FINAL DRAFT FOR THE DRINKING WATER
  CRITERIA DOCUMENT ON TRIHALOMETHANES
             April 8,  1994
             Prepared For

Health and Ecological Criteria Division
   Office of Science and Technology
            Office of Water
 U.S.  Environmental Protection Agency
        Washington,  D.C.   20460
                 Under

      EPA Contract No. 68-C2-0139

                  by

   Clement International Corporation

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3. REPORT TYPE AND DATES COVERED
4. TITLE AND SUBTITLE
Final Draft for the Drinking Water
Criteria Document on Trihalomethanes
6. AUTHOR(S)
7. PERFORMING ORGANIZATION NAME(S) AND AOORESS(ES)
Clement International Corporation
9. SPONSORING /MONITORING AGENCY NAME(S) AND ADDRESS(ES)
U.S. EPA
Office of Water
401 M St., SW
Washington, DC 20460
5. FUNDING NUMBERS
68-C2-0139
8. PERFORMING ORGANIZATION
REPORT NUMBER
10. SPONSORING /MONITORING
AGENCY REPORT NUMBER
11. SUPPLEMENTARY NOTES
12a. DISTRIBUTION /AVAILABILITY STATEMENT
12b. DISTRIBUTION CODE
13. ABSTRACT (Maximum 200 words)
This document provides the health effects basis to be considered
art establishing the maximum contaminant level goals for four
trihalomethanes found in chlorinated drinking water.
y
3
14. SUBJECT TERMS
health effects, trihalomethanes
17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION
OF REPORT OF THIS PAGE

15. NUMBER OF PAGES
309
16. PRICE CODE
#N . A03
19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACT
OF ABSTRACT
NSN 7540-01-280-5500
Standard Form 298 (Rev. 2-89)
Prescribed by ANSI Std. Z39-18
298-102

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                                   FOREWORD
Section 1412 (b) (3) (A) of the Safe Drinking Water Act, as amended in 1986
requires the Administrator of the Environmental Protection Agency to publish
Maximum Contaminant Level Goals (MCLGs) arid promulgate National Primary
Drinking Water Regulations for each contaminant, which, in the judgment of the
Administrator,  may have an adverse effect on public health and which is known
or anticipated to occur in public water systems.  The MCLG is nonenforceable
and is set at a level at which no known or anticipated adverse health effects
in humans occur and which allows for an adequate margin of safety.  Factors
considered in setting the MCLG include health effects data and sources of
exposure other than drinking water.

This document provides the health effects basis to be considered in
establishing the MCLGs for four trihalomethanes found in chlorinated drinking
water.  To achieve this objective, data on pharmacokinetics,  human exposure,
acute and chronic toxicity to animals and humans,  epidemiology and mechanisms
of toxicity were evaluated.  Specific emphasis is placed on literature data
providing dose-response information.  Thus, while the literature search and
evaluation performed in support of this document was comprehensive, only the
reports considered most pertinent in the derivation of the MCLGs are cited in
this document.   The comprehensive literature search in support of this
document includes information published up to 1989, however,  more recent
information may .have been added during the review process.

When adequate health effects data exist, Health Advisory values for less than
lifetime exposure (One-day, Ten-day and Longer-term, approximately 10% of an
individual's lifetime) are included in this document.  These values are not
used in setting the MCLGs, but serve as informal guidance to municipalities
and other organizations when emergency spills or contamination situations
occur.
                                                Tudor T. Davies
                                                Director, Office of Science
                                                and Technology
                                                Office of Water

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                               TABLE OF CONTENTS
1.     SUMMARY	    1-1

II.    PHYSICAL AND CHEMICAL PROPERTIES 	   II-l
       A.  Chloroform	   II-l
       B.  Brominated Trihalomethanes 	   II-l
       C.  Occurrence	   II-3
       D.  Summary	   II-5

III.   TOXICOKINETICS	    III-l
       A.  Absorption	    III-l
       B.  Distribution .-.	    III-6
       C.  Metabolism	'	    Ill-9
       D.  Excretion	_	   111-23
       E.  Bioaccumulation and Retention  	   111-24
       F.  Summary	   Ill-25

IV.    HUMAN EXPOSURE	   VI-1
       A.  Drinking Water Exposure	   VI-1
           1.  Total Trihalomethanes	   VI-1
           2.  Chloroform .  .	   VI-4
           3.  Brominated Trihalomethanes	VI-12
       B.  Exposure to Sources Other Than Drinking Water  .	VI-26
           1.  Dietary Intake	VI-26
               a.   Chloroform	VI-26
               b.   Brominated Trihalomethanes	VI-29
           2.  Air Intake	VI-31
               a.   Chloroform	VI-31
               b.   Brominated Trihalomethanes	VI-38
       C.  Overall Exposure	VI-42
           1.  Chloroform	VI-42
           2.  Brominated Trihalomethanes	VI-42
       D.  Body Burden	VI-43
           1.  Chloroform	VI-43
               a.   Breath   	VI-43
               b.   Blood	VI-44
               c.   Mother's Milk	VI-45
               d.   Adipose Tissue	VI-45
           2.  Brominated Trihalomrthanes	VI-46
               a.   Blood	VI-46
               b.   Mother's Milk	VI-46
       E.  Summary	VI-46

V.     HEALTH EFFECTS IN ANIMALS   	    V-l
       A.  Short-Term Exposure  	    V-l
           1.  Lethality	    V-l
           2.  Other Effects	    V-l
               a.   Chloroform	    V-l
               b.   Brominated Trihalomethanes  	    V-9
       B.  Longer-Term Exposure 	   V-24
           1.  Chloroform	   V-24
           2.  Brominated Trihalomethanes 	   V-41

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                           TABLE OF CONTENTS (cont.)
       C.  Reproductive and Developmental Effects  	   V-60
           1.  Chloroform	   V-60
           2.  Brominated Trihalomethanes 	   V-63
       D.  Mutagenicity and Genotoxicity	   V-65
           1.  Chloroform	   V-65
               a.  In Vitro Studies	   V-66
               b.  .In Vivo Studies	   V-69
           2.  Brominated Trihalomethanes 	   V-73
               a.  .In Vitro Studies	   V-73
               b.  In Vivo Studies	   V-85
       E.  Carcinogenicity	   V-87
           1.  Chloroform	   V-87
           2.  Brominated Trihalomethanes 	  V-102
       F.  Summary	  .  V-110
           1.  Health Effects of Acute and Short-Term Exposure of
               Animals	V-110
           2.  Health Effects of Longer-Term Exposure of Animals   .  .  .  V-lll
           3.  Reproductive/Developmental Effects  in Animals   	  V-112
           4.  Mutagenicity and Genotoxicity Studies  	  V-113
           5.  Carcinogenicity Studies in Animals  	  V-113

VI.    HEALTH EFFECTS IN HUMANS	   VI-1
       A.  Clinical Case Studies	   VI-1
       B.  Epideraiological Studies	   VI-2
       C.  High-Risk Populations   	   VI-6
       D.  Summary	   VI-9

VII.  MECHANISM OF TOXICITY	    VII-1
       A.  Role of Metabolism	    VII-1
       B.  Biochemical Basis of Toxicity	    VII-3
       C.  Mechanism of Carcinogenesis	:	    VII-5
       D.  Interactions	    VII-6
       E.  Summary	    VII-8

VIII.  QUANTIFICATION OF TOXICOLOGICAL EFFECTS   	   VIII-1
       A.  Noncarcinogenic Effects   	   VIII-6
           1.  Chloroform  	   VIII-6
               a.  One-day Health Advisory  	   VIII-6
               b.  Ten-day Health Advisory for Chloroform  	   VIII-9
               c.  Longer-term Health Advisory for Chloroform  ....  VIII-12
               d.  Reference Dose and Drinking Water Equivalent
                   Level for Chloroform  	  VIII-14
           2.  Bromodichloromethane   	  ....  VIII-17
               a.  One-day Health Advisory for
                   Bromodichloromethane  	  VI11-17
               b.  Ten-day Health Advisory for
                   Bromodichloromethane  	  VIII-18

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                          TABLE OF CONTENTS  (cont.)
               c.   Longer-term Health Advisory for
                   Bromodichloromethane 	   VIII-20
               d.   Reference Dose and Drinking Water Equivalent
                   Level for Bromodichloromethane 	   VIII-23
           3.   Dibromochloromethane	'	VIII-26
               a.   One-day Health Advisory for
                   Dibromochloromethane 	  	   VIII-26
               b.   Ten-day Health Advisory for
                   Dibromochloromethane 	   VIII-26
               c .   Longer-'term Health Advisory for
                   Dibromochloromethane 	   VI11-29
               d.   Reference Dose and Drinking Water Equivalent
                   Level for Dibromochloromethane 	   VIII-31
           4.   Bromoform  	   VIII-34
               a.   One-day Health Advisory for Bromoform  	   VIII-34
               b.   Ten-day Health Advisory for Bromoform  	   VIII-35
               c.   Longer-term Health Advisory for Bromoform  ....   VIII-37
               d.   Reference Dose and Drinking Water Equivalent
                   Level for Bromoform  	   VIII-40
       B.   Carcinogenic Effects 	  	   VIII-43
           1.   Categorization of Carcinogenic  Potential 	   VIII-43
               a.   Chloroform 	   VIII-43
               b.   Brominated Trihalomethanes  	   VIII-43
           2.   Quantitative Carcinogenic Risk  Estimates 	   VIII-44
               a.   Chloroform 	   VIII-44
               b.   Brominated Trihalomethanes  	   VIII-46
       C.   Summary  	   VIII-50

IX.     REFERENCES	   IX-1

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                                LIST OF FIGURES




                                                                          Page




III-l   Metabolic Pathways of Trihalomethane Biotrar.sforaation   .  .  .    111-10




IV-1    THMs in Water:  Means--Results From National Surveys   ....     IV-3

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                                LIST OF TABLES

                                                                          Page

1-1     Summary of Quantification of Toxicologicai Effects for
        Trihalomethanes 	   1-13

II-1    Physical and Chemical Properties of Chloroform and
        Brominated Trihalomethanes	   II-2

III-l   Recovery of Label 8 Hours after Oral Administration of
        14C-Labeled  Trihalomethanes  to  Male Rats  or Male  Mice   ....    Ill-3

III-2   Cumulative Excretion of Label after Oral Administration of
        KC-Labeled  Bromndichloromethane  to Male  Rats	    Ill-4

III-3   In Vitro Binding of 14C-Chloroform  Metabolites to
        Microsomes of Male Mice	   Ill-11

IV-1    Comparison of Results of National Surveys	   IV-2

IV-2    Chloroform in Drinking Water from the EPA TEAM study (jig/D  .  .  IV-12

IV-3    Bromodichloromethane in Drinking water from the EPA TEAM
        Study (/ig/L)  	IV-20

IV-4    Dibromochloromethane in Drinking water from the EPA TEAM
        Study (jug/L)  	IV-21

IV-5    Bromofrom in Drinking water from the EPA TEAM Study (/xg/L)   .  .  IV-22

IV-6    Chloroform in Personal Air Samples: EPA TEAM Study (jig/m3)   .  .  IV-33

IV-7    Chloroform in Outdoor Air Samples From the EPA TEAM
        Study (jug/m3)	IV-34.

V-l     Summary of Oral Lethality of Trihalomethanes  	    V-2

V-2     Summary of Short-Term Health Effects Data on Chloroform  ....   V-10

V-3     Effects of Brominated Trihalomethanes in Mice Dosed by
        Gavage for 14 Days	   V-14

V-4     Incidence and Severity of Microscopic Changes in Kidney
        and Liver of Mice Dosed with Brominated Trihalomethanes  ....   V-15

V-5     Serum Biochemical Levels in Rats Fed Brominated
        Trihalomethanes For One Month	   V-21

V-6     Incidence and Severity of Microscopic Changes in the Liver
        of Rats Dosed with Brominated Trihalomethanes for
        One Month .'	   V-22

V-7     Summary of Short-term Health Effects Data  on
        Bromodichloromethane   	   V-25

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                          LIST OF TABLES (Continued)

                                                                          Page
V-8     Summary of Short-term Health Effects Data on
        Dibromochloromethane  	    V-26

V-9     Summary of Short-term Health Effects Data on Bromoform  ....    V-27

V-10    Effects of Vehicle on the Subchronic Toxicity of Chloroform
        in B6C3F1 Mice	    V-29

V-ll    Comparison of Clinical Chemistry Parameters of Mice Treated
        with Chloroform in Corn Oil Versus Emulphor for 90 Days ....    V-30

V-12    Effect of Vehicle on Chloroform-Induced Accumulation of
V-13
V-14
V-15
V-16
V-17
V-18
V-19
V-20
V-21
V-22
V-23
V-24
V-25
V-26
V-27
Hepatic Changes Observed in Dogs Administered Chloroform
Orally for 7.5 Years 	
Summary of Longer-term Studies of Chloroform 	
Incidence and Severity of Liver and Thyroid Lesions in Rats
Fed Brominated Trihalomethanes for 90 Days 	
Serum Biochemical Levels in Rats Fed Brominated
Trihalomethanes for 18 to 24 Months . 	
Liver Lesions in Rats Fed Bromodichlorome thane for 2 Years . .
Nonneoplastic Lesions in Rats and Mice Exposed to
Bromodichlorome thane for 2 Years 	
Summary of Longer-term Studies of Bromodichloromethane ....
Summary of Longer-term Studies of Dibromochloromethane ....
Summary of Longer-tern1 Sfurfiec of BromofoTi . ......
S'munary of Tn vitro Gpnor^xiciry Data on Chloroform
Summary of In Vivo Genotoxicity Data on Chloroform 	
Summary of Genotoxicity Data on Bromodichloromethane 	
Summary of Genotoxicity Data on Dibromochloromethane 	
Summary of Genotoxicity Data on Bromoform .... 	
Liver and Kidney Necrosis and Hepatoraas in Strain A Mice
Following Repeated Oral Administration of Chloroform in
Olive Oil 	
V-35
V-39
V-42
V-49
V-50
V-54
V-57
V-58
V-59
V-70
V-74
V-75
V-77
V-79
V-88

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                          LIST OF TABLES (Continued)

                                                                          Page
V-28    Summary of Tumor Frequencies in Rats Administered
        Chloroform for 78 Weeks	   V-91

V-29    Summary of Tumor Frequencies in Mice Administered
        Chloroform for 78 Weeks	  .   V-92

V-30    Kidney Tumor Incidence in Male ICI Mice Treated with
        Chloroform	   V-94

V-31    Compound-Related'"Increased Incidences of Neoplasms in Rats
        and Mice Exposed to Chloroform in the Drinking Water for
        Two Years	"	   V-.96

V-32    Effects of Chloroform Exposure on Liver Tumors Initiated
        by Ethylnitrosourea Exposure in Male CD-I Mice	   V-99

V-33    Effect of Chloroform Exposure on Liver and Lung Tumors in
        Male B6C3F1 Mice	V-101

V-34    Tumor Frequencies in Rats and Mice Exposed to
        Bromodichloromethane in Corn Oil for 2 Years	V-103

V-35    Frequencies of Liver Tumors in Mice Administered
        Dibromochloromethane in Corn Oil for 104 Weeks	V-107

V-36    Tumor Frequencies in Rats Exposed to Bromoform in Corn Oil
        for 2 Years	V-109

VI-1    Epidemiological Studies Investigating an Association Between
        Cancer and Chlorinated Water  	   VI-7

VIII-1  Summary of Candidate Studies for Derivation of the One-day
        Health Advisory for Chloroform  	   VIII-7

VIII-2  Summary of Candidate Studies for Derivation of the Ten-day
        Health Advisory for Chloroform  	  VIII-10

VIII-3  Summary of Candidate Studies for Derivation of the
        Longer-term Health Advisory for Chloroform  	  VIII-13

VIII-4  Summary of Candidate Studies for Derivation of the DWEL
        for Chloroform  	  VIII-15

VIII-5  Summary of Candidate Studies for Derivation of the Ten-day
        Health Advisory for Bromodichloromethane  	  VIII-19

VIII-6  Summary of Candidate Studies for Derivation of the
        Longer-term Health Advisory for Bromodichloromethane  ....  VIII-21

VIII-7  Summary of Candidate Studies for Derivation of the DWEL
        for Bromodichloromethane  	  VIII-24

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                          LIST OF TABLES (Continued)

                                                                          Paee
VIII-8  Summary of Candidate Studies for Derivation of the Ten-
        day Health Advisory for Dibromochloromethane  	  VIII-27

VIII-9  Summary of Candidate Studies for Derivation of the.
        Longer-term Health Advisory for Dibromochloromethane   ....  VIII-30

VIII-10 Summary of Candidate Studies for Derivation of the DUEL
        for Dibromochloromethane  	  VIII-32

VIII-11 Summary of Candidate Studies for Derivation of the Ten-
        day Health Advisory for Bromoform 	  VIII-36

VIII-12 Summary of Candidate Studies for Derivation of the
        Longer-term Health Advisory for Bromoform 	  VIII-38

VIII-13 Summary of Candidate Studies for Derivation of DWEL
        for Bromoform 	  VIII-41

VIII-14 Upper-Bound Estimates of Cancer Risk of 1 mg/kg/day of
        Chloroform, Calculated by Four Models on the Basis of
        Various Data Sets	VIII-45

VIII-15 Carcinogenic Risk Estimates for Chloroform Calculated
        by NAS	VIII-47

VIII-16 Carcinogenic Risk Estimates for Brominated
        Trihalomethanes 	  VIII-49

VIII-17 Summary of Quantification of Toxicological Effects for
        Trihalomethanes 	  VIII-51

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I.      SUMMARY









        Trihalomethanes are volatile organic liquids that have a number of




industrial and chemical uses.  The chief reason for health concern is that




they are generated as by-products during the chlorination of drinking water.




The principal trihalomethanes that have been observed in water are chloroform




(CHClj),  bromodichloromethane (CHBrCl2),  dibromochloromethane  (CHBr2Cl),  and




bromoform (CHBr3) .   Chloroform is formed by the action of hypochlorous acid on




endogenous organic molecules (e.g., humic or fulvic acids) present in the




water.  Hypochlorous acid also oxidizes any bromide ion present to form




hypobromous acid,  which reacts with the organic material to form the bromi-




nated trihalomethanes.  Chloroform has been detected in finished drinking




water samples at. mean concentrations typically ranging from 9 to 100 jjg/L,




while average values of brominated trihalomethanes typically range from about




0.2 to 25 ^g/L.  Chloroform and bromodichloromethane levels tend to be higher




in. systems using surface water sources than those using groundwater sources,




whereas dibromochloromethane and bromoform levels are comparable in systems




using either source.









Toxicokinetics









        Measurements of gastrointestinal absorption of trihalomethanes in




mice,  rats and monkeys indicate that absorption is rapid (peak blood levels at




1 hour)  and extensive (64% to 98%).  Limited data indicate that gastrointes-




tinal absorption of chloroform (and presumably other trihalomethanes) is also




rapid and extensive (at least 90%) in humans.  Most studies of trihalomethane




absorption have used oil-based vehicles and gavage dosing.  One study in rats




found higher chloroform blood levels following oral gavage administration of




                                      1-1

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chloroform in water than after administration of chloroform in an oil vehicle.




This was interpreted as being due to higher absorption from water than from




oil, but the possible influence of first-pass metabolism was not taken into




account.  Dermal absorption of chloroform in water by rats and hairless guinea




pigs is rapid and extensive.  Absorption has also been reported in humans




dermally exposed to chloroform in water.









        Absorbed trihalomethanes appear to distribute widely throughout the




body.  Chloroform was detected in a number of postmortem tissues from humans,




with highest levels (5 to 68 /ig/kg) in body fat and lower levels (1 to




10 jig/kg) in kidney, liver,  and brain.  Radiolabeled trihalomethanes were




detected in a variety of tissues following oral dosing in rats and mice,  with




somewhat higher J.evels in stomach,  liver, blood, and kidney than in lung,




muscle, or brain.  Chloroform crosses the placenta and may be detected in




fetal tissues following inhalation exposure of pregnant rats.









        Trihalomethanes are extensively metabolized by both humans and




animals.  The main site of metabolism is the liver, but metabolism also occurs




in the kidney.   Both the oxidative and reductive metabolism of trihalomethanes




are mediated by cytochrome P-450.  The oxidative pathway requires NADPH and




oxygen, whereas the reductive pathway can utilize NADPH or NADH and is




inhibited by oxygen.  In the presence of oxygen (oxidative metabolism), the




reaction product is trihalomethanol (CX3OH),  which then decomposes to yield a




reactive dihalocarbonyl (CX20)  such as phosgene (CC120).   Dihalocarbonyls are




relatively reactive species, and may undergo a variety of reactions including




adduct formation with various cellular nucleophiles, hydrolysis to yield




carbon dioxide, or glutathione-dependent reduction to yield carbon monoxide.




If oxygen is lacking (reductive metabolism), the metabolic reaction products




                                      1-2

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appear to be free radical species such as the dihalomethyl radical (CHX2-).




These radicals are extremely reactive and also may form covalent adducts with




a "ariety of cellular molecules.  Metabolism via the reductive pathway appears




to occur more readily for brominated trihalomethanes than for chloroform.









        Both in vivo and in vitro studies indicate that the pattern of




trihalomethane metabolism may differ between animal species and sexes.  In




vivo, one study reported that mice metabolize trihalomethanes to carbon




dioxide more extensively than do rats (40% to 80% versus 4% to 18%),  although




another study indicated that rats metabolized bromodichloromethane to carbon




dioxide to an extent that was comparable to that reported for mice.  In vitro.




the capacity for reductive metabolism of trihalomethanes has been found to be




greater in hepat.ic microsomes from mice than rats, and the incorporation of




label into- covalent adducts in renal microsomes has been found to be greater




in male mice than female mice.   These metabolic differences may underlie some




of the important toxicological differences that have been noted between sexes




and species.









        Excretion of trihalomethanes occurs primarily via the lungs.   In




humans, approximately 90% of an oral dose of radiolabeled chloroform was




exhaled as the end metabolite,  carbon dioxide, or as the parent compound,




chloroform.  Levels in the urine were below the limit of detection (0.1%).  In




mice and rats, 45% to 88% of an oral dose of chloroform or brominated trihalo-




methane was excreted from the lungs either as the parent trihalomethane or as




carbon dioxide, with 1% to 5% excreted in the urine.  Intraperitoneal injec-




tion of rats with 36C1-chloroform resulted in the appearance of both  inorganic




and organic forms of chloride in the urine, but the total amount was not




quantified.




                                      1-3

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        No data were located regarding the bioaccumulation and retention of




the trihalomethanes following chronic exposure.  However, based on the rapid




metabolism and excretion of chloroform and the brcminsted trihalomethanes,




along with the low levels of chloroform in human autopsy samples, marked




accumulation and retention of these compounds are not anticipated.









Health Effects of Acute and Short-term Exposure of Animals









        Large oral doses of trihalomethanes are lethal to laboratory animals.




Acute LD50  values  range  from 119  to  2,000  mg/kg for chloroform,  450  to




969 mg/kg for bromodichloromethane,  800 to 1,200 mg/kg for dibromochloro-




methane, and 1,388 to 1,550 mg/kg for bromoform.  Death from acute high-dose




trihalomethane exposure was usually found to be due to central nervous system




depression and cardiac effects,  and was usually accompanied by histopatholo-




gical changes in the liver and kidney.









        Acute oral exposure to sublethal doses of trihalomethanes can also




produce effects on the liver, kidney, and central nervous system.  In mice,




single oral doses of 60 to 89 mg/kg chloroform produced kidney damage,  with




doses of 140 to 250 mg/kg producing liver damage.  Organ damage was character-




ized by fatty infiltration, cellular necrosis, vacuolization, enzyme level




changes, and/or organ weight changes.  Ataxia and sedation were noted in mice




receiving 500 mg/kg chloroform,  500 mg/kg bromodichloromethane, 500 mg/kg




dibromochloromethane, or 1,000 mg/kg bromoform.









        Short-term exposures of laboratory animals to trihalomethanes has been




observed to cause effects on the liver, kidney, central nervous system, and




immune system.  Hepatic effects, including organ weight changes,  elevated




                                      1-4

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serum enzyme levels, and histopathological changes were reported in mice




and/or rats administered 37 to 290 rag/kg/day chloroform, 148 to 250 mg/kg/day




bromodichloromethane, 147 to 500 mg/kg/day dibromochloromethane, or 187 to




289 mg/kg/day bromoform for 14 to 30 days.  Kidney effects, characterized by




decreased p-aminohippurate (PAH) uptake, histopathological changes and organ




weight changes, were reported in mice and/or rats administered  37 to




148 mg/kg/day chloroform, 148 to 600 mg/kg/day bromodichloromethane, 147 to




500 mg/kg/day dibromochloromethane, or 289 mg/kg/day bromoform  for 14 days.




Hyperactivity and/or a decreased operant response were observed in mice and/or




rats after ingesting 100 to 600 mg/kg/day bromodichloromethane, 400 mg/kg/day




dibromochloromethane, or 100 mg/kg/day bromoform for 14 to 60 days.









Health Effects of Longer-term Exposure of Animals









        The predominant effects of longer-term oral exposure to trihalo-




methanes occur in the liver and kidney.  The effects on these two organs are




similar to those described for short-term exposures, with liver appearing to




be the most sensitive target organ.  Hepatic effects were reported in mice




and/or rats administered 15 to 180 mg/kg/day chloroform, 24 to  300 mg/kg/day




bromodichloromethane, 39 to 250 mg/kg/day dibromochloromethane, or 50 to




250 mg/kg/day bromoform.  In general, these dose ranges are slightly lower




than those reported to cause effects following short-term exposures.  Renal




effects were reported in mice and/or rats administered 25 to 300 mg/kg/day




bromodichloromethane or 250 mg/kg/day dibromochloromethane.
                                      1-5

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Reproductive/Developmental Effects in Animals









        Data concerning the developmental effects of trihalomethanes indicate




that these chemicals are toxic to the mother and fetus at high doses and




suggest that reproductive and developmental toxicity may occur as well.  Signs




of maternal toxicity (decreased body weight and changes in organ weight) were




reported in rats, rabbits and/or mice administered 50 to 100 mg/kg/day




chloroform, 200 mg/kg/day bromodichloromethane, or 171 to 200 mg/kg/day




dibromochloromethane.   Fetotoxicity,  as indicated by decreased fetal body




weights, was evident in the offspring of rats administered 121 to




400 mg/kg/day chloroform or in mice administered 685 mg/kg/day dibromochloro-




methane.  Oral exposure of pregnant animals to trihalomethanes has caused




variations in the offspring,  mainly in the skeletal system.  Delayed




ossification and sternebral aberrations have been reported in rats and/or




rabbits administered 20 to 200 mg/kg/day chloroform, 50 to 200 mg/kg/day




br.omodichloromethane,  or 50 to 200 mg/kg/day bromoform.  However, malforma-




tions and variations (cleft plate, imperforate anus, acaudia, delayed




ossification) have been observed in inhalation studies in which mice and/or




rats were exposed to 30 to 100 ppm chloroform.









        Two studies were located which investigated the effects of trihalo-




methanes on reproduction.  In one study, oral doses of 685 mg/kg/day of




dibromochloromethane administered to mice for two generations led to decreased




fertility and gestational indices.  In the second study, bromoform adminis-




tered at 200 mg/kg/day had no effect on the fertility of mice.
                                      1-6

-------
Mutagenicity and Genotoxicitv Studies









        In vitro and in vivo studies on the mutagenic and genotoxic potential




of the trichloromethanes have yielded mixed results.  Interpretation of the




overall weight of evidence from these studies is complicated by the use of a




variety of testing protocols, different strains' of test organisms, different




activating systems, different dose levels, different exposure methods (gas




versus liquid), and in some cases, problems due to evaporation of the test




chemical.   Overall, a majority of studies yielded more positive results for




bromoform and bromodichloromethane,  and evidence of mutagenicity is considered




adequate for these chemicals.  Studies on the mutagenicity of dibromochloro-




methane and chloroform were mixed, and the overall evidence for mutagenicity




of these two chemicals is judged to be inconclusive.









Carcinogenicity Studies in Animals









        The carcinogenic potential of each of the four trihalomethanes has




been investigated in chronic oral exposure studies in animals.  Ingestion of




chloroform in oil has been found to cause liver tumors in male and female




mice, but hepatic tumors were not detected in mice exposed to chloroform in




drinking water.  Renal tumors were detected in male rats exposed to chloroform




in either oil or water, and renal tumors have been reported in male mice




exposed to chloroform in a toothpaste base.









        Ingestion of bromodichloromethane in oil has been found to cause liver




tumors in female mice, renal tumors in male mice and in male and female rats,




and tumors of the large intestine in male and female rats.  Ingestion of




dibromochloromethane in oil has been found to cause liver tumors in male and




                                      1-7

-------
female mice, and ingestion of bromoform in oil has been found to cause tumors




in the large intestines of exposed male and female rats.









Health Effects in Humans









        In a case study of a young man who ingested 4 ounces of chloroform (a




dose of about 2,500 mg/kg),  prominent clinical findings included jaundice, an




enlarged liver,  increased serum levels of bilirubin,  alkaline phosphatase




(AP),  and serum glutamic oxaloacetic transferase (SCOT) along with albumi-




nuria, glucosuria,  ketonuria and the presence of red cells and granular casts




in the urine.  These observations indicated that in humans, as in animals, the




liver and kidneys are the organs most affected by chloroform ingestion.









        In the past, bromoform was given orally as a sedative to children




suffering from whooping cough.  Doses of 50 to 100 mg/kg/day usually produced




sedation without any apparent adverse effects.  Some cases of severe toxicity




or death were reported, but these were generally attributed to accidental




overdoses.  No data were located on human exposure to either bromodichloro-




methane or dibromochloromethane.









        Workers exposed to chloroform by inhalation at levels of 112 to




1,158 mg/m3 for 1 or more years complained of nausea,  lassitude,  dry mouth,




flatulence, thirst, depression, irritability, and "scalding" micturition, but




clinical examination and tests of liver function (serum enzyme levels) failed




to detect any abnormalities.  Inhalation exposure of workers to chloroform at




levels of about 10 to 1,000 mg/m3 for 1 to 4 years was reported to be




associated with an increased incidence of viral hepatitis and enlarged liver.
                                      1-8

-------
        Some epidemiological studies suggest there may be an association




between water chlorination and increased cancer mortality rates.  An associ-




ation has also been reported between exposure to water chloroform levels of at




least 10 /ig/L and intrauterine growth retardation.  However, since chlorinated




water contains many by-products, it cannot be directly concluded from such




studies that trihalomethanes are human carcinogens or developmental toxicants.









        No data were located regarding whether any human subpopulation may be




at greater risk to trihalomethane exposure than the general population.  Data




from studies on mice suggest males may be more sensitive than females to




kidney effects, that alcohol consumption may increase toxicity in the liver.




and that diabetics may be more sensitive to liver effects.









Mechanism of Toxicity









        Three lines of evidence indicate that trihalomethane metabolism is




essential for toxicity:  (1) the tissues that most actively metabolize the




trihalomethanes (liver, kidney) are also the chief target tissues;




(2) chemical treatments that increase or decrease metabolism also tend to




increase or decrease toxicity in parallel; and (3) species- and sex-related




differences in metabolism are paralleled by similar differences in toxicity.




The detailed biochemical mechanisms by which trihalomethane metabolism leads




to toxicity are not certain, but covalent binding of reactive metabolites to




cellular macromolecules is one likely mechanism.  Such metabolites are




produced both by oxidative metabolism to dihalocarbonyls and reductive




metabolism to free radicals.  Free radical production may also lead to cell




injury by inducing lipid peroxidation in cellular membranes.
                                      1-9

-------
        Formation of DNA adduces might also account for the genotoxic and




carcinogenic potential of the trihalomethanes.   Alternatively, carcinogenesis




may be related, at least in part, to increased cell proliferation following




direct tissue injury.  However,  neither of these potential mechanisms have




been definitively linked to trihalomethane carcinogenesis.









        Several chemicals,  including various ketones,. dichloroacetic acid, and




carbon tetrachloride, potentiate the toxic effects of chloroform.  The




raechanism(s) of the potentiation by ketones is  not known but appears to




include a process other than induction of microsomal enzymes.  The vehicle




(corn oil versus aqueous) used for oral dosing also affects toxicity, with




toxicity generally being more severe following administration in oil.









Quantification of Noncarcinogenic Effects









        For chloroform, a One-day Health Advisory (HA) value of 4 mg/L was




calculated from an acute oral No-Observed-Adverse-Effect Level (NOAEL) value




of 35 mg/kg in mice.   A NOAEL value of 35 mg/kg/day, identified in pregnant




rabbits dosed by gavage on days 6 to 15 of gestation, was used to calculate a




Ten-day HA value of 4 mg/L.  No adequate data were located for calculating




Longer-term HA values for chloroform, so the DWEL (0.4 mg/L) may be taken as a




conservative Longer-term HA for adults, and the adjusted DWEL (0.1 mg/L) as a




conservative Longer-term HA for children.  A Reference Dose  (RfD) of




0.01 mg/kg/day and a Drinking Water Equivalent Level (DWEL) of 0.4 mg/L wer-e




derived based on a Lowest-Observed-Adverse-Effect Level (LOAEL) value of




15 mg/kg/day identified in a 7.5-year study in dogs.
                                     I-10

-------
        For bromodichloromethane, no suitable study was located for calcu-




lating a One-day HA value.  A NOAEL of 62 mg/kg/day, identified in a 28-day




feeding study in rats, was used  to calculate a Ten-day HA value of 7 mg/L.  A




NOAEL of 50 mg/kg/day, identified in a 13-week gavage study in male mice, was




used to calculate Longer-term HA values of 4 and 13 mg/L for a 10-kg child and




a 70-kg adult, respectively.  The calculations for the RfD of 0.02 mg/kg/day




and DWEL of 0.7 mg/L employed a LOAEL of 25 mg/kg/day, identified in a 102-




week gavage study in mice.  The RfD is currently under review in light of




recent data.









        For dibromochloromethane, no suitable study was located for the




calculation of a One-day HA value.  The Ten-day HA value of 6 mg/L was




calculated using, a NOAEL of 56 mg/kg/day, identified in a 28-day feeding study




in rats.   A NOAEL value of 30 mg/kg/day,  identified in a 13-week gavage study




in rats,  was used to calculate Longer-term HA values of 2 and 8 mg/L for a




lOrkg child and a 70-kg adult, respectively.  The NOAEL value of 30 mg/kg/day




identified in the 13-week gavage study in mice was also used to calculate an




RfD of 0.02 mg/kg/day and a DWEL of 0.7 mg/L.









        For bromoform, an estimated dose of 54 mg/kg/day that caused sedation




in children was used to calculate a One-day HA value of 5 mg/L.  A NOAEL of




25 mg/kg/day, identified in a 13-week study in rats, was used to calculate a




value of 2 mg/L for both the Ten-day HA and the Longer-term HA for the




10-kg child.  This value was also used to calculate a value .of 6 mg/L for the




Longer-term HA for the 70-kg adult.  The NOAEL value of 25 mg/kg/day was also




used to calculate an RfD of 0.02 mg/kg/day and a DWEL of 0.7 mg/L.
                                     1-11

-------
Quantification of Carcinogenic Effaces









        Chloroform has been reported tc be carcinogenic in several different




chronic animal studies, increasing the frequency of liver tumors in male  and




female mice and kidney tumors in male rats.  Because the formation of liver




tumors in mice appears to be dependent upon the use of an oil vehicle,  the




U.S. EPA has calculated the cancer risk estimate for chloroform based on  the




incidence of renal tumors in male rats exposed to chloroform in drinking




water.  The resulting unit risk value is 1.7xlO~7
        Long-term oral studies in rats and mice performed by the National




Toxicology Program provide adequate data to derive quantitative cancer risk




estimates for the three brominated t rihalome thane s , although the chemicals




were administered in a corn oil vehicle.  For bromodichloromethane , a unit




risk of l.SxlO"6 (/ig/L)"1 was derived, based on the incidence of renal tumors




in male mice.   For dibromochloromethane , a unit risk of 2.4xlO"6 (^g/L)"1  was




derived, based  on liver tumors in female mice.  For bromoform, a unit risk of




2.3xlO"7 (/xg/L)"1 was derived, based on  tumors of the large  intestine  in




female rats.









        Table 1-1 summarizes the quantification of noncarcinogenic  and




carcinogenic effects for trihaloraethanes .
                                     1-12

-------
             TABLE 1-1  Summary of Quantification of Toxicological
                          Effects for Trihalomethanes
                                     Bromodi-        Dibromo-
Value                Chloroform   chloromethane   chloromethane   Bromoform
One-day HA for          4            6a        -        6a            5
10-kg child
(mg/L)

Ten-day HA for          4            6                 6             2b
10-kg child
(mg/L)

Longer-term HA for      0.1          4                 2             2
10-kg child
(mg/L)

Longer-term HA for      0.4          13                8             6
70-kg adult
(mg/L)

DUEL (70-kg adult)      0.4          0.7               0.7           0.7
(mg/L)

Concentration (jjg/L)    6            0.6               0.4           4
equivalent to 10"6
risk level
aData are insufficient or inappropriate for calculation of a One-day HA.  The
 value for the Ten-day HA is used as a conservative estimate.
bData are insufficient or inappropriate for calculation of a Ten-day HA.  The
 value for the Longer-term HA is used as a conservative estimate.
                                      1-13

-------
II.     PHYSICAL AND CHEMICAL PROPERTIES









        A.  Chloroform









        Chloroform (CHC13)  is a clear,  colorless volatile liquid with a




nonirritating odor and a sweet taste (Hardie 1964; Windholz 1976).  Some




important physical and chemical properties of chloroform are summarized in




Table II-l.









        Chloroform was used as an anesthetic as early as 1847 but is no longer




employed for this purpose.   It is manufactured and used as a solvent and as an




intermediate in the production of refrigerants, plastics and other solvents




(U.S. EPA 1980a)..  Approximately 120,000 tons of chloroform were produced in




1977 (U.S. EPA 1977).









        Because of its volatility, chloroform has the potential for




evaporation from water or other sources.  Chloroform is stable in water, but




light,  aeration or the presence of metals such as iron promote degradation




(Hardie 1964).









        B.  Brominated Trihalomethanes









        Bromodichloromethane (CHBrCl2) ,  dibromochloromethane (CHBr2Cl)  and




bromoform (CHBr3) are clear liquids with higher densities than chloroform. 




They have limited solubility in water but are very soluble in organic solvents




(Windholz 1976).  Some important physical and chemical properties of these




bromine-containing trihalomethanes are summarized in Table II-l.
                                     II-l

-------
          Table  II-1   Physical  and Chemical  Properties of Chloroform and Brominated Trihalomethanes

Information
Property
Chemical Abstracts Registry No.
Registry of Toxic Effects of
Chemical Substances Number
Synonyms
Chemical formula
Molecular weight
Boiling point
Melting point
Specific gravity (20C)
Vapor pressure
Stability in water
Water solubility
Chloroform
67-66-3
FS9100000
trichloro-
me thane
CHC13
119.39
61-62C
-63.5C
1.485
160 mm (20C)
200 mm (25C)
245 mm (30C)
volatile
8,000 mg/L
(20C) .
Bromodichloro-
ine thane
75-27-4
PA 5310000
dichlorobromo-
me thane
CHBrCl2
163.83
90C
-57.1C
1.980
50 mm (20C)
volatile
3,032 mg/L.
(30C)
Dibromochloro-
me thane
124-48-1
PA 6360000
i
chlorodibromo-
me thane
CHBr2Cl
208.29
116C
--
2.38
15 mm (10C)
volatile
1,050 mg/L
(30C)
Bromoform
75-25-2
PB 5600000
tribromo-
me thane
CHBrj
252.77
149-150"C
6-7C
2.887
5.6 mm (25C)
vol. at i le
3,190 mg/L
(30C)

Adapted from Windholz 1976; Verschueren 1977; Hawley 1981; U.S. EPA 1980b; and U.S. EPA (1985a).

-------
        Bromoform, bromodichloromethane and dibromochloromethane have been




used in pharmaceutical manufacturing and chemical synthesis,  as ingredients in




fire-resistant chemicals and gauge fluids and as solvents for waxes, greases,




resins and oils (U.S. EPA 1975a).









        Brominated trihalomethanes,  although less volatile than chloroform,




are considered to be volatile enough to evaporate from drinking water (Jolley




et al. 1978) .









        C.   Occurrence









        Chloroform occurs in the drinking water mainly as a by-product of the




chlorination process.  The hypochlorite ion (OC1~)  reacts with naturally




occurring organic substances in water (e.g., humic and fulvic acids) to form a




variety of by-products, including chloroform (Jolley et al. 1978).  A number




of national drinking water surveys performed in the United States between 1975




and 1981 revealed that chloroform was detectable in a majority of systems




using a surface water source.  Average levels usually ranged from 20 to




90 Mg/L (U.S. EPA 1975a, 1975b; Brass et al. 1977; U.S. EPA 1985a).   Chloro-




form was also detectable in systems using groundwater as a source, but usually




at lower levels (1 to 10 jig/L) .  This is presumably because surface water




typically contains higher levels of organic precursors than groundwater, and




may also require more extensive chlorination than groundwater.  Concentration




values have also been noted to vary as a function of season,  being higher in




warm weather and lower in cold weather (Wallace et al. 1987,  1988).









        Brominated trihalomethanes also occur in drinking water as by-products




of chlorination.  Bromide (Br~), a common constituent of natural waters, is




                                     II-3

-------
oxidized by hypochlorous acid to form hypobromous acid  (HOBr)  in the following




reaction:









     Br'  +  HOC1   '   HOBr  +  Cl"









        Hypobromous acid then reacts with organic precursors to yield bromine -




containing trihalome thanes such as bromoform, dibromochlorome thane and bromo-




dichlorome thane (in increasing order of formation rates) (Jolley et al.,




1978) .   A number of national surveys of brominated trihalome thane levels in




drinking water systems have revealed average levels typically ranging from 1




to 17 Mg/L (Boland 1981;  Brass 1981; Brass et al. 1981; Bull and Kopfler 1990;




Symons et al. 1975; U.S.  EPA 1991;  Westrick et al .  1983).  As noted above for




chloroform, mean levels of bromodichlorome thane tend to be somewhat higher for




systems using surface water sources (6 to 17 jig/L)  than those using ground-




water sources (1 to 8 jzg/L) .   Mean levels of dibromochlorome thane and




bromoform were comparable in systems using waters from either source (2 to




12
        Trihalome thanes may be produced not only by reaction of chlorine with




humic materials in water, but also by reaction of sodium hypochlorite  (NaOCl)




with endogenous organic material in the gut.  Mink et al.  (1983) dosed adult




male Sprague-Dawley rats (three fasted and three nonfasted per dose group)




with distilled water or 56 mg of NaOCl (8 mg/mL, pH 7.9) by gavage.  One hour




after dosing, the researchers sacrificed all rats and collected blood  and




stomach samples.  Chloroform was detected in the plasma of one nonfasted,




treated rat, and in the stomachs of. all treated rats.  In a second experiment,




rats were given a single oral dose of 100 mg NaOCl (48 mg Cl) along with 32 mg




Br"  (KBr) .   This led to the detection of all three brominated trihalome thanes




                                     II-4

-------
as well as chloroform in the stomach contents of nonfasted rats (Mink et al.




1983).  Bromoform and dibromochloromethane were also detected in the plasma.









        D.  Summary









        Trihalomethanes are volatile organic liquids that have a number of




industrial and chemical uses.   The chief reason for health concern is that




they are generated as by-products during the chlorination of drinking water.




The principal trihalomethanes that have been observed in water are chloroform,




bromoform, dibromochloromethane and bromodichloromethane.   Chloroform is




formed by the action of hypochlorous acid on endogenous organic molecules




(e.g., humic or fulvic acids)  present in the water.  Hypochlorous acid also




oxidizes any bro.mide ion present to form hypobromous acid, which reacts with




the organic material to form the brominated trihalomethanes.  Chloroform has




been detected in finished drinking water samples at concentrations typically




ranging from 20 to 90 /xg/L, while average values of brominated trihalomethanes




typically range from about 1 to 20 jig/L.  Chloroform and bromodichloromethane




levels tend to be higher in systems using surface water sources than those




using groundwater sources,  whereas dibromochloromethane and bromoform levels




are comparable in systems using either source.
                                     II-5

-------
III.    TOXICOKINETICS









        A.  Absorption









        Fry et al. (1972) administered chloroform orally to male and female




human subjects (18 to 50 years of age, 58 to 80 kg).  In eight subjects who




ingested 500 mg of  'C-chloroform  in olive  oil  in gelatin capsules,  pulmonary




excretion of unchanged chloroform at 8 hours ranged from 18% to 67% of the




administered dose (mean value = 40.3%).  In a separate experiment with two




subjects administered a gelatin capsule containing 500 mg of 13C-chloroform  in




olive oil, 49% and 51% of the total dose was expired as 13C-carbon  dioxide. 




Taken together, the data on carbon dioxide and chloroform excretion suggest




that at least 9Q% of an oral dose of chloroform is absorbed from the human




gastrointestinal tract.









        Jo et al.  (1990b) investigated chloroform absorption from inhalation




and dermal exposure during showers.  Chloroform breath levels were measured in




one female and five male volunteers pre- and post-exposure in a normal shower




(using a defined set of parameters) and following inhalation-only shower




exposure.   Breath levels measured 5 minutes after completion of the shower




correlated with tap water levels in both the normal shower and inhalation-only




shower protocols.   The inhalation-only exposure resulted in breath levels that




were about half those following a normal shower.  These data indicate that




humans absorb chloroform by both the dermal and inhalation routes when




showering.









        Brown et al. (1974a) and Taylor et al.  (1974) observed that UC-




chloroform (60 mg/kg) administered orally in olive oil to mice, rats, and




                                     III-l

-------
monkeys was 93% to 98% absorbed, as measured by the recovery of radioactivity




in exhaled air, urine, and the carcass.  Absorption was rapid, with peak blood




levels (3 to 10 ^g chloroform equivalent/mL) reached ac about 1 hour in both




mice and monkeys.









        Mink et al. (1986) compared the absorption of chloroform, bromo-




dichloromethane,  dibromochloromethane, and bromoform in male rats and male




mice.  The rats and mice_were given single oral doses of 14C-labeled compound




in corn oil by gavage at dose levels of 100 rag/kg (rats) or 150 mg/kg (mice).




Total recovery of label in breath, urine, or tissues after 8 hours ranged from




62% to 94% (Table III-l),  indicating that gastrointestinal absorption was high




for all four compounds.  However, this study has been criticized since the




level of labeled, carbon monoxide in exhaled air was not determined.









        Mathews et al. (1990) administered 14C-bromodichloromethane  by gavage




in corn oil to male F344 rats at doses of 1, 10, 32, or 100 mg/kg, and




monitored the radiolabel in exhaled air, urine, f eces, and tissues..




Absorption was extensive--at least 86% of the dose was recovered in expired




volatiles, C02,  or CO.  Only small amounts were recovered in urine (<5%)  or in




feces (<3%)  within 24 hours of dosing in all dosing groups (Table III-2).









        Withey et al. (1983) compared the rate and extent of gastrointestinal




absorption of chloroform given orally in either aqueous or corn oil vehicles.




Twelve male Wistar rats (approximately 400 g, six rats per vehicle) were given




single oral doses of  75 mg/kg 'by gastric intubation.  The peak chloroform




concentration in blood was reached at approximately the same time for each




vehicle (5.6 and 6.0  minutes for water and corn oil, respectively).  However,




the concentration levels differed by a factor of approximately 6.7  (39.3




                                     III-2

-------
       TABLE III-l  Recovery of Label 8 Hours after Oral Administration
           of 14C-Labeled Trihalomethanes to Male Rats or Male Mice
Chloroform
                         Percent of Label Recovered
Chemical
Expired Expired
Species C02 Parent
Urine
Total
Organs
Recovery
Rat
Mouse
 6.5
49.5
64.8
26.1
2.6
4.9
 3.6
13.5
78.2
94.5
Bromodichloromethane    Rat        14.2     41.7      1.4      3.3      62.7
                        Mouse      81.2      7.2      2.2      3.2      92.7
Dibromochloromethane
Rat
Mouse
18.2
71.6
48.1
12.3
1.1
1.9
 1.4
 5.0
70.3
91.6
Bromoform
Rat
Mouse
 4.3
39.7
66.9
 5.7
2.2
4.6
 2.1
12.2
78.9
62.2
Adapted from Mink et al. (1986).
                                     III-3

-------
     TABLE III-2  Cumulative Excretion of Label after Oral Administration
               of 14C-Labeled Bromodichloromethane to Male Rats

Percent of Dose

Dose
1 mg/kg




10 mg/kg




100 mg/kg




Time to
Sampling
1 hr
4 hr
8 hr
16 hr
24 hr
1 hr
4 hr
8 hr
16 hr
24 hr
1 hr
2 hr
6 hr
8 hr
24 hr
Expired

9
37
62
76
77
8
39
66
81
82
1
5
co2
.5+1.1
.0+3.2
.92.2
. 4^-3 . 2
.53.3
.0+2.0
.9+3.2
. 0+4 . 0
.3+1.7
.11.8
.9+0.9
.51.8
NR
33
71
.4+7.4
.0+1.7
Expired
CO
NRb
1.5+0
2.7+1
NR
3.3+1
NR
1.9+0
3.4+0
NR "
4.3+1
0.1+0
0.3+0
NR
2.3+0
5.2+0
Expired
Volatiles

.7
.1

.5

.4
.9

.0

.1

.7
.3
2.1+1.
2.71.
NR
NR
3.0+1.
2.0+0.
2.7+1.
NR
NR
2.8+1.
1.5+1.
4.21.
NR
5.7+2.
6.3+2.
5
8


6
8
1


1
2
9

1
1
Urine
NR
NR
NR
NR
4.1+0.2
NR
NR
NR
NR
4.3+0.2
NR
NR
0.6+0.4
NR ~
4.1+0.2
Feces
NR
NR
NR
NR
2.7+1.5
NR
NR
NR
NR
0.7+0.2
NR
NR
NR
NR
0.7+0.3
Total
Recovery
11
41
68
81
90
10
44
72
87
94
4.
10
10
42
87
.6+1.3
.1+2.8
.+1.7
.8+2.9
.7+1.8
.0+1.5
.5+3.0
.1+3.9
.4+1.5
.2+1.6
6+1.8
.6+2.9
.6+3.0
.0+8.3
.3+1.6

aResults from 4 rats/dose.
''Not reported
Adapted from Mathews et al.
(1990).
                                     III-4

-------
versus 5.9 /ig/mL for water and corn oil, respectively).  At all times, the




concentration of chloroform in blood was lower when given in an oil vehicle




than when given in vatsr.  The chloroform blood levels fell below the limit of




detection after approximately 200 minutes when given in oil and 240 minutes




when given in water.  These data suggest that administration of chloroform in




corn oil might not be a good model for oral exposure in water.   The authors




noted that the majority of toxicity studies using the oral route of dosing




have been carried out using oil-based vehicles.  The study did not determine




whether the vehicle-related difference was due to differences in absorption or




in first-pass metabolism.









        Staats et al.  (1991) found that a two-compartment model of gastro-




intestinal absorption was better than a one-compartment absorption model at




predicting blood levels following oral administration of chloroform.  Using




this model, they also found that blood levels of chloroform were higher




following gavage administration in water than in corn oil.  Peak levels were




about sevenfold higher following administration in water.









        Morgan et al.  (1991) measured chloroform blood levels in male F344




rats during 24-hour dermal exposures of a 3.1 cm2 shaved region of the back to




2 mL of neat chloroform,  or aqueous chloroform solutions that were saturated




(4,709 /ig/mL), two-thirds saturated (2,733 /xg/mL) ,  or one-third saturated




(1,180 /ig/mL).  The chloroform blood level peaked at 51 ng/mL after exposure




to the neat chemical for 4 to 8 hours, and remained about constant for the .




duration of the exposure period.  The plateau was attributed to the attainment




of an equilibrium between the toxicokinetics and the depletion of the chemical




from the exposure cell.  Exposure to the aqueous solutions resulted in peak




chloroform blood levels after about 2 hours, followed by a rapid return to




                                     III-5

-------
control levels by 24 hours of exposure.  The decreasing blood levels were




attributed to depletion of chemical from the exposure cell.  The study authors




stated that hydration of the skin could have increased the rate of absorption.









        Bogen et al.  (1992) investigated the dermal absorption of dilute




aqueous   C-chloroform  solutions  by hairless guinea pigs.   Animals  were




exposed by immersion to concentrations of 10 to 100 ppb chloroform in water




for 70 minutes.   Dermal uptake was estimated by comparing the rate of




radiolabel loss from water in systems with or without animals.  Dermal uptake




was confirmed by measuring the radiolabel content of the urine and feces.  A




pe.rme ability coefficient of 0.13 cm/hr was obtained for chloroform.  The




authors concluded that dermal absorption of chloroform from water may be an




important route .of human exposure.









        B.  Distribution









        McConnell et al.  (1975) analyzed chloroform levels in postmortem




tissue from eight persons (four males and four females, 48 to 82 years old)




living in nonindustrial areas of the United Kingdom.  Chloroform levels  (jtg/kg




wet tissue weight) observed were:  body fat, 5 to 68 (average of 51); liver, 1




to 10 (average of 7.2); kidney, 2 to 5; and brain, 2 to 4.  The source of the




chloroform in these tissues was noc discussed.









        Phillips and Birchard (1991) reported on a nationwide survey of  the




general population by EPA's National Human Adipose Tissue Survey (NHATS).




Several hundred fat samples were pooled into 46 composite samples by age and




geographic region, and analyzed.  Chloroform was measured at  levels ranging
                                     III-6

-------
from 5 co 580 ng/g in 29 of the composite samples, and the other three




trihalomethanes were not detected in any samples (Wallace 1992).









        Pellizzari et al. (1982) measured trihalomethanes in 42 samples of




human milk taken from women in urban areas.  Chloroform was detected in seven




samples, and dibromochloromethane was detected in one sample.  Neither the




levels measured nor the detection limit were reported.









        Roth (1904) measured the bromoform content of tissues of a man who




died from an accidental oral overdose of bromoform.  Levels in stomach and




lung (rag bromoform/kg wet weight) were 130 and 90 mg/kg,  respectively.   Lower




levels were reported in the intestine, liver, kidney, and brain.









        Mink et al. (1986) compared the distribution of chloroform, bromo-




dichloromethane,  dibromochloromethane, and bromoform in male rats and male




mice.  The rats and mice were given single oral doses of uC-labeled  compound




in corn oil by gavage at dose levels of 100 mg/kg (rats)  or 150 mg/kg (mice).




After 8 hours,  the researchers measured the tissue levels of radioactivity.




