Page 1 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*1.2“Chemicaland PhysicalInformation”GlobalSince Cr6+ and Cr3+ are the most common valencestates of chromium naturally occurring in the earth’scrust, we believe that it would be helpful if the textdiscussed the state-of-the-science related toanalytical methods. It would be especially usefultodiscuss whether methods are available for thedetermination of Cr+6 in various environmental/biological media and in foods. This discussiondetailing the potential analytical problems, detectionlimits and the uncertainties related to the analyseswould be very useful and help explain why in manycases only total chromium was reported in some ofthe studies cited in the Toxicological Review. As anexample, the recent Soares M.E. et al. 2010 paperentitled, “Chromium speciation analysis in breadsamples,” (J. Agric. Food Chem., 2010,58(2), pp1366–1370) discusses a new method to determinetotal 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 dryweight for white and whole bread samples,respectively; those for Cr+6 were 5.65 +/- 5.44 and6.82 +/- 4.88 microgram/kg of dry weight. Thesedata show the difficulty in isolating potentialimpacts from Cr+6 at low levels of potentialconcern.We recommend some discussion of thelimitations of analytical speciationmethodology for Cr+6 in environmental andbiological media be included in this section asgeneral chemical information. We believe thiswould provide the environmental communitywith information necessary to characterize thepotential health risks from ingestion ofCr+6and better explain why chemical speciationmay be required instead of total chromiumanalyses of environmental media.SPage 2 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*2.3.1.“Absorptionfollowingingestion”;6.1. “HumanHazardPotential.”9, lines 27-35; 10, lines1-6;204, line 22.NTP (2008).Sutherland et al. (2000) reported elevatedconcentrations of chromium in various tissues withhigher levels in bone and kidney after ingestion ofCr+6, demonstrating that chromium was taken upfrom systemic circulation (bioavailability of aportion of the chromium). The text states that theauthors proposed two “non-mutually exclusive”possibilities to explain their results: (1) a portion ofthe ingested Cr+6 escaped reduction, enteredsystemic circulation, and was available for cellularabsorption, or (2) the Cr+3 formed in the gut wasabsorbed and was not cleared by the kidneys butwas taken up by the cells. As the chromium wasreported as total chromium and not speciated, wequestion EPA’s comment that “In any event, thisstudy suggests that even at relatively lowconcentrations, hexavalent chromium is likelyabsorbed and retained in the body”. It appears thatEPA inferred this conclusion and not the studyauthors, but the text is not clear. Regardless, webelieve that the word “likely” should be changed to“possibly” based on the authors’ second proposalinvolving Cr+3 being absorbed and taken up by thecirculatory system, and not Cr+6. Also, latersections of the EPA draft and the references thereinindicate that Cr+6 is poorly absorbed via ingestion(only about 2-11%; Donaldson and Barreras, 1966;etc.).Although we understand that the focus of thissection is on the toxicokinetics of Cr+6following ingestion, nevertheless, we believe itis important to describe alternate possibilitiesinvolving how Cr+3 can form complexes withorganic ligands, which then allows Cr+3 topass more easily across cell membranes andpotentially enter systemic circulation and/orhow the Cr+6 can be reduced inside the redblood cells to Cr+3. Thus, we recommend thatthe EPA text describing the opinions of theseresearchers (Sutherland et al., 2000) and othersshould more clearly distinguish between thestudy researchers’ conclusions and those fromthe EPA’s(page10, lines 5-6). As the speciesof chromium absorbed is dependent on variousconditions, we believe that it is important topresent alternate theories even if they differfrom the EPA’s final conclusions, especially asanalytical speciation was usually not feasible.In keeping with a more realistic approach, werecommend that page 204, line 22 “HumanHazard Potential” should be reworded toindicate that not just “some” Cr+6 is reduced toCr+3 in the GI tract, but that available datasuggests that the majority of ingested Cr+6 isreduced to Cr+3 in the GI tract.S3.4.2.1.Subchronic43, lines 5-7;49, lines 9-Table 4-4 summarizes the hematological effects forratsexposed toCr+6in drinking water for 23 daysThe inconsistencies in the text noted should beresolved. Moreover, we believe the NTPSPage 3 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*OralExposure;NTP (2007,2008).14; 42-49;Table 4-4,44-45,Hematological effects inmale andfemaleF344/Nrats...up to 3months;45, Lines 7-8;Table 4-5,50, Clinicalchemistryeffects inmale andfemaleF344/Nrats..., 47;Table 4-7,50.