Page 1 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

No. Section Page & Line, or Global Comment Suggested Action, R

and References (if ne

1. 2 “Chemical 

and Physical Information” 

Global Since Cr6+ and Cr3+ are the most common valence 

states of chromium naturally occurring in the earth’s crust, we believe that it would be helpful if the text discussed the state-of-the-science related to analytical methods. It would be especially useful to discuss whether methods are available for the determination of Cr+6 in various environmental /biological media and in foods. This discussion detailing the potential analytical problems, detection 

limits and the uncertainties related to the analyses would be very useful and help explain why in many cases only total chromium was reported in some of the studies cited in the Toxicological Review. As an 

example, the recent Soares M.E. et al. 2010 paper entitled, “Chromium speciation analysis in bread samples,” (J. Agric. Food Chem., 2010, 58 (2), pp 

1366–1370) discusses a new method to determine total and Cr+6 in bread samples. The authors’ reported that the total chromium contents were 47.3

+/- 20.0 and 50.9 +/- 22.2 microgram/kg of dry weight for white and whole bread samples, respectively; those for Cr+6 were 5.65 +/- 5.44 and

6.82 +/- 4.88 microgram/kg of dry weight. These data show the difficulty in isolating potential impacts from Cr+6 at low levels of potential concern.

We recommend some discusslimitations of analytical specimethodology for Cr+6 in envibiological media be included general chemical informationwould provide the environmewith information necessary to potential health risks from ing

and better explain why chemimay be required instead of totanalyses of environmental me
Page 2 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

No. Section Page & Line, or Global Comment Suggested Action, R

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2. 3.1. 

“Absorption following ingestion”; 

6.1. “Human Hazard Potential.” 

9, lines 27-35; 10, lines 1-6; 204, line 22. NTP (2008). 

Sutherland et al. (2000) reported elevated concentrations of chromium in various tissues with higher levels in bone and kidney after ingestion ofCr+6, demonstrating that chromium was taken up from systemic circulation (bioavailability of a portion of the chromium). The text states that the

authors proposed two “non-mutually exclusive” 

possibilities to explain their results: (1) a portion of the ingested Cr+6 escaped reduction, entered systemic circulation, and was available for cellular 

absorption, or (2) the Cr+3 formed in the gut was absorbed and was not cleared by the kidneys but was taken up by the cells. As the chromium was reported as total chromium and not speciated, we question EPA’s comment that “In any event, this study suggests that even at relatively low concentrations, hexavalent chromium is likely absorbed and retained in the body”. It appears that EPA inferred this conclusion and not the study authors, but the text is not clear. Regardless, we

believe that the word “likely” should be changed to 

“possibly” based on the authors’ second proposal involving Cr+3 being absorbed and taken up by the circulatory system, and not Cr+6. Also, later sections of the EPA draft and the references therein indicate that Cr+6 is poorly absorbed via ingestion(only about 2-11%; Donaldson and Barreras, 1966; etc.). 

Although we understand that section is on the toxicokineticfollowing ingestion, neverthelis important to describe alterninvolving how Cr+3 can form organic ligands, which then alpass more easily across cell mpotentially enter systemic circhow the Cr+6 can be reduced blood cells to Cr+3. Thus, we the EPA text describing the opresearchers (Sutherland et al., should more clearly distinguis

study researchers’ conclusionthe EPA’s (page10, lines 5-6)

of chromium absorbed is depeconditions, we believe that it present alternate theories even from the EPA’s final conclusianalytical speciation was usuaIn keeping with a more realistrecommend that page 204, lin

Hazard Potential” should be rindicate that not just “some” CCr+3 in the GI tract, but that asuggests that the majority of ireduced to Cr+3 in the GI trac

3. 4.2.1. 

Subchronic 

43, lines 5-7; 49, lines 9-

Table 4-4 summarizes the hematological effects for rats exposed to Cr+6 in drinking water for 23 days 

The inconsistencies in the texresolved. Moreover, we believ
Page 3 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

No. Section Page & Line, or Global Comment Suggested Action, R

and References (if ne

Oral Exposure;NTP (2007, 2008). 