Interpretation of these data is limited because the chemical form of the label




measured in the tissues (parent or metabolite, bound or free) was not




determined.  In the rat, the total organ content of label ranged from 1.4% to




3.6% for the various compounds.  The stomach, liver, and kidneys contained




higher levels than other tissues analyzed (bladder, brain, lung, muscle,




pancreas,  and thymus).   Similar levels of label (4% to 5% of total dose) were




recovered in the organs of mice.  However, for bromoform and chloroform, an




additional 10% of the label was recovered in the blood of mice, yielding total




organ levels of 12% to 14%.  The authors suspected that this was due to




carboxyhemoglobin formation, but this was not measured.




                                     III-7

-------
        Taylor et al. (1974.) administered an oral gavage dose of 60 mg/kg of




UC-chloroform in olive  oil to  mice and studied distribution by whole-body




autoradiography.   The highest radioactivity levels were detected in the liver




and kidney.  The radioactivity level in the kidneys of males was 3.5 times




greater than that in the kidneys of females.  Castration of the males or




testosterone administration to female mice abolished this difference.  This




observation may provide insight as to why male mice are more sensitive to the




nephrotoxic effects of chloroform than females (see Section V).  Sex




differences in distribution were not noted by Brown et al.  (1974a) in rats or




squirrel monkeys.









        Mathews et al.  (1990) investigated the distribution of bromodichloro-




methane following oral exposure in rats.  Animals were given a single oral




gavage dose of 1, 10, 32,  or 100 mg/kg of 14C-bromodichloromethane  dissolved




in corn oil.  Approximately 3% to 4% of the administered dose was detected in




tissues after 24 hours.   The highest levels (1% to 3%) were measured in liver.




Repeated doses (10 or 100 mg/kg/day for 10 days) showed no bioaccumulation




(0.9% to 1.1% total retention of label) and had no effect on the tissue




distribution of bromodichloromethane.









        Withey and Karpinski (1985) exposed pregnant rats to 100 to 2,000 ppm




chloroform in air for 5 hours on day 17 of gestation.  A good linear relation-




ship was observed between the exposure level and maternal and fetal blood




concentrations of chloroform.  Fetal blood levels of chloroform were less than




maternal levels.   This study indicates that chloroform can cross the placental




barrier and distribute to the fetus.
                                     III-8

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        C.  Metabolism









        The metabolism of trihalomethanes has been studied in a number




of laboratories, both in vitro and in vivo.  Figure III-l presents a. metabolic




scheme based on these studies.  In brief, metabolism of trihalomethanes occurs




via two pathways, one in the presence of oxygen (the oxidative pathway) and




the other in the absence of oxygen (the reductive pathway).  Both pathways are




mediated by cytochrome P-450 and NADPH (or NADH).   In the presence of oxygen,




the initial reaction product is trihalomethanol (CXjOH),  which spontaneously




decomposes to yield the corresponding dihalocarbonyl (CXgO) .   These dihalo-




carbonyl species (e.g., phosgene) are quite reactive and may undergo a variety




of reactions with cellular molecules.  Under conditions when intracellular




oxygen levels are low, the trihalomethane is metabolized via the reductive




pathway, resulting in a highly reactive dihalomethyl radical ('CHXg),  which




may also form covalent adducts with cellular molecules.   Evidence supporting




this metabolic scheme and information on species differences in the rate and




extent of trihalomethane metabolism are presented below.









In Vitro Studies









        The differences between oxidative and reductive pathways for




trihalomethane metabolism have been studied mainly in vitro, where oxygen




levels can be controlled.









        Ilett et al.  (1973) investigated the in vitro metabolic activation of




14C-chloroform  using microsomal preparations from  the livers and kidneys of




male mice.  As shown  in Table III-3, incorporation of label into microsome




protein was much more extensive in liver than in kidney.   Uptake of label into




                                     III-9

-------
                                                                           RH
    REDUCTIVE PATHWAY
        CHXr
       OXIDATIVE PATHWAY
                   P-450
CHXa
          P-450
 f   " ^\    vn/vj f     f   ^\"^

X'  NADPH   2 NADPH O2   H2O
       or
     NADH
                                         >
V
 HX
CX2Q
                                                                                  RCX2OH
                                                                           2GSH
                                                                           V	
                                                            >
                                                            2HX
                                                                                  CO+GSSG
                                                                           H2O
                                                                            v
                                                                              2HX

                                                                           Cysteine
                                                                              \

                                                                              2HX
                                                                                  OTZ
X - halogen atom (chlorine, bromine); R  cellular nucleophile (protein, nucleic acid);
GSH - reduced glutathtone; GSSG - oxidized glulalhione; OTZ  oxolhiazolidine
carboxylic acid; P-450 - cytochrome P-450
(a) Adapted from Stevens and Anders (1981). Tomasi el al. (1985)
         FIGURE I1I-1.  Metabolic Pathways of  Trihalomethane Biotransformntlon

-------
TABLE III-3  In Vitro Binding of 14C-Chloroform  Metabolites
                 to Microsomes of Male Mice
                              Label Incorporation
                                (pmol/mg/5 min)
       Assay Conditions      Liver          Kidney
Complete
-------
liver microsomes was almost entirely blocked by removal of the NADPH




generating system, supporting the concept that the reaction was mediated by




cytochrome P-450-dependent metabolism.  This is further supported by the




finding that pretreatment of animals with phenobarbital (a known inducer of




cytochrome P-450) in three daily intraperitoneal injections of 80 mg/kg/day




led to a threefold increase in binding of label.  Under anaerobic conditions




(produced either by flushing with nitrogen or by adding carbon monoxide to




block oxygen binding to cytochrome P-450),  incorporation of label into




microsomal protein was reduced but was not eliminated.   This observation is




consistent with the concept that metabolism may proceed by both reductive and




oxidative pathways.  Removal of oxygen or NADPH affected binding of label to




renal microsomes less than it did binding to liver microsomes.  Phenobarbital




pretreatment did. not increase binding to renal microsomes.  This suggested to




the authors that a different metabolic pathway may play a significant role in




the kidney.









        Evidence for cytochrome P-450-dependent oxidative metabolism of




chloroform to dichlorocarbonyl (phosgene) was reported by Pohl et al. (1977).




Cysteine was used to inhibit covalent binding of radiolabeled chloroform to




microsomal proteins by trapping a reactive intermediate as 2-oxothiazolidine-




4-carboxylic acid (OTZ).   These authors suggested that unstable trichloro-




methanol forms initially via the cytochrome P-450 system, and hydrogen




chloride is eliminated spontaneously to yield the reactive phosgene, which




then binds with cysteine or protein thiol groups.  Pohl and Krishna  (1978) and




Pohl et al.  (1979) reported that deuterium-labeled chloroform was less toxic




and less readily metabolized than unlabeled chloroform, suggesting that the




enzymic cleavage of the carbon-hydrogen bond might be the rate-limiting step




in the metabolism of chloroform to a hepatotoxic product.




                                    111-12

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        Ahmed et al.  (1977) investigated the in vitro oxidative (aerobic)




metabolism of trihalomethanes to carbon monoxide by the rat liver microsomal




fraction.  Metabolism of bromoform resulted in the highest level of carbon




monoxide formation, followed by dibromochloromethane,  bromodichloromethane,




and chloroform, in decreasing order.  Glutathione, NADPH and oxygen were




required for maximal carbon monoxide production.  This activity was inducible




by phenobarbital or 3-methylcholanthrene pretreatment (agents which are known




to increase cytochrome P;.450 activity) and was inhibited by the cytochrome




P-450 inhibitor, SKF 525-A.  Similar results were later reported by Stevens




and Anders (1979).   In addition, Stevens and Anders (1979) reported the




formation of 2-oxothiazolidine-4-carboxylic acid (OTZ) when bromoform was




incubated in the presence of cysteine.  Dihalocarbonyls can react with




cysteine to form OTZ; therefore, detection of OTZ provides evidence that a




dihalocarbonyl intermediate was formed during bromoform metabolism.









        Smith and Hook (1984) reported that microsomal preparations from the




kidneys of male mice metabolized chloroform to carbon dioxide and reactive




intermediates,  while microsomal preparations from the kidneys of female mice




showed little or no activity for chloroform metabolism.  This enzymic




difference in metabolism between sexes is in agreement with the greater




nephrotoxicity of chloroform in male mice than in female mice (Smith et al.




1983).  The metabolism of chloroform by male kidney preparations required




oxygen and an NADPH regenerating system for maximal activity.  Metabolism was




shown to depend on the concentration of microsomal protein, the concentration




of chloroform,  and the time of incubation.  Addition of carbon monoxide (an




inhibitor of cytochrome P-450) resulted in decreased chloroform metabolism.




The metabolism of chloroform to carbon dioxide obeyed Michaelis-Menten
                                    111-13

-------
kinetics, and the authors reported values of 2.78 ^M for Km and a Vmax of




0.391 fjmol/mg microsomal protein/min.









        Uehleke and Werner (1975) investigated the extent of irreversible




label binding to proteins and lipids resulting from the in vitro'oxidative




(aerobic) metabolism of  C-chloroform  in hepatic  microsomes  prepared from




rabbits, rats, mice, and humans.  The level of irreversible reactive inter-




mediate binding to protein was highest for the rabbit and human liver




preparations.  The mouse and rat had considerably lower capacities.  Similar




results were reported for the extent of label bound to lipids for the animal




liver preparations,  although data were not presented on label binding to




lipids from human microsomes.  These results indicate that there are consider-




able species differences in the production of reactive intermediates from




chloroform.   Binding under anaerobic conditions to protein and lipids of




hepatic microsomes prepared from phenobarbital-pretreated rabbits was about




50% that observed in the presence of oxygen, suggesting that the reductive




metabolism of chloroform can also generate reactive intermediates.









        Corley et al. (1990) compared the in vitro oxidative metabolism of




14C-chloroform  in hamsters,  mice,  rats,  and humans.   Microsomal  preparations




from the liver and kidney of these species were incubated with 0.049 to




0.058 mM chloroform, and the generation of carbon dioxide was measured.  In




the liver, the relative levels of metabolic activity (nmol oxidized/min/mg




protein) were as follows:  hamster > mouse > rat > human.  .In the kidney the




activities were lower, and the following order was observed:  mouse > hamster




> rat > human.  The authors used these data to develop a pharmacokinetic model




for describing the disposition of chloroform in mice, rats, and humans.

-------
        Reitz et al. (1990) used the pharmacokinetic model of Corley et al.




(1990) to develop a model for the cytotoxic effects of chloroform.  The model




for oral, exposure was verified by administering com oil gavage doses of 100,




200, or 500 mg/kg to male B6C3F1 mice.  Cytotoxicity was determined by




directly measuring hepatocyte death histologically.   For inhalation exposure,




the model was verified by exposing male B6C3F1 mice to 10, 30, or 100 ppm




chloroform in air for 5 to 6 hours and measuring stimulation of compensatory




cell regeneration as an index of cell killing.  The model was then used to




compare predicted macromolecular binding and cytotoxicity for various long-




term chloroform bioassays with the observed liver tumor incidence.  The




bioassays used were those of NCI (1976) (oil gavage), Jorgenson et al.  (1985)




(drinking water), and Roe et al. (1979) (gavage in toothpaste).  The model




predicted that tumor incidence was correlated with the instantaneous rate of




chloroform metabolism and with cytotoxicity, but not with the total amount of




chloroform metabolized in the liver.  Thus the predicted peak rate of




chloroform metabolism following bolus gavage dosing was much higher than the




rate predicted for continuous exposure in drinking water, and liver tumors




were observed only under the former dosing protocol.  Conolly and Andersen




(1991) extended these results to predict that, corn oil gavage administration




of chloroform results in a much higher instantaneous rate of chloroform




metabolism in male B6C3F1 mice (in which liver tumors are observed--see




Chapter 5) than in nonsensitive male Osborne-Mendel rats.









        Testai and Vittozzi (1986) studied the in vitro metabolism of 14C-  .




chloroform by rat liver microsomal preparations under aerobic and anaerobic




conditions.   The production of a reactive intermediate was quantitated by




measuring the binding of radiolabel to microsomal proteins and lipids.  Under




aerobic conditions, pretreatment with phenobarbital produced a two- to three-




                                    111-15

-------
fold increase in binding, whereas p-naphchoflavone pretreatment had little




effect.  While binding to microsomes prepared from untreated rats was




relatively low under anaerobic, conditions, pretreatment with p-naphthoflavor.e




or phenobarbital resulted in markedly increased binding (approximately 9- to




15-fold and 50- to 90-fold, respectively).  These results indicate that




cytochrome P-450 also plays a role in the anaerobic metabolism of chloroform.




Under both aerobic and anaerobic conditions, the addition of glutathione or




cysteine reduced binding to both proteins and lipids, although glutathione




decreased binding to proteins more than it decreased binding to lipids.  The




aerobic metabolism of chloroform concentrations of 1 to 15 mM decreased the




cytochrome P-450 activity by about 5% to 30%, but glutathione or cysteine




reduced this effect.  The anaerobic metabolism of chloroform produced a loss




of cytochrome P-450 activity somewhat greater than that observed under aerobic




conditions, and the reduced activity was not affected by the addition of




glutathione or cysteine.   This study indicates that chloroform is metabolized




via a cytochrome P-450-mediated process to a reactive intermediate that can be




scavenged by glutathione or cysteine.  Since binding to uninduced microsomes




under anaerobic conditions was negligible, the study authors were able to rule




out significant residual oxygen contamination.  Therefore, they concluded that




most of the covalent binding to induced microsomes under anaerobic conditions




was produced by reductive metabolism of chloroform.









        Testai et al. (1987) investigated the in vitro anaerobic metabolism of




chloroform by liver microsomes from untreated and phenobarbital or p-naphtho-




flavone-pretreated B6C3F1 mice.  The authors reported that, unlike untreated




rats, untreated mice had a significant capacity to produce covalent binding of




14C-labelled  intermediates under anaerobic conditions.   Both NADPH and NADH




could provide the reducing equivalents needed for the metabolic .-activation of




                                    111-16

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chloroform.  This activity was not significantly increased by pretreatment




with either phenobarbital or p-naphthoflavone.   These differences in baseline




metabolic activity and enzyme inducibility may underlie some of the toxicclo-




gical differences observed between rats and mice (see Chapter 5).   As with rat




microsomes, the loss of cytochrome P-450 in mouse microsomes exposed to




chloroform was greater under anaerobic conditions than under aerobic




conditions.









        Species differences were also observed by Vittozzi et al.  (1991).




Radiolabeled chloroform was found to bind under hypoxic conditions to the




lipids of liver microsomes prepared from B6C3F1 mice but essentially not to




microsomes prepared from Sprague-Dawley rats.  Binding under aerobic condi-




tions was also lower in rat microsomes than in mouse microsomes.  Minimal




binding was seen with human liver microsomes under aerobic conditions,  while




binding was observed in 2/4 samples of microsomes prepared from human colon




mucosa biopsies, but only under reducing conditions.









        Wolf et al.  (1977) studied the in vitro metabolism of chloroform and




bromoform to carbon monoxide under anaerobic conditions using liver prepara-




tions from phenobarbital-induced rats.  Sromoform metabolism resulted in much




greater levels of carbon monoxide production than did the metabolism of




chloroform.  Similarly, Gao and Pegram (1992) reported that binding of




reactive intermediates to rat hepatic microsomal lipid and protein under




reductive (anaerobic) conditions was more than twice as high for bromo-




dichloromethane as for chloroform.  These data suggest that reductive




metabolism may be more important for brominated trihalomethanes than




chloroform.
                                    111-17

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        Tomasi et al.  (1985) studied the anaerobic activation of chloroform.




bromodichloromethane,  and bromoform to free radical intermediates .in vitro




using rat hepatocytes isolated from phenobarbital-induced male rats.  The




production of a free radical intermediate was measured by electron spin




resonance (ESR) spectroscopy using the spin trap compound phenyl-t-butyl-




nitrone.  The intensity of the ESR signal was greatest for bromoform, followed




by bromodichloromethane and. then chloroform.  The largest ESR signal was




detected when hepatocytes.were incubated under anaerobic conditions.




Incubation in the presence of air resulted in a reduction of the signal, as




did addition of cytochrome P-450 inhibitors such as SKF-525A, metyrapone, and




carbon monoxide.   These data were interpreted to indicate that free-radical




formation depended on reductive metabolism of the trihalomethanes mediated by




the cytochrome P.-450 system.  Comparison of the ESR spectra for chloroform,




deuterated chloroform, and bromodichloromethane indicated that the free




radical intermediate produced by chloroform metabolism was -CHClj.   The




authors speculated that the other trihalomethanes are also metabolized by




transfer of an electron directly from the cytochrome to the halocompound with




the successive formation of the dihalomethyl radical ('CHXj)  and a halide ion




(x-).









        Testai et al.  (1990) investigated the chloroform and oxygen dependence




of covalent binding of label from UC-chloroform into mouse liver microsomal




proteins and lipids.  Label was incorporated into protein and lipid in both




the presence and absence of oxygen, confirming the finding described above




that both oxidative and reductive pathways were involved.  However, protein




binding in the presence of oxygen appeared to occur by two processes.  The




first process was saturated at a low chloroform concentration (0.1 mM) and was




strongly inhibited by the reduction of the oxygen level from 20%  (air) to 1%.




                                    Ill-'18

-------
The second process occurred at higher chloroform concentrations (0.1 to 5 mM)




and was inhibited only by complete anoxia.  The authors interpreted these




observations as indicating that oxidative incorporation of label occurred by




two pathways:  the first by an enzyme system with high affinity for chloroform




and low affinity for oxygen,  and the second by an enzyme system with low




affinity for chloroform but high affinity for oxygen.  They did not speculate




on the enzymatic or biochemical mechanisms underlying these findings.   Both




oxidative processes produced reactive metabolites that bound mainly to




proteins,  in a process that was strongly inhibited by reduced glutathione.




Metabolites formed by the reductive pathway mainly bound to lipid, and their




production was not blocked by reduced glutathione.  The authors concluded that




phosgene is the chief reactive intermediate of both of the oxidative pathways,




while free radicals are the chief reactive product of the reductive pathway.




They noted that hypoxic conditions are generally present in the liver, with




oxygen levels of about 1% to 5%,  and that reduced glutathione is usually




present.  These conditions could favor the formation of adducts via the




reductive pathway.









        Testai et al. (1991)  measured the in vitro binding of radiolabeled




chloroform to microsomes prepared from human colon or ileum mucosa recovered




from surgical biopsies.  Covalent binding to lipid was observed with.about




half the colon and ileum specimens, but only under anoxic conditions.   No




binding to protein was observed in the presence or absence of oxygen.   By




contrast,  colonic microsomes from rats did not metabolize chloroform in the




presence or absence of oxygen to a form that reacted with lipid or protein.




The study authors noted that low oxygen tension is present in the outer layers




of the colonic mucosa, and suggested that their observations support the
                                    111-19

-------
claims in some epidemiological studies of an association between chloroform




exposure and colonic cancer (see Chapter 6).









In Vivo Studies









        In vivo studies on trihalomethane metabolism demonstrate that both the




oxidative production of carbon dioxide and the reductive production of free




radicals occur in animals.  The production of carbon monoxide has been




reported and can result from either pathway.  In addition,  considerable




species differences exist in the extent of trihalomethane metabolism to carbon




dioxide.









        Fry et a1.  (1972) reported that the extent of oxidative chloroform




metabolism, to carbon dioxide in humans was dependent on the oral dose




administered.  The subjects were healthy male and female volunteers, 18 to




50 years of age (a total of 11 subjects), with body weights ranging from 60 to




80 kg.  Chloroform was administered in gelatin capsules in an olive oil




vehicle.   The dose levels were 0.5 g (7.5 mg/kg) for eight subjects and 0.1 g




(1.5 mg/kg), 0.25 g (3.8 mg/kg), and 1.0 g (15.4 mg/kg) for one subject each.




Based on repeated gas chromatographic analyses of exhaled breath over 8 hours,




the authors estimated that expired chloroform accounted for 0%, 12%, 40% and




65% of the dose in the 1.5-, 3.8-, 7.5- and 15.4-mg/kg dose groups, respec-




tively.  Based on the authors' observation that nearly all of the administered




chloroform could be accounted for in the expired air as either chloroform or




carbon dioxide, it was estimated that approximately 100%, 88%, 60% and 35% of




the dose was metabolized to carbon dioxide, respectively.  These data indicate




that metabolism of chloroform to carbon dioxide occurs by a saturable




(presumably enzymic) system in humans.




                                    111-20

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        Mink et al.  (1986) compared the metabolic products of chloroform,




bromodichloromethane,  dibromochloromethane,  and bromoform in male rats and




male mice (strain not reported).   Animals were given a single oral dose of




KC-labeled  compound in corn  oil  by  gavage at  dose  levels  of  100  mg/kg for




rats and 150 mg/kg for mice.   In rats, expired carbon dioxide accounted for




4.5% to 18.2% of the label (Table III-l), indicating that the parent compound




had undergone limited metabolism and oxidation.  In mice,  the fraction of




label excreted as carbon dioxide was higher, ranging from 40% to 81%.  These




data indicate that oxidative metabolism of trihalomethanes to carbon dioxide




was more rapid and extensive (by a factor of four- to ninefold) in mice than




in-rats.









        Mathews et al.  (1990) studied the metabolism of 14C-bromodichloro-




methane in male Fischer rats.  Animals were given a single oral dose of 1, 10,




32,  or 100 mg/kg of bromodichloromethane dissolved in corn oil.  Levels of




labeled carbon dioxide and carbon monoxide in exhaled air were measured for




24 hours.   Approximately 70% to 80% of the dose was metabolized and exhaled as




UC02 and  3%  to 5% of the  dose  as 14CO.  However, KC02 production was  slower




following a single dose of 100 mg/kg than after the administration of a single




dose of 32 mg/kg or lower, suggesting saturation of metabolism.  Repeated




doses of 100 mg/kg/day for 10 days resulted in an increased rate of '^COj




production,  compared with the initial rate.   The authors concluded that




bromodichloromethane may induce its own metabolism.









        Anders et al.  (1978)  investigated the in vivo formation of carbon




monoxide from trihalomethanes administered to rats by intraperitoneal




injection in corn oil.   At a dose of 1 mmol/kg  (119 to 252 mg/kg), bromoform




administration produced the highest levels of blood carbon monoxide,  followed




                                    111-21

-------
by dibromochloromechane,  chloroform, and bromodichloromechane in decreasing




order.  A dose-response relationship was noted for bromoform.  Carbon monoxide




production was inducible by phenobarbital pretreacment but not by 3-methyi-




cholanthrene pretreatment,  and was significantly inhibited by SKF-525-A.




Administration of 3H-bromoform resulted in decreased carbon monoxide




formation, indicating that the carbon-hydrogen bond breakage may be the rate-




limiting step under aerobic conditions.  Similar results were later reported




by Stevens and Anders (19.81).  In addition, Stevens and Anders (1981) reported




that the co-administration of chloroform and cysteine (which reacts with




dihalocarbonyls) to rats resulted in decreased carbon monoxide formation.




These data suggest that trihalomethanes are metabolized oxidatively to carbon




monoxide via a dihalocarbonyl intermediate, such as in the mechanism proposed




on the basis of j.n vitro data.









        Ilett et al.  (1973) investigated the covalent binding of metabolites




to proteins in the liver and. kidney in mice after in vivo exposure to




chloroform by intraperitoneal injection.  The animals were pretreated with




phenobarbital and then given 300 to 740 mg/kg 14C-chloroform in sesame oil.




The authors reported centrilobular hepatic necrosis in both sexes and renal




necrosis in male mice.   Autoradiographic analysis of tissue slices indicated




that the amount of covalent binding of radiolabel paralleled the extent of




renal and hepatic necrosis.  These data suggest a possible causal relationship




between tissue necrosis and covalent binding.









        Tomasi et al. (1985) studied the in vivo metabolism of chloroform,




bromodichloromethane, and bromoform.to free radical intermediates in  rats.




Phenobarbital-induced rats were given  intraperitoneal injections of




1,100 mg/kg chloroform, 820 mg/kg bromodichloromethane, or  1,260 mg/kg




                                    111-22

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bromoform dissolved in olive oil.  The animals were sacrificed and the livers




were homogenized.  The production of a free radical intermediate by the livers




was determined by ESR spectroscopy.  . Free radicals were detected in the livers




of all treated rats.  The intensity of the ESR signal followed a ranking




similar to that observed in in vitro experiments (bromoform > bromodichloro-




methane > chloroform),  confirming that the reductive formation of free




radicals is greater for brominated trihalomethanes than for chloroform.









        D.  Excretion









        Fry et al. (1972) administered a single oral dose of chloroform




(500 mg) in gelatin capsules to male and female human subjects (18 to 50 years




of age, 60 to 80. kg).  Pulmonary excretion of unchanged chloroform at 8 hours




ranged from 18% to 67% in eight subjects (mean value = 40.3%).  Two subjects




who were administered a gelatin capsule containing 500 mg 13C-chloroform  in




olive oil excreted 48.5% and 50.6% of 13C-carbon  dioxide  in 7.5 hours.   Taken




together, these data suggest that nearly all of an oral dose is excreted via




the lungs, either as chloroform or as carbon dioxide.  A linear relationship




was noted between the rate of pulmonary excretion of chloroform and the




chloroform concentration in the blood (levels of up to 5 jig/mL within 1 hour




after exposure).  Urinary levels of chloroform were below 0.01% (the level of




detection).    Fecal chloroform content was not measured.









        Mink et al.  (1986) compared the excretion of chloroform, bromodichloro-




methane, dibromochloromethane,  and bromoform in male rats and male mice.




Animals were given single oral doses of 14C-labeled compound in corn oil by




gavage at dose levels of 100 mg/kg and 150 mg/kg for rats and mice, respec-




tively.  The lung was the principal route of excretion in both species,




                                    111-23

-------
accounting for 45% to 88% of the administered label, either as carbon dioxide




or as parent compound.  Small amounts of label  (1.1% to 4.9%) were recovered




in urine, but the chemical form of the label, was not investigated.









        Mathews et al. (1990) exposed rats to either a single oral dose of 1,




10, 32, or 100 mg/kg, or 10-day repeated doses  of 10 or 100 mg/kg/day bromo-




dichloromethane dissolved in corn oil.  Approximately 70% to 80% of the




administered dose was excreted in exhaled air as 14C-carbon  dioxide,  with 3%




to 5% as UC-carbon  monoxide.   In general,  less  than 5%  of the  dose was




excreted in the urine or feces.









        Van Dyke et al. (1964) administered 0.1 mL of 14C-chloroform




(1.06xl08 dpm/mL_)  by intraperitoneal injection to 30 rats (200 g, strain and




sex not specified).   This corresponded to a dose of about 740 mg/kg.  After




12 hours, a total of 0% to 2% of the injected 14C-label  was  recovered as




urinary metabolites and 4% to 5% was recovered  as expired COj.   The




disposition of the other 93% to 96% of the dose was not determined.  Adminis-




tration of a comparable dose of 36C1-chloroform  (specific activity not




reported) resulted in excretion of 36C1-urinary  metabolites,  but the




percentage of the dose excreted in the urine was not reported.   Of the  total




urinary label, 73% appeared as 36C1",  with  27% as  organic  forms.









        E.  Bioaccumulation and Retention









        No data were located regarding the bioaccumulation or retention of




chloroform or brominated trihalomethanes following repeated exposures.




However, based on the rapid excretion and metabolism of chloroform and  the




brominated trihalomethanes, along with the low  levels of chloroform detected




                                    111-24

-------
in human post-mortem tissue samples, marked accumulation and retention of




these compounds are not anticipated.









        F.  Summary









        Measurements of gastrointestinal absorption of trihalomethanes in




mice, rats, and monkeys indicate that absorption is rapid (peak blood levels




at 1 hour) and extensive.-(64% to 98%).   Limited data indicate that gastro-




intestinal absorption of chloroform (and presumably other trihalomethanes) is




also rapid and extensive (at least 90%) in humans.   Most studies of trihalo-




methane absorption have used oil-based vehicles.  One study in rats found




higher chloroform blood levels following oral gavage administration of




chloroform in wa.ter than after administration of chloroform in an oil vehicle.




This was interpreted as being due to higher absorption from water than from




oil, but the possible influence of first-pass metabolism was not taken into




account.  Dermal absorption of chloroform in water by rats and hairless guinea




pigs is rapid and extensive.  Dermal absorption by humans of chloroform in




water has also been demonstrated.









        Absorbed trihalomethanes appear to distribute widely throughout the




body.  Chloroform was detected in a number of postmortem tissues from, humans,




with the highest levels (5 to 68 jig/kg) in body fat and lower levels (1 to




10 jig/kg) in kidney, liver, and brain.   Radiolabeled trihalomethanes were




detected in a variety of tissues following oral dosing in rats and mice, with




somewhat higher levels in stomach, liver, blood, and kidney than in lung,




muscle, or brain.  Chloroform crosses the placenta and may be detected in




fetal tissues following inhalation exposure of pregnant rats.
                                    111-25

-------
        Trihalomethanes are extensively metabolized by both humans and




animals.  The main site of metabolism is the liver, but metabolism also occurs




in the kidney.  Beth oxidative metabolism and reductive metabolism of trihalo-




methanes are mediated by cytochrome P-450.  The oxidative pathway requires




NADPH and oxygen, whereas the reductive pathway can utilize NADPH or NADH and




is inhibited by oxygen.  In the presence of oxygen (oxidative metabolism), the




reaction product is trihalomethanol (CXjOH),  which decomposes to yield a




reactive dihalocarbonyl .CX20) such as phosgene (CC120).   Dihalocarbonyls are




relatively reactive species, and may undergo a variety of reactions, such as




the formation of adducts with various cellular nucleophiles,  hydrolysis to




yield carbon dioxide, or glutathione-dependent reduction to yield carbon




monoxide.  If oxygen is lacking (reductive metabolism), the metabolic reaction




products appear .to be free radical species such as the dihalomethyl radical




(CHXj')-   These  radicals are extremely reactive and may also  form covalent




adducts with a variety of cellular molecules.  Metabolism via the reductive




pathway appears to occur more readily for brominated trihalomethanes than for




chloroform.









        Both in vivo and in vitro studies indicate that the pattern of




trihalomethane metabolism may differ between animal species and  sexes.   In




vivo, a single study reported that mice metabolize trihalomethanes to carbon




dioxide more extensively than do racs (40% to 80% versus 4% to 18%).  However,




data from another study indicate that another strain of rat was  capable  of




oxidizing bromodichloromethane to carbon dioxide to an extent that was




comparable to that reported for mice.  I_n vitro, the capacity for reductive




metabolism of trihalomethanes has been found to be greater in hepatic




microsomes from mice than rats, and the incorporation of label into covalent




adducts in renal microsomes has been  found to be greater in male mice than




                                    111-26

-------
female mice.  These metabolic differences may underlie some of the important;




toxicological differences that have been noted between sexes and species.




Reductive metabolism of chloroform to reactive intermediates has also been




observed with microsomes prepared from human ileum and colon samples.









        Excretion of trihalomethanes occurs primarily via the lungs.   In




humans, approximately 90% of an oral dose of radiolabeled chloroform was




exhaled as the end metabolite, carbon dioxide, or the parent compound,




chloroform.  Levels in the urine were below the limit of detection (0.1%).




In mice and rats, 45% to 88% of an oral dose of chloroform or brominated




trihalomethane was excreted from the lungs either as parent trihalomethane or




as carbon dioxide, with 1% to 5% excreted in the urine.  Intraperitoneal




injection of rats with 36C1-chloroform  resulted in variable  levels  of labeled




chloride in the urine.  The total amount of label excreted in the urine was




not reported, but 73% of the urinary label appeared as ionic chloride (36C1~)




and 27% was present in organic compounds.









        No data were located regarding the bioaccumulation and retention of




the trihalomethanes following chronic exposure.  However, based on the rapid




metabolism and excretion of chloroform and the brominated trihalomethanes,




along with the low levels of chloroform in human autopsy samples, marked




accumulation and retention of these compounds are not anticipated.
                                    111-27

-------
IV.     HUMAN EXPOSURE









        A.  Drinking Water Exposure




            SamplinE Methodology




        Several national surveys have measured THMs in drinking water at the




treatment plant or in the distribution system. ' These surveys had different




purposes; therefore, the sampling methods and location of sample collection




varied.  Three different ^sampling methods were used in the national surveys,




and each method resulted in different outcomes.  The three methods are as




follows:  (1) if the actual concentration of THMs at the time of sample




collection is needed, then sodium thiosulfate, a reducing agent, is added to




the sample to prevent or "quench" further THM formation; (2) if the maximum




level of THM concentration in water is required, then the quenching process is




not employed, and THM formation is allowed to'continue unrestricted after




collection; and (3)  if the predicted level of THMs in the water at the tap




(after  spending time in the distribution system) is needed then the samples




are refrigerated to  slow the THM formation until analysis is performed.




Comparison of the results of each survey should take into consideration  the




sampling method employed (Wallace 1992).  Table IV-1 summarizes the results of




the national THM studies. Figure 1 graphically displays how different  sampling




techniques affect mean THM concentrations in  the national surveys.









         1.  Total Trihalomethanes









         The American Water Works Association  Research  Foundation  Study




 (AWWARF)  conducted  a nationwide  survey  of  727 utilities,  representing more




 than  105 million customers,  that collected  total  trihalomethane data between




 1984  and 1986.  Concentration data  for  the  specific  trihalomethane components




                                      IV-1

-------
             TABLE IV-1 Comparison of Results of National Surveys


Survey
NORS*
NOMS -
NOMS -
NOMS -
NOMS -
NOMS -
AWWARFC



Phase 1
Phase 2C
Phase 3Qd
Phase 3TC
All phases

Number of
Cities
80
111
113
106
105
105/113
727
THM
Mean
68
68
117
53
100
84
42
Concentration
Median
41
45
 87
37
74
55
39
.ue/L
Range
NDb-482
ND-457
ND-784
ND-295
ND-695
ND-784
ND-360
Modified from Wallace,  1992.

"Samples shipped and stored at 2-8C for one to two weeks prior to analysis;
 sample method 3
b Not detected (detection limits varied among the three surveys).
c Samples stored at 20-25C for three to six weeks prior to analysis; sample
 method 2
d Sodium thiosulfate added; sample method 1
                                     IV-2

-------
              THMs  in  Water:  Means
              Results from  National Surveys
         ug/L
           NORS    NOMS 1   MOMS 2  NOMS 3T  MOMS 3Q AWWARF
              Max THM Formation
Moderate
Minimum (Quenched)
        Mean values of THMs from three nationwide surveys. Different storage techniques (refrigerated; not
refrigerated; refrigerated with a reducing agent) resulted in widely varying THM concentrations.

-------
analyzed were not provided.  A quenching agent, sodium thiosulface, was used



prior to analysis.  The median trihalomethane values ranged from 30 jig/L  (ppb)



in uhe winter to 44 jjg/L (ppb) in the summer.  The overall median value wa;,



39 M/L (PPb). similar to the results of the NOMS Survey (Wallace 1992).






        2.  Chloroform







        A large number of. surveys have analyzed drinking water for chloroform
                        ~     / .


content;  nine describe the occurrence of chloroform in drinking water on  a



national level.  In 1975, the National Organics Reconnaissance Survey (NORS),



conducted by the EPA,  collected drinking water samples from 80 cities nation-.



wide. The survey sampled for several organics,  including trihalomethanes, at



the water treatment facilities.   Eighty percent of the systems had surface



water sources, and the remaining 20% had ground water sources.  The median



concentration for chloroform was 23 ^g/L (ppb), and the maximum level found



was 311 pg/L (ppb) (Symons et al.  1975).  The detection limit was 0.05 pig/L



(ppb).   The sampling method employed in the NORS survey was refrigeration



without quenching; therefore, chloroform concentrations may have increased



following collection.   NORS was performed prior to the promulgation of the



total trihalomethane regulation;  therefore, these results may be higher than



current levels.







        The National Organics Monitoring Survey (NOMS) was conducted by the



EPA from March 1976 to January 1977.  For NOMS, 113 community water supplies



were sampled at the treatment plants.  Surface water was the major source for



92 of the systems, and ground water was the major source for the remaining 21



systems.   The NOMS used all three sampling methods.  During Phase 1, all



samples were refrigerated at 2-8C for 1-2 weeks prior to analysis.  In



                                     IV-4

-------
Phase 2, the samples were allowed to stand at 20-25C for two to three weeks




to maximize THM formation.  In Phase 3T, one set of samples was allowed to




stand 3-5 weeks.  In Phase 3Q, a' quenching agent, sodium thiosulfate, was




added.  As expected, the highest chloroform values occurred in Phases 2 and




3T.  Chloroform was detected in 92-100% of the systems sampled in all three




phases.   The median chloroform concentrations of the three phases ranged from




22 to 54.5 jjg/L (ppb).   The maximum value found was 540 fig/L (ppb) .   Like




NORS,  NOMS was conducted .before the promulgation of the total trihalomethane




regulation; therefore,  these results may be higher than current levels.









        The Community Water Supply Survey (CWSS) was conducted by the EPA in




1978.   The survey examined over 1,100 samples, representing over 450 water




supply systems. , Samples were taken at the treatment plants and in the




distribution systems.  Concentration data were combined; therefore,  results




can be separated by treatment plant or distribution system.  In the CWSS, 97%




of the surface water supplies and 34% of the ground water supplies were




positive for chloroform.  For surface water supplies, the mean of the




positives and the overall median were 90 ^g/L (ppb) and 60 fig/L (ppb),




respectively.  The mean of the positives for ground water supplies was




8.9 /ig/L (ppb). and the overall median was below the minimum reporting limit




(MRL)  of 0.5 ftg/L (ppb) (Brass et al. 1981).  The MRL is the lowest value of a




contaminant that can be detected based on sampling methodology, analytical




method,  and laboratory and environmental conditions at the time of sampling.




It may or may not be identical to the detection limit.









        The Rural Water Survey (RWS) was conducted between 1978 and 1980 by




the EPA to evaluate the status of drinking water in rural America.  Samples




from over 2,000 households, representing more than 600 rural water supply




                                     IV-5

-------
systems, were examined.  In Che RWS, 82% of the surface water supplies and 17%




of the ground water supplies were positive for chloroform.  For the surface




vatsr supplies, the mean of the positives and the overall median




concentrations were 84 jig/L (ppb) and 57 jtg/L (ppb) ,  respectively.  For the




ground water supplies, the mean of the positives was 8.9 /zg/L (ppb), and the




overall median was below the minimum reporting limit of 0.5 ^g/L  (ppb) (Brass




1981).









        The Ground Water Supply Survey (GWSS) was conducted from December 1980




to December 1981 by the EPA to develop data on the occurrence of volatile




organic chemicals in ground water supplies.  Out of 945 ground water systems




that were sampled, 466 systems were chosen at random, while the remaining




479 systems were chosen on the basis of location near industrial, commercial,




and waste disposal activities.  Samples were collected at or near the entry to




the distribution system, and chloroform formation was allowed to proceed




unrestricted.  For chloroform, the median concentration of the positives for




the randomly chosen systems serving greater than 10,000 people was 1.4 jig/L




(ppb), and the occurrence rate was 37%.  For randomly chosen systems serving




less than 10,000 people, the median of the positives was 1.6 ^g/L (ppb), with




an occurrence rate of 57%.  The nonrandomly chosen systems had a median




concentration for the positives of 1.9 /ig/L (ppb) and an occurrence rate of




53% (Westrick et-al. 1983).









        The National Screening Program for Organics in Drinking Water (NSP).




sponsored by the EPA, was conducted from June 1977 to March 1981.  The survey




sampled 169 systems nationwide.  Samples were collected at the treatment faci-




lities.  For chloroform, the mean and median concentrations for 132 positives
                                     IV-6

-------
were 45 ng/L (ppb) and 29 jtg/L  (ppb) , respectively, with a maximum concentra-




tion of 204 ftg/L  (ppb) (Boland  1981).
        Thirty- five water utilities nationwide, of which 10 were located in




California, were sampled for chloroform in clearwell effluent  (after final




disinfection, but before distribution).  Source water used by  the 35 utilities




included groundwater (7), lake/reservoir (17), and flowing stream (11).




Samples were taken for four quarters (spring, summer, and fall in 1988 and




winter in 1989).  The median for all four quarters was 14 /ig/L (ppb), with the




medians of the individual quarters reported as 15, 15, 13 and 9.6 jtg/L (ppb),




respectively.  The maximum value was .130 pg/L (ppb).  For all four quarters,




75% of the data were below 33 jig/L (ppb) .  The detection limit was not




reported (Krasne.r et al. 1989; U.S. EPA 1989a, 1989b) .









        The Technical Support Division (TSD) of the Office of Ground Water and




Drinking Water (OGWDW) maintains a ground water contaminant database.  For




chloroform, the database contains 5,806 measurements taken at  treatment




facilities from 19 states between 1984 and 1991.  The mean and median




chloroform concentrations were determined to be 17 ^g/L (ppb) and 5 jjg/L




(ppb), respectively (U.S. EPA 1991).









        The EPA's Technical Support Division  (TSD) has compiled a database




from its disinfection by-products field studies.  The field studies  included a




chlorination by-products survey, conducted from October 1987 to March  1989..




In this survey, chloroform in finished water  from the treatment plant  and  in




the distribution system was sampled.  For surface water systems, the mean




concentrations, in finished water for systems  serving greater than and  less




than 10,000 people were 38.9 jjg/L (ppb) (90th percentile based on 42 samples,




                                     IV- 7

-------
74.4 jtg/L  (ppb)) and 42.8 ^g/L  (ppb)  (90th percentile based on 20 samples.




63.5 /ig/L  (ppb)), respectively.  In the distribution system, the means were




58.7 Mg/L  (ppb)  (90th percentile, 141 Mg/L(ppb)) for 39 samples from plants




serving greater  than 10,000 people and 77.2 jig/L (ppb) (90th percentile,




110 jig/L(ppb)) for 11 samples from plants serving fewer than 10,000 people.




The ground water systems serving less than 10,000 people had mean chloroform




concentrations in seven finished water samples and five distribution system




samples of 2.8 ^g/L (ppb) (90th percentile, 10.3 ^g/L (ppb)) and 3.6 Mg/L




(ppb) (90th percentile, 9.4 ^g/L (ppb)), respectively.  Only one ground water




system serving greater than 10,000 people was sampled; the concentrations at




the plant and in the distribution system were 0.6 and 0.8 /ig/L (ppb),




respectively (U.S. EPA 1992).









        The Rhode Island Private Well Study was conducted in 1986 by the Rhode




Island Department of Environmental Management (RIDEM).  Private wells that




were located in  land use areas that posed a threat to groundwater (e.g.




industrial areas, unsewered areas,  junkyards, and agricultural areas) were




sampled for contamination.  A total of 485 samples were taken from 463 private




wells.  Chloroform concentrations ranged from below the detection limit of




5 Mg/L (ppb) to  8 Mg/L (ppb) (mean. 3.9 Mg/L (ppb)) (RIDEM 1990).









        Fair et  al. (1988) analyzed drinking water from three community water




supplies for chlorination by-products.  Chloroform concentrations reported for




each of the plants ranged from 11 to  100 jig/L (ppb) in finished water at  the




plants and from  21 to 160 ^g/L  (ppb)  in the distribution systems.









        The EPA's five-year Total Exposure Assessment Methodology (TEAM)  study




measured the personal exposures of urban populations  to a number of  organic




                                      IV-8

-------
chemicals in the air and drinking water of several U.S. cities between  1981




and 1987.  As part of the study, running tap water samples, collected from




residences of nearly 850 study participants during the icrning and the




evening, were analyzed for chloroform content.  Table IV-2 shows chloroform




concentrations found in drinking water from the six cities surveyed.









        Uden and Miller (1983) sampled drinking water from two treatment




plants in Amherst, Massachusetts.  Two sets of tap water samples were




collected at each plant;, the first set of samples was analyzed immediately




following collection, and the second set was allowed to sit for 24 hours to




mimic distribution levels.   Chloroform concentrations for the two treatment




plants were 39.6 jig/L (ppb) and 87.4 ng/L (ppb) immediately after disinfection




and 139 /ig/L (ppb) and 190 /jg/L (ppb) after standing for 24 hours.









        Howard (1990) reported results from several additional surveys  in a




literature review.  In a Federal survey of finished water supplies, chloroform




was found to occur in 70% of ground water supplies (Dyksen and Hess 1982).




Coleman et al.  (1976) reported that a survey of drinking water in five  cities




found concentrations ranging from 1 to 301 ^g/L (ppb) (mean, 85 ^g/L (ppb)).




In a nine-city survey,  chloroform was found in eight of nine water supplies,




with concentrations ranging from below the detection limit to 58 jig/L (ppb);




the mean of the positive samples was 19.2 /ig/L (ppb) (Heikes 1987).  Furlong




and D'itri (1986) reported that a survey of chlorinated drinking water  from




40 plants in Michigan had a chloroform detection rate of 80%, with




concentrations ranging from below the detection limit to 201.4 jjg/L (ppb).




The mean of the positive samples was 41.8 ^tg/L (ppb), and the median was




16.2 Mg/L (ppb).
                                     IV-9

-------
              Table IV-2  Chloroform Concentrations in Drinking Water from the EPA TEAM study

Locat ion
El izabeth/Bayonne
New Jersey

Los Angeles,
California


Ant loch/Pittsburgh
California
Devils Lake,
North Dakota
Greensboro,
North Carolina
Baltimore,
Maryland
Date
Sampled
Fall 1981
Summer 1982
Winter 1983
Winter 1984
Summer 1984
Winter 1987
Summer 1987
Spring 1984

Fall 1982

Fall 1982

Spring 1987

Sample
Size
355
157
49
117
52
9
7
71

24

24

10

Concentration g/L (ppb)
Mean
70
61
17
14
29
6.8
11
42

0.46

43

24

Median
67
55
16
14
33
7.5
9.6
49

0.38

44

24

Maximum
170
130
33
60 '
52
13
18
99

1.4

91

35

Percent Measured
25% 75% 95%
83 102
77 104
24 30
5.6 20 32
9.2 42 49
......

13 65 91

- - . - - -

56

. .


Adapted from Hartwell et al. 1987b; Wallace et al. 1988; Wallace et al. 1987; and Wallace 1992

-------
        As part of a study to determine the exposure of university students to




volatile organics during normal daily activities, tap water at two




universities was analyzed for chloroform.  The University of North Carolina




(Chapel Hill, NC) was chosen to represent a non-industrial site, and Lamar




University (Beaumont, TX) was chosen to represent a petrochemical industry




area.  Chloroform concentrations in 14 samples of tap water at the University




of North Carolina ranged from 180 to 260 /xg/L (ppb) (mean, 220 Mg/L (ppb)).




At Lamar University, chloroform concentrations in 25 tap water samples ranged




from 99 to 550 /xg/L (ppb) (mean, 150 /ig/L (ppb)).  The levels of detection at




The University of North Carolina and Lamar University were 1.0 and 0.05 /ig/L




(ppb), respectively (Wallace et al. 1982).









        A 1987 analysis of drinking water in Nassau County, New York, which




takes its drinking water from underground aquifers, found chloroform to be




present in approximately 3% of the drinking water samples (detection limit




1 ppb); however, the chloroform concentration exceeded 10 ppb in only 1% of




the samples (Moon et al. 1990).









        The exposure to chloroform in drinking water from ground water sources




has been estimated from the median levels found in the GWSS.  Based on the




range of median levels, 1.4-1.9 /ig/L (ppb), and a consumption rate of two




liters per day for adults, the median exposure to chloroform was determined to




be 2.8-3.8 /ig/day.  The data from NOMS,  which sampled mainly surface water




systems, has been used to estimate exposure to chloroform in drinking water




from surface water supplies.  Based on the range of medians, 22-54.5 ng/L




(ppb), the exposure is estimated to range from 44 to 109 jjg/day.  The NOMS




data, however, were collected prior to promulgation of the total




trihalomethane rule; therefore, the exposures based on this data may be




                                     IV-11

-------
overestimated.  Exposure to chloroform in drinking water also has been




estimated from mean levels found at the tap in the TEAM studies.  The mean




concentrations ranged from 24 to 49 ^g/L (ppb) for the four TEAM study cities




using surface water, and the mean concentration for Devils Lake, which used




ground water exclusively, was 0.46 /jg/L (ppb).  The mean concentration in Los




Angelos, where both surface and ground water were used, was 15 /ig/L (ppb).




The exposure to chloroform from the tap is estimated to be from 0.48 to




98 jig/day for surface water users, but only 1 ^g/day for ground water users in




the TEAM study.  The exposure estimates assume a consumption rate of two




liters per day.   The data used in estimating chloroform exposure is limited




and contains considerable uncertainty.  There may be sampling errors that




arise from uncertainties relating to the representativeness of actual samples




being measured, .and there may be measurement errors, that arise from random




and systematic error in a given measurement technique.









        3.   Brominated Trihalomethanes









        The occurrence of brominated THMs,  including bromodichloromethane and




bromoform,  in U.S. drinking water was described in eight national surveys, and




dibromochloromethane occurence has been described in nine national surveys.




In 1975, the National Organics Reconnaissance Survey (NORS),  conducted by the




EPA, collected drinking water samples from 80 cities nationwide.  The survey




sampled for several organics,  including brominated trihalomethanes, at the




water treatment facilities.  The sampling method employed was refrigeration




without quenching; therefore,  brominated THM concentrations may have increased




following collection. Dibromochloromethane was found in 90X of the systems




sampled at a median concentration of 2 ^S/L (ppb)  Bromodichloromethane was




found in 98% of the systems sampled.  The median concentration was 8 /ig/L




                                     IV-12

-------
(ppb), and the maximum level was 116 ^g/L (ppb).  The detection limit  for




dibromochloromethane and bromodichloromethane was 0.1 ^g/L (ppb). The  median




concentration for bromoform was below the detection limit of approximately




5 jzg/L (ppb), and the maximum level found was 92 /xg/L (ppb) (Symons et al.




1975) .  NORS was performed prior to the promulgation of the total trihalo-




methane regulation; therefore, these results may be higher than current




levels.









        The National Organics Monitoring Survey (MOMS) was conducted by the




EPA from March 1976 to January 1977.   In NOMS,  113 community water supplies




were sampled.  Surface water was the major source for 92 of the systems, and




ground water was the major source for the remaining 21 systems.  The NOMS used




all three sampling methods.  During Phase 1, all samples were refrigerated.




In Phase 2, the samples were allowed to stand at 20-25C for 2-3 weeks to




maximize THM formation.  In Phase 3T, one set of samples was allowed to stand




an additional 2-3 weeks.   In Phase 3Q, a quenching agent, sodium thiosulfate,




was added.  As expected,  the highest THM values occurred in Phases 2 and 3T.




Bromodichloromethane was detected in over 90% of the systems sampled.  The




median concentration under the various sampling conditions ranged from 5.9 to




14 /jg/L (ppb) ,  and the maximum concentration was 183 ng/'L (ppb) .  Dibromo-




chloromethane was detected in 73% of the systems sampled.  The median




concentration ranged from below the detection limit to 3 jig/L  (ppb), and the




maximum value was 280 ^g/L (ppb).   The median bromoform concentration  under




all sampling conditions was below the detection limit of 0.3 jig/L (ppb); the




maximum value was 280 /ig/L (ppb).   NOMS was conducted before the promulgation




of the total trihalomethane regulation; therefore, these results may be higher




than current levels.
                                     IV-13

-------
        The Community Water Supply Survey  (CWSS) was conducted by the EPA in




1978.  The survey examined over 1,100 samples, representing over 450 water




supply systems.  The samples were taken at the treatment plants and in the




distribution systems.  In the CWSS, 94% of the surface water supplies and 33%




of the ground water supplies were positive for bromodichloromethane.   For




surface water supplies, the mean of the positives and the overall median were




12 and 6.8 ng/L (ppb),  respectively.  The mean of the positives for ground




water supplies was 5.8 ng/L (ppb), and the overall median was below the




minimum reporting limit of 0.5 jig/L (ppb).  For dibromochloromethane, 97% of




the surface water supplies and 34% of the ground water supplies were positive.




For surface water supplies, the mean of the positives and the overall median




were 5.0 and 1.5 ^g/L (ppb), respectively.  The mean of the positives for




ground water supplies was 6.6 ^tg/L (ppb), and the overall median was below the




minimum reporting limit of 0.5 ptg/L (ppb).  For bromoform, 13% of the surface




water supplies and 26% of the ground water supplies were positive.  The mean




concentration of the positives in surface water supplies was 2.1 fig/L (ppb),




and the overall median was less than 1.0 jig/L (ppb).  The mean of the




positives for ground water supplies was 11 /jg/L (ppb), and the overall median




was below the minimum reporting limit of 0.5 /xg/L (ppb) (Brass et al. 1981).









        The Rural Water Survey (RWS) was conducted between 1978 and 1980 by




the EPA to evaluate the status of drinking water in rural America.  Samples




from over 2,000 households, representing more than 600 rural water supply




systems,  were examined.  In the RWS, 76% of the surface water supplies and 13%




of the ground water supplies'were positive for bromodichloromethane, 56% of




the surface water supplies and 13% of the ground water supplies were positive




for dibromochloromethane, and 18% of the surface water supplies and 12% of the




ground water supplies were positive for bromoform.  For the surface water




                                     IV-14

-------
supplies, the mean of the positives and the overall median concentrations were




17 ^g/L  (ppb) and 11 ng/L (ppb) for bromodichloromethane, 8.5 ng/L (ppb) and




0.3 /ig/L (ppb) for dibromochloromethane, and 8.7 ng/L (ppb) and <0.5  ^g/L




(ppb) for bromoform.  For the ground water supplies, the mean of the positives




was 7.7 fig/L (ppb) for bromodichloromethane,  9.9 /xg/L (ppb) for dibromochloro-




methane, and 12 /ig/L (ppb) for bromoform.  The overall median was below the




minimum reporting limit of 0.5 /xg/L (ppb) for all three brominated trihalo-




methanes (Brass 1981).









        The Ground Water Supply Survey (GWSS) was conducted from December 1980




to December 1981 by the EPA to .develop data on the occurrence of volatile




organic chemicals in ground water supplies.   Out' of a total of 945 ground




water systems that were sampled, 466 systems were chosen at random, and the




remaining 479 systems were chosen on the basis of location near industrial,




commercial, and waste disposal activities.  Samples were collected at or near




the entry to the distribution system,  and THM formation was allowed to




continue without quenching after sample collection.  For bromodichloromethane,




the median of the positives for the randomly chosen systems serving greater




than 10,000 people was 1.4 /ig/L (ppb), and the occurrence rate was 36%.  For




the randomly chosen smaller systems,  the median positive concentration was




1.6 /ig/L (ppb), and the occurrence rate was 54%.  The nonrandomly chosen




systems had a median positive concentration of 2.1 /ig/L (ppb) and an




occurrence rate of 51%.  For dibromochloromethane, the median positive




concentration and the occurrence rate for the randomly chosen systems serving




greater than 10,000 people were 2.1 ^g/L  (ppb) and 31%, respectively; these




values for the smaller systems were 2.9 >ig/L (ppb) and 52%.  The nonrandomly




chosen systems had a median positive concentration of of 3.9 jig/L  (ppb) and an




occurrence rate of 46%.  For bromoform, the median positive concentration was




                                     IV-15

-------
2.4 MS/L (ppb) for the randomly chosen systems serving  greater  than and




3.8 jjg/L (ppb)-for the randomly chosen systems serving  fewer  than




10,000 people, with occurrence rates of 16% and  31%,  respectively.  The




nonrandomly chosen systems had a median positive concentration  of 4.2




(ppb) and an occurrence rate of 31%  (Westrick et al.  1983).
        The Technical Support Division  (TSD) of the Office of Ground Water and




Drinking Water (OGWDW) maintains a ground water contaminant database.  For




both bromodichloromethane and dibromochloromethane , the database contains




4,439 samples taken at the treatment facilities from nineteen states between




1984 and 1991.  For bromodichloromethane, the mean concentration was 5.6 ng/L




(ppb), and the median was 3 /xg/L (ppb).  For dibromochloromethane, the mean




concentration was 3.0 /ig/L (ppb), and the median was 1.7 jig/L (ppb).  For




bromoform, the database contains 1,409  samples from 19 states taken at treat-




ment facilities between 1984 and 1991.  The mean and median concentrations




were determined to be 2.5 jig/L (ppb) and 1 /ig/L (ppb), respectively (U.S. EPA




1991).









