NTP TECHNICAL REPORT

ON THE

CARCINOGENESIS STUDIES OF 

2,2-BIS(BROMOMETHYL)-1,3-PROPANEDIOL,

NITROMETHANE, 

AND 1,2,3-TRICHLOROPROPANE

(CAS NOS. 3296-90-0, 75-52-5, and 96-18-4)

IN GUPPIES (Poecilia reticulata)

AND MEDAKA (Oryzias latipes)

(WATERBORNE STUDIES)

NATIONAL TOXICOLOGY PROGRAM

P.O. Box 12233

Research Triangle Park, NC 27709

October 2005

NTP TR 528

NIH Publication No. 06-4464

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

National Institutes of Health
FOREWORD 

The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of Health and Human 

Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; the National Institute of Environmental 

Health Sciences (NIEHS), National Institutes of Health; the National Center for Toxicological Research (NCTR), Food and 

Drug Administration; and the National Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control 

and Prevention. In July 1981, the Carcinogenesis Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP 

coordinates the relevant programs, staff, and resources from these Public Health Service agencies relating to basic and applied

research and to biological assay development and validation. 

The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous chemicals. 

This knowledge is used for protecting the health of the American people and for the primary prevention of disease. 

The studies described in this Technical Report were performed under the direction of the NIEHS and were conducted in 

compliance with NTP laboratory health and safety requirements and must meet or exceed all applicable federal, state, and local 

health and safety regulations. Animal care and use were in accordance with the Public Health Service Policy on Humane Care 

and Use of Animals. The prechronic and chronic studies were conducted in compliance with Food and Drug Administration 

(FDA) Good Laboratory Practice Regulations, and all aspects of the chronic studies were subjected to retrospective quality 

assurance audits before being presented for public review. 

These studies are designed and conducted to characterize and evaluate the toxicologic potential, including carcinogenic activity, 

of selected chemicals in laboratory animals (usually two species, rats and mice). Chemicals selected for NTP toxicology and 

carcinogenesis studies are chosen primarily on the bases of human exposure, level of production, and chemical structure. The 

interpretive conclusions presented in this Technical Report are based only on the results of these NTP studies. Extrapolation of 

these results to other species and quantitative risk analyses for humans require wider analyses beyond the purview of these 

studies. Selection per se is not an indicator of a chemical's carcinogenic potential. 

Details about ongoing and completed NTP studies, abstracts of all NTP Technical Reports, and full versions of the completed 

reports are available at the NTP's World Wide Web site: http://ntp.niehs.nih.gov. In addition, printed copies of these reports are 

available from NTP as supplies last by contacting (919) 541-3419. 
NTP TECHNICAL REPORT

ON THE

CARCINOGENESIS STUDIES OF 

2,2-BIS(BROMOMETHYL)-1,3-PROPANEDIOL,

NITROMETHANE, 

AND 1,2,3-TRICHLOROPROPANE

(CAS NOS. 3296-90-0, 75-52-5, and 96-18-4)

IN GUPPIES (Poecilia reticulata)

AND MEDAKA (Oryzias latipes)

(WATERBORNE STUDIES)

NATIONAL TOXICOLOGY PROGRAM

P.O. Box 12233

Research Triangle Park, NC 27709

October 2005

NTP TR 528

NIH Publication No. 06-4464

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

National Institutes of Health
2 Fish Studies, NTP TR 528

CONTRIBUTORS

National Toxicology Program

Evaluated and interpreted results and reported findings

N.J. Bernheim, M.S., Study Scientist

G.A. Boorman, D.V.M., Ph.D., Study Scientist

R.A. Herbert, D.V.M., Ph.D., Study Pathologist

D.W. Bristol, Ph.D.

J.R. Bucher, Ph.D.

J.R. Hailey, D.V.M.

J.K. Haseman, Ph.D.

G.E. Kissling, Ph.D.

R.R. Maronpot, D.V.M.

A. Nyska, D.V.M.

C.S. Smith, Ph.D.

G.S. Travlos, D.V.M.

K.L. Witt, M.S.

University of Southern Mississippi,

Gulf Coast Research Laboratory

Conducted study

W.E. Hawkins, Ph.D., Principal Investigator

M. Brouwer, Ph.D.

Experimental Pathology Laboratories, Inc.

Evaluated pathology findings

J.F. Hardisty, D.V.M., Principal Investigator

M.J. Wolfe, D.V.M., Ph.D.

Pathology Associates International 

A Charles River Company

Provided pathology quality assurance

M.P. Jokinen, D.V.M.

Dynamac Corporation

Prepared quality assurance audits

S. Brecher, Ph.D., Principal Investigator

NTP Pathology Working Group

Evaluated slides and prepared pathology reports for the 

2,2-bis(bromomethyl)-1,3-propanediol, nitromethane, 

and 1,2,3-trichloropropane studies in guppies and medaka

(July 11, 2002)

M.P. Jokinen, D.V.M., Chairperson

Pathology Associates International, A Charles River Company

G.A. Boorman, D.V.M., Ph.D.

National Toxicology Program

J.W. Fournie, Ph.D.

Environmental Protection Agency

J.C. Harshbarger, Ph.D.

Georgetown University

W.E. Hawkins, Ph.D.

Gulf Coast Research Laboratory

R.A. Herbert, D.V.M., Ph.D.

National Toxicology Program

J.M. Law, D.V.M., Ph.D.

North Carolina State University

M.J. Wolfe, D.V.M., Ph.D.

Experimental Pathology Laboratories, Inc.

Analytical Sciences, Inc.

Provided statistical analyses

P.W. Crockett, Ph.D., Principal Investigator

L.J. Betz, M.S.

K.P. McGowan, M.B.A.

J.T. Scott, M.S.

Biotechnical Services, Inc.

Prepared Technical Report

S.R. Gunnels, M.A., Principal Investigator

P.H. Carver, B.A.

L.M. Harper, B.S.

E.S. Rathman, M.S.

D.C. Serbus, Ph.D.
3

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

TECHNICAL REPORTS REVIEW SUBCOMMITTEE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS . . . . . . . . . . . . 12

OVERVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

DISCUSSION AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

APPENDIX A Summary of Lesions in Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

APPENDIX B Summary of Lesions in Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

APPENDIX C Summary of Lesions in Guppies in the 16-Month Waterborne Study 

of Nitromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

APPENDIX D Summary of Lesions in Medaka in the 13-Month Waterborne Study 

of Nitromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

APPENDIX E Summary of Lesions in Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

APPENDIX F Summary of Lesions in Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

APPENDIX G Chemical Characterization and Generation of Aquaria Concentrations . . . . . . . . . . . . . 175

APPENDIX H Nutrient Composition and Contaminant Levels in Fish Feed and Aquaria Water . . . . 189

CONTENTS
4 Fish Studies, NTP TR 528

SUMMARY

Background 

The National Toxicology Program has used rats and mice to test if chemicals can cause cancer in animals. We

wanted to see if fish could be used as a test animal for cancer testing. In this study, we exposed two species of

fish, guppies and medaka, to three different chemicals that caused cancer in rodents to see if fish had the same

response.

Methods 

We held groups of approximately 110 guppies or 170 medaka in aquaria each containing a specific concentration

of a chemical. Three different chemicals were tested, and three different concentrations of each chemical were

used. Similar groups of fish were held in aquaria containing only clean water and served as the control groups.

The three chemicals used were 2,2-bis(bromomethyl)-1,3-propanediol, nitromethane, or 1,2,3-trichloropropane.

After nine months of exposure, some of the fish were transferred into aquaria with just clean water, and the others

remained exposed to chemical for another four to seven months. At the end of the studies, about 20 tissue sites

from each fish were examined microscopically. 

Results 

At higher concentrations, 2,2-bis(bromomethyl)-1,3-propanediol was toxic to guppies, and many of the females

died. Some of the male guppies and male medaka developed liver cancer, but few female medaka did.

Nitromethane in the water promoted the growth of algae in the aquaria, and the tanks had to be cleaned often. The

cleaning process may have resulted in the death of several of the fish. Not enough male guppies were left to evaluate, and very few tumors were seen in the surviving female guppies or male or female medaka.

Male and female guppies exposed to 1,2,3-trichloropropane had higher rates of liver tumors than the control guppies did, and male and female medaka developed tumors of the liver and gallbladder. 

Conclusions

While all three test chemicals caused cancer in several different tissues in laboratory rodents, the response in fish

was not as consistent, as cancer was seen only mainly in the liver. Fish that died during the study often were eaten

by the other fish or dissolved and could not be evaluated. We conclude that several technical problems remain to

be solved before fish can be a reliable replacement for rodents in cancer testing.
ABSTRACT

2,2-BIS(BROMOMETHYL)-1,3-PROPANEDIOL

(FR-1138®)

(Technical Grade: 78.6% 2,2-bis(bromomethyl)-1,3-propanediol, 6.6% 2,2-bis(hydroxymethyl)-1-bromo-3-hydroxypropane, 6.9% 

2,2-bis(bromomethyl)-1-bromo-3-hydroxypropane, 0.2% pentaerythritol, and 7.7% dimers and structural isomers)

CAS No. 3296-90-0

Chemical Formula: C5H10Br2O2 Molecular Weight: 261.94

Synonyms: 2,2-Bis(2-bromomethyl)-1,3-propanediol; 1,3-dibromo-2,2-dihydroxymethylpropane; 

1,3-dibromo-2,2-dimethylolpropane; 2,2-dibromomethyl-1,3-propanediol; dibromopentaerythritol; 

dibromoneopentyl glycol; pentaerythritol dibromide; pentaerythritol dibromohydrin 

CH3NO2

NITROMETHANE

CAS No. 75-52-5

Molecular Weight: 61.04

Synonym: Nitrocarbol

1,2,3-TRICHLOROPROPANE

CAS No. 96-18-4

Chemical Formula: C3H5Cl3 Molecular Weight: 147.44

Synonyms: Allyl trichloride, glycerol trichlorohydrin, glyceryl trichlorohydrin, trichlorohydrin

HOH2C C CH2OH

CH2Br

CH2Br

5

Cl C C C Cl

H

H Cl H

H H
6 Fish Studies, NTP TR 528

The NTP chose to initiate studies in fish as an exploration of alternate or additional models for examining

chemical toxicity and carcinogenicity. The use of small

fish species in carcinogenicity testing offered potential

advantages as a bioassay test system, including significant savings in cost and time over rodent studies. Large

numbers of small fish could be easily maintained in a

limited area. The two species chosen for study were

guppy (Poecilia reticulata) and medaka (Oryzias

latipes), both of which are hardy, easily maintained, and

have a low occurrence of background lesions.

The three chemicals chosen for study in fish had already

been studied by the NTP in rodents, permitting a comparison of results between the two models. Two of the

chemicals used (2,2-bis(bromomethyl)-1,3-propanediol

and 1,2,3-trichloropropane) were mutagenic and multisite carcinogens in rats and mice. The third chemical,

nitromethane, was nonmutagenic with a more modest

carcinogenic response in rodents. Male and female guppies and medaka were exposed to 2,2-bis(bromomethyl)-

1,3-propanediol (greater than 99% pure), nitromethane,

(greater than 99% pure), or 1,2,3-trichloropropane (99%

pure) in aquaria water for up to 16 months. 

OVERALL STUDY DESIGN

Groups of approximately 220 guppies (two replicates of

110) were maintained in aquaria water containing 

nominal concentrations of 0, 24, 60, or 150 mg/L

2,2-bis(bromomethyl)-1,3-propanediol; 0, 10, 30, or

70 mg/L nitromethane; or 0, 4.5, 9.0, or 18.0 mg/L

1,2,3-trichloropropane. Groups of approximately

340 medaka (two replicates of 170) were maintained in

aquaria water containing 0, 24, 60, or 150 mg/L

2,2-bis(bromomethyl)-1,3-propanediol; 0, 10, 20, or

40 mg/L nitromethane; or 0, 4.5, 9.0, or 18.0 mg/L

1,2,3-trichloropropane. The overall study durations

were 16 months for all guppy studies, 14 months for

2,2-bis(bromomethyl)-1,3-propanediol-exposed

medaka, and 13 months for nitromethane- and

1,2,3-trichloropropane-exposed medaka.

Ten guppies and 10 medaka from each group replicate

were sacrificed at 9 months for histopathologic analysis.

Approximately one third of the remaining fish from each

group were placed in chemical-free water at 9 months

and constituted a stop-exposure study component. The

remainder of the fish were exposed for the duration of

the study and constituted the core study component. A

stop-exposure component was added to determine if

stopping the exposure at 9 months and transferring to

chemical-free aquaria might allow for better survival and

tumor development. The sex of guppies and medaka

was determined at histopathologic analysis.

2,2-BIS(BROMOMETHYL)-

1,3-PROPANEDIOL

16-MONTH STUDY IN GUPPIES

2,2-Bis(bromomethyl)-1,3-propanediol was chronically

toxic to guppies in the 60 and 150 mg/L core and

stop-exposure groups. Due to mortality, exposure of

core study animals in the 150 mg/L group was terminated on day 443, after approximately 64 weeks on

study, and fish were maintained in 2,2-bis(bromomethyl)-1,3-propanediol-free water in the exposure system until the end of the study at 69 weeks. Nominal

exposure concentrations of 24, 60, and 150 mg/L provided actual aquaria water exposure concentrations of

20.0, 53.5, and 139.0 mg/L 2,2-bis(bromomethyl)-

1,3-propanediol, respectively. There were no treatmentrelated differences between the control and exposed

groups in body weights or lengths.

At 9 months, hepatocellular adenomas occurred in one

24 mg/L male and in one 150 mg/L male. In the core

study, the incidence of hepatocellular adenoma or carcinoma (combined) in 150 mg/L males was greater than

that in the controls; multiple adenomas occurred in

two 150 mg/L males and in one 150 mg/L female.