14; 42-49; Table 4-4, 44-45, Hematological effects in male and female F344/N rats...up to 3 months; 45, Lines 7-8; Table 4-5, 50, Clinical chemistry effects in male and female F344/N rats..., 47; Table 4-7, 50. “Incidence of none-plastic lesions observed in male and female F344/N rats.”

to up to three months at various treatment levels. Page 43 states that although more severe, doserelated effects were observed in several hematological parameters, severity at 3 months was generally less than that observed at 23 days. The text does not mention that a number of the hematological parameters were within the limits of the experimental accuracy cited in the Table. We also noted variability in the direction of the changes in rat mean cell hemoglobin at 23 days compared to treatment time 3 months that was also not mentioned in the text. Lines 5-6 on page 43 only stated that MCH decreased. We believe it is noteworthy that NTP (2007) considered the data to reflect a compensatory hematopoietic response.

Page 45 notes that “A consistent relationship between severity and dose was not observed in the clinical chemistry results” (T 4-5). This also appears to apply to Table 4-4. Page 46, lines 5-9 notes that NTP (2007) suggested that the clinical chemistry analyses’ results indicate that exposure of rats to sodium dichromate dihydrate in drinking water induced hepatocellular membrane damage or cytotoxicity at doses greater than or equal to 1.7 mg Cr+6/kg-day. We believe that the hematological data from rats after 3 months compared to 23 days exposures help support NTP’s (2007) conclusions concerning a compensatory response. Sex-related differences in whether organ weight increased (females) or decreased (males) were noted. NTP (2007) concluded that the changes in body weight 

scientists who conducted the sgood position to review and inimpartially the results, and thea minimum be presented alonhoc analysis. Specifically, the should be amended to support Table 4-4, i.e., to better demoapparent compensatory mechamonths exposure in rats. The variations in hematological pafrequently within the range of accuracy at 3 months should bSection 4.6.3. This also appliresponses for the data at 23 damean cell hemoglobin (MCH) mg Cr+6/kg-day in male rats. discussed in the text. The rationale for organ weighbetween the rat sexes at some and exposure durations and thimpact of weight loss, especiadosed animals, would be of inother sex and/or species-relate(NTP, 2007). We also recommend that the pcompensatory adaptive responincreased hematopoiesis in re

exposure from ingestion be ad

greater detail. 
Page 4 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

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. may have impacted some organ weight changes. 

The text on pages 48 and 49 presents the significant histopathological changes noted from microscopic slide examination, to include “In males [rats] a dose-dependent increase in the incidence of histocytic cellular infiltration of pancreatic lymph 

nodes was observed at 1.7 mg hexavalent chromium/kg-day, whereas increased pancreatic lymph node sinusoidal ectasia and lymphoid hyperplasia were only increased in the highest dose- group...” (page 48, lines 18-19; page 49, lines 1-2). 

The text does not mention the positive control responses noted in the female rat for two effects and the lack of a dose-response for some of the endpoints noted in Table 4-7. 

4. 4.2.1. 

“Subchronic Oral Exposure”; NTP (2007); 5.1.1.; NTP (2008). 

54, lines 3-13; Table 4-9, 54. “Incidence of nonneoplastic lesions observed in male and female B6C3F1...3 months”; Table 4-11, 58. 

The text states that based on histopathological changes (histiocytic cellular infiltration) in the 

duodenum, a LOAEL of 3.1 mg Cr+6/kg-day was identified for both sexes of B6C3F1 mice (3-month NTP (2007) study. Table 4-11 in the EPA draft (NTP, 2007) provides the incidence of nonneoplastic lesions observed in another mouse study comparing effects in three different strains of 

mice, one of which was the same B6C3F1 strain. It is interesting to compare the results of the study 

using B6C3F1 mice given in Table 4-9 with the same endpoint (duodenum (histiocytic cellular infiltration) and same strain (B6C3F1) of mouse from the comparative study depicted in Table 4-11. 