        Thirty-five water utilities nationwide, of which 10 were located in




California, were sampled for bromodichloromethane, dibromochloromethane, and




bromoform in the clearwell effluent.  Samples were taken for four  quarters




(spring, summer, and fall in 1988 and winter in 1989).  The median bromo-




dichloromethane concentration for all four quarters was 6.6 jig/L  (ppb), with




the medians of the individual quarters  reported as 6.9, 10, 5.5 and 4.1 /xg/L




(ppb), respectively, and with a maximum value of 82 fjg/L (ppb).  For all four




quarters, 75% of the data were less than 14 jig/L (ppb).  The median dibromo-




chloromethane concentration for all four quarters was 3.6 /ig/L  (ppb), with the




medians of the individual quarters reported as 2.6, 4.5, 3.8 and 2.7 /ig/L




                                     IV-16

-------
(ppb) , respectively, and with a. maximum value of 63 /ig/L  (ppb) .   For  all  four




quarters, 75% of the data were below 9.1 yg/L (ppb).  The median  bromoform




concentration for all four quarters was 0.57 /ig/L  (ppb), with  the medians of




the individual quarters reported as 0.33, 0.57, 0.88, and 0.51 /ig/L  (ppb),




respectively, and with a maximum value of 72 /ig/L  (ppb).  For  all four




quarters, 75% of the data were below 2.8 /ig/L (ppb)   (Krasner  et  al.  1989;




U.S. EPA 1989a 1989b).









        The EPA's Technical Support Division (TSD) has compiled a database




from its disinfection by-products field studies.   The studies  included a




chlorination by-products survey, conducted from October 1987 to March 1989.




In this survey, concentrations of bromodichloromethane, dibromochloromethane,




and bromoform were determined in finished water from  the treatment plant  and




in the distribution system.  Systems using both surface water  sources and




ground water sources were analyzed.









        Mean concentrations of bromodichloromethane,  dibromochloromethane,  and




bromoform in finished water at the treatment plants were determined  for




surface water systems serving both greater than and less than  10,000  people.




The mean concentration of bromodichloromethane was 12.7 /ig/L (ppb) in




42 samples from systems serving more than 10,000 people (90th  percentile,




25.0 /tg/L (ppb)) and 17.0 /ig/L (ppb) for 20 samples from the smaller  systems




(90th percentile, 29.5 /ig/L (ppb)).  The mean dibromochloromethane concentra-




tions was 4.7 /ig/L (ppb) for 42 samples from the larger systems (90th percen-




tile, 13.8 /ig/L (ppb)) and 6.9 /ig/L (ppb) for 20 samples from  the smaller




systems (90th percentile, 24.2 /ig/L (ppb)).  The mean concentrations  for




bromoform were 0.7 /ig/L (ppb) (90th percentile, 1.5 /ig/L  (ppb)) and  0.9 /ig/L
                                     IV-17

-------
(ppb) (90th percentile, 4.9 /ig/L  (ppb))  in 42  samples  from  the  larger systems




and 20 samples from the smaller systems, respectively  (U.S.  EPA 1992).









        Mean bromodichloromethane, dibromochloromethane,  and bromoform




concentrations in distribution systems of these surface water systems also




were analyzed.  Thirty-nine samples were taken from systems  serving  greater




than 10,000 people, and 11 samples were  from systems serving less  than




10,000 people.  The mean.bromodichloromethane  concentrations in the  larger




systems and the smaller systems were 17.4 /ig/L (ppb)   (90th  percentile,




35.3 ng/L (ppb)) and 24.8 Ag/L (ppb) (90th percentile, 51.0  Mg/L (ppb)),




respectively.  The mean dibromochloromethane concentrations  were 6.3 /jg/L




(ppb) (90th percentile, 17.3 /jg/L (ppb)) and 10.4 jig/L (ppb) (90th percentile,




35.0 ^tg/L (ppb))., respectively.  Mean bromoform concentrations  were  0.8  fig/L




(ppb) (90th percentile, 3.1 /ig/L  (ppb))  and 1.4 j*g/L (ppb)   (90th  percentile,




5.1 Mg/L (ppb)), respectively (U.S. EPA  1992).









        Ground water systems serving less than 10,000  people were  analyzed for




bromodichloromethane, dibromochloromethane, and bromoform in 7  finished  water




samples and in 5 distribution system samples..  Mean bromodichloromethane




concentrations were in the finished water samples and  in  the distribution




system samples were 1.1 /ig/L (ppb) (90th percentile, 2.6  fig/L (ppb)) and




2.2 ng/L (ppb) (90th percentile,  3.4 jig/L (ppb)), respectively.   Mean




dibromochloromethane concentrations were 0.6 /ig/L (ppb)  (90th percentile,




1.0 ng/L (ppb)) and 1.8 Mg/L (ppb) (90th percentile, 3.6  Mg/L  (ppb)),




respectively.  Mean bromoform concentrations were 0.6  /ig/L  (ppb) (90th




percentile,  2.6 jjg/L (ppb)) and 2.3 /ig/L (ppb) (90th percentile, 10  /ig/L




(ppb)),  respectively.
                                     IV-18

-------
        For  ground water systems  serving greater than  10,000 people, dibromo-




chloromethane and bromoform were  not detected in single samples taken at  the




plant and  from the distribution system, based on a detection limit of 0.2 ^tg/L




(ppb).  Bromodichloromethane concentrations in the plant and distribution




system samples were 0.2 and 0.4 jig/L (ppb), respectively (U.S. EPA 1992).









        Several less comprehensive surveys have analyzed drinking water for




bromodichloromethane and Jsromoform content.  The EPA's 5-year Total Exposure




Assessment Methodology (TEAM) study measured the personal exposures of urban




populations  to various organic chemicals in air and drinking water in several




U.S. cities  between 1981 and 1987.  As part of the study, running tap water




samples were collected from residences of nearly 850 study participants during




the morning  and .the evening to test for bromodichloromethane and dibromo-




chloromethane concentrations.  Tables IV-3, IV-4, and  IV-5 show bromodichloro-




methane, dibromochloromethane, and bromoform concentrations found in drinking




water from the six cities surveyed.









        Furlong and D'itri (1986) reported that a survey of water treatment




plants in Michigan detected"bromodichloromethane in 35 of 40 plants at a




median concentration of 2.7 /ig/L  (ppb) and a maximum of 54.2 ng/L (ppb);  the




mean of the  positive samples was  7.4 /ig/L (ppb).  Dibromochloromethane also




was detected in 30 plants at a median concentration of 2.2 ng/L (ppb) and a




maximum of 39.6 pg/L (ppb); the mean of the positives was 5.1 /ig/L (ppb).




Bromoform was detected at three of 40 plants sampled at concentrations of 0.9.




1.3, and 1.6 ng/l--  The EPA Region V Organics Survey sampled finished water




from 83 sites in a region that includes Illinois, Indiana, Michigan,




Minnesota, Ohio, and Wisconsin.   Bromoform was found at a median concentration




of 1 fig/L  (ppb) and a maximum level of 7 jtg/L (ppb).  A total of 14% of the




                                     IV-19

-------
                       Table  IV-3  Bromodichloromet.hane  Concentrations  in  Drinking Water from the  EPA

                                                         TEAM  Study
Ni
o

Local ion
El i zabe th/Bayonne ,
New Jersey

Los Angeles,
California


Antioch/Pittsburgh,
California
Devils Lake,
North Dakota
Greensboro ,
North Carolina
Baltimore ,
Maryland
Date
Sampled
Fall 1981
Summer 1982
Winter 1983
Winter 1984
Summer 1984
Winter 1987
Summer 1987
Spring 1984

Fall 1982

Fall 1982

Spring 1987

Sample
Size
355
157
49
117
52
9
7
71

24

24

10

Concentration
Mean
13.6
13.6
5.4
11
20
19
26
21

0.21

7.1

10

Median
13
12
5.8
12
24
24
27
17

0.18

7.8

10

uR/L (ppb) Percent Measured
Maximum 25% 757. 95X
23 -- 15 18
54 --15 20
16 , -- 7.1 8.3
23 5.1 16 20
38 7.7 31 37
31 	
36 	
47 2.4 36 47

1.0 	

11 -- 9.2 --

13 	


         Adapted from Hartwell et al. 1987b; Wallace et al. 1988; Wallace et al.  1987; and Wallace 1992.

-------
              Table  IV-4   Dibromochloromethane  Concentrations  in Drinking Water  from  the EPA
                                                TEAM  study

Location
El izabeth/Bayonne
New Jersey

Los Angeles,
California


Aritioch/Pittsburgh
Cal ifornia
Devils Lake,
North Dakota
Greensboro,
North Carolina
Baltimore,
Maryland
Date
Sampled
Fall 1981
Summer 1982
Winter 1983
Winter 1984
Summer 1984
Winter 1987
Summer 1987
Spring 1984

Fall 1982

Fall 1982

Spring 1987

Sample
Size
355
157
49
117
52
9
7
71

24

24

10

Concentration u/L (ppb) Percent Measured
Mean
2.4
2.1
1.4
9.4
28
10
24.7
8

0.09

1.2

2.7

Median
2.4
1.9
1.6
11
32
12
18
6.4

0.06

1.2

2.6

Maximum 25%
8.4
7.2
3.0 , --
19 2.4
55 15
17
70
21 0.98

0.45

1.9

3.5

75%
2.7
2.4
1.8
15
42


15

0.06

1.5



95%
3.4
3.8
2.1
18
48


19








Adapted from Hartwell et al. 1987b; Wallace et al. 1988; Wallace et al. 1987; and Wallace 1992.

-------
               Table IV-5  Bromoforin Concentrations in Drinking Water from the EPA TEAM Study


Locat ion
Los Angeles,
Cal i fornia


Ant ioch/Pittsburgh ,
California
Date
Sampled
Winter 1984
Summer 1984
Winter 1987
Summer 1987
Spring 1984

Sample
Size
117
52
9
7
71

Concentration R/L (ppb)
Mean
0.78
8.08
3.2
25.5
0.78

Median
0.54
3.0
3.2
9.6
0.58

Maximum
12
78
4.7
113
2.0

Percent Measured
25% 75% 95%
0.34 0.92 1.5
2.00 5.9 53
. .

0.19 1.2 1.9


Adapted from Wallace, 1992.

-------
locations sampled contained detectable  levels of bromoform  (U.S.  EPA  1980b).




Kelley (1985) surveyed 18 drinking water plants in Iowa for  trihalomethanes,




detecting bromoform in five water supplies at concentrations ranging  from  1.0




to 10 /ig/L (ppb) .









        Fair et  al. (1988) analyzed drinking water from three community water




supplies for chlorination by-products.  Bromodichloromethane concentrations




ranged from 7.5  to 30 /ig/-L (ppb) in finished water and from  9.9  to  36  /ig/L




(ppb) in the distribution systems.  Dibromochloromethane concentrations ranged




from less Chan 0.5 to 19 jig/L  (ppb) in  finished water at the plant  and from




less than 0.5 to 23 fig/L (ppb) .in the distribution systems.  Bromoform




concentrations ranged from less than 0.5 to 2.5 j*g/L  (ppb)  in finished water




and from less than 0.5 to 3.1  jxg/L (ppb) in the distribution systems.









        The National Screening Program  for Organics in Drinking  Water  (NSP),




sponsored by the EPA,  was conducted from June 1977 to March  1981.   The survey




sampled 169 systems nationwide.  Samples were collected at  the treatment




facilities.  For dibromochloromethane,  the mean and median  for 130  positives




were 17.2 and 10 /ig/L (ppb), respectively.  The maximum concentration found




was 131 ^g/L (ppb) (Boland 1981).









        As part  of a study to  determine individual exposure  to volatile




organics during  normal daily activities of students at the  University of North




Carolina, Chapel Hill, tap water was analyzed for bromodichloromethane.




Bromodichloromethane concentrations in  tap water ranged from 15  to  20 /ig/L




(ppb), with a mean of 17 /ig/L  (ppb).  The level of detection was 0.1  /ig/L




(ppb) (Wallace et al.   1982).
                                     IV-23

-------
        Chang and Singer (1984) analyzed the bromoform concentration in




drinking water samples prepared by the desalination of seawater.  After




pretireatment using either activated carbon or ultrafiltration, but prior to




the reverse osmosis treatment, bromoform concentrations were 13+14 and




110+59 ^g/L (ppb),  respectively.  After reverse osmosis was completed, the




finished water product contained bromoform concentrations ranging from 2.0 to




51 /ig/L (ppb) (mean, 20.17 /jg/L (ppb)) when activated carbon was used as a




pretreatment and 127 jig/L (ppb) when ultrafiltration was used.  In the reverse




osmosis treatment,  three reverse osmosis membranes were evaluated.  The




cellulose triacetate filter resulted in concentrations of 51 /ig/L (ppb),




making it less efficient in removing bromoform, compared to the polyether/urea




thin film spiral wound membrane and the polysulfone membrane filters  whcih




resulted in concentrations of 5.0 jig/L (ppb) and 2.25 /ig/L (ppb),




respectively.









        Broraodichloromethane,  dibromochloromethane,  and bromoform were




detected in 9.5-12.8% of drinking water samples collected in 1987 in Nassau




County, New York.  The county draws its drinking water from underground




aquifers.   Bromodichloromethane and dibromochloromethane had similar concen-




tration profiles, with approximately 10% and 8.5% of the samples containing




less than 4.9 ppb of the respective chemicals.  The detection limit was  1 ppb




for each chemical.   Bromoform was detected at less than 4.9 ppb in 8% of the




samples,  at 5-9.9 ppb in 2.5% of the samples, and 10-49.9 ppb in less than 1%




of the samples.  The detection limit was 2 ppb.  None of the drinking water




samples contained more than 50 ppb of any of the trihalomethanes, and less




than 1% of the samples contained between 10 and 49.9 ppb of the brominated




compounds (Moon et al. 1990).
                                     IV-24

-------
        Exposure co bromodichloromethane, dibromochloromethane, and bromoform




in drinking water from ground water supplies can be estimated  from the median




levels found in the GWSS.  Based on the range of median levels  (1.4-2.1 /ig/L




(ppb)) and a consumption rate of two liters per day, the median exposure to




bromodichloromethane may range from 2.8 to 4.2 /ig/day.  Similarly, median




exposure to dibromochloromethane may range from 4.2 to 7.8 ^g/L (ppb), and for




bromoform, median exposure may range from 4.8 to 8.4 .>ig/day.   Exposure to




bromodichloromethane from, ground water can be estimated based  on the range of




medians observed under different conditions in NOMS, which mainly sampled




surface water systems.  Based on a range of 5.9-14 ^g/L (ppb), exposure to




bromodichloromethane from surface water is estimated, to be between 12 and




28 /jg/day.  Similarly, based on the range of medians reported  for




dibromochloromethane concentrations, the median exposure is estimated to be up




to 6 /jg/day.  The median levels of bromoform in the surface water supplies




have been found to be less than the EPA Drinking Water minimum reporting




levels (MRLs) of 0.5-1 jig/L (ppb).  An estimate of exposure based on the MRLs




will be overly conservative because the actual concentration of bromoform is




not detectable.  Based on the range of MRLs, 0.5-1 ^g/L (ppb), the exposure to




bromoform is estimated to range from 1 to 2 /ig/day for surface water supplies.




Exposure to bromodichloromethane,  dibromochloromethane and bromoform in




drinking water has also been estimated from mean or median levels found at the




tap in the TEAM studies.  For the six cities in the TEAM study, the mean




concentrations for bromodichloromethane and dibromochloromethane ranged from




0.21 to 22.5 jig/L (ppb) and from 0.09 to 17.3 ^g/L  (ppb), respectively.  The




exposure to bromodichloromethane and dibromochloromethane from the tap is




estimated to be 0.42-45 /ig/day and 0.18-34.6 ^g/day, respectively, assuming a




consumption rate of two liters per day.  If samples were taken over more than




one season, the medians of the seasons were averaged to reflect temporal




                                     IV-25

-------
changes.  To estimate exposure to bromoform at the tap, the median




concentrations were used instead of the mean to negate the effect of one




sample with a very high level of bromoform.  Over 90% of all samples had




bromoform levels below the detection limit; therefore, only data from two of




the six cities are available to estimate exposure.  The median concentrations




ranged from 0.58 to 6.4 ^g/L (ppb),  and based on a tap water consumption rate




of two liters per day, the exposure to bromoform from the tap is estimated to




be 1.16-12.8 fig/day.  The data used in estimating bromodichloromethane,




dibromochloromethane, and bromoform exposure is limited and contains consider-




able uncertainty.  There may be sampling errors that arise from uncertainties




relating to the representativeness of actual samples being measured, and there




may be measurement errors, that arise from random and systematic error in a




given measurement technique.









        B.  Exposure to Sources Other Than Drinking Water









        1.  Dietary Intake









        a.  Chloroform









        No exposure estimates were found for chloroform in food.  Several




studies, however, analyzed foods for chloroform content.  Daft  (1987, 1988,




1989) analyzed foods in three studies.  The most recent and comprehensive of




these surveys analyzed 549 food items.  Foods were chosen from  the FDA's




market basket and were made table-ready (i.e cooked, peeled, etc.) prior to




analysis.  Chloroform was detected in 302 of the foods at an average




concnetration of 71 ng/g (ppb), with concentrations ranging from 2 to 830 ng/g




(ppb).  In an earlier study,  231 food items obtained from the FDA's market -




                                     IV-26

-------
basket collection were analyzed.  Chloroform was detected in 94 samples at an




average concentration of 52 ng/g (ppb) with concentrations ranging from  4 to




312 ng/g (ppb).  Ir- -he third study, 16 unprepared, uncooked, or off-the-shelf




food items were analyzed.  Chloroform was detected in three items: instant hot




cereal at 30 ng/g (ppb),  golden cake mix at 10 ng/g (ppb),  and pancake mix at




70 ng/g (ppb).









        A Total Exposure Assessment Methodology (TEAM) study measured




chloroform in five composite food samples.  In accordance with the FDA's Total




Diet Market Basket Study, 39 different food items were purchased at retail




markets in three geographical areas (Elizabeth, NJ; Chapel Hill, NC;  and




Washington, D.C.) (Entz et al.  1982) .  Twenty food composites,' comprising




four different fpod groups (dairy,  meats, oils-and-fats, and beverages), were




analyzed.   Chloroform was detected in one dairy composite at 17 ng/g (ppb),




one oils-and-fats composite at trace levels (less than 12 ng/g (ppb)) and




three beverage composites at concentrations of less than 12, 6, and 12 ng/g




(ppb).   Subsequent analysis of individual food items detected chloroform in




four cola soft drink samples (178,  22, 9, and 36 ng/g (ppb)), two non-cola




soft drink samples (32 and 14.5 ng/g (ppb)), milk  (17 ng/g (ppb)), ice cream




(23 ng/g (ppb)), processed American cheese (17 ng/g (ppb)), natural cheese




(15 ng/g (ppb)), butter (56 ng/g (ppb)), and mayonnaise (34 ng/g (ppb)).









        Heikes (1987) analyzed 18 table-ready food items, representative of




the 234 items in the FDA Total Diet Study.  A total of 56% of the foods tested




were positive for chloroform-at the following levels: chocolate chip cookies,




22 ng/g (ppb);  plain granola, 57 ng/g (ppb); cheddar cheese, 80 ng/g (ppb);




peanut butter,  29 ng/g (ppb); butter, 670 ng/g (ppb); fried breaded shrimp,




24 ng/g (ppb);  scalloped potatoes,  7.1 ng/g (ppb); cream styled corn, 6.1 ng/g




                                     IV-27

-------
(ppb) ; frozen fried chicken dinner, 29 ng/g (ppb);  and high meat baby food




dinner, 17 ng/g (ppb).  Those items shown to be high in volatile halocarbons




were  further studied.  Samples cf individual food items, representative of




2 or  3 regions of the country, were analyzed and found to have the following




mean  concentrations: 14 butter and margarine samples, 364 ng/g (ppb);




8 samples of 4 types of cheeses, 182 ng/g (ppb); 11 samples of ready-to-eat




cereal products, 60.1 ng/g (ppb); 7 samples of peanut butter, 51.3 ng/g (ppb);




and 12 samples of highly processed foods, 122 ng/g (ppb).









        Uhler and Diachenko (1987) sampled food products from 15 food




processing plants located in nine states.  Plants were chosen from areas where




contamination of water used in processing would be most probable.  Results




of analysis showed that out of 37 food items tested,  chloroform was detected




in 13 samples at the foloowing levels: 2 samples of clear sodas, 2.3 and




15.6  ng/g (ppb); 1 sample of dark cola, 12.3 ng/g (ppb); 4 samples of cheese,




2.4 to 10.9 ng/g (ppb); and 6 samples of ice cream, 4.6 to 31.2 ng/g (ppb).




Abdel-Rahman (1982) analyzed various soft drinks for chloroform and found




average levels ranging from 9 to 61 ^g/L (ppb) for colas and 2.7 to 10.9 jig/L




(ppb) for clear soft drinks.









        Wallace (1992) reported che results of sampling margarine products for




volatile organic carbons in 18 grocery stores and 19 manufacturing plants.




The FDA study detected chloroform in 5 of 18 samples in grocery stores and in




13 of 19 finished samples at the manufacturing plants.  Levels in the samples




at the manufacturing plants were higher than at the stores.  Two samples had




concentrations of 100 and 150 ppb; however, the remaining samples from the




manufacturing plants ranged from 15-50 ppb.
                                     IV-28

-------
        Kroneld and Reunanen (1990) sampled human milk and pasteurized and




unpasteurized cow's milk from a suburban area of Turku, Finland.  The




chloroform concentration in pasteurized milk ranged from unde.tectable to




3.1 ng/L (ppb) (mean, 2.2 M/L (PPM).  Chloroform was not detected in either




the human or unpasteurized cow's milk.  The detection limit was'not reported.









        Toyoda et al. (1990) analyzed the dietary chloroform intake of




30 Japanese housewives in Nagoya and Yokohama, Japan.  Duplicate portions of




3 meals (the type of food was not reported) were sampled for chloroform.  The




detection limit was 0.5 ppb.  Chloroform concentrations ranged from undetect-




able to 106.8 ppb (mean, 19.6+18.1 ppb); the mean dietary intake was




40.0+25.4 Mg/day.









        No exposure estimates concerning the use of chloroform as a food




additive are available.   Chloroform has been approved for use as an indirect




food additive.  Chloroform may be used as a component in adhesives for




packaging foods (FDA 1977 (21 CFR 175.105 4-1-93 Edition)), and it may be used




in the production of polycarbonate resins.  Polycarbonate resins are articles




or components of articles intended for use in producing, manufacturing,




packing, processing, preparing,  treating, packaging, transporting, or holding




food (FDA 1988).









        b.   Brominated Trihalomethanes









        No information is available concerning the occurrence of dibromochloro-




methane or bromoform in food in the United States.  The FDA does not analyze




for dibromochloromethane or bromoform in foods.  However, chlorine is used in




food production for applications such as the disinfection of chicken in




                                     IV-29

-------
poultry plants and the superchlorination of water at soda and beer bottling




plants (Borum 1991).  Therefore, the possibility exists for dietary exposure




from the by-products of chlorination in foods.









        Entz et al. (1982) analyzed food samples from Elizabeth, NJ,  Chapel




Hill, NC, and Washington, D.C. for bromodichloromethane.   A total of




39 different food items from each city were collected according to standards




set for the FDA's Total Diet Market Basket Study.  From 20 food composites




covering four food groups, bromodichloromethane was detected in one dairy




composite at 1.2 ppb and two beverage composites at 0.3 ppb and 0.6 ppb.




Analysis of individual foods found bromodichloromethane in three samples of




cola soft drinks at concentrations of 2.3 ppb, 3.4 ppb, and 3.8 ppb and in one




sample of butter at 7 ppb.  In a second study, analysis of various soft drinks




found average levels ranging from 0.2 to 6.6 /ig/L (ppb) for'colas and from




0.1 to 0.2 ng/L (ppb) for clear soft drinks (Abdel-Rahman 1982).  Uhler and




Diachenko (1987) sampled food products from 15 food processing plants in nine




states.  Plants were chosen from areas where contaminated water would most




likely be used in processing.  Bromodichloromethane was detected in six out of




37 food items tested at the following levels:,  two samples of clear sodas at




1.2 and 2.3 ng/g (ppb); one sample of dark cola at 1.2 ng/g (ppb); and three




samples of ice cream at 0.6 to 2.3 ng/g (ppb).  Bromodichloromethane also was




identified in bacon, but no concentrations were given  (U.S. EPA 1985b).









        Toyoda et al. (1990) analyzed the dietary intake of bromodichloro.-




methane,  dibromochloromethane, and bromoform  for 30 Japanese housewives in




Nagoya and Yokohama, Japan.  Duplicate portions of 3 meals (the type of food




was not reported) were sampled for all three chemicals.  The detection limits




were 0.1, 0.2, and 0.5 ppb, for bromodichloromethane,  dibromochloromethane,




                                     IV-30

-------
and bromoform, respectively.  The concentration of bromodichloromethane ranged




from undetectable to 1.7 ppb (average, 0.3+0.3 ppb).   The detection limit was




0.1 ppb.  The mean daily intake of broroodichlorcice thane was 0.6+0.5 ^g/dav.




The concentration of dibromochloromethane ranged from undetectable to 0.6 ppb




(average, 0.1+0.2 ppb), and the mean dietary intake was 0.3+0.3 ^g/day.  The




detection limit was 0.2 ppb.  The concentration of bromoform ranged from




undetectable to 8.1 ppb (average, 0.5+1.3 ppb).  The mean dietary intake of




bromoform was 0.91.3 fig/day.   The detection limit was 0.5 ppb.









        In a study that determined volatile organic compounds in human milk




and pasteurized and unpasteurized cow's milk in Turku, Finland, the average




concentration of bromodichloromethane measured in pasteurized milk was




0.008 ^g/L (ppb). (range, undetectable to 0.03 ^ig/L (ppb)).  Dibromochloro-




methane was detected in only one sample of pasteurized milk at 5 /ig/L (ppb).




Traces of bromoform also were detected but were not quantified.  Bromo-




dichloromethane and dibromochloromethane were not detected in human or




unpasteurized milk.   The presence of the brominated trihalomethanes in




pasteurized milk was most likely due to the use of' chlorinated water during




processing (Kroneld and Reunanen 1990).









        2.  Air Intake









        a.  Chloroform









        Inhalation exposure to chloroform in indoor and outdoor air has been




surveyed in several studies.  The major source of these data is the EPA's




5-year Total Exposure Assessment Methodology (TEAM) study measuring the




personal exposures of urban populations to a number of organic chemicals in




                                     IV-31

-------
the air and drinking water of several U.S. cities between 1981 and 1987.  As




part of the study, personal air samples and outdoor air samples were analyzed




for chloroform.  Personal air samples were collected for each individual for




two 12-hour periods, excluding showers, and outdoor samples also were




collected in two 12-hour periods.  Table IV-6 shows chloroform concentrations




in personal air samples, and Table IV-7 shows concentrations found in outdoor




air samples.  Based on personal air results, the median exposure to chloroform




from air intake would range from 0.6 to 64 /xg/day, using an inhalation rate of




20 m3/day.









        Additional studies have measured exposure levels similar to those




found in the TEAM studies.  Wallace (1992) reported results from short-term




measurements of ambient air in 10 cities between 1979 and 1981.  For a total




of 2,577 measurements, the 25th percentile, median, and 75th percentile air




concentrations were 0.14, 0.20, and 0.78 /ig/m3,  corresponding to exposures of




2.8,  4.0,  and 16 /jg/day, respectively.  Ambient air samples at four urban/in-




dustrial sites in the California South Coast air basin were surveyed from




November 1982 to December 1983 for the presence of halogenated hydrocarbons.




Chloroform was detected above the quantitation limit of 0.02 ppb in 96% of the




samples.  Concentrations generally increased by a similar degree at all sites




during the summer months.  The highest concentration, 3.0 ppb, was recorded




for downtown Los Angeles, as was the highest monthly mean (0.78 ppb) and the




highest composite mean (0.13 ppb) (Shikiya et al. 1984).  Using an inhalation




rate of 20 m3/day,  potential human exposure could be as high as 14 jig/day,




based on the highest observed composite mean of 0.13 ppb.









        Singh et al.  (1981) reported results from field studies conducted in




three cities: Los Angeles, California; Oakland, California; and Phoenix,




                                     IV-32

-------
                   Table  IV-6   Chloroform in Personal  Air  Samples  from the  EPA TEAM Study'

Concentration ita/m3
Location
Elizabeth/Bayonne ,
New Jersey

Los Angeles,
California


Antioch/Pittsburgh,
Cal ifornia
Devils Lake,
North Dakota
Greensboro,
North Carolina
Baltimore,
Maryland
Date
Sampled
Fall 1981
Summer
Winter
Winter
Summer
Winter
Summer
Spring

1982b
1983
1984
1984
1987
1987 .
1984

Fall 1982


Fall 1982

Spring


1987

Sample
Size
340
150
49
117
52
88
78
68

23

24

116

Mean
8
4
4
1
1
1
2
0

2

2

5

.0
.3
.0
.9
.1
.6
.4
.64

.02

.2

.8

Median
3.
0.
2.
1.
0.
0.
0.
0.

0

1

3.

2
8
2
25
49
71
48
03

.38

.7

05

Maximum
Percent
Measured
(Night/Day) 25% 75%
210/89
35/140
16/40
9.7/92
20/12
42/4.2
7.6/76
6.3/4.2

2.8/50

5.5/7.5

38/51

7
6
' -- 5
0.69 2
0.16 1
0.23 1
0.15 0
0.03 0

0

3

0

.8
.0
.3
.3
.2
.7
.99
.72

.77

.2

.75

95%
26
14.5
16.5
4.8
4.4
6.5
47.5
2.65





18


Adapted from Wallace 1986; Wallace et al.  1986a; and Wallace et al.  1986b.
 Average of night and day measurements
6 These values were affected by contamination of sampling cartridges.

-------
                  Table  IV-7   Chloroform  in  Outdoor Air Samples  From  the  EPA TEAM Study *

Concentration ug/m3

Location
Elizabeth/Bayonne ,
New Jersey


Los Angeles,
California
Antioch/Pittsburgh,
California
Devils Lake,
North Dakota
Greensboro,
North Carolina
Date
Sampled
Fall 1981
Summer 1982b
Winter 1983

Winter 1984
Summer 1984
Spring 1984

Fall 1982

Fall 1982

Sample
Size
86
60
9

24
23
10

5

6

Maximum Percent Measured
Mean Median
1.4 0.63
13 0.11
0.3 0.07

0.7 0.3
0.3 0.03
0.3 0.11

0.05

0.14

Night/Day 25%
22/8.8
130/230
1.2/1.2
1
5.9/1.5
2.4/2.4
1.5/0.23

0.78

11.0

75% 95%
1.7 5.9
9.8 78
0.63 --

0.45 1.2
0.03 1.1
0.33 0.6

. .

. .

v
Adapted from Wallace 1986;  Wallace et al.  1986a;  and Wallace et al.  1986b.

 Average of night and day measurements
bThese values were affected by contamination of sampling cartridges.

-------
Arizona.  Ambient air samples were collected at each site over 2-week periods




in 1979 and analyzed for chloroform.  Results of the analyses indicated  that




for iihe Los Angeles area, Che mean concentration was 0.088 ppb and the range




was 0.024-223 ppb, while Oakland had a mean of 0.032 ppb and a range of




0.013-0.060 ppb.  In Phoenix, a mean of 111 ppb and range of 0.027-514 ppb was




reported.  Based on the range of means, the average exposure to chloroform due




to inhalation would be from 3.2-10.8 /jg/day, using an inhalation rate of




20 m3/day.









        Although TEAM studies do not collect personal air samples during




showers, attempts have been made to measure the contribution to airborne




chloroform levels.  Wallace (1992) reported results from a 1987 EPA study




which found chloroform levels in the air of a shower to increase from 2  to




100 ppb during a 10-minute shower with chlorinated water.









        Jo et al. (1990a, 1990b) reported separate inhalation and dermal




exposure concentrations for chloroform exposure in the shower.  Volunteers




took showers with and without wetsuits to estimate the fraction of chloroform




absorbed dermally.  Chloroform concentrations in the tap water prior to  the




shower ranged from 5.3 to 35.9 /ig/L (ppb) (mean, 23.5 ;ig/L (ppb)).  The  total




mean absorbed chloroform dose for a 10-minute shower was 0.47 ^g/kg/day.  A




chloroform absorption efficiency rate of .0.77 was used for inhalation.   Body




burden from dermal exposure was estimated to be 93% of the body burden from




inhalation exposure.   The inhalation and dermal exposure components were  




0.24 jjg/kg/day and 0.23 MAg/day. respectively.  Chloroform concentrations




were slightly higher in air in the shower stall in the presence of a showering




individual than in an empty shower with the water running.  In the absence of




a showering individual, chloroform concentrations ranged 58.1 to 326.9




                                     IV-35

-------
(11.9 to 66.9 ppb) (mean, 157 /jg/m  (32.2 ppb)), and in the presence of a




showering individual, chloroform concentrations ranged from 119 to 313.4




(24.4 to 64.18 ppb)  (mean, 186 H&/K*  (38.1 ppb)) (Jo et al.,  1990b).









        Tancrede et al.   (1992) reported that volatilization of chloroform




during a shower increased by 21% when  the water temperature was increased from




25 to 42C.   Additionally, the fraction of chloroform in the shower water that




volatilized (42 to 64%)   increased by 15% when the water flow rate was




increased from 9.7 to 13.5 L/min.  Wallace (1992) reported that 55% of the




chloroform in tap water volatilized during an 11 minute shower with a 5 L/min




rate of flow.   The initial chloroform  concentration in the water was 580 ^ig/L




(ppb),  and the shower water effluent contained 260 jig/L (ppb)  chloroform.




Furthermore,  the. longer  the shower was on, the greater the volatilization




rate.  The low-volatility compounds rapidly reached a steady state, while the




high-volatility compounds reached a steady state more slowly.   Therefore, the




concentration of chloroform in the air increases over time.  An example of a




high-exposure scenario is a shower facility in a locker room where showers are




on for long periods of time (Little 1992).









        Heating and boiling water reduced chloroform levels;  this reduction




increased with the temperature and duration of boiling.  Chloroform was




reduced by SOX after one minute ac 80C, by approximately 70% immediately




after the water began to boil, approximately 80% after the water had boiled




for about one minute, and by 90% when  the water had boiled for five minutes.




Dibromochloromethane, bromodichloromethane, and bromoform decreased by 86, 90,




and 81%, respectively, when the water  had been boiled for five minutes (Lahl




et al.  1982).'
                                     IV-36

-------
        Wallace  (1992) analyzed  indoor air concentrations of chloroform




resulting from the use of hot water.  Seven volunteers  in four homes  took  long




showers, boiled water, and washed clothes while ir.dcor  air concentrations  were




being monitored.  Washing clothes and dishes increased  levels of chloroform  in




the house from undetectable (<4.5 /ig/m3  (0.92 ppb))  to 44 jig/m3  (9.00  ppb)




over a period of 5-11 hours.  Limited data indicate that taking long  showers




and boiling water did not increase chloroform levels  in the rest of the house.




Further measurements are required to determine whether  long showers or boiling




water increases chloroform concentrations outside of  the bathroom or  kitchen.









        The TEAM program analyzed personal air concentrations of chloroform




for lifeguards at three swimming pools (2 outdoor and 1  indoor).  The personal




air levels of ch.loroform at the  indoor pool were 95,  46, and 68 jig/m3 (19.5,




9.4, and 13.9 ppb) for the three lifeguards. Chloroform concentrations




measured at the homes of the lifeguards were 2.2, 5.2 and 2.0 ^g/m3,   (0.45,




1.00,  and 0.41 ppb).  The chloroform levels in the air  at outdoor pools did




not differ from the concentrations measured at the lifeguards' homes.  Levels




of chloroform in the pool water ranged from 48 to 153 jig/L (ppb) for  all pools




(Wallace 1992).









        Armstrong and Golden (1986) measured chloroform concentrations in  the




water and surrounding air of four indoor swimming pools, five outdoor swimming




pools, and four hot tubs.  Concentrations in air were measured two centimeters




above the water.  Chloroform concentrations in the water- in the outdoor pools




ranged from 4 to 402 jig/L (ppb)  (mean, 128 ^ig/L (ppb)), while levels  in the




indoor pools ranged from 3 to 580 /ig/L (ppb) (mean, 133 ^g/L (ppb)).  The




levels of chloroform in the hot  tubs ranged from <0.1 to 530 /xg/L (ppb) (mean,




115 jig/L (ppb)).  Means and ranges of concentrations  (in parentheses) of




                                     IV-37

-------
chloroform measured two meters above the water surface for outdoor pools.




indoor pools, and hot tubs, respectively, were: <0.1 /ig/m3 (0.02 ppb)




(<0.1-1 /ig/m3 (0.02-0.2  ppb)),  90  Mg/m3  (13.4  ppb)   (<0.1-260 Mg/m3




(0.02-53.2 ppb)), and 12 /jg/m3 (2.5 ppb) (<0.1-47 Mg/m3 (0.02-9.6 ppb)).









        b.  Brominated Trihalomethanes









        Brominated trihalomethanes usually are found in air at  low concentra-




tions.  Brodzinsky and Singh (1983) reported  ambient outside air concentra-




tions for several urban locations across the  United States.  Ambient air




samples also were analyzed for bromodichloromethane and dibromochloromethane




at Magnolia, AZ, El Dorado, TX, Chapel Hill,  NC, and Beaumont,  TX.  Bromo-




dichloromethane was detected at mean concentrations of 0.76 ppt, 1.40 ppt,




120 ppt,  and 180 ppt,  respectively.  Based on an inhalation rate of 20 m3/day,




daily intake of bromodichloromethane was estimated to range from 0.12 to




26 ^tg/day.  Dibromochloromethane was detected in the air  samples from




Magnolia, AZ, El Dorado, TX, Chapel Hill, NC, Beaumont TX, and  Lake Charles,




LA at concentrations of 0 ppt, 0.48 ppt, 14 ppt, 14 ppt,  and 19 ppt,




respectively.  From 1976 to 1977 at El Dorado., Texas, bromoform was detected




in 76% of 46 samples at a mean concentration  of 0.81 jjg/m3 (0.08 ppb),  with




concentrations ranging from below the detection limit  to  2.7 /ig/m3 (0.3 ppb).




In air samples collected in 1978 at Lake Charles, Louisiana, bromoform was




detected in all of four samples, with a mean  of 50 ng/m3  (4.8 ppt) and a range




of 6.6-7.1 ng/m3 (0.66-0.71 ppt).   In 1977,  bromoform was detected in 89.3% of




28 ambient air samples from Magnolia, AZ.  Concentrations ranged from not




detected to 8.3 ng/m3  (0.83 ppt) , with a mean of 1.5 ng/m3 (0.15 ppt).




Overall,  the average bromoform concentration  was 37 ng/m3  (0.36 ppt)




(Brodzinsky and Singh 1983).




                                     IV-38

-------
        From November 1982 Co December 1983, ambient air samples at four




urban/industrial sites in the California South Coast air basin were surveyed




for  the presence of halogenated hydrocarbons.  Thirty-five percent of the




samples had bromodichloromethane levels above the quantitation limit of




0.01 ppb, 17% percent had dibromochloromethane levels above the quantitation




limit of 10 ppt, and 31% had bromoform levels above.the quantitation limit of




10 ppt.  Peaks  in the concentration of bromodichloromethane were observed at




various sites in June and-July, with downtown Los Angeles and Dominguez




registering the highest monthly means, 30 ppt.  The maximum reported value was




40 ppt, while the composite means ranged from 20 to 100 ppt.   The composite




sample was a collection of several individual samples mixed together before




analysis, and the concentration of any chemical in a composite sample is equal




to or greater than the concentration in individual samples.  For dibromo-




chloromethane,  the highest reported concentration, monthly mean, and mean




composite, respectively,  were 290 ppt, 280 ppt, and 50 ppt; all were recorded




in-downtown Los Angeles in July.  Mean concentrations ranged from 10 to




50 ppt.  Peaks  in the concentration of bromoform were observed at various




sites in May and June, with the downtown Los Angeles site registering the




highest composite mean (40 ppt) and the highest monthly mean (310 ppt) in June




1983 (Shikiya et al. 1984).  Based on the range of mean composites, daily




intake was estimated to range from 2.8 to 15 ^g/day  (Shikiya et al. 1984).









        Replicate air samples were collected at various locations on the




Island of Hawaii during a month-long field experiment to test an analytical




method for determining halocarbons in ambient air.  Dibromochloromethane was




found at a mean level of 0.27 ppt, .and bromoform was found at a mean




concentration of 1.9 ppt (Atlas and Schauffler 1991).
                                     IV-39

-------
        Wallace et al.  (1982) conducted  a pilot  study  designed to  field test




personal air-quality monitoring methods.  .Personal  air samples were  collected




frorr. students at two universities: Laraar University, Tor.as,  located  near a




petrochemical manufacturing area, and the University of North  Carolina  (UNC),




located in a nonindustrialized area.  Bromodichloromethane was detected in 64%




of personal air samples from 11 Lamar students,'  with a mean  of 1.23  /ig/m3




(0.18 ppb),  a median of 1 ng/m2 (0.15 ppb),  and  a range of 0.12-3.72 ^g/m3




(0.017-0.56 ppb).  The limit of detection was  0.24  jig/m3 (0.04 ppb).  At UNC,




17% of the samples from 6 students had detectable levels of  bromodichloro-




methane.  Concentrations ranged from 0.12-4.36 /jg/m3 (0.017-0.65 ppb) (mean,




0.83 /ig/m3 (0.12 ppb); median,  0.12 jig/m3 (0.017 ppb)).  Based on the above




information, the average daily intake of bromodichloromethane  from air  using




an inhalation rate of 20 nr/day was estimated  to be 25  ^g/day  for  Lamar




students and 17 /ig/day for UNC students.  Dibromochloromethane was not




detected above 0.12 /ig/m3 at either site.









        Armstrong and Golden (1986) measured bromodichloromethane, dibrorao-




chloromethane, and bromoform concentrations in the  water and surrounding air




of four indoor swimming pools, five outdoor swimming pools,  and four hot tubs.




Concentrations in air were measured two  centimeters from the water.  The




bromodichloromethane concentrations of water in  the outdoor  pools  ranged from




1 to 72 fig/L (mean, 33 /ig/m3) .   Levels in the  indoor pools ranged  from  1 to




90 jig/L (mean, 16 /ig/L) .  The levels of  bromodichloromethane in the  hot tubs




ranged from S0.1 to 105 /ig/L (mean, 17 jtg/L) .  Means and ranges of the  bromo-




dichloromethane concentration two meters above the  water surface for outdoor




pools, indoor pools, and hot tubs, respectively, were:  <0.1  /xg/nr  (0.02  ppb)




(mean only), 1.7 ng/m* (0.25 ppb) (<0.1-10 Mg/m3 (0.25-1.49 ppb)),  and




1.4 jig/m3 (0.21 ppb) (<0.1-10 /ig/m3 (0.25-1.49  ppb)).   The dibromochloro-




                                     IV-40

-------
methane concentration  of water  in  the  outdoor pools  ranged from <0.1  to 8  jjg/L




(ppb) (mean, 4.2 jig/m3, (ppb)).  Levels  in  the  indoor  pools  ranged from 0.3  to




30 fig/L (ppb)  (mean, 9.5 /ig/L  (ppb)).  The  level  of  dibromochloromethane ir.




the hot tubs ranged from sO.l to 48  /ig/L (ppb)  (mean,  14.4 |ig/L (ppb)).  Means




and ranges of  the dibromochloromethane concentration two meters above the




water surface  for outdoor pools, indoor  pools,  and hot tubs,  respectively,




were: <0.1 jtg/m3 (0.01 ppb)  (mean only),  0.9 /ig/m3 (0.1 ppb) (<0.1 to 5 ng/n?




(0.011-0.59 ppb)), and 0.3. ;ig/m3 (0.08 ppb)  (<0.1  to 5 /ig/m3 (0.01-0.59 ppb)).




The mean bromoform concentration in  the  outdoor pools  was less  than 0.1 jig/L




(ppb).  Levels  in the  indoor pools ranged from less  than 0.1 to 20 /ig/L (ppb)




(mean, 6 ^ig/L  (ppb)).  The levels of bromoform in the  hot tubs  ranged from




less than 0.1  to 62 ^g/L (ppb)  (mean,  13 /ig/L (ppb)).   Means and ranges of the




bromoform concentration two meters above the water surface for  outdoor pools,




indoor pools,  and hot  tubs, respectively, were: <0.1 jig/m3 (9.6 ppt)  (mean




only), 9 Mg/m3  (87 PPC) (<0.1-14 ng/m3 (9.6-1354 ppt)), and 8 ^g/m3 (773 ppt)




(<0.1-14 Mg/m3  (9.6-1354 ppb)).









        Based  on the above surveys,  exposure to bromodichloromethane,  dibromo-




chlorome thane  and bromoform due to inhalation can be estimated  based on an




inhalation rate of 20  m3/day.   Based on  an  ambient air concentration  of




0.76-180 ppt,  daily intake of bromodichloromethane was estimated to range from




0.12 to 26 fig/day.  For dibromochloromethane,  assuming an ambient air concen-




tration of 1-50 ppt, exposure may be as  low as 0.18  /ig/day or as high as




9.4 /ig/day.  Using an  ambient air concentration of 0.021-3.5 /jg/m,




(0.002-0.34 ppb), mean exposure may  be as low as  0.42  ^g/day or as high as




70 ^g/day for  bromoform.  The use of ambient air  concentrations,  due to the




lack of data on indoor air concentrations,  will underestimate the exposure.
                                     IV-41

-------
Brominaced THM levels are higher in indoor air compared to oucdoor air due co




confined space and additional indoor air sources of THMs.









        C.  Overall Exposure









        1.  Chloroform









        The EPA has set the maximum contaminant level goal (MCLG) for




chloroform at zero because it has been determined that it  is a probable human




carcinogen.  Therefore, a relative source contribution (RSC) analysis is not




relevant because it is the Agency's policy to perform RSC  analyses for only




non-carcinogens.









        2.  Brominated Trihalomethanes









        The EPA has set the maximum contaminant level goal (MCLG) for dibromo-




chloromethane and bromoform at zero because it has been determined that these




chemicals are probable human carcinogens.  Therefore, a relative source




contribution (RSC) analysis is not relevant for these chemicals because it is




the Agency's policy to perform RSC analyses for only non-carcinogens.









        A relative source contribution (RSC) analysis for bromodichloromethane




cannot be performed due to the limited occurrence and exposure data.  Based on




the available water and air data, it appears that, on average, drinking water




may be the predominant source of bromodichloromethane exposure, but  that air




may contribute a significant amount.  Based on these anecdotal data, an RSC




value of 80% for drinking water is believed to be on the high side.
                                     IV-42

-------
        D.  Body Burden









        1.  Chloroform









        a.  Breath









        The largest existing data set on chloroform concentrations  in breath




is from the TEAM study which reports measurements on 800 people taken from




fall 1981 to summer 1987; the study provides more than 1,200 breath samples.




Mean concentrations ranged from 0.5 to 3 /ig/m3 (0.1 to  0.6  ppb) ,  with the




lowest levels in California.  Wallace et al. (1982) analyzed chloroform




concentrations in breath for two groups of university students.  Students at




Lamar University., Texas, resided in the vicinity of oil wells  and oil storage




tanks,  and students at the University of North Carolina (UNC)  resided in a




nonindustrial area.  Chloroform was detected in the breath of  all students at




Lamar University, with concentrations ranging from 0.22 to 2.48 jig/m3




(0.05-0.51 ppb) (mean, 0.42 ng/m3 (0.09 ppb)).   At UNC, 100% of the students




had detectable levels of chloroform in their breath with concentrations




ranging from 1.70 to 5.06 ng/v? (0.35-1.04 ppb) (mean,  2.86 ng/mz  (0.56 ppb)).




The detection limit was 0.11 ^g/m3 (0.02 ppb)
        Jo et al. (1990a, 1990b) measured breath concentrations of chloroform




pre- and post-exposure in the shower and for  inhalation -only exposure  in  the




shower.  The post-exposure breath concentrations after a normal (10-minute)




shower ranged from 6.0 to 21 /ig/m3 and from 2.4 to 10 jig/m3 for inhalation-




only exposure showers.  The chloroform body burden after showering was 14 to




49 times higher than background chloroform body burden.  The pre -shower breath




concentrations of chloroform were all below the detection  limit of 0.86 jig/m3 .




                                     IV-43

-------
        b.  Blood









        Wallace (1992) reported that chloroform concentrations  in blood were




measured  (in triplicate) in 25 female volunteers using a purge  and trap




method.   Five subjects had blood concentrations below 10 ^ig/L (ppb),  6




subjects  ranged from 10 to 25 /ig/L (ppb), with a small standard deviation




(<10 Mg/L (ppb)),  and another 5 subjects were in the same range with a large




standard  deviation (>20 fig/L (ppb)).   Seven subjects exceeded 25 jig/L (ppb).




All values above 20 ppb were confirmed with gas chromatography/mass




spectrography.   The author noted that the analytical method used thermally




decomposes trichloroacetic acid (TCA) in the blood to chloroform.  Subse-




quently,  the measured chloroform may include original chloroform in  the blood




and "derived"  chloroform from the decarboxylation of TCA.   Therefore, the




measured values are upper limits with respect to chloroform exposure  and may




be due to chlorinated VOC's as well as chloroform.  Hajimiragha et al. (1986)




analyzed blood chloroform concentrations in 39 subjects with no known




occupational exposure.  Chloroform was detected in 60-95% of the samples




analyzed, with concentrations ranging from <0.1 to 7 ^ig/L (ppb) (mean,




0.2 jig/m3 (ppb)).   The analytical  method used .was  headspace gas




chromatography.









        Antoine et al. (1986) analyzed the blood of 250 environmentally




sensitive patients for 18 volatile organic compounds.  Chloroform




concentrations ranged from undetectable to 7.0 /zg/L (ppb)  (mean, 1.5  pg/L




(ppb)).  Hajimiragha et al.  (1986) measured chloroform concentrations in the




blood of  dry cleaners.  Concentrations ranged from 0.2 to 51.2  jjg/L  (ppb)




(mean, 12.2 ^g/L (ppb)).  Wallace (1992) reported that plasma chloroform
                                     IV-44

-------
levels in 127 swimmers using indoor pools in Italy ranged from 0.1 to 3.1




(ppb) shortly after swimming.









        c.  Mother's Milk
        Pellizzari et al. (1982) analyzed mother's milk for chloroform by




using gas chromatography/mass spectrometry .   Samples were collected from




49 lactating women living, in the vicinity of chemical manufacturing plants




and/or industrial user facilities in Bridgeville, PA, Bayonne ,  NJ ,  Jersey




City, NJ , and Baton Rouge,  LA.  Seven of the 49 samples contained detectable




levels of chloroform.  Actual concentrations and detection limits were not




reported.









        d.  Adipose Tissue









        The EPA's National Human Adipose Tissue Survey (NHATS) ,   in 1982    I




quantified the prevalence of toxic compounds detected in the general public of




the United States.  Several hundred samples of adipose tissue were collected,




pooled into 46 composite samples by age and geographic re-gion,  and analyzed.




Chloroform was detected at 5-580 ng/g (ppb)  in 29 of the samples (Phillips




1992; Wallace 1992).  The results were divided into nine geographical regions,




and chloroform exposure was ranked highest in the East North Central Region,




which consisted of Ohio, Indiana, Illinois,  Michigan and Wisconsin (Phillips




and Birchard 1991) .
                                     IV-45

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        2.  Brominaced Trihalomethanes









        a.  Blood









        Antoine et al. (1986) analyzed the blood of 250 environmentally




sensitive patients for 18 volatile organic compounds.   Bromoform




concentrations ranged from undetectable to 3.4 jjg/L (ppb),  with a mean of




0.6 Mg/L  (ppb).









        b.  Mother's Milk









        Pellizzari et al. (1982) analyzed mother's milk for dibromochloro-




methane using gas chromatography/mass spectrometry.   Samples were collected




from 49 lactating women living in the vicinity of chemical manufacturing




plants and/or industrial user facilities in Bridgeville,  PA, Bayonne,  NJ,




Jersey City, NJ ,  and Baton Rouge, LA.  One of the 49 samples contained




detectable levels of dibromochloromethane.   Actual concentrations and




detection limits were not reported.









        E.  Summary









        Trihalomethanes are found in virtually all treated drinking water;




however, concentrations of individual forms vary widely depending on the type




of water treatment, locale, time of year, and source of the drinking water.




Chloroform is the most commonly detected THM and usually occurs at the




greatest concentrations.   Chloroform concentrations in drinking water have




ranged from less than 0.5 to 550 ^ig/L (ppb).  Bromodichloromethane concen-




trations in drinking water ranged from 0.2 to 183 jzg/L (ppb), while dibromo-




                                     IV-46

-------
chloromethane and bromoforra concentrations ranged from less than 0.5 to




280 ^g/L (ppb).   Concentrations of all trihalomethanes in drinking water were




generally lower when the raw water is derived from Around water sources rat-her




than surface water sources.  In any study analyzing THMs in water, the type of




sampling method used should be considered because some methods restrict THM




formation by refrigeration or the use of quenching agents, whereas others




maximize THM formation.









        Chloroform has been detected in food although data on the brominated




forms is not available in the U.S..  Chloroform was detected in food at




concentrations ranging from nondetectable to 830 ng/g (ppb),  and concentra-




tions of bromodichloromethane detected in food ranged from nondetectable to




70 ng/L (0.07 ppb).  No studies were located that analyzed dibromochloro-




methane and bromoform in food in the United States.   Bromodichloromethane,




dibromochloromethane,  and bromoform concentrations in various foods in studies




conducted in Japan and Finland ranged from undetectable to 1.7 ppb, undetect-




able to 0.6 ppb,  and undetectable to 8.1 ppb, respectively.  Chloroform is




approved by FDA for use in food packaging.