Cholangioma occurred in a small number of exposed

males and females. In the stop-exposure study, incidences of hepatocellular adenoma (including multiple)

and of hepatocellular carcinoma were greater in

150 mg/L males than in controls. One cholangioma and

one cholangiocarcinoma occurred in the 150 mg/L

female group.

14-MONTH STUDY IN MEDAKA

Exposure to 2,2-bis(bromomethyl)-1,3-propanediol did

not result in any significant reduction in survival,

although the mortality of fish was somewhat greater in

the 60 and 150 mg/L core study groups than in the

control and 24 mg/L groups. After reallocation, mortality of medaka in the 60 and 150 mg/L core groups was

slightly increased over the corresponding stop-exposure

groups.
Fish Studies, NTP TR 528 7

Nominal exposure concentrations of 24, 60, and

150 mg/L provided actual exposure concentrations of

19.4, 56.9, and 137.8 mg/L 2,2-bis(bromomethyl)-

1,3-propanediol, respectively. Core study animals in the

60 and 150 mg/L groups were significantly larger, in

both body length and weight, than control group fish.

In the core study, the incidence of hepatocellular

adenoma or carcinoma (combined) was increased in

150 mg/L males. Cholangiocarcinomas occurred in a

few exposed males and females, with all but one occurring in 150 mg/L fish. One cholangioma occurred in a

150 mg/L female, and one occurred in a control female.

In the stop-exposure study, incidences of hepatocellular

adenoma or carcinoma (combined) were marginally

increased in the 150 mg/L group of males and in the 60

and 150 mg/L groups of females as compared with controls. Cholangiocarcinoma occurred in one male and

one female in the 150 mg/L groups and in one control

female.

NITROMETHANE

16-MONTH STUDY IN GUPPIES

Although the cause of death could not be confirmed in

many cases, mortality in the 70 mg/L groups appeared to

indicate that this level of nitromethane exposure was

chronically toxic. This is confirmed by the similar survival rate of guppies from all treatments following

removal from treatment aquaria and placement in stopexposure. Due to the high mortality of fish in the

70 mg/L core study groups, these fish were removed

from treatment (day 396) and fixed for histological

analyses after approximately 57 weeks on study. The

controls and other exposed groups were sacrificed at

70 weeks. Nominal exposure concentrations of 10, 30,

and 70 mg/L provided actual exposure concentrations of

9.9, 28.7, and 66.4 mg/L nitromethane, respectively.

There were no treatment-related differences between the

control and exposed groups in body lengths or weights.

13-MONTH STUDY IN MEDAKA

Nitromethane in the aquaria supported a substantial

microfaunal growth which, without frequent cleaning,

affected water quality and treatment concentrations. To

maintain acceptable water quality and treatment concentrations potentially affected by the rapid microfaunal

growth, the study aquaria were brushed once and

siphoned three times each day. Due to this frequent

activity, a number of fish probably died due to mechanical injury. Unfortunately, the cause of death could not

be confirmed in many cases; the mortality from this

activity is believed to have been approximately uniform

among treatments and should not have affected the 

comparison of survival between treatments. Based on

mortality in this study and the previous life-span evaluation, the life phase of this study was terminated approximately 13.5 months after hatching.

Nominal exposure concentrations of 10, 20, and

40 mg/L resulted in actual exposure concentrations of

9.3, 20.8, and 41.7 mg/L nitromethane, respectively. No

differences between control and exposed groups were

found in body lengths or weights at the 9-month interim

evaluation. Due to mortality, unequal numbers of fish

were distributed among the core study and stop-exposure

aquaria at 9 months. Differences in lengths and weights

were found at 13 months. The biological significance of

this finding is unknown.

At 9 months, a single cholangiocarcinoma occurred in a

40 mg/L male. Hepatocellular adenomas occurred in

two 20 mg/L males and in one 40 mg/L female. In the

core study, one cholangioma occurred in a 20 mg/L

male, and cholangiocarcinomas were seen in a few

exposed males, but none occurred in control males.

1,2,3-TRICHLOROPROPANE

16-MONTH STUDY IN GUPPIES

The survival of exposed guppies was less than that of the

control group at 9 months. Reduced survival was evident at 6 months in the 18.0 mg/L groups and at

7 months in the 4.5 and 9.0 mg/L groups. Survival was

significantly reduced in the 18.0 mg/L core study group

within 1 month of the 9-month interim evaluation, and

mortality in this group was 42.6% between 9 months and

study termination. Nominal exposure concentrations of

4.5, 9.0, and 18.0 mg/L resulted in actual exposure concentrations of 4.4, 8.8, and 18.2 mg/L 1,2,3-trichloropropane, respectively. Guppies in the 18.0 mg/L core

study group were significantly longer and weighed more

than the controls. Fish in the 18.0 mg/L stop-exposure

group also weighed more than the controls. Mortality of

fish during the study resulted in unequal numbers of

individuals distributed to core study and stop-exposure

aquaria at 9 months. This appears to have influenced the

length and weight of fish measured at study termination

(i.e., the smaller tank population allowed the fish to grow
8 Fish Studies, NTP TR 528

more). Observed differences in weight and length

between controls and 18.0 mg/L fish was most likely an

artifact of the reduced numbers of fish in the 18.0 mg/L

aquaria. 

At 9 months, multiple hepatocellular adenomas occurred

in one 4.5 mg/L male, and one hepatocellular adenoma

occurred in a control male. In the core study, increased

incidences of cholangiocellular (bile duct) and hepatocellular neoplasms occurred in exposed groups of males

and females. Cholangioma and cholangiocarcinoma

were seen in several exposed males and females. In the

stop-exposure study, increased incidences of hepatocellular neoplasms occurred in 18.0 mg/L males and

increased incidences of cholangiocellular (bile duct)

neoplasms occurred in 18.0 mg/L females.

13-MONTH STUDY IN MEDAKA

Survival in the 9.0 and 18.0 mg/L groups indicated that

1,2,3-trichloropropane was chronically toxic to the

medaka; reduced survival was evident beginning at

9 months of exposure. Mortality in the 18.0 mg/L core

study group was 26.3% and mortality in the 9.0 mg/L

group was 17.3% between 9 months and study termination at 13 months. Survival was also reduced in stopexposure fish from the 9.0 and 18.0 mg/L groups at the

end of the study. Nominal exposure concentrations of

4.5, 9.0, and 18.0 mg/L resulted in actual exposure concentrations of 4.6, 9.2, and 18.0 mg/L 1,2,3-trichloropropane, respectively. At 9 months, the weights of

medaka in the 9.0 and 18.0 mg/L groups were significantly increased. Core study medaka in the 18.0 mg/L

group were both longer and weighed more than the controls at the end of the study. Observed differences in

length and/or weight between controls and 9.0 or

18.0 mg/L fish were most likely an artifact of the

reduced numbers of fish in these exposure aquaria.

The incidences of cholangiocarcinomas in the 9.0 and

18.0 mg/L groups of males were significantly increased

at 9 months. In the core study, the incidences of cholangiocarcinoma were significantly increased in all exposed

groups of males and females; the incidences of hepatocellular neoplasms and hepatocholangiocarcinomas

were significantly increased in 18.0 mg/L medaka.

In the stop-exposure study, increased incidences of

cholangiocarcinoma occurred in all exposed groups of

males and females. The incidence of hepatocellular

carcinoma was significantly increased in 18.0 mg/L

females.

At 9 months, papillary adenomas of the gallbladder

occurred in two 18.0 mg/L males. No gallbladder neoplasms were seen in the controls or any of the other

exposed groups. In the core study, papillary adenoma of

the gallbladder occurred in a number of exposed males

and females, and incidences were significantly increased

in the 9.0 and 18.0 mg/L groups. In the stop-exposure

study, the incidence of papillary adenoma in males

exposed to 18.0 mg/L was significantly increased; papillary adenoma and carcinoma were observed in most

exposed groups of males and females.

CONCLUSIONS

Under the conditions of these waterborne studies,

2,2-bis(bromomethyl)-1,3-propanediol at concentrations

of up to 150 mg/L for 16 months was considered carcinogenic for male guppies based on increased incidences of hepatocellular adenomas or carcinomas. The

study in female guppies was considered inadequate

based on reduced survival. Under the conditions of

these waterborne studies, 2,2-bis(bromomethyl)-

1,3-propanediol at concentrations of up to 150 mg/L for

14 months was considered carcinogenic for male

medaka based on increased incidences of hepatocellular

adenomas or carcinomas. The study in female medaka

was considered negative.

Under the conditions of these waterborne studies, the

study of nitromethane in male guppies was considered

inadequate based on reduced survival. The study in

female guppies at concentrations up to 70 mg/L for

16 months was considered negative. The study in male

and female medaka was considered negative.

Under the conditions of these waterborne studies, 

1,2,3-trichloropropane at concentrations of up to

18 mg/L for 16 months was considered carcinogenic for

male and female guppies based on increased incidences

of a variety of liver neoplasms. Under the conditions of

these waterborne studies, 1,2,3-trichloropropane at

concentrations of up to 18 mg/L for 13 months was

considered carcinogenic for male and female medaka

based on increased incidences of a variety of liver

neoplasms and papillary adenoma of the gallbladder.
a Inadequate based on loss of animals early in study which hindered interpretation of results.

Summary of the Carcinogenesis Studies of 2,2-Bis(bromomethyl)-1,3-propanediol 

in Guppies and Medaka 

Guppies Medaka

Concentrations in

aquaria water

Study duration

Survival

Neoplastic effects

Carcinogenic response

Male

Liver (Core Study):

hepatocellular adenoma

(3/61, 4/50, 4/41, 8/38);

hepatocellular adenoma or

carcinoma (4/61, 6/50,

6/41, 10/38) 

Liver (Stop-Exposure

Study): hepatocellular

adenoma (0/28, 3/22,

3/21, 6/24); hepatocellular

adenoma or carcinoma

(0/28, 3/22, 3/21, 9/24) 

Positive

Female 

None

Inadequate studya

Male 

Liver (Core Study):

hepatocellular adenoma

(1/47, 0/59, 0/56, 6/59);

hepatocellular adenoma or

carcinoma (1/47, 0/59,

0/56, 8/59)

Liver (Stop-Exposure

Study): hepatocellular

adenoma (1/27, 0/21,

1/31, 3/29); hepatocellular

adenoma or carcinoma

(1/27, 0/21, 1/31, 4/29)

Positive

Female 

None

Negative

0, 24, 60, and 150 mg/L 

16 months

24 mg/L group similar to the control group. 60 

and 150 mg/L groups decreased.

0, 24, 60, and 150 mg/L 

14 months

Exposed groups similar to the control group.

Fish Studies, NTP TR 528 9

Summary of the Carcinogenesis Studies of Nitromethane in Guppies and Medaka

Guppies Medaka

a Inadequate based on loss of animals early in study which hindered interpretation of results.

Concentrations in

aquaria water

Study duration

Survival

Neoplastic effects

Carcinogenic response

Male

None

Inadequate studya

Female

None 

Negative

Male

None 

Negative

Female

None 

Negative

0, 10, 30, and 70 mg/L

16 months

70 mg/L group less than the control group. 

Core study animals in the 70 mg/L group were 

sacrificed at 57 weeks.

0, 10, 20, and 40 mg/L

13 months

Exposed groups similar to the control group.
10 Fish Studies, NTP TR 528

Summary of the Carcinogenesis Studies of 1,2,3-Trichloropropane in Guppies and Medaka

Guppies Medaka

Concentrations in

aquaria water

Study duration

Survival

Neoplastic effects 

Carcinogenic response

Male

Liver (Core Study):

cholangiocarcinoma

(0/61, 0/47, 0/67, 3/27);

hepatocellular adenoma

(3/61, 8/47, 17/67, 9/27);

hepatocellular carcinoma

(0/61, 1/47, 0/67, 7/27);

hepatocellular adenoma

or carcinoma (3/61, 9/47,

17/67, 15/27)

Liver (Stop-Exposure

Study):

cholangiocarcinoma

(0/32, 0/19, 0/26, 1/19);

hepatocellular adenoma

(3/32, 4/19, 5/26, 7/19);

hepatocellular carcinoma

(0/32, 0/19, 1/26, 4/19);

hepatocellular adenoma

or carcinoma (3/32, 4/19,

5/26, 10/19)

Positive

Female

Liver (Core Study):

cholangioma (0/64, 3/64,

4/47, 2/33); 

cholangiocarcinoma

(0/64, 0/64, 0/47, 6/33);

hepatocellular adenoma

or carcinoma (5/64, 4/64,

4/47, 8/33)

Liver (Stop-Exposure

Study): cholangioma

(2/31, 2/31, 0/31, 2/27);

cholangiocarcinoma

(0/31, 0/31, 0/31, 5/27);

hepatocellular adenoma

or carcinoma (2/31, 1/31,

2/31, 3/27)

Positive 

Male

Liver (Core Study):

cholangioma (0/95, 6/94,

2/65, 1/78); 

cholangiocarcinoma (0/95,

32/94, 43/65, 45/78); 

hepatocellular adenoma or

carcinoma (0/95, 2/94,

1/65, 6/78); 

hepatocholangiocarcinoma

(0/95, 0/94, 2/65, 6/78)

Liver (Stop-Exposure

Study): cholangioma

(0/42, 0/47, 3/33, 1/46);

cholangiocarcinoma (0/42,

19/47, 16/33, 26/46); 

hepatocellular adenoma

(0/42, 1/47, 0/33, 0/46); 

hepatocellular adenoma or

carcinoma (0/42, 2/47,

0/33, 1/46)

Gallbladder (Core Study):

adenoma, papillary (0/91,

0/83, 4/61, 9/72)

Gallbladder (StopExposure Study):

adenoma, papillary (0/41,

0/42, 3/30, 4/46)

Positive 

Female

Liver (Core Study):

cholangiocarcinoma (0/95,

29/93, 53/94, 41/67); 

hepatocellular adenoma

(1/95, 1/93, 5/94, 8/67);

hepatocellular carcinoma

(1/95, 1/93, 2/94, 5/67);

hepatocellular adenoma or

carcinoma (2/95, 2/93,

7/94, 13/67); 

hepatocholangiocarcinoma

(0/95, 0/93, 4/94, 7/67)

Liver (Stop-Exposure

Study):

cholangiocarcinoma (1/57,

15/44, 31/49, 21/31);

hepatocellular adenoma

(4/57, 2/44, 2/49, 1/31); 

hepatocellular carcinoma

(0/57, 2/44, 1/49, 4/31); 

hepatocellular adenoma

carcinoma (4/57, 4/44,

2/49, 5/31); 

hepatocholangiocarcinoma

(0/57, 0/44, 0/49, 1/31); 

Gallbladder (Core Study):

adenoma, papillary (0/85,

1/80, 5/88, 6/65)

Gallbladder (StopExposure Study):

adenoma, papillary (0/56,

1/40, 1/45, 1/30)

Positive 

0, 4.5, 9.0, and 18.0 mg/L

16 months

All groups less than control groups

0, 4.5, 9.0, and 18.0 mg/L

13 months

9.0 and 18.0 mg/L groups less than control groups
Fish Studies, NTP TR 528 11

NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS

TECHNICAL REPORTS REVIEW SUBCOMMITTEE

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on Fish Studies on February 18,

2004, are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, company, or governmental agency. In this capacity, subcommittee members have five major responsibilities in reviewing the NTP studies:

• to ascertain that all relevant literature data have been adequately cited and interpreted,

• to determine if the design and conditions of the NTP studies were appropriate,

• to ensure that the Technical Report presents the experimental results and conclusions fully and clearly,

• to judge the significance of the experimental results by scientific criteria, and

• to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses.