Although we acknowledge thmonth and two-year NTP toxi(NTP, 2007a; 2008) of sodium dihydrate, histiocytic cellular consistently observed in severincluding the liver, duodenumand pancreatic lymph nodes owe believe that the increased same mouse strain (B6C3F1) 4-11 compared to Table 4-9 (Nsubchronic studies) deserves ashould be acknowledged and appropriate section(s) of the Emay wish to consider includin
Page 5 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

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“Incidence of nonneoplastic lesions observed in male B6C3F1, Balb/C, AND AM3-c57bl/6 mice ...3 months”; 186-186. 

Of the three strains of mice, the 4/10 number of positive lesions observed at 3.1 mg Cr+6/kg-day (LOAEL) (Table 4-9) for mouse strain B6C3F1 had fewer lesions than at the lower treatment dose of 2.8 mg Cr+6 /kg-day LOAEL given in Table 4-11, 

which reported 8/10 nonneoplastic lesions. Both studies were for 3 month durations. A similar comparison for the next higher treatment group of B6C3F1 mice yields a similarly great difference in the number of lesions reported between the two studies. The reproducibility of the results for the lower level treatment groups does not lend to theirsuitability for quantitative analyses. This variability in the number of positive findings should be addressed in the appropriate sections of the text where the results are discussed (and the uncertainty analyses) and may impact the ability to draw meaningful conclusions from the lower concentration response data. 

inconsistency in the uncertainindicate whether these variatiosignificance, particularly as inincidence of histopathologicaduodenum in both male and feconsidered as possible critical derivation of the oral referencchronic exposure (NTP, 2008

5. 4.4.1. 

“Genotoxicity Studies”; 7.0. 

“References.” 

115, lines 11-14. NTP, 2008; Wise et al., 2008; 2010; NJ, “Derivation 

of IngestionBased Soil Remediation Criterion for 

Page 115 discusses the mutagenicity of Cr+6 and states that it is mediated through the generation of highly reactive chromium intermediates and reactiveoxygen species, citing Wise et al., 2008. It was our understanding that the postulated mechanism(s) 

underlying chromium-induced genotoxicity and carcinogenicity is still the subject of much research and some scientific debate (NTP, 2008). The Wise et al. paper cited states, “In the particulate form, Cr(VI) dissolves slowly in vivo, leading to an extended exposure of lung cells...Hexavalent chromium is taken into the cell and rapidly reduced 

We believe that the text on paindicate that the authors belieinhaled as particulate. EPA sconsider how this might affecregarding mode of action of cwell as conclusions regarding We acknowledge that there mdate for considering publicatioin the Toxicological Review, wish note that the following arecent publications: Wise et a“Chronic exposure to zinc chr
Page 6 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

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Cr

+6 

Based 

on the NTP Chronic Bioassay Data for Sodium Dichromate Dihydrate”(April, 2009). 

to Cr(V), Cr(IV), Cr(III), and reactive oxygen species. Cells treated with Cr(VI) are subject to several types of DNA damage resulting from this reduction...These types of damage, if left unrepairedor are misrepaired, can lead to growth arrest, cytotoxicity, and apoptosis, as well as mutations leading to neoplastic trans-formation and ultimately tumorigenesis.” (emphasis added) Thus, the Wise et al. (2008) study appears to suggest a sequential progression of events that may lead to tumor formation after long-term inhalation exposure of particulate. This is in contrast to the rapid absorption of a small portion of the ingested Cr+6 and its known reactivity at the site of contact (GItract). A more recent paper by Wise et al.(2010), entitled, “Comparative genotoxicity and cytotoxicity of four hexavalent chromium compounds in human bronchial cells,” also discusses the importance of solubility in Cr+6 to its carcinogenic potential and states that particulate Cr+6 compounds slowly dissolve in the lung and are thus the most carcinogenic. Although no studies were located regarding genotoxic effects in humans after oral exposure to Cr+6, recent examinations of 

chromosomal damage after in vivo exposures of 

animals of Cr+6 in drinking water have given mixed results (DeFlora et al., 2006; NTP 2007a).Although the referenced documents cite numerous 

studies providing experimental evidence in support of the role of reactive oxygen species in the genotoxicity of chromium, other studies raise 

centrosome amplification and checkpoint bypass in human lSoares, M.E. et al, 2010, Chranalysis in bread samples; an

al.(2010), “Comparative genocytotoxicity of four hexavalencompounds in human bronchi
Page 7 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