        THMs are ubiquitous in air, although the concentrations are highly




variable depending on the ambient environment.  Chloroform concentrations tend




to be higher in indoor air compared to outdoor air because of the confined




space and release of chloroform from various indoor sources.   Chloroform




concentrations in personal air and outdoor air ranged from 0.06 to 215 jig/m3




(0.01-44 ppb) ,  and from 0.04 to 21.5 ^ig/m3 (0.008-44 ppb), respectively.  One




major source of chloroform in indoor air appears be tap water that releases




chloroform when it is used for showers or washing.   One study indicated  that




concentrations of chloroform in shower stall air samples during a 10-minute




                                     IV-47

-------
shower ranged from 10 to 500 Mg/m3 (2.05-112 ppb).   The absorbed inhalation




and dermal doses were 0.24 and 0.23 jig/kg/day, respectively for a combined




absorbed chloroform dose for the 10-rcinute shower of 0.47 /ig/kg/day.




Concentrations of other THMs, in indoor or outdoor air are generally lower




than chloroform.  Bromodichloromethane concentrations in personal air ranged




from 0.12 to 4.36 /jg/m3 (0.017-0.65 ppb),  whereas concentrations in outdoor




air ranged from 0.006 to 1.3 ^g/m3 (0.9-194 ppt).  Bromoform and




dibromochloromethane concentrations in outdoor air ranged from below the




detection limit to 3.5 Mg/m3 (0.34 ppb),  and from 0.00 to 0.47 jig/m3




(0-0.06 ppb), respectively.









        The use of chlorine to disinfect swimming pools and hot tubs also




results in the release of THMs to the overlying air.  One study indicated that




chloroform, bromodichloromethane,  dibromochloromethane, and bromoform




concentrations in swimming pool and hot tub water ranged from less  than 1 to




530 /ig/L (ppb),  from 1 to 105 jig/L (ppb),  from 0.1 to 48 ^g/L (ppb), and from




less than 0.1 to 62 ^g/L (ppb), respectively.  Concentrations of these same




THMs in the air two meters above the pool water ranged from 0.1 to  260 ng/m3




(0.2-53 (ppb)),  less than 0.1 to 14 /ig/m3  (0.15-2.00 (ppb)),  from less than




0.1-10 ng/nf  (0.01 to 1.17 (ppb)),  and from less  than 0.1 to 5.0 /^g/m3 (0.014




33-0.48 ppb), respectively.









        Chloroform exhaled in breath is related to body burden of chloroform




and recent exposure to chloroform in air or water.  Background chloroform




concentrations measured in breath have ranged from 0.22 to 5.06 ^ig/m3




(0.05-1.04 ppb), and reported breath concentrations after a 10-minute shower




ranged from 6 to 21 ^g/m3 (1.23-4.3 ppb).   THMs also have been detected in the




blood, milk and adipose tissue of humans.   Chloroform concentrations  in blood




                                     IV-48

-------
have ranged from less chan 0.1 to greater than 25 ^g/L (ppb),  and bromoform




has been detected in blood at levels up to 3.4 jig/L (ppb).  Chloroform has




been detected in the milk of 7 of 49 lactacing women living in industrial




areas; however, actual concentrations were not reported.
                                     IV-49

-------
V.      'HEALTH EFFECTS IN ANIMALS









        A.  Short-Term Exposure









        1.  Lethality









        Table V-l summarizes reported oral LDSO values in rats and mice for




the trihalomethanes.   Reported values for chloroform range from 119 to




2,000 mg/kg, with most of the values falling in the range of  908 to




2,000 mg/kg.  Values for the brominated trihalomethanes range from 450 to




1.-550 mg/kg.  Bowman et al. (1978) concluded that male mice were more sensi-




tive to the lethal effects of trihalomethanes  than females (see Table V-l).




Hill (1978) repo.rted single-dose LD50  values  for chloroform ranging from 119




to 490 mg/kg in three strains of mice.  Intraperitoneal LD50  values for




chloroform in mice range from 1,780 to 3,280 mg/kg (Kutob and Plaa 1962;




Klaassen and Plaa 1967b),  and an intraperitoneal LD50  of 1,490 mg/kg has been




reported in dogs (Klaassen and Plaa 1967a).









        2.  Other Effects  









        a.  Chloroform









        A number of studies have indicated that the organs most affected by




acute exposure to chloroform are the liver and kidneys.  Jones et al.  (1958)




administered single oral doses of chloroform (in olive oil) by gavage  to male




and female Swiss mice at levels from 7 to 1,100 mg/kg  (10 animals/dose).




Animals were sacrificed after 72 hours and livers were examined histolo-




gically.  The authors observed minimal midzonal fatty  infiltration at  35 mg/kg




                                      V-l

-------
TABLE V-l  Summary of Oral Lethality of Trihalomethanes

Compound
CHC13
CHBr3
CHBr2Cl
CHBrCl2
^50
Mouse3
Male Female
1,120 1,400
119-490C
1,400 1,550
800 1,200
450 900
(mg/ke)
Ratb
Male Female
908 1,117
2,000d
1,388 1,147
1,186 848
916 969

aBowman et al. (1978) .
bChu et al. (1980).
cHill (1978).
dTorkelson et al . (1976).
                          V-2

-------
and minimal cencral fatty changes at 70 mg/kg.  These fatty changes, in the




absence of other histopathological signs, are not considered to be adverse,




and the NOAEL for liver damage was 35 to 70 mg/kg.  However, the 3-day period




between dosing and observation may have been too long to detect transient




effects at the lower doses.  The authors observed moderate to severe hepatic




injury (massive fatty infiltration with necrosis) at doses of 140 to




350 mg/kg.  Based on this, 140 mg/kg was identified as the LOAEL.









        Hill (1978) investigated strain and sex differences in chloroform-




induced toxicity in mice.  Male mice of three strains (DBA/2J,  B6D2F1/J,  and




C57BL/6J) were given single oral doses of chloroform in oil.  No clear




difference in hepatotoxicity between strains was observed; centrilobular




necrosis occurre.d at doses greater than 250 mg/kg in all three strains.  In




contrast, there were strain-specific differences in renal toxicity.  Doses of




89 mg/kg caused glucosuria and/or proteinuria in half of the DBA/2J animals,




while doses of 119 and 163 mg/kg were required to produce these effects in




half the B6D2F1/J and C57BL/6J animals, respectively.  Plasma levels of




testosterone in resistant strains tended to be lower than levels in




susceptible strains.  The author conjectured that testosterone may act by




sensitizing the renal proximal convoluted tubules to chloroform through a




testosterone receptor mechanism.  This study identified a LOAEL of 89 mg/kg,




based on renal injury in male mice of sensitive strains.









        Reitz et al. (1980) administered single oral doses of 15, 60, or




240 mg CHCl3/kg to male B6C3F1 mice (two/group) and examined liver and kidney




tissues 48 hours later.  Severe diffuse renal necrosis occurred after a. single




dose of 240 mg/kg, and focal tubular regeneration occurred after single doses




of 60 or 240 mg/kg. .These effects were not observed at 15 mg/kg.  Liver




                                      V-3

-------
damage  (hepatocellular necrosis and swelling with inflammatory cell infiltra-




tion) was observed only at the highest dose.  This study identified a NOAEL of




15 nig/kg and a LOAEL of 60 mg/kg for renal injury in male mice, but too few




animals (two/dose) were used to allow a firm conclusion.









        Larson et al.  (1993) administered a single oral gavage dose of




chloroform in corn oil to male F344 rats (three to five/group) and to female




B6C3F1 mice (three to five/group).   Doses of 0, 34, 180, or 477 mg/kg were




administered to the rats, and 0, 34, 238, or 477 mg/kg was administered to the




mice.  The kidney and liver were examined histologically 1 day posttreatment.




As an indirect quantitative measure of necrosis, cell proliferation was




determined in the mid-dose mice and the mid- and high-dose rats by determining




the labeling index, a measure of the percentage of nuclei in S-phase.   Mild to




severe proximal tubular necrosis was observed in the kidney of the rats at all




treatment levels, in a dose-related manner.   There was no effect on BUN or




urinary protein or glucose at any dose.  The labeling index was increased




20-fold at 180 mg/kg.   Centrilobular necrosis was observed in the livers of




rats in all treatment groups with a dose-related severity; the severity in the




mid- and high-dose groups was characterized as slight to moderate.  The




labeling index was increased 10-fold in the livers of high-dose rats but was




unaffected in the mid-dose group.   In mice,  renal lesions were not observed,




but centrilobular hepatic lesions were observed with a dose-related severity




in mice treated with at least 238 mg/kg.  In a separate experiment, female




mice received an oral gavage dose of 350 mg/kg and were necropsied at varying




times after treatment.  The renal labeling index in these mice was increased




only 2-fold over controls, but the hepatic labeling index was increased




38-fold.  The study authors noted that the organ- and species-specific pattern




of cytotoxicity and resulting cell proliferation was the same as the pattern




                                     V-4

-------
of tumorigenicity at similar doses.  Because the kidney and liver effects




observed in the low-dose rats were limited to scattered necrotic tubules and




soatt-ered necrotic foci iu the liver, the NOAEL for tats in this study was




identified as 34 mg/kg; the LOAEL was 180 mg/kg, based on liver and kidney




histopathology.  In mice, the NOAEL was 34 mg/kg and the LOAEL was 238 mg/kg,




based on liver histopathology.









        Larson et al.  (19.94) measured cell proliferation in the liver




following administration of chloroform by gavage in corn oil or in drinking




water.  Female B6C3F1 mice were administered doses of 0, 3, 10, 34, 90, 238,




or 477 mg/kg for 4 days or 5 days/week for 3 weeks, or provided drinking water




containing 0,  60,  200, 400, 900, or 1,800 ppm for 4 days or 3 weeks.  The




average daily doses in the 4-day drinking water study were 0,  16, 26, 54, 81,




or 105 mg/kg/day and the average doses in the 3-week study were 0, 16, 43, 83,




184,  or 329 mg/kg/day.  Mice administered 34 or 90 mg/kg/day by oil gavage for




4 days exhibited mild degenerative changes in centrilobular hepatocytes;  more




severe changes were observed at 3 weeks.  Centrilobular necrosis was observed




at the higher doses (238 or 477 mg/kg/day in oil),  with increased severity




following the longer treatment period.  The hepatic labeling index was




significantly (p <  0.05)  increased at both dosing durations following gavage




dosing with 238 or 477 mg/kg/day, and following 3 weeks of dosing with




90 mg/kg/day.   No histopathological changes and no increase in the labeling




index was observed in any mice receiving chloroform in drinking water for




either duration.  Thus, cytolethality and cell proliferation were observed-




following gavage dosing in oil, but not following drinking water administra-




tion.  The study authors noted that sustained cell proliferation was observed




only under the conditions that induce tumors (oil gavage dosing; see Section




V.E.I.).  Using the pharmacokinetic model of Corley et al. (1990), they




                                      V-5

-------
predicted that the bolus dosing in oil results in a higher rate of chloroform




metabolism in the liver than would be seen following drinking water




adminis tration.









        Culliford and Hewitt (1957) (reviewed in U.S. EPA 1985a) reported sex-




and strain-dependent differences in mice exposed to chloroform by inhalation.




Adult male mice of two strains (CBA and WH) developed extensive necrosis of




the renal tubules after exposure to low concentrations of chloroform vapor




(7 to 10 mg/L for 2 hours),  while adult females showed no renal damage after




equivalent exposure.  Assuming an inhalation absorption fraction of 0.63




(Lehmann and Hasegawa 1910), a body weight of 0.03 kg, and an inhalation rate




of 0.052 m3/day  (U.S.  EPA 1986),  this concentration corresponds to an absorbed




dose range of 63.7 to 910 mg/kg.  Adult females became fully susceptible to




renal necrosis after treatment with androgens,  and the susceptibility of males




was greatly reduced by treatment with estrogens.   Castration removed the




susceptibility of the males  of one strain, but did not completely remove it in




another.  The residual susceptibility of castrates was abolished by




adrenalectomy.   Male mice under 11 days old were not susceptible to renal




necrosis, even after massive doses of androgen.  Between 11 and 30 days, they




were susceptible if given androgen.  Thereafter,  they became spontaneously




susceptible.   In contrast to thest- sex hormone-dependent effects on the




kidney, liver damage occurred in nearly all exposed mice and was not




correlated with sex hormone  status.









        Klaassen and Plaa (1967a) investigated the effects of ethanol pre-




treatment on chloroform-induced liver damage in dogs.  Groups of four to six




mongrel dogs (7 to 14 kg) were administered either a-single dose of 4 g/kg of




ethanol by gavage or an equicaloric dose of dextrose solution.  Twenty-four




                                      V-6

-------
hours later, the animals received an intraperitoneal injection of chloroform




(300 mg/kg) or corn oil; serum glutamic pyruvic transaminase (SGPT) activity




was measured 24 hours after that.  In control animals, the average SGPT level




was 29 units.  The values increased slightly following exposure to either




ethanol alone (42 units) or chloroform alone (44 units).  When chloroform was




given to animals pretreated with ethanol, the values increased dramatically




(1,760 units).   The authors concluded that ethanol pretreatment markedly




increased the potential fo.r chloroform hepatotoxicity.  These authors obtained




similar results in mice (Klaassen and Plaa 1966).  The mechanism of this




potentiation is not known,  but may be related to induction of enzymes by




ethanol that result in increased metabolism of chloroform.









        Munson e.t al.  (1982) administered chloroform (aqueous) by gavage to




male and female CD-I mice.   Dosage levels of 0, 50, 125, or 250 mg/kg/day were




administered to groups of 14 to 24 animals for either 14 or 90 days.




Parameters measured included body weights, organ weights, hematology, clinical




chemistry, hepatic microsomal enzymes,  and cell-mediated and humoral immunity.




In the 14-day study, dose-related increases in liver weight (both absolute and




relative to total body weight) were observed in mid- and high-dose males.




Increased relative liver weights (not dose related) were observed in all




female groups.   Increases of 2- to 36-fold (mean 8.4-fold) were reported in




SGPT values in high-dose males and females and in serum glutamic oxaloacetic




transferase (SCOT) levels in high-dose females.  The authors observed a




decrease in spleen antibody-forming cells at all doses in males and females




(p < 0.05),  but no effects  on hemagglutination titers or alterations in cell-




mediated immunity.  This study identified a NOAEL of 125 mg/kg/day and a LOAEL




of 250 mg/kg/day, based on elevated serum enzyme levels.  In the 90-day study,




the authors viewed differences or improvements in a number of clinical




                                     V-7

-------
chemistry parameters compared to the 14-day study as indicative of recovery




from or induction of tolerance to chloroform during the longer experiment.









        Condie et al. (1983) reported dose-dependent increases in the




incidence and severity of centrilobular cytoplasmic pallor, mitotic figures,




and focal inflammation of the liver in mice given doses of 0, 37, 74, or




148 mg/kg/day chloroform in oil for 14 days.  Body weights were decreased in




animals at the highest dojse.  Decreased renal uptake of PAH, accompanied by an




increased incidence of microscopic changes,  were observed in the two highest




dose groups.  Blood urea nitrogen and SGPT were increased in the high-dose




group.








        Chu et a.l.  (1982a) exposed male Sprague-Dawley rats to 0, 5,  50, or




500 ppm chloroform in drinking water for 28 days (ten animals/group).  Based




on measured water intake, these doses corresponded to 0, 0.74, 7.4, or




62.9 mg/kg/day.   At the end of the experiment, animals were sacrificed and the




authors measured serum biochemical and hematological parameters and hepatic




microsomal and soluble enzyme activities and performed gross histopathological




examinations.   No changes were observed in any of these parameters, except a




decreased number of neutrophils in the high-dose rats (1.00.27 versus




0.520.3xl03 cells/jiL).   The authors stated that they observed no treatment-




related histopathological changes, but they provided no data or details.  This




study identified a NOAEL of 7.4 mg/kg/day.  The significance of the decrease




in neutrophil count in the high-dose rats is not certain.









        Plummer et al. (1990) compared the liver effects of exposing male




Wistar rats (12/group) for 4 weeks to 50 ppm in air for 24 hours/day,




7 days/week (continuous profile) or 275 ppm for 6 hours/day, 5 days/week




                                      V-8

-------
(fluctuating profile).  The total exposure was about 31,600 ppm-hour in each




group.  Minor liver pathology was observed in both exposed groups, but quali-




tative and quantitative assessment revealed more injury in the continuously




exposed group.  The continuous exposure group had microvesicular fatty change,




largely in zone 3 hepatocytes (cells furthest from the terminal" afferent




vessel),  while only scattered hepatocytes containing small fat droplets were




observed in the fluctuating profile group.  Necrosis scores were also higher




in the continuous profile group.  The difference in toxicity was attributed to




saturation of chloroform metabolism in the fluctuating profile group.  This




hypothesis was supported by data regarding the kinetics of chloroform uptake




(two rats/dose) that suggested saturation of chloroform metabolism at levels




above 100 ppm.









        Table V-2 summarizes the short-term ef-fects of chlotoform.









        b.  Brominated Trihalomethanes









        Bowman et al. (1978) studied the acute oral toxicity of bromoform,




bromodichloromethane, and dibromochloromethane in mice.  Groups of ten male




(30 to 35 g) and ten female (25 to 30 g) ICR Swiss mice were treated with




doses ranging from 500 to 4,000 mg/kg.  Compounds were solubilized in




emulphor: alcohol:saline (1:1:8) and administered by gavage.  Ataxia,




sedation, and anesthesia occurred with bromodichloromethane or dibromochloro-




methane at 500 mg/kg, but a 1,000-mg/kg dose of bromoform was required to




produce these effects.  These endpoints are insufficiently sensitive to




identify useful acute NOAEL or LOAEL values.
                                      V-9

-------
    TABLE  V-2   Summary  of  Short-Term Health Effects  Data on Chloroform

Reference Species
Jones et al. House
(1958)
Hill (1978) Mouse
Reitz et al. Mouse
(1980)
Larson et al. Rat
(1993)
Mouse
Route
Gavage
(oil)
Gavage
(oil)
Gavage
Gavage
(oil)
Gavage
(oil)
dose
Sex Duration (mg/kg/day)
M,F One dose 35-70
140-350
M One dose 89
250
M One dose 15
60
240
M One dose 34
180
F One dose 34
238
Results
Fatty liver (NOAEL)
Fatty liver, hepatic
necrosis
Renal damage (LOAEL)
Liver damage
NOAEL
Focal renal
necrosis (LOAEL)
Diffuse renal
necrosis, liver
damage
Trace liver and kidney
necrosis (NOAEL)
Liver and kidney necrosis
(LOAEL)
NOAEL
Liver necrosis (LOAEL)
Klaassen and    Dog
Plaa (1967a)
        Intraperi-   M,F   One dose
        toneal
        injection
Munson et al.   Mouse    Gavage     M,F   14 days
(1982)                 (aqueous)
Condie et al.   Mouse
(1983)
        Gavage
        (oil)
14 days
Chu et  al.
(1982a)
Rat     Drinking    M,F   28 days
        water
300          Slight increase in
              SCOT levels;
              potential ion
              by ethanol
              pretreatment

125          Increased liver
              weight (NOAEL)
250          Increased serum
              enzymes (LOAEL)

37           Decreased PAH uptake,
              moderate renal and
              hepatic histo-
              pathotogy (LOAEL)

0.74-7.4      NOAEL (histologic,
              hematologic,
              biochemical
              evaluation)
63           Decreased
              neutrophils
                                         V-10

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        NTP (1987) administered single oral doses of bromodichloromechane




(150, 300, 600, 1,250, or 2,500 mg/kg) by gavage in corn oil to F344/N rats




and S6C3F1 mice (five/sex/'dose).   Animals were observed for 14 days, and a




necropsy was performed on at least one male and one female in each dose group.




All animals dosed with 1,250 or 2,500 mg/kg died before the end of the study.




At 600 mg/kg,  deaths occurred in two of five male rats, one of five female




rats, five of five male mice, and two of five female mice.  Clinical signs




included lethargy and labored breathing.  At necropsy, the liver from animals




dosed with 1,250 or 2,500 mg/kg appeared pale.  No dose-related effects were




seen on body-weight gain in animals that survived.  This study is not




appropriate for the identification of a reliable NOAEL or LOAEL because no




histopathological or biochemical endpoints were monitored.









        In a preliminary range-finding study by NTP (1985), F344/N rats and




B6C3F1 mice (five/sex/dose) were administered single doses of 160, 310, 630,




1,250, or 2,500 mg/kg dibromochloromethane in corn oil by gavage.  All rats




that received 2,500 mg/kg were dead by day 3.  At 1,250 mg/kg, only one of




five male rats survived, while four of five females survived.  A dose of




310 mg/kg caused no deaths but produced lethargy in all animals.  All mice




receiving 2,500 mg/kg, and all male mice and four of five female mice




receiving 1,250 mg/kg, died betwem days 2 and 8.  No females died at




630 mg/kg or lower, while three of five males died at 630 mg/kg and one of




five males died at 310 mg/kg.  Livers with discolored foci and kidneys with




dark red or pale medullae were seen more frequently in dosed animals than in




control animals.  Single oral doses of 310 mg/kg of dibromochloromethane were




hazardous in this study, but mortality is an insufficiently sensitive endpoint




for the identification of a useful NOAEL or LOAEL.
                                     V-ll

-------
        NTP  (1989a) investigated the acute oral toxicity of bromoform in




F344/N rats  and B6C3F1 mice.  Groups of five males and five females of each




species were administered a single oral dose of bromoforra (by gavage,  in corn




oil) at dose levels of 125, 250, 500, 1,000, or 2,000 mg/kg.  There were no




controls.  In rats, mortality was 10/10 at 2,000 mg/kg, 6/10 at 1,000 mg/kg,




and 0/10 at  500 mg/kg or lower.  In mice, mortality was 0/10 at 2,000 mg/kg,




6/10 at 1,000 mg/kg, 1/10 at 500 mg/kg, and 0/10 at 250 mg/kg or lower.   The




main clinical sign observed was shallow breathing.  This study is also not




appropriate  for identification of a reliable NOAEL or LOAEL because no




histopathological or biochemical endpoints were monitored.









        Munson et al.  (1982) administered bromodichloromethane, dibromochloro-




methane,  and bromoform (aqueous) by gavage to CD-I male and female mice  (14 to




25/group) for 14 days at levels of 0, 50, 125, or 250 mg/kg/day.  Parameters




observed included body and organ weights, hematology, serum enzyme levels




(SCOT, SGPT), and humoral and cell-mediated immune system functions.  At the




high dose (250 mg/kg/day) of all three compounds,  body weights were generally




decreased and SCOT and SGPT levels were clearly increased (p < 0.05).  These




effects were not significant at the mid or low doses.  Bromodichloromethane




and dibromochloromethane appeared to affect the humoral immune system, as




judged by decreased antibody-forming (ABF) cells in serum and by decreased




hemagglutination titers.  These changes were clearly significant (p <  0.05) at




the high dose in both males and females, and decreased ABF cells were also




noted at the mid dose (125 mg/kg/day) in females.   The authors judged that  the




humoral immune system was not significantly affected by bromoform (although a




decrease in ABF cells was reported for high-dose males).  On the basis of




decreased immune function in females, this study identified a NOAEL of




50 mg/kg/day and a LOAEL of 125 mg/kg/day for bromodichloromethane and




                                     V-12

-------
dibromochloromethane.   For bromoform, this study identified a NOAEL of




125 mg/kg/day and a LOAEL of 250 mg/kg/day, based on elevated serum enzymes.









        Condie et al.  (1983) investigated the renal and hepatic toxicity of




the brominated trihalomethanes.   Groups of 8 to 16 male CD-I mice were admin-




istered the chemicals by gavage, using corn oil as the vehicle, for 14 days.




Doses tested were 0, 37, 74 or 148 mg/kg/day for bromodichloromethane; 0, 37,




74, or 147 mg/kg/day for _dibromochloromethane;  and 0, 72, 145, or 289 mg/kg/




day for bromoform.  Biochemical evidence of liver and kidney damage (decreased




PAH uptake by kidney slices and elevated SGPT release from liver) was consis-




tently observed at the high dose but not at the mid or low doses (Table V-3).




Similarly, histological examinations revealed no consistent or important




changes at the low or mid doses, with slight to moderate liver and kidney




injury at the high dose (Table V-4).   On this basis, this study identified




NOAEL values of 74,  74,  and 145 mg/kg/day and LOAEL values of 148,  147 and




289 mg/kg/day for bromodichloromethane, dibromochloromethane, and bromoform,




respectively.









        NTP (1987) administered doses of 0, 38, 75, 150, 300, or 600 mg/kg/day




of bromodichloromethane in corn oil by gavage to F344/N rats (five/sex/dose)




for 14 days.  A necropsy was performed on all animals.  One of five female




rats that received 38 mg/kg and one of five female rats that received




600 mg/kg died before the end of the study.  All rats receiving 600 mg/kg were




hyperactive after dosing and either lost weight or gained no weight during the




study.  Final mean body weights were not significantly affected in groups




given 38, 75 or 150 mg/kg/day.  In the 300-mg/kg groups, body weights in males




and females decreased by 21% and 7%,  respectively.  At 600 mg/kg, body weights




decreased by 44% and 22%, respectively.  Renal medullae were reddened in five




                                     V-13

-------
              TABLE  V-3   Effects  of Brominated Trihalomethanes  in
                       Mice Dosed by Gavage for 14 Days

Chemical
CHBrCl2



CHBr2Cl



CHBr3



Dose
(mg/kg)
0
37
74
148
0
37
74
147
0
72
145
289
Mean
Body Weight
Gain (g)
2
0
1
-0
-0
0
0
-1
0
0
1
2
.0
.7
.2
.3
.1
.1
.2
.3
.8
.8
.1
.7
PAH Uptake
(slice/ BUN
medium ratio) (mg%)
18.
18.
13.
8.
14.
12.
14.
10.
19.
18.
18.
13.
3
1
4a
8a
3
5
0
4a
1
5
1
3a
24
20
20
24
20
24
26
25
22
22
23
25
.6
.7a
.5a
.0
.1
.6
.2
.2
.9
.4
.9
.0
Creatinine SGPT
(mg%) (Units/L)
0
0
0
0
0
0
0
0
0
0
0
0
.44
.41
.47
.51
.49
.49
.55
.48
.42
.46
.38
.42
20
26
21
51a
16
16
21
35a
21
21
17
30a

ap <  0.05  compared to control.

Adapted from Condie et al. (1983).
                                     V-14

-------
                  Table V-4  Incidence and Severity of Microscopic Changes  in Kidney and Liver
                            of Mice  Dosed with Brominated Trihalomethanes for  14 Days

Kidney
Dose
Compound (mg/kg/day)
Number
of
Animals
Examined
Intra-
tubular
Mineral-
ization
Epithe-
lial
Hyper-
plasia
Cyto-'
megaly
Mesan-
gial
Hyper-
trophy
Mesan-
gial
Neph-
rosis
Centri-
lobular
Pallor
Liver
Mitotic
Figures
Focal
Inflam-
mation
Cyto-
plasmic
Vacuo-
lation
Severity of Chanae3

Corn oil
(vehicle)
CHBrCl2

CHBr2Cl
CHBr3

0

37
74
148
37
74
147
72
145
289

16

9
10
10
5
10
10
5
10
10
ABCD
0000

0000
0100
2111
0000
0000
0000
0000
0000
1000
ABCD
2000

1000
0100
1521
0000
0000
2110
0000
1000
3200
ABCD
0000

0000
1000
5100
0000
0000
0000
0000
0000
0000
ABCD
0000

0000
0000
0000
4000
7000
4300
0000
2000
1000
ABCD
0000

0000
0000
0000
0000
0000-
0000
0000
4100
6100
ABCD
0000

2000
6210
5220
0000
0000
0000
0000
6000
5111
ABCD
0000

0000
1320
0100
0000
1010
3100
0000
1000
1010
ABCD
1000

2000
4000
5110
0000
1000
1000
0000
1000
3300
ABCD
1000

0000
0000
1000
2100
4000
3500
2000
0000
0100

Categories of severity ratings:
     A ~ minimal
     B = slight
     C = moderate
     D = moderately severe/severe.
 Numbers of animals in each category are listed in columns.

Adapted from Condie et al. (1983).

-------
of five male rats in the 600-mg/kg group, one of  five female vehicle controls.




one of five females in the 38-mg/kg group, and one of five females in the




600-mg/kg group.  Based on body weight; gain, this study identified a NOAEL of




150 mg/kg/day and a LOAEL of 300 mg/kg/day in rats.









        In a similar experiment, NTP  (1987) administered doses of 0, 19, 38,




75, 150,  or 300 mg/kg/day broraodichloromethane in corn oil by gavage to




B6C3F1 mice (five/sex/doae) for 14 days.  All male mice that received 150 or




300 ntg/kg bromodichloromethane died before the end of the study.  Clinical




signs included lethargy, dehydration, and hunched posture.  The final mean




body weights of the mice that survived were not significantly different from




the controls.   The renal medullae were reddened in four of five males




receiving 150 mg/kg, five of five males receiving 300 mg/kg, and one of five




females receiving 150 mg/kg.  Based on behavior, appearance, gross necropsy,




and mortality,  this study identified  a NOAEL of 75 mg/kg/day in mice.









        In a 14-day study by NTP (1985), groups of F344/N rats (five/sex/dose)




were administered 0, 60, 125, 250, 500, or 1,000 mg/kg/day dibromochlorome-




thane in corn oil by gavage.  Animals were observed twice daily for mortality




and were  weighed once per week.  Necropsies were performed on all animals.




All of the rats of each sex that rt-ceived 1,000 mg/kg dibromochloromethane and




all of the female rats that received  500 mg/kg were dead by day 6.  No deaths




occurred at 250 mg/kg/day.  Lethargy, ataxia, and labored breathing were




observed in rats of each sex that received 500 or 1,000 'mg/kg.  Mottled livers




and reddened,  darkened renal medullae were seen at gross necropsy in male and




female rats administered 500 or 1,000 mg/kg, but no gross lesions were obser-




ved in animals dosed with 250 mg/kg/day or less.  Based on behavior, gross




pathology, and mortality, this study  identified a NOAEL of 250 mg/kg/day.




                                      V-16

-------
        In a parallel study in B6C3F1 mice (NTP 1985), groups of five animals/




sex/dose were administered doses of 0, 30, 60, 125, 250,  or 500 mg/kg/day of




dibromochloromethane in corn oil by gavage for 14 dzys.   Deaths of four of




five males and three of five females that received 500 mg/kg were considered




to be compound related.  Clinical signs at this dose included lethargy,




ataxia, and labored breathing.  Mottled livers and reddened renal medullae




were seen in male and female mice administered 500 mg/kg; these changes were




considered to be compound related.   Stomach lesions (white papillomatous




nodules) were seen in male mice that received 125, 250,  or 500 mg/kg and in




female mice that received 250 or 500 mg/kg.  Based on gross lesions,  this




study identified a NOAEL of 60 mg/kg/day and a LOAEL of 125 mg/kg/day in mice.









        NTP (1989a) investigated the subacute oral toxicity of bromoform in




F344/N rats and B6C3F1 mice.  Groups of male and female rats (five/sex/dose)




and female mice (five/dose) were administered doses of 0, 100, 200, 400, 600,




or 800 mg/kg/day of bromoform in corn oil by gavage for 14 days.  Male mice




were administered 0, 50, 100,  200,  400, or 600 mg/kg.  All rats that received




600 or 800 mg/kg/day and one male rat that received 400 mg/kg/day died before




the end of the study.  These rats exhibited lethargy, labored breathing, and




ataxia.  Rats receiving 400 mg/kg/day had final body weights 14% less than




controls.   In mice, no effects were detected at 200 mg/kg/day, but ataxia and




lethargy were noted at 600 mg/kg/day.  One male died at 600 mg/kg/day, and one




female died at 800 mg/kg/day.   Raised stomach nodules were observed in males




and females at doses of 400 mg/kg/day or higher.  Based on body weight and




mortality in rats and stomach nodules in mice, this study identified a NOAEL




of 200 mg/kg/day and a LOAEL of 400 mg/kg/day.
                                     V-17

-------
        Chu et al. (1982a) administered bromodichloromethane,  dibromochloro-




methane, or bromoform to male Sprague-Dawley rats (150 to 200 g) in drinking




water for 28 days at dose levels of 0, 5, 50, or 500 ppm (10 animals per




group).  Based on recorded fluid intake, these levels corresponded to doses of




0.8, 8.6, or 68 mg/kg/day, as calculated by the authors.  The authors observed




no effects on growth rate or food consumption from any of the compounds and no




signs of toxicity throughout the exposure.  No dose-related biochemical or




histological changes were, detected (no data provided).  This study identified




a NOAEL of 68 mg/kg/day for each of the brominated trichloromethanes,  but the




reported data were too limited to allow a. firm conclusion.









        Aida et al. (1992a) investigated the effects of administering




bromoform, dibrojnochloromethane,  or bromodichloromethane to groups of seven




male and seven female Slc:Wistar rats for 1 month.  The test material was




microencapsulated and mixed with powdered feed; placebo granules were used for




the control groups.  Clinical effects, body weight, food consumption,  hemato-




logical parameters, serum chemistry, and histopathology of all major organs




were determined.









        Bromodichloromethane was administered at dietary levels of 0%, 0.024%,




0.072%, or 0.215% of diet for males, and 0%, 0.024%, 0.076%, or 0.227% of diet




for females.  Based on the mean food intakes, the study authors reported the




mean compound intakes for the 1-month period as 0, 20.6, 61.7, or 189.0 mg/kg/




day for males and 0, 21.1, 65.8,  or 203.8 mg/kg/day for females.  Body weight




gain was significantly (p < 0.01) decreased in the high-dose groups.  The




high-dose animals also exhibited slight piloerection and emaciation.  Relative




liver weight was increased only in high-dose females.  Significant, dose-




related biochemical findings in the low-dose groups were limited to decreased




                                     V-18

-------
Laccate dehydrogenase (LDH) levels, but the biological significance of this




effect is unclear.  As shown in Table V-5, other significant, dose-related




changes included decreased serum triglycerides (high-dose groups), decreased




serum cholinesterase activity (high-dose males and mid- and high-dose




females), and increased total cholesterol (mid- and high-dose males).  The




cholesterol levels were within normal ranges at all doses.  Chemical-related




lesions were limited to the liver and were rated as very slight or slight.




Histopathology was mostly.-..confined to the high-dose groups, and the incidence




was lower than for the other two brominated trihalomethanes (Table V-6).  The




vacuolization observed in mid-dose females,  and in single low-dose males was




not considered an adverse effect.  Other observed effects included swelling of




hepatocytes, single cell necrosis, hepatic cord irregularity, and bile duct




proliferation.  JExcept for very slight to slight changes in individual low-




dose males, these lesions were observed only in high-dose males and females.




There was no effect on any hematological parameter.  Based on the histopath-




ology observed in high-dose males and females, the LOAEL identified in this




study for bromodichloromethane in rats was 189 mg/kg/day in males and




204 mg/kg/day in females; the NOAEL was 62 (males) and 66 (females) mg/kg/day.









        Dibromochloromethane was administered at dietary levels of 0%, 0.020%.




0.062%, or 0.185% of diet for males, and 0%,  0.038%, 0.113%, or 0.338% of diet




for females.  Based on.the mean food intakes, the study authors reported the




doses as 0, 18.3, 56.2,  or 173.3 mg/kg/day for males and 0, 34.0, 101.1, or




332.5 mg/kg/day for females.  Body weight gain was significantly  (p < 0.01)




reduced in high-dose females.  High-dose females also exhibited slight




piloerection and emaciation.  Dose-related increases in relative  liver weight




were observed in males at the high dose and females of all dosing groups, and




relative kidney weight increased in the high-dose females.  Significant




                                     V-19

-------
decreases in alkaline phosphatase  (AP) and LDH were observed, but the biolo-




gical significance of these changes is unclear (Table V-5).  Significant.




dcss-related changes in serum biochemistry included reduced nonesterified




fatty acids (FAA) in high-dose males, reduced total serum  triglycerides




(T-GLY) in high-dose males and females, and increased cholesterol in mid- and




high-dose males and in females at all dosing levels.  However, the cholesterol




levels were within normal ranges at all dose levels.  Serum cholinesterase




activity was also significantly decreased in high-dose males and mid- and




high-dose females.  Liver cell vacuolization was noted at  a similar incidence




in the controls and all dosing groups, but dose-related increases in severity




were observed in mid- and high-dose males and females.  The incidence and




severity of the effects at the low dose were similar to those observed in the




control groups, and were not considered adverse.  Swelling and single cell




necrosis were also observed, largely in the high-dose groups (Table V-6).




There was no effect on any hematological parameter.  Based on the




histopathology and serum biochemistry results,  NOAELs of 56 (males) and 101




(females) mg/kg/day and LOAELs of 173 (males) and 332 (females) mg/kg/day were




identified for dibromochloromethane in rats.









        Bromoform was administered at dietary levels of 0%, 0.068%, 0.204%, or




0.612% of diet for males, and 0%, 0.072%, 0.217%, or 0.651% of diet for




females.  Based on the mean food intakes, the study authors reported the mean




compound intakes as 0, 61.9, 187.2, or 617.9 mg/kg/day for males and 0, 56.4,




207.5, or 728.3 mg/kg/day for females.  Body weight gain was significantly.




(p < 0.01)  reduced in high-dose males, and high-dose animals of both sexes




exhibited slight piloerection and emaciation.  Relative liver weight was




significantly (p < 0.05)  increased in mid- and high-dose males and females.




Serum glucose, T-GLY, blood urea nitrogen (BUN), and cholinesterase levels




                                     V-20

-------
                          Table V-5  Serum Biochemical Levels8 in Rats  Fed Brominated
                                         Trihalomethanes for One Month

Dietary
Chemical Sex Level (X)
CHBrCl2 M 0
0.024
0.072
0.215
F 0
0.024
0.076
0.227
CHBr2Cl. M 0
0.020
0.062
0.185
F 0
0.038
0.113
0.338
CHBr3 M 0
0.068
0.204
0.612
F 0
0.072
0.217
0.651
Blood
Urea
Nitrogen
162
151
142
141
151
152
153
152
182
171
182
172
191
191
181
201
172
193
182
172
183
153
132*
123**
Tri-
glycerides
17051
14213
12920
49 18**
9225
8924
6012
386**
12924
12416
11216
6520**
6314
6210
6010
586*
9926
8425
7416
314"
7514
62 14
454"
364**
Cholesterol
525
583
653**
607*
9260
678
692
625
582
594
684**
746**
595
677*
74 5**
746**
464
525
576**
587**
628
673
766**
716**
Nonesterif led
Fatty
Acids
0.370.12
0.370.06
0.460.15
0.250.09
0.250'.04
0.290.07
0.270.05
0,220.07
0.52 0.14
0.500.04
0.400.09
0.310.06*
0.540.17
0.500.13
0.560.11
0.390.15
0.340.07
0.300.05
0.330.09
0.290.07
0.430.07
0.400.10
0.320.08
0.360.10
Serum
Glucose Cholinesterase
16517
15915
15726
1389*
11436
12610
1306
1195
1199
1137
1166
1176
8512
8212
769
8712
15114
13610*
13910
1176**
12825
11413
10614*
1059*
547208
46342
48979
328131*
1449257
1300172
939lll"
498118**
600108
62473
54255
39976"
145967
132073
100431**
748124"
53797
46687
37850
30122**
1306161
1029105**
72360**
62483**

aResults from seven animals/sex/dose.   Cholinesterase activity expressed as mU/mL; other parameters as mg/clL.
*p less than 0.05 compared to control
**p  less than 0.01  compared to control

Adapted from Aida et al.  (1992a).

-------
                            Table V-6   Incidence and Severity of Microscopic Changes  in  the  Liver

                                 of Rats Dosed with Brominated Trihaloinethanes  for One Month
ro
to

Hepatocyte
Dietary Vacuoli- Hepatocyte Focal
Compound Sex Level (%) zation Swelling Necrosis
Single
Cell
Necrosis
Hepatic Bile Duct
Cord Prolifer.1-
Irregularity tion
Severity of Change3

CHBrCl2 M 0.024
0.072
0.215
F 0.024
0.076
0.227
CHBr2Cl M 0
0.020
0.062
0.185
F 0
0.038
0.113
0.338
CHBr3 M 0.068
0.204
0.612
F 0
0.072
0.217
0.651
ABCD
0100
0000
2400
0000
3200
2300
3000
4100
4300
0061
6000
4100
1600
0052
1000
0430
0025
2000
2410
0016
0016
ABCD
0100
0000
2300
0000
0000
1100
0000
0000
0000
0006
0000
0000
2000
0700
0000
2000
2210
0000
2100
4300
0700
ABCD
0000
0000
0001
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
ABCD
pooo
0000
4100
.0000
' 0000
0000
0000
1000
0000
3300
0000
0000
0000
4000
0000
2000
1000
0000
0000
0000
0000
ABCD
0000
0000
3000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
4100
6100
0000
0000
0000
0000
ABCD
0100
0000
1100
0000
0000
1100
0000
0000
0000
0000
0000
0000
0000
0000
0000
6000
511.1
0000
0000
0000
0000

         aCat.egories  of  severity  ratings:

              A = very slight change   B = slight change    C = moderate change   D = remarkable change

          Numbers of  animals in each category are listed in cplumns; seven animals were tested per dose.


          Adapted from Aida et al.  (1992a).

-------
were decreased  in males and females in a dose-related manner, while




phospholipids (males only) and cholesterol were increased in the mid- and




high-dose groups (Table V-5).  The only change of clear biological signifi-




cance at the low dose was a decrease in cholinesterase activity in females.




LDH and AP activity and creatinine levels were decreased at the low dose in




females, but the physiological significance of these effects is unclear.




There was no effect on any hematological parameter. .Chemical-related lesions




were limited to the liver^ (Table V-6).  Discoloration was observed in all




males and females in the high-dose group.  The incidence and severity of liver




cell vacuolization and swelling were dose-related.  Females were more




sensitive, but  the histopathology observed in all low-dose females was not




considered an adverse effect.  Based on the histopathology and serum chemistry




changes in the mid-dose animals, NOAELs of 62 mg/kg/day for males and




56 mg/kg/day for females, and LOAELs of 187 and 208 mg/kg/day for males and




females, respectively, were identified for bromoform in rats.









        Balster and Borzelleca (1982) investigated the effects of trihalo-




methanes on behavior in mice.  Groups of six to eight adult male ICR mice  (19




to 24 g) were dosed (aqueous) by gavage for 14 or 90 days with bromodichloro-




methane (1.2 or 11.6 mg/kg/day), dibromochloromethane (1.0 or 10.0 mg/kg/day),




or bromoform (0.9 or 9.2 mg/kg/day). .These treatments caused no significant




effects in a. number of behavioral tests (swimming endurance, screen test,




hole-board).  Dosing with these compounds for 30 days at 100 mg/kg/day also




produced no effect on passive avoidance learning.  Animals dosed for 60 days




with 100 mg/kg/day of bromodichloromethane or bromoform exhibited decreased




response rates  (p < 0.05) in an operant behavior test; all three brominated




trihalomethanes produced this decrease after dosing for 60 days at




400 mg/kg/day.  The greatest effects were observed early in the regimen, with




                                     V-23

-------
no evidence of progressive deterioration.  In fact, partial tolerance was




observed in all cases.  Based on behavioral effects, this study identified a




subacute (30-day) NOAEL of 100 mg/kg/day for all three brominated




trihalomethanes.









        Tables V-7, V-8, and V-9 summarize the short-term effects of the




brominated trihalomethanes.









        B.   Longer-Term Exposure









        1.   Chloroform









        Chu et aJL. (1982b) administered chloroform in drinking water




to weanling Sprague-Dawley rats (94 to 100 g) for 90 days.  Animals




(20/sex/group) received 0, 5,  50,  500, or 2,500 ppm chloroform, corresponding




to approximately 0, 0.7, 6, 50, or 180 mg/kg/day.  After 90 days, one-half of




each group was sacrificed; the remaining rats were observed for an additional




90 days.  At the high dose, increased mortality, decreased growth rate, and




decreased food intake were reported.   Mild to moderate liver and thyroid




lesions were observed in the prerecovery groups, but these were not signifi-




cantly different from controls except the thyroid lesions at the high dose in




males.  After the 90-day recovery period, these changes became very mild and




did not differ significantly from controls.  No significant changes were




observed in serum biochemical profiles or hematological parameters.  This




study identified a NOAEL of 50 mg/kg/day.









        Bull et al. (1986) studied the effect of vehicle on the hepatotoxicity




of chloroform in mice.  Chloroform was administered by gavage to 80 male




                                     V-24

-------
TABLE V-7  Summary of Short-term Health Effects Data
               on  Bromodichloromethane

Reference
Bowman et a I.
(1978)
NTP
(1987)

Munson et al.
(1982)



Condie et al.
(1983)




NTP
(1987)






Chu et al.
Aida et al.
(1992a)


Aida et al.
(1992a)


Balster and
Borzelleca
(1982)

Species
Mouse

Rat
Mouse

Mouse




Mouse





Rat




Mouse


Rat
Rat



Rat



Mouse



Route Sex
Gavage M,F
(aqueous)
Gavage M.F
(oil)

Gavage M,F
(aqueous)
---.


Gavage M
(oil)




Gavage M,F
(oil)



Gavage M,F


Drinking M
Diet M



Diet F



Gavage M
(aqueous)


Duration
One dose

One dose


. ". days




U days





14 days




14 days


28 days
1 month



1 month



30 days
60 days

90 days
Dose
(mg/kg/day)
500

150-300
600
1,250-2,500
50
125

250

37-74
148




38-150
300

600

19-75
150-300

0.8-68
21-62
189


21-66
204


100
100-400

12
Results
Ataxia, sedation

No deaths
Some deaths
Complete mortality
NOAEL
Decreased immune
function
Increased serum
enzymes
NOAEL
Decreased PAH
uptake, moderate
liver and kidney
histopathology
(LOAEL)
NOAEL
Decreased body
weight gain (LOAEL)
Hyperactivity,
renal pathology
NOAEL
Mortality, renal
histopathology
NOAEL
NOAEL
Liver
histopathology
(LOAEL)
NOAEL
Liver
histopathology
(LOAEL)
NOAEL
Decreased operant
response
NOAEL

                        V-25

-------
TABLE V-8  Sununary of Short-term Health Effects  Data
               on Dibromochloromethane

Reference
Bowman et al.
(1978)
NTP (1985)


NTP (1985)

Munson
et al.
(198Z)


Condi e
et. al.
(1983)



NTP (1985)









Chu et al.
(1982a)
A Ida et al.



Aida et al.
(1992a)




Balster and
Borzelleca
(1982)

Species Route
Mouse Gavage
(aqueous)
Rat Gavage
(oil)

Mouse Gavage
(oil)
Mouse Gavage
(aqueous)



Mouse Gavage
(oil)




Rat Gavage
(oil)


Mouse Gavage
(oil)




Rat Drinking
water
Rat Diet



Rat Diet





Mouse Gavage
(aqueous)


Sex Duration
M,F One dose

M,F One dose


M,F One dose

' M,F U days




M 14 days





M,F 14 days



M,F 14 days





M 28 days

M 1 month



F 1 month





M 30-60 days
60 days

90 days
Dose
(mg/kg/day)
500

160
310
630-2.500
160
310
50
125

250

37-74
147




60-250
500-1,000


30-60
125

500


0.8-68

18-56
173


34-101


332


100
400

10
Results
Ataxia, sedation

No clear effects
Lethargy
Mortality
No clear effects
20% mortality
NOAEL
Decreased immune
f unct i on
Increased serum
. enzymes
NOAEL
Decreased PAH
uptake, moderate
liver and kidney
histopathology
(LOAEL)
NOAEL
Mortality; liver
and renal gross
pathology
NOAEL
Stomach lesions
(LOAEL)
Mortality, liver
and renal gross
pathology
NOAEL .

NOAEL
Liver histo-
pathology, serum
enzymes (LOAEL)
Relative liver
weight increased
(NOAEL)
Liver histo-
pathology, serum
enzymes (LOAEL)
NOAEL
Decreased operant
response
NOAEL

                        V-26

-------
TABLE V-9  Summary  of Short-term Health  Effects Data
                      on Bromoform

Reference
Bowman et a I .
(1978)
NTP (1989a)
NTP <1989a)
Munson et a I .
(1982)
Condi e
et al.
(1983)
Species Route Sex
Mouse Gavage M,F
(aqueous)
Rat Gavage M,F
(oil)
House Gavage M,F
(oil)
House Gavage M,F
(aqueous)
House Gavage M
(oil)
Duration
One dose
One dose
One dose
14 days
14 days
Dose
(mg/kg/day)
500
1.000
'500
1,000
250
500
50-125
250
72-145
289
Results
NOAEL
Ataxia, sedation
No deaths
60% mortality
No deaths
10% mortality
NOAEL
Increased serum
enzymes
NOAEL
Decreased PAH
uptake, moderate
                                                     histopathological
                                                     changes (LOAEL)
NTP (1989a)



NTP (1989a)



Chu et al.
Aida et al.
(1992a)



Balster and
Borzelleca
(1982)

Rat Gavage
(oil)


House Gavage
(oil)


Rat Drinking
Rat Diet




House Gavage
(aqueous)


H,F 14 days



M,F 14 days



H 28 days
M,F 1 month




H 30 days
60 days

90 days
200
400

600
200
400
600

0.8-68
62 (M)
56 (F)
187 (M)
208 (F)

100
100-400

9
NOAEL
Decreased body
weight gain, 1/5 died
100% mortality
NOAEL
Stomach nodules
Ataxia, lethargy.
1/5 died
NOAEL
NOAEL

Hepatic vacuol-
ization, serum
chemistry (LOAEL)
NOAEL
Decreased operant
response
NOAEL

                          V-27

-------
(8 weeks old) and 80 female (6 weeks old) B6C3F1 mice at doses of 60, 130, or




270 mg/kg/day for 90 days, using either corn oil or 2% emulphor as the




vehicle.  At sacrifice, body and organ weights were measured (Table V-10), and




blood was recovered to perform several serum chemistry measurements.  The




liver was sectioned for histopathological examination.  In the group given




chloroform in corn oil, mice administered 270 mg/kg/day displayed significant-




ly elevated SCOT levels (p < 0.05,  see Table V-10),  a significant degree of




diffuse parenchymal degeneration (5 of 10 males and 1 of 10 females), and mild




to moderate early cirrhosis (5 of 10 males and 7 of 10 females).  Serum tri-




glyceride levels were significantly decreased (p a 0.05) for males adminis-




tered 130 or 270 mg/kg/day and for females administered 270 mg/kg/day




(Table V-ll).  Total liver lipid was elevated in males (not significantly) and




females (p & 0.0.1) at 60 mg/kg/day (Table V-12) .  Significant pathological




lesions were not observed in the animals administered corn oil without chloro-




form or in mice receiving chloroform in 2% emulphor.  These data suggested to




the authors that administration of chloroform by corn oil gavage results in




more marked hepatotoxic effects than when it is provided in an aqueous suspen-




sion.   The authors could not determine if this was due to differences in




absorption kinetics or if the effect resulted from an interaction between




chloroform and the corn oil vehicle.  These data identified 270 mg/kg/day




chloroform as a LOAEL when given in oil but as a NOAEL when given in aqueous




vehicle.









        Jorgenson and Rushbrook (1980) exposed 6-week-old male Osborne-Mendel




rats weighing 190 g to chloroform in drinking water for 30, 60, or 90 days.




Animals received chloroform at levels of 200, 400, 600, 900, or 1,800 ppm




(30 animals/dose).  There were two control groups of 40 animals each; one
                                     V-28

-------
           TABLE V-10  Effects of Vehicle on the Subchronic Toxicity
                         of Chloroform  in B6C3F1 Mice
Chloroform Dose   	Male	   	Female	
  (mg/kg/day)     2% Emulphor       Corn Oil       2%  Emulphor       Corn Oil
                          Final Body Weight (g + SEM)

       0          33.6 + 0.5     35.5 + 0.4b-c     26.5    0.6      25.4    0.4
      60          31.0 + 0.8     31.7  0.4        25.4    0.6      25.5    0.7
     130          31.6 + 0.8     30.6 + 0.5        26.4    0.6      26.0    0.3
     270          29.3 +~0.6C    26.6  0.3b-c     25.4    0.3      24.0+0.7

                            Liver Weight  (g + SEM)

       0          1.29  0.03    1.17 + 0.03b      0.98+0.03     1.00    0.04
      60          1.18  0.03    1.32 + 0.03b-c    1.09    0.04C    1.19  +  0.05C
     130          1.33 + 0.05    1.36  0.03C      1.13    0.04C    1.22  +  0.03C
     270          1.36 + 0.03    1.50  0.02b-c    1.22  +  0.03C    1.39    0.03b-c

                    Liver/Body Weight Ratiod (g/lOOg + SEM)

       0          4.02  0.49    3.58  0.10b-     4.32    0.10     4.38  +  0.15   .
      60          4.28 + 0.06    4.67  0.06b-c    4.84  +  0.10C    5.25  +  0.10b-c
     130          4.84+0.11   5.09+0.09C      4.94  +  0.12C    5.47  +  0.08b-c
     270          5.34 + 0.07C   6.64 + 0.09b'c    5.58  +  0.12C    6.86  +  0.16b-c

                     Liver/Brain Weisht Ratio (g/g + SEM)
0
60
130
270
2.31 + 0.30
2.47 + 0.04
2.72 + 0.10
2.87 + 0.06C
2.33 + 0.05b
2.72 + 0.07b-c
2.74 + 0.05b
3.10 + 0.07c-e
1.90 + 0.05
2.14 + 0.08C
2.15 + 0.08C
2.42 + 0.05C
1.96 + 0.07
2.31 + O.llc
2.39 + 0.05c-e
2.83 + 0.03b-c

aFor each sex and dose level, significant differences between  emulphor  and
 corn oil groups of p & 0.05 or p  * 0.01, based on Student's  t-test.
 Number of animals per group = 9 or 10.
bp s. 0.01,  as in footnote a.
cSignificantly different from corresponding control  at p  s  0.05  by  one-way
 analysis of variance and pairwise Student's  t-test.
dLiver/body weight ratios were determined using the  fasted  body  weights rather
 than the final body weights.
ep s. 0.05,  as in footnote a.

Adapted from Bull et al. (1986).
                                      V-29

-------
    TABLE  V-ll   Comparison  of  Clinical  Chemistry  Parameters  of Mice  Treated
            with Chloroform in Corn  Oil Versus  Emulphor  for  90 Days

Chloroform Dose Male
(mg/kg/day) 2% Emulphor
Corn
Oil
Female
2% Emulphor
Corn Oil
SCOT (mU/mL)
Control
60
130
270
284  53a
142 -- 32b
176  43
167 + 31
181 
158 
116 
298 
36
31
14
31b,d
151  27
113  26
169  25
117  17
193  22
126  16
127  14C
335  60b-d
Triglvcerides (rag, %)
Control
60
130
270
58  8
75  12
54  6
60  4
80 
75 
60 
43 
10
4
5b
3b,d
63  10
62  9
50  4
55  3
64  5
65  5
55  5
41  3b-d

aMean  Standard error of the mean (SEM).   Four to 10 animals per group.
bSignificantly different from corresponding control at p s. 0.05.
cFor each sex and dose level, significant  differences of p s 0.05 between
 emulphor and corn oil groups were based on Student's t-test.
dSignificant differences at p <  0.01,  as in footnote c.