Mary Anna Thrall, D.V.M., Chairperson

Department of Pathology 

Colorado State University

Fort Collins, CO

Larry S. Andrews, Ph.D.

Toxicology Department

Rohm and Haas Company

Spring House, PA

Diane F. Birt, Ph.D. 

Department of Food Science & Human Nutrition

Iowa State University

Ames, IA

Kim Boekelheide, M.D., Ph.D., Principal Reviewer

Division of Biology and Medicine

Department of Pathology and Laboratory Medicine

Brown University

Providence, RI

Michael R. Elwell, D.V.M., Ph.D.

Pathology, Drug Safety Evaluation

Pfizer Global Research and Development

Groton, CT

Thomas A. Gasiewicz, Ph.D.*

Department of Environmental Medicine

Environmental Health Sciences Center

University of Rochester School of Medicine

Rochester, NY

Shuk-Mei Ho, Ph.D.

Department of Surgery, Division of Urology

University of Massachusetts Medical School

Worcester, MA

* Did not attend.

Special Ad Hoc Reviewers

George S. Bailey, Ph.D.

Department of Environmental and Molecular Toxicology

Oregon State University

Corvallis, OR

James E. Klaunig, Ph.D., Principal Reviewer

Division of Toxicology

Indiana University School of Medicine

Indianapolis, IN

Charlene A. McQueen, Ph.D.*

Department of Pharmacology and Toxicology,

College of Pharmacy

University of Arizona

Tucson, AZ

Walter W. Piegorsch, Ph.D., Principal Reviewer

Department of Statistics

University of South Carolina

Columbia, SC

Stephen M. Roberts, Ph.D.

Center for Environmental & Human Toxicology

University of Florida

Gainesville, FL

Richard D. Storer, M.P.H., Ph.D.

Department of Genetic and Cellular Toxicology

Merck Research Laboratories

West Point, PA

Mary Vore, Ph.D. 

Graduate Center for Toxicology

University of Kentucky 

Lexington, KY

Cheryl Lyn Walker, Ph.D.*

Department of Carcinogenesis

M.D. Anderson Cancer Center

The University of Texas

Smithville, TX

Jerry M. Law, D.V.M., Ph.D.

Department of Environmental and Molecular Toxicology

North Carolina State University

Raleigh, NC
12 Fish Studies, NTP TR 528

On February 18, 2004, the draft Technical Report on the

carcinogenesis studies of 2,2-bis(bromomethyl)-

1,3-propanediol, nitromethane, and 1,2,3-trichloropropane received public review by the National

Toxicology Program’s Board of Scientific Counselors’

Technical Reports Review Subcommittee. The review

meeting was held at the National Institute of

Environmental Health Sciences, Research Triangle

Park, North Carolina.

Dr. G.S. Bailey, a special reviewer, presented an

overview of the history of carcinogenesis research in

fish models. Dr. Bailey summarized the results of a

variety of studies in different fish species, noting there

were certainly variations in responses to given carcinogens for different species. He cited advantages of fish

compared to rodents, including the smaller cost and

space requirement per individual, which allow studies to

have large sample numbers per dose group. Drawbacks

include the absence of certain organ systems found in

mammalian species.

Dr. G.A. Boorman, NIEHS, introduced the carcinogenesis studies of 2,2-bis(bromomethyl)-1,3-propane-diol,

nitromethane, and 1,2,3-trichloropropane in guppies and

medaka by explaining that the intent of these studies was

to test the feasibility of the fish models. Thus, the test

chemicals chosen were three chemicals characterized as

carcinogenic in NTP rodent studies. For each chemical

study, he discussed the survival and compared the

responses of the two fish species with the results of the

rodent studies.

For these studies, the conclusions were not framed in the

standard Levels of Evidence categories. The proposed

conclusions were:

Under the conditions of these waterborne studies,

2,2-bis(bromomethyl)-1,3-propanediol at concentrations

of up to 150 mg/L for 16 months was considered carcinogenic for male guppies based on increased incidences of hepatocellular adenomas or carcinomas. The

study in female guppies was considered inadequate

based on reduced survival. Under the conditions of

these waterborne studies, 2,2-bis(bromomethyl)-

1,3-propanediol at concentrations of up to 150 mg/L for

14 months was considered carcinogenic for male

medaka based on increased incidences of hepatocellular

adenomas or carcinomas. The study in female medaka

was considered negative.

Under the conditions of these waterborne studies, the

study of nitromethane in male guppies was considered

inadequate based on reduced survival. The study in

female guppies at concentrations up to 70 mg/L for

16 months was considered negative. Under the conditions of these waterborne studies, the study of

nitromethane at concentrations of up to 40 mg/L for

13 months was considered equivocal for male medaka

based on the occurrence of cholangiomas or cholangiocarcinomas. The study in female medaka was considered negative.

Under the conditions of these waterborne studies,

1,2,3-trichloropropane at concentrations of up to

18 mg/L for 16 months was considered carcinogenic for

male and female guppies based on increased incidences

of a variety of liver neoplasms. Under the conditions of

these waterborne studies, 1,2,3-trichloropropane at concentrations of up to 18 mg/L for 13 months was considered carcinogenic for male and female medaka based on

increased incidences of a variety of liver neoplasms and

papillary adenoma of the gallbladder.

Dr. Boorman also noted that the performance of the

studies was not as inexpensive as anticipated, and the

models were less sensitive than rodent studies, as

gauged by the number of sites eliciting responses.

Interpretation was also limited by lack of time-to-tumor

data and some of the fish dying early.

Dr. Boekelheide, the first principal reviewer, had several

concerns about the technical limitations of these studies.

The fish were not initially sexed and were potentially

reproducing, their sizes varied with the number of fish

per tank, and the occurrence of algal blooms and a

background infection of granulomata presented other

confounders. He did concur with the proposed concluding assessments.

Dr. Piegorsch, the second principal reviewer, applauded

the effort to explore new test systems but agreed that

many design and control issues need to be resolved. For

the studies at hand, he noted a number of cases where

very low tumor incidences were presented as supporting

an equivocal conclusion. He inquired about the

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS
Fish Studies, NTP TR 528 13

inclusion of stop-exposure study data and discussed the

fundamental flaw of fish dying and being cannibalized,

resulting in a loss of key data.

Dr. Klaunig, the third principal reviewer, also mentioned

the issues of survival and husbandry and expressed concern about the possibility of fish dying from lesions and

then disappearing. He felt larger fish species might offer

better opportunities to observe tumors, particularly

tumor multiplicity.

Dr. Bailey said that doing sex determinations at the

beginning of a study would not be possible, but he did

not consider that a severe limitation. Based on these

studies, he felt optimistic about the prospects for fish

research.

Dr. J.M. Law was the second special reviewer. Dr. Law

suggested that fish studies offered some economy of

scale for tests involving very large numbers of animals.

He was concerned about the extent of the granuloma

infection and the effects of the resultant inflammation.

He felt the pathology diagnoses could have been completed more quickly with fewer slides per fish.

Dr. Boorman acknowledged the recommendations of the

reviewers and suggested that this report be part of the

Technical Report series rather than a summary article to

permit full reporting of the study details and lessons

learned. Dr. J.R. Hailey, NIEHS, indicated that stopexposure study data were becoming a regular part of

NTP studies.

Dr. Bailey pointed out that no false positives had ever

been reported in fish studies. He also mentioned that the

survival rates were much higher for larger fish like trout.

Dr. Boekelheide moved, and Dr. Klaunig seconded, that

the proposed conclusions be accepted as written.

Dr. Piegorsch offered an amendment that the proposed

conclusion for nitromethane in male medaka be changed

from equivocal to negative. Dr. Birt seconded the

amendment. The amendment was approved with seven

members in favor and three opposing. The overall conclusion as amended was approved with eight members in

favor and two opposing.

In further discussion, Dr. C.J. Portier, NIEHS, suggested

presenting these studies in the existing Technical Report

series rather than creating a new series for each study

design. Dr. Klaunig said that fish were a valid model for

carcinogenicity. Dr. Andrews recalled that discussions

about the vision for the future of the program included

exploring different and predictive models, and this study

was one such attempt. Dr. Storer distinguished the fish

studies from the transgenic mouse studies, noting that

some of the latter might be characterized as a reporter

gene model, whereas for fish cancer, itself is the endpoint. Dr. Piegorsch noted that many of the technical

and analytic refinements had not been developed at the

beginning of the rodent bioassay series either. Dr. Ho

favored keeping this report in the Technical Report

series because fish have long been used as a model for

carcinogenesis.

Drs. Storer and Klaunig noted that most of the fish studies involved testing carcinogens rather than noncarcinogens. Dr. Boekelheide said that if more studies were

performed using fish models, a major investment would

be required. Dr. Portier felt most confounders could be

overcome, but the key problem was the loss of fish and

data during studies. Dr. Bailey felt this problem could be

resolved with an appropriate species and husbandry

system. Drs. Birt and Storer felt these models could be

promising for low-dose extrapolation studies requiring

very large numbers of animals.
14 Fish Studies, NTP TR 528
15

In the past 20 years, the National Toxicology Program

(NTP) has primarily used rodents in studies to determine

the potential carcinogenic hazard from chemicals that

occur in the workplace or environment (Page et al.,

1977; Weisburger and Williams, 1981; Weisburger et al.,

1984; Goodman and Wilson, 1991; Faccini et al., 1992).

Because rodent cancer studies are costly and require

years to complete, the NTP continues to investigate

other animal models that may offer advantages in terms

of time, cost, and mechanistic information.

Several small fish species have been suggested to be

more sensitive for detecting carcinogens in assays that

would be less expensive and faster than rodent studies

(Sinnhuber et al., 1978; Ishikawa and Takayama, 1979;

Bailey et al., 1984; Simon and Lapis, 1984;

Walker et al., 1985). Since large numbers of small fish

can be easily maintained in a limited space, this model

appeared to offer real advantages to the NTP. However,

the NTP has found that other types of short-term tests

have limitations for predicting carcinogenicity when

systematically evaluated under controlled conditions in a

contract laboratory situation (Zeiger et al., 1990).

To examine fish as a test species for cancer hazard identification, the NTP decided to evaluate two small fish

models using three chemicals under standard study conditions. Most fish cancer studies reported in the literature used potent carcinogens that caused liver tumors in

rodents and also in the fish models (Hawkins et al.,

1998; Law et al., 1998; Brown-Peterson et al., 1999;

Okihiro and Hinton, 1999; Liu et al., 2003). The spectrum of tumors in organs and tissues in rodents has often

provided clues as to the mechanisms of toxicity and carcinogenicity of the test chemicals. Therefore,

1,2,3-trichloropropane and 2,2-bis(bromomethyl)-

1,3-propanediol, both mutagens that caused a broad

spectrum of cancers in rodents, were selected to test the

spectrum of tissue response in the fish models.

1,2,3-Trichloropropane was shown to cause increased

incidences of tumors of the oral cavity, forestomach, kidney, harderian gland, Zymbal’s gland, liver, uterus, pancreas, and other sites in rodents. 2,2-Bis(bromomethyl)-

1,3-propanediol caused increased incidences of tumors

of the mammary gland, skin, oral cavity, forestomach,

harderian gland, Zymbal’s gland, lung, urinary bladder,

seminal vesicle, and other sites in rodents.

To date, most carcinogens tested in fish studies are also

hepatocarcinogens in rodents. To test the sensitivity of

the small fish models, nitromethane, a nonmutagen with

a modest carcinogenic response in rodents, was selected.

Nitromethane did not cause increased tumors in male

rats, but was clearly carcinogenic for female rats with

increased mammary gland neoplasms, and clearly carcinogenic for male and female mice with increased lung

and harderian gland tumors plus increased liver tumors

in female mice. Another factor for the selection of

nitromethane was that it was a rodent carcinogen that did

not primarily target the liver.

On December 2 and 3, 1991, a “Workshop of

Carcinogenesis Testing with Fish” was held at the

National Institute of Environmental Health Sciences to

evaluate the utility of the small fish model. A National

Toxicology Program Pathology Working Group (PWG)

was convened, and diagnostic criteria for degenerative,

inflammatory, proliferative neoplastic and nonneoplastic

lesions in the liver of medaka were published

(Boorman et al., 1997). This report consolidated hepatic

lesion criteria into a simplified scheme for comparison

of findings from different studies and laboratories.