“Incidence ofnone-plasticlesionsobserved inmale andfemaleF344/N rats.”to up to three months at various treatment levels.Page 43 states that although more severe, doserelated effects were observed in severalhematological parameters, severity at 3 months wasgenerally less than that observed at 23 days. Thetext does not mention that a number of thehematological parameters were within the limits ofthe experimental accuracy cited in the Table. Wealso noted variability in the direction of the changesin rat mean cell hemoglobin at 23 days compared totreatment time 3 months that was also notmentioned in the text. Lines 5-6 on page 43 onlystated that MCH decreased. We believe it isnoteworthy that NTP (2007) considered the data toreflect a compensatory hematopoietic response.Page 45 notes that “A consistent relationshipbetween severity and dose was not observed in theclinical chemistry results”(T 4-5). This alsoappears to apply to Table 4-4. Page 46, lines 5-9notes that NTP (2007) suggested that the clinicalchemistry analyses’ results indicate that exposureofrats to sodium dichromate dihydrate in drinkingwater induced hepatocellular membrane damage orcytotoxicity at doses greater than or equal to 1.7mgCr+6/kg-day. We believe that the hematologicaldata from rats after 3 months compared to 23 daysexposures help support NTP’s (2007) conclusionsconcerning a compensatory response. Sex-relateddifferences in whether organ weight increased(females) or decreased (males) were noted. NTP(2007) concluded that the changes in body weightscientists who conducted the study are in agood position to review and interpretimpartially the results, and therefore should ata minimum be presented alongside EPA’sposthocanalysis. Specifically, the text on page 43should be amended to support the actual data inTable 4-4, i.e., to better demonstrate theapparent compensatory mechanism after 3months exposure in rats. The fact that thevariations in hematological parameters werefrequently within the range of experimentalaccuracy at 3 months should be noted inSection 4.6.3. This also applied to some of theresponses for the data at 23 days (for example,mean cell hemoglobin (MCH) at 1.7 and 3.5mg Cr+6/kg-day in male rats. This should bediscussed in the text.The rationale for organ weight differencesbetween the rat sexes at some treatment levelsand exposure durations and the potentialimpact of weight loss, especially in the highestdosed animals, would be of interest as wouldother sex and/or species-related differences(NTP, 2007).We also recommend that the potential for acompensatory adaptive response related toincreased hematopoiesis in response toCr+6exposure from ingestion be addressed ingreater detail.Page 4 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*. may have impacted some organ weight changes.The text on pages 48 and 49 presents the significanthistopathological changes noted from microscopicslide examination, to include “In males [rats] adose-dependent increase in the incidence ofhistocytic cellular infiltration of pancreatic lymphnodes was observed at 1.7 mg hexavalentchromium/kg-day, whereas increased pancreaticlymph node sinusoidal ectasia and lymphoidhyperplasia 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 controlresponses noted in the female rat for two effects andthe lack of a dose-response for some of theendpoints noted in Table 4-7.4.4.2.1.“SubchronicOralExposure”;NTP (2007);5.1.1.; NTP(2008).54, lines 3-13; Table 4-9, 54.“Incidence ofnonneoplasticlesionsobserved inmale andfemaleB6C3F1...3months”;Table 4-11,58.The text states that based on histopathologicalchanges(histiocytic cellular infiltration) in theduodenum, a LOAEL of 3.1 mg Cr+6/kg-day wasidentified for both sexes of B6C3F1 mice (3-monthNTP (2007) study. Table 4-11 in the EPA draft(NTP, 2007) provides the incidence ofnonneoplastic lesions observed in another mousestudy comparing effects in three different strainsofmice, one of which was the same B6C3F1 strain.It is interesting to compare the results of the studyusing B6C3F1 mice given in Table 4-9 with thesame endpoint (duodenum (histiocytic cellularinfiltration) and same strain (B6C3F1) of mousefrom the comparative study depicted in Table 4-11.Although we acknowledge that in both the 3-month and two-year NTP toxicity studies(NTP, 2007a; 2008) of sodium dichromatedihydrate, histiocytic cellular infiltration wasconsistently observed in several tissuesincluding the liver, duodenum, and mesentericand pancreatic lymph nodes of rats and mice,we believe that the increased response in thesame mouse