Adapted from Bull et al. (1986).
                                     V-30

-------
      TABLE  V-12   Effect of Vehicle on Chloroform-Induced Accumulation of
                       Lipid in the Liver of B6C3F1 Mice

Chloroform Dose
(mg/kg/day)
0
60
130
270
Male Female
2% Emulphor Corn Oil 2% Emulphor Corn Oil
6.6  1.1 7.9  0.7 6.8  0.4 7.5  0.8
6.8+0.6 9.4+0.8 7.5+0.3 13.1 + l.lb
7.6_.+ 0.7 7.8 + 0.4 6.9 + 0.3 9.2 + 0.8
5.3 + 0.6 8.0 + 0.3_ 6.0 + 0.4 8.4 + 0.4

aAverage lipid content in percent net-weight  S.D.  Nine or 10 animals per
 group.
bSignificantly different from corresponding control at p s 0.01.

Adapted from BuJ.1 et al. (1986).
                                     V-31

-------
group was an ad libitum control, and the second group matched the water con-




sumption of the high-dose group.  Based on reported water intakes, the levels




of chloroform administered corresponded to doses of 0, 20, 38, 57, 81, or




160 mg/kg/day.  The authors then examined body weight, kidney-fat-to-kidney-




weight ratios, serum biochemical parameters, and gross and microscopic




pathology findings.  Treatment decreased the body weight gain of rats receiv-




ing the high concentration by 25% compared to the ad libitum control, but




there was no clear decrease compared to the matched control.   Therefore,  the




decreased body weight was probably secondary to decreased water consumption,




probably due to poor palatability.   No effect was observed on the percentage




of kidney fat.  Most serum biochemical parameters were unchanged, but some




showed changes at doses of 400 to 1,800 ppm.  The authors judged these changes




to also be secondary to reduced water intake.  Gross and microscopic pathology




findings generally were slight or mild in severity, not dose related, and




either appeared adaptive (occurred in rats sacrificed after 30 or 60 days, but




not in those sacrificed after 90 days) or were sporadic (by nature and/or




incidence) and not considered related to treatment.  These data indicate that




dose's up to 160 mg/kg/day are without significant adverse effect, identifying




160 mg/kg/day as the NOAEL.-









        Jorgenson and Rushbrook (1980) performed a similar experiment using




6-week-old B6C3F1 female mice (19 g).   Groups of 30 animals were given water




containing 200, 400, 600, 900, 1,800,  or 2,700 ppm chloroform for 30 to




90 days.  Body weight and water consumption were monitored throughout.  Using




reported water intakes, calculated dose levels were approximately 0, 32, 64.




97, 145, 290, or 436 mg/kg/day.  Groups of 10 animals were sacrificed at 30.




60, or 90 days, organ fat/organ weight ratios were measured, and gross and




microscopic pathologic examinations were performed.  Mice receiving  900,




                                     V-32

-------
1,800, or 2,700 ppm sustained body weight losses during the first week, but




all body weights thereafter were comparable to those of controls.  Because




there was considerable variability in water consumption, dose-dependency was




not evident.  Liver fat increased significantly at the 2,700-ppm level




throughout the study,  ranging from 160% to 250% of the control value.  Gross




pathological examinations revealed occasional, Very slight hemorrhaging in the




lungs of mice from all dose levels.  Histologically,.mild centrilobular fatty




changes in mouse livers appeared in the two highest dose groups.  Reversible




fatty changes also appeared at doses as low as 64 mg/kg/day at the 30-day




sacrifice.  Values for SCOT and LDH (lactate dehydrogenase) in plasma were




highly variable but tended to decrease at the high-dose levels.  The authors




also observed extramedullary hematopoiesis in the liver, but did not consider




this effect to be related to treatment because it was sporadic and not dose-




related.   Lymphoid atrophy of the spleen at the high dose was considered




treatment-related.  These data indicate that the dose of chloroform of




290 mg/kg/day produced mild adverse effects on liver and other tissues




(LOAEL),  while doses of 145 mg/kg/day or lower were without significant effect




(NOAEL).









        Heywood et al.  (1979) administered chloroform to beagle dogs orally in




a toothpaste base in gelatin capsules 6 days/week for 7.5 years, followed by a




20- to 24-week recovery period.  Groups of 16 males and females received




0.5 mL/kg/day of the vehicle (toothpaste without chloroform), and eight dogs




of each sex.remained untreated.  Treated groups, comprising eight dogs of each




sex, received doses equivalent to 15 or 30 mg chloroform/kg/day in the tooth-




paste vehicle; another group of the same size received an alternative




nonchloroform toothpaste (0.5 mL/kg/day).  Eleven of the 96 dogs died during




the study, one from each chloroform-treated group.  SGPT levels rose moder-




                                     V-33

-------
ately in che high-dose group during the treatment period.  The effect was




detectable after 6 weeks of exposure  (34 versus 29 mU/mL, p < 0.05) and




reached a peak in the sixth year of the study (134 versus 49 mU/mL,




p < 0.001).  Slight,  but statistically significant,  increases in SGPT were




also detected after 130 weeks in the  low-dose group.  The authors concluded




that this effect probably corresponded to minimal liver damage.  The most




prominent histopathological effect was the presence of hepatic "fatty cysts"




which, although observed .in the control groups, were larger and more numerous




in the treated animals (Table V-13).  Nodules of altered hepatocytes were also




observed in all groups, but the frequency of nodules was not obviously dose




related (Table V-13).  Based on SGPT  levels and increased frequency of fatty




cysts, this study identified a LOAEL  of 15 mg/kg/day.









        Palmer et al. (1979) dosed groups of Sprague-Dawley rats (50/sex/dose)




by gavage (toothpaste vehicle) with 0 or 60 mg/kg/day chloroform, 6 days/week,




for 80 weeks.  Toxic effects attributable to treatment were absent except for




a marginal, though consistent and progressive, retardation of weight gain




(=10%) in both sexes.  A decrease in  plasma, but not in erythrocyte, cholines-




terase activity, was observed in the  treated female rats with a maximum at




week 52.  There were no significant differences in the timing or numbers of




deaths during the study between treated and control groups.  The only notewor-




thy finding in organ-weight analyses  was a significant (p < 0.01) decrease in




relative liver weight for treated female rats.  The authors observed minor




histological changes in the liver but no severe fatty infiltration, fibrosis,




or marked bile duct abnormalities.  There was no evidence of any treatment-




related toxic effect in the liver or  any macroscopic or microscopic treatment-




related changes in the brain.  Slight increases in the incidence of moderately
                                     V-34

-------
          TABLE V-13  Hepatic Changes Observed in Dogs Administered
                        Chloroform Orally  for  7.5 Years

Chloroform
Treatment
(mg/kg/day)
Control

Vehicle
control
Nonchloroform
toothpaste
15

30



Sex
M
F
M
F
M
F
M
F
M
F
Number of Animals
Examined
Histopathologically
7
5
15
12
8
7
7
8
7
8
Number of
Animals with
Nodules
1
1
0
3
0
1
1
1
0
4
Number of
with Fattv
Minimal
2
1
7
3
2
0
0
2
1
0
Animals
Cvsts
Moderate
0
0
1
0
0
0
6
3
6
7

Adapted from Heywood et al.  (1979).
                                     V-35

-------
severe glomerulonephritis were of uncertain significance.  This study identi-




fied a LOAEL of 60 mg/kg/day for decreases observed in body weight gain,




relative liver weight, and plasma cholinesterase activity.









        Jorgenson et al.  (1982) administered chloroform to male Osborne-Mendel




rats at concentrations of 0, 200, 400, 900, or 1,800 ppm in drinking water for




23 months.  Corresponding dose groups contained 330, 330, 150, 50, and




50 animals, respectively^..  Based on measured water consumption and body




weights, the time-weighted 23-month average doses were 0, 19,  38,  81, or




160 mg/kg/day  (Jorgenson et al. 1985).  Water consumption was lower in




chloroform-exposed animals than in controls, especially at the two highest




doses.  In order to compensate for the effect of decreased water consumption,




therefore, the authors included a matched control group of 50 animals, for




which water consumption was restricted to that of the high-dose group.  Body




weight gain was inversely proportional to chloroform dose, probably as a




result of reduced water consumption, since there was no consistent difference




between the high-dose and matched control groups.  Survival was proportional




to dose (lowest in controls, highest in high-dose animals).  The authors




indicated that this result may have been due to the beneficial effect of




reduced body weight.  Groups of 10 rats/dose were sacrificed at 3 or 6 months,




and liver triglyceride levels were- measured.  No increase in mean percent




liver fat was observed at any dose level, except for a small increase (from




4.5% to 5.1%) at the 1,800-ppm dose at 6 months (p < 0.05).  Groups of 20 rats




were sacrificed at 6,  12, or 18 months of exposure, and a number of blood




parameters were evaluated.  White blood cell values were lower in the




1,800-ppm group and in the matched controls at 6 and 12 months.  Differences




in erythrocyte and hemoglobin parameters at 12 months suggested hemoconcen-




tration in the treated groups, but no significant differences were apparent  at




                                     V-36

-------
18 months.  Various blood chemistry parameters differed significantly from the




control values, according to a number of apparent trends.   Chloride, potas-




sium, phosphorus, bilirubin, alkaline phosphatase (A?),  total iron, albumin,




and the albumin/globulin ratio tended to be higher in treated groups than in




the controls.  Cholesterol, triglycerides, LDH, and globulin tended to be




lower in treated groups than in the controls.  With the exception of serum




triglyceride levels, changes in hematologic and blood chemistry parameters




observed in the treated .groups were also evident in the matched control group;




the authors, therefore, 'concluded that the changes observed were secondary to




reduced water and food consumption.  However, decreased water intake alone




produced a 40% decline in serum triglyceride levels,  compared to an 89%




decrease observed in the high-dose group.  The significance of these observa-




tions is uncertain.









        Jorgenson et al. (1982) also exposed female B6C3F1 mice to chloroform




in drinking water for 23 months.   Drinking water concentrations were 0, 200.




400, 900,  or 1,800 ppm.  Based on measured water consumption and body weight,




these doses corresponded to time-weighted average doses of 0, 34, 65, 130, and




263 mg/kg/day (Jorgenson et al. 1985).  Dose groups contained 430, 430, 150.




50, and 50 animals, respectively.  A matched control group of 50 animals was




included,  in which water consumption was restricted to that of the high-dose




group.  Decreased survival at 3 weeks (99%, 94%, 74%, and 76% for the 200-,




400-, 900-, and 1,800-ppm groups, respectively) and behavioral effects (e.g..




lassitude, lack of vigor) in the 900- and 1,800-ppm dose groups were




attributed to markedly decreased fluid consumption.  The percent fat in the




liver of the mice was significantly increased  (p & 0.01) in the 400- to




1,800-ppm dose groups at 3 months and in the 900- and 1,800-ppm dose groups




(p s 0.05) at 6 months.  These data indicate that doses of 65 to 263 mg/kg/dav




                                     V-37

-------
may produce adverse effects in mice, possibly secondary to decreased water




consumption.  However, because no sensitive index of liver toxicity was




monitored, the data were not adequate to identify a useful NOAEL or LOAEL




value.









        Klaunig et al. (1986) examined the effect of chronic chloroform




exposure in mice.  Two groups of 35 male B6C3F1 mice (4 weeks old) were




supplied with drinking water containing 600 or 1,800 ppm of chloroform




(equivalent to 86 or 258 mg/kg/day).  Animals were sacrificed after 24 weeks




(10 mice) or 52 weeks (25 mice), and body weight and water consumption were




monitored throughout the study.  Both groups displayed a statistically signi-




ficant (p < 0.05) decrease in drinking water intake.   The 1,800-ppm group




exhibited statistically significant (p < 0.05) decreases in mean body weight,




but this effect may have been due to decreased water intake; no matched




control was included.  No deaths occurred in the control group during the




52-week study period, but 2/35 mice in the low-dose group and 3/35 in the




high-dose group died between weeks 40 and 52.  Focal areas of cellular




necrosis were found in the kidneys and liver of chloroform-treated mice at 24




and 52 weeks (no data reported).  Focal areas of hepaticlipid accumulation




were also seen in the 1,800-ppm mice.  This study suggested that 86 mg/kg/day




may be a LOAEL in mice,  but the data were not adequate to provide a firm




conclusion.









        Table V-14 summarizes the longer-term health effects data on




chloroform.
                                     V-38

-------
               TABLE  V-14   Summary  of  Longer-term  Studies of Chloroform
Reference
 Species     Route
                    Dose
Sex    Duration   (mg/kg/day)
                                                                                       Results
Chu et al.
(1982b)
Bull et al.
(1986)
                   Rat
Jorgenson and
Rushbrook
(1980)
Heywood
et al.
(1979)
Palmer
et al.
(1979)
Jorgenson
et al.
(1982)
                   Rat
                   House
                   Rat
                   Rat
           Drinking
           water
Mouse      Gavage
           (oil)
           Drinking
           water
M,F    90 days      0.7-50
                   180
M,F    90 days     -60-130


                   270



M,F    90 days      60-270


M      30-90 days   20-160
                                30-90 days   32-145
                                            290
M,F    7.5  years    15
           Gavage
           (aqueous)

           Drinking
           water
                             Drinking
                             water
           Oral
           (toothpaste
           base
           gelatin
           capsule)
           Gavage         M,F    80 weeks
           (toothpaste
           base)
                                                               60
                                                   23 months    19-160
NOAEL
Increased mortality;
  mild thyroid lesions

Decreased serum
  triglycerides,
  elevated liver fat
Elevated SCOT,
  diffuse liver
  degeneration

NOAEL
Serum chemistry
  changes (judged
  secondary to
  reduced water
  intake) (NOAEL)

NOAEL
Mild hepatic
  fatty change
  (LOAEL)

Rise in SGPT and
  fatty cysts (LOAEL)
                                    Decreased body weight
                                       liver  weight and
                                       plasma cholinesterase
                                       activity.  Minor
                                       liver  changes.
                                       (LOAEL)

                                    Minimal changes
                                       in various blood
                                       chemistry
                                       parameters and
                                       mean percent liver
                                       fat,  probably
                                       secondary to
                                       decreased water
                                       consumption
                                                  V-39

-------
                                      Table  V-14  (Continued)
                                                                 Dose
Reference          Species     Route         Sex    Duration   (mg/kg/day)               Results


Jorgenson          Mouse      Drinking        F      23 months   65                Increased mean
et at.  (1982)                 water                                                 percent  liver fat
(continued)                                                                         (3 months)
                                                               130-263           Rejection of
                                                                                   chloroform
                                                                                   solutions leading
                                                                                   to high  initial
                                                                                   mortality;
                                                                                   Increased mean
                                                                                   percent  liver
                                                                                   fat (3 and 6
                                                                                   months).

Klaunig            Mouse      Drinking        M      52 weeks    86-258            Kidney and  liver
et al.                        water                                                 histological
(1986)                                                                              changes;  LOAEL (?)
                                                  V-40

-------
        2.  Brominated Trihalomethanes









        Chu et al. (19S2b) administered bromodichloromethane,  dibromochloro-




methane or bromoform to weanling Sprague-Dawley rats for 90 days.  The




compounds were added to drinking water at levels of 0, 5, 50,  500, or




2,500 ppm (20 rats/sex/dose).  Half of each group (10/sex/dose) was sacrificed




at the end of the exposure period, and the remaining animals were given tap




water for another 90 days.-...  Using the authors' calculations of trihalomethane




ingested per rat per day and reported average body weights, these levels




corresponded to doses of approximately 0, 0.6, 7, 52, or 250 mg/kg/day.  Food




consumption was depressed in all of the 2,500-ppm groups, although growth




suppression occurred only in the 2,500-ppm bromodichloromethane group.  Mild




histological changes occurred in the liver and thyroid.  Although neither




incidence nor severity were clearly dose related, these parameters did tend to




increase with dose (Table V-15).   Observed lesions included increased




cytoplasmic volume and vacuolation due to fatty infiltration.   Statistically




significant (p < 0.05)  increases  in the severity of hepatic lesions were




observed at the highest dose for all three chemicals.  The authors indicated




that in both sexes the hepatotoxicity of the brominated trihalomethanes




followed the order:  bromoform > bromodichloromethane > dibromochloromethane.




The only serum biochemical parameter affected was LDH, which was significantly




reduced in rats given 2,500 ppm bromoform (both sexes).  Ninety days after




cessation of treatment, histological changes were reversed, although LDH




levels in plasma remained reduced.  These data identified a NOAEL of




52 mg/kg/day and a LOAEL of 250 mg/kg/day for all three brominated




trihalomethanes.
                                     V-41

-------
                    Table  V-15   Incidence  and  Severity  of  Liver  and Thyroid  Lesions  in  Rats
                                   Fed Brominated Trihalomethanes for 90 Days

Liver
Male
Group
Control (H20)
Control (Vehicle)
(1% Emulphor)
Bromodichlorome thane
5 ppm
50 ppm
500 ppm
2,500 ppm
Dibromochl or ome thane
5 ppm
50 ppm
500 ppm
2,500 ppm
Bromoform
5 ppm
50 ppm
500 ppm
2,500 ppm
Female
Incidence Severity3 Incidence
0/10
2/9


1/10
8/10
8/10
9/10

3/10
4/10
5/10
6/9

5/10
4/10
7/10
9/9
1.0
1.2


1.1
2.2
2.0
2.3

1.3
1.4
1.7
2.0

1.6
1.4
1.8
2.7

 0.4


0.3
 0.8b
 0.7b
 0.7b

0.5
0.5
 0.8
 0.9b

0.7
0.5
 0.6
 0.7b
0/10
0/10


3/10
5/10
3/10
4/10

3/10
5/10
3/10
4/10

3/10
0/10
4/10
6/10
Male
Soever ity Incidence
1.0
1.0


1.3
1.6
1.3
1.5

1.3
1.6
1.3
1.6

1.3
1.0
1.5
1.7




0.5
0.7b
0.5
 0.7b

0.5
 0.7b
0.5
 0.8b

0.5

 0.7b
 0.7b
3/10
3/9
1

2/10 .
5/10
5/10
4/10

2/10
3/10
6/10
3/10

3/10
5/10
4/10
5/10
Thyroid

Female
Severity Incidence
1.3
1.3


1.2
1.7
1.8
1.5

1.3
1.3
1.9
1.4

1.3
1.6
1.5
1.7
0.5
0.5


 0.4
 0.8
 0.9
0.7

0.7
0.5
 0.9
0.7

0.5
0.7
0.7
0.7
2/10
0/10


0/10
1/10
3/10
2/10

3/10
1/10
0/10
3/10

I/ 10
0/10
3/10
5/10
Severity
1.2
1.0


1.0
1.1
1.3
1.2

1.3
1 .1
1.0
1.3

1.1
1.0
1.4
1.7
0.4




0.3
0.5
0.4

0.5
0.3

0.5

 0.3

0.7
0.8

aSeverity rating for lesions range from 1 (no effect)  to 10 (malignant/complete necrosis).
Significantly different from vehicle control (p <  0.05).

Adapted from Chu et al.  (1982b).

-------
        NTP  (1987) administered doses of 0, 19, 38, 75, 150, or 300 mg/kg/day




of bromodichloromethane to F344/N rats  (10/sex/dose) by gavage in corn oil,




5 days/week, for 13 weeks.  (The IS-mg/kg/day group was administered




1.9 mg/kg/day for the first 3 weeks of  the study.)  A necropsy was performed




on all animals.   Five of ten male rats  and two of ten female rats that




received 300 mg/kg died before the end  of the study.  Final mean body weights




of male rats that received 150 or 300 mg/kg and of f.emale rats that received




300 mg/kg were 30% to 55%.. or more lower than those of the vehicle controls;




the final mean body weight of female rats at 150 mg/kg was 12% lower than that




of the vehicle controls.  No other compound-related clinical signs were




reported.  Compound-related lesions were observed at 300 mg/kg but not at




150 mg/kg.  In high-dose males, centrilobular degeneration of the liver and




occasional necro.tic cells were observed in four of nine animals.   Mild bile




duct hyperplasia was also observed in these animals.  Degeneration of renal




proximal tubular epithelial cells was observed in four of nine males, and two




of nine males had definite foci of coagulative necrosis of the tubular




epithelium.  Lymphoid atrophy of the thymus, spleen, and lymph nodes and mild




to moderate atrophy of the seminal vesicles and/or prostate were present in




four of nine high-dose males.   In high-dose females, enlarged hepatocytes were




observed in two  of nine animals.   Atrophy of the thymus,  lymph nodes, and




spleen occurred in high-dose female rats but was much less than that observed




in males.  Rats  in the high-dose groups were emaciated and appeared to consume




less feed on dosing days (although actual feed consumption was not measured).




Based on reduced body weight gain, this study identified a NOAEL of




75 mg/kg/day and a LOAEL of 150 mg/kg/day.









        In a similar experiment,  NTP (1987) administered bromodichloromethane




in corn oil by gavage to B6C3F1 mice (10/sex/dose), 5 days/week, for 13 weeks.




                                     V-43

-------
Doses were 6.25, 12.5, 25, 50, or 100 mg/kg/day for males, and 0, 25, 50, 100.




200, or 400 mg/kg/day for females.  All animals survived to the end of the




study.  The final mean body weight of males that received 100 mg/kg was 9%




lower than that of the vehicle controls.  The final mean body weight of




females that received 200 or 400 mg/kg was 4% to 5% lower than that of the




vehicle controls.  Focal necrosis of the proximal renal tubular epithelium




occurred in 6/10 males at 100 mg/kg, and nephrosis of minimal severity




occurred in 2/10 males at this level.  No compound-related lesions were seen




in the 50-mg/kg groups. . Hepatocytes in the centrilobular area of the liver of




8/10 female mice at 400 mg/kg and 7/10 female mice at 200 mg/kg were greatly




enlarged with vacuolated or foamy cytoplasm characteristic of lipid accumu-




lation.  Microgranulomas were present in the liver of 7/10 female mice that




received 200 mg/kg,  and no compound-related lesions were noted in females at




100 mg/kg.  No compound-related clinical signs-were noted.  Based on renal




histopathology, this study identified a NOAEL of 50 mg/kg/day and a LOAEL of




100 mg/kg/day in male mice.









        NTP (1985) administered dibromochloromethane by gavage (in corn oil)




to F344/N rats (10/dose/sex).   Doses of 0, 15, 30, 60, 125, or 250 mg/kg were




given 5 days/week for 13 weeks.   Animals were weighed weekly and a thorough




histological examination was performed on all animals, either at the time of




death or at the end of the study.  Only one male and one female in the high-




dose group survived, with most deaths occurring in weeks 8 to 10.  Histo-




logical examination revealed severe lesions and necrosis in kidney, liver, and




salivary glands.  Body weight gain of males receiving 125 mg/kg/day was 85% of




the corresponding control value.  A dose-dependent increase in the frequency




of clear cytoplasmic vacuoles indicative of fatty metamorphosis was observed




in males; this effect was statistically significant at doses of 60 mg/kg/day




                                     V-44

-------
or higher.  On this basis, this study identified a. NOAEL of 30 mg/kg/day and a




LOAEL of 60 mg/kg/day in rats for dibrompchloromethane.









        NTP (1985) performed a similar 13-week gavage study with dibromochloro-




methane in male and female B6C3F1 mice (10/sex/dose).   The doses and dosing




schedule were the same as for the study with rats.  No effects on body weight




or histopathology were observed at doses of 125 mg/kg/day or lower.  Fatty




liver and toxic nephropathy were observed in males, but not females,  receiving




250 mg/kg/day.  Both males and females gained less weight at this dose level.




This study identified a NOAEL of 125 mg/kg/day and a LOAEL of 250 mg/kg/day in




mice.









        NTP (1989a) exposed F344/N rats to bromoform by gavage, 5 days/week




for 13 weeks.   Animals (10/sex/dose) received doses of 0, 12, 25, 50,  100, or




200 mg/kg/day.  None of the rats died before the end of the study, and body




weights were not significantly affected.  Animals receiving 100 or




200 mg/kg/day were lethargic.  At sacrifice, tissues were examined for gross




and histological changes.  A dose-dependent increase in the frequency of




hepatocellular vacuolation was observed in male rats,  which became statis-




tically significant (p < 0.05) at 50 mg/kg/day (IRIS 1988).   These hepatic




effects were not observed in females.  On the basis of the hepatic vacuolation




seen in male rats, this study identified a NOAEL of 25 mg/kg/day and a LOAEL




of 50 mg/kg/day.









        In a parallel study, NTP (1989a) exposed B6C3F1 mice to bromoform by




gavage, 5 days/week for 13 weeks.  Animals  (10/sex/dose) received doses of 0,




25, 50. 100, 200, or'400 mg/kg/day.  One female died at 100 mg/kg/day, but




there were no other deaths, even at  the higher doses.  At sacrifice, tissues




                                     V-45

-------
were examined for gross and histological changes.  Body weights were not




significantly affected, although males receiving 400 mg/kg/day had body




weights about 8% less than controls.  An increase in the number of hepato-




cellular vacuoles was seen in male mice (5/10 at 200 mg/kg and 8/10 at




400 mg/kg) but not in females.  Based on hepatocellular vacuolation, this




study identified a NOAEL of 100 mg/kg/day and a LOAEL of 200 mg/kg/day in male




mice.









        Daniel et al.  (1990) administered gavage doses (in corn oil) of 0, 50,




100, or 200 mg/kg/day of dibromochloromethane to male and female Sprague-




Dawley rats (10/sex/dose) for 90 consecutive days.   Individual dosages were




adjusted weekly based on individual body weights.  During the final week of




the study, urinalysis was conducted following an overnight fast.  Ophthalmo-




scopic examinations were performed prior to treatment and during the last week




of the study,  and hematological analysis,  serum clinical chemistry, and a




thorough histopathological examination were conducted.  There were no deaths,




clinical signs of toxicity, or treatment-related changes in the ophthalmo-




scopic examinations or hematology.   Body weight gain was significantly reduced




in the high-dose groups, to less than 50% of the controls (males) and less




than 70% of the controls (females).  Clinical chemistry values indicative of




hepatotoxicity and suggestive of nephrotoxicity included increased levels of




AP (high-dose males and females), alanine aminotransferase (also known as




SGPT)  (mid- and high-dose males) and creatinine (mid- and high-dose males and




high-dose females), and decreased potassium levels (high-dose males).  Centri-




lobular lipidosis (vacuolization) was observed in the liver of almost all




high-dose males and females and all mid- and low-dose males (with one




exception at 'each level), but in only one mid-dose female.  The severity of




the effect was dose related.  Centrolobular necrosis was also observed in




                                     V-46

-------
high-dose males and females.  Slight-to-moderate degeneration within the




proximal kidney tubular cells occurred in all high-dose males and females, and




to a lesser extent in mid-dose males and low- and inid-do3e females.  Based on




the liver histopathology in males and kidney histopathology in females, the




LOAEL for dibromochloromethane in this study was 50 mg/kg/day.









        Tobe et al. (1982) evaluated the chronic effects of bromodichloro-




methane,  dibromochloromethane, and bromoform administered in the diet to




groups of 40 male and 40 female Slc:Wistar SPF rats for 24 months.  The




histopathology data from this study for the bromodichloromethane-exposed




animals were reported by Aida et al. (1992b).  No histopathology data has been




published for the other two chemicals.  The animals were 5 weeks old at the




start of the tes.t and weighed approximately 100 g.  Each trihalomethane was




microencapsulated, and an appropriate amount was mixed with powdered feed.




Control groups (70 rats/sex) received microcapsules without trihalomethanes.




Body weights and food consumption were monitored weekly for the first




6 months, every 2 weeks from 6 to 12 months, and every 4 weeks during the




second year of the study.  Interim sacrifices of at least 9 animals/sex/




control group and 5 animals/sex/dose group were conducted for bromodichloro-




methane at 6,  12, and 18 months; all surviving animals were sacrificed at




2 years.   For bromoform and dibromochloromethane, data were reported from the




sacrifices of 9 animals/sex in the control group and 5/sex/dose group at




18 months; all surviving animals were sacrificed at 24 months.  At each time




of sacrifice,  necropsies, hematological studies, and serum biochemical studies




were conducted.









        Bromodichloromethane was administered in the diet at levels of 0.0%.




0.014%, 0.055%, or 0.22%.  Based on mean food intakes, the reported average




                                     V-47

-------
doses were about 0, 6, 26, or 138 mg/kg/day for males and 0, 8, 32, or




168 mg/kg/day for females (Aida et al.  1992b).   Marked suppression of body




weight gain was seen in males and females of the high-dose group.  Males and




females of the high-dose group exhibited mild piloerection and emaciation.




Relative liver weight was significantly increased in all dose groups, and




relative kidney weight was significantly increased (p < 0.01) in males and




females in the high-dose group.  Dose-dependent reductions in T-GLY levels and




increases in vglucaniyl transpeptidase (y-GTP)  activity (indicative of bile




duct proliferation) were observed at all sacrifice times in both sexes




(Table V-16).   Serum cholinesterase activity was also decreased in a dose-




related manner in both sexes.  The most sensitive markers at 24 months were




T-GLY and serum cholinesterase, with significant (p < 0.05)  changes seen in




low-dose males. .Gross necropsy revealed dose-related yellowing and roughening




of the surface of the liver.   Treatment-related.lesions were limited to the




liver and included fatty degeneration and granuloma in all dosage groups, and




cholangiofibrosis in the high-dose groups (Table V-17).  Bile duct prolifera-




tion was observed in most high-dose animals at 6 months, but by 24 months it




was prevalent in the controls and all dosage groups.   Histopathology was




observed in all dose groups,  except for low-dose females, as early as




6 months.   At 24 months,  fatty degeneration and granuloma were observed in




low-dose males, but not control males,  and fatty degeneration was observed in




low-dose females at a higher rate (8/19) than in control females (2/32).




Based on the results of Tobe et al. (1982) alone, the NOAEL was 6 mg/kg/day in




males and 8 mg/kg/day in females and the LOAEL was identified as 26  (males), to




32 (females) mg/kg/day, based on serum enzyme changes and altered liver




appearance.  However, taking into consideration the histopathology data




reported for this study by Aida et al.  (1992b),  the entire study identified a
                                     V-48

-------
                                        Table  V-16   Serum Biochemical  Levels8  in  Rats  Fed Brominated
                                                        Trihalomethanes  for 18  to  24 Months
<
I
vD

SCOT
Dietary
Chemical Sex Level (X)
CHBrCl2 M 0
0
0
0,
F 0
0
0
0.
CHBr2Cl M 0
0
" 0
0
F 0
o
o
0
CHBr3 M 0
o
0
0
F 0
0
0
0

.on
.055
.220

.014
.055
220

.022
.088
.350

.022
.068
.350

.04
.160
.650

.04
. 160
.650
18
106
77'
122
85
92
100
103
96
106
92
110
175
92
121'
148"
124"
106
115
202
229'
92
112
112
21B'
24
123
95
95
119
95
156
88
102
123
110
107
187
95
94
123
no
123
83
96
198
95
121
127
182*
SGPT
18
42
39
59
47
37
56
53
43
42
45
57'
92"
41
52
81*
42
42
47
106
100"
41
53
48
100"
24
35
45
34
44
46
47
42
39
35
36
45
58
46
42
54
42
35
31
40
92"
46
44
69
75"
yGTP
18
0.90
0.35
3.38
6.08"
0.27
0.50
1.85"
9.14"
0.90
0.78
3.13"
21.59"
0.27
1.48*
5.03'
11.87"
0.90
0.34
2.84
4.94"
0.26
1.11"
1.07"
7.66"
24
2.25
2.45
3.50
9.51"
1.83
3.92
2.72
9.49"
2.25
3.73
5.46*
17.26"
1.83
2.11
4.09*
8.57"
2.25
0.19
0.53
7.50"
1.83
2.18
4.06"
6.61"
LDH
18
2610
1900
1925
1220"
1770
1250
1400
1440
2610
1670
1370"
mo"
1770
1880
2120
1940
2610
2850
3160
2830
1770
2210
2130
2210
24
1890
1910
2260
2480"
1300
1440
1300
1340
1890
2160
2640
2000
1300
1250
1600
1430
1890
1560
2050
1350"
1300
1420
1460
1570
TGLY
18
153
168
112"
54"
117
96
68*
43"
153
160
94"
69"
117
164"
79"
37"
153
263
168
52"
117
173"
120
34"
24
321
164"
106"
77"
334
194
172"
97"
321
209"
195
50"
235
178
106"
50"
321
232
231
46"
234
238
179
47"
CHL
18
954
877
786
556"
1962
1338"
1133"
758"
954
831
839
568"
1962
1456"
1202"
737"
954
1067
798
467"
1962
1729
1436"
674"
24
1538
994"
772"
627"
1739
1469
1293"
865"
153B
1150
1044*
582"
1739
1465
1235"
866"
1538
1313
1179
393"
1739
1720
1443
837"
FAA
18
0.59
0.44
0.43
0.36"
0.74
0.52"
0.48
0.46"
0.59
0.62
0.50
0.40"
0.74
0.87
0.59
0.49
0.59
0.66
0.39"
0.39"
0.74
0.91
0.54
0.37"

0
0
0
0
0
0
0
0
0.
0.
0
0
0
0
0
0
0
0
0.
0.
0.
0.
0.
0.
24
.60
.56
.53
.52
.83
.81
. 79
57"
60
63
.55
.30"
.83
.84
.64
.47"
.60
.46"
40"
34"
83
99
52"
39"

             'Results from nine animals/sex in the control group and five/sex/dose  In the treatment groups at the 18-month sacrifice,  and  12 animals/sex in the
              control group and seven/sex/dose in the treatment groups at 24 months.  Enzyme activities expressed as mU/mL, TGLY  in mg/dl.. and NEFA in mEq/L
             "p less than 0.05 compared to control
             "p less than 0.01 compared to control

             Adapted from Tobe et al.  (1982).

-------
                            Table V-17  Liver Lesions In Rats Fed Bromodichloromethane for 2 Years
Ul
o



Dose

Control
0.014
0.055
0.22

Control
0.014
0.055
0.22

Control
0.014
0.055
0.22

Control
0.014
0.055
0.22
Number of
Animals/Sex
Examined
Histologically

10
6
6
6

9
5
5
5

9
5
5
5

24a (32)b
14 (19)
13 (18)
19 (18)
: Fatty
Bile
Duct Altered Cholangio-
Degeneration Granuloma Proliferation Cell Foci fibrosis
M

- .
6
6
6

.. .
5
5
5

1
3
5
5


5
12
19
F

	
--
5
6

	
--
5
4


1
5
5

2
8
18
18
M
6 Months
	
--
1
--
12 Months


--
1
18 Months

--
2
4
24 Months

4
9
19
F

_ .

3
6


--
5
5


--
5
5


--
17
18
M

	

--
5

1
1
--
5

9
5
5
5

24
13
13
19
F M F M

- - - - - - _.
~~l ~"

6 .... 6

	 * 	 . __

3 	
5 5

6 2
2 	
4 	
5 -- --3

28 12 15
16 69--
17 7 6 --
18 734
F

_ _


6

	


5


--

4




12

       aNuinber  of  males
       bNumber  of  females
       Adapted from Aida et al.
(1992b).

-------
LOAEL of 6 mg/kg/day for bromodichloromethane in male rats and 8 mg/kg/day in




female rats, based on histopathology and serum biochemistry.









        Dibromochloromethane was administered at dietary levels of 0.0%,




0.022%, 0.088%, or 0.35%.  Based on reported body weights (150 to 475 g) and




food consumption (15 to 20 g/day),  these levels correspond to doses of




approximately 0, 10, 39, or 210 mg/kg/day for males and 0, 17, 66, or




350 mg/kg/day for females.  Marked suppression of body weight gain was seen in




males and females of the high-dose group, and mild (about 10%) suppression of




body weight gain was seen in males and females of the mid-dose group.




D.ecreased T-GLY and serum cholinesterase activity and increased y-GTP were




seen in the mid-dose males and females (Table V-16).   Yellowing and roughening




(high-dose males_ only)  of the surface of the liver were noted in the mid- and




high-dose groups.  Based on the serum biochemistry data, decreased body




weight, and gross necropsy results, this study suggests a NOAEL of




10 mg/kg/day in males and 17 mg/kg/day in females, and a LOAEL of 39 (males)




and 66 (females) mg/kg/day.  However, no histopathology results were




presented.









        Bromoform was administered at dietary levels of 0.0%, 0.04%, 0.16%,




or 0.65%.  Based on reported bodv weights (150 to 475 g) and food consumption




(15 to 20 g), these levels correspond to doses of about 0, 18, 71, or




480 mg/kg/day for males and 0, 30,  120, or 870 mg/kg/day for females.  Marked




suppression of body weight gain was seen in males and females of the high-dose




group, and mild (about 15%) suppression of body weight gain was seen in males




and females of the mid-dose group.   Dose-related decreases in FAA (non-esteri-




fied fatty acids) were observed in all treated males, and mid- and high-dose




females had increased levels of y-GTP.  Other serum biochemistry changes  in




                                     V-51

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the high-dose groups included decreased T-GLY and increased SCOT and SGPT




activity (Table V-16).  Elevations in SCOT and SGPT are indicative of hepato-




cellular necrosis.  Yellowing, small white spots, and roughening of the




surface (high-dose only) were seen in the livers of the mid- and high-dose




animals.  Based on the necropsy findings and the serum biochemistry data, this




study indicates a NOAEL of 18 mg/kg/day in males and 30 mg/kg/day in females,




and a LOAEL of 71 (males) and 120 (females) mg/kg/day.   However, no histo-




pathology results were presented.









        The authors concluded that serum biochemical changes and the necropsy




findings in the liver among the groups administered high doses of the three




brominated trihalomethanes were primarily a result of abnormal lipid




metabolism.









        NTP (1987) administered doses of 0, 50,  or 100 mg/kg/day of bromo-




dichloromethane in corn oil by gavage to F34A/N rats (50/sex/dose),




5 days/week for 102 weeks.  The authors observed all animals for clinical




signs and recorded body weights (by cage) once per week for the first 12 weeks




of the study and once per month thereafter.  A necropsy was performed on all




animals, including those found dead, unless they were excessively autolyzed or




cannibalized.  During necropsy, all organs and tissues were examined for




grossly visible lesions.  Complete histopathologic examinations were performed




on all female rats and on high-dose and vehicle-control male rats.  Male rats




in the low-dose group that died early in the study were also examined histolo-




gically.  Survival of dosed rats was comparable to that of vehicle controls.




Mean body weights of high-dose male and female rats were decreased during the




last 1.5 years of the study; body weight gains of high-dose male and female




rats were 86% and 70% of the corresponding vehicle-control values.  Body




                                     V-52

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weight gains of low-dose male and female rats were comparable to those of the




vehicle control group.  No compound-related clinical signs were observed.




Compound-related nonneoplastic lesion?; arp shown in Table V-18.  In males,




effects included renal cytomegaly, tubular cell hyperplasia, hepatic necrosis,




and fatty metamorphosis.  In females, changes included eosinophilic cyto-




plasmic change, clear cell change, focal cellular change, fatty metamorphosis




of the liver, and tubular cell hyperplasia of the kidney.  Based on these




histological findings, this study identified a LOAEL of 50 mg/kg/day in rats.









        NTP  (1987) administered bromodichloromethane in corn oil by gavage to




B6C3F1 mice  (50/sex/dose),  5 days/week for 102 weeks.  For males, doses were




0, 25, or 50 mg/kg/day; for females, doses were 0, 75, or 150 mg/kg/day.




Final survival o.f dosed male mice was comparable to that of vehicle controls.




At week 84,  survival of female mice was greater than 50% in all dose groups.




After week 84, survival of dosed and vehicle-control female mice was reduced




(final survival:  26/50; 13/50;  15/50), and this decreased survival was




associated with ovarian abscesses (8/50; 19/47; 18/49).  Body weight gain of




high-dose male mice was 87% that of the vehicle control group; the body weight




gain of low-dose male mice was comparable to that of the vehicle control




group.  Mean body weights of high-dose female mice were decreased during the




last 1.5 years of the study.  The body weight gain was reduced 55% compared to




the controls at the high dose and by 25% among low-dose females.  Compound-




related nonneoplastic lesions are shown in Table V-18.  In males, changes




included fatty metamorphosis of the liver, renal cytomegaly, and folliculap




cell hyperplasia of the thyroid gland.  In females, hyperplasia of the thyroid




gland was observed.  Based on these histological findings, this study




identified a LOAEL of 25 mg/kg/day in male mice.
                                     V-53

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              TABLE V-18  Nonneoplastic Lesions in Rats and Mice
                  Exposed to Bromodichloromethane  for 2  Years

Frequency
Animal
Male rat

Female rat

Male mouse


Female mouse
Tissue
Kidney
Liver
Liver
Kidney
Liver
Kidney
Thyroid
Thyroid
Lesion
Cytomegaly
Tubular hyperplasia
Necrosis
" Fatty -metamorphosis
Fatty metamorphosis
Focal cellular change
Clear cell change
Eosinophilic cytoplasm
Tubular hyperplasia
Fatty metamorphosis
Cytomegaly
Follicular cell hyperplasia
Follicular cell hyperplasia
Control
0/50
0/50
1/50
36/50
7/50
4/50
4/50
0/50
0/50
4/49
0/49
0/48
6/50
Low
Dose
18/50
0/50
4/50
48/50
22/50
. 4/50
6/50
1/50
1/50
8/50
41/50
3/44
18/45
High
Dose
44/50
3/50
6/50
47/50
13/50
11/50
39/50
11/50
4/50
19/50
47/50
5/49
21/48

Adapted from NTP (1987).
                                     V-54

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        NTP  (1985) investigated the chronic oral toxicity of dibromochloro-




methane in F344/N rats.  Groups of 50 animals/sex/dose were administered doses




of 0, 40, or 80 mg/kg/day by gavage (in corn oil), 5 days/week for 104 weeks.




Survival was comparable in all dose groups.  Body weight gain was decreased  in




high-dose males after week 20; final weight gain was 88% of the control value.




Females in both dose groups gained more weight than did the controls.




Histologic lesions in liver were observed in both males and females at both




dose levels  (p < 0.05).  _Qhanges included fat accumulation,  "ground glass"




appearance of the cytoplasm,  and altered basop^hilic staining.  This study




identified a LOAEL of 40 mg/kg/day for dibromochloromethane in rats.









        NTP  (1985) performed a similar chronic oral study with dibromochloro-




methane using male and female B6C3F1 mice.  Groups of 50 animals/sex/dose were




administered doses of 0, 50,  or 100 mg/kg/day by gavage (in corn oil),




5 days/week for 105 weeks.  Survival in females was not different from




controls, while survival in high-dose males was decreased (p < 0.05).  An




overdosing accident at week 58 killed 35/50 male mice in the low-dose group,




and this group was not considered further.  Mean body weights were decreased




in high-dose males and females but not in low-dose females.  Treatment-related




hepatocytomegaly and focal necrosis were observed (p < 0.05) in livers of




males (high dose).  Females showed liver calcification (high dose) and fatty




metamorphosis (both low and high doses) .   This study identified a LOAEL of




50 mg/kg/day for dibromochloromethane in mice.









        NTP.  (1989a) exposed groups of 50 male and 50 female F344/N rats to




bromoform by gavage for 103 weeks (5 days/week) at doses of 0, 100, or




200 mg/kg/day.  Animals were observed for clinical signs throughout the study




(2 days/week).  At termination, necropsy was performed on all animals, as was




                                     V-55

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a thorough histological examination of tissues.  Body weight gain was




decreased by 37% in high-dose females and by 29% in high-dose males, compared




to the respective controls.  Survival of the high-dose males was also




decreased.  Both males and females were lethargic.  Hepatic fatty change and




chronic inflammation were noted in both males and females at both doses, and




minimal necrosis was increased in high-dose males.  Nonneoplastic changes were




not reported in other tissues.  This study identified a LOAEL of 100 mg/kg/day




in both male and female rats.









        NTP (1989a) exposed groups of 50 male B6C3F1 mice by gavage to doses




of 0, 50, or 100 mg/kg/day of bromoform for 103 weeks (5 days/week).  Groups




of 50 female mice were administered doses of 0, 100, or 200 mg/kg/day.




Animals were observed for clinical signs 2 days/week throughout the study.  At




termination, all animals were necropsied, and a thorough histological




examination of tissues was performed.  Decreased survival was observed in




females but not males.  This was at least partly due to a utero-ovarian




infection.  No clinical signs were noted.  Body weight gains were 82% and 72%




of the control values for low- and high-dose females, respectively, but body




weight gain was not affected in males.  An increased incidence of minimal to




mild fatty changes was noted in the livers .of both low- and high-dose females




but not males.  Nonneoplastic changes were not detected in other tissues.




This study identified a LOAEL of 100 mg/kg/day for female mice, and a NOAEL of




100 mg/kg/day for male mice.









        Tables V-19, V-20, and V-21 summarize the longer-term studies of




brominated trichloromethane toxicity.
                                     V-56

-------
        TABLE  V-19   Summary of  Longer-term  Studies  of  Bromodichloromethane
Reference
Species     Route
                    Pose
Sex    Duration   (mg/kg/day)
                                                                                     Results
Chu et  al.
(1982b)
NTP
(1987)
                  Rat
                  Rat
                  Mouse
                  Rat
          Drinking
          water
          Gavage
          (oil)
                  Mouse      Gavage
                             (oil)
                             Gavage
                             (oil)
                             Gavage
                             (oil)
                  Mouse      Gavage
                             (oil)
M,F    90 days     0.6-52

                  250
M,F    13 weeks    19-75
                  150

                  300
                        M      13  weeks    6.25-50
                                          100
                                                  13 weeks
                        M,F    2  years
                                          25-100
                                          200-400
                                                             50
                        M.F    2  years     25
Mild and reversible
  changes (NOAEL)
Hepatic lesions
  (LOAEL)

NOAEL
Decreased weight
  gain (M)UOAEL)
Hepatic and renal
  pathology

NOAEL
Decreased body
  weight, renal  and
  hepatic pathology
  (LOAEL)

NOAEL
Hepatic pathology
  (LOAEL)

Renal and hepatic
  histopathology
  (LOAEL)

Renal and hepatic
  histopathology
  (LOAEL)
Aida et al. Rat Diet
(1992b); Tobe
et al. (1982)
M,F 2 years 6 (M)
8 (F)
Hepatic vacuol-
ization, serum
chemistry (LOAEL)

                                                 V-57

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        TABLE V-20   Summary  of  Longer-term  Studies  of  Dibromochloromethane
Reference
Species
                               Route
Sex    Duration   (nig/kg/day)
                                                                                       Results
Chu et. al.
(1982b)
NTP
(1985)
                   Rat
                   Rat
          Drinking
          water
          Gavage
          (oil)
Daniel  et  al.
(1990)
NTP
(1985)
                   House
                   Rat
                   Rat
                   Mouse
                             Gavage
                             (oil)
          Gavage
          (oil)
          Gavage
          (oil)

          Gavage
          (oil)
Tobe et  al.
(1982)
                   Rat
                             Diet
M,F    90 days      0.6-52

                   250


M,F    13 weeks     30
                   60

                   125

                   250
                        M.F     13 weeks    15-125
                                           250
M,F    90 days






M,F    2 years


M,F    2 years
50


100

200

40


50


100
                         M,F    2 years      10 (M)
                                            17 (F)
                                            39 (M)
                                            66 (F)
Mild and reversible
   changes (NOAEL)
   Hepatic lesions
   (LOAEL)

NOAEl
Hepatic vacuolation
   (LOAEL)
Decreased body
   weight (males)
Severe hepatic and
   renal  lesions,
   mortality

NOAEL
Fatty liver,  toxic
   nephropathy in
   males  (LOAEL)

Hepatic vacuolization
   (M),  renal lesions (F)
   (LOAEL)
Hepatic and  renal
   lesions
Decreased body weight

Histologic changes
   in  liver (LOAEL)

Liver calcification,
   fatty metamorphosis
   (F)  (LOAEL)
Decreased body
   weight (M,F),
   decreased survival
   (M)

NOAEL

Enzyme changes and
   altered liver
   appearance at
   necropsy
   (LOAEL)
                                                  V-58

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               TABLE  V-21   Summary of Longer-term  Studies of Bromoform

Reference
Chu et al.
(1982b)
Species Route
Rat Drinking
water
Dose
Sex Duration (mg/kg/day)
M,F 90 days 0.6-52
250
Results
Mild and reversible
changes (NOAEL)
Hepatic lesions
. UOAEL)
NTP (1989a)
Tobe et  al.
(1982)
NTP (1989a)
                  Rat        Gavage
                  Mouse      Gavage
                  Rat        Diet
                  Rat        Gavage
                  Mouse      Gavage
M,F    13 weeks    25
       (5 day/wk)  50
M,F
13 weeks     100
(5 day/wk)   200
M,F    2  years      18 (M)
                   30 (F)
                   71 CM)
                   120 (F)
M,F    103 weeks    100
       (5 day/wk)
       103 weeks    100
       (5 day/wk)

       103 weeks    100
       (5 day/wk)
NOAEL
Hepatic vacuolation
   in males UOAEL)

NOAEL
Hepatic vacuolation
   in males (LOAEL)

NOAEL

Enzyme changes and
   altered liver  appear-
   ance at necropsy
   (LOAEL)

Decreased body weight,
   lethargy,  mild
   hepatotoxicity (LOAEL)

NOAEL
                                                                               Decreased body weight,
                                                                                 mild hepatotoxicity
                                                                                 (LOAEL)
                                                 V-59

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        C.  Reproductive and Developmental Effaces
        1.  Chloroform









        Thompson et al.  (1974) studied the effects of chloroform on the




embryonic and fetal development of Sprague-Dawley rats.  In a preliminary




range-finding study, groups of six pregnant females given chloroform in corn




oil by intubation at doses of 79, 126, 300, 316, or 516 mg/kg/day on days 6 to




15 of gestation developed alopecia, rough hair, and eczema.  At levels of




126 mg/kg/day and greater, food consumption and body weight gain were signifi-




cantly suppressed.  Doses of 316 mg/kg/day caused acute toxic nephrosis,




hepatitis, and maternal death, as well as fetotoxicity.  In the main study,




groups of 25 pregnant rats (181 to 224 g) were administered chloroform in corn




oil at doses of 0, 20, 50, or 126 mg/kg/day by oral intubation on days 6 to 15




of gestation.  Dams receiving 50 or 126 mg/kg/day displayed signs of maternal




toxicity (decreased weight gain,  mild fatty changes in the liver).   Fetuses




were removed by Caesarean section 1 or 2 days prior to expected parturition




and examined for external, skeletal, and/or soft tissue abnormalities.  There




were no fetal malformations, but the incidence of bilateral extra lumbar ribs




was significantly (p < 0.05) increased at the high dose.   Fetal weight was




also reduced at the high dose (p < 0.05).  This study identified a NOAEL of




20 mg/kg/day and a LOAEL of 50 mg/kg/day in rats.









        In the same study, Thompson et al. (1974) administered chloroform  (in




corn oil)  to Dutch-Belted rabbits.  In a preliminary range-finding study,




doses of 0, 25, 63, 100, 159, 251, or 398 mg/kg/day were administered to




pregnant rabbits on days 6 to 18 of gestation.  High levels of maternal death




(60% to 100%) were observed at doses of  100 mg/kg/day and above.  Adverse




                                     V-60

-------
effects at 63 mg/kg/day included anorexia, weight loss, diarrhea, abortion.




and one maternal death.  No overt signs of toxicity other than mild diarrhea




and intermittent anorexia were observed in dams dosed with 25 mg/kg/day.  In




the main study, groups of 15 dams (1.7 to 2.2 kg) were dosed by oral




intubation with chloroform at 0,  20, 35, or 50 mg/kg/day on days 6 to 18 of




gestation.  Decreased maternal weight gain was observed in dams given




50 mg/kg/day.  Four high-dose dams died from hepatotoxicity.   Four high-dose




dams aborted, but this was not considered to be a treatment-related effect




because three control animals aborted.  No evidence of maternal toxicity was




noted at 35 mg/kg/day.  Small reductions in body weights were observed in




fetuses from dams administered 20 or 50 mg/kg/day (p < 0.05),  and an increased




incidence of incompletely ossified skull bones (usually parietals) was




observed at 20 ajid 35 mg/kg/day (p < 0.05).   The authors did  not consider




these effects to be evidence of teratogenicity or fetotoxicity.   These data




indicate doses resulting in maternal toxicity are lower than those resulting




in fetotoxicity.  On this basis,  this study identified a NOAEL of 35 mg/kg/day




and a LOAEL of 50 mg/kg/day.









        Ruddick et a-1. (1983) investigated the potential developmental




toxicity of chloroform in groups  of 15 mated rats.  Pregnant dams (8 to




14 animals per dose group) were given 0, 100, 200, or 400 mg/kg chloroform in




corn oil on days 6 to 15 of gestation.  Maternal weight gain was depressed by




at least 20% at all dose levels.   In addition, all dose levels of chloroform




produced maternal liver enlargement, decreased hemoglobin, and decreased




hematocrit.  Levels of serum inorganic phosphorus and cholesterol were ele-




vated in the dams at the highest exposure level.  Fetal weight was decreased




by about 19% at the highest dose level.  There were no fetal malformations,




but sternebra aberrations were observed with a dose-dependent incidence at




                                     V-61

-------
200 mg/kg/day and 400 mg/kg/day.  Interparietal deviations also occurred ac




the high dose.  Although there was a clear increase in the incidence of these




variations, no statistical analysis v;as performed.









        In a study using inhalation exposure, Schwetz et al.  (1974) exposed




pregnant Sprague-Dawley rats (approximately 250 g) to chloroform at 30, 100,




or 300 ppm (147, 488, or 1,466 mg/m3)  for 7 hours daily on days 6 to 15 of




gestation.  The number of animals exposed in the three dose groups was 31, 28,




and 20, respectively.  At the highest dose level, fertility was decreased (15%




compared with 88% to 100% in controls, p < 0.05).  At 100 ppm,  there was an




increase in acaudia (short tail) and imperforate anus (p < 0.05).   At 30 ppm.




there was an increased incidence of delayed skull ossification and wavy ribs




(p < 0.05), but no other adverse developmental effects occurred when compared




to controls.  The authors concluded that chloroform was not highly teratogenic




but was highly embryotoxic.









