of DNA-protein cross-linking by chromium. Chern. Res. 

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Mantel-Hagen A, Kinet S, Mane! N, Mongellaz C, Prohaska R, Battini JL, Delaunay J, Sitbon M, Taylor N. 

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Pillay K, von Blottnitz H, Petersen J. Ageing of chromium(III)-bearing slag and its relation to the 

atmospheric oxidation of solid chromium(III)-oxide in the presence of calcium oxide. Chemosphere 

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Quievryn G. and Zhitkovich A. Loss of DNA-protein crosslinks from formaldehyde-exposed cells occurs 

through spontaneous hydrolysis and an active repair process linked to proteosome function. 

Carcinogenesis 21: 1573-1580 (2000). 

Quievryn G., Peterson E., Messer J. and Zhitkovich A. Genotoxicity and mutagenicity of 

chromium(VDfascorbate-generated DNA adducts in human and bacterial cells. Biochemistry 42: 1062-

1070 (2003). 

Reynolds MF, Peterson-Roth EC, Bespalov lA, Johnston T, Gurel VM, Menard HL, Zhitkovich A. Rapid 

DNA double-strand breaks resulting from processing of Cr -DNA cross-links by both MutS dimers. 

Cancer Res. 69(3): 1071-1079 (2009). 

Slade R, Crissman K, Norwood J, Hatch G. Comparison of antioxidant substances in bronchoalveolar lavage 

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Standeven A.M. and Wetterhahn K.E. Ascorbate is the principal reductant of chromium (VI) in rat liver and 

kidney ultrafiltrates. Carcinogenesis 12: 1733-1737 (1991). 

Standeven A.M. and Wetterhahn K.E. Ascorbate is the principal reductant of chromium(VI) in rat lung 

ultrafiltrates and cytosols, and mediates chromium-DNA binding in vitro. Carcinogenesis 13: 1319-

1324 (1992). 
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Stern A.H. A quantitative assessment of the carcinogenicity of hexavalent chromium by the oral route and its 

relevance to human exposure. Env. Res. 110: 798-807 (2010). 

Suchy B, Waldmann V, Klugbauer S, Rabes HM. Absence of RAS and p53 mutations in thyroid carcinomas 

of children after Chernobyl in contrast to adult thyroid tumours. Br J Cancer. 77(6): 952-955 (1998). 

Sugiyama M., Patierno S. R., Cantoni 0. and Costa M. Characterization of DNA lesions induced by CaCr04 

in synchronous and asynchronous cultured mammalian cells. Mol. Pharmacal. 29: 606-613 (1986). 

Sun H, Zhou X, Chen H, Li Q, Costa M. Modulation of histone methylation and MLHI gene silencing by 

hexavalent chromium. Toxicol. Appl. Pharmacal. 237(3): 258-266 (2009). 

Tsapakos M. J., Hampton T. H. and Wetterhahn K. E. Chromium(VI)-induced DNA lesions and chromium 

distribution in rat kidney, liver, and lung. Cancer Res. 43: 5662-5667 (1983). 

Williams D. Cancer after nuclear fallout: lessons from the Chernobyl accident. Nat Rev Cancer. 2(7): 543-

549 (2002). 

Zecevic A., Hagan E., Reynolds M., Poage G., Johnston T. and Zhitkovich A. XPA impacts formation but 

not proteasome-sensitive repair of DNA-protein crosslinks induced by chromate. Mutagenesis 25: 381-

388 (2010). 
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POST-MEETING COMMENTS SUBMITTED BY 

Yiliang Zhu, Ph.D. 
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Post Meeting Review Comments on EPA's 

Toxicological Review of Hexavalent Chromium 

Yiliang Zhn, PhD 

College of Pnblic Health 

University of South Florida 

Yiliang Zhu, Ph.D. 

Gl. Is the Toxicological Review logical, clear and concise? Has EPA clearly presented and synthesized 

the scientific evidence for noncancer and cancer hazard? 