strain (B6C3F1) depicted in Table4-11 compared to Table 4-9 (NTP, 2007)subchronic studies) deserves attention andshould be acknowledged and discussed in theappropriate section(s) of the EPA draft. EPAmay wish to consider including this apparentSPage 5 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*“Incidence ofnonneoplasticlesionsobserved inmaleB6C3F1,Balb/C, ANDAM3-c57bl/6mice ...3months”;186-186.Of the three strains of mice, the 4/10 number ofpositive lesions observed at 3.1 mg Cr+6/kg-day(LOAEL) (Table 4-9) for mouse strain B6C3F1 hadfewer lesions than at the lower treatment dose of 2.8mgCr+6/kg-day LOAEL given in Table 4-11,which reported 8/10 nonneoplastic lesions. Bothstudies were for 3 month durations. A similarcomparison for the next higher treatment group ofB6C3F1 mice yields a similarly great difference inthe number of lesions reported between the twostudies. The reproducibility of the results for thelower level treatment groups does not lend to theirsuitability for quantitative analyses. This variabilityin the number of positive findings should beaddressed in the appropriate sections of the textwhere the results are discussed (and the uncertaintyanalyses) and may impact the ability to drawmeaningful conclusions from the lowerconcentration response data.inconsistency in the uncertainty section andindicate whether these variations may be ofsignificance, particularly as increasedincidence of histopathological changes in theduodenum in both male and female mice wereconsidered as possible critical effects inderivation of the oral reference dose based onchronic exposure (NTP, 2008).5.4.4.1.“GenotoxicityStudies”; 7.0.“References.”115, lines 11-14.NTP, 2008;Wise et al.,2008; 2010;NJ,“Derivationof IngestionBased SoilRemediationCriterion forPage 115 discusses the mutagenicity of Cr+6 andstates that it is mediated through the generation ofhighly reactive chromium intermediates and reactiveoxygen species, citing Wise et al., 2008. It was ourunderstanding thatthe postulated mechanism(s)underlying chromium-induced genotoxicity andcarcinogenicity is still the subject of much researchand some scientific debate (NTP, 2008). The Wiseet al. paper cited states, “In the particulate form,Cr(VI) dissolves slowly in vivo, leading to anextended exposure of lung cells...Hexavalentchromiumis taken into the cell and rapidly reducedWe believe that the text on page 115 shouldindicate that the authors believed the Cr+6 wasinhaled as particulate. EPA should alsoconsider how this might affect its conclusionregarding mode of action of carcinogenicity, aswell as conclusions regarding absorption.We acknowledge that there must be a cut-offdate for considering publications for inclusionin the Toxicological Review, but EPA maywish note that the following additional, morerecent publications: Wise et al., 2010,“Chronic exposure to zinc chromate inducesSPage 6 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*Cr+6Basedon the NTPChronicBioassayData forSodiumDichromateDihydrate”(April, 2009).to Cr(V), Cr(IV), Cr(III), and reactive oxygenspecies. Cells treated with Cr(VI) are subject toseveral types of DNA damage resulting from thisreduction...These types of damage, if left unrepairedor are misrepaired, can lead to growth arrest,cytotoxicity, and apoptosis, as well as mutationsleading to neoplastic trans-formation and ultimatelytumorigenesis.” (emphasis added) Thus, the Wise etal. (2008) study appears to suggest a sequentialprogression of events that may lead to tumorformation after long-term inhalation exposure ofparticulate. This is in contrast to the rapidabsorption of a small portion of the ingested Cr+6and its known reactivity at the site of contact (GItract). A more recent paper by Wise et al.(2010),entitled, “Comparative genotoxicity and cytotoxicityof four hexavalent chromium compounds in humanbronchial cells,”also discusses the importance ofsolubility in Cr+6 to its carcinogenic potential andstates that particulate Cr+6 compounds slowlydissolve in the lung and are thus the mostcarcinogenic. Although no studies were locatedregarding genotoxic effects in humans after oralexposure to Cr+6,recent examinations ofchromosomal damage afterin vivoexposures ofanimals of Cr+6 in drinking water have given mixedresults (DeFlora et al., 2006; NTP 2007a).Althoughthe referenced documents cite numerousstudies providing experimental evidence in supportof the role of reactive oxygen species in thegenotoxicity of chromium, other studies raisecentrosome amplification and spindle assemblycheckpoint bypass in human lung fibroblasts;”Soares, M.E.et al, 2010,Chromium speciationanalysis in bread samples; andWise etal.