        Murray et al. (1979) observed teratogenic effects in mice exposed to




chloroform in air.  Groups of 34 to 40 pregnant females were exposed to 0 or




100 ppm for 7 hours/day on days 8 to 15 of gestation.  Exposure to chloroform




resulted in a significant increase (p < 0.05) in the incidence of cleft




palate.  Exposure on gestational days 1 to 7 or 6 to 15 caused reduced litter




size but no malformations, suggesting the possibility that lethality to the




early embryo obscured other effects.  In this study, the cleft palates were




seen predominately in fetuses with retarded growth, suggesting to the authors




that chloroform might have induced an indirect, rather than a direct,




teratogenic effect.
                                     V-62

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        2.  Brominated Trihalomethanes









        Borzelleca and Carchman (1982) conducted a two-generation reproductive




study in ICR Swiss mice.  Nine-week-old mice (10 males and 30 females per dose




group) were continuously maintained on drinking water containing 0, 0.1, 1.0,




or 4.0 mg/mL dibromochloromethane (0, 17, 171, 'or 685 mg/kg/day).  After




35 days on the test solutions, the F/0 mice were randomly mated to produce the




F/la litters.  Rematings to produce F/lb and F/lc generations occurred 2 weeks




after weaning the previous generation.  Thus, the F/0 mice were exposed for a




total of 27 weeks prior to sacrifice and necropsy.  Body weight gain was




significantly reduced in both males and females at the high dose (685 mg/kg/




day) and in females at the mid dose (171 mg/kg/day).  Animals in both of these




groups exhibited^ enlarged livers with gross morphological changes interpreted




by the authors to indicate hepatotoxicity.  Fertility (mating index) was




reduced in the high-dose group for the F/lc generation but not the F/la or




F/lb generations.  Gestational index was significantly reduced for all three




F/l generations at the high dose but not at the lower doses.









        The F/lb generation was treated with test solutions for 11 weeks prior




to mating for the F/2a generation.  Remating to produce the F/2b generation




occurred 2 weeks after weaning.  The F/lb generation was sacrificed after




weaning of the F/2b generation, resulting in a total exposure period for the




F/lb generation of 24 weeks.  Effects on body weight and liver morphology were




very similar to those seen in the F/0 group.  Fertility was reduced at the.




highest dose between the F/lb and F/2a generation, but not in the F/2b




generation.  Gestational index was not affected at any dose.
                                     V-63

-------
        A 21-day litter survival study was performed on all matings.  In




addition, the final matings of both generations  (F/lc and F/2b) were utilized




for dominant lethal and teratology studies.  Parental ingestion of dibromo-




chloromethane at a dosage level of 685 mg/kg/day caused decreased litter size




in all generations (F/la, F/lb,  F/lc, F/2a, and F/2b), a decreased viability




index in four of the five generations (F/la, F/lb, F/lc and F/2a), a decreased




lactation index in the F/2b generation, and a decrease in the postnatal body




weight of the F/2b generation.  Ingestion of 171 mg/kg/day decreased the




litter size of the F/lc generation, decreased the viability index of the F/lb




generation, decreased the lactation index of the F/lb and F/2b generations,




and decreased the postnatal body weight of the F/2b generation.  The only




effect observed at 17 mg/kg/day was decreased postnatal body weight in the




F/2b generation..  No dominant lethal or teratogenic effects were seen in the




F/lc or F/2b generations.









        Based on maternal toxicity (weight loss, liver pathology) and possible




fetotoxicity (decreased pup weight and viability in some generations), this




study identified a NOAEL of 17 mg/kg/day and a LOAEL of 171 mg/kg/day for




dibromochloromethane.









        Ruddick et al. (1983) investigated the potential developmental toxi-




city of bromoform, bromodichlorotnr thane,  and dibromochloromethane in groups of




15 mated rats.  Pregnant dams (9 to 15 animals per dose group) were admini-




stered 0, 50, 100, or 200 mg/kg/day on days 6 to 15 of gestation.  Maternal




weight gain was depressed by over 25% in the high-dose groups for bromodi-




chloromethane and dibromochloromethane.  Relative maternal liver weight was




increased in 'all bromodichloromethane-exposed groups, and the kidney weight




was increased at the highest dose.  There were no fetal malformations, but




                                     V-64

-------
scernebra aberrations were observed with a dose-dependent incidence in all




bromodichloromethane and bromoform groups.  Interparietal deviations also




occurred at the mid and high doses of bromoform.   Although there was a clear




increase in the incidence of these variations, no statistical analysis was




performed.









        NTP (1989b) investigated the effect of bromoform on fertility and




reproduction in Swiss CD-I mice.  Twenty male-female pairs were dosed by




gavage with 0, 50, 100, or 200 mg/kg/day for 105  days.  There was no detect-




able effect of treatment on fertility, litters per pair, live pups per litter,




proportion of pups born alive, sex of live pups,  or pup body weights.   This




study identified a reproductive NOAEL of 200 mg/kg/day.









        D.  Mutagenicitv and Genotoxicity









        1.  Chloroform









        The overall evidence regarding chloroform genotoxicity is




inconclusive.   In vitro.  chloroform has yielded mixed but mainly negative




results in a number of assays of mutagenic activity.  Some of these results,




however, are inconclusive because of inadequacies in experimental protocols,




especially in the failure to use an appropriate (reconstituted) activation




system or to take precautions to prevent the escape of volatilized chloroform




(U.S. EPA 1985a; Rosenthal 1987).  In vitro assays for early evidence of DNA




damage (sister chromatid exchanges or DNA damage  in yeast) and in vivo assays




for chromosomal damage tend to give positive results.  Specific studies are




summarized below.  Unless specified otherwise, S9 fractions were prepared from




the liver of Aroclor 1254-induced male rats.




                                     V-65

-------
        a.  In Vitro Studies









        Chloroform has been reported to be nonmutagenic in both Salmonella




typhimurium and Escherichia goli reverse mutation assays under conditions




designed to reduce chloroform loss through volatilization.  The inclusion of




exogenous metabolic activation derived from the livers or kidneys of untreated




or Aroclor-pretreated mice or rats did not alter these findings.









        Gocke et al. (1981) assessed the mutagenicity of chloroform in .




typhimurium strains TA1535, TAlOO, TA1538, TA98 and TA1537 in the presence and




absence of S9.   Chloroform at levels up to 30 jimol/plate (3,600 /ig/plate) did




not increase the mutation frequency in any tested strain.









        Van Abbe et al. (1982) also tested chloroform mutagenicity in these




five tester strains of S^.  typhimurium.  In a standard mutation assay, there




was no evidence of mutagenicity at 84 ^mol/plate (10,000 ^g/plate) in the




presence or absence of microsomal preparations from the liver or kidney of




rats and mice.  There was also no evidence of mutagenicity in strains TA1535




or TA1538 exposed to a stream of chloroform vapor at 32 mL/hour in the




presence or absence of rat liver S9.









        Van Abbe et al. (1982) also tested chloroform mutagenicity in these




five tester strains of S..  typhimurium. using a closed system to reduce




volatilization of the test material.   There was no evidence of mutagenicity at




84 ^mol/plate in the presence or absence of microsomal preparations from the




liver or kidney of rats and mice.
                                     V-66

-------
        Uehleke et al. (1977) found that 5 mM chloroform was not mutagenic in




. tvphimurium strains TA1535 or TA1538 in the presence or absence of




exogenous metabolic activation.  Liver micrcscmes prepared from male rats and




mice were used as the activation system.  Similar negative results were also




reported by Simmon et al. (1977).









        Kirkland et al.  (1981) investigated the mutagenicity of chloroform in




E. coli over a wide range of concentrations (8.4 x 10"^ to 84 /jmol/plate;  0.1




to 10,000 jig/plate), using a closed system to prevent  chloroform




volatilization.  Negative results were reported in a standard assay and in a




preincubation assay, both in the presence and in the absence of S9 microsomes.









        In contr.ast to the sizable number of negative  results, Varma et al.




(1988) reported that 0.2 or 0.4 /unol/plate chloroform  induced a mutagenic




response in four strains of S_. tvphimurium and the response was lower with




activation than without.   Normal background levels were observed at higher




doses (0.8 to 4.2 /xmol/plate).  This spike in mutation frequency at the low




dose is very unusual, especially since the number of revertants was almost




identical in strains that detect frameshifts and those that detect base




substitutions.  It is possible that the reported data  may have resulted from




cytotoxicity,  although the number of revertants at the norunutagenic doses was




comparable Co background levels.  A methanol/water mixture was used as a




solvent and the authors did not specify whether the assay was conducted in a




closed system.









        Sturrock (1977)  investigated the mutagenic potential of chloroform in




Chinese hamster lung fibroblasts at the 8-azaguanine locus in the absence of




exogenous metabolic activation.  Chloroform was tested as a vapor at




                                     V-67

-------
concentrations of 10,000 to 25,000 ppm.   These concentrations did not produce




any significant increase in the incidence of mutation.









        Kirkland et al.  (1981) did not observe an increase in the frequency of




chromosomal aberrations  or sister chromatid exchanges in human lymphocytes




exposed to 0.21 to 3.35  mM of chloroform.  Incubations  were performed in a




closed system to prevent chloroform volatilization.   However, the positive




control was not run concurrently.









        Morimoto and Koizumi (1983) also used human lymphocytes to test




chloroform for its potential to induce sister chromatid exchanges.  No




activation system was used.   Significant increases were observed at chloroform




concentrations o.f 10 mM  or higher.









        Sobti (1984) assessed the potential of S9-activated chloroform to




induce sister chromatid  exchanges in human lymphocytes.  Microsomes prepared




form the liver of phenobarbital-induced rats were used  as the activating




system.  Significant increases (p < 0.01) in sister  chromatid exchanges were




observed at concentrations of 0.1 mM or higher.









        White et al. (1979)  investigated the ability of chloroform to induce




sister chromatid exchanges in Chinese hamster ovary (CHO) cells in the




presence of an activation system.  Chloroform was tested as a vapor at only




one concentration (7,100 ppm).  This concentration did not significantly




increase the incidence of sister chromatid exchanges.









        Callen et al. (1980) used the D7 strain of Saccharomyces cerevisiae to




study the genotoxicity of chloroform in the absence of S9 activation.




                                     V-68

-------
Incubations were carried out in screw-capped glass tubes to prevent




volatilization of the chloroform.  Positive results were obtained for gene




conversion, gene reversion and mitotic crossing over at the highest dose




(54 mM) .   At lower concentrations (21 mM and 41 mM),  small dose-related




increases in gene convertants were observed.









        Crebelli et al.  (1988) used the fungus Aspergillus nidulans to




investigate the ability of chloroform to cause chromosome malsegregation




(nondisjunctional diploid and haploid segregants).   The incidence of these




events was increased at a very high concentration (20 mM; 0.16% by volume).









        Table V-22 summarizes data on the genotoxic potential of chloroform




from these in vitro studies.









        b.  In Vivo Studies









        Reitz et al.  (1980) studied the potential of chloroform to cause DNA




alkylation in vivo in mice and rats.  Animals were given a single oral dose of




240 mg/kg 14C-chlorofonn  and were  sacrificed four hours later.   DNA was




isolated from the livers and kidnevs and evaluated for label binding.  No




significant increase in DNA alkylation was observed.









        Colacci et al.  (1991) administered chloroform to rats and mice




intraperitoneally,  and observed covalent binding of radiolabeled chloroform to




DNA isolated from the liver, kidney, lung, and stomach.  Chloroform binding to




calf thymus DNA was also observed in vitro in the presence of mouse liver




microsomes, but only at very low levels in the absence of microsomes or




cytosol.




                                     V-69

-------
        TABLE V-22   Summary of In Vitro  Genotoxicity Data  on Chloroform
Endpoint
                  Assay System
                      Results (w/wo
                      Activation)
References
Gene mutation
Salmonella
tvphimurium
TA1535, TA100,
TA1538, TA98,
TAl537b

TA98, TA100,
TA1535, TA1537
TAl538b

TA1535;' TA153-83

TA1535, TA15383

TA1535, TA1537,
TA1538, TA98,
TA1003

TAlOOb

TA1537, TA1535,
TA98, TA100b
                                                          Gocke et al .  1981
                                                          Van Abbe et al. 1982



                                                          Van Abbe et al. 1982
                                                          Uehleke et al. 1977

                                                          Simmon et al. 1977



                                                          Rapson et al. 1980

                                                          Varma et al. 1988

Chromosome
aberration
Sister
chromatid
exchange



DNA damage
Chromosome
malsegregatio-
Ischerichia colia
Chinese hamster
lung fibroblasts3
Human
lymphocytes3
Chinese hamster
ovary cells3
Human
lymphocytes3
Human
lymphocytes'5
Human
lymphocytes'*
Saccharomvces
cerevisiae3
Aspergillus
nidulans3
V-
NTC/-
( vapor)
-/NT
-/NT
(vapor)
-/NT
NT/+
+/NT
NT/+
NT/+
Kirkland et al
Sturrock 1977
Kirkland et al
. 1981
. 1981
White et al . 1979
Kirkland et al
Morimoto and
Koizumi 1982
Sobti 1984
Callen et al .
Crebelli et al
. 1981


1980
. 1988

3Assay was conducted in a closed system.
^he authors did not specify whether or not the assay was conducted in a
 closed system.
Not tested.
                                     V-70

-------
        Gocke et al.  (1981) conducted studies on the potential genotoxicity of




chloroform in two in vivo assay systems.  Ir. the first assay, Drosophila were




exposed to one chloroform concentration (25 mM), and chromosomes were




evaluated for sex-linked recessive lethal mutations.  No increased incidence




of this type of mutation was detected.  In the  clastogenicity assay, mice were




given two doses of 238 to 952 mg/kg chloroform, and polychromatic erythrocytes




from the bone marrow were scored for micronucleus formation.  No significant




increase was detected.   However, the protocol (two treatments separated by 24




hours with sampling at 6 hours after the second treatment) was incomplete.




The sample should have been collected at least 48 hours after the first




treatment (Mavournin et al. 1990).









        Agustin and Lim-Sylianco (1978) investigated the mutagenicity of




chloroform in a host-mediated assay.  Salmonella strains TA1535 and TA1537




were used as indicator organisms and injected into male and female mice.  The




authors reported that male, but not female, mice metabolized chloroform to a




form that was mutagenic in strain TA1537.  Although the results suggest a




positive response in males, incomplete reporting of the data and the




procedures used prohibit a definitive conclusion (U.S. EPA 1985a).









        Fujie et al.  (1990) analyzed chromosome aberrations in bone marrow




cells from Long-Evans rats (three/sex/dose) following oral  (males only) or




intraperitoneal (males and females) exposure to chloroform.  Oral administra-




tion was by gavage in saline for five consecutive days, and the animals were




sacrificed 18 hours after the last dose.  Dose-related increases in the inci-




dence of aberrant cells were observed at all levels (1.2 to 119.4 mg/kg/day),




but the effect was significant  (p < 0.01) only at the high dose.  More pro-




                                     V-71

-------
nounced increases in clastogenic activity were noted when comparable doses




were administered once by intraperitoneal injection.  Regardless of the route,




the predominant types of induced aberrations were chromatid and chromosome




breaks.









        Liang et al. (1983) investigated the ability of chloroform to produce




chromosome aberrations in grasshopper embryos exposed in vivo.  The incidence




of chromosome aberrations was increased in embryos exposed to 620 to




1,200 ^mol/jar (0.05 to. 0.1 mL/jar).









        Land et al.  (1981) exposed mice to 400 or 800 ppm chloroform in air




for 4 hours/day for 5 days, and observed an increase in the frequency of sperm




head abnormalities when compared to the negative controls (p < 0.01).




Although the results suggested a positive response, the appropriateness of the




statistical analysis has been questioned (U.S. EPA 1985a).   Topham (1980) did




not observe any sperm-head abnormalities in mice given 0.037 to 0.37 mg/kg/day




by intraperitoneal injection.









        Morimoto and Koizumi (1983) investigated the potential of chloroform




to cause sister chromatid exchanges in mice.  The mice were given 25 to




200 mg/kg/day chloroform by gavage (in olive oil) for four days.  Significant




increases (p < 0.05) were detected at doses of 50 mg/kg/day or more.









        Vogel and Nivard (1993) found that chloroform at concentrations




ranging from 2,000 ppm to a lethal dose of 16,000 ppm did not increase the




frequency of interchromosomal mitotic recombination at the white locus in




Drosophila melanogaster.
                                     V-72

-------
        Table V-23 summarizes data on the genotoxic potential of chloroform




from these in vivo studies.









        2.  Brominated Trihalomethanes









        A number of in vitro and in vivo studies have been performed to inves-




tigate the genotoxicity of brominated trihalomethanes.  In general, these




studies have yielded mixed results, and in some cases are subject to uncer-




tainty due to the tendency of these compounds to volatilize from the test




systems.  However, a number of positive findings have been reported, both in




vitro and in vivo. for all three compounds.  These data generally indicate




that bromodichloromethane and bromoform are genotoxic.  The data on dibromo-




chloromethane are less conclusive, but are also suggestive of genotoxicity.




Data are summarized in Tables V-24 (bromodichloromethane), V-25 (dibromo-




chloromethane), and V-26 (bromoform).  More detailed descriptions of these




studies are provided below.









        a.  In Vitro Studies









        Simmon and Tardiff (1978) reported that nonactivated bromodichloro-




methane, dibromochloromethane,  and bromoform were mutagenic in S^. tvphimurium




strain TA100 when assayed in a desiccator containing an atmosphere with the




test compound.  The minimal amount added to the dessicator resulting in a




mutagenic response was 600, 57, and 570 jjmol (50, 5, and  50 /tL) for




bromodichloromethane, dibromochloromethane, and bromoform, respectively.









        Zeiger (1990) found that bromoform vapor was mutagenic in S_.




tvphimurium strain TA98 when tested as a vapor in a closed system, but not




                                     V-73

-------
        TABLE V-23  Summary of In Vivo Genotoxicicy Data on Chloroform
Endpoint
Assay System
Results
Reference
DNA alkylation

DNA binding

Gene mutation
Micronuclei

Chromosome
aberrations
Sperm
abnormalities
Sister
chromatid
exchange

Mitotic
recombination
Mouse and rat

Mouse and rat

Drosophila

Host (mouse) mediated
assay (. tvptiimurium
TA 1537)

Host (mouse) mediated
assay (S.. typhimurium
TA1535)

Mouse

Rat
Rat
(intraperitoneal)

Grasshopper
embryo

Mouse
(vapor)

Mouse
(intraperitoneal)

Mouse
Drosophila melanogaster
(vapor)
            Reitz et al. 1982

            Colacci et al.  1991

            Gocke et al. 1981

            Agus t in and
            Lim-Sylianco
            1978

            Agustin and
            Lim-Sylianco
            1978

            Gocke et al. 1981

            Fujie et al. 1990


            Fujie et al. 1990


            Liang et al. 1983


            Land et al. 1981


            Topham 1980
            Morimoto and
            Koizumi 1983
            Vogel and Nivard 1993
aPositive in male mice but not in female mice.
                                     V-74

-------
       TABLE V-24  Summary of Genotoxicity Data on Bromodichloromethane
Endpoint
Assay System
Results (w/wo
 Activation)
  References
In Vitro Studies

Gene mutation
Chromosome
aberration
DNA damage
Sister
chromatid
exchange
Salmonella
typhimurium
TAIOO3

TA98, TAIOO, -
TA1535, TA1537C

TA100C

TA15370

TA1535, TA98,
TA100C

Mouse lymphoma
cellsc

Chinese hamster
fibroblastsc

Chinese hamster
ovary cells0

Saecharomyces
cerevisiae3

Human
lymphocytes3
                  Human
                  lymphocytes3

                  Rat liver cells9

                  Chinese hamster
                  ovary cellsc
    NTb/+
    V-


    V-


    NT/+



    +/NT


    +/NT
Simmon and Tardiff
1978
                  NTP 1987


                  Ishidate et al.  1982

                  Varma et al.  1988

                  Varma et al.  1988


                  NTP 1987


                  Ishidate et al.  1982
NTP 1987; Anderson
et al. 1987

Nestmann and Lee
1985

Morimoto and Koizumi
1983
                                        Sobti 1984
                                        Sobti  1984

                                        NTP  1987; Anderson
                                        et al.  1987
                                     V-75

-------
                            Table V-24 (Continued)

Endpoint
In Vivo Studies
Micronuclei

Chromosome
aberrations

Sister
chromatid
exchange
Assay System Results
Mouse bone
marrow cells (i.p.)
Mouse bone
marrow cells (i.p.)
Rat bone marrow +
cells
Rat bone marrow +
cells (i.p.)
Mouse bone +
marrow cells
References
Ishidate
Hayashi
Fuj ie et
Fuj ie et
Morimoto
1983
et al. 1982
et al. 1988
al. 1990
al. 1990
and Koizumi

aAssay was conducted in a closed system.
kNot tested.
cAuthors did not specify whether or not the assay was conducted in a closed
 system.
dquivocal
                                     V-76

-------
       TABLE V-25  Summary of Genotoxicity Data on Dibromochloromethane
Endpoint
Assay System
Results (w/wo
 Activation)
  References
In Vitro Studies

Gene mutation
Salmonella
typhimurium
TA1003

TA98, TA100, "
TA1535, TA1537C

TA1537, TA1535C
TA98, TA100C

TA100C
    NTb/+
Simmon and Tardiff
1978
                                                          NTP 1985
                                                          Varma et al. 1988
                                                          Varma et al. 1988

                                                          Ishidate et al. 1982
Chromosome
aberration
DNA damage
Sister
chromatid
exchange
Mouse lymphoma
cells3

Chinese hamster
fibroblastsc

Chinese hamster
ovary cellsc

Saccharomyces
cerevisiae3

Human
lymphocytes3
                  Human
                  lymphocytes3

                  Rat liver cellsc

                  Chinese hamster
                  ovary cells0
                                            NT/+
    NT/+



    +/NT


    +/NT
                  McGregor et al. 1991
                  Ishidate et al. 1982
                                                          Loveday et al. 1990
Nestmann and Lee
1985

Morimoto and Koizumi
1983


Sobti 1984
                                        Sobti 1984

                                        Loveday  et al.  1990
                                     V-77

-------
                            Table V-25  (Continued)

Endpoint
In Vivo Studies
Micronuclei

Chromosome
aberrations

Sister
chromatid
exchange
Assay System Results

Mouse bone
marrow cells (i.p.)
Mouse bone
marrow cells (i.p.)
Rat bone marrow +
cells
Rat bone marrow +
cells (i.p.)
Mouse bone +
marrow cells
References

Ishidate
Hayashi
Fuj ie et
Fuj ie et
Morimoto
1983

ec-al. 1982
et al. 1988
al. 1990
al. 1990
and Koizumi

aAssay was conducted in a closed system.
''Not tested.
cAuthors did not specify whether or not the assay was conducted  in  a.  closed
 system.
dNot specified.
                                      V.-78

-------
            TABLE V-26   Summary  of  Genotoxicity  Data  on  Bromoform
Endpoint
Assay System
Results (w/wo
 Activation)
                References
In Vitro Studies

Gene Mutation
Chromosome
aberration
Sister
chromatid
exchange
Salmonella
typhimurium
TA1535, TA1003

TAl535riAl537c
TA100.
TA98, TA98

TA100C
TA98a
TA100, TA15383

Mouse lymphoma
cells0

Chinese hamster
fibroblastsc

Chinese hamster
ovary cellsc

Toadfish
leukocytes3
                  Human
                  lymphocytes0

                  Chinese hamster
                  ovary cells0
NTb/+
    ?"/-

    -/+
    +/+
    -/-
+/-
NT/-



NT/+


-/mixed
                  Simmon and Tardiff
                  1978
                                                          NTP 1989a
                                                          NTP 1989a
                                                          NTP 1989a

                                                          Ishidate et al .
                                                          1982

                                                          Zeiger 1990
                                                          Zeiger 1990

                                                          NTP 1989a
                  Ishidate et al . 1982
                                                          NTP 1989a
                  Maddock and Kelly
                  1980
                                        Morimoto and Koizumi
                                        1983

                                        NTP 1989a
                                     V-79

-------
Table V-26 (Continued)

Endpoint
In Vivo Studies
Micronuclei


Chromosome
aberration


Sister
chromatid
exchange


Sex -linked
recessive
lethal
mutations
Assay System

Mouse bone
marrow cells (i.p.)
Mouse bone
marrow cells (i.p.)
Mouse bone
marrow cells (i.p.)
Rat bone marrow
cells
Rat bone marrow
cells (i.p.)
Mouse bone
marrow cells

Mouse bone
marrow cells (i.p.)
Drosophila



Results References

Ishidate et al. 1982

Hayashi et al. 1988
NTP 1989a

+ Fuj ie et al . 1990
+ Fujie et al . 1990
+ Morimoto and Koizumi
1983

+ NTP 1989a

+ NTP 1989a




aAssay was conducted in a closed system.
''Not tested.
cAuthors did not
system.
dEquivocal
specify whether or not

the assay was conducted in a closed

         V-80

-------
when tested in an open system using a preincubation protocol.  Positive




results were observed at levels of at least 114 ^mol/desiccator (0.01 mL/




dessicator),  in the presence and absence of S9 prepared from rat or hamster




liver.  Bromoform was negative in the closed system with strains TAlOO and




TA1538 +/- rat or hamster liver S9.









        Ishidate et al. (1982) conducted studies on the mutagenicity of




bromodichloromethane,  dibromochloromethane,  and bromoform with .  typhimurium




strain TAlOO.   Increased mutation frequencies were observed with all three




nonactivated compounds, but not with the compounds in the presence of S9.  All




three compounds also induced chromosomal aberrations in Chinese hamster




fibroblasts,  in the presence, but not the absence, of rat liver S9.   The




concentrations tested in these two assays were not reported.









        Varma et al.  (1988) tested bromodichloromethane and dibromochloro-




methane for mutagenicity in J5. typhimurium strains TA1535, TA1537, TA98, and




TAlOO.  Bromodichloromethane at nonactivated concentrations of 2.4 to




3.2 /xmol/plate induced mutations in strain TA1537.  There was no effect in the




other strains.  Dibromochloromethane produced a significantly increased




mutation frequency at the lowest S9-activated dose (0.12 ^mol/plate) in all




four strains.   Dosing with nonactivated dibromochloromethane at this level




also resulted in increased mutation frequencies in strains TA1535 and TA1537.




Higher concentrations had no clear effect on mutation frequency.  This spike




in mutation frequency at the low dose with similar responses in strains that




detect frameshifts and those that detect base substitutions is very unusual.




It is possible that the reported data may have resulted from cytotoxicity,




although the number of revertants at the nonmutagenic doses was comparable to




background levels.




                                     V-81

-------
        NTP (1987) reported that bromodichloromethane was not mutagenic when




tested using a preincubation protocol in S.. typhimurium strains TA1535,




TA153"?, TA98, or TA100 at concentrations reaching cytotoxic levels




(20 /jmol/plate; 3,333 jig/plate).  Testing was done  in the absence of  S9 and in




the presence of S9 prepared from Aroclor-induced male hamster or rat  liver.




NTP concluded that the negative results may have been due to volatilization of




the test compound from the test system.  Bromodichloromethane was not




mutagenic in the mouse lymphoma L5178Y/TK*/"  assay in the  absence  of S9, but




it did induce dose-related increases in forward mutations at S9-activated




concentrations greater than or equal to 2,000 jiM  (300 ^g/mL).  Cytogenetic




tests with CHO cells were reported here and by Anderson et al. (1990).  There




was no evidence of induction of chromosomal aberrations following treatment




with up to 30,00.0 /iM (5,000 jjg/mL) in either the presence or absence  of




exogenous metabolic activation.  There was also no  evidence of sister




chromatid exchanges induced by the nonactivated material.  In the presence of




S9 activation, one of three assays resulted in a positive response  at doses




greater than or equal to 24,400 /xM (4,000 ^g/mL).   However, these results are




difficult to interpret because cytotoxicity was observed at similar levels in




the other trials.









        NTP (1985) reported that dibromochloromethane (0.5 to 50 ^mol/plate;




100 to 10,000 /ig/plate) was not mutagenic in strains TA1535, TA1537,  TA98, or




TAlOO when tested in the presence or absence of Aroclor-induced Sprague-Dawley




rat or Syrian hamster liver S9 fractions.  The volatilization of the  test




compound was again considered to be a possible explanation for the  negative




results.
                                     V-82

-------
        NTP (L989a) studied the genotoxic potential of bromoform in several




test systems.  Concentrations of 0.04 to 13 ^mol/plate (10 to 3,333 fig/plate)




produced no evidence of mutager.icity in j5. typhiguriun: strains TA1535 or




TA1537, with or without exogenous metabolic activation derived from rat or




hamster liver.  Equivocal evidence of mutagenicity was noted in strain TAlOO




(without activation) and in strains TA97 and TA98 (in the presence of liver




microsomes prepared from Aroclor-induced Syrian hamsters).  Exposure of mouse




L5178Y cells to nonactivated bromoform concentrations greater than or equal to




2,300 /iM (200 nL/mL) or S9-activated concentrations of at least 300 ^M




(25 nL/mL) resulted in forward mutations at the thymidine kinase (tk) locus.




One of two laboratories reported increased sister chromatid exchanges in CHO




cells exposed to 3,800 fM (290 jjg/mL) bromoform in the absence of exogenous




activation; neither laboratory observed increases in the presence of S9.  S9-




activated bromoform did not induce chromosome aberrations in CHO cells;




results in the absence of exogenous activation were equivocal.









        Nestmann and Lee (1985) investigated the mutagenicity of bromodichloro-




methane at 12 to 1,200 /JU (0.001 to 0.1 /*L/mL) and dibromochloromethane at 11




to 5,700 jiM (0.001 to 0.5 /zL/mL) in S. cerevisiae strains .07 and XV185-14C in




the presence or absence of S9 activation.  No clear increase in convertants or




in revertants of strain XV185-14C were observed with bromodichloromethane +/-




S9 or S9-activated dibromochloromethane.  Nonactivated dibromochloromethane




produced an increased incidence of gene convertants in strain D7 at




concentrations greater than 1,140 /^M (0.1 jxL/mL), but there was no effect on




revertants.  The high dose was cytotoxic.









        Dibromochloromethane induced mutations at the tk locus of L5178Y mouse




lymphoma cells when tested at concentrations greater than or equal to 480 jiM




                                     V-83

-------
(100 ^g/rnL) in screw-capped tubes.  The material was  tested  only  in  the




absence of S9 activation (McGregor et al.  1991).









        Loveday et al.  (1990) found that dibromochloromethane did not  induce




chromosome aberrations  in CHO cells at S9-activated levels that caused cell-




cycle delay (12,200 /iM; 2,540 jig/mL) or at nonactivated levels that  were  cyto-




toxic with a standard harvest time (6,000  /iM; 1,240 /ig/mL) .  Sister  chromatid




exchange was induced in CHO cells by S9-activated 3,600 jiM (740 jjg/mL) with a




delayed cell harvest, while the nonactivated test material had no effect  up to




cytotoxic levels (1,200 /jM; 247 jig/mL) .









        Morimoto and Koizumi (1983) investigated the  ability of the  brominated




trihalomethanes .to induce sister chromatid exchanges  in human lymphocytes in




vitro in the absence of S9 activation.  All three compounds  (bromodichloro-




methane, dibromochloromethane,  and bromoform) caused  a dose-dependent  increase




in sister chromatid exchanges.   Bromoform  was more potent than bromodichloro-




methane or dibromochloromethane.  The increases were  significant  (p  <  0.05) at




concentrations greater  than or equal to 400 /iM, 400 /jM, and  80 /jM for  bromo-




dichloromethane, dibromochloromethane, and bromoform, respectively.









        The potential of S9-activared bromodichloromethane and dibromochloro-




methane to induce sister chromatid exchanges in vitro was also investigated by




Sobti (1984).   A dose of 100 /iM of either  trihalomethane produced an increased




frequency of sister chromatid exchange in  rat liver cells.   Bromodichloro-




methane produced the same effect in human  lymphocytes at a concentration  of




100 jiM, as did dibromochloromethane at 1 jiM.  Maddock and Kelly  (1980)




reported that bromoform did not induce an  increase in sister chromatid




exchanges in toadfish leukocytes exposed to concentrations of 0.4 to 400  /iM.




                                     V-84

-------
        b.  In Vivo Studies









        Fujie et al. (1990) analyzed chromosome aberrations in bone marrow




from Long-Evans rats (3/sex/dose) following oral (males only) or intraperi-




toneal (males and females) exposure to bromoform, dibromochloromethane,  or




bromodichloromethane.  Oral administration was by gavage in saline for five




consecutive days, and the animals were sacrificed 18 hours after the last




dose.  Bromoform induced a dose-related increase in the incidence of aberrant




cells, with a significant (p < 0.01) increase at 253 mg/kg/day.   The other two




chemicals induced smaller, dose-related increases in breaks.  More pronounced




increases in clastogenic activity were observed following a single intraperi-




toneal dose, with significant (p < 0.05)  effects at 16.4,  20.8,  and 25.3 mg/kg




for bromodichlorpmethane,  dibromochloromethane, and bromoform respectively.




Regardless of the route, the predominant types of induced aberrations were




chromatid and chromosome breaks.









        Hayashi et al.  (1988) measured induction of micronucleated polychro-




matic erythrocytes in ddY mice by intraperitoneal administration of bromodi-




chlorome thane, dibromochloromethane, or bromoform at single doses up to 500,




1,000 and 1,400 mg/kg in corn oil, respectively.  No evidence of clasto-




genicity was observed with any of the chemicals.  However, the sampling time




was not long enough for dibromochloromethane and bromoform, and there was no




clear evidence of toxicity or cytotoxicity in the target tissue.









        Ishidate et al.  (1982) investigated the in vivo clastogenicity of




bromodichloromethane, dibromochloromethane, and bromoform using ddY and MS




mice and Wistar rats.  Animals were administered 125 to 500 mg/kg/day trihalo-




methane in olive oil by intraperitoneal injection, and were sacrificed at  18,




                                     V-85

-------
24, 30, 48, and 72 hours after dosing.  No significant induction of micro-




nucleus formation was reported in mice or rats given any of the brominated




trihalomethanes.









        Morimoto and Koizumi (1983) used male ICR/SJ mice to investigate the




potential of trihalomethanes to produce sister chromatid exchanges i.n vivo.




Animals were given 0, 25, 50, 100, or 200 mg/kg/day of bromodichloromethane,




dibromochloromethane, or bromoform for four days by olive oil gavage.  All




three brominated trihalomethanes were similarly potent and produced a roughly




linear dose - dependent increase in sister chromatid exchange frequency.  These




increases were statistically significant (p < 0.05)  at 50,  25,  and




25 mg/kg/day for bromodichloromethane, dibromochloromethane, and bromoform,




respectively.  The authors noted that the concentrations required to produce




an increased incidence of sister chromatid exchange were on the order of 1,000




to 10,000 times higher than the concentrations typically found in drinking




water, although only short-term exposure periods were investigated.









        NTP (1989a) studied the genotoxic potential of bromoform in several




test systems.  Feeding adul-t male Drosophila with a 1,000-ppm solution of




bromoform increased the frequency of sex-linked recessive lethal mutations but




had no significant effects on reciprocal translocations.  Intraperitoneal




injection of mice with 200 to 800 mg/kg bromoform caused an increase  in sister




chromatid exchange but not in chromosomal aberrations in bone marrow  cells.




NTP concluded that the genotoxicity of bromoform has been demonstrated in a




variety of test systems, both in vivo and in vitro (see above), and indicated




that this property may be involved in the carcinogenicity of bromoform.  This




is consistent with the general observation that sister chromatid exchanges are




usually observed at  lower doses than are chromosome aberrations.




                                     V-86

-------
        E.  Carcinogenicitv









        1.  Chloroform









        Eschenbrenner and Miller  (1945) induced hepatomas in mice with




chloroform.  Three-month-old Strain A mice (historical spontaneous hepatoma




rate of <1% at 16 months of age) were administered chloroform at dose levels




of 150, 300, 600, 1,200, or 2,400 mg/kg in olive oil by gavage (five/sex/




group).  "Chemically pure" chloroform was used, but chemical analysis was not




indicated.  Controls received olive oil only.  The animals were dosed every




4 days for a total of 30 doses  (120 days) and were examined for hepatomas




30 days after the last dose.  Twenty-four hours before necropsy the animals




were given an additional dose of chloroform.  Tissues and organs were examined




histopathologically.   Five other groups of mice (one male and two females per




group) were given single doses of chloroform (same dose levels as used




previously) 24 hours before liver removal.  No males administered doses of at




least 600 mg/kg and no females in the high-dose group survived the study.  All




deaths occurred 24 to 48 hours after the first or second chloroform dose.  All




surviving females dosed with chloroform at 600 or 1,200 mg/kg developed




hepatomas.  Liver necrosis was observed in both sexes in the three highest-




dose groups.  Males in all treatment groups developed kidney necrosis, whereas




kidney necrosis was not apparent  in any females (Table V-27).









        Necrosis was not observed in hepatoma cells.  Hepatomas contained




cords of enlarged liver-like cells that formed disorganized anastomosed




columns.  The hepatomas did not appear invasive and no metastasis was found.




Renal necrosis in males was localized in the areas of the proximal and distal




tubules; glomeruli and collecting tubules appeared normal.  The severity of




                                     V-87

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  TABLE V-27  Liver and Kidney Necrosis and Hepatomas in Strain A Mice
    Following  Repeated  Oral  Administration of Chloroform in Olive  Oil

Chloroform Dose (mg/kg)
Observation
Liver necrosis3

Kidney necrosis

Deaths

Hepatomasb

Sex
F
M
F
M
F
M
F
M
150
0
0
0
+ '
0/5
0/5
0/5C
0/5
300
0
0
0
+
0/5
2/5
0/5
0/3
600 1,200 2,400 Control
+ + + 0
+ + + 0
000 0
+ + + 0
2/5 1/5 5/5 0/5
5/5 5/5 5/5 0/5
3/3 4/4 --d 0/5
0/5

a+ = necrosis.;  0 = no indication of necrosis.
bln animals surviving the dosing regimen.
cPositive occurrences/animals observed.
"^No animals survived.

Adapted from Eschenbrenner and Miller  (1945)
                                  V-88

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renal necrosis was dose related.  The authors speculated that the different




renal responses by males and females to chloroform treatment may have been due




to the unique lining of the Bowman's capsules with flat or cuboidal epithelium




in females and males, respectively (an anatomic sexual dimorphism in mice).









        In the single-dose experiment, there was a sharp distinction between




normal and necrotic cells in the liver.  Doses of 1,200 and 2,400 mg/kg




produced extensive necrosis in all liver lobules, and the 600 mg/kg dose




produced necrosis in some lobes.  U.S. EPA (1985a) stated that the Eschen-




brenner and Miller study (1945) indicated that hepatomas in female mice were




induced at chloroform doses that also produced liver necrosis.  Early




mortality precluded the development of hepatomas in all animals administered




doses that produced liver necrosis.  The observation of kidney necrosis in




males without tumor formation and the lack of necrosis in hepatomas suggested




to the authors that livers in Strain A mice were uniquely sensitive to tumor




induction at necrotizing doses of chloroform, or that there might be other




factors affecting liver tumor formation, in addition to necrosis.  Also,




because a dose of chloroform was given 1 day before sacrifice (a factor which




in itself could have been responsible for producing necrosis), the extent of




necrosis during the last month of observation while the animals were untreated




is not clear.









        The National Cancer Institute (NCI 1976) performed a carcinogenic




bioassay of chloroform on Osborne-Mendel rats.  Chloroform was administered by




oral gavage (in corn oil) to 50 animals/sex five times per week for 78 weeks.




Male rats (52 days old) were administered doses of 90 or 180 mg/kg/day.




Female rats ('52 days old) were initially administered doses of 125 or




250 mg/kg/day, but the doses were reduced to 90 or 180 mg/kg/day after




                                     V-89

-------
22 weeks (average doses of 100 or 200 mg/kg/day).   All animals were sacrificed




after 111 weeks.  Decreases in survival rates and weight gain were evident for




all treated groups.  The incidence of renal epithelial tumors in male rats was




24% in the high-dose groups (p = 0.0016).   An increase in thyroid tumors was




observed in treated female rats, but this finding was not considered




biologically significant.  Table V-28 summarizes tumor frequencies for all




groups in the study.









        NCI (1976) performed a similar carcinogenic bioassay of chloroform in




B6C3F1 mice.  Animals (35 days old)  were administered initial dose levels of




100 or 200 mg/kg/day for males and 200 or 400 mg/kg/day for females.  These




doses were increased after 18 weeks  to 150 or 300  mg/kg/day for males and 250




or 500 mg/kg/day for females (average doses of 138 or 277 mg/kg/day for males,




238 or 477 mg/kg/day for females).  The animals were sacrificed after 92 to




93 weeks.   Survival rates and weight gains were comparable for all groups




except high-dose females.  Significant increases (p < 0.001)  in hepatocellular




carcinomas were observed in all treated groups.  Table V-29 summarizes tumor




frequencies for all groups in the study.  Nodular  hyperplasia of the liver was




observed in many low-dose male mice  that had not developed hepatocellular




carcinomas.









        Roe et al. (1979) studied the carcinogenicity of chloroform in




toothpaste in four strains of mice (C57BL, CBA, CF/1, and ICI) .  In three




different studies, 10-week-old mice  were administered chloroform by gavage




6 days per week for 80 weeks, followed by a 13- to 24-week observation period.




In the first study, ICI mice (52/sex/dose) were given chloroform (in




toothpaste) at 17 or 60 mg/kg/day.  One control group per sex of ICI mice was




given toothpaste without chloroform.  The second study included 260 ICI mice




                                     V-90

-------
                 TABLE V-28 Summary of Tumor Frequencies in Rats
                       Administered Chloroform for 78 Weeks
                          Hepatocellular
                         Carcinomas/Number
                            of Animals
Kidney Epithelial
  Tumors/Number
    of Animals
 Thyroid Tumors/
Number of Animals
Treatment
Control -colony
Control -matched
Low dose
High dose
Sex
M
F
M
F
M
F
M
F
Examined (%)
1/99
0/98
0/19.
0/20
0/50
0/49
1/50
0/48
(1)
(0)
(0)
(0)
(0)
(0)
(2)
(0)
Examined (%)
0/99
0/98
0/19
0/20
4/50
0/49
12/50
2/48
(0)
(0)
(0)
(0)
(8)
(0)
(24)
(4)
Examined
6/99
1/98
4/19
1/19
3/49
8/49
4/48
10/46
(%)
(8)
(1)
(21)
(5)
(6)
(16)
(8)
(22)

Adapted from NCI (1976).
                                       V-91

-------
                 TABLE  V-29   Summary  of  Tumor  Frequencies  in Mice
                       Administered Chloroform for 78 Weeks
                          Hepatocellular
                          Carcinomas/Number
                            of Animals
Kidney Epithelial
   Tumors/Number
    of Animals
  Thyroid Tumors/
Number of Animals
Treatment
Control -colony
Control -matched
Low dose
High dose
Sex
M
F
M
F
M
F
M
F
Examined (%)
5/77
1/80
" 1/18
0/20
18/50
36/45
44/45
39/41
(6)
(1)
(6)
(0)
(36)
(80)
(98)
(95)
Examined (%)
1/77
0/80
1/18
0/20
1/50
0/45
2/45
0/40
(1)
(0)
(6)
(0)
(2)
(0)
(4)
(0)
Examined (%)
0/77
0/80
0/17
0/20
0/48
0/41
0/43
0/36
(0)
(0)
(0)
(0)
(0)
(0)
(0)
(0)

Adapted from NCI (1976)
                                       V-92

-------
given toothpaste without chloroform and a group of 52 mice administered




chloroform at 60 mg/kg/day in toothpaste;.a second control group consisted of




52 untreated mice.  In the third study, four groups oT 32 male mice (one




group/strain) were administered chloroform in toothpaste at a dose of




60 mg/kg/day.  A fifth group (52 male ICI mice) was given the same dose of




chloroform in arachis oil.  This study contained three control groups:  an




untreated control (100 ICI mice), a group given toothpaste without chloroform




(52 mice), and a group given arachis oil only (52 ICI mice).  Body weights




were recorded in each study, and food consumption was estimated in the second




and third studies.  In each study the animals were necropsied, and tumors,




lesions, and selected tissues and organs were examined histopathologically.




Adrenals, kidneys, livers, lungs, and spleens were weighed.








        Differences in survival, body weight, and food consumption between




control and treatment groups were not statistically significant (p > 0.05).




Median survival was approximately 73 weeks in the first and second studies.




In the third study, 52% to 79% of the C57BL and CBA mice and 12% to 31% of the




CF/1 and ICI mice were alive at the end of the observation period.  Liver and




kidney weights were slightly lower (data not reported) in male ICI mice given




chloroform in toothpaste.









        The incidence of tumors did not differ significantly between control




and chloroform-treated groups of male or female C57BL, CBA or CF/1 mice, or




between control and treated female ICI mice.  Increased frequency of kidney




tumors was observed in treated male ICI mice (Table V-30).  Malignant tumors




were identified as hypernephromas, and benign kidney tumors were characterized




as cortical adenomas.  The authors observed a significantly higher  incidence




of moderate to severe kidney "changes" in treated CBA and CF/1 males




                                     V-93

-------
              TABLE  V-30   Kidney  Tumor  Incidence  in Male  ICI  Mice
                            Treated with Chloroform
Dose Group
Numbers of Mice
   Examined
Histologically
 Number of Mice with Kidnev Tumors
Benign       Malignant        Total
First study:

Vehicle-control3
17 mg/kg/dayb
60 mg/kg/day*

Second study:

Untreated control
Vehicle-control3
60 mg/kg/dayf

Third study:
     72
     37
     38
     45
    237
     49
   0
   0
   5C
   1
   6
   7C
0
0
3d
0
0
2d
0
0
8e
1
6
9e
Untreated control
Vehicle -control9
60 mg/kg/dayh
Vehicle -control1
60 mg/kg/dayj
83
49
47
50
48
0
1
2
1
3
0
0
3
0
9C
0
1
5
0
12e

toothpaste-base vehicle without chloroform, eucalyptol or peppermint oil.
bChloroform given in toothpaste base with eucalyptol and peppermint oil.
cStatistically significant versus vehicle-control (p < 0.05).
dStatistically significant versus vehicle-control (p < 0.01).
Statistically significant versus vehicle-control (p < 0.001).
*Chloroform given in toothpaste base without eucalyptol or peppermint oil.
9Toothpaste-base vehicle without chloroform.
hChloroform given in toothpaste base.
'Arachis oil.
^Chloroform given in arachis oil.

Adapted from Roe et al. (1979)
                                     V-94

-------
(p < 0.001,  chi-square test) and moderate to severe kidney disease (p < 0.05.




chi-square test) in treated ICI males compared to controls.  (These results




"ere described by the authors without presentation cf data.)









        Jorgenson et al. (1985) (also detailed in SRI 1985) studied the




carcinogenic activity of chloroform administered at 0, 200, 400, 900, or




1,800 mg/L in drinking water to male Osborne-Mendel rats and female B6C3F1




mice for 104 weeks.   Based on measured water intake and body weights, these




exposures corresponded to time-weighted average doses of 0, 19, 38, 81, or




160 mg/kg/day in rats and 0, 34, 65, 130, or 263 mg/kg/day in mice.  The water




consumption of a second control group included in the study was restricted to




that of the high-dose group.  Group sizes at low doses were adjusted so that a




detectable tumor, response would result at the lowest dose if there were a




linear relationship with dose.   (Group sizes were 330, 150, 50, and 50 for the




low- to high-dose rat groups,  and 430, 150, 50, and 50 for the low- to high-




dose mice groups.)  Chloroform increased the incidence of renal tumors




(metastatic carcinomas, transitional cell carcinomas, tubular cell adenomas,




and a'denocarcinomas and nephroblastomas) in male rats in a dose-related manner




(Table V-31).  The incidences for all kidney tumors were 5/301 (2%), 1/50




(2%), 6/313 (2%),  7/148 (5%),  3/48  (6%), and 7/50 (14%) for the control,




matched control, 200-, 400-, 900-,  and 1,800-mg/L groups, respectively.




Chloroform in the drinking water did not increase the incidence of hepato-




cellular carcinomas in female B6C3F1 mice.  The combined incidence of hepato-




cellular adenomas and carcinomas was 2% in the high-dose group compared to 6%




in the control groups.  The authors speculated that the differences observed




between this study and the NCI (1976) bioassay may be related to differences




in the mode of administration (in drinking water versus in corn oil by




gavage).




                                     V-95

-------
                       Table V-31  Compound-Re la ted Increased Incidences of Neoplasms in Rats and Mice
                                        Exposed  to  Chloroform  in  the  Drinking Water  for  2 Years
<

o>

Animal
Male rat






Female mice



Tissue/Tumor
Kidney
Tubular cell adenoma
Tubular cell adenocarclnoma
Nephroblastoma
Metastatic carcinoma
Transitional cell carcinoma
Combined
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Combined
Control

4/301
0/301
0/301
0/301
1/301
5/301

19/415
2/415
21/415
Matched
Control

0/50
1/50
0/50
0/50
0/50
1/50

0/47
0/47
0/47
Tumor
200
mg/L

2/313
,2/313
5/313
0/313
0/313
1 6/313

8/410
7/410
15/410
Frequency
400
mg/L

3/148
1/148
3/148
1/148
0/148
7/148

8/142
1/142
9/142
900
mg/L

2/48
1/48
0/48
0/48
0/48
3/48

0/47
0/47
0/47
1 , 800
mg/L

5/50
2/50
0/50
0/50
0/50
7/50

0/44
1/44
1/44

        Adapted  from Jorgenson  et  al.  (1985)  and SRI  (1985).

-------
        Tumasonis et al.  (1987) exposed groups of 32 male and 45 female Wistar




rats to chloroform in drinking water until all of the animals died (up to




185 weeks).  The exposure level was 2,900 mg/L for 72 weeks and was reduced to




1,450 mg/L for the remaining 113 weeks.  Based on a graph presented by the




authors,  the average dose over the course of the experiment was probably about




200 mg/kg/day for females and about 150 mg/kg/day for males.  Exposed animals




of both sexes gained significantly less weight than did control animals.




There was a statistically significant  (p < 0.01)  increase in incidence of




hepatic neoplastic nodules in exposed  females compared to control females (25%




versus 0%), but not in males (18% versus 23%).   Hepatic adenofibrosis occurred




in 61% of the males and 85% of the females, but not in control animals.




However,  neoplas.tic nodules and adenofibrosis are not specific pathology




terms,  and it is unclear whether these lesions were actually tumors.




Increases in tumor frequency were not noted in other tissues, although




statistically significant decreases occurred in several tumor types (mammary




tumors and pituitary tumors in females, and lymphosarcomas in males).  This




study is  also limited because the study ran until all of the animals died,




rather than having a scheduled sacrifice.  SLnce the control animals had




shorter lifespans than did the exposed animals, age-related tumors could not




be distinguished from treatment-related ones.









        Voronin et al. (1987) investigated the carcinogenicity of chloroform




in mice when administered in oil or water.  Chloroform produced an increased




incidence of tumors (tissue not specified) in mice given 250 mg/kg/day chloro-




form in oil but not in those given 15  mg/kg/day.   No such increase was




reported in mice given 0.0042 to 42 mg/kg/day chloroform in drinking water.
                                     V-97

-------
Interpretation of this study is limited since the only information available




was obtained from an abstract.









        A number of studies have investigated the carcinogenic potential of




chloroform using initiation-promotion protocols.  Pereira et al. (1985) and




Herren-Freund and Pereira (1987) administered 0, 5, or 20 mg/kg




ethylnitrosourea (EMU) to groups of male and female 15-day-old CD-I Swiss




mice.  At weaning, groups.,of 23 to 45 per dose level were supplied with




drinking water containing 0 or 1,800 ppm chloroform, equivalent to about




0 or 270 mg/kg/day,  assuming consumption of 150 mL/kg/day (Arrington 1972).




After 46 weeks of exposure (at 51 weeks of age), animals were sacrificed and a




complete histopathological examination was performed on liver, lung, kidney,




and all gross le.sions.  In males,  ENU treatment alone resulted in a dose-




dependent production of liver adenomas and hepatocellular carcinomas




(Table V-32).  Subsequent administration of chloroform inhibited the




occurrence of tumors both in animals that did not receive ENU and in those




that did (Table V-31).  No significant effect of chloroform exposure was




observed in female mice.   The authors concluded that, in Swiss mice,




chloroform in drinking water acts as an inhibitor of hepatocarcinogenesis.   In




considering why the results of this study differed from those reported by NCI




(1976), the authors speculated that the difference may be due either to the




toxicokinetic difference between administration of chloroform as a bolus by




gavage in corn oil and continuous dosing in water or, alternatively, to a




possibly synergistic interaction between chloroform and corn oil.









        Klaunig et al. (1986) examined the effects of chronic oral chloroform




exposure on liver and lung tumor incidence in mice.  Thirty-five male B6C3F1




mice (4 weeks old) were initiated by treatment with diethylnitrosamine  (DENA)




                                     V-98

-------
    TABLE  V-32   Effects  of  Chloroform Exposure  on  Liver  Tumors  Initiated  by
                  Ethylnitrosourea  Exposure  in  Male  CD-I Mice

Exposure
ENU CHC13
Sex (mg/kg) (1,800 ppm)
Males 0
0 +
5
5 + ""
20
20 +
Females 0
0 +
5
5 . +
20
20 +
Animals Animals with
with Adenomas Adenomas Carcinomas Carcinomas
(%) per Animal (%) per Animal
5
0
21
5
73
41
0
0
0
4
0
4
0.19
0
0.51
0.04
3.13
1.00
0
0
0
0.24
 0
0.11
5
0
5
0
33
17
0
0
0
0
0
0
0.
0
0.
0
0.
0.
0
0
0
0
0
0
08

10

83 .
21







Adapted from Pereira et al. (1985) and Herren-Freund and Pereira (1987).
                                     V-99

-------
(10 mg/L) in drinking water for 4 weeks, while a second group of 35 received




deionized drinking water.  Each group was subsequently supplied with drinking




water containing 0, 600, or 1,800 mg chloroform/L, corresponding to doses of




about 0, 0.7, or 1.8 mg/kg/day.  Animals were sacrificed after 24 weeks




(10/dose) or 52 weeks (25/dose) and were examined grossly and histologically




for liver and lung tumors.  In mice not initiated with DENA, exposure to




chloroform did not cause an increase in the incidence of these tumors.  In




mice initiated with DENA.,. chloroform inhibited lung and liver tumorigenesis.




These results are shown-in Table V-33.  The authors noted that these results




were different from earlier studies that had reported carcinogenic effects of




chloroform.   Like Pereira et al. (1985), they speculated that the reason




chloroform did not appear to be carcinogenic in'this study was that water (as




opposed to corn .oil) was used as the vehicle.