This EPA's Review is well organized overall and for most part well presented. The literature review 

is extensive and thorough. However, the Review does not contain a set of clearly-stated criteria under 

which the literature was searched, critiqued, and synthesized. Specifically, was each published study 

judged with respect to design (including sample size), exposure assessment, choice of dose metrics, 

choice of endpoints, adequate dose-response data, dose-response modeling, and positive findings? 

Whereas some of these criteria may have been used in the Review, the lack of a systematic approach 

may have compromised the consistency and transparency of this review process. In its independent 

review of the EPA's IRIS Documents on Formaldehyde and Dioxins, for example, the National 

Academies of Science and National Research Council have strongly advocated the adoption of a 

systematic review approach to EPA's IRIS risk assessment process (NAS 2006, 2011). The present 

Review of Hexavalent Chromium once again demonstrates the need for adopting a systematic review 

approach. 

G2. Please identify any additional studies that would make a significant impact on the conclusions of the 

Toxicological Review. 

NA 

Chemical-Specific Charge Questions: 

(A) Oral Reference Dose (RID) for Hexavalent Chromium 

Al. A two-year drinking water study of sodium dichromate dihydrate in rats and mice (NTP, 2008) was 

selected as the basis for the derivation of the RID. Please comment on whether the selection of this 

study as the principal study is scientifically supported and clearly described. Please identify and 

provide the rationale for any other studies that should be selected as the principal study. 

The Review offers EPA's rational for selecting the NTP's two-year drinking water study of sodium 

dichromate dihydrate in rats and mice (NTP, 2008). EPA's justification includes the lack of reliable 

epidemiological data, solid design of the NTP' s experiment, its controlled exposure regimens, the 

sensitivity of the endpoint, the availability of dose-response data, and consistency with hypothesized 
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genotoxicity MOA. These justifications are acceptable. There are still merits to include other studies, 

particularly the 3-month sodium dichromate dehydrate drinking water exposure of rats and mice 

(NTP 2007) in calculating RIDs. Inclusion of additional and all qualified studies is especially 

beneficial for better quantifying uncertainties and variations arising from different studies due to 

different study designs, strain/species of animals, and exposure regimens. As in a systematic review, 

studies meeting selection criteria should all be included for review and for analysis. Selecting a final 

RID then becomes a risk management decision. 

A2. Diffuse epithelial hyperplasia in the duodenum of female mice was selected as the critical effect for 

the RID. Please comment on whether the selection of this critical effect is scientifically supported 

and clearly described. Please identify and provide the rationale for any other endpoints that should 

be selected as the critical effect. 

EPA considered seven non-cancer endpoints for deriving RIDs (Table 5-1): chronic liver 

inflammation in female rats, histiocytic cellular infiltration in the liver of female mice, diffuse 

epithelial hyperplasia in the duodenum of the male and female mice, histiocytic cellular infiltration in 

the mesenteric lymph nodes of male and female mice, and cytoplasmic cellular alteration of acinar 

epithelial cells in the pancreas of female mice. All seven are quanta] response from the NTP's 2-year 

chronic exposure study. The selections were largely driven by the dose-response data these effects 

exhibited. After dose-response modeling and the estimation of benchmark dose (BMD) for each of 

these select effects, diffuse epithelial hyperplasia in the duodenum of the female mice was chosen as 

the critical endpoint simply because it yielded the smallest BMD and its corresponding lower 

confidence limit (BMDL). It must be noted that the dose-response model for this critical effect was 

done only after deleting the two highest doses. As a result, the dose-response modeling relied on only 

three dose level (including the control), leaving little room for any flexible dose-response forms other 

than the "linear" multi-stage model with a polynomial of 1 degree of freedom. 