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questions about the relative contribution of this mechanism (O’Brien et al., 2003; Zhitkovich, 2005; 

Quievryn et al., 2006). In particular, if chromium 

mutagenicity is mediated through reactive oxygen, it would not be considered a mutagenic mode of action, as the chemical (or its metabolite) would not be directly mutagenic. NTP (2008) concluded that “There was evidence of systemic exposure to Cr VI following oral administration in the drinking water based on the tissue distribution data, toxicity to the hematopoietic system, and the presence of microscopic changes in multiple tissues.” Advances in analytical detection methods involving speciation of total chromium may help resolve this scientific debate. 

6. Table 4-23. 

“In vivo genotoxicity studies of hexavalent chromium...”; 

142 The table incorrectly labels the second study 

reported in NTP Technical Report on the Toxicity 

Studies of Sodium Dichromate Dihydrate (NTP, 2007) as “±” for the B6C3F1 mice. Although the text of that report says that the results were “[a]n equivocal increase,” that same sentence concludes “based on a small increase in micronucleated normochromatic erythrocytes that did not reach statistical significance.” Following standard 

practice, an increase that is not statistically significant should be summarized as negative, “−”. 

The notation in the table shouas well as any references to thequivocal rather than a negati

7. 4.6.3. “Mode 

of Action 

Global; 134-143; 

The document correctly notes that in vivo

mutagenicity by oral exposure in mice is highly dependent on the strain used. After correcting the

The mode of action analysis fused for deriving the cancer pchanged, as the data for this s
Page 8 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

No. Section Page & Line, or Global Comment Suggested Action, R

and References (if ne

Information” Table 4-23, 

142. “In vivo genotoxicity studies of hexavalent chromium...”; Table 5-5, 196. “Incidence of adenoma and carcinomas combined in the small intestine of male B6C3Fi mice...” 

notation for the results of two studies in the male

mice in the strain used for cancer studies (see comment #6 on Table 4-23, below), this strain does 

not produce mutations by the oral route. Therefore, 

the biological plausibility is lacking for a mutagenic mode of action for the tumors produced, as oral exposure to Cr+6 in these mice does not produce mutations. The ability of a chemical to cause mutations in some biological systems is necessary, but not sufficient to establish a mutagenic mode of action for carcinogenesis. In this case, we are fortunate to have in vivo mutagenicity and cancer bioassays performed in the same strain of mouse by the same Federal testing facility, thus obviating considerations of both quality of testing, genetic drift of inbred strains, and quality of analysis of the results. The conclusion is clear: The tumors 

produced in the male B6C3F1 mice can not have a mutagenic mode of action because this strain of mice did not produce mutations under the same conditions tested at the same time in other strains of mice.

can not support that finding. plausible mode of action for tproposed in comment #3, althnot allow us to perform the anthe data will support that modthe data support that mode of be reasonable to use a nonlinefrom the point of departure, aaction requires high concentrachemical at a point proximal texposure. A nonlinear, low-dis further supported by the lacsignificant tumors at the first experiment (Table 5-5). 

8. 4.6.3.1. 

“Hypothesized Mode of Action.” 

168, line 32; 169, lines 18-20. 

If “carcinogenicity can be induced directly by reduced forms of chromium interacting with DNA to form adducts and crosslinks that can lead to DNA breaks and mutations,” it is not completely evident 

why overall Cr+3 less carcinogenic. In particular,EPA’s IRIS analysis of Cr+3 states, “The data from 

oral and inhalation exposures of animals to 

The document should explain mutagenic mode of action depreduction of Cr+6 to Cr+3, thcarcinogenic by both routes oalso less mutagenic. If EPA’s conclusions regarding Cr+6 dupdated analysis of Cr+3, the 
Page 9 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