        Herren-Freund and Pereira (1986) studied the cancer initiating and




promoting activity of chloroform in a short-term assay in rats.   The assay




used an increase in the number of gamma-glutamyltranspeptidase (GGT) foci in




regenerating liver as an indicator of carcinogenicity.  This system has been




found to detect the activity of both hepatic and nonhepatic carcinogens.  Rats




underwent a two-thirds partial hepatectomy, followed 18 to 24 hours later by




administration of the initiator.  Exposure to promoter was begun 7 days after




the hepatectomy and was continued for at.least 10 weeks.  After sacrifice,




liver slices were prepared and examined for the presence of GGT-positive foci




and any other lesions.  Chloroform was negative in both'the initiation assay




(a single [presumably oral] dose of 130 or 260 mg/kg [1.1 or 2.2 mmol/kg] of




chloroform in an unspecified vehicle, followed by 500 mg/L phenobarbital in




drinking water as the promoter) and the promotion assay (a single oral dose of




0.3 mmol/kg of DENA as initiator followed by 1,800 mg/L chloroform in drinking




                                     V-100

-------
           TABLE V-33  Effect of Chloroform Exposure on Liver
                   and Lung  Tumors  in Male  B6C3F1 Mice

Exposure
CHC13
(mg/L)
0
0
600
600
1,800
1,800
DENA Liver Tumor
(10 mg/L) 24 weeks
0/10
+ 7/10
0/10
+ 4/10
0/10
+ 3/10
Incidence
52 weeks
5/25
25/25
3/25
25/25
4/25
20/25
Lung Tumor
24 weeks
0/10
1/10
0/10
0/10
0/10
1/10
Incidence
52 weeks
2/25
18/25
0/25
13/25
0/25
6/25

Adapted from Klaunig et al. (1986).
                                  V-101

-------
water).  When chloroform (1,800 mg/L) was given concurrently with weekly doses




of DENA (8.2 mg/kg),  there was a slight increase in the incidence of liver




tumors and a slight decrease in the mean number cf GGT-foci, but neither of




these effects was statistically significant.









        Demi and Oesterle (1987) administered one oral dose of DENA (8 mg/kg)




to female Sprague-Dawley rats.  One week later, chloroform was administered by




gavage in oil to groups of 4 to 6 rats at doses of 0, 25,  100, 200 or




400 mg/kg, twice per week for 11 weeks.  After this time,  animals were




sacrificed and the livers were examined histochemically for GGT-foci.   No




significant effect was observed in low-dose animals,.but increases in




preneoplastic islands were observed in animals dosed with 100 mg/kg or above.




Chloroform alone, at these doses did not result in increases in preneoplastic




islands.   The observation of a promoting effect in this study, where




chloroform was administered in oil, but not in studies where chloroform was




administered in drinking water, supports the hypothesis that the tumor-




promoting activity of chloroform is vehicle-dependent.









        2.  Brominated Trihalomethanes









        NTP (1987) administered doses of 0, 50, or 100 mg/kg of bromodichloro-




methane in corn oil by gavage to F344/N rats (50/sex/dose), 5 days/week for




102 weeks.  In a similar experiment, B6C3F1 mice (50/sex/dose) were adminis-




tered doses of 0, 25, or 50 mg/kg/day  (males) or 0, 75, or  150 mg/kg/day




(females).  All animals were examined grossly and microscopically for




neoplastic lesions.  As shown in Table V-34, bromodichloromethane caused




compound-related increases in the  incidences of neoplasms of the large




intestine and kidney in male and female rats, the kidney in male mice, and the




                                     V-102

-------
                 TABLE V-34   Tumor  Frequencies  in Rats  and Mice  Exposed to
                       Bromodichloromethane in Corn Oil for 2 Years
Animal
Tissue/Tumor
Tumor Frequency
Male rat
Large intestine3
Adenomatous polyp
Adenocar c inoma
Combined
Kidney3
Tubular cell adenoma
Tubular cell adehocar cinema
Combined
Large intestine and/or kidney combined3
Female rat
Large intestineb
Adenomatous polyp
Adenocarc inoma
Combined
Kidney
Tubular cell adenoma
Tubular cell adenocarcinoma
Combined
Large intestine and/or kidney combined0
Male mouse
Kidneyd
Tubular cell adenoma
Tubular cell adenocarcinoma
Combined
Female mouse
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Combined
Control

0/50
0/50
0/50

0/50
0/50
0/50
0/50
Control

0/46
0/46
0/46

0/50
0/50
0/50
0/46
Control

1/46
0/46
1/46
Control

1/50
2/50
3/50
50 mg/kg

3/49
11/49
13/49

1/49
0/49
1/49
13/49
50 mg/kg

0/50
0/50
0/50

1/50
0/50
1/50
1/50
25 mg/kg

2/49
0/49
2/49
75 mg/kg

13/48
5/48
18/48
100 mg/kg

33/50
38/50
45/50

3/50
10/50
13/50
46/50
100 mg/kg

7/47
6/47
12/47

6/50
9/50
15/50
24/48
50 mg/kg

6/50
4/50
9/50
150 mg/kg

23/50
10/50
29/50

aOne  rat died at week 33 in the low-dose group and was eliminated from the cancer
 risk calculation.
blntestine  not examined in four rats from control group and three rats from high-dose
 group.
C0ne  rat in high-dose group not examined for intestinal tumors had kidney tumors.
dln the  control group,  two mice died during the first week, one mouse died during
 week, nine and one escaped in week 79.  One mouse in the  low-dose group died  in the
 first week.  All of these mice were eliminated from the cancer risk calculations.

Adapted from NTP  (1987).
                                           V-103

-------
liver in female mice.  The authors noted that neoplasms of the large intestine




and kidney are uncommon tumors in F344/N rats and B6C3F1 mice, and concluded




that under the conditions of these 2-year gavage studies, there was clear




evidence of carcinogenic activity for male and female rats and mice.









        Tumasonis et al. (1987) exposed groups of 58 male and female Wistar




rats to bromodichloromethane in drinking water until all of the animals died




(185 weeks).   The exposure, level was 2,400 mg/L for 72 weeks and was reduced




to 1,200 mg/L for the remaining 113 weeks.  Based on a graph presented by the




authors, the average dose over the course of the experiment was probably about




150 mg/kg/day for females and about 100 mg/kg/day for males.  Exposed animals




of both sexes gained significantly less weight than control animals.  There




was a statistically significant (p < 0.01) increase in incidence of hepatic




neoplastic nodules in exposed females compared to control females (32% versus




0%),  but not in males (13% versus 23%).  Increases in tumor frequency were not




noted in other tissues,  although statistically significant decreases occurred




in several tumor types (mammary tumors and pituitary tumors in females, and




lymphosarcomas in males).  This study is limited because the study ran until




all of the animals died, rather than having a scheduled sacrifice.  Since the




control animals had shorter lifespans than did the exposed animals, age-




related tumors could not be distinguished from treatment-related ones.









        Aida et al. (1992b) administered bromodichloromethane to Sic:Wistar




rats (40/sex/dose for the treatment groups and 70/sex/dose for the controls)




at dietary levels of 0%, 0.014%, 0.055%, or 0.22% for up to 24 months.  The




test material was microencapsulated. and mixed with powdered feed.  Based on




the mean food intakes, the mean doses were 0, 6.1, 25.5, or 138.0 mg/kg/day




(males) and 0, 8.0, 31.7, or 168.4 mg/kg/day (females).  The observed  liver




                                     V-104

-------
tumors were three cholangiocarcinomas and two hepatocellular adenomas in the




high-dose females, one hepatocellular adenoma in a control female, one




cholar.giocarcinoma in a high-dose male, and one hepatocellular adenoma each in




a low-dose male and a high-dose male.  Based on these results, the study




authors concluded that there was no clear evidence that microencapsulated




bromodichloromethane in feed was carcinogenic in Wistar rats.  The study




authors further noted that the first indication of liver changes in this study




was fatty degeneration, although bile duct proliferation and cholangiofibrosis




were also observed.  This was in contrast with the usual progression leading




to cholangiocarcinomas of bile duct cell proliferation, cholangiofibrosis,




benign cystic cholangiomas, cholangiofibromas,  and eventually




cholangiocarcinomas.









        Theiss et al.  (1977) examined the carcinogenic activity of bromoform




and bromodichloromethane in Strain A mice.  Male animals, 6 to 8 weeks old,




were injected intraperitoneally up to three times weekly over a period of




8 weeks.  Three dose levels (20 mice/group) were used (4, 48, or 100 mg/kg




bromoform and 20, 40,  or 100 mg/kg bromodichloromethane).  A positive and a




negative control group each contained 20 animals.  Mice were sacrificed




24 weeks after the first injection and the frequency of lung tumors in each




test group was compared with vehicle-treated controls.  Bromoform produced a




significant increase (p = 0.041) in tumor frequency only at the intermediate




dose.  Bromodichloromethane produced a marginally significant (p = 0.062)




increase at the high dose.  U.S. EPA (1980b) concluded that these results were




suggestive of carcinogenic activity but were not an adequate basis for the




development of a risk assessment.
                                     V-105

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        In a carcinogenesis study reported by NTP  (1985), dibromochloromethane




was administered by gavage in corn oil five times per week for 104 weeks to




groups of 50 male and 50 female F344/N rats at 0, 40, or 80 mg/kg/day and to




groups of 50 male and 50 female B6C3F1 mice for 105 weeks at doses of 0, 50,




or 100 mg/kg/day.  Survival of dosed male and female rats and female mice was




comparable to that of the corresponding vehicle-control groups.  High-dose




male mice had lower survival rates than the vehicle controls.   At week 82,




nine high-dose male mice died of an unknown cause.  High-dose male rats and




male and female mice had lower body weights compared with the vehicle




controls.  An inadvertent overdose of dibromochloromethane given to low-dose




male and female mice at week 58 killed 35 male mice, but apparently did not




affect the female mice.   The low-dose male mouse' group was therefore




considered to be. inadequate for analysis of neoplasms.  Compound-related




nonneoplastic lesions were found primarily in -the livers of male and female




rats (fatty metamorphosis and ground-glass cytoplasmic changes),  male mice




(hepatocytomegaly,  necrosis, fatty metamorphosis) and female mice (calcifi-




cation and fatty metamorphosis).   Nephrosis was observed in male mice and




female rats.  A summary of tumor frequencies in the mice is presented in




Table V-35.  Administration of dibromochloromethane significantly increased




the incidence of hepatocellular adenomas and the combined incidences of




hepatocellular adenomas or carcinomas in high-dose female mice.  The incidence




of hepatocellular carcinomas was significantly increased in high-dose male




mice.  Significant increases in the combined incidence of hepatocellular




adenomas or carcinomas was detected by the life table test, but not by the




incidental tumor test.  Negative trends in several common tumors were found in




dosed animals in the 2-year study.  These neoplasms included fibroadenomas of




the mammary gland and endometrial stromal polyps of the uterus in female rats
                                     V-106

-------
         TABLE V-35  Frequencies of Liver Tumors in Mice Administered
                Dibromochloromethane in Corn Oil for 104 Weeks

Treatment
(mg/kg/day)
Vehicle Control

50

100

Sex
M
F
M
F _.'
M.
F

Adenoma
14/50
2/50
_ _a
4/49
10/50
ll/50b

Carcinoma
10/50
4/50

6/49
19/50b
8/50
Adenoma or
Carcinoma (combined)
23/50
6/50

10/49
27/50=
19/50d

aMale low-dose group was inadequate for statistical analysis.
bp <  0.05 relative to controls.
cp <  0.01 (life table analysis);  p = 0.065 (incidental tumor test) relative
 to controls.
^ <  0.01 relative to controls.

Adapted from NTP  (1985).
                                     V-107

-------
and malignant lymphomas in male mice.  There was no evidence of carcino-




genicity in rats administered dibromochloromethane.   Fatty metamorphosis and




ground-glass cytoplasmic changes observed in the rat livers were,  however,




related to administration of dibromochloromethane.









        The authors concluded that this study provided equivocal evidence of




dibromochloromethane carcinogenicity in male B6C3F1 mice, some evidence of




carcinogenicity in female. B6C3F1 mice and no evidence of carcinogenicity in




male or female F344/N rats.









        NTP (1989a) exposed groups of 50 male B6C3F1 mice by gavage (corn oil)




to doses of 0, 50, or 100 mg/kg/day of bromoform for 103 weeks (5 days/week).




Groups of 50 females received doses of 0, 100,  or 200 mg/kg/day.   At




termination, all animals underwent gross necropsy and thorough histological




examinations of tissues.  An increased incidence of follicular cell hyper -




plasia was.noted in high-dose females, but no increase in tumors was reported




in any tissue in any group.   A decreased incidence of lung tumors was noted in




males.   The NTP concluded there was no evidence of carcinogenic activity in




male or female mice.









        NTP (1989a) exposed groups of 50 male and 50 female F344/N rats to




bromoform by gavage for 103 weeks (5 days/week) at doses of 0, 100, or




200 mg/kg/day.  At termination, all animals were necropsied, and a thorough




histological examination of tissues was performed.  Adenomatous polyps or




adenocarcinomas of the large intestine were noted in three high-dose male




rats, eight high-dose female rats, and one low-dose female rat (Table V-36).




Despite the small number of tumors found, the increase was considered to be




significant because these tumors are very rare in the rat.  The NTP concluded




                                     V-108

-------
Animal
               TABLE V-36  Tumor Frequencies in Rats Exposed to
                       Bromoform in Corn Oil  for  2  Years
                                                       Tumor Frequency
Tissue/Tumor
Control   100 mg/kg   200 mg/kg
Male rat


Female rat


Large intestine
Adenocar c inoma
Polyp (adenomatous)
Large intestine
Adenocarc inoma
Polyp (adenomatous)

0/50
0/50

0/48
0/48

0/50
0/50

0/50
1/50

1/50
2/50

2/50
6/50

Adapted from NTP (1989a).
                                     V-109

-------
that there was some evidence for carcinogenic activity in male rats and clear




evidence in female rats.









        Herren-Freund and Pereira (1986) used the rat liver GGT-foci assay to




study the initiating activity of oral exposure to bromoform.  The authors




reported that bromoform at 250 mg/kg (1 mmol/kg) in an unspecified vehicle did




not initiate GGT-foci in this test.









        F.   Summary









        1.   Health Effects of Acute and Short-Term Exposure of Animals









        Large oral doses of trihalomethanes are lethal to laboratory animals.




Reported acute LD50 values  range  from 119  to  2,000 mg/kg  for chloroform,  450




to 969 mg/kg for bromodichloromethane, 800 to 1,200 mg/kg for dibromochloro-




methane, and 1,388 to 1,550 mg/kg for bromoform.  Death from acute high-dose




trihalomethane exposure was usually found to be due to central nervous system




depression and cardiac effects,  and .was usually accompanied by histopatho-




logical changes in the liver and kidney.









        Acute oral exposure to sublethal doses of trihalomethanes can also




produce effects on the liver, kidney and central nervous system.  In mice,




single oral doses of 60 to 89 mg/kg chloroform produced kidney damage, with




doses of 140 to 250 mg/kg producing liver damage.  Organ damage was character-




ized by fatty infiltration, cellular necrosis, vacuolization, enzyme level




changes, and/or organ weight changes.  Ataxia and sedation were noted in mice




receiving 500 mg/kg chloroform,  500 mg/kg bromodichloromethane,  500 mg/kg




dibromochloromethane, or 1,000 mg/kg bromoform.




                                     V-110

-------
        Short-term exposures of  laboratory animals to trihalomethanes has been




observed to cause effects on the  liver, kidney, central nervous system, and




immune system.  Hepatic effects,  including organ weight changes, elevated




serum enzyme levels, and histopathological changes, were reported in mice




and/or rats administered 37 to 290 mg/kg/day chloroform, 148 to 250 mg/kg/day




bromodichloromethane, 147 to 500  mg/kg/day dibromochloromethane, or 187 to




289 mg/kg/day bromoform for 14 to 30 days.  Kidney effects, characterized by




decreased p-arainohippurate uptake, histopathological changes, and organ weight




changes, were reported in mice and/or rats administered 37 to 148 mg/kg/day




chloroform, 148 to 600 mg/kg/day  bromodichloromethane, 147 to 500 mg/kg/day




dibromochloromethane, or 289 mg/kg/day bromoform for 14 days.  Hyperactivity




and/or a decreased operant response were observed in mice and/or rats after




ingesting 100 to. 600 mg/kg/day bromodichloromethane, 400 mg/kg/day dibromo-




chloromethane, or 100 mg/kg/day bromoform for 14 to 60 days.









        2.  Health Effects of Longer-Term Exposure of Animals









        The predominant effects of longer-term oral exposure to trihalo-




methanes occur in the liver and kidney.  The .effects produced on these two




organs are similar in nature to  those described for short-term exposures, with




liver appearing to be the most sensitive target organ.  Hepatic effects were




reported in mice and/or rats administered 15 to 180 mg/kg/day chloroform, 6 to




300 mg/kg/day bromodichloromethane, 39 to 250 mg/kg/day dibromochloromethane,




or 187 to 250 mg/kg/day bromoform.  In general, these dose ranges are slightly




lower than those reported to'cause effects following short-term exposures.




Renal effects were reported in mice and/or rats administered 25 to




'300 mg/kg/day bromodichloromethane or 250 mg/kg/day dibromochloromethane.
                                     V-lll

-------
        3.  Reproductive/Developmental Effects in Animals









        Data concerning the developmental effects of trihalomethanes indicate




that these chemicals are toxic to the mother and fetus at high doses and




suggest that reproductive and developmental effects may occur as well.  Signs




of maternal toxicity (decreased body weight and changes in organ weight) were




reported in rats, rabbits and/or mice administered 50 to 100 mg/kg/day chloro-




form, 200 mg/kg/day bromodichloromethane,  or 171 to 200 mg/kg/day dibromo-




chloromethane.   Fetotoxicity,  as indicated by decreased fetal body weights,




was evident in the offspring of rats administered 121 to 400 mg/kg/day




chloroform or in mice administered 685 mg/kg/day dibromochloromethane.  Oral




exposure of animals to trihalomethane has caused variations, mainly in the




skeletal system..  Delayed ossification and sternebral aberrations have been




reported in rats and/or rabbits administered 20 to 200 mg/kg/day chloroform,




50 to 200 mg/kg/day bromodichloromethane or 50 to 200 mg/kg/day bromoform.




The study authors generally considered these effects to be secondary to




maternal toxicity.  Malformations (cleft plate, imperforate anus, acaudia,




delayed ossification) have been observed in inhalation studies in which mice




and/or rats were exposed to 30 to 100 ppm chloroform, suggesting that




chloroform may be weakly teratogenic.









        Two studies were located which investigated the effects of trihalo-




methanes on reproduction.  In one study, oral doses of 685 mg/kg/day of




dibromochloromethane administered to mice for two generations led to decreased




fertility and gestational indices.  In the second study, doses of




200 mg/kg/day bromoform had no effe.ct on the fertility of mice.
                                     V-112

-------
        4.  Mutagenicicy and Genotoxicity Studies









        In vitro and in vivo studies or> the mutagenic a.nd genotoxic potential




of the trichloromethanes have yielded mixed results.  Interpretation of the




overall weight of evidence from these studies is complicated by the use of a




variety of testing protocols, different strains' of test organisms, different




activating systems, different dose levels, different exposure methods (gas




versus liquid), and in some cases, problems due to evaporation of the test




chemical.   Overall, a majority of studies yielded positive results for




bromoform and bromodichloromethane,  and evidence of mutagenicity is considered




adequate for these chemicals.  Studies on the mutagenicity of dibromochloro-




methane and chloroform were mixed, and the overall evidence for mutagenicity




of these two chemicals is judged to be inconclusive.









        5.  Carcinogenicity Studies in Animals









        The carcinogenic potential of each of the four trihalomethanes has




been investigated in chronic oral exposure studies in animals.  Ingestion of




chloroform in oil has been found to cause liver tumors in male and female




mice, but these tumors were not detected in mice exposed to chloroform in




drinking water.  Renal tumors were detected in male rats exposed to chloroform




in either oil or. water, and renal tumors have been reported in male mice




exposed to chloroform in a toothpaste base.









        Ingestion of bromodichloromethane in oil has been found to cause liver




tumors in female mice, renal tumors in male mice and in male and female rats.




and tumors of the large intestine in male and female rats.  Ingestion of




dibromochloromethane in oil has been found to cause liver tumors in male and




                                     V-113

-------
female mice,  and ingestion of bromoform in oil has been found to cause




intestinal tumors in male and female rats.
                                     V-114

-------
VI.     HEALTH EFFECTS IN HUMANS









        A.  Clinical Case Studies









        The mean lethal oral dose of chloroform in humans is estimated to be




about 630 mg/kg (Gosselin et al. 1976).  Fatalities may occur at doses as low




as 211 mg/kg, with death attributable to respiratory or cardiac arrest (U.S.




EPA 1985a).









        Schroeder (1965) reported a case history of a 27-year-old white male




who drank 4 fluid ounces of chloroform.  Assuming a body weight of 70 kg, the




approximate dose ingested was 2,500 mg/kg.  He was deeply unconscious and




cyanosed, and relaxation of his jaw had obstructed his upper respiratory




tract.  His pupils were dilated and did not react to light.  The patient




responded to medical treatment which included lavage.  Blood urea reached high




levels in the first few days and then returned to normal.  Urinary output was




scanty during the first 2 days, and urinalysis indicated albuminuria,




glucosuria,  ketonuria,  and the presence of bilirubin, red cells, and granular




casts.  Excessive excretion of leucine, alanine,  glutamine, glutamic acid,




serine, and tyrosine occurred during the acute phase.  Liver function tests




indicated elevated levels of bilirubin, AP, and SCOT, indicating liver damage.




Clinical sequelae included slight jaundice and an enlarged liver.  A liver




biopsy was performed 26 days later; the histological appearance of the




specimen was reported as consistent with recovering from toxic  liver damage




(no other data were provided in this report).









        In the past, oral dosing with bromoform was used as a sedative for




children with whooping cough.  Typical doses were usually around one drop




                                     VI-1

-------
 (about 180 mg), given three to six times/day  (Burton-Fanning 1901).  This




 dosing usually resulted in mild sedation in children, although a few rare




 instances of death or near-death were reported (e.g., Dwelle 1903; Benson




 1907).  These cases were believed to be due to accidental overdoses.  Based on




 these clinical observations, the estimated lethal dose for a 10- to 20-kg




 child is probably about 300 mg/kg, and the LOAEL for mild sedation is about




 54 mg/kg/day.









        No clinical case studies relevant to  the ingestion of bromodichloro-




 methane or dibromochloromethane were located.









        B.  Epidemiological Studies









        Challen et al.  (1958) studied the inhalation exposure of industrial




workers to chloroform in a confectionary firm in England.  In 1950, the




wqrkers exposed to chloroform vapor given off during the production of




 lozenges were placed on a reduced work week to alleviate complaints of




 lassitude, flatulence,  dry mouth,  thirst, depression, irritability, and




 "scalding" micturition.   This action was not  successful and the employees




refused to work on that particular process.   In 1954, a new team of operators




was engaged, and in 1955,  the firm installed  an exhaust ventilation system,




 after which manufacturing proceeded without interruption.









        Within this study, one group of eight employees, "long service




operators," refused to continue in the lozenge department after experiencing




 the previously described symptoms.  This group of workers had been observed




 staggering about the work area when exposed to chloroform vapor in concen-




 trations ranging from 376 to 1,158 mg/m3.   After terminating work in the




                                     VI-2

-------
lozenge department, this group reported experiencing nausea after even short




exposures to chloroform.









        A second group of nine employees, the "short service operators,"




replaced the "long service operators" and worked in locations where the




chloroform concentrations ranged from 112 to 347 mg/m3.   Two of these nine




employees did not report unpleasant experiences from chloroform exposure.




Among the other seven, fiwe reported dryness of the mouth and throat at work;




two were subject to lassitude in the evening; one complained of lassitude and




flatulence at work; and the two others experienced similar symptoms to those




of the "long service operators."









        A third group of five employees who worked in other departments of the




firm served as controls and exhibited no symptoms.  Tests of the liver




function (thymol flocculation, direct van den Bergh, and indirect serum




bilirubin),  clinical examinations and urinary urobilinogen measurements failed




to show significant differences among the three groups of workers.









        Bomski et al.  (1967) reported liver injury from inhalation exposure to




chloroform among workers in a pharmaceutical factory in Poland.  The




concentration of chloroform ranged from 9.8 to 1,002 mg/m3.   Sixty-eight




workers were exposed to chloroform for 1 to 4 years and were still in contact




with chloroform; 39 had been exposed to chloroform previously, but were no




longer exposed; 23 had viral hepatitis with jaundice 2 to 3 years earlier and




were designated as posticteric controls and were working in a germ-free area:




and 165 worked in a germ-free area and had no history of viral hepatitis




(controls).   Blood pressure, blood morphology, urinalysis, blood albumin,




serum protein, thymol turbidity, zinc sulfate turbidity, urobilinogen, SCOT




                                     VI-3

-------
and SGPT were measured in all workers; Che "Takata-Ara" sulfate (colorimetric)




test was performed, and a complete medical history was taken.  Sixty workers




were hospitalized for determination of bromosulfophthalein (BSP) clearance and




urinary urobilinogen.









        The frequency of viral hepatitis and jaundice among the 68 pharmaceu-




tical workers currently exposed to chloroform was compared with that of a




group of city inhabitants, 18 years of age and older.  In 3 successive years,




the incidence of viral hepatitis among the pharmaceutical workers was




significantly higher than the incidence of hepatitis within the city group.




The authors suspected that the toxic liver changes that occurred as a result




of chloroform exposure promoted a viral infection; however, the incidence of




viral hepatitis among the other groups of plant workers was not reported.  The




majority of the workers exposed to chloroform during this study complained of.




headaches, nausea, belching and loss of appetite.  Among the 68 workers




working with chloroform,  19 cases of splenomegaly were reported; none were




observed in the controls.  The frequency of enlarged livers (25%) among the




chloroform-exposed workers exceeded that of the other two groups (13% and 9%).




In 3 of the 17 chloroform workers with enlarged livers, toxic hepatitis was




diagnosed on the basis of elevated serum enzyme activities and elevated serum




gamma globulin.  In the remaining 14 cases of liver enlargement, fatty liver




was diagnosed.









        Kramer et al. (1992) conducted a population-based case-control




analysis to determine if exposure to trihalomethanes in drinking water is




associated with low birthweight, prematurity, or  intrauterine growth




retardation (lower than the 5th percentile of weight for gestational age).  A




separate analysis was conducted for each endpoint, using five randomly




                                     VI-4

-------
selected controls for each affected newborn.  Data were collected from Iowa




birth certificates from January 1, 1989, to June 30, 1990; the study




population was restricted to residents of small towns where all of the




drinking water was derived from a single source (surface water, shallow wells,




or deep wells).  Exposure data were based on a 1987 municipal water survey;




birth certificate data from 1987 were not used because data on maternal




smoking status first became available in 1989.  The study authors adjusted for




maternal age, number of previous children,  marital status, education, adequacy




of prenatal care, and maternal smoking.  A significant association was




observed between exposure to water chloroform levels of at least 10 jig/L and




intrauterine growth retardation (odds ratio = 1.8,  95% confidence interval =




1.1-2.9).  Associations were also observed between exposure to at least




10 fig/L chloroform and low birthweight (odds ratio = 1.3), exposure to




intermediate levels of chloroform (1 to 9 ^g/L) and intrauterine growth




retardation, and exposure to at least 10 ^g/L bromodichloromethane and




intrauterine growth retardation (odds ratio = 1.7).  However, the confidence




interval in these cases included one, indicating that the increases were not




statistically significant.  The elevated risk of intrauterine growth retar-




dation associated with high chloroform levels remained when only chlorinated




water sources were included.  The study authors could not control directly for




the presence of other chemicals.  However,  the effect remained when the only




water source was deep wells.  Since deep wells are separated from groundwater,




they are less likely to be contaminated by pesticides and other chemicals,




aside from disinfectant byproducts.  The study authors noted that the




association with chloroform may result at least partially from chloroform




acting as a marker for other organic halides.  They also noted that ambient




conditions may- have led to higher trihalomethane levels in the water from  1989
                                     VI-5

-------
to 1990 than in 1987, so the relative chloroform concentrations should be




interpreted qualitatively rather than quantitatively.









        Shaw et al.  (1991) conducted a case-control study of 141 mothers of




children born with a cardiac anomaly and 176 controls.  No association was




found between a first trimester residence that received chlorinated water, or




drinking of water potentially containing trihalomethanes,  and babies born with




cardiac anomaly.  This study was reported in an abstract.









        A number of epidemiological studies  suggest there may be an associ-




ation between water chlorination and cancer mortality rates (see Table VI-1).




These studies are discussed in more detail in the Drinking Water Criteria




Document on Chlorine (EPA 1994).  It cannot be concluded from these studies,




however, that trihalomethanes are carcinogenic-in humans,  since chlorinated




water contains many chemical by-products in addition to the trihalomethanes.




Morris et al. (1992) conducted a meta-analysis of nine case-control studies




and one cohort study investigating an association between chlorinated water or




trihalomethane exposure and cancer.  The overall relative risk estimate for




bladder cancer was 1.21 (95% CI:  1.09, 1.34), and the risk estimate for




rectal cancer was 1.38 (95% CI:  1.01, 1.87).  Although objective criteria




were used to assess  the quality of the studies and a pre-determined protocol




was used for the analysis, it is unclear whether meta-analysis can be




productively applied to observational epidemiological studies.









        C.   High-Risk Populations









        No data were located that indicate any human subpopulation may be at




greater risk from exposure to trihalomethanes than the general population,




                                     VI-6

-------
       TABLE VI-1   Epidemiological  Studies  Investigating an Association
                     Between Cancer and Chlorinated Water
Reference
     Study Description
      Observation
Alavanja et al.
(1978)
Case control study in seven
New York State counties.
Cantor et al.
(1978)
Hogan et al.
(1979)
Ecological study using age-
standardized cancer mortality
rates,  1968-71; and halomethane
levels  from U.S. EPA surveys.
Ecological study using NCI
cancer mortality data 1950-
1969.  Chloroform levels in
finished drinking water
from U.S. EPA surveys.
Struba (1979)
Brenniman et
al. (1980)
Case-control study of mortality
in North Carolina, 1975-1978.
Case-control study in 70
Illinois communities, 1973-76.
Questionnaires sent to water
treatment plants to verify
1963 inventory data on chlorine
levels.
Greater risk of
gastrointestinal and
urinary tract cancer
mortality, both sexes,
in chlorinated water
areas of the counties.

Strongest correlation
between bromine -
containing trihalo-
methanes and bladder
cancer.

Significant positive
correlations between
chloroform levels and
cancer mortality in
white females for
bladder, rectum and
large intestine; in
white males for stomach
cancer.

Small but significant
odds ratios for rectum,
colon and bladder
cancers in rural areas
but not in urban areas.

Statistically signifi-
cant relative risks of
cancer of gall bladder,
large intestine, and
total gastrointestinal
and urinary tract  in
females served by
systems with chlorinated
versus.nonchlorinated

- continued
                                     VI-7

-------
                            Table VI-1 (Continued)
Reference
        Study Description
         Observation
Brenniman - continued
Gottlieb et
al. (1981)
Young et
al. (1981)
Cragle et
al. (1985)
Young et
al. (1987)
Morris et
al. (1992)
Case-control study using
mortality data in Louisiana
and estimations of exposure.
Case-control, State of
Wisconsin, 1972-1977.
Questionnaires sent to
waterworks superintendents on
chlorine content.

Case-control study using
colon cancer cases from
seven hospitals in North
Carolina.
Case-Control study of colon
cancer cases in Wisconsin.
Water consumption was deter-
mined by interview, and
chloroform levels by
historical records and
measurement

Meta-analysis of nine
case-control studies and one
cohort study analyzing cancer
and consumption of chlorinated
water or water containing high
significant increase in
chloroform levels.
ground water.   Due to
many uncontrolled
confounding factors,
authors concluded that
chlorination was not a
major factor in the
etiology of gastro-
intestinal and urinary
tract cancers.

Rectal cancer
significantly
elevated with
respect to surface
or Mississippi River
water consumption.

Colon cancer showed
significant (p < 0.05)
association with
chlorine intake in all
three dosage categories

Consumption of
chlorinated water
strongly associated
with colon cancer,
above age 60.

No association found
between trihalomethane
exposure and colon
cancer incidence.
Statistically signifi-
cant relative risk of
rectal cancer and
bladder cancer in
exposed groups.   No
colon cancer.
                                     VI-8

-------
although studies in animals suggest that adult males may be at a greater risk




from chloroform exposure than young males or females (Culliford and Hewitt




1957; Eschenbrenner and Miller 1945).   Also, studies in animals suggest that




ethanol may potentiate the hepatotoxic effects of chloroform (Klaassen and




Plaa 1966,  1967a),  so alcoholics may be at increased risk.   As described in




Chapter VII, chloroform-induced liver hepatotoxicity was potentiated in a rat




model of diabetes (Hanasono et al. 1975).  This suggests that diabetics may




also be at increased risk.









        D.   Summary









        In a case study of a young man who ingested 4 ounces of chloroform (a




dose of about 2,.500 mg/kg),  prominent clinical findings included jaundice, an




enlarged liver, increased serum levels of bilirubin, alkaline phosphatase and




SCOT along with albuminuria, glucosuria, ketonuria and the presence of red




cells and granular casts in the urine.  These observations indicated that in




humans, as in animals, the liver and kidneys are the organs most affected by




chloroform ingestion.









        In the past, bromoform was given orally as a sedative to children




suffering from whooping cough.  Doses of 50 to 100 mg/kg/day usually produced




sedation without any apparent adverse effects.  Some cases of severe toxicity




or death were reported, but these were generally attributed to accidental




overdoses.   No data were located on human exposure to either bromodichloro-




methane or dibromochloromethane.









        Workers exposed to chloroform by inhalation at levels of 112 to




1,158 mg/m3 for 1 or more years complained of nausea,  lassitude, dry mouth,




                                     VI-9

-------
flatulence, thirst, depression, irritability and "scalding" micturition, but




clinical examination and tests of liver function failed to detect any




abnormalities.  Inhalation exposure of workers to chloroform at levels of




about 10 to 1,000 mg/m3 for 1 to 4 years was reported to be associated with an




increased incidence of viral hepatitis and enlarged liver.









        Some epidemiological studies suggest there may be an association




between water chlorination and increased cancer mortality rates.   An




association has also been reported between exposure to water chloroform levels




of at least 10 ^g/L and intrauterine growth retardation.   However, since




chlorinated water contains many by-products, it cannot be directly concluded




from such studies that trihalomethanes are human carcinogens or developmental




toxicants.









        No  data were located regarding whether any human subpopulation may be




at. greater  risk to trihalomethane exposure than the general population.  Data




from animal studies suggest that males may be more sensitive than females to




kidney effects, that alcohol consumption may increase toxicity, and that




diabetics may be at increased risk.
                                     VI-10

-------
VII.  MECHANISM OF TOXICITY









        A.  Role of Metabolism









        It is generally believed Chat the toxicity of the trihalomethanes is




related to their metabolism.  This conclusion is based mainly on the




observation that the chief target tissues for these compounds (liver, kidney)




are also the primary sites of their metabolism.   Moreover,  treatments which




increase or decrease metabolism also tend to increase or decrease trihalo-




methane-induced toxicity in parallel.  Representative studies that demonstrate




correlations between metabolism and toxicity are presented below.









        Brown et. al. (1974b) compared the effects of inhalation exposure to




chloroform (0.5% or 1%) in rats with and without pretreatment with phenobar-




bital (an inducer of cytochrome P-450).   The authors reported that pretreat-




ment led to increased chloroform metabolism (as measured by formation of




covalent adducts),  and this was paralleled by increased hepatotoxicity (as




measured by triglyceride levels, destruction of cellular microsomal enzymes,




and histopathological examination).  Thorton-Manning et al. (1993) found that




pretreatment with acetone, a cytochrome P4502E1  inducer, potentiated  the




hepatotoxicity of bromodichloromerhane in male rats.









        Gopinath and Ford (1975) reported that various inducers of microsomal




hydroxylases (phenobarbitone,  phenylbutazone,  and chlorpromazine) potentiated




the hepatotoxicity of chloroform in male rats.  Conversely, inhibitors of




microsomal oxidase (SKF-525A,  sodium diethyl-dithiocarbamate, and carbon




disulfide) acted to protect against the hepatotoxic effect of chloroform.  The
                                     VII-1

-------
cycochrome P450 inhibitor' 1-aminobenzotriazole prevented bromodichloromethane-




induced hepatotoxicity in rats (Thorton-Manning et al. 1993).









        Smith and Hook (1983) reported that 2H-chloroform (which is less




readily metabolized than 1H-chloroform)  was less effective in inducing




nephrotoxicity in male mouse renal cortical slices than was an equimolar




concentration of chloroform.  Nephrotoxicity was assessed iu vitro by




measuring decreased PAH and tetraethylammonium accumulation by kidney slices.









        Ruch et al. (1986)  reported that chloroform-induced release of LDH




from cultured B6C3F1 mouse hepatocytes into the culture medium (an index of




cell injury or death) was decreased by adding SKF-525A to the cultures, and




could also be decreased by adding antioxidants such as N-N'-diphenyl-




p-phenylenediamine, a-tocopherol (Vitamin E) or superoxide dismutase.




Addition of diethylmaleate,  which depletes intracellular glutathione, led Co




increased chloroform toxicity.









        Gao et al.  (1993) evaluated the effects of glutathione on bromo-




dichloromethane toxicity in vivo and in vitro.  Depletion of glutathione by




pretreatment of male rats with the glutathione synthesis inhibitor buthionine




sulfoximine led to increased hepatotoxicity in vivo.  The addition of




glutathione to a reaction mixture of rat hepatic microsomal fraction and




radiolabeled bromodichloromethane resulted in a 90% reduction in protein




binding by bromodichloromethane.









        Sex and species differences in the metabolism of trihalomethanes also




appear to correlate with sex and species differences  in toxicity.  Taylor




et al. (1974) reported that the kidneys from male mice have a higher capacity




                                     VII-2

-------
to metabolize chloroform than kidneys from female mice, and male mice are also


more sensitive than female mice to the renal toxic effects of chloroform


(Culliford and Hewitt 1957; Eschenbrenner and Miller 1945).  Similarly,  mice


appear to have a higher capacity to metabolize trihalomethanes than rats , both


via the oxidative pathway  (Mink et al. 1986) and the reductive pathway (Testai


and Vittozzi 1986; Testai et al .  1987),  and mice also appear to be more


sensitive than rats to chloroform, at least for renal toxicity (Klaunig et al.


1986;  Munson et al. 1982).



                                             *


        B.  Biochemical Basis of Toxicity
                        /

                        I



        The precise biochemical mechanisms which link metabolism to toxicity


are not certain,, but many researchers have proposed that toxicity results from


the production of reactive intermediates, either from the oxidative pathway


(dihalocarbonyls) or the reductive pathway (free radicals).  These reactive


intermediates are known to form covalent adducts with various cellular


molecules (see Section III.C), which could presumably impair the function of


those molecules and cause cell injury.  However, direct evidence showing a


relation between the degree of covalent binding and the extent of toxicity is


limited.
        Ilett et al. (1973) reported that in mice administered 10 /iCi of


14C- chloroform,  the amount of label bound to proteins in the liver and kidneys


paralleled the extent of hepatic and renal necrosis, both in normal animals


and in animals pretreated with an  inducer (phenobarbital) or inhibitor


(piperonyl butoxide) of cytochrome P-450.  Furthermore, autoradiograms


revealed that incorporation of radioactivity occurred primarily at the sites


of necrotic lesions.


                                     VII-3

-------
        Another mechanism chat has been proposed for trihalomethane toxicicv




is lipid peroxidation induced by free radical metabolites.  Although evidence




is limited that lipid peroxidation actually accounts for observed cellular




toxicity, several studies have established that lipid peroxidation does occur




in conjunction with trihalomethane metabolism.









        Brown et al.  (1974b) exposed rats to chloroform in air at levels of




0.5% or 1% for 2 hours.   Livers were removed and levels of conjugated dienes




were measured in hepatic microsomes.   This endpoint was taken to reflect the




level of hepatic lipid peroxidation.   Chloroform exposure resulted in a




significant increase in diene conjugation in phenobarbital-treated rats, but




not in  untreated rats.   The authors speculated that chloroform metabolism




leads to free radicals which induce destructive lipid peroxidation.









        More recently, de Groot and Noll (1989) reported that all four




trihalomethanes induced lipid peroxidation in rat liver microsomes in vitro.




and that this was maximal at low oxygen levels (between 1 and 10 mm Hg of 02) .




The authors interpreted these data to support the concept that lipid




peroxidation is caused by free radical metabolites that are generated by the




reductive metabolism of trihalomethanes.









        Cohen and Chance (1990) measured hepatic chemiluminescence as an in




vivo measure of lipid peroxidation in two male phenobarbital-induced Wistar




rats.  Increased luminescence was observed within about 15 minutes after the




initiation of inhalation exposure to an unspecified concentration of




chloroform.
                                     VII-4

-------
        C.  Mechanism of Carcinogenesis









        The mechanism by which trihalomethanes induce i-umors in laboratory




animals is not known.  However, two possible mechanism have been proposed.




Metabolism of trihalomethanes to reactive intermediates could lead to




formation of covalent DNA-trihalomethane adduct's.  As discussed in




Section V.D., there is some direct evidence that chloroform can bind to DNA




Colacci et al. (1991).  This is consistent with positive findings in




genotoxicity tests, although the overall evidence regarding chloroform




genotoxicity is inconclusive.









        Alternatively, the induction of tumors by trihalomethanes could




involve an epigenetic mechanism.  Induction of tumors in animal studies has




been noted to occur primarily at sites where significant cytotoxicity was




observed (i.e., liver and kidney), and there is a correlation between




hepatotoxic.ity and liver tumorigenicity of trihalomethanes in mice (bromo-




dichloromethane > chloroform = dibromochloromethane > bromoform).  This raises




the possibility that regenerative hyperplasia caused by the cytotoxic effects




of the trihalomethanes may be important in the tumorigenic potential of these




chemicals.  However, because of the apparent genotoxicity of the trihalo-




methanes (evidence is considered adequate for bromoform and bromodichloro-




methane and marginal for dibromochloromethane and chloroform), EPA does not




believe it is appropriate at present to assume that an epigenetic pathway  is




operating.
                                     VII-5

-------
        D.  Interactions









        A variety cf chemicals have been shown to potentiate the toxicity of




chloroform.  The potentiating activity of inducers of microsoraal hydroxylases




was described earlier in this chapter.  Nontoxic levels of several ketones and




compounds that are metabolized to ketones also increase the hepatotoxicity of




chloroform, but this effect cannot be attributed solely to induction of




hepatic enzymes.   Although pretreatment with the insecticide mirex induced




hepatic mixed function oxidases to a greater extent than did pretreatment with




its ketone analog, chlordecone, pretreatment with chlordecone,  but not mirex,




ma-rkedly increased chloroform binding to hepatic constituents (Cianflone et




al. 1980).  Furthermore, chlordecone pretreatment resulted in a different




histological pat.tern of lesions than that observed following dosing with




chloroform alone at a level that produce a similar level of total abnormal




hepatocytes (Hewitt et al.  1979).  Potentiation has also been observed with




various alcohols,  which can be metabolized to ketones, and with other ketones




(Hewitt et al.  1980, 1986;  Ray and Mehendale 1990).  Rats with metabolic




ketosis due to the induction of diabetes by alloxan are also more sensitive to




the hepatotoxic effects of chloroform (Hanasono et al. 1975).  Possible




mechanisms for potentiation by kerones include an effect on calcium pump




activity (Moore and Ray 1983) and increased susceptibility of organelles




resulting from ketone exposure (Hewitt et al. 1990).









        Davis (1992) found that dichloroacetic acid administered at nontoxic




levels (three doses of 2.45 mmol/kg/dose [315.8 mg/kg] in 24 hours) increased




chloroform hepatotoxicity (measured as increased plasma alanine amino-




transferase) and nephrotoxicity (measured as increased BUN) in female Sprague-




Dawley rats.  Trichloroacetic acid at the same molar level increased chloro-




                                     VII-6

-------
form nephrotoxicity.   Histopathology was not conducted, and no mechanism of




interaction was proposed.  Borzelleca et al.  (1990) used a statistical




technique called response surface methodology to predict the synergistic




interactions between chloroform and carbon tetrachloride at varying




concentrations of each chemical, based on measurements of plasma enzyme levels




following oral administration of these chemicals singly and in combination.









        Lilly et al.  (1992) found that concurrent oral administration of




chloroform at doses up to 1.5 mg/kg (1 mL/kg) and trichloroethylene at 1 mL/kg




to adult male F344 rats resulted in decreased toxicity relative to that seen




following chloroform administration alone.  Hepatic and renal toxicity were




observed at this dose of chloroform alone, while trichloroethylene alone was




not overtly toxic to either organ.  Trichloroethylene antagonism of chloroform




toxicity appeared to be independent of dosing vehicle, and was observed




following administration in oil or an aqueous vehicle.









        The severity of trihalomethane toxicity is markedly affected by the




vehicle of administration.  Several s-tudies showing that chloroform was more




toxic following administration in oil than in an aqueous vehicle were




discussed in Chapter V (e.g., Bull et al. 1986; Jorgenson et al. 1985).  Lilly




et al.  (1992) found that chloroform administration in corn oil caused




substantially greater hepatic and renal toxicity in adult male F344 rats than




did administration in an aqueous vehicle.  In a study of vehicle effects on




the acute toxicity of bromodichloromethane, a high dose (400 mg/kg) of the




chemical was more hepato- and nephrotoxic when given in corn oil compared  to




aqueous administration, but this difference was not evident at a lower dose




(200 mg/kg) (Lilly et al. 1994).
                                     VII-7

-------
        E.  Summary









        Three lines of evidence indicate that trihalomethane metabolism is




essential for toxicity:  (1) the tissues that most actively metabolize the




trihalomethanes (liver, kidney) are also the chief target tissues;




(2) chemical treatments that increase or decrease metabolism also tend to




increase or decrease toxicity in parallel; and (3) species- and sex-related




differences in metabolism are paralleled by similar differences in toxicity.




The detailed biochemical mechanisms by which trihalomethane metabolism leads




to toxicity are not certain, but covalent binding of reactive metabolites to




key cellular molecules is one likely mechanism.   Such metabolites are produced




both by oxidative metabolism to dihalocarbonyls and reductive metabolism to




free radicals.  Free radical production may also lead to cell injury by




inducing lipid peroxidation in cellular membranes.









        Formation of DNA adducts might also account for the genotoxic and




carcinogenic potential of the trihalomethanes.  Alternatively, carcinogenesis




may be related, at least in part,  to increased cell proliferation following




direct tissue injury.  However, neither of these potential mechanisms have




been definitively linked to trihalomethane carcinogenesis.









        Chloroform toxicity is potentiated by various chemicals.  At least




some of the potentiation by ketones appears to occur by a mechanism other than




induction of microsomal enzymes.  Some quantitative data are available




regarding the interactions between chloroform and dichloroacetic acid, carbon




tetrachloride, or trichloroethylene.   The vehicle (corn oil versus aqueous)




used for oral dosing also affects toxicity, with toxicity generally being more




severe following administration in oil.




                                     VII-8

-------
VIII.   QUANTIFICATION OF TOXICOLOGICAL EFFECTS









        The quantificaticn of Lexicological effects of a chemical consists of




separate assessments of noncarcinogenic and carcinogenic health effects.




Chemicals that do not produce carcinogenic effects are believed to have a




threshold dose below which no adverse, noncarcinogenic health effects occur,




while carcinogens are assumed to act without a threshold.








Quantification of NoncarcinoEenic Effects








        In the quantification of noncarcinogenic effects, a Reference Dose




(RfD),  (formerly called the Acceptable Daily Intake (ADI)), is calculated.




The RfD is an es.timate of a daily exposure to the human population that is




likely to be without appreciable risk of deleterious health effects, even if




exposure occurs over a lifetime.  The RfD is derived from a NOAEL, or LOAEL,




identified from a subchronic or chronic study, and divided by an uncertainty




factor(s).   The RfD is calculated as follows:
                 D_     (NOAEL or LOAEL)             ..   ,   ..
                 RfD = rr-4	:	"Tr = 	 mg/kg bw/day
                       Uncertainty Factor(s)   	  &/ e>   /  J
        Selection of the uncertainty factor to be employed in the calculation




of the RfD is based on professional judgment and consideration of the entire




data base of toxicological effects for the chemical.  To ensure that




uncertainty factors are selected and applied in a consistent manner, the




Office of Water (OW) employs a modification of the guidelines proposed by the




National Academy of Sciences (NAS 1977, 1980), as follows:
                                    VIII-1

-------
          An uncertainty factor of 10 is generally used when good chronic or




           subchronic human exposure data identifying a NOAEL are available




           and are supported by good chror.ic or subchronic tcxicity data in




           other species.









          An uncertainty factor of 100 is generally used when good chronic




           toxicity data identifying a NOAEL are available for one or more




           animal species., (and human data are not available),  or when good




           chronic or subchronic toxicity data identifying a LOAEL in humans




           are available.









          An uncertainty factor of 1,000 is generally used when limited or




           incomplete chronic or subchronic toxicity data are available, or




           when good chronic or subchronic toxicity data that identify a LOAEL




           but not a NOAEL for one or more animal species are available.









        The uncertainty factor used for a specific risk assessment is based




principally on scientific judgment rather than scientific fact and accounts




for possible intra- and interspecies differences.  Additional considerations




not incorporated in the NAS/OW guidelines for selection of an uncertainty




factor include the use of a less-rhan-lifetime study for deriving a RfD, the




significance of the adverse health effect and the counterbalancing of




beneficial effects.









        From the RfD, a Drinking Water Equivalent Level (DWEL) can be




calculated.  The DWEL represents a medium specific (i.e., drinking water)




lifetime exposure at which adverse, noncarcinogenic health effects are not




anticipated to occur.  The DWEL assumes 100% exposure from drinking water and




                                    VIII-2

-------
provides the noncarcinogenic health effects basis for establishing a drinking



water standard.  For ingestion data, the DWEL is derived as follows:
              ntTPT      (RfD)  x (Body Weight in ks)             ,,
              DWEL = TT7r~r	77	'  , . n	:-~~ =     mg/L
                     Drinking Water Volume  in L/day    	  
where:








        Body weight is as'sumed to be 70 kg for an adult.




        Drinking water volume is assumed to be 2 L per day for an adult.








        In addition to the RfD and the DWEL, Health Advisories (HAs) for




exposures of shorter duration (One-day, Ten-day and Longer-term) are




determined.  The HA values are used as informal guidance to municipalities and




other organizations when emergency spills or contamination situations occur.




The HAs are calculated using a similar equation to the RfD and DWEL; however.




the NOAELs or LOAELs are identified from acute or subchronic  studies.  The HAs




are derived as follows:
                    (NOAEL or LOAEL) x  (bw)          /T

                       (UF) x (	 L/day)   = 	 mg/L
        Using the above equation, the following drinking water HAs  are



developed for noncarcinogenic effects:








        1.  One-day HA for a 10-kg child  ingesting  1  L water  per  day.



        2.  Ten-day HA for a 10-kg child  ingesting  1  L water  per  day.



        3.  Longer-term HA for a  10-kg  child  ingesting 1 L water  per day.



        4.  Longer-term HA for a  70-kg  adult  ingesting 2 L water  per day.



                                    VIII-3

-------
        The One-day HA calculated for a 10-kg child assumes a single acute


exposure to the chemical and is generally derived from a study of less than


7 days' duration.  The Ten-day HA assumes s. limited exposure period of 1 to


2 weeks and is generally derived from a study of less than 30-days' duration.


A Longer-term HA is derived for both the 10-kg child and a 70-kg adult and


assumes an exposure period of approximately 7 years (or 10% of an individual's


lifetime).   A Longer-term HA is generally derived from a study of subchronic


duration (exposure for 10% of an animal's lifetime).




Quantification of Carcinogenic Effects




        The EPA categorizes the carcinogenic potential of a chemical based on


the overall weight-of-evidence,  according to the following scheme:




          Group A:  Known Human Carcinoeen.  Sufficient evidence exists from


           epidemiology studies to support a causal association between


           exposure to the chemical and human cancer.




          Group B:  Probable Human Carcinogen.   Sufficient evidence of

    /
           carcinogenicity in animals with limited (Group Bl) or inadequate


           (Group B2) evidence in humans.




          Group C:  Possible Human Carcinogen.   Limited evidence of


           carcinogenicity in animals in the absence of human data.




          Group D:  Not Classified as to Human Carcinogenicity.  Inadequate


           human and animal evidence of carcinogenicity or for which no data


           are available.


                                    VIII-4

-------
          Group E:  Evidence of Noncarcinogenicitv for Humans.  No evidence




           of carcinogenicity in at least two adequate animal tests in




           different species or in both adequate epidemiologic and animal




           studies.









        If toxicological evidence leads to the classification of the




contaminant as a known, probable or possible human carcinogen, mathematical




models are used to calculate the estimated excess cancer risk associated with




the ingestion of the contaminant in drinking water.  The data used in these




estimates usually come from lifetime exposure studies in animals.  In order to




predict the risk for humans from animal data, animal doses must be converted




to equivalent human doses.  This conversion includes correction for




noncontinuous exposure, less-than-lifetime studies and differences in size.




The factor that compensates for the size difference is the cube root of the




ratio of the animal and human body weights.  It is assumed that the average




adult human body weight is 70 kg and that the average water consumption of an




adult human is 2 L of water per day.









        For contaminants with a carcinogenic potential, chemical levels are




correlated with a carcinogenic risk estimate by employing a cancer potency




(unit risk) value together with the assumption for lifetime exposure via




ingestion of water.  The cancer unit risk is usually derived from a linearized




multistage model with a 95% upper confidence limit providing a low-dose




estimate; that is,  the true risk to humans, while not identifiable, is not




likely to exceed the upper limit estimate and, in fact, may be lower.  Excess




cancer risk estimates may also be calculated using other models such as the




one-hit, Weibull, logit and probit.  There is little basis in the current




understanding of the biological mechanisms involved in cancer to suggest that




                                    VIII-5

-------
any one of these models is able to predict risk more accurately than others.




Because each model is based upon differing assumptions, the estimates derived




for each model can differ by several orders of magr.itv.de.









        The scientific data base used to calculate and support the setting of




cancer risk rate levels has an inherent uncertainty due to the systematic and




random errors in scientific measurement.  In most cases, only studies using




experimental animals have been performed.  Thus, there is uncertainty when the




data are extrapolated to humans.  When developing cancer risk rate levels,




several other areas of uncertainty exist, such as incomplete knowledge con-




cerning the health effects of contaminants in drinking water; the impact of




the experimental animal's age, sex and species; the nature of the target organ




system(s) examined; and the actual rate of exposure of the internal targets in




experimental animals or humans.  Dose-response data usually are available only




for high levels of exposure, not for the lower levels of exposure closer to




where a standard may be set.  In exposures to more than one contaminant,




additional uncertainty results from a lack of information about possible




synergistic or antagonistic effects.