Instead of relying on a select "critical" effect, EPA could report a range of RIDs based on a set of 

qualified and select effects. A.~ a result, EPA will be able to a range of RIDs, projecting the 

uncertainty and variation of RIDs arising from man y sources and affording risk management the 

opportunity to make an informed choice of a final RID (NAS, 2010). This is important as EPA is 

moving towards enhancing analysis of uncertainty and variation in risk assessment 

To this end, EPA could have benefited greatly by including additional endpoints from this principle 

study as well as other qualified studies. Potential candidates include histiocytic cellular infiltration in 

the duodenum of female rats and male mice, histiocytic cellular inflammation in pancreatic lymph 

nodes of male rats, and histiocytic cellular infiltration in the liver of female rats. EPS considered 

only the quanta( responses in this Review for the purpose of computing RIDs. It is unclear whyeffects of continuous measurement scale were not considered. Many of these effects show 
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Yiliang Zhu, Ph.D. 

unequivocal dose-response (e.g. Tables 4-12 and 4-13) and seem to be relevant to the hypothesized 

MOA of hexavalent chromium. The availability of EPA's software BMDS for dose-response 

modeling and benchmark dose computation makes it practical and useful to consider continuous 

effects for RID derivation as well. 

A3. Benchmark dose (BMD) modeling was applied to the incidence of diffuse epithelial hyperplasia in 

the duodenum of female mice to derive the point of departure (POD) for the RID. Has the BMD 

modeling been appropriately conducted and clearly described? Is the benchmark response (BMR) 

selected for use in deriving the POD (i.e., a 10% increase in the incidence of diffuse epithelial 

hyperplasia) scientifically supported and clearly described? 

EPA should be commended for conducting BMD modeling for multiple effects with different model 

forms. For the modeling of the incidence of diffuse epithelial hyperplasia in the duodenum of female 

mice, EPA should provide a more detailed discussion on the limitation of the dose-response 

modeling (See A2). Uncertainties due to model choice, variation in the shape of seemingly equally 

well-fit models also can be quantified to a degree by considering multiple benchmark response levels 

(BMR) for each model. EPA used only BMR=IO%. It makes perfect sense to also consider 

BMR=5% or even BMR= I% when such a choice is supported by the data. This is the general 

recommendation of EPA's own guideline (EPA, 2000). By doing so EPA would be able to 

quantitatively demonstrate uncertainty and variations due to the choice of different models and 

different BMR levels. Additionally, EPA should briefly but clearly define the BMD concept and 

methodology in an appendix to improve the readability for readers unfamiliar with the process. 

A4. Please comment on the rationale for the selection of the uncertainty factors (UFs) applied to the POD 

for the derivation of the RID. Are the UFs scientifically supported and clearly described? If changes 

to the selected UFs are proposed, please identify and provide a rationale. 

The use of uncertainty factors (UFs) in this review is well described and is consistent with EPA's 

guidance documents for RIDs. Exposure in earlier stage of life was discussed. 

(B) Carcinogenicity of Hexavalent Chromium 

Bl. Under EPA's 2005 Guidelines for Carcinogen Risk Assessment (www.epa.gov/iris/backgrd.htmll, 

hexavalent chromium is likely to be carcinogenic to humans by the oral route of exposure. Is the 

cancer weight of evidence characterization scientifically supported and clearly described? 

The descriptor of "likely to be carcinogenic to humans" for hexavalent chromium is consistent with 

EPA's Guidelines for Carcinogen Risk Assessment. EPA gave a clear description of the hypothesized 

MOA. 
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82. A mutagenic mode of carcinogenic action by all routes of exposure is proposed as the primary mode 

of action for hexavalent cbromium. Please comment on whether this determination is scientifically 

supported and clearly described. Please comment on data available for hexavalent cbromium that 

may support an alternative primary mode of action. 

A mutagenic mode of action was proposed as the primary mode of action. On the one hand, EPA 

discussed data gap and uncertainties about the mutagenic MOA and other possible MOAs. On the 

other hand, EPA defended the mutagenic MOA despite the lack of data evidence. For example, the 

only animal study that investigated target tissue genotoxicity (De Flora eta!. 2008) reported negative 

results for DNA-protein crosslinks and DNA adducts in forestomach, glandular stomach, and 

duodenum of mice exposed to hexavalent chromium in drinking water for 9 month. EPA dismisses 

the negative finding on the basis of a shorter duration of the study compared with the 2-year NTP 

study. 