(2010), “Comparative genotoxicity andcytotoxicity of four hexavalent chromiumcompounds in human bronchial cells.”Page 7 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*questions about the relative contribution of thismechanism (O’Brienet al., 2003; Zhitkovich, 2005;Quievrynet al., 2006). In particular, if chromiummutagenicity is mediated through reactive oxygen, itwould not be considered a mutagenic mode ofaction, as the chemical (or its metabolite) would notbe directly mutagenic.NTP (2008) concluded that “There was evidence ofsystemic exposure to Cr VI following oraladministration in the drinking water based on thetissue distribution data, toxicity to the hematopoieticsystem, and the presence of microscopic changes inmultiple tissues.” Advances in analytical detectionmethods involving speciation of total chromiummay help resolve this scientific debate.6. Table 4-23.“In vivogenotoxicitystudies ofhexavalentchromium...”;142 The table incorrectly labels the second studyreported inNTPTechnical Report on the ToxicityStudies of Sodium Dichromate Dihydrate(NTP,2007) as “±” for the B6C3F1 mice. Although thetext of that report says that the results were“[a]nequivocal increase,” that same sentence concludes“based on a small increase in micronucleatednormochromatic erythrocytes that did not reachstatistical significance.” Following standardpractice, an increase that is not statisticallysignificant should be summarized as negative, “−”.The notation in the table should be corrected,as well as any references to this as anequivocal rather than a negative result.S/M7. 4.6.3. “Modeof ActionGlobal;134-143;The document correctly notes thatin vivomutagenicity by oral exposure in mice is highlydependent on the strain used. After correcting theThe mode of action analysis for the tumorsused for deriving the cancer potency should bechanged, as the data for this strain of mouseS/MPage 8 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*Information”Table 4-23,142. “In vivogenotoxicitystudies ofhexavalentchromium...”;Table 5-5,196.“Incidence ofadenoma andcarcinomascombined inthe smallintestine ofmale B6C3Fimice...”notation for the results of two studies in the malemice in the strain used for cancer studies (seecomment #6 on Table 4-23, below), this strain doesnot produce mutations by the oral route.Therefore,the biological plausibility is lacking for a mutagenicmode of action for the tumors produced, as oralexposure to Cr+6 in these mice does not producemutations.The ability of a chemical to cause mutations in somebiological systems is necessary, but not sufficienttoestablish a mutagenic mode of action forcarcinogenesis. In this case, we are fortunate tohavein vivomutagenicity and cancer bioassaysperformed in the same strain of mouse by the sameFederal testing facility, thus obviatingconsiderations of both quality of testing, geneticdrift of inbred strains, and quality of analysis oftheresults. The conclusion is clear: The tumorsproduced in the male B6C3F1 mice can not have amutagenic mode of action because this strain ofmice did not produce mutations under the sameconditions tested at the same time in other strainsofmice.can not support that finding. An alternative,plausible mode of action for these tumors isproposed in comment #3, although time willnot allow us to perform the analysis to see ifthe data will support that mode of action. Ifthe data support that mode of action, it wouldbe reasonable to use a nonlinear extrapolationfrom the point of departure, as the mode ofaction requires high concentrations of thechemical at a point proximal to that ofexposure. A nonlinear, low-dose extrapolationis further supported by the lack of statisticallysignificant tumors at the first two doses of theexperiment (Table 5-5).8. 4.6.3.1.“HypothesizedMode ofAction.”168, line 32;169, lines 18-20.If “carcinogenicity can be induced directly byreduced forms of chromium interacting with DNA toform adducts and crosslinks that can lead to DNAbreaks and mutations,” it is not completely evidentwhy overall Cr+3 less carcinogenic. In particular,EPA’s IRIS analysis of Cr+3 states, “The data fromoral and inhalation exposures of animals toThe document should explain why, if itsmutagenic mode of action depends on thereduction of Cr+6 to Cr+3, the latter is lesscarcinogenic by both routes of exposure, andalso less mutagenic. If EPA’s currentconclusions regarding Cr+6 depend on anupdated analysis of Cr+3, the two wouldS/MPage 9 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*trivalent chromium do not support determination ofthe carcinogenicity of trivalent chromium. IARC(1990) concluded that animal data are inadequatefor the evaluation of the carcinogenicity of Cr (III)compounds... In general, trivalent chromium wasnot mutagenicin