Later, a competitive contract was awarded to Gulf Coast

Research Laboratory to conduct up to 16-month

exposure studies in two species of small fish. The

contract was a part of the NTP’s effort to develop

alternative test methods. The species of fish used for

these studies were the medaka (Oryzias latipes) and the

guppy (Poecilia reticulata).

The ultimate test of the small fish models is whether the

data generated allow an equally informed decision of the

potential carcinogenicity of the test chemical.

Evaluating three chemicals already evaluated in NTP

rodent studies allows some assessment of these two fish

models.

Rodent cancer evaluations have benefited from careful

attention to study details. Therefore, the NTP attempted

OVERVIEW
16 Fish Studies, NTP TR 528

,to follow standard chemistry, pathology, statistical, and

QA evaluations of the data for the fish studies. It was

found that, in many cases, the rodent standards could not

be applied to the fish studies for a variety of reasons.

NTP scientists were asked to evaluate the data for

carcinogenicity determinations as would be done for an

unknown chemical. Thus, for each chemical, the

carcinogenic response was judged to be positive, negative, or equivocal for each sex and species combination.

In one instance, the study was judged to be inadequate

because of poor survival. This report details the experience with the medaka and guppy for three rodent

carcinogens.
17

INTRODUCTION

2,2-BIS(BROMOMETHYL)-1,3-PROPANEDIOL

(FR-1138®)

(Technical Grade: 78.6% 2,2-bis(bromomethyl)-1,3-propanediol, 6.6% 2,2-bis(hydroxymethyl)-1-bromo-3-hydroxypropane, 6.9% 

2,2-bis(bromomethyl)-1-bromo-3-hydroxypropane, 0.2% pentaerythritol, and 7.7% dimers and structural isomers)

CAS No. 3296-90-0

Chemical Formula: C5H10Br2O2 Molecular Weight: 261.94

Synonyms: 2,2-Bis(2-bromomethyl)-1,3-propanediol; 1,3-dibromo-2,2-dihydroxymethylpropane; 

1,3-dibromo-2,2-dimethylolpropane; 2,2-dibromomethyl-1,3-propanediol; dibromopentaerythritol; 

dibromoneopentyl glycol; pentaerythritol dibromide; pentaerythritol dibromohydrin 

CH3NO2

NITROMETHANE

CAS No. 75-52-5

Molecular Weight: 61.04

Synonym: Nitrocarbol

1,2,3-TRICHLOROPROPANE

CAS No. 96-18-4

Chemical Formula: C3H5Cl3 Molecular Weight: 147.44

Synonyms: Allyl trichloride, glycerol trichlorohydrin, glyceryl trichlorohydrin, trichlorohydrin

HOH2C C CH2OH

CH2Br

CH2Br

Cl C C C Cl

H

H Cl H

H H
18 Fish Studies, NTP TR 528

CHEMICAL AND PHYSICAL PROPERTIES

2,2-Bis(bromomethyl)-1,3-propanediol

2,2-Bis(bromomethyl)-1,3-propanediol is a white solid

material with a slight, mild, musty odor. It has a melting

point of 75° to 95° C for technical grade material and

109° to 110° C for pure material. It is soluble in acetone,

ethanol, and ether, and slightly soluble in water

(2 g/1,000 g water at 25° C). Complete information on

chemical and physical properties of 2,2-bis(bromomethyl)-1,3-propanediol is available in NTP Technical

Report 452 (NTP, 1996).

Nitromethane

Nitromethane is a colorless, oily liquid with a moderately strong, disagreeable odor (Merck Index, 1989).

Nitromethane has a melting point of –29° C, a boiling

point of 101.2° C, a flash point of 112° F, and a lower

explosive limit of 7.3%. Its density is 1.1322 at 25° C;

the vapor density is 2.11, and the vapor pressure is

27.8 mm Hg at 20° C. Nitromethane is soluble in alcohol, ether, N,N-dimethylformamide, acetone, and alkali

and is slightly soluble in water (9.5 g/L at 20° C).

Complete information on chemical and physical properties of nitromethane is available in NTP Technical

Report 461 (NTP, 1997).

1,2,3-Trichloropropane

1,2,3-Trichloropropane is a colorless liquid with a strong

acidic odor. It has a boiling point of 156° C (760 mm

Hg), a vapor pressure of 3 mm Hg at 25° C, a specific

gravity of 1.370 g/mL, and a flash point of 71.1° C

(Hawley’s, 1987). 1,2,3-Trichloropropane is only

slightly soluble in water but freely soluble in alcohol and

ether. Complete information on chemical and physical

properties of 1,2,3-trichloropropane is available in NTP

Technical Report 384 (NTP, 1993).

PRODUCTION, USE, 

AND HUMAN EXPOSURE

2,2-Bis(bromomethyl)-1,3-propanediol

2,2-Bis(bromomethyl)-1,3-propanediol is used as a

flame retardant in unsaturated polyester resins, for

molded products, and in rigid polyurethane foam. This

flame retardant may enter the environment as fugitive

dust and through wastewater. 2,2-Bis(bromomethyl)-

1,3-propanediol is expected to remain for long periods of

time in water (USEPA, 1983). Complete information on

production, use, and human exposure of 2,2-bis(bromomethyl)-1,3-propanediol is available in NTP Technical

Report 452 (NTP, 1996).

Nitromethane

In the past, nitromethane was used extensively as a

chemical stabilizer to prevent the decomposition of various halogenated hydrocarbons such as metal degreasers

and aerosol propellants such as 1,1,1-trichloroethane.

Nitromethane is used as a fuel or fuel additive to

increase the power output of rockets, race cars, boats,

and model engines. Nitromethane is also used as a synthesis intermediate for a variety of chemicals, such as

trichloronitromethane (chloropicrin), an agricultural soil

and grain fumigant; the nitroalcohol 2-hydroxymethyl2-nitro-1,3-propanediol, which is used as a biocide for

cutting fluids and as a source of formaldehyde for crosslinking amino resins; and the alkanolamine 2-hydroxymethyl-2-amino-1,3-propanediol, which is used as a

formaldehyde scavenger in resin curing and polyester

resin modification and as a buffer. Nitromethane is used

in a variety of solvent applications, such as solventextraction separation of aromatics from aliphatic compounds, in the crystallization of nitrofurantoin, as a reaction medium for aluminum chloride in Friedel-Crafts

reactions, and as a solvent for resins such as "-cyanoacrylate. Nitromethane is used in mixtures with ammonium nitrate as an explosive in mining, oil-well drilling,

and seismic exploration (Biocides, U.S.A., 1974;

Remington’s Pharmaceutical Sciences, 1975; KirkOthmer, 1978; SRI International, 1980).

Nitromethane is fairly reactive and therefore does not

persist in the environment; the half-life (t1⁄2) of

nitromethane is from 4 to 9 hours in air and about 1 day

in water (HSDB, 1995). However, because nitromethane is slightly soluble in water and evaporates at

about the same rate as water, the t1⁄2 is somewhat dependent on the rate of evaporation. In the atmosphere and in

water, nitromethane is degraded through its reaction with

hydroxyl radicals; it may also undergo aerobic or

anaerobic degradation. It may react with chlorine in

water to form trichloronitromethane if the pH of the

medium is high (Wade et al., 1977). Information on production, use, and human exposure of nitromethane is

available in NTP Technical Report 461 (NTP, 1997).
Fish Studies, NTP TR 528 19

1,2,3-Trichloropropane

1,2,3-Trichloropropane is commonly used as a paint and

varnish remover, solvent, and degreasing agent, but the

extent of these uses is uncertain. 1,2,3-Trichloropropane

is used as a crosslinking agent in the synthesis of polysulfides and hexafluoropropylene, and it may be found

as an impurity in certain nematicides and soil fumigants

(Aharonson, 1987). The general routes of exposure are

inhalation, ingestion, and skin and eye contact (Sittig,

1985). Complete information on production, use, and

human exposure of 1,2,3-trichloropropane is available in

NTP Technical Report 384 (NTP, 1993).

ABSORPTION, DISTRIBUTION,

METABOLISM, AND EXCRETION

Information on the absorption, distribution, metabolism,

and excretion of 2,2-bis(bromomethyl)-1,3-propanediol,

nitromethane, and 1,2,3-trichloropropane is available in

NTP Technical Reports 452 (NTP, 1996), 461 (NTP,

1997), and 384 (NTP, 1993), respectively.

TOXICITY

2,2-Bis(bromomethyl)-1,3-propanediol 

and Nitromethane

Toxicity information for 2,2-bis(bromomethyl)-

1,3-propanediol and nitromethane can be found in NTP

Technical Reports 452 and 461, respectively (NTP,

1996, 1997).

1,2,3-Trichloropropane

Acute and subchronic toxicity of 1,2,3-trichloropropane

has been studied by inhalation, gavage, dermal exposure,

and ingestion of drinking water. Inhalation studies are

particularly relevant due to the similarity of aquatic total

exposures and inhalation total exposures. Complete toxicity information about 1,2,3-trichloropropane can be

found in NTP Technical Report 384 (NTP, 1993).

REPRODUCTIVE

AND DEVELOPMENTAL TOXICITY

Reproductive and developmental toxicity information

for 2,2-bis(bromomethyl)-1,3-propanediol can be found

in NTP Technical Report 452 (NTP, 1996). No information on the reproductive or developmental toxicity of

nitromethane or 1,2,3-trichloropropane in humans has

been reported in the literature.

CARCINOGENICITY

Experimental Animals

2,2-Bis(bromomethyl)-1,3-propanediol

In a 2-year toxicity/carcinogenicity study, SpragueDawley rats were administered the flame retardant

2,2-bis(bromomethyl)-1,3-propanediol [FR-1138®:

80% dibromopentyl glycol(2,2-bis(bromomethyl)-

1,3-propanediol); 8% tribromoneopentyl alcohol

(bis(bromomethyl)-1-bromo-3-hydroxypropane) and 6%

monobromoneopentyl triol (2,2-bis(hydroxymethyl)-

1-bromo-3-hydroxypropane)] in feed at concentrations

that delivered 0, 5, or 100 mg 2,2-bis(bromomethyl)-

1,3-propanediol/kg body weight per day (Keyes et al.,

1979). No carcinogenic effect was observed. However,

degenerative changes in the liver and lens of the eye

were attributed to chemical exposure. The article did not

provide details on the preparation or stability of the

chemical in the feed. No dose-related effects on the feed

consumption, weight gain, clinical signs, or mortality

were observed, suggesting that the animals may have

been able to tolerate higher doses.

2,2-Bis(bromomethyl)-1,3-propanediol was administered in feed for 13-week and 2-year studies (NTP,

1996). In the 2-year study increased tumors were found

in F344/N rats and B6C3F1 mice. In both male and

female rats, there were increased incidences of neoplasms of the oral cavity, esophagus, thyroid gland and

mammary gland. Male rats also had increased incidences of neoplasms of the skin, subcutaneous tissue,

Zymbal’s gland, forestomach, small and large intestines,

mesothelium, urinary bladder, lung, seminal vesicle and

of mononuclear cell leukemia. Slight increases in incidences of neoplasms of the pancreas and kidney of male

rats many have also been related to treatment.

Male and female mice had increased incidences of

neoplasms of the harderian gland and lung, male mice

had increased neoplasms of the kidney, and female mice

had increased neoplasms of subcutaneous tissue (NTP,

1996). Slight increases in the incidences of neoplasms

in the forestomach, mammary gland and circulatory

system in female mice may have also been related to

treatment.
20 Fish Studies, NTP TR 528

Nitromethane

Nitromethane was evaluated for toxicity and carcinogenicity in 16-day, 13-week, and 2-year studies in

F344/N rats and B6C3F1 mice, with whole body

inhalation as the route of exposure (NTP, 1997).

Increased incidences of several tumors were found in the

2-year inhalation studies. Increased incidences of mammary gland fibroadenomas and carcinomas were found

in female rats. In male and female B6C3F1 mice there

were increased incidences of harderian gland adenomas

and carcinomas. Female mice had an increased

incidence of liver neoplasms (primarily adenomas).

Male and female mice also had increased incidences of

adenomas and carcinomas in the lung that were considered to be related to chemical administration. Male and

female mice also had increased incidences of nasal

lesions including degeneration and metaplasia of the

olfactory epithelium and degeneration of the respiratory

epithelium.

Chemicals that are structurally related to nitromethane

are also carcinogenic in rodents. Sprague-Dawley rats

exposed to 207 ppm 2-nitropropane by inhalation for

6 months had multiple hepatocellular carcinomas

(Lewis et al., 1979). Male and female Long-Evans rats

exposed to 100 or 200 ppm nitroethane by inhalation for

2 years (Griffin et al., 1988) or to 100 ppm 1-nitropropane by inhalation for 21 months (Griffin et al.,

1982) had no neoplasms or nonneoplastic lesions associated with treatment. 

1,2,3-Trichloropropane

1,2,3-Trichloropropane was administered in corn oil by

gavage to F344/N rats and B6C3F1 mice in 17-week and

2-year studies (NTP, 1993). Increased incidences of a

variety of hyperplasias and tumors were found in rats in

the 2-year studies. Increased incidences of squamous

cell papillomas and carcinomas of the oral mucosa and

forestomach and carcinomas of the Zymbal’s gland were

seen in male and female rats. Male rats also had

increased incidences of adenomas of the pancreas and

kidney and adenomas or carcinomas of the preputial

gland. Female rats had increased incidences of adenomas or carcinomas of the clitoral gland and adenocarcinomas of the mammary gland. A few adenocarcinomas

of the intestine found in male and female treated rats

may have been related to trichloropropane administration. Increased severity of renal disease in male rats was

associated with exposure to trichloropropane.