83. A two-year drinking water study in rats and mice (NTP, 2008) was selected for the derivation of an 

oral slope factor. Please comment on whether the selection of this study for quantification is 

scientifically supported and clearly described. Please identify and provide the rationale for any other 

studies that should be considered. 

The selection of the NTP's 2-year drinking water study in rats and mice (NTP, 2008) is justified. 

Reasons for why existing epidemiological data were not used for estimating cancer slope factor are 

acceptable. 

84. The incidence of adenomas and carcinomas combined in the small intestine of male mice from the 

NTP (2008) two-year drinking water study were selected to serve as the basis for the quantitative 

cancer assessment. Please comment on whether this selection is scientifically supported and clearly 

described. Please identify and provide the rationale for any other endpoints that should be selected to 

serve as the basis for the quantitative cancer assessment. 

For dose-response assessment EPA considered the incidences of adenoma and carcinoma combined 

in the small intestine of male and female B6C3F mice (Tables 5.3 and 5.4), in the oral cavity 

(mucosa and tongue) (Tables 5.3 and 5.4) in rats (NTP 2008). (Note the denominators that determine 

the tumor incidence in small intestine are not consistent with those in Table 4.19). EPA did not 

consider the incidence of other neoplasm because the incidence is not dose-dependent. 

The incidence of adenoma or carcinoma in the oral cavity in both male and female rats elevated only 

at the highest dose, but not at the tbree lower doses (up to 2.1 and 2.4 mg/kg-d for male and female 

respectively). To fit a dose-response model to these incidence data that exhibited hockey-sticker 

shape of dose-response requires a nonlinear (curve-linear) functional form or even a threshold model. 

Such curve-linear pattern seems inconsistent with the hypothesized genotoxic MOA. The lack of 

dose-response in these two endpoints was cited as reason for not advancing these two endpoints for 

final dose-response analysis. Better justification is needed. 
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Yiliang Zhu. Ph.D. 

BS. The oral slope factor was calculated by linear extrapolation from the POD (i.e., the lower 95% 

confidence limit on the dose associated with l 0% extra risk of tumors of the small intestine in male 

mice). Has the modeling been appropriately conducted and clearly described? 

EPA carried out dose-response modeling and BMD estimation for the incidences of adenoma and 

carcinoma of small intestine in male and female mice separately. EPA stated that it relied on the 

multi-stage model because the model is preferred by the agency. but gave no justification or 

explanation. It went on to report an estimated slope of 0.09 (mg/kg-day) and 0.10 (mg/kg-day) for 

male and female mice respectively. In section 5.3.4 of the Review, EPA reported the CSFs derived 

on the basis of the cancer incidence of small intestine in male and female mice, and chooses that of 

male mice because of "the poor fit of the multistage model to the female mouse data". EPA did not 

provided adequate detail on the modeling efforts, or a discussion and justifications for its final 

selection (section 5.3.3 and 5.3.4). EPA did provide some detail in Appendix B2, which is essentially 

the direct output from running the BMDS software, but again no discussion or explanation of the 

output. It would be helpful and necessary that EPA substantially expand sections 5.3.3 and 5.3.4 to 

report in greater detail the modeling process, the issues encountered, and justify the decision and 

choice therein. 

On a more technical side, examination of Appendix B2 reveals that (1) no standard error is reported 

for the estimate of model coefficient, and (2) the coefficient of the second order in the polynomial 

was set to be zero, not estimated. The model did not fit the data at all despite a non-significant pvalue for goodness-of-fit test. EPA did not give any explanation or discussion in this regard. The 

significance of a goodness-fit-test depends on sample size, and a non-significant result does not 

imply a correct model, especially within a range where there are no data. EPA should explore 

different options: trying different models, considering omitting the highest dose, or considering 

combine male and female mice. 