bacterial assays when tested withor without a mammalian activation system (Venittand Levy, 1974; Petrilli and Deflora, 1977,1978a,b). In one study, trivalent chromium wasmutagenic in Baccillus subtilis, but this activitywaslow compared with compounds of hexavalentchromium(Nakamuro et al., 1978).” (emphasisadded).As currently written, differences between Cr+3reduced from Cr+6 activity versus administeredCr+3 are not clear in the document. Thesedifferences should be clearly described in order forrisk managers and the public to have a clearunderstanding of the proposed mechanism,especially since chromium is an essential nutrient.optimally be reviewed together.Conversely,if the differences incarcinogenicity are due to differences incellular absorption of Cr+6 and Cr+3 it shouldbe more clearly described. If there aredifferences in mutagenic activity of Cr+3 whenit enters the cell with an organic ligand itshould be described.9.4.6.3.2“ExperimentalSupport forthehypothesizedMode ofAction”;Table4-23; Section4.2.1,“Subchronic55, line 10;142 and 171,line 21The text in section 4.2.1 states that though five am3-C57BL/6 mice were exposed to sodium dichromatedehydrate for a mutagenicity study, but the studieswere not carried out due to technical problems. Thisseems to be in conflict with the results reported inTable 4-23 and the results described in Section4.6.3.2.The conflicting text needs to be corrected orthe text in Section 4.2.1 needs to be made moreclear.S,EPage 10 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*oralExposure”10.4.6.3. “Modeof ActionInformation”;5.1.1, “Choiceof PrincipalStudy andCriticalEffect.”168-181; 186 EPA should reconsider its conclusion that Cr+6’spotential carcinogenicity is related to a mutagenicmode of action, specifically via an oral exposurepathway. The results of NTP and others suggestthat a site of contact (or chromium accumulation)produces cell damage, progression to cytotoxicity,and regenerative proliferation as a more plausiblemode of action. Page 174, lines 19-24, state that“Only one study examined tumor target tissue forevidence of mutagenicity (De Flora et al., 2008). DeFlora et al. (2008) found negative results for DNAprotein crosslinks and DNA adducts in theduodenum in mice following drinking waterexposures. Other available drinking water exposurestudies of hexavalent chromium that measuredmutagenicity in mice failed to show evidence ofmicronucleus induction in the blood or bonemarrow (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 ofcontact toxicity of exposure to sodium dichromatedihydrate, a significantly increased incidence ofulcer and epithelial hyperplasia and metaplasia ofthe glandular stomach occurred in rats in the 1,000mg/L group. Coincident with the focal ulcers wasevidence of inflammation within the stomach wall.The increases in blood neutrophil and monocytecounts were consistent with an inflammatoryEPA should reconsider its evaluation of thestrength of evidence for a mutagenic mode ofaction for carcinogenicity specifically relatedto oral exposure via the ingestion exposurepathway, “within the reductive capacity of theGI tract” based on the likelihood of alternatemodes of action, as presented in the citedreferences and discussed in the precedingcomments pertaining to data and rationalesuggestive of an alternate mode of action. Wenote that the New Jersey Department ofEnvironmental Protection’s risk assessment (onwhich EPA states they based their Cr+6dose/response; Forward, pagex) recentlyconcluded that the NTP data indicated otheralternate modes of action related to potentialoral carcinogenicity of Cr+6 (April, 2009).As an aside, it would be helpful if EPA clearlystate the results of the “key” studies in an“Executive Summary.” This would helpidentify which endpoints, species, studies EPAconsidered critical and thus, were used to helpEPA derive their Cr+6 proposed oral cancerpotency value and other toxicity values forIRIS.S, EPage 11 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*response. These lesions occurred at the junctionbetween the forestomach and the glandularstomach, where the reduction of Cr VI to Cr V andto Cr IV and subsequent radical formation would beenhanced by the increased acidity. Histiocyticinfiltration was consistently noted as a minor lesionin the duodenum of the small intestine of rats andmice receiving sodium dichromate dihydrate.Hyperplasia, characterized by villi that were tallerthan normal and with tightly packed basophilicepithelial cells, was an additional finding in thesmall intestine in all strains of mice.” Thesefindings are characteristic of a non-mutagenic modeof action.11. 4.6.3.2.