1,2,3-Trichloropropane exposure in male and female

mice was associated with increased incidences of squamous cell papillomas and carcinomas of the forestomach, hepatocellular adenomas or carcinomas of the liver

and harderian gland adenomas (NTP, 1993). Female

mice also had increased incidences of uterine adenomas,

adenocarcinomas, and stromal polyps. In female mice

there were increased incidences of squamous cell carcinomas of the oral mucosa. A few squamous cell papillomas of the oral mucosa in male mice may have been

related to trichloropropane administration.

Humans

No epidemiological studies of 2,2-bis(bromomethyl)-

1,3-propanediol, nitromethane, or 1,2,3-trichloropropane exposure in humans were found in the literature.

GENETIC TOXICITY

2,2-Bis(bromomethyl)-1,3-propanediol 

The genotoxicity test data for 2,2-bis(bromomethyl)-

1,3-propanediol were recently reviewed (IARC, 2000).

2,2-Bis(bromomethyl)-1,3-propanediol gave positive

results in a limited number of genetic toxicity assays

under specific experimental conditions. In the two

Salmonella typhimurium gene mutation assays presented

in NTP (1996), 2,2-bis(bromomethyl)-1,3-propanediol

gave a positive response only in strain TA100 in the

presence of 30% induced hamster liver S9 activation

enzymes (Zeiger et al., 1992); 10% hamster S9 was

ineffective, as was 10% or 30% S9 derived from livers

of pretreated rats. No other Salmonella strain/activation

combination was responsive to the effects of 2,2-bis(bromomethyl)-1,3-propanediol (Mortelmans et al., 1986;

Zeiger et al., 1992).

In cytogenetic tests with Chinese hamster ovary cells,

2,2-bis(bromomethyl)-1,3-propanediol did not induce

sister chromatid exchanges with or without induced rat

liver S9, but a dose-related increase in chromosomal

aberrations was observed in cultured Chinese hamster

ovary cells when testing occurred in the presence of S9

(Galloway et al., 1987). A majority of the breaks that

were observed in the aberrations assay were located in

the heterochromatic region of the long arm of the

X chromosome. The reason for this preferential

breakage site is not known (Galloway et al., 1987). The
Fish Studies, NTP TR 528 21

pattern of damage seen with 2,2-bis(bromomethyl)-

1,3-propanediol (induction of chromosomal aberrations

with no concomitant induction of sister chromatid

exchanges) is unusual (Galloway et al., 1987).

2,2-Bis(bromomethyl)-1,3-propanediol was shown to be

genotoxic in vivo. Significant increases in micronucleated erythrocytes were seen in peripheral blood

samples obtained from male and female B6C3F1 mice

administered 2,2-bis(bromomethyl)-1,3-propanediol

(625 to 10,000 ppm) for 3 months in dosed feed

(Witt et al., 2000). Equivocal results were obtained in

an acute bone marrow micronucleus test with

2,2-bis(bromomethyl)-1,3-propanediol administered

over a dose range of 100 to 400 mg/kg to male mice by

gavage, three times at 24-hour intervals (NTP, 1996). A

second micronucleus test was conducted in which

2,2-bis(bromomethyl)-1,3-propanediol was administered as a single intraperitoneal injection (150 to

600 mg/kg) to male and female mice; bone marrow was

sampled 24 hours after treatment, and significant

increases in micronucleated erythrocytes were seen in

both male and female mice.

Nitromethane

The genetic toxicity test data for nitromethane were

reviewed in NTP (1997); results of all in vivo and

in vitro tests were negative. Tests for mutagenicity

induction using a number of different strains of

Salmonella typhimurium, with and without exogenous

metabolic activation (S9 liver enzymes), were negative

(Chiu et al., 1978; Mortelmans et al., 1986; Dayal et al.,

1989; Dellarco and Prival, 1989). The nitronate form of

nitromethane was also reported to be negative for induction of mutations in S. typhimurium strains TA100 and

TA102 (Dayal et al., 1989). No induction of sister chromatid exchanges or chromosomal aberrations was

observed in cultured Chinese hamster ovary cells treated

with up to 5,000 μg/mL nitromethane, with and without

S9 (NTP, 1997). No significant increases in sex-linked

recessive lethal mutations were noted in germ cells of

male Drosophila melanogaster after administration of

nitromethane by feeding (Gocke et al., 1981). No induction of micronuclei was observed in bone marrow

erythrocytes of male NMRI mice administered two

intraperitoneal injections of 205 to 1,830 mg/kg

nitromethane (Gocke et al., 1981). In this test, bone

marrow was sampled 6 hours after the second injection,

so the effect of the second treatment is not likely to be

reflected in these results; however, this negative result is

in agreement with the results of the 3-month micronucleus study conducted by the NTP (1997). In this latter

study, male and female B6C3F1 mice showed no significant increases in micronucleated erythrocytes in peripheral blood following 3 months of inhalation exposure to

nitromethane at levels up to 1,500 ppm (Witt et al.,

2000).

1,2,3-Trichloropropane

1,2,3-Trichloropropane contains two chlorinated methyl

groups that are structural alerts to potential DNA reactivity (Ashby and Tennant, 1988); this molecular structure is consistent with the results of a limited number of

genetic toxicity assays showing significant activity for

1,2,3-trichloropropane. All mutagenic responses were

obtained in in vitro assays and were observed only in the

presence of metabolic activation provided by induced rat

or hamster S9 liver enzymes. 1,2,3-Trichloropropane

induced gene mutations in Salmonella typhimurium

strains TA97, TA98, TA100, and TA1535 (Stolzenberg

and Hine, 1980 Haworth et al., 1983; Ratpan and

Plaumann, 1988) and in mouse lymphoma L5178Y tk+/–

cells (NTP, 1993). In cytogenetic tests with cultured

hamster cells, 1,2,3-trichloropropane induced both sister

chromatid exchanges (von der Hude et al., 1987; NTP,

1993) and chromosomal aberrations (NTP, 1993).

In contrast to these positive results, 1,2,3-trichloropropane did not induce unscheduled DNA synthesis in

hepatocytes of male F344/N rats treated in vitro

(Mirsalis et al., 1983; Williams et al., 1989) or in vivo

(Mirsalis et al., 1983). Negative results were also

obtained in an in vivo test for induction of chromosomal

damage measured as dominant lethal mutations in male

Sprague-Dawley rats treated once daily for 5 consecutive days with 80 mg/kg 1,2,3-trichloropropane (SaitoSuzuki et al., 1982). 

DOSE SELECTION RATIONALE

2,2-Bis(bromomethyl)-1,3-propanediol

2,2-Bis(bromomethyl)-1,3-propanediol has been identified as a multisite carcinogen in both male and female

rats and mice (Dunnick et al., 1997). The purpose of this

study was to determine the carcinogenic potential of

chronic exposure to sublethal concentrations of

2,2-bis(bromomethyl)-1,3-propanediol in two small fish

species; guppies (Poecilia reticulata) and medaka

(Oryzias latipes). Following 2-day static tests and 7-day

flow-through tests, 28-day range finding studies using

guppies and medaka were conducted to determine
22 Fish Studies, NTP TR 528

maximum 2,2-bis(bromomethyl)-1,3-propanediol concentrations to use in the subsequent chronic exposure

studies. Two replicates containing each of six nominal

concentrations (0.0, 19.4, 32.4, 54.0, 90.0, and

150.0 mg/L) were employed. The maximum

2,2-bis(bromomethyl)-1,3-propanediol concentration for

the chronic studies was established at 150 mg/L based

upon the sublethal responses observed from the range

finding study. The low concentration and the mid-level

concentration were selected as 60% of the next higher

concentration for even spacing and adequate separation

of treatments. Guppies and medaka were exposed to

2,2-bis(bromomethyl)-1,3-propanediol in a flowthrough system to each of four nominal concentrations

(0, 24, 60, and 150 mg/L; measured concentrations were

0.0, 20.0, 53.5, and 139.0 mg/L, respectively for guppies, and 0.0, 19.4, 56.9, and 137.8 mg/L, respectively

for medaka). Nine months after test initiation, approximately one-third of the fish from each exposure group

was transferred from the exposure system to aquaria

receiving 2,2-bis(bromomethyl)-1,3-propanediol-free

water under flow-through conditions for an additional

7 (guppies) or 5 (medaka) months. Fish remaining in the

exposure system continued to be exposed to 2,2-bis(bromomethyl)-1,3-propanediol at the medium and low

treatment for a total of 16 months (488 days; guppies) or

14 months (428 days; medaka). Guppies in the high

treatment were exposed for approximately 14.5 months

(443 days) when the treatment was terminated due to

increasing mortality of fish. Surviving fish in both the

exposure system and those placed in 2,2-bis(bromomethyl)-1,3-propanediol-free aquaria at 9 months

were examined for clinical signs of toxicity and carcinogenicity, sacrificed, and preserved for histopathologic

evaluation. To assess the toxic and carcinogenic effects

of 2,2-bis(bromomethyl)-1,3-propanediol, histopathologic evaluations were conducted on major tissues and

organs in each fish.

Nitromethane

The purpose of this study was to determine the carcinogenic potential of chronic exposure to sublethal concentrations of nitromethane in two small fish species, guppies (Poecilia reticulata) and medaka (Oryzias latipes).

Following 2-day static tests and 7-day flow-through

tests, 28-day range finding studies using guppies and

medaka were conducted to determine maximum

nitromethane concentrations to use in the subsequent

chronic exposure studies. The treatment concentration

selection was adjusted to incorporate the approximate

highest no-observable-effect concentration from the

28-day range finding studies as the high concentration in

the chronic studies; 70 mg/L for guppies and 40 mg/L

for medaka. The low concentration selected was

10 mg/L and the mid-level concentration selected was

30 mg/L for guppies and 20 mg/L for medaka, each a

mid point between the high and low concentrations.

Guppies were exposed to nitromethane in a flow-through

system to each of four nominal concentrations (0, 10, 30,

and 70 mg/L; measured concentrations were 0.0, 9.9,

28.7, and 66.4 mg/L, respectively). Medaka were similarly exposed to nitromethane at four nominal concentrations (0, 10, 20, and 40 mg/L; measured concentrations were 0.0, 9.3, 20.8, and 41.7 mg/L, respectively).

Nine months after the test initiation, approximately

one-third of the fish from each exposure group was

transferred from the exposure system to aquaria

receiving nitromethane-free water under flow-through

conditions for an additional 7 (guppies) or 4 (medaka)

months. Fish remaining in the exposure system continued nitromethane exposure for approximately 16 months

(495 days; guppies) or 13 months (398 days; medaka).

Surviving fish in both the exposure system and those

placed in nitromethane-free aquaria at 9 months were

examined for clinical signs of toxicity and carcinogenicity, sacrificed, and preserved for histopathologic evaluation. Histopathologic evaluations were conducted on

major tissues and organs in each fish to assess the toxic

and carcinogenic effects of nitromethane.

1,2,3-Trichloropropane

The National Toxicology Program tested 1,2,3-trichloropropane because it is a high production chemical which

has been found as an impurity in certain nematicides and

soil fumigants. This chemical has also been detected in

drinking water and ground water in the United States.

The purpose of this study was to determine the carcinogenic potential of chronic exposure to sublethal concentrations of 1,2,3-trichloropropane in two small fish

species, guppies (Poecilia reticulata) and medaka

(Oryzias latipes). Following 2-day static tests and 7-day

flow-through tests, 28-day range finding studies using

guppies and medaka were conducted to determine maximum 1,2,3-trichloropropane concentrations to use in the

subsequent chronic exposure studies. Two replicates

containing each of six nominal concentrations (0.0, 6.4,

10.8, 18.0, 30.0, and 50.0 mg/L) were employed.

Exposure concentrations above 18 mg/L compromised

the growth rates of study animals. The 18 mg/L, though

it produced lethargy in the study animals, did not seem
Fish Studies, NTP TR 528 23

to produce any visible health effects; thus, 18 mg/L was

selected as the high exposure concentration for guppies

and medaka. Each of the two lower concentrations were

set at 4.5 and 9 mg/L, each being 50% of the next higher

concentration. These concentrations, it was hoped,

would produce at least one effect and one no-effect

1,2,3-trichloropropane treatment.

Guppies and medaka were exposed to 1,2,3-trichloropropane in a flow-through system to each of four nominal concentrations, 0, 4.5, 9.0, and 18.0 mg/L (measured

concentrations were 0.0, 4.4, 8.8, and 18.2 mg/L for

guppies and 0.0, 4.6, 9.2, and 18.0 mg/L for medaka).

Nine months after the test initiation, approximately

one-third of the fish from each exposure group were

transferred from the exposure system to aquaria receiving 1,2,3-trichloropropane-free water under flowthrough conditions. They remained in this system for an

additional 7 (guppies) or 4 (medaka) months. Fish

remaining in the exposure system continued

1,2,3-trichloropropane exposure for a total of approximately 16 months (487 days; guppies) or 13 months

(396 days; medaka). Surviving fish in both the exposure

system and those placed in 1,2,3-trichloropropane-free

aquaria at 9 months were examined for clinical signs of

toxicity and carcinogenicity, sacrificed, and preserved

for histopathologic evaluation. Histopathologic evaluations were conducted on major tissues and organs in

each fish to assess the toxic and carcinogenic effects of

1,2,3-trichloropropane.
24 Fish Studies, NTP TR 528
25

PROCUREMENT

AND CHARACTERIZATION

2,2-Bis(bromomethyl)-1,3-propanediol

2,2-Bis(bromomethyl)-1,3-propanediol was obtained

from Aldrich Chemical Company (Milwaukee, WI) in

three lots (16728PG, 11215BQ, and 00713BQ) that were

used in the 14- and 16-month studies. Lots 11215BQ

and 00713BQ were manufactured in the same batch but

were assigned different lot numbers by the supplier

because they were received on different dates. Identity,

purity, and stability analyses were conducted by the

study laboratory, University of Southern Mississippi,

Gulf Coast Research Laboratory (Gulf Springs, MS).