Inclusion of multiple studies, multiple endpoints, multiple model choices, and various BMR levels 

for deriving a POD is increasingly desirable towards a more systematic and quantitative risk 

assessment paradigm. It will afford an opportunity to quantify the underlying uncertainties and 

variations associated with the choices and options made each many stages of the risk assessment 

process. Within the context of CSF for hexavalent chromium, EPA is in a position to conduct a more 

thorough and comprehensive assessment by including multiple endpoints, different model forms that 

allow for nonlinear dose-response, various BMR levels. The outcome will then demonstrate a range 

for POD and CSF to permit a better quantification and better understanding of uncertainties and 

variations. 
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Appendix B: List of Reviewers 
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ft EA~ United States :W:.: Environmental Protection Agency 

~3' National Center for Environmental Assessment 

Peer Review Workshop for EPA's Draft 

Toxicological Review of Hexavalent Chromium 

Hilton Crystal City Hotel 

Arlington, VA 

May 12,2011 

Peer Reviewers 

Janusz Z. Byczkowski, Ph.D. 

Independent Consultant 

Fairborn, OH 

Joshua W. Hamilton, Ph.D. 

Senior Scientist, Bay Paul Center 

Marine Biologiallaboratory 

Woods Hole, MA 

Monica Nordberg, Ph.D. 

Professor, Karolinska lnstitutet 

Institute Environmental Medicine 

Stockholm, Sweden 

Steven R. Patierno, Ph.D. 

Executive Director of the GW Cancer Institute 

The George Washington University 

Washington, DC 

Toby G. Rossman, Ph.D. 

Professor, Department of Environmental 

Medicine 

New York University School of Medicine 

Tuxedo, NY 

Konstantin Salnikow, Ph.D. 

Program Director, Division of Cancer 

Biology 

National Institutes of Health 

Bethesda, MD 

John P. Wise Sr., Ph.D. 

Professor of Toxicology and Molecular 

Epidemiology 

University of Southern Maine 

Portland, ME 

Anatoly Zhitkovich, Ph.D. (Chair) 

Brown University 

Providence, Rl 

Viliang Zhu, Ph.D. 

Professor 

University of South Florida 

Tampa, Fl 
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Appendix C: List of Observers 
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ft EA~ United States ~ Environmental Protection Agency 

~J' National Center for Environmental Assessment 

Peer Review Workshop for EPA's Draft Toxicological 

Review of Hexavalent Chromium 

Hilton Crystal City Hotel 

Arlington, VA 

May 12,2011 

Final List of Observers 

David Andrews 

Environmental Working Group 

Washington, DC 

Andrea Auerbach 

ERG 

Arlington, VA 

Jim Ball 

U.S. Environmental Protection Agency 

Washington, DC 

Deborah Barsotti {via teleconference} MACTEC Engineering and Consulting 

Hamilton, NJ 

Barbara Beck 

Gradient 

Cambridge, MA 

Nancy Beck 

Office of Management and Budget 

Washington, DC 

Susan Belman {via teleconference} 

Thompson Hine 

Cleveland, OH 

Ted Berner 

U.S. Environmental Protection Agency 

Washington, DC 

Norman Birchfield 

U.S. Environmental Protection Agency 

Washington, DC 

Amanda Boone-Edwards 

U.S. Environmental Protection Agency 

Washington, DC 

Kevin Bromberg 

U.S. Small Business Administration 

Washington, DC 

Erica Brown 

Association of Metropolitan Water Agencies 

Washington, DC 

Laura Brust 

American Chemistry Council 

Washington, DC 

Sharon Campleman 

Electric Power Research Institute 

Palo Alto, CA 

Jennifer Cheung 

U.S. Government Accountability Office 

Washington, DC 

Becki Clark 

U.S. Environmental Protection Agency 

Washington, DC 

Jan Connery (Facilitator) 

ERG 

lexington, MA 

Glinda Cooper 

U.S. Environmental Protection Agency 

Washington, DC 
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Lon Couillard (via teleconference) 