“ExperimentalSupport fortheHypothesizedMode ofAction.”170, line 30 Thein vivosection emphasizes the positive resultsin fruit flies, while only mentioning afterwards theinconsistent results in mammals.We suggest discussing the mammalian datafirst, as these data are more relevant for thetumors of interest, in particular, the speciesdependent results.E12. 4.6.3.3 Global As organs affected by the cancers are directlyassociated with the route of exposure, it would seemthat 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) themutagenicity and carcinogenicity are associatedwith high levels of exposure and not at lower levelsof exposure.This alternative mode of action, i.e., thathexavalent chromium is carcinogenic only athigh levels of exposure, should be discussed insection 4.6.3.3. “Other Possible Modes ofAction.” Ideally in the absence of a knownmode of action, if sufficient biologicalinformation exists to infer that the chemicalmay be nonlinear at low doses, both procedures(linear and nonlinear extrapolation) would beSPage 12 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*presented as part of risk characterization. IfEPA does not provide this information in thissection, it can not easily be brought forwardinto the risk characterization. We thereforerecommend that EPA also provide a nonlinearextrapolation from the point of departure inaddition to the linear extrapolation.13. 5.1.1 “ChronicStudies”;4.6.3.3.“OtherPossibleModes ofaction”;NTP (2008)185, Lines 1-27;Table 5-1,187.“IncidenceData forLesions FromAll TreatmentGroups ofFemaleF344/N Rateand Male andFemaleB6C3F1Mice....”;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 and0.77 mg Cr+6/kg/day, respectively, based onincreased incidences of nonneoplastichistopathological changes to the liver, duodenum(histiocytic cellular infiltrate), and mesenteric lymphnodes. In female rats, a LOAEL for noncancereffects of 0.24 mg Cr+6 /kg-day was identifiedbased on the increased incidence (21/50) of chronicinflammation of the liver (observed in all treatmentgroups). Lines 7-8 state that a NOAEL was notidentified because effects observed were at thelowest dose tested. The text does not mention that12/50 of the control female rats also showed chronicliver inflammation, as shown in Table 5-1. Aneffect is a NOAEL if it is not observed to have astatistically higher incidence in dosed animals thanin control animals. EPA should determine if this isthe case. This study identified a LOAEL fornoncancer effects of 0.38 mg hexavalentWe suggest that this section provide the controlpositive response data from the NTP (2008)study, particularly for the male mice, andcompare them to the number of mice withpositive histopathological responses attributedto the toxicity of Cr+6, as the positive micedata (and not the rat data) were considered“critical” to EPA’s derivation of their proposedCr+6 ingestion-related toxicity values.The potential effects that may not be directlyrelated to the toxicity of chromiumper seshould also be included, such as the potentialimpact of the palatability of the drinking waterand weight changes, as applicable, should bediscussed, as well as other sex-relateddifferences noted that may impact the resultsshould be discussed in greater detail (NTP,2008).Also, the potential for formation ofhyperplasia. resulting from cytotoxicitywithout progression to GI tract tumors in ratsS, MPage 13 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*chromium/kg-day in both male and female B6C3F1mice; a NOAEL value was not identified becauseeffects seen were at the lowest dose administered.Table 5-1 also showed that 14/47 of the controlmale mice were positive for histiocytic cellularinfiltration. In males, the LOAEL was based onincreased incidences (11/50) of histopathologicalchanges to the duodenum (diffuse epithelialhyperplasia) and mesenteric lymph nodes(histiocytic cellular infiltration);in females, theLOAEL was based on increased incidences ofhistopathological changes to the duodenum. Again,EPA should determine if the response in the dosedanimals is a NOAEL rather than a LOAEL, giventhe high level of responses in the control animals.The NTP (2008) 2-year study involved the use ofmultiple dose groups, and included a comprehensiveevaluation of multiple endpoints. Also, this bioassayused lower doses than the subchronic (90-day)studies also conducted by NTP (2007), and thusprovides dose-response information at lowerexposure levels than the 90-day studies. When theadenomas and carcinomas were combined for allsites of the small intestine, including the duodenum,jejunum, and ileum, there was a clear exposureresponse relationship, and the incidences werestatistically significant in the two highest exposuregroups of male and female mice.compared to mice data (NTP, 2008) should bediscussed in greater detail in section 4.6.3.3 ofthe document on alternate modes of action(page 179). We recommend that this sectionshould be expanded to include the potentiallink between noncancer and cancer effectsinmice and the potential role of cytotoxicity forboth species in the GI tract, as it relates to analternate mode of action, should be discussedin greater detail.14. 