Lots 16728PG and 11215BQ were identified as

2,2-bis(bromomethyl)-1,3-propanediol by the University

of Mississippi (Oxford, MS) (lot 16728PG) and by the

University of Southern Mississippi (Hattiesburg, MS)

(lot 11215BQ) using infrared spectroscopy. The spectra

were consistent with the structure of 2,2-bis(bromomethyl)-1,3-propanediol. 

The purities of lots 16728PG and 11215BQ were determined by the study laboratory using gas chromatography

(GC). GC revealed that both lots contained no contaminants. Information provided by the supplier indicated

that the purity of lot 16728PG was greater than 99%.

The overall purity of lots 16728PG and 11215BQ was

determined to be greater than 99%.

The bulk chemical was stored at approximately 4° C (lot

16728PG) or at room temperature (lots 11215BQ and

00713BQ). The stability of bulk 2,2-bis(bromomethyl)-

1,3-propanediol was monitored monthly during the 14-

and 16-month studies using GC. No contaminants were

found during the course of the studies.

Nitromethane

Nitromethane was obtained from Aldrich Chemical

Company in one lot (CG02304HF) that was used in the

13- and 16-month studies. Identity, purity, and stability

analyses were conducted by the study laboratory.

Lot CG02304HF, a colorless liquid, was identified as

nitromethane by the University of Mississippi using

infrared spectroscopy. The spectrum was consistent

with the structure of nitromethane.

The purity of nitromethane was determined by the study

laboratory using GC; lot CG02304HF contained no contaminants other than water. Information provided by the

supplier indicated that the purity of lot CG02304HF was

greater than 99%. The overall purity of lot CG02304HF

was determined to be greater than 99%.

The bulk chemical was stored at approximately 4° C.

The stability of bulk nitromethane was monitored

monthly during the 13- and 16-month studies using GC.

No contaminants were found during the course of the

studies.

1,2,3-Trichloropropane

1,2,3-Trichloropropane was obtained from Aldrich

Chemical Company in one lot (01628HG) that was used

in the 13- and 16-month studies. Identity and purity

analyses were conducted by the study laboratory.

Lot 01628HG, a colorless to straw-colored liquid, was

identified as 1,2,3-trichloropropane by the University of

Southern Mississippi and by the supplier using infrared

spectroscopy. The spectra were consistent with a reference spectrum (Aldrich, 1985).

The purity of lot 01628HG was determined by the

University of Southern Mississippi, the chemical

supplier, and the study laboratory using GC. GC by several methods indicated that lot 01628HG was either

highly pure or 99% pure. The overall purity of lot

01628HG was determined to be 99%. The bulk chemical was stored at approximately 4° C.

MATERIALS AND METHODS
26 Fish Studies, NTP TR 528

EXPOSURE SOLUTION GENERATION

AND EXPOSURE SYSTEM

Exposure was conducted in a closed chamber similar to

that described by Walker et al. (1985) (Figure G4). A

schematic of the exposure solution delivery systems

used in the 13-, 14-, and 16-month studies is shown in

Figure G5. Stock solutions of 2,2-bis(bromomethyl)-

1,3-propanediol, nitromethane, or 1,2,3-trichloropropane were prepared by adding neat chemical to

diluent well water in glass carboys to produce concentrations sufficient to provide the required concentrations

in exposure aquaria. A water partitioner was regulated

to perform at least five volume additions per day to each

exposure aquarium. Diluent well water entered the

water partitioner through a solenoid-controlled valve

immediately after filtration and ultraviolet light sterilization. A float switch within the water partitioner

activated liquid dispensing pumps (Hamilton Company,

Reno, NV) which were preset to remove the appropriate

volume of stock solution from the carboy containers.

The dispensing pumps injected the stock solution

through Teflon® microbore tubing into glass

mixing/splitter boxes immediately prior to delivery of

two liters of diluent water. Diluent water alone was

delivered to the control aquaria mixing/splitter boxes.

Exposure solutions were delivered from mixing/splitter

boxes to duplicate 35 L exposure aquaria through calibrated glass delivery lines. Aquaria volumes were maintained by an overflow drain siphon designed to remove

water from near the bottom of each aquarium.

The resin-coated plywood exposure chamber measured

333 cm long, 118 cm wide, and 244 cm high. Access to

the chamber was through sliding polycarbonate doors on

each side of the chamber. Aquaria were placed at random in a central waterbath maintained at 26° ± 1° C.

Timer-controlled fluorescent lights with dimmer ballasts

provided 16 hours of light per 24 hours with 30-minute

transitions simulating dawn and dusk. The chamber was

maintained at a slightly negative pressure.

Individual test aquaria measured 48 cm long, 35.5 cm

wide, and 24 cm high. A water depth of 20.5 cm was

maintained. Aquaria were individually covered and covers of adjacent tanks were not raised simultaneously.

EXPOSURE SOLUTION CONCENTRATION

MONITORING

Concentrations of 2,2-bis(bromomethyl)-1,3-propanediol, nitromethane, and 1,2,3-trichloropropane in the

exposure aquaria were monitored using GC, approximately 3 times each week. Duplicate samples were

analyzed from each aquarium. Summaries of the aquaria solution concentrations are given in Tables G2

through G4.

EXPOSURE SOLUTION

CHARACTERIZATION

2,2-Bis(bromomethyl)-1,3-propanediol

Exposure solution characterization was performed prior

to the start of the 14- and 16-month studies. The generation of target concentrations of 2,2-bis(bromomethyl)-

1,3-propanediol exposure solution in exposure aquaria

was determined with and without fish present. Duplicate

aquaria at target concentrations of 10, 35, and 100 mg/L

were sampled prior to the first injection and 3, 6, 9, 12,

14, and 24 hours after the initial injection. Samples were

analyzed using GC. All aquaria reached their approximate target 2,2-bis(bromomethyl)-1,3-propanediol concentrations within 24 hours of the initial injection.

The uniformity of distribution of 2,2-bis(bromomethyl)-

1,3-propanediol in single 10 and 100 mg/L aquaria was

determined following 6-day intermittent flow-through

generation and stability studies. Samples were collected

from nine locations at 2 cm below the surface of each

aquarium solution and from nine locations within 2 cm

of the bottom of each aquarium. Samples were analyzed

using GC and uniformity of distribution was established.

The persistence of 2,2-bis(bromomethyl)-1,3-propanediol in the exposure aquaria was determined by monitoring the exposure solution concentration with intermittent

flow through of diluent water only after stable target

concentrations were reached. Samples were collected

every 3 hours for 12 hours and analyzed using GC. The

measured concentration was below the limit of detection

after 6 hours in the 10 mg/L aquaria, after 12 hours in

the 35 mg/L aquaria, and was less than 6 mg/L after

12 hours in the 100 mg/L aquaria.
Fish Studies, NTP TR 528 27

Stability of 2,2-bis(bromomethyl)-1,3-propanediol in the

intermittent flow-through exposure solution was monitored with and without fish present for 132 hours after

approximate target concentrations were reached in 10,

35, and 100 mg/L exposure aquaria. Samples were collected approximately every 4 hours and analyzed using

GC. Stability was confirmed for at least 132 hours. The

stability of 2,2-bis(bromomethyl)-1,3-propanediol in

exposure solution without intermittent flow-through was

monitored with and without fish present for 24 hours.

The 2,2-bis(bromomethyl)-1,3-propanediol exposure

solution was stable for up to 24 hours in the absence of

intermittent flow-through.

Nitromethane

Exposure solution characterization was performed prior

to the start of the 13- and 16-month studies. The generation of target concentrations of nitromethane exposure

solution in exposure aquaria by intermittent flowthrough was determined with and without fish present.

Duplicate aquaria at target concentrations of 8.6, 24.5,

and 70.0 mg/L were sampled prior to the first injection

and every 2 hours for 14 hours after the initial injection.

Samples were analyzed using GC. All aquaria reached

their approximate target nitromethane concentrations

within 14 hours of the initial injection. 

The uniformity of distribution of nitromethane in a single 70.0 mg/L aquarium was determined during 6-day

intermittent flow-through generation and stability studies. Samples were collected from nine locations 2 cm

below the surface of the aquarium solution and from

nine locations within 2 cm of the bottom of the

aquarium. Samples were analyzed using GC and uniformity of distribution was established.

The persistence of nitromethane in the exposure aquaria

was determined by monitoring the exposure solution

concentration of a 100 mg/L exposure solution prepared

by injection of neat nitromethane into the mixing/splitter

box. Intermittent flow-through contained diluent water

only after stable target concentrations were reached.

Samples were collected every hour for 13 hours and analyzed using GC. The nitromethane concentration was

below the limit of detection after 13 hours.

Stability of nitromethane in the intermittent flowthrough exposure solution was monitored with and without fish present for 132 hours after approximate target

concentrations were reached in 8.6, 24.5, and 70.0 mg/L

exposure aquaria. Samples were collected approximately every 4 hours and analyzed using GC. Stability

was confirmed for up to 132 hours. The stability of

nitromethane stock carboy solutions was monitored for

144 hours. Samples were collected approximately every

24 hours and analyzed using GC. The results indicated

that nitromethane solution was stable in stock carboys

for at least 144 hours. The stability of nitromethane in

exposure solution without intermittent flow-through was

monitored for approximately 70 hours with and without

fish present in 100 mg/L exposure solutions prepared by

injection of neat nitromethane into the mixing/splitter

box. Nitromethane concentrations decreased to approximately 60% of initial target concentrations after

3.5 hours.

1,2,3-Trichloropropane

Exposure solution characterization was performed prior

to the start of the 13- and 16-month studies. The generation of target concentrations of 1,2,3-trichloropropane

exposure solution in exposure aquaria by intermittent

flow-through was determined with and without fish present. Duplicate aquaria at target concentrations of 10, 35,

and 100 mg/L were sampled prior to the first injection

and 3, 6, 9, 12, 14, and 24 hours after the initial injection.

Samples were analyzed using GC. All aquaria reached

their approximate target 1,2,3-trichloropropane concentrations within 24 hours of the initial injection.

The uniformity of distribution of 1,2,3-trichloropropane

in single 10 and 100 mg/L aquaria was determined during 6-day intermittent flow-through generation and stability studies. Samples were collected from nine locations 2 cm below the surface of the aquarium solution

and from nine locations within 2 cm of the bottom of the

aquarium. Samples were analyzed using GC and uniformity of distribution was established.

The persistence of 1,2,3-trichloropropane in the exposure aquaria was determined by monitoring the exposure

solution concentration with intermittent flow-through of

diluent water only after stable target concentrations were

reached. Samples were collected every 3 hours for

12 hours and analyzed using GC. After 12 hours, the

measured concentrations were 0.96 mg/L in the 10 mg/L

aquaria, 3.63 mg/L in the 35 mg/L aquaria, and

9.16 mg/L in the 100 mg/L aquaria.

Stability of 1,2,3-trichloropropane in the intermittent

flow-through exposure solution was monitored with and
28 Fish Studies, NTP TR 528

without fish present for 120 hours after approximate target concentrations were reached in 10, 35, and 100 mg/L

exposure aquaria. Samples were collected approximately every 4 hours and analyzed using GC. Stability was

confirmed for at least 120 hours. The stability of

1,2,3-trichloropropane in exposure solution without

intermittent flow-through was monitored with and without fish present for 24 hours. After 24 hours, the average 1,2,3-trichloropropane exposure solution concentrations in all aquaria declined to approximately 54% of the

measured concentrations before flow-through was

terminated.

16-MONTH STUDIES IN GUPPIES

AND 13- AND 14-MONTH STUDIES

IN MEDAKA

Study Design

The results of previous studies of life span and dose

range-finding studies (2-day static, 7-day flow-through,

and 28-day range-finding studies) were used to determine the overall study lengths and the individual study

dose ranges. Groups of approximately 220 guppies (two

replicates of 110) were maintained in aquaria water

containing 0, 24, 60, or 150 mg/L 2,2-bis(bromomethyl)-1,3-propanediol; 0, 10, 30, or 70 mg/L nitromethane; or 0, 4.5, 9.0, or 18.0 mg/L 1,2,3-trichloropropane. Groups of approximately 340 medaka (two

replicates of 170) were maintained in aquaria water

containing 0, 24, 60, or 150 mg/L 2,2-bis(bromomethyl)-1,3-propanediol; 0, 10, 20, or 40 mg/L nitromethane, or 0, 4.5, 9.0, or 18.0 mg/L 1,2,3-trichloropropane. The overall study durations were 16 months

for all guppy studies, 14 months for 2,2-bis(bromomethyl)-1,3-propanediol-exposed medaka, and

13 months for nitromethane- and 1,2,3-trichloropropaneexposed medaka. More medaka were used because of

the expected shorter lifespan.

Nine months after initiation of exposure, all treatment

and control fish were removed from the test aquaria and

counted. Ten guppies and 10 medaka from each group

replicate were sacrificed for histopathologic analysis.

The remaining fish from each group were distributed

into either continuing exposure groups or stop-exposure

groups in chemical-free water. This distribution was

performed such that roughly two-thirds of the remaining

fish from each group were placed in continuing exposure

groups and the remaining third were placed into stopexposure groups for the remainder of the studies. The

stop-exposure component was added since this was a

new system for NTP and for fish studies, and it was

thought that stopping the exposure at 9 months and

transferring to exposure chemical-free aquaria would

allow for better survival and tumor development. The

sex of guppies and medaka was not determined until

histopathologic analysis.