        A.  Noncarcinogenic Effects









        1.  Chloroform









        a.  One-day Health Advisory









        Table VIII-1 summarizes studies that were considered for calculating




the One-day HA for chloroform.  All four studies (Jones et al. 1958; Hill




1978; Reitz et al. 1980; Larson et al. 1993) indicate moderate to severe




                                    VIII-6

-------
                         Table VIII-1  Summary of Candidate Studies for Derivation
                               of the One-day Health Advisory for Chloroform

Reference
Jones et al.
(1958)
Hill (1978)
Rei r.2 et al .
(1980)
Larson et al.
(1993a)

Species Route
Mouse Gavage
(oil)
Mouse Gavage
' (oil)
Mouse Gavage8
Rat Gavage
(oil)
Mouse Gavage
(oil)
Exposure
Duration
Single
dose
Single
dose
Single
dose
Single
dose
Single
dose
Endpoints
Histology
(liver)
Histology
(liver),
urinalysis
Histology
(liver,
kidney)
Histology
(liver,
kidney)
Histology
(liver,
kidney)
NOAEL
(mg/kg/day)
35-70
(Mild fatty
infiltration
of liver)
~ ~
15
34
34
LOAEL
(mg/kg/day)
140
(Marked fatty
infiltration,
necrosis)
89
(Glucosuria,
proteinuria)
60
(Renal tubular
regeneration)
180
(Liver and kidney
necrosis)
238
(Mild liver
necrosis)
' 
BThe  vehicle  was  not  reported by  Reitz  et  al.  (1980) but  is presumed to be oil.

-------
liver and/or  kidney  damage  following single  oral doses of 140 to 250 mg/kg


chloroform  in oil.   The  study  by Jones  et al.  (1958)  indicates that only mild


hepatic, fat accumulation, without overt liver  injury,  occurs following doses


of 35 mg/kg.   The  data of Hill (1978)  indicate that renal effects (evidenced


by urinary  loss  of glucose  or  protein)  occur at doses  of 89 to 163 mg/kg, and


liver histopathology occurs at 250 mg/kg.  Reit'z et al.  (1980) reported


regenerative  changes in  renal  tubules  of mice  given 60 mg/kg (although only


two animals per  dose were used).   Larson et  al.  (1993) reported scattered


necrotic tubules in  rats at 34 mg/kg, with no  consistent effects on BUN or


urinary protein  or glucose.  Taken together, these studies indicate that renal


and hepatic injury begins to become manifest following doses of about 60 mg/kg


and becomes severe following doses of  140 to 250 mg/kg.   However, confidence


in these values  ;Ls limited  because of  the use  of an oil vehicle,3 which may


have contributed to  the  observed hepatotoxicity.   Nevertheless, the NOAEL of


35 mg/kg identified  by Jones et al.  (1958) is  selected as the most appropriate


basis for calculation of the One-day HA value.





        Using this value, the  One-day HA for the 10-kg child is calculated as


follows:
      ^   j   u.    (35 mg/kg/day) (10 kg)   _ c    ..  .     ,  .  .   ,    ,T,
      One-day HA -     (100)  (1  L/day)           mg/   
where:
     35 mg/kg/day = NOAEL, based on absence of hepatotoxicity in mice given


                    single oral  doses  of chloroform via gavage
    'The vehicle  was not reported by Reitz  et al. (1980) but is presumed  to be oil.


                                     VIII-8

-------
            10 kg = assumed weight of a child









              100 = uncertainty factor; chosen in accordance with NAS/OW




                    guidelines in which a NOAEL from a study in animals is




                    employed









          1 L/day = assumed water consumption by a 10-kg child









        No existing guidelines or standards were located for acute oral




exposure to chloroform.   A concentration of 10 ppm in air has been proposed as




a Time-Weighted Average-Threshold Limit Value (TWA-TLV) by the American




Conference of Governmental Industrial Hygienists (ACGIH 1991) .   A concentra-




tion of 2 ppm in air has been proposed as a Short-Term Exposure Limit (STEL)




(NIOSH 1990).   The Occupational Safety and Health Administration (OSHA)




established an 8-hour time-weighted average (TWA) permissible exposure limit




of 2 ppm (54FR2332, January 19, 1989).









        b.  Ten-day Health Advisory for Chloroform









        Table VIII-2 summarizes studies that were considered for calculation




of the Ten-day HA for chloroform.  The studies by Munson et al. (1982) and by




Thompson et al. (1974) identify hepatotoxicity as the most appropriate




endpoint of short-term exposure to chloroform.  Munson et al. (1982) detected




no significant changes in serum enzymes in mice given up to 125 mg/kg/day




chloroform in water for  14 days,  and Chu et al. (1982a) reported no change in




serum enzymes or gross pathology in the liver of rats given 63 mg/kg/day in




the drinking water for 28 days.  However, Thompson et al. (1974) noted




maternal toxicity (decreased weight gain and fatty liver) in pregnant rats and




                                    VIII-9

-------
Table VIII-2  Summary of Candidate Studies for Derivation
      of the Ten-day Health Advisory for Chloroform

Reference
Munson et al.
(1982)
Chu et al.
(1982a)
Thompson
et al. (1974)
Thompson
et al. (1974)
Species Route
Mouse Gavage
(aqueous)
Rat Drinking
water
Rat Gavage
(oil)
Rabbit Gavage
(oil)
Exposure .
Duration
14 days
28 days
Days
6-15 of
gestation
Days
6-15 of
gestation
NOAEL
Endpoints (mg/kg/day)
Liver weight, 125
serum enzymes,
immune function. ,
Serum 7.4
biochemistry ,
hematology ,
gross pathology
Maternal 20
toxicity ,
fetotoxicity ,
teratogenicity
Maternal 35
toxicity,
fetotoxicity,
teratogenicity
LOAEL
(rag/kg/day)
250
(Elevated
serum
enzymes)
63
(Decreased
neutrophil
count; no liver
toxicity)
50
(Decreased
maternal
weight gain;
fatty liver)
50
(Decreased
maternal
weight gain;
fatty liver)

-------
rabbits given 50 mg/kg/day chloroform in oil on days 6 to 15 of gestation;


this dose was identified as the LOAEL.  Doses of 20 mg/kg/day in rats and


35 mg/kg/day in rabbits were without significant maternal toxicity,


fetotoxicity,  or teratogenicity.   The dose of 35 mg/kg/day from the rabbit


study was selected as the NOAEL.   Even though this study employed the use of a


corn oil vehicle that may have contributed to maternal hepatotoxicity,  these


NOAELs are supported by the drinking water study by Chu et al. (1982a).




        Using this value, the Ten-day HA for the 10-kg child is calculated as


follows:


      _    ,   .    (35 mg/kg/dav)  (10  kg)    ,                ,
      Ten-day HA =     (100) (1 L/day)         '5  mg/L  (rounded  to 4  mS/L)
where:
     35 mg/kg/day = NOAEL,  based on absence of maternal or fetal toxicity in

                    rabbits exposed to chloroform via gavage during days 6 to

                    15 of gestation




            10 kg = assumed weight of a child




              100 = uncertainty factor; chosen in accordance with NAS/OW

                    guidelines in which a NOAEL from a study in animals is

                    employed




          1 L/day = assumed water consumption by a 10-kg child
                                    VIII-11

-------
        No existing guidelines or standards were located for short-term oral



exposure to chloroform.  OSHA currently limits occupational exposure to



chloroform Co a ceiling level of 50 ppm (29 GFR 1910.1000).








        c.  Longer-term Health Advisory for Chloroform








        Table VIII-3 summarizes studies that were considered for calculating



the Longer-term HA values for chloroform.  These studies indicate that



exposure of rats and mice to chloroform at doses of 50 to 270 mg/kg/day in



water or 130 mg/kg/day in oil does not result in significant liver injury or



other evidence of toxicity, while doses of 270 to 290 mg/kg/day produce mild



changes that may reflect incipient injury.  Decreased growth rate was observed



at a lower dose .(180 mg/kg/day).  This conclusion is not consistent with



short-term studies (see Tables VIII-1 and VIII-2), which indicate that doses



of 50 mg/kg/day or higher produce hepatic or renal injury.  The reasons for



this discrepancy are not certain, but the difference may be due in part to



metabolic adaptation during continued exposure or to differences in exposure



route (drinking water versus gavage) or vehicle (aqueous versus corn oil).



Since the data identify a longer-term NOAEL that is higher than short-term



LOAEL values, it is recommended that the DWEL for chloroform (0.4 mg/L,



calculated below) be taken as an appropriate estimate of the Longer-term  HA



value for adults.  For children, che Longer-term HA value may be estimated by



calculation of an adjusted DWEL value that accounts for the higher water



intake of children, as follows:
                           UA  . ,  .. ..    (0.01 mg/kE/dav)(10 kg)
               Longer-term HA  (child) =  	:	~ T'	  	
                                               i. Li/ o.ay
                                      =0.1 mg/L  (rounded  to  0.1 mg/L)





                                    VIII-12

-------
Table VIII-3  Summary of Candidate Studies for Derivation
    of the Longer-term Health Advisory for Chloroform  -

Reference Species
Chu et al . Rat
(1982b)
Jorgenson Rat
and Rushbrook
(1980)
Jorgenson Mouse
and Rushbrook
(1980)
Bull et al. Mouse
(1986)

Bull et al. Mouse
(1986)
Exposure
Route Duration
Drinking 90 days
water
Drinking 90 days
water
Drinking 90 days
water
Gavage 90 days
(oil)

Gavage  90 days
(aqueous)
Endpoints
Serum
biochemistry,
hematology ,
histology,
growth
Growth, serum
biochemistry,
histopathology,
organ fat
Growth, serum
biochemistry,
histopathology ,
organ fat
Serum
biochemistry,
. liver histology

Serum
biochemistry,
NOAEL
(mg/kg/day)
50
160
145
130
(Increased
liver fat,
decreased
serum tri-
glycerides)
270
LOAEL
(mg/kg/day)
180
(Thyroid
lesions ,
decreased
growth)

290
(Increased
liver fat)
270
(Elevated
serum
enzymes ,
diffuse
liver
pathology)

                               liver histology

-------
where:
        0.01 mg/kg/day = RfD (see below)



                 10 kg = assumed weight of a child



            1 L/day = assumed water consumption by a 10-kg child






        The Longer-term HA for a 70-kg adult consuming 2 L/day of water is


calculated as follows:
T           UA   (0.01 mg/kg/day) (70 kg)    _ ,,      ,     .  ,    . .
Longer-term HA = J	?2 L/d  (  	^~ = -35 m/L (rounded to 0.4 mg/L)
        d.  Reference Dose and Drinking Water Equivalent Level for Chloroform






        Table VIII-4 summarizes studies that were considered for deriving the


RfD and DWEL for chloroform.  The studies by Palmer et al. (1979) and



Jorgenson et al. (1982) indicate that chronic exposure of rats to chloroform



at doses ranging from 60 mg/kg/day in toothpaste to 160 mg/kg/day in drinking


water did not result in any clear hepatotoxicity, although decreased weight



gain (probably secondary to reduced water consumption) was noted in both



studies.  The study by Heywood et al. (1979) indicated that chronic exposure



of dogs (eight/sex) to doses of 15 mg/kg/day (6 days/week) resulted in minimal


liver injury (evidenced by slightly elevated SCOT levels and an increased



number of fatty cysts in the liver).   This study is selected as the most  .



appropriate for derivation of the DWEL because exposure was chronic



(7.5 years), there were 16 animals per dose group, and sensitive indices of



hepatotoxicity (serum enzyme levels,  liver histology) were monitored.






                                   VIII-14

-------
Table VIII-4  Summary of Candidate Studies for Derivation
                of.the  DWEL for  Chloroform

Reference
Heywood
et al .
(1979)
Palmer
et al.
(1979)
Jorgenson
et al.
(1982)
Exposure
Species Route Duration
Dog Oral 7.5 years
(toothpaste
base in
capsules)
Rat Cayage 80 weeks
(toothpaste
base)
Rat Drinking 23 months
water
Endpolnts
Serum enzymes ,
hlstopathology
Weight gain,
organ weight,
histology
Weight gain,
liver fat,
hematology ,
serum
chemistry
NOAEL
(mg/kg/day)
1
60
(Decreased
body
weight)
160
(Decreased
.body weight
secondary to
decreased water
LOAEL
(mg/kg/d,ay)
15
(Elevated
SCOT, fatty
cysts in
liver)


                                                consumption;
                                                hemoconcentration)

-------
        Using this study, the DWEL  is derived as  follows:
Step 1:  Determination of Reference Dose  (RfD)
      T,.rrv   (15 mg/kg/davH6/7)   -. _.n _    ..  ..    .     ,  ,       ,    
      RfD = J	?1 000)      " 0.013 mg/kg/day  (rounded  to  0.01  mg/kg/day)
where:
      15 mg/kg/day - LOAEL, based on mild hepatotoxicity  in  dogs  exposed to



                     chloroform via gelatin  capsules  for  7.5 years






             1,000 = uncertainty factor; chosen  in  accordance with  NAS/OW



                     guidelines in which a LOAEL from a study in  animals is



                     employed






Step 2:  Determination of DWEL
    DWEL =   -               (70 kg.) _ ^ mg/L  (rounded  to  Q  4 mg/L)
where:
     0.01 mgAg/day = RfD
              70 kg = assumed weight  of  an  adult
            2 L/day = assumed water  consumption by a 70-kg adult
                                    VII1-16

-------
        An interim Maximum Contaminant Level (MCL) for total trihalomethanes




of 100 jig/L was established by the U.S. EPA in November 1979 (44FR68624).




Although some estimates of cancer risk were performed, this limit was set




primarily on the basis of technological and economic feasibility.  The basis




and purpose of this regulation are discussed in a report prepared by OW




(U.S. EPA 1979).  Based on its carcinogenic potential, the U.S. EPA (1980a)




proposed an ambient water quality criterion of zero for chloroform.   On the




basis of reports of carcinogenicity and embryotoxicity, the ACGIH classified




chloroform as a suspected human carcinogen (ACGIH 1991) and recommended a




threshold limit value (TLV) of 10 ppm.  Based on positive carcinogenic




findings (NCI 1976), NIOSH (1977) recommended that exposure to chloroform be




limited to 2 ppm (the lowest detectable level using the recommended sampling




and analysis techniques).









        2.  Bromodichloromethane









        a.  One-day Health Advisory for Bromodichloromethane









        Studies by Bowman et al.  (1978) and NTP (1987) indicate that single




oral doses of 500 mg/kg or higher of bromodichloromethane produce profound




central nervous system (CNS) depression and may lead to death.  However, no




studies were located that identified a NOAEL or LOAEL based on more sensitive




endpoints suitable for derivation of the One-day HA value.  In the absence of




adequate data, it is recommended that the Ten-day HA of 6 mg/L be taken as a




conservative estimate of the One-day HA value.









        No existing guidelines or standards for acute oral or inhalation




exposure to bromodichloromethane were located.




                                    VIII-17

-------
        b.  Ten-day Health Advisory for Bromodichloromethane









        Table VIII -5 summarizes studies that were considered for calculating




the Ten-day HA for bromodichloromethane .   The 30-day study by Aida et al .




(1992a) was selected for the calculation of the Ten-day HA since it resulted




in a lower LOAEL than the only other study of appropriate duration that




included a histological examination (Condie et al. 1983).  This study




identified a NOAEL (in males) of 62 mg/kg/day and a LOAEL of 189 mg/kg/day




when microencapsulated bromodichloromethane was administered in feed to rats,




based on histopathology of the liver,  along with sensitive biochemical tests




of liver and kidney injury.   This NOAEL value is strongly supported by




drinking water studies (Munson et al.  1982; NTP 1987; Chu et al. 1982a) and a




study using oil gavage administration (Condie et al. 1983), all of which




identified NOAEL values from 50 to 150 mg/kg/day.









        Using the NOAEL identified by Aida et al. (1992a) ,  the Ten-day HA for




the 10-kg child is calculated as follows:
      Ten-day HA =  <62 ffffi           **>  =  6.2  mg/L (rounded  to  6  mg/L)
where:
     62 mg/kg/day = NOAEL, based on absence of reduced body weight and




                    histopathological effects on the liver of rats fed




                    bromodichloromethane for 30 days




            10 kg = assumed weight of a child
                                    VIII-18

-------
Table V1II-5  Summary of Candidate Studies for Derivation
 of the Ten-day Health Advisory for Bromodichloromethane

Reference
Munson et al.
(1982)
Condie et al.
(1983)
NTP
(1987)
NTP
(1987)
Chu et al.
(1982a)
Aida et al.
(1992a)
Exposure
Species Route Duration
Mouse Gavage 14 days
(aqueous)
Mouse Gavage 14 days
(oil)
Rat Gavage 14 days
(oil)
Mouse Gavage 14 days
(oil)
Rat Drinking 28 days
water
Rat Feed 1 month
NOAEL
Endpoints (mg/kg/day)
"Body and organ 50
weight, serum
chemistry ,
hematology ,
immune function '.
Serum enzymes, 74
histopathology ,
PAH uptake in
vitro
Body weight, 150
clinical signs,
gross necropsy
Body weight, 75
clinical signs,
gross necropsy
Clinical signs, 68
serum chemistry,
histology
Clinical signs, 62
body weight, serum
LOAEL
(mg/kg/day)
125
(Depressed
humoral
immunity)
148
(Elevated SCOT, 
decreased PAH,
histopathology in
liver, kidney)
300
(Decreased
weight gain)
150
(Mortality,
lethargy,
gross renal
pathology)

189
(Hi stopathology
                               biochemistry,
                               histology,
                               hematology
in liver)

-------
              100 = uncertainty factor; chosen in accordance with NAS/OW




                    guidelines in which a NOAEL from a study in animals is




                    employed









          1 L/day = assumed water consumption by a 10-kg child









        No existing guidelines or standards for short-term oral or inhalation




exposure to bromodichloromethane were located.









        c.  Longer-term Health Advisory for Bromodichloromethane









        Table VIII-6 summarizes studies that were considered for calculation




of the Longer-term HA values for bromodichloromethane.   The studies by Chu




et al. (1982b) and NTP (1987) are in good agreement, each identifying NOAEL




values of 50 to 75 mg/kg/day in oil or water.  The study by NTP (1987) is




selected as the most appropriate for deriving the Longer-term HA values,




because clear dose-response trends were observed and the doses given




(separated by factors of 2.0) defined the threshold more sharply than in the




Chu study (where doses were separated by factors of 10.0).  The NOAEL of




50 mg/kg/day in male mice is selected as the most appropriate value because




the LOAEL in this group (100 mg/kg/day) was lower than that for female mice




(LOAEL = 200 mg/kg/day) and that for rats (LOAEL - 150 mg/kg/day).




        Using the NTP (1987) study, the Longer-term HA for the 10-kg child is




calculated as follows:
Longer-term HA - <5                          " 3'6 mS/L 
-------
Table VIII-6  Summary of Candidate Studies for Derivation of
  the Longer-term Health Advisory for Bromodichloromethane

Reference
Chu et al .
(1982b)
NTP
(1987)

NTP
(1987)
Species Route
Rat Drinking
water
Rat Gavage
(oil)

Mouse Gavage
(oil)
Exposure
Duration
90 days
13 weeks
(5 days/
week)

13 weeks
(5 days/
week)
NOAEL
Endpoints (mg/kg/day)
Histology, 52
serum chemistry
Body weight, 75
clinical signs,
histology

Body weight, 50
clinical signs,
histology
LOAEL
(mg/kg/day)
250
(Mild hepatic
lesions)
150
(Decreased
weight gain)
300
(Hepatic,
renal lesions)
100
(Renal
lesions)

-------
where:
     50 mg/kg/day = NOAEL, based en absence of clinical signs or histological




                    changes in male mice exposed to bromodichloromethane via




                    gavage for 13 weeks









            10 kg = assumed weight of a child









              5/7 = correction factor to account for exposure 5 days/week









              100 = uncertainty factor; chosen in accordance with NAS/OW




                    guidelines in which a NOAEL from a study in animals is




                    employed









          1 L/day =  assumed water consumption by a 10-kg child









        The Longer-term HA for a 70-kg adult consuming 2 L of water per day is




calculated as follows:
Longer-term HA = (5                          = 12'5 ^/L 
-------
        d.  Reference Dose and Drinking Water Equivalent Level for
            Bromodichloromethane
        Table VIII-7 summarizes studies that were considered for derivation of

the RfD and DWEL for bromodichlorome thane .   The dietary study by Tobe et al.

(1982) identified a NOAEL of 6 mg/kg/day (males) based on gross pathology and

serum biochemistry.  Histological examination of tissues in the Tobe et al.

(1982) study was recently reported in Aida et al. (1992b) ,  and a LOAEL of

6 mg/kg/day was identififed, based on fatty degeneration and granuloma of the

liver.  The oil gavage study by NTP (1987)  included a thorough histological

examination of control and exposed animals.  The authors observed histological

lesions in the liver, kidney, and thyroid of rats exposed to 50 mg/kg/day and

of mice exposed to 25 mg/kg/day chloroform in oil.  Before the publication of

the histology data in Aida et al .  (1992b),  the NTP (1987) study was considered

most appropriate for the derivation of the RfD and DWEL.  Since a NOAEL value

was not observed in either species, the lowest LOAEL value (25 mg/kg/day in

mice) is selected as the most appropriate basis for deriving the RfD and the

DWEL.  This choice is currently under review in light of Aida et al. (1992b) .



        Using the NTP (1987) value, the DWEL is derived as follows:



Step 1: Determination of Reference Dose (RfD)
   RfD  =  (25  "ft         (5/7)  = 0.0179 mg/kg/day (rounded to 0.02 mg/kg/day).
                                    VIII-23

-------
Table VI11-7  Summary of Candidate Studies for Derivation
          of  the DWEL  for Bromodichloromethane

Reference
NTP
(1987)
NTP
(1987)
Aida et al.
(1992b)
Species Route
Rat Gavage
(oil)
Mouse Gavage
(oil)
Rat Diet
Exposure . NOAEL
Duration Endpoints (mg/kg/day)
102 weeks Clinical signs,
body weight,
gross necropsy, .
histology
102 weeks Clinical signs,
body weight,
gross necropsy,
histology
24 months Clinical signs,
body weight,
LOAEL
(mg/kg/day)
50
(Renal and
hepatic
lesions)
25
(Lesions of
liver, kidney,
thyroid)
6.1
(Liver fatty
                               serum biochemistry,
                               gross necropsy,
                               histology
degeneration
and granuloma)

-------
where:
     25 mg/kg/day - LOAEL, based on histological lesions in liver, kidney and

                    thyroid in mice exposed to bromodichloromethane via gavage

                    for 102 weeks



              5/7 = correction for exposure 5 days/week



            1,000 = uncertainty factor; chosen in accordance with NAS/OW

                    guidelines in which a LOAEL from a study in animals is

                    employed



Step 2:  Determination of DWEL
                  DWEL = (0.02 ms/ka/day) <70     - 0.7 mg/L
                                2 L/day
where:
     0.02 mg/kg/day =    RfD


              70 kg =    assumed weight of an adult


            2 L/day -    assumed water consumption by a 70-kg adult




        No existing guidelines or standards were located for chronic oral

exposure to bromodichloromethane.  The interim MCL for total trihalomethanes

is 100 /xg/L (44FR68624).   Although some estimates of cancer risk were


                                   VIII-25

-------
performed, this limit was set primarily on the basis of technological and




economic feasibility.  The basis and purpose of this regulation are discussed




in a report that was prepared by the OW (U.S. EPA 1979").









        3.  Dibromochloromethane









        a.  One-day Health Advisory for Dibromochloromethane









        Studies by Bowman et al. (1978) indicate that single oral doses of




500 mg/kg or higher of dibromochloromethane produce profound CNS depression,




and NTP (1985) data show that doses from 310 to 2,500 mg/kg/day may lead to




death in rats and mice.  However, no studies were located that identified a




NOAEL or LOAEL based on more sensitive endpoints suitable for derivation of




the One-day HA value.  In the absence of adequate data, it is recommended that




the Ten-day HA of 6 mg/L be considered as a conservative estimate of the One-




day HA value.









        No existing guidelines or standards for acute oral or inhalation




exposure to dibromochloromethane were located.









        b.  Ten-day Health Advisory for Dibromochloromethane









        Table VIII-8 summarizes studies that were considered for calculating




the Ten-day HA for dibromochloromethane.  The study by Aida et al.  (1992a) was




selected as the basis for the calculation of the Ten-day HA.  In this study,




microencapsulated dibromochloromethane was administered in feed to rats for




one month.  Based on liver histopathology and increased cholesterol levels in




male rats, this study identified a NOAEL of 56 mg/kg/day and a LOAEL of




                                    VIII-26

-------
Table VIII-8  Summary of Candidate Studies for Derivation
 of the Ten-day Health Advisory for Dibromochloromethane

Reference
Munson et al .
(1982)



Condie et al.
(1983)




NTP (1985)



NTP (1985)


Chu et al.
(1982a)

Aida et al .
(1992a)
Exposure
Species Route Duration
Mouse Gavage 14 days
(aqueous)



Mouse Gavage 14 days
(oil)




Rat Gavage 14 days
(oil)


Mouse Gavage 14 days
(oil)

Rat Drinking 28 days
water

Rat Feed 1 month

NOAEL
Endpoints (mg/kg/day)
"Body and organ 50
weight, serum
chemistry,
hematology ,
immune function '
Serum enzymes, 74
histopathology ,
PAH uptake in
vitro


Body weight, 250
clinical signs,
gross necropsy

Body weight, 60
clinical signs,
gross necropsy
Clinical signs, 68
serum chemistry,
histology
Clinical signs, 56
body weight, serum
LOAEL
(mg/kg/day)
125
(Decreased
humoral
immunity)

147
(Elevated SCOT,
decreased PAH,
histopathology
in liver,
kidney)
500
(Lethargy,
gross pathology,
mortality)
125
(Stomach
lesions)



173
(Histopathology
                              biochemistry,
                              histology,
                              hematology
in liver,
increased
cholesterol |M|)

-------
173 mg/kg/day.  Histopathology was also assessed in the oil gavage study of




Condie et al. (1983), which identified a NOAEL of 74 rag/kg/day.  These data




are strongly supported by the other drinking water and corn oil vehicle




studies (Munson et al. 1982; NTP 1985; Chu et al.  1982a),  all of which




identified NOAEL values from 50 to 250 mg/kg/day.









        Using the NOAEL identified by Aida et al.  (1992a), the Ten-day HA for




the 10-kg child is calculated as follows:
                                                     (rounded to 6 ms/L)
where:
     56 mg/kg/day = NOAEL, based on absence of hepatic effects in rats exposed




                    to microencapsulated dibromochloromethane in feed for one




                    month









            10 kg = assumed weight of a child









              100 = uncertainty factor; chosen in accordance with NAS/OW




                    guidelines in which a NOAEL from a study in animals is




                    employed









          1 L/day = assumed water consumption by a 10-kg child









        No existing guidelines or standards for short-term oral or inhalation




exposure to dibromochloromethane were located.






                                    VIII-28

-------
        c.  Longer-term Health Advisory for Dibromochloromethane






        Table VIII-9 summarizes studies that were considered for calculation


of the Longer-term HA values for dibromochloromethane.   The 90-day drinking


water study by Chu et al. (1982b) and the corn oil study by NTP (1985) are in



good agreement, each identifying NOAEL values of about 30 to 50 mg/kg/day and


LOAEL values of 60 to 250 mg/kg/day.  The drinking water study by Borzelleca



and Carchman (1982) is also consistent with these values, identifying a NOAEL



of 17 mg/kg/day and a LOAEL of 171 mg/kg/day after somewhat longer exposure


(24 to 27 weeks).   The LOAEL of 50 mg/kg/day identified by Daniel et al.


(1990) in a corn oil study is consistent with the threshold suggested by NTP



1985).  The NOAEL value of 30 mg/kg/day chloroform in oil identified in rats


by NTP (1985) is. selected as the most appropriate value for derivation of the



Longer-term HA values and is supported by two drinking water studies.






        Using this study, the Longer-term HA for the 10-kg child is calculated



as follows:
 T      -    UA   (30 mg/kg/dav) (10 kg) (5/7)          ...                .
 Longer-term HA = J	 (100) (1 L/dav)      =2.1 mg/L (rounded to 2 mg/L)
where:



     30 mg/kg/day = NOAEL, based on absence of clinical signs or histological



                    changes in rats exposed to dibromochloromethane via gavage



                    for 13 weeks






            10 kg = assumed weight of a child








                                    VIII-29

-------
Table VIII-9  Summary of Candidate Studies for Derivation  of
  the Longer-term Health Advisory for Dibromochloromethane

Reference
Chu et al.
(1982b)
NTP (1985)
NTP (1985)
Daniel et al.
(1990)
Borzelleca
and Carchman
(1982)
Exposure
Species Route Duration
Rat Drinking 90 days
water
Rat Gavage 13 weeks
(oil)
Mouse Gavage 13 weeks
(oil)
Rat Gavage 90 days
(oil)
Mouse Drinking 27 weeks
water
NOAEL
Endpoints (mg/kg/day)
Histology, 52
serum chemistry
t
Body weight, 30
clinical signs,
histology
Body weight, 125
clinical signs,
histology
Body weight, ' --
clinical signs,
histology, serum
biochemistry, gross
necropsy
Maternal body 17
weight, gross
pathology, fetal
LOAEL
(mg/kg/day)
250
(Mild hepatic
lesions)
60
(Hepatic
vacuolation)
250
(Renal and
hepatic
lesions)
50
(Hepatic
vacuolizat ion,
serum
biochemistry)
171
(Maternal
toxic. ity ,
                                weight, survival,
                                teratogeriicity
possible
fetotoxicity)

-------
              5/7 = correction factor to account for exposure 5 days/week






              100 = uncertainty factor;  chosen in accordance with NAS/OW


                    guidelines in which a NOAEL from a study in animals is


                    employed






          1 L/day = assumed water consumption by a 10-kg child






        The Longer-term HA for a 70-kg adult consuming 2 L/day of water is


calculated as follows:
T            UA   (30 mg/kg/dav) (70 kg) (5/7)          ...                ...
Longer-term HA = J	  (100) (2 L/dav)    =7.5 mg/L (rounded to 8 mg/L)
        No existing guidelines or standards were located for longer-term


(subchronic) oral or inhalation exposure to dibromochloromethane.
        d.   Reference Dose and Drinking Water Equivalent Level for

            Dibromochloromethane
        Table VIII-10 summarizes studies that were considered for derivation



of the RfD and DWEL for dibromochloromethane.   The dietary study by Tobe



et al. (1982) identified a NOAEL of 10 mg/kg/day based on gross pathology and



serum biochemistry,  but mild weight suppression did occur, and histological



examination of tissues was not performed.  The chronic corn oil vehicle study



by NTP (1985) indicated that doses of 40 and 50 mg/kg/day produce histological



lesions in the liver of rats and mice, respectively.  However, the chronic



studies do not identify a reliable NOAEL.  The subchronic (13-week) study by



NTP (1985) identified a LOAEL and NOAEL of 60 and 30 mg/kg/day, respectively.



                                    VIII-31

-------
Table VIII-10  Summary of Candidate Studies for Derivation
           of the DWEL for Dibromochloromethane

Reference
Tobe et al.
(1982)
NTP (1985)
NTP (1985)
Species Route
Rat Diet
Rat Gavage
(oil)
Mouse Gavage
(oil)
Exposure NOAEL
Duration Eridpoints (mg/kg/day)
24 months Body weight, 10
serum
biochemistry, ,
gross
pathology
102 weeks Clinical signs,
body weight,
gross necropsy,
histology
102 weeks Clinical signs,
body weight,
gross necropsy, 
LOAEL
(mg/kg/day)
39
(Serum enzyme
changes and
altered liver
appearance)
40
(Hepatic
lesions)
50
(Hepatic
lesions)
                               histology

-------
Use of the chronic LOAEL of 40 mg/kg/day would result in a slightly less

protective DWEL of 1 mg/L.   Therefore, greater confidence is placed in the

NOAF.L of 30 mg/kg/day identified in rats in the subchronic (13-week) study by

NTP (1985).



        Using this value, the DWEL is derived as follows:



Step 1: Determination of Reference Dose (RfD)
   RfD  =  (30  ^As/day)  (5/7) _  Q  02U mg/kg/day  (rounded  to  0.02  mg/kg/day)
               (1,000)
where:
     30 mg/kg/day = NOAEL,  based on absence of histological lesions in liver

                    of rats exposed to dibromochloromethane via gavage for

                    13 weeks



              5/7 = correction factor to account for exposure 5 days/week



            1,000 = uncertainty factor; chosen in accordance with NAS/OW

                    guidelines in which a NOAEL from a less-than-lifetime

                    study in animals is employed to derive an RfD



Step 2:  Determination of DWEL
                   DWEL  =
                                    VIII-33

-------
where:
     0.02 mg/kg/day -  RfD









              70 kg =  assumed weight of an adult









            2 L/day -  assumed water consumption by a 70-kg adult









        No existing guidelines or standards were located for chronic oral




exposure to dibromochloromethane.   The interim MCL for total trihalomethanes




is 100 /ig/L (44FR68624).   Although some estimates of cancer risk were




performed, this limit was set primarily on the basis of technological and




economic feasibility.  The basis and purpose of this regulation are discussed




in a report that was prepared by the OW (U.S. EPA 1979).









        4.  Bromoform









        a.  One-day Health Advisory for Bromoform









        Although the data are limited, information from reports on the use of




bromoform as a sedative in children is adequate to establish that the normal




therapeutic dosing regimen (about one drop given three to four times per day,




equivalent to a total daily dose of about 540 mg) produced mild sedation, with




more severe reactions typically being associated with accidental overdoses




(Burton-Fanning 1901; Dwelle.1903; Benson 1907).  On this basis, a dose of




54 mg/kg/day (540 mg/day given to a 10-kg child) is identified as a LOAEL in




humans, and the One-day HA for the 10-kg child  is calculated as follows:









                                    VIII-34

-------
      n   A*  UA   (54 mg/kg/dav) (10 kg)   = .     ...     ,  .
      One-day HA =          (1 L/day)     = 5'4 mS/L (rounded to 5 mg/L)
where:
     54 mg/kg/day = LOAEL, based on sedation in children given bromoform by


                    mouth





            10 kg  assumed weight of a child





              100 = uncertainty factor; selected in accordance with NAS/OW


                    guidelines in which a LOAEL from a study in humans is


                    employed





          1/L day = assumed water consumption by a 10-kg child





        No existing guidelines or standards for acute oral exposure to


bromoform were located.  A concentration of 0.5 ppm in air has been proposed


as a Time-Weighted Average-Threshold Limit Value (TWA-TLV) by the American


Conference of Governmental Industrial Hygienists (ACGIH 1991).   The OSHA


permissible exposure limit (PEL) for bromoform is 0.5 ppm as the TWA exposure


that is not to be exceeded in any up 8-hour work shift of a 40-hour work week


(54FR2332, January 19, 1989).





        b.  Ten-day Health Advisory for Bromoform





        Table VIII-11 summarizes studies that were considered for calculating


the Ten-day HA for bromoform.  The study by Condie et al. (1983) identified a




                                    VIII-35

-------
Table V11I-11  Summary of Candidate Studies for Derivation
       of the Ten-day Health Advisory for Bromoform

Reference
Munson et al.
(1982)
Condie et al.
(1983)

Chu et al.
(1982a)
NTP (1989a)
Aida et al.
(1992a)
Exposure
Species Route Duration
Mouse Gavage 14 days
(aqueous)
Mouse Gavage 14 days
(oil)

Rat Drinking 28 days
water
Rat Gavage 14 days
(oil)
Rat Feed 1 month
NOAEL
Endpoints (mg/kg/day)
Body and organ 125
weight, serum
chemistry,
hematology,
immune function
Serum enzymes, 145
histopathology ,
PAH uptake in
vitro

Clinical signs,' 68
serum chemistry,
histology
Body weight, 200
clinical signs
Clinical signs, 62
body weight, serum
LOAEL
(mg/kg/day)
250
(Elevated
serum enzymes)
289
(Elevated SCOT,
decreased PAH,
histopathology
in li.ver,
kidney)
 -
400
(Decreased
body weight)
187
(Histopathology
                               biochemistry,
                               histology,
                               hematology
in liver,
decreased
cholinesternse)

-------
NOAEL of 145 mg/kg/day and a LOAEL of 289 mg/kg/day chloroform in oil,  based




on histological examination of the liver and kidney along with sensitive




biochemical tests of liver and kidney injury.   This NOAEL value is supported




by the drinking water and corn oil vehicle studies of Munson et al.  (1982),




Chu et al.  (1982a) and NTP (1989a) ,  which identified NOAEL values of 125,  68,




and 200 mg/kg/day, respectively.   A NOAEL of 62 mg/kg/day and a LOAEL of




187 mg/kg/day was determined in a recent feed study based on liver




histopathology and decreased serum cholinesterase (Aida et al. 1992a).









        However, based on a NOAEL of 62 mg/kg/day in animals,  the resulting




Ten-day HA for a 10-kg child would be 6 mg/L.   Since this is larger than the




One-day HA calculated from human data (above),  it is recommended that the




Longer-term HA value of 2 mg/L (calculated below) be taken as a conservative




estimate of the Ten-day HA value.









        No existing guidelines or standards for short-term oral or inhalation




exposure to bromoform were located.









        c.   Longer-term Health Advisory for Bromoform









        Three studies were located chat are candidates for calculating the




Longer-term HA values for bromoform (Table VIII-12).  Chu et al. (1982b)




identified a NOAEL of 52 mg/kg/day and a LOAEL of 250 mg/kg/day chloroform in




the drinking water, based on mild histologic lesions in liver.  The study by




NTP (1989a) identified a NOAEL of 25 mg/kg/day and a LOAEL of 50 mg/kg/day




chloroform administered by corn oil.gavage, based on histological signs of




hepatotoxicity in rats, while mice appeared to be somewhat less sensitive.









                                    VIII-37

-------
                                Table VIII-12  Summary of Candidate Studies for Derivation of
                                        the Longer-term Health Advisory for Broraoform
00

Reference
Chu et al .
(1982b)
NTP (1989a)
NTP (1989a)
Species Route
Rat Drinking
water
Rat Gavage
(oil)
Mouse Gavage
(oil)
Exposure 
Duration
90 days
13 weeks
(5 days/
week)
13 weeks
(5 days/
week)
NOAEL
Endpoints (mg/kg/day)
Histology, 52
serum chemistry
i
Clinical signs, 25
body weight,
necropsy,
histopathology
Clinical signs, 100
body weight,
necropsy,
histopathology
LOAEL
(mg/kg/day)
250
(Mild hepatic
lesions)
50 (Hepato-
cellular
vacuolation)
200 (Hepato-
cellular
vacuolation)

-------
The NOAEL of 25 mg/kg/day in rats is selected as the most appropriate basis



for derivation of the Longer-term HA, which is calculated as follows:





            UA   (25 mg/kg/dav)  (10 kg) (5/7)   . 0    .. ,   '   _,
Longer-term HA -         (100) (1 L/day)      = 1  8 mg/L (rounded to 2 mg/L)
where:
     25 mg/kg/day = NOAEL, based on absence of clinical or histological



                    effects in rats exposed to bromoform by gavage for



                    13 weeks







            10 kg = assumed weight of a child







              5/7 = correction for exposure 5 days/week







              100 = uncertainty factor; chosen in accordance with NAS/OW



                    guidelines in which a NOAEL from a study in animals is



                    employed







          1 L/day = assumed water consumption by a 10-kg child







        The Longer-term HA for a 70-kg adult consuming 2 L/day of water is



calculated as follows:
T            UA   C25 mg/kg/dav)  (70 kg)  (5/7)                 ^ A  r-   f. m  n ^
Longer-term HA = J	  (100)  (2 L/day)       =6.2 mg/L  (rounded  to  6 mg/L)
                                    VIII-39

-------
        No existing guidelines or standards were located for longer-term




(subchronic) oral exposure to bromoform.  The OSHA has established an 8-hour




TWA permissible exposure limit of 0.5 ppm (54FR2322, January 19,  1989).









        d.  Reference Dose and Drinking Water Equivalent Level for Bromoform









        Three studies were located that are candidates for deriving the RfD




and DWEL for bromoform (Table VIII-13).  Tobe et al. (1982) identified a NOAEL




of 18 mg/kg/day, based on gross pathology and serum biochemistry in rats




exposed to bromoform in the diet for 2 years.  However, this study did not




include any histological examination of tissues following bromoform exposure,




and the data have not been published or formally peer reviewed.  On this




basis, this study is judged as not adequate for derivation of an RfD and a




DWEL for bromoform.   A LOAEL of 100 mg/kg/day was identified by the chronic




(2-year) corn oil gavage study by NTP (1989a) for decreased body weight and




liver effects in rats and mice.  A reliable NOAEL was not identified by this




study since only high doses (100 or 200 mg/kg/day) were tested.  The subchro




nic (13-week) study by NTP (1989a) tested lower doses and identified NOAEL and




LOAEL values of 25 and 50 mg/kg/day, respectively.  Use of the chronic LOAEL




of 100 mg/kg/day would result in a less protective DWEL of 2.5 mg/L.




Therefore, the subchronic NOAEL of 23 mg/kg/day is selected as the most




appropriate basis for the derivation of an RfD and DWEL.









        Based on this value,  the DWEL is calculated as follows:
                                    VIII-40

-------
                        Table VIII-13
                     Summary of Candidate  Studies  for  Derivation of
                           DWEL for Bromoform

Reference
Tobe et al.
(1982)
Exposure
Species Route Duration
Rat Diet 2k months
NOAEL
Endpoints (mg/kg/day)
Body weight, 18 (M)
serum chemistry, 30 (F)
gross pathology ,
LOAEL
(mg/kg/day)
71 (M)
120 (F)
(Decreased
NTP (1989a)
Rat
NTP (1989a)
Mouse
Gavage       103 weeks    Clinical signs,
(oil) .       (5 days/     body weight,
             week)        survival, gross
                          necropsy, histo-
                          pathology
Gavage       103 weeks    Clinical signs,  100 (M)
(oil)        (5 days/     body weight,
             week)        survival, gross
                          necropsy, histo-
                          pathology
body weight,
elevated serum
enzymes)

100 (M)(F)
(Decreased
body weight,
lethargy, mild
liver histo-
pathology)

100 (F)
(Decreased
body weight,
mild liver
histopathology)

-------
Seep 1:  Determination of RfD
   RfD = (25 mR/kfi/dav) (5/7) _ Q Qlg mg/kg/day (rounded to 0.02 mg/kg/day)
               (1,000)
where:
     25 mg/kg/day = NOAEL,  based on absence of clinical or histological

                    effec-ts in rats exposed to bromoform in the diet for

                    13 weeks



              5/7 = correction factor to account for exposure 5 days/week



            1,000 = uncertainty factor;  chosen in accordance with NAS/OW

                    guidelines in which a NOAEL from a less-than-lifetime

                    study in animals is employed to derive an RfD



Step 2:  Determination of DWEL
                  DWEL =  (0-02 ,R/kK/d.y)  (70     =  Q  ? mg/L
                                2 L/day
where:
      0.02 mg/kg/day = RfD
               70 kg = assumed weight of an adult



             2 L/day = assumed water consumption by a 70-kg adult


                                    VIII-42

-------
        B.  Carcinogenic Effects









        1.  Categorization of Carcinogenic Potential









        a.  Chloroform









        The International Agency for Research on Cancer (IARC) has performed




an assessment of the degree of evidence for the carcinogenicity of chloroform




to humans and experimental animals (IARC 1982, 1987).  This assessment




concluded that chloroform is a Group 2B chemical (sufficient evidence of




carcinogenicity in animals but inadequate evidence of carcinogenicity in




humans).









        The U.S. EPA (1985a) has reviewed the evidence on the carcinogenicity




of chloroform and has ranked it as a Group B2 chemical (probable human




carcinogen) (IRIS 1990).









        b.  Brominated Trihalomethanes









        The Carcinogenic Risk Assessment Verification Endeavor (CRAVE) group




of the U.S. EPA has recently reviewed the available evidence on the carcino-




genicity of the brominated trihalomethanes.  Based on this review, CRAVE has




assigned bromoform and bromodichloromethane to Group B2 and dibromochloro-




methane to Group C (IRIS 1991).   IARC has evaluated the carcinogenic




potentials of brominated trihalomethanes (IARC 1991a, 1991b, 1991c).  IARC




concluded that there is sufficient .evidence of carcinogenicity for




bromodichloromethane in experimental animals, but inadequate evidence in




humans.  For dibromochloromethane and bromoform, IARC concluded that there  is




                                    VIII-43

-------
limited-evidence of carcinogenicity in experimental animals and  inadequate




evidence in humans.  IARC suggested that bromodichloromethane  is a Group  2B




carcinogen (possibly carcinogenic to humans) and r.h^t dibromochloromethane and




bromoform are Group 3 carcinogens (not classifiable as to their  carcinogen-




icity to humans).









        2.   Quantitative Carcinogenic Risk Estimates









        a.   Chloroform









        Five data sets were used by the U.S. EPA (1985a) to estimate the




carcinogenic potency of chloroform, as shown in Table VIII-14.   The unit  risks




at 1 mg/kg/day, calculated by the linearized multistage model, were judged to




be comparable for each of these data sets.   The values of q^ derived from




these five data sets were 2.0xlCT1,  3.3xlO'2, 2.35xlO"2,  l.OxlO"1  and




4.41xlO"3  (mg/kg/day)"1, respectively.  The geometric mean of  the slope




estimates calculated from liver tumors in male and female mice (S.lxlO"2




[mg/kg/day]"1)  was  taken to  represent  the carcinogenic  potency.  Based on




this, the upper-bound estimate of the cancer risk due to 1 ^g/L  in water  was




2.3xlO"6.   This estimate was judged to be consistent with the limited




epidemiologic data available for humans  (U.S. EPA 1985a).  Since risk is




linear in this range, risk factors of 10"4,  10"5, and 10"6 correspond to




concentrations of 40, 4, and 0.4 /ig/L, respectively.









        More recently, the U.S. EPA (1987) reviewed these studies and




concluded that the quantitative risk estimate for chloroform  should be  based




only on the incidence of renal tumors in rats reported in the  study by




Jorgenson et al. (1985) and should not consider the incidence  of liver  tumors




                                    VIII-44

-------
      TABLE VIII-14  Upper-Bound Estimates of Cancer Risk of 1 mg/kg/day
               of Chloroform, Calculated by Four Models on the
                          Basis of Various Data Sets

Data Base Multistage Probit
Liver tumors in female 1.8 x 10"1 2.1 x 10"1
mice (NCI, 1976)
Liver tumors in male 3.3 x 10"2 6.7 x 10"2
mice (NCI, 1976)
Kidney tumors in male 2.4 x 10"2 3.9 x 10"5
rats (NCI, 1976)
Kidney tumors in male 1.0 x 10"1 NAb
mice (Roe et al. , 1979)
Kidney tumors in male 4.4 x 10~3 9.0 x 10"5
rats (Jorgenson et al.,
1985)
Weibull One-Hit
4.8 x 10'1 1.8 x 10'1

3.2 x 10-3 1.6 x lO'1

3.0 x 10'3 2.5 x ID'2

NA 1.0 x 10'1

4.8 x ID'4 5.4 x 10'3



aUpper-bound estimates are calculated by the one-sided 95% confidence limit.
''NA = not applicable;  models are not applicable because there is only one
 dosed group

Adapted from U.S. EPA (1985a).
                                    VIII-45

-------
reported in mice by NCI (1976).  This decision was based on the observation




that liver tumors were not increased in mice exposed to chloroform via




drinking water, suggesting that the vehicle may have played a role ir. the




hepatic tumors following exposure in oil.  This concept is supported by the




observation that corn oil appears to potentiate the hepatotoxic effects of




chloroform in B6C3F1 mice (Bull et al.  1986).  The slope factor based on renal




tumors in rats was recalculated employing the incidence of tubular cell




adenomas, tubular cell adenocarcinomas,  and nephroblastomas,  but excluding




metastatic and transitional tumors (see Table V-31).  The resulting slope




factor is 6.IxlO'3  (mg/kg/day)'1 (IRIS 1992).  Based on this, the unit risk




for chloroform is 1.7xlO~7  (jig/L)'1, and the drinking water concentration




corresponding to the 10'6 risk level  is  6  ^g/L (IRIS 1992) .









        The NAS (1987) has  also reviewed available data on the carcinogenicity




of chloroform and calculated several different risk estimates using the




linearized multistage model.   The results of their calculations are shown in




Table VIII-15.  Because of the same concern discussed above regarding the




apparent oil vehicle effect on liver tumors in mice, the NAS Subcommittee on




Health Effects of Disinfectants and Disinfectant By-Products also recommended




that the cancer slope factor for chloroform be based on the kidney tumor data




in rats reported by Jorgenson et al (1985).









        b.  Brominated Trihalomethanes









        The NAS (1987) utilized the data reported by NTP (1985) on the




frequency of liver tumors in female B6C3F1 mice exposed to dibromochloro-




methane to calculate an excess lifetime cancer unit risk of 8.3xlO"7,  using




the linearized multistage model and assuming consumption of 1 L of water per




                                    VIII-46

-------
                TABLE VIII-15   Carcinogenic  Risk  Estimates  for
                         Chloroform Calculated bv NAS



Reference
Jorgenson et al .
(1985)
NCI (1976)

NCI (1976)

Roe et al .
(1979)
Concentration
Equivalent to
Unit Cancer Riskb Risk of 10'6
Species Sex Tumor (Upper 95% Limit) (/ig/L)c
Osborne -Mendel M Kidney 1.1 x 10"7 4.5
rat
B6C3F1* F Liver 1.9 x 10'6 0.3
mouse
Osborne -Mendel M Kidney 4.7 x 10'8 10.6
rat
ICI M Kidney 3.7 x 10'7 1.4
mouse

aAdapted from NAS (1987).
bAssuming consumption of 1 L/day of water.
Assuming consumption of 2 L/day of water.
                                    VIII-47

-------
day containing 1 pig/L of dibromochloromethane.  Based on this calculation, the




concentration associated with a risk of 10"6 is 0.6 ng/L,  assuming consumption




of 2 L of water per day.









        More recently, the U.S. EPA has calculated quantitative cancer risk




estimates for bromodichloromethane, dibromochloromethane, and bromoform, based




on the data from the three 2-year oral exposure studies performed by NTP




(1987, 1985, 1989a) (see Tables V-31, V-32, V-33).   The resulting slope




factors, unit risks, and risk-specific concentration values are summarized in




Table VIII-16, along with a summary of the  tumor data used to derive the




values.









        Evaluation of the quantitative cancer risk estimates for the




brominated trihalomethanes based on the three NTP rodent studies (1985, 1987,




1989a) is complicated by the use of a corn  oil vehicle in these studies.




Although a vehicle effect has not been investigated for brominated




trihalomethanes, it can be inferred from studies of chloroform carcinogenicity




(see above) that such an effect might exist, at least for hepatic tumors in




mice.  Therefore, in the case of bromodichloromethane, the U.S. EPA believes




that the most appropriate basis of the cancer risk estimate is the incidence




of renal tumors in male mice.  Renal tumors are considered to be appropriate




because these tumors were increased in a dose-dependent manner in both mice




(male) and rats (both sexes).  Therefore, the slope factor based on renal




tumors in male mice (6.2xlO~2  per mg/kg/day) is the recommended value.









        In the case of dibromochloromethane,  the only tumor data available are




for liver tumors in mice.  Therefore, the slope factor (8.4xlO'2




[mg/kg/day]"1)  is based on these data,  but some uncertainty exists regarding




                                    VIII-48

-------
   TABLE VIII-16  Carcinogenic  Risk  Estimates for  Brominated  Trihalomethanes


Trihalomethane Tumor Site
Sromodichloro- Liver
methane
Kidney


Large
intestine
Large intestine
and kidney
(combined)

Spec i es
House

Rat

Mouse
Rat

Rat



Sex
Female

Male
Female
Male
Male
Female
Male
Female

Slope Factorl
(mg/kg-day)"
1.3 x 10'1

8.7 x 10^
9.5 x 10
6.2 x 10"2
2.5 x 10"*
4.9 x 10"3
2.4 x 10'*
7.9 x 10 

Unit
Risk
((ig/L)"1
3.7 x 10"6

2.5 x 10"'
2.7 x 10
1.8 x 10"6
7.1 x 10"'
1.4 x 10
6.9 x 10''
2.3 x 10"7

10"5 Risk
Concen-
tration
Ug/D
3

40
37
6
14
72
15
44

Oibromochloro- Liver
  methane
Bromoform
Large intestine
                                Mouse
                                Rat
                                          Female
                                          Female
                                                      8.4 x 10"
                                                      7.9 x 10"
                                                         2.4 x 10"
                                                         2.3 x 10"
                                                                                   40
Adapted from IRIS (1992).
                                          VIII-49

-------
the relevance of this value to exposure via drinking water.  The U.S. EPA




plans to seek data on the tumorigenicity of dibromochloromethane in water  in




order ro clarify this issue.









        In the case of bromoform, the cancer risk estimate  (7.9xlO~3 [tng/kg/




day]"1)  is  based on the  incidence of intestinal"tumors in rats.   There are no




data to suggest that tumor incidence in this tissue is influenced by the use




of an oil vehicle, so thi.s risk estimate is believed to be  applicable to




drinking water exposures.









        C.   Summary









        Table VIII-17 summarizes HA and DWEL values (calculated on the basis




of noncarcinogenic endpoints) and the 10~6  excess cancer risk levels




(calculated using the linearized multistage model) for chloroform,




bromodichloromethane, dibromochloromethane, and bromoform.
                                    VIII-30

-------
              TABLE VIII-17
Summary of Quantification of Toxicological
Effects for Trihalomethanes

Value
Chloroform
One-day HA for 10-kg child
Ten-day HA for 10-kg child
Longer-term HA for 10-kg child
Longer-term HA for 70-kg adult
OWEL (70-kg adult)
Excess cancer risk (10" )
--.
Bromodi ch 1 oromethane
One-day HA for 10-kg child
Ten-day HA for 10-kg child
Longer-term HA for 10-kg child
Longer-term HA for 70-kg adult
DUEL (70-kg adult)
Excess cancer risk (10~6)
Dibromoch I oromethane 
One-day HA for 10-kg child
Ten-day HA for 10-kg child
Longer-term HA for 10-kg child
Longer-term HA for 70-kg adult
DWEL (70-kg adult)
Excess cancer risk (10~6)

Drinking Uater
Concentration
4 mg/L
4 mg/L
0.1 mg/L
0.4 mg/L
0.4 mg/L
6 ig/L
4.5 A9/L

6 mg/L*
6 mg/L
4 mg/L
13 mg/L
0.7 mg/L
0.6 jig/L

6 mg/L*
6 mg/L '
2 mg/L
8 mg/L
0.7 mg/L
0.4 
-------
IX.     REFERENCES
Abdel-Rahman, M.S.  1982.  The presence of trihalomechanes in soft: drinks.  J.
Appl. Toxicol.  2(3) :165-166.

ACGIH.   1980.  American Conference of Governmental Industrial Hygienists.
Threshold limit values for chemical substances and physical agents in the
workroom environment with intended changes for 1980.   Cincinnati, OH:  ACGIH.
pp. 90-91.

ACGIH.   1989.  American Conference of Governmental Industrial Hygienists.
Threshold limit values and biological exposure indices for 1989-1990.
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