Milwaukee Water Works 

Milwaukee, WI 

Helen Fahy (via teleconference) 

Fahy Associates 

Morristown, NJ 

David Garcia (via teleconference) 

City of Riverside Public Utilities 

Riverside, CA 

Catherine Gibbons 

U.S. Environmental Protection Agency 

Washington, DC 

Helen Goeden (via teleconference) 

Minnesota Department of Health 

St. Paul, MN 

Robert Grace 

u.s. Government Accountability Office 

Washington, DC 

Mohammad T. Habibian (via teleconference) 

wssc 

Laurel, MD 

Joseph Haney 

Texas Commission on Environmental Quality 

Austin, TX 

Mark Harris 

ToxStrategies, Inc. 

Katy, TX 

Laurie Haws 

ToxStrategies, Inc. 

Austin, TX 

Sean Hays 

Summit Toxicology 

Lyons, CO 

Maria Hegstad 

Inside EPA/Risk Policy Report 

Arlington, VA 

Donald Hintz 

Electric Environmental Services 

Glen Allen, VA 

Annie Jarabek (via teleconference) 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 

Julie Kabel 

AECOM 

Manchester, NH 

Patricia Kablach Casano 

GE 

Washington, DC 

David Kahane (via teleconference) 

Forensic Analytical 

Hayward, CA 

Connie Kang-Sickel 

U.S. Environmental Protection Agency 

Washington, DC 

Troy Kennedy (via teleconference) 

Honeywell 

Morristown, NJ 

Chris Kirman 

Summit Toxicology, LLP 

Orange Village, OH 

Katharine Kurtz (via teleconference) 

Navy and Marine Corps Public Health Center 

Portsmouth, VA 

Mollie Lemon 

U.S. Environmental Protection Agency 

Washington, DC 

Gerald Llewellyn 

Delaware Division of Public Health 

Dover, DE 

April Luke 

U.S. Environmental Protection Agency 

Washington, DC 

Ann Mason 

American Chemistry Council 

Washington, DC 

Anita Meyer (via teleconference) 

U.S. Army Corps of Engineers 

Omaha, NE 

Sherrie Morris 

Booz Allen Hamilton 

McLean, VA 

Kathleen Newhouse 

U.S. Environmental Protection Agency 

Washington, DC 
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Gunnar Nordberg 

Umea University 

Umea, Sweden 

Margaret Pratt 

U.S. Environmental Protection Agency 

Washington, DC 

Deborah Proctor 

T oxStrategies, Inc. -

Rancho Santa Margarita, CAResha Putzrath 

Navy and Marine Corps Public Health Center 

Washington, DC 

Patrick Quinn 

The Accord Group 

Washington, DC 

Susan Rieth 

U.S. Environmental Protection Agency 

Washington, DC 

Pat Rizzuto 

Daily Environment Report 

Arlington, VA 

Jennifer Sass 

Natural Resources Defense Council 

Washington, DC 

Randy Schumacher 

Schumacher Partners lnt'l 

Washington, DC 

Cheryl Siegel Scott 

U.S. Environmental Protection Agency 

Washington, DC 

Eugenia Sosa 

Global Communications 

Washington, DC 

William Vance (via teleconference} 

Davis, CA 

Steve Via 

American Water Works Association 

Washington, DC 

Hui-Min Yang 

U.S. Environmental Protection Agency 

Washington, DC 
376 


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Appendix D: Agenda 
377 


23819.330

ft 

~ EA~ United States Environmental Protection Agency 

~J' National Center for Environmental Assessment 

Peer Review Workshop for EPA's Draft Toxicological 

Review of Hexavalent Chromium 

Hilton Crystal City Hotel 

Arlington, VA 

May 12,2011 

Agenda 

8:00a.m. 

8:30a.m. 

8:40a.m. 

8:45a.m. 

9:30a.m. 

10:30a.m. 

10:45 a.m. 