Source and Specification of Animals

Guppy (Poecilia reticulata) and medaka (Oryzias

latipes) fry used in the 13-, 14-, and 16-month studies

were obtained from established Gulf Coast Research

Laboratory (GCRL) cultures. Guppy cultures originating from Aqua World (St. Louis, MO) and medaka cultures originating from Carolina Biological Supply

(Burlington, NC) had been maintained at GCRL for

more than 10 years prior to the initiation of the studies.

Brood cultures were established from the GCRL cultures

for the purpose of producing fry for use in these studies.

Fry were collected from brood cultures to create the

various dose groups in the studies. Dose groups were

established by randomly pooling fry into transfer vessels

that were in turn randomly assigned to exposure aquaria.

Fry were 14 days old (nitromethane and 1,2,3-trichloropropane) or 16 days old [2,2-bis(bromomethyl)-

1,3-propanediol] on the first day of the studies.

Animal Maintenance

Animals were maintained in all glass aquaria throughout

the studies. Aquaria were cleaned at least once weekly

except for the nitromethane study aquaria which were

cleaned 2 (guppy) or 3 (medaka) times per day due to

microfauna growth. Aquaria water was carbon-filtered,

aerated, unchlorinated well water containing the appropriate chemical concentration. All exposure aquaria for

each study and species were partially submerged in a

single water bath to maintain consistent temperature

within and between exposure concentrations and their

replicates. For the first 9 months, fish were maintained

in aquaria that were 48 cm long H 38 cm wide H 24 cm

high, containing approximately 35 L of test solution. To

adjust for loss of fish volume at 9 months, fish were

moved to smaller aquaria that were 44 cm long H 25 cm

wide H 23 cm high, containing approximately 21 L of

test solution. A water depth of 19 cm was maintained by

a drain siphon that removed water from near the bottom

of each aquarium. Throughout the studies, aquaria were

covered with polycarbonate covers to prevent the escape
Fish Studies, NTP TR 528 29

of fish and aerosol mixing of test chemical between

exposure groups. Covers were raised only for feeding,

observations, tank maintenance, and sample collection.

Covers were not raised from adjacent aquaria simultaneously. Animals were fed flake food and rehydrated

brine shrimp larvae once daily. Flake food was withheld

24 to 48 hours before sacrifice and brine shrimp larvae

were withheld during the last week of study to clear the

gut. Information on feed composition and contaminants

is provided in Appendix H. Further details of animal

maintenance are given in Table 1.

Visual External Examinations 

and Pathology

All animals were observed twice daily. Visual external

findings were recorded daily and body weights and

lengths were recorded at the time of sacrifice. Sacrifice

was via tricane methanesulfonate (MS222). Individual

animal worksheets were prepared prior to the start of

mounting and sectioning. These sheets were used

throughout the entire histopathology process. Whole

fish were fixed in Bouin’s fixative solution for 96 hours,

preserved in 10% neutral buffered formalin, processed,

and embedded in paraffin. Whole fish were processed

unless trimming was required to fit a female guppy into

a cassette or to capture a gross lesion. Ten longitudinal

step sections (two serial sections at each of five steps)

through the whole fish at a thickness of 4 to 6 μm were

prepared for microscopic examination by staining with

hematoxylin and eosin. Fish that died early were generally not evaluated histologically due to severe autolysis

or cannibalism. Tissues examined microscopically are

listed in Table 1.

Microscopic evaluations were completed by a study

pathologist. Following the evaluation, the slides and

blocks were sent to the NTP Archives for inventory. The

slides, individual animal data records, pathology tables,

and study pathologists reports were sent to an independent quality assessment pathologist who conducted a

pathology quality assessment review of each study of

each exposure chemical and both fish species. The quality assessment reviews were conducted in two phases. In

the first phase, the quality assessment pathologist

reviewed all diagnoses, neoplastic and nonneoplastic,

from all tissues from a randomly selected 40% of the fish

each from the control and high exposure groups from

each of the studies. Only fish that survived to study termination were examined. No fish that died early and

none of the interim evaluation fish were reviewed. In

each study, the quality assessment review of all diagnoses in all tissues from 40% of the fish from the control

and high exposure groups confirmed the study pathologist’s findings concerning potential chemical-related

neoplastic and nonneoplastic effects. The primary focus

of these studies was to assess the performance of this

model relative to the neoplastic responses, with less concern for the nonneoplastic responses. For animals in

which tissues were reviewed for nonneoplastic lesions

there was good agreement. However, because of the

magnitude of the studies, the nonneoplastic diagnoses

were not reviewed with the same rigor as the neoplastic

diagnoses. The most important nonneoplastic lesions

were included in the results section of the report, and

there is confidence in the accuracy of the presented data.

Consequently, in the second phase another review was

undertaken for each study in which all neoplasms diagnosed by the study pathologist in potential target organs

that had not been reviewed previously during the first

phase of the review were examined by the quality assessment pathologist in order to confirm the diagnoses.

Only diagnosed neoplasms were reviewed since during

the initial review of tissues from 40% of the control and

high exposure fish no additional proliferative lesions in

any of the potential target organs were observed. Thus it

was considered highly unlikely that the review of target

organs from fish in which no neoplasm had been diagnosed by the study pathologist would reveal any additional neoplasms.

Following completion of the quality assessment reviews

for all studies, an NTP Pathology Working Group

(PWG) was convened with the quality assessment

pathologist now serving as the PWG chairperson. A single PWG was held which included all the studies of all

three exposure chemicals in both fish species.

Representative histopathology slides containing examples of lesions related to chemical administration, disagreements in diagnoses between the study pathologist

and quality assessment pathologist/PWG chairperson, or

lesions of general interest were presented to the PWG by

the chairperson for review. The PWG consisted of

pathologists experienced in fish and/or rodent pathology.

This group examined the tissues without any knowledge

of exposure groups or previously rendered diagnoses.

Overall, the PWG confirmed the study pathologist’s

findings in each study.
30 Fish Studies, NTP TR 528

Study Laboratory

University of Southern Mississippi, 

Gulf Coast Research Laboratory 

(Ocean Springs, MS)

Strain and Species

Guppy (Poecilia reticulata)

Medaka (Oryzias latipes)

Animal Source

University of Southern Mississippi,

Gulf Coast Research Laboratory 

(Ocean Springs, MS)

Time Held Before Studies

16 Days

Average Age When Studies Began

16 Days

Date of First Exposure

August 16, 1995

Duration of Exposure

Guppy: 9 months (stop-exposure 

and interim evaluation groups), 16 months

(core group), or 14.5 months (150 mg/L 

group)

Medaka: 9 months (stop-exposure 

and interim evaluation groups) or

14 months (core group)

Date of Last Exposure

Guppy: December 15, 1996

Medaka: October 16, 1996

Sacrifice Dates

Guppy: June 5 (interim evaluation group),

November 2 (150 mg/L core group),

December 16 (remaining core groups), or 

December 17 (stop-exposure group), 1996

Medaka: June 5 (interim evaluation group),

October 17 (core group), or

October 18 (stop-exposure group), 1996

Average Age at Sacrifice

Guppy: 57 weeks (150 mg/L core group),

72 weeks (remaining core groups and 

stop-exposure group), or 45 weeks 

(interim analysis group)

Medaka: 64 weeks (core and stop-exposure 

groups) or 45 weeks (interim analysis 

group)

TABLE 1

Experimental Design and Materials and Methods in the Waterborne Studies 

of 2,2-Bis(bromomethyl)-1,3-propanediol, Nitromethane, 1,2,3-Trichloropropane

2,2-Bis(bromomethyl)- Nitromethane 1,2,3-Trichloropropane

1,3-propanediol

University of Southern Mississippi, 

Gulf Coast Research Laboratory

(Ocean Springs, MS)

Guppy (Poecilia reticulata)

Medaka (Oryzias latipes)

University of Southern Mississippi,

Gulf Coast Research Laboratory 

(Ocean Springs, MS)

14 Days

14 Days

May 17, 1995

Guppy: 9 months (stop-exposure 

and interim evaluation groups), 16 months

(core group), or 13 months (70 mg/L 

group)

Medaka: 9 months (stop-exposure 

and interim evaluation groups) or

13 months (core group)

Guppy: September 23, 1996

Medaka: June 17, 1996

Guppy: March 1 (interim evaluation group),

June 17 (70 mg/L group), or September 24

(remaining core and stop-exposure

groups), 1996

Medaka: February 29 (interim evaluation

group), June 18 (core group), or

June 19 (stop-exposure group), 1996

Guppy: 74 weeks (core and stop-exposure 

groups), 57 weeks (70 mg/L core group), 

or 44 weeks (interim analysis group)

Medaka: 60 weeks (core and stop-exposure

groups) or 44 weeks (interim analysis 

group)

University of Southern Mississippi,

Gulf Coast Research Laboratory

(Ocean Springs, MS)

Guppy (Poecilia reticulata)

Medaka (Oryzias latipes)

University of Southern Mississippi,

Gulf Coast Research Laboratory 

(Ocean Springs, MS)

14 Days

14 Days

May 30, 1996

Guppy: 9 months (stop-exposure 

and interim evaluation groups) or

16 months (core group)

Medaka: 9 months (stop-exposure 

and interim evaluation groups) or

13 months (core group)

Guppy: September 28, 1997 

Medaka: June 30, 1997

Guppy: March 12 (interim evaluation

group) or September 29 (core and stopexposure groups), 1997

Medaka: March 11 (interim evaluation 

group) or July 1, (core and stop-exposure 

groups), 1997

Guppy: 72 weeks (core and stop-exposure

groups) or 44 weeks (interim analysis 

group)

Medaka: 59 weeks (core and stop-exposure

groups) or 43 weeks (interim analysis 

group)
Fish Studies, NTP TR 528 31

Approximate Size of Study Groups

Guppy: 220 fish

Medaka: 340 fish

Method of Distribution

Animals were distributed randomly into

groups of equal numbers.

Approximate Animals per Aquarium

Guppy: 110

Medaka: 170

Diet

Aqua-Tox (Specialized Formula) Flake

(Ziegler Brothers, Inc., Gardners, PA) once a

day until 24 hours before sacrifice and

rehydrated brine shrimp (Artemia) larvae

(Aquarium Products, Glen Burnie, MD) once

daily except during the last week of the

study.

Aquaria Water

Aged, aerated, carbon-filtered, unchlorinated

well water (University of Southern

Mississippi, Ocean Springs, MS)

Aquaria

Glass (Oceanview Aquarium Products, Inc.,

Pascagoula, MS) with polycarbonate covers;

brushed daily and siphoned at least once

weekly.

Aquatic Environment

Temperature: 26° ± 1.0° C, pH: 8.9 ± 0.1 

Room fluorescent light: 16 hours light/

8 hours dark with 30 minute transition to

simulate dawn and dusk.

Dissolved oxygen: $50% saturation

Exposure Concentrations

0, 24, 60, or 150 mg/L

Type and Frequency of Observation

Observed twice daily at intervals at least

6 hours apart. Body weights and lengths

were recorded at necropsy.

Guppy: 220 fish

Medaka: 340 fish

Animals were distributed randomly into

groups of equal numbers.

Guppy: 110

Medaka: 170

Aqua-Tox (Specialized Formula) Flake

(Ziegler Brothers, Inc., Gardners, PA) once a

day until 24 hours before sacrifice and

rehydrated brine shrimp (Artemia) larvae

(Aquarium Products, Glen Burnie, MD) once

daily except during the last week of the

study.

Aged, aerated, carbon-filtered, unchlorinated

well water (University of Southern

Mississippi, Ocean Springs, MS)

Glass (Oceanview Aquarium Products, Inc.,

Pascagoula, MS) with polycarbonate covers;

brushed two (guppy) or three (medaka) times

per day.

Temperature: 26° ± 0.3° C, pH: 8.8 ± 0.2 

8.9 ± 0.2 (Medaka)

Room fluorescent light: 16 hours light/

8 hours dark with 30 minute transition to

simulate dawn and dusk.

Dissolved oxygen: $40% saturation

Guppy: 0, 10, 30, or 70 mg/L

Medaka: 0, 10, 20, or 40 mg/L

Observed twice daily at intervals at least

6 hours apart. Body weights and lengths

were recorded at necropsy.

Guppy: 220 fish

Medaka: 340 fish

Animals were distributed randomly into

groups of equal numbers.

Guppy: 110

Medaka: 170

Aqua-Tox (Specialized Formula) Flake

(Ziegler Brothers, Inc., Gardners, PA) once a

day until 24 hours before sacrifice and

rehydrated brine shrimp (Artemia) larvae

(Aquarium Products, Glen Burnie, MD) once

daily except during the last week of the

study.

Aged, aerated. carbon-filtered, unchlorinated

well water (University of Southern

Mississippi, Ocean Springs, MS)

Glass (Oceanview Aquarium Products, Inc.,

Pascagoula, MS) with polycarbonate covers;

brushed daily and siphoned at least once

weekly.

Temperature: 26° ± 0.3° C, pH: 8.8 ± 0.2 

Room fluorescent light: 16 hours light/

8 hours dark with 30 minute transition to

simulate dawn and dusk.

Dissolved oxygen: $40% saturation

0, 4.5, 9.0, or 18.0 mg/L 

Observed twice daily at intervals at least

6 hours apart. Body weights and lengths

were recorded at necropsy.

TABLE 1

Experimental Design and Materials and Methods in the Waterborne Studies 

of 2,2-Bis(bromomethyl)-1,3-propanediol, Nitromethane, 1,2,3-Trichloropropane

2,2-Bis(bromomethyl)- Nitromethane 1,2,3-Trichloropropane

1,3-propanediol
32 Fish Studies, NTP TR 528

Method of Sacrifice

Anesthetization with tricaine

methanesulfonate

Histopathologya

Complete histopathology was performed on

all animals sacrificed at 9 months and at the

end of the studies. In addition to gross

lesions and tissue masses, the following

tissues were examined: bone, brain,

chromaffin tissue, corpuscle of Stannius,

esophagus, eye, gallbladder, gill, heart,

hematopoietic tissue, interrenal tissue,

intestine, kidney, liver, olfactory tissue, oral

cavity, ovary, pancreas, peripheral nerve,

pineal gland, pituitary gland, psuedobranch,

skeletal muscle, skin, spinal cord, spleen,

statoacoustic organ, swim bladder, testes,

thymus, thyroid tissue, and urinary bladder.