No. Section Page & Line, or Global Comment Suggested Action, R

and References (if ne

trivalent chromium do not support determination of the carcinogenicity of trivalent chromium. IARC (1990) concluded that animal data are inadequate for the evaluation of the carcinogenicity of Cr (III) compounds... In general, trivalent chromium was not mutagenic in bacterial assays when tested with 

or without a mammalian activation system (Venitt and Levy, 1974; Petrilli and Deflora, 1977, 1978a,b). In one study, trivalent chromium was mutagenic in Baccillus subtilis, but this activity was 

low compared with compounds of hexavalent chromium (Nakamuro et al., 1978).” (emphasis 

added). As currently written, differences between Cr+3 reduced from Cr+6 activity versus administered Cr+3 are not clear in the document. These differences should be clearly described in order for risk managers and the public to have a clear understanding of the proposed mechanism, especially since chromium is an essential nutrient.

optimally be reviewed togetheConversely, if the differences 

carcinogenicity are due to diffcellular absorption of Cr+6 anbe more clearly described. If differences in mutagenic activit enters the cell with an organshould be described. 

9. 4.6.3.2 

“Experimental Support for the hypothesized Mode of Action”;Table 4-23; Section 4.2.1, “Subchronic 

55, line 10; 142 and 171, line 21 

The text in section 4.2.1 states that though five am3-C57BL/6 mice were exposed to sodium dichromate dehydrate for a mutagenicity study, but the studies

were not carried out due to technical problems. This 

seems to be in conflict with the results reported in 

Table 4-23 and the results described in Section 4.6.3.2. 

The conflicting text needs to bthe text in Section 4.2.1 needs clear. 
Page 10 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

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oral Exposure” 

10. 4.6.3. “Mode 

of Action Information”; 5.1.1, “Choice of Principal Study and Critical Effect.” 

168-181; 186 EPA should reconsider its conclusion that Cr+6’s 

potential carcinogenicity is related to a mutagenicmode of action, specifically via an oral exposure pathway. The results of NTP and others suggest that a site of contact (or chromium accumulation) produces cell damage, progression to cytotoxicity, and regenerative proliferation as a more plausible mode of action. Page 174, lines 19-24, state that “Only one study examined tumor target tissue for 

evidence of mutagenicity (De Flora et al., 2008). De Flora et al. (2008) found negative results for DNAprotein crosslinks and DNA adducts in the duodenum in mice following drinking water exposures. Other available drinking water exposure studies of hexavalent chromium that measured mutagenicity in mice failed to show evidence of micronucleus induction in the blood or bone marrow (De Flora et al., 2008, 2006; NTP, 2007; Mirsalis et al., 1996).”

The following excerpt is from NTP (2007): “Regarding other evidence for systemic or site of contact toxicity of exposure to sodium dichromate dihydrate, a significantly increased incidence of ulcer and epithelial hyperplasia and metaplasia of the glandular stomach occurred in rats in the 1,000mg/L group. Coincident with the focal ulcers was evidence of inflammation within the stomach wall. The increases in blood neutrophil and monocyte counts were consistent with an inflammatory 

EPA should reconsider its evastrength of evidence for a mutaction for carcinogenicity speto oral exposure via the ingestpathway, “within the reductivGI tract” based on the likelihomodes of action, as presented references and discussed in thcomments pertaining to data asuggestive of an alternate modnote that the New Jersey DepEnvironmental Protection’s riwhich EPA states they based dose/response; Forward, page 

concluded that the NTP data ialternate modes of action relaoral carcinogenicity of Cr+6 (As an aside, it would be helpfstate the results of the “key” s“Executive Summary.” This identify which endpoints, speconsidered critical and thus, wEPA derive their Cr+6 propospotency value and other toxicIRIS. 
Page 11 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

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response. These lesions occurred at the junction between the forestomach and the glandular stomach, where the reduction of Cr VI to Cr V and to Cr IV and subsequent radical formation would be enhanced by the increased acidity. Histiocytic infiltration was consistently noted as a minor lesion in the duodenum of the small intestine of rats and mice receiving sodium dichromate dihydrate. Hyperplasia, characterized by villi that were taller than normal and with tightly packed basophilic epithelial cells, was an additional finding in the small intestine in all strains of mice.” These findings are characteristic of a non-mutagenic modeof action. 