5.1.1 189The choice of diffuse epithelial hyperplasia of theThe possibility that duodenal lesions in miceS,MPage 14 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*duodenum in female mice as the critical effect isquestionable because of decreased drinking waterintake due to decreased palatability of chromatetainted water (see Page 75 para 2). Decreased waterconsumption and resulting abrasion of the luminallining on ingestion of pelleted dry rodent chow isapossible cause of the observed duodenal lesionsfollowing chromate administration in drinkingwater. (see DeSesso, J.M., A.L. Lavin, S.M. Hsia,and R. D. Mavis. 2000, “Assessment of theCarcinogenicity Associated with Oral Exposures toHydrogen Peroxide,” Food and ChemicalToxicology, 38, pp. 1021-1041.) If the duodenallesions are a result of abrasion of the lumen by dryrodent chow, this effect is not appropriate forderivation of a human RfD.arose from abrasion of the luminal lining bydry rodent chow due to decreased waterconsumption should be discussed; the impactof this possibility on the appropriateness of thiseffect as the critical effect should be addressed.A different critical effect should be chosen ifthis possibility cannot be eliminated.15. 5.3.3. “DoseAdjustmentsandExtrapolationMethod(s).”197, line 7. The statement that “The dose response is assumed tobe linear in the low-dose range when evidencesupports a mutagenic mode of action because ofDNA reactivity” is not accurate. Our search of thecancer guidelines shows it states: [emphasis added]“Agents that are generally consideredto be linear inthis region include:• agents that are DNA-reactive and have directmutagenic activity.”These cancer guidelines also state “Special attentionis important when the data support a nonlinearThe sentence should be revised so that“assumed” is replaced by “generallyconsidered”.SPage 15 of 15Department of Defense Comments on Toxicological Review of Hexavalent ChromiumComments submitted by: Office of theSecretary of Defense Chemical and MaterialRisk Management DirectorateOrganization: Department of DefenseDate Submitted: May 5, 2010*Comment categories: Science or methods (S); Editorial, grammar/spelling, clarifications needed (E);or Other (O). Also please indicate if Major i.e. affects the outcome,conclusions or implementation of the assessment.CommentNo.SectionPage & Line,or GlobalCommentSuggested Action, Revisionand References (if necessary)Category*mode of action but there is also a suggestion ofmutagenicity. Depending on the strength of thesuggestion of mutagenicity, the assessment mayjustify a conclusion that mutagenicity is notoperative at low doses and focus on a nonlinearapproach, or alternatively, the assessment may useboth linear and nonlinear approaches.” Thus, ourread of the cancer guidelines is that it allows fortheoccurrence of tumors by a chemical that can causemutations but does not act by a mutagenic mode ofaction and/or that are nonlinear at low doses evenwith mutagenicity.16.General,Sections 5 and6GlobalPage 66 of the NTP (2008) report states that the ratoral mucosa carcinomas were highly aggressiveneoplasms that invaded other tissues, such as thesoft tissue around the nose. The human relevance ofthe rat squamous epithelial cell carcinomas of theoral mucosa and tongue, etc., from ingestion ofdrinking water containing Cr+6 should be discussedeven though these data were not used to derive theoral cancer slope factor. NTP also stated that thesecarcinomas are relatively rare in rats and were notseen in the mouse studies.The National CancerInstitute (NCI) has stated that oral cancer accountsfor about 3% of cancers in men and 2% of cancersin women. Thus, it does not appear to be a commonhuman cancer.We recommend providing epidemiologicaldata concerning the statistical incidence of theoral cancers discussed to help risk assessorsrelay the current estimates for these types ofcancer in humans to risk managers and theconcerned public who may misinterpret theneed for chromium as an essential nutrientbased on the rodent cancer data presented.This would be consistent with the “realitycheck” proposed by EPA’s SAB whenreviewing EPA’s toxicity evaluation of arsenic,where the SAB member indicated that EPA’sprojection of human lung cancers due toarsenic would exceed those caused bysmoking.S