Registration/ check in 

Welcome, Introductions, Meeting Purpose & Agenda .......................... Jan Connery, ERG (contractor) 

EPA Welcome Remarks ............... ........................ Vincent Cogliano, IRIS Program Director, EPA NCEA 

Public Comment ............................................................................................................. Jon Connery 

General Questions ......................................................................... Anatoly Zhitkovich (Chair) & Panel 

G1. Is the Toxicological Review logical, dear and concise? Has EPA dearly presented and synthesized the 

scientific evidence for noncancer and cancer hazard? 

G2. Please identify any additional studies that would make a significant impact on the conclusions of the 

Toxicological Review. 

.. ....................................... BREAKChemical-Specific Charge Questions 

(A) Oral Reference Dose (RfD) for Hexavalent Chromium ... Anatoly Zhitkovich & Panel 

Al. A two-year drinking water study of sodium dichromate dihydrate in rats and mice (NTP, 2008) was 

selected as the basis for the derivation of the RfO. Please comment on whether the selection of this 

study as the principal study is scientifically supported and dearly described. Please identify and provide 

the rationale for any other studies that should be selected as the principal study. 

A2. Diffuse epithelial hyperplasia in the duodenum of female mice was selected as the critical effect for the 

RfD. Please comment on whether the selection of this critical effect is scientifically supported and 

dearly described. Please identify and provide the rationale for any other endpoints that should be 

selected as the critical effect. 

A3. Benchmark dose (BMD) modeling was applied to the incidence of diffuse epithelial hyperplasia in the 

duodenum of female mice to derive the point of departure (POD) for the RfD. Has the BMD modeling 

been appropriately conducted and clearly described? Is the benchmark response (BMR) selected for 
378 

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Noon 

1:00 p.m. 

use in deriving the POD (i.e., a 10% increase in the incidence of diffuse epithelial hyperplasia) 

scientifically supported and clearly described? 

A4. Please comment on the rationale for the selection of the uncertainty factors (UFs) applied to the POD 

for the derivation of the RfD. Are the UFs sclentifically supported and dearly described? If changes to 

the selected UFs are proposed, please identify and provide a rationale. 

LUNCH 

(B) Carcinogenicity of Hexavalent Chromium ....................... , .......................... Anatoly Zhitkovich & Panel 

81. Under EPA's 2005 Guidelines for Carcinogen Risk Assessment (www.epa.gov/iris/backgrd.html), 

hexavalent chromium is likely to be carcinogenic to humans by the oral route of exposure. Is the cancer 

weight of evidence characterization scientifically supported and clearly described? 

82. A mutagenic mode of carcinogenic action by all routes of exposure is proposed as the primary mode of 

action for hexavalent chromium. Please comment on whether this determination is scientifically 

supported and clearly described. Please comment on data available for hexavalent chromium that may 

support an alternative primary mode of action. 

83. A two.year drinking water study in rats and mice {NTP, 2008) was selected for the derivation of an oral 

slope factor. Please comment on whether the selection of this study for quantification is scientifically 

supported and clearly described. Please identify and provide the rationale for any other studies that 

should be considered. 

3:00p.m. BREAK 

3:15p.m. 

4:30p.m. 

4:55p.m. 

5:00p.m. 

(B) Carcinogenicity of Hexavalent Chromium (continued} ... ................. Anatofy Zhitkovich & Panel 

84. The incidence of adenomas and carcinomas combined in the small intestine of male mice from the NTP 

(2008) two·year drinking water study were selected to serve as the basis for the quantitative cancer 

assessment. Please comment on whether this selection is scientifically supported and clearly described. 

Please identify and provide the rationale for any other endpoints that should be selected to serve as the 

basis for the quantitative cancer assessment. 

85. The oral slop-e factor was calculated by linear extrapolation from the POD (i.e., the lower 95% 

confidence limit on the dose associated with 10% extra risk of tumors of the small intestine in male 

mice}. Has the modeling been appropriately conducted and dearly described? 

Reviewer Final Comments .. ................................................................ Anatofy Zhitkovich & Panel 

Closing Remarks .. . ...................... Jan Connery 

ADJOURN