11. 4.6.3.2. 

“Experimental Support for the Hypothesized Mode of Action.” 

170, line 30 The in vivo section emphasizes the positive results 

in fruit flies, while only mentioning afterwards the inconsistent results in mammals. 

We suggest discussing the mafirst, as these data are more retumors of interest, in particuladependent results. 

12. 4.6.3.3 Global As organs affected by the cancers are directly 

associated with the route of exposure, it would seem that the cancers are likely to be caused by a high (bolus) dose proximal to site of exposure. This isalso consistent with the findings that (1) the mutagenicity and carcinogenicity are associated with high levels of exposure and not at lower levels 

of exposure. 

This alternative mode of actiohexavalent chromium is carcihigh levels of exposure, shoulsection 4.6.3.3. “Other PossibAction.” Ideally in the absencmode of action, if sufficient binformation exists to infer thamay be nonlinear at low dose(linear and nonlinear extrapol
Page 12 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

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presented as part of risk charaEPA does not provide this infsection, it can not easily be brinto the risk characterization. recommend that EPA also proextrapolation from the point oaddition to the linear extrapol

13. 5.1.1 “Chronic 

Studies”; 4.6.3.3. “Other Possible Modes of action”; NTP (2008) 

185, Lines 1-27; Table 5-1, 187. “Incidence Data for Lesions From All Treatment Groups of Female F344/N Rate and Male and Female B6C3F1 Mice....”; 

179. 

Page 185 states that NTP (2008) study identified “No Observable Adverse Effect Level” (NOAEL) 

and “Lowest Observable Effect Level” (LOAEL) 

values for noncancer effects in male rats of 0.21 and 0.77 mg Cr+6/kg/day, respectively, based on increased incidences of nonneoplastic histopathological changes to the liver, duodenum (histiocytic cellular infiltrate), and mesenteric lymph nodes. In female rats, a LOAEL for noncancer effects of 0.24 mg Cr+6 /kg-day was identified based on the increased incidence (21/50) of chronic

inflammation of the liver (observed in all treatment 

groups). Lines 7-8 state that a NOAEL was not identified because effects observed were at the lowest dose tested. The text does not mention that

12/50 of the control female rats also showed chronic 

liver inflammation, as shown in Table 5-1. An effect is a NOAEL if it is not observed to have a statistically higher incidence in dosed animals than in control animals. EPA should determine if this isthe case. This study identified a LOAEL for noncancer effects of 0.38 mg hexavalent 

We suggest that this section ppositive response data from thstudy, particularly for the malcompare them to the number opositive histopathological respto the toxicity of Cr+6, as the data (and not the rat data) wer“critical” to EPA’s derivation Cr+6 ingestion-related toxicitThe potential effects that may related to the toxicity of chrom

should also be included, such impact of the palatability of thand weight changes, as applicdiscussed, as well as other sexdifferences noted that may imshould be discussed in greater 2008). Also, the potential for formatihyperplasia. resulting from cywithout progression to GI trac
Page 13 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

No. Section Page & Line, or Global Comment Suggested Action, R

and References (if ne

chromium/kg-day in both male and female B6C3F1 mice; a NOAEL value was not identified because effects seen were at the lowest dose administered. Table 5-1 also showed that 14/47 of the control male mice were positive for histiocytic cellular infiltration. In males, the LOAEL was based on increased incidences (11/50) of histopathological changes to the duodenum (diffuse epithelial hyperplasia) and mesenteric lymph nodes (histiocytic cellular infiltration); in females, the 

LOAEL was based on increased incidences of histopathological changes to the duodenum. Again, EPA should determine if the response in the dosed animals is a NOAEL rather than a LOAEL, given the high level of responses in the control animals. The NTP (2008) 2-year study involved the use of multiple dose groups, and included a comprehensive evaluation of multiple endpoints. Also, this bioassay used lower doses than the subchronic (90-day) studies also conducted by NTP (2007), and thus provides dose-response information at lower exposure levels than the 90-day studies. When the adenomas and carcinomas were combined for all sites of the small intestine, including the duodenum, jejunum, and ileum, there was a clear exposure response relationship, and the incidences were statistically significant in the two highest exposure groups of male and female mice. 

compared to mice data (NTP, discussed in greater detail in sthe document on alternate mo

(page 179). We recommend tshould be expanded to include link between noncancer and cmice and the potential role of both species in the GI tract, as alternate mode of action, shouin greater detail. 

14. 5.1.1 189 The choice of diffuse epithelial hyperplasia of the The possibility that duodenal 
Page 14 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

No. Section Page & Line, or Global Comment Suggested Action, R

and References (if ne

duodenum in female mice as the critical effect is questionable because of decreased drinking water intake due to decreased palatability of chromatetainted water (see Page 75 para 2). Decreased waterconsumption and resulting abrasion of the luminal lining on ingestion of pelleted dry rodent chow is a possible cause of the observed duodenal lesions following chromate administration in drinking water. (see DeSesso, J.M., A.L. Lavin, S.M. Hsia, and R. D. Mavis. 2000, “Assessment of the Carcinogenicity Associated with Oral Exposures to Hydrogen Peroxide,” Food and Chemical Toxicology, 38, pp. 1021-1041.) If the duodenal lesions are a result of abrasion of the lumen by dry rodent chow, this effect is not appropriate for derivation of a human RfD.

arose from abrasion of the lumdry rodent chow due to decreaconsumption should be discusof this possibility on the approeffect as the critical effect shoA different critical effect shouthis possibility cannot be elim

15. 5.3.3. “Dose 

Adjustments and Extrapolation Method(s).” 

197, line 7. The statement that “The dose response is assumed to 

be linear in the low-dose range when evidence supports a mutagenic mode of action because of DNA reactivity” is not accurate. Our search of the cancer guidelines shows it states: [emphasis added] 

“Agents that are generally considered to be linear in 

this region include: • agents that are DNA-reactive and have direct mutagenic activity.” These cancer guidelines also state “Special attention is important when the data support a nonlinear 

The sentence should be revise“assumed” is replaced by “ge

considered”. 
Page 15 of 15 

Department of Defense Comments on Toxicological Review of Hexavalent Chromium 

Comments submitted by: Office of the Secretary of Defense Chemical and Material Risk Management Directorate 

 Organization: Department of Defense Date Submitted: May 5, 2010 

*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E); or Other (O). Also please indicate if Majconclusions or implementation of the assessment. 

Comment 

No. Section Page & Line, or Global Comment Suggested Action, R

and References (if ne

mode of action but there is also a suggestion of mutagenicity. Depending on the strength of the suggestion of mutagenicity, the assessment may justify a conclusion that mutagenicity is not operative at low doses and focus on a nonlinear approach, or alternatively, the assessment may use both linear and nonlinear approaches.” Thus, our read of the cancer guidelines is that it allows for the occurrence of tumors by a chemical that can cause mutations but does not act by a mutagenic mode of action and/or that are nonlinear at low doses even with mutagenicity.

16. General, 

Sections 5 and 6 

Global Page 66 of the NTP (2008) report states that the rat 

oral mucosa carcinomas were highly aggressive neoplasms that invaded other tissues, such as the soft tissue around the nose. The human relevance of

the rat squamous epithelial cell carcinomas of the oral mucosa and tongue, etc., from ingestion of drinking water containing Cr+6 should be discussed even though these data were not used to derive the oral cancer slope factor. NTP also stated that these carcinomas are relatively rare in rats and were not

seen in the mouse studies. The National Cancer 

Institute (NCI) has stated that oral cancer accounts 

for about 3% of cancers in men and 2% of cancers in women. Thus, it does not appear to be a common human cancer. 

We recommend providing epidata concerning the statistical oral cancers discussed to help relay the current estimates for cancer in humans to risk manconcerned public who may mneed for chromium as an essebased on the rodent cancer daThis would be consistent with check” proposed by EPA’s SAreviewing EPA’s toxicity evawhere the SAB member indicprojection of human lung canarsenic would exceed those casmoking.