Anesthetization with tricaine

methanesulfonate

Complete histopathology was performed on

all animals sacrificed at 9 months and at the

end of the studies. In addition to gross

lesions and tissue masses, the following

tissues were examined: bone, brain,

chromaffin tissue, corpuscle of Stannius,

esophagus, eye, gallbladder, gill, heart,

hematopoietic tissue, interrenal tissue,

intestine, kidney, liver, olfactory tissue, oral

cavity, ovary, pancreas, peripheral nerve,

pineal gland, pituitary gland, psuedobranch,

skeletal muscle, skin, spinal cord, spleen,

statoacoustic organ, swim bladder, testes,

thymus, thyroid tissue, and urinary bladder.

Anesthetization with tricaine

methanesulfonate

Complete histopathology was performed on

all animals sacrificed at 9 months and at the

end of the studies. In addition to gross

lesions and tissue masses, the following

tissues were examined: bone, brain,

chromaffin tissue, corpuscle of Stannius,

esophagus, eye, gill, gallbladder, heart,

hematopoietic tissue, interrenal tissue,

intestine, kidney, liver, olfactory tissue, oral

cavity, ovary, pancreas, peripheral nerve,

pineal gland, pituitary gland, psuedobranch,

skeletal muscle, skin, spinal cord, spleen,

statoacoustic organ, swim bladder, testes,

thymus, thyroid tissue, and urinary bladder.

TABLE 1

Experimental Design and Materials and Methods in the Waterborne Studies 

of 2,2-Bis(bromomethyl)-1,3-propanediol, Nitromethane, 1,2,3-Trichloropropane

2,2-Bis(bromomethyl)- Nitromethane 1,2,3-Trichloropropane

1,3-propanediol

a A gross observation list was employed to record all gross abnormalities which included skeletal abnormalities and the number 

and description of tissue masses.
Fish Studies, NTP TR 528 33

STATISTICAL METHODS

Survival Analyses

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Fish

that were sacrificed or transferred to another tank were

censored; fish found dead before the 9-month and terminal sacrifices were not censored. Possible dose-related

trends were tested with Tarone’s (1975) life table test;

pairwise comparisons with the control group were made

with Cox’s (1972) method for testing the equality of two

groups.

Incidence of Lesions

For each chemical, data were collected from the two

replicate tanks in the control group and at each dose

level. For each combination of sex, species, and chemical, tank effects were tested using Fisher’s exact test for

the neoplasm of interest. The frequency of significant

differences in tumor incidences between the two replicates was consistent with what would be expected by

chance. Therefore, lesions and tissue counts from each

replicate were pooled within each sex, species, chemical

study, and treatment (9-month, core, or stop-exposure

study) combination for the control groups and for each

dose group. Each combination of chemical study, sex,

species, and treatment were analyzed separately. Lesion

incidences are based on the number of tissues examined

in which the lesion was present. 

Analysis of Lesions

Because lesions were generally examined only in fish

that were sacrificed and not those that died early,

survival adjustments were not made.

Neoplastic and nonneoplastic lesions were analyzed as

follows. Dose-related trends were tested with the

Cochran-Armitage trend test (Armitage, 1971;

Gart et al., 1979). When a small number of lesions were

present and they were disproportionately distributed at

one extreme of the dose range, an exact permutation

trend test was used (Plackett, 1981). Fisher’s exact test

was used for pairwise comparisons of each dose group to

the control group. P-values for the trend tests and

Fisher’s exact test are one sided.

Analysis of Continuous Variables

Body weight and body length, which have approximately normal distributions, were analyzed by analysis

of variance followed by Dunnett’s multiple comparison

procedure (1955) to compare each dose group to the

control group.

QUALITY ASSURANCE METHODS

The 13-, 14-, and 16-month studies were conducted in

compliance with Food and Drug Administration Good

Laboratory Practice Regulations (21 CFR, Part 58). In

addition, as records from the studies were submitted to

the NTP Archives, these studies were audited retrospectively by an independent quality assurance contractor.

Separate audits covered completeness and accuracy of

the pathology data, pathology specimens, final pathology tables, and a draft of this NTP Technical Report.

Audit procedures and findings are presented in the

reports and are on file at NIEHS. The audit findings

were reviewed and assessed by NTP staff, and all comments were resolved or otherwise addressed during the

preparation of this Technical Report.
34 Fish Studies, NTP TR 528
35

RESULTS

2,2-BIS(BROMOMETHYL)-

1,3-PROPANEDIOL

16-MONTH STUDY IN GUPPIES

Survival

2,2-Bis(bromomethyl)-1,3-propanediol was chronically

toxic to guppies in the exposed core and stop-exposure

groups (Table 2). Due to mortality, exposure of core

study fish in the 150 mg/L group was terminated on

day 443, after approximately 64 weeks on study, and

fish were maintained in 2,2-bis(bromomethyl)-

1,3-propanediol-free water in the exposure system until

the end of the study.

Exposure Concentrations, Body Lengths,

and Body Weights

Nominal exposure concentrations of 24, 60, and

150 mg/L (analyzed three times per week) provided

actual aquaria water exposure concentrations of 20.0,

53.5, and 139.0 mg/L 2,2-bis(bromomethyl)-1,3-propanediol, respectively. The only treatment-related difference between the control and exposed groups

occurred at the 9-month interim evaluation at which

time the 150 mg/L fish were longer than the control fish

(Tables 3 and 4). 

TABLE 2

Survival of Guppies in the 16-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Animals initially in study 220 220 220 220

Died before the 9-month interim evaluation 3 9 13 16

Unaccounted fora 6 286

9-Month interim evaluation 20 20 20 20

Died after reallocationb

Core study 4 8 18 40

Stop-exposure study 1 3 10 22

Survivors

Core study 123 117 101 78d

Stop-exposure study 63 61 50 38

Survival analysisc

Core study <0.0001 0.0360 <0.0001 <0.0001

Stop-exposure study <0.0001 0.0429 0.0001 <0.0001

a The laboratory report did not account for these fish at 9 months. b At 9 months, approximately one third of the fish in each replicate was reassigned to stop-exposure aquaria; the remaining fish (core study)

were returned to their original exposure aquaria. c The result of life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparison (Cox, 1972)

between the controls and exposed animals appear in the exposure concentration columns. d Exposure was stopped during week 64 (day 443), and fish were maintained in exposure chemical-free aquaria until the end of the study.
36 Fish Studies, NTP TR 528

TABLE 3

Mean Body Lengths of Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediola

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 28.4 ± 1.0 34.2 ± 1.1 34.5 ± 1.16

24 mg/L 28.9 ± 1.1 33.8 ± 1.2 36.5 ± 1.2

60 mg/L 30.4 ± 1.2 34.7 ± 1.2 35.2 ± 1.3

150 mg/L 30.2 ± 1.7 34.1 ± 1.2 34.2 ± 1.6

a Data are presented as mean length ± standard error (mm).

TABLE 4

Mean Body Weights of Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 0.55 ± 0.07 1.01 ± 0.09 1.08 ± 0.01

24 mg/L 0.55 ± 0.07 1.03 ± 0.11 1.26 ± 0.11

60 mg/L 0.63 ± 0.07 1.16 ± 0.02 1.23 ± 0.13

150 mg/L 0.66 ± 0.09 1.16 ± 0.12 1.22 ± 0.18

a Data are presented as mean wet weight ± standard error (g).
Fish Studies, NTP TR 528 37

Pathology and Statistical Analyses

This section describes the statistically or biologically

noteworthy changes in incidences of neoplasms and/or

nonneoplastic lesions of the liver, gallbladder, kidney,

and pituitary gland. Summaries of incidences of neoplasms by replicate are presented in Appendix A.

Liver (9-Month Interim Evaluation): Hepatocellular

adenomas occurred in one 24 mg/L male and in one

150 mg/L male (Table 5 and A1). A single cholangioma

occurred in a control female (Table A3). 

Liver (Core Study): Incidences of hepatocellular adenoma or carcinoma (combined) in 150 mg/L males were

greater than those in the controls (Tables 5, A1, and A3).

The incidence of basophilic focus was significantly

greater in 150 mg/L males than in controls.

Hepatocellular adenoma was a distinct, well demarcated, nodular lesion that produced some degree of compression of adjacent normal liver. Adenomas often were

relatively small but some were quite large and replaced

much of the normal liver. Adenomas consisted of a relatively uniform population of polygonal cells, generally

with increased eosinophilic to basophilic cytoplasmic

staining, arranged in normal appearing to slightly irregular, hypercellular hepatic tubules. Sometimes varying

numbers of cells with slightly to highly vacuolated clear

cytoplasm were present. Hepatocellular carcinoma was

generally a large lesion that replaced much or, occasionally, all of the observable liver parenchyma. The border

was irregular with varying numbers of projections of

neoplastic cells into the surrounding parenchyma that

appeared to represent localized invasion. Neoplastic

cells within carcinomas stained eosinophilic to

basophilic, were polygonal to elongated, and were

arranged in irregular, highly cellular, densely packed

cords that were thicker than the normal tubules.

Carcinomas also often tended to form solid clusters of

cells. The neoplastic cells within some carcinomas were

moderately to markedly pleomorphic with nuclear

atypia. Cholangioma was a discrete, small to

moderately large, nodular lesion, consisting of a cluster

of variably sized, often irregular bile ducts surrounded

by varying amounts of stroma, that replaced and often

compressed adjacent hepatic parenchyma. The bile duct

structures were composed of normal-appearing flattened

to cuboidal to low columnar biliary epithelial cells.

Basophilic focus was generally a small, round to irregular cluster of hepatocytes, with increased basophilic to

amphophilic cytoplasmic staining, that caused little or

no evidence of compression of the adjacent liver. Often

a basophilic focus had indistinct borders although some

foci, especially those with more prominent staining,

appeared well demarcated from the surrounding liver.

Liver (Stop-Exposure Study): Incidences of hepatocellular adenoma (including multiple) and of hepatocellular

carcinoma were greater in 150 mg/L males than in

controls (Tables 5 and A2).
38 Fish Studies, NTP TR 528

TABLE 5

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Guppies 

in the 16-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol

Trend

0 mg/L 24 mg/L 60 mg/L 150 mg/L Test

P-Valuea

Male 

9-Month Interim Evaluation

Number Examined Microscopically 13 13 10 11

Hepatocellular Adenomab 0 1 0 1 0.2104

Core Study

Number Examined Microscopically 61 50 41 38

Basophilic Focus 3 4 3 8* 0.0039

Cholangioma 0 0 0 1 0.2000

Hepatocellular Adenoma, Multiple 0 0 0 2 0.0392

Hepatocellular Adenoma (includes multiple) 3 4 4 8* 0.0040

Hepatocellular Carcinoma 1 2 2 2 0.1933

Hepatocellular Adenoma or Carcinoma 4 6 6 10** 0.0027

Stop-Exposure Study

Number Examined Microscopically 28 22 21 24

Hepatocellular Adenoma, Multiple 0 0 1 2 0.0565

Hepatocellular Adenoma (includes multiple) 0 3 3 6** 0.0062

Hepatocellular Carcinoma 0 0 0 3 0.0146

Hepatocellular Adenoma or Carcinoma 0 3 3 9** 0.0001

Eosinophilic Focus 0 0 1 0

Female 

Core Study

Number Examined Microscopically 61 69 58 46

Basophilic Focus 5 2 4 8 0.0109

Cholangioma 0 2 0 0 0.2323

Hepatocellular Adenoma, Multiple 0 0 0 1 0.1966

Hepatocellular Adenoma (includes multiple) 2 1 2 5 0.0106

Eosinophilic Focus 0 1 0 0

Stop-Exposure Study

Number Examined Microscopically 35 36 28 13

Cholangioma 0 0 0 1 0.1161

Cholangiocarcinoma 0 0 0 1 0.1161

Cholangioma or Cholangiocarcinoma 0 0 0 2 0.0126

Hepatocellular Adenoma 1 1 1 0 0.3206

Basophilic Focus 0 1 0 0

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion
Fish Studies, NTP TR 528 39

TABLE 6

Incidences of Neoplasms and Nonneoplastic Lesions of the Gallbladder in Guppies 

in the 16-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol

Trend

0 mg/L 24 mg/L 60 mg/L 150 mg/L Test

P-Valuea

Male

Core Study

Number Examined Microscopically 56 48 41 36

Dilatationb 0 1 4* 5** 0.0010

Enlargement 0 4* 5* 9** <0.0001

Epithelium, Hyperplasia 1 0 0 9** <0.0001

Stop-Exposure Study

Number Examined Microscopically 27 21 21 23

Enlargement 0 2 0 6** 0.0008

Epithelium, Hyperplasia 0 1 0 3 0.0147

Inflammation, Granulomatous 0 1 1 3 0.0235

Female

Core Study

Number Examined Microscopically 61 68 57 46

Dilatation 0 15** 18** 17** <0.0001

Enlargement 0 3 12** 10** <0.0001

Epithelium, Hyperplasia 0 6* 4 7** 0.0043

Inflammation, Granulomatous 0 0 1 3 0.0074

Carcinoma 0 0 1 0 0.4369

Stop-Exposure Study

Number Examined Microscopically 35 34 28 13

Dilatation 2 4 4 3 0.0473

Enlargement 2 2 3 3 0.0243

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion

Gallbladder (Core Study): Incidences of dilatation,

enlargement, and epithelial hyperplasia were greater in

exposed core study males and females than in the controls (Table 6). Rupture of the gallbladder wall, accompanied by wall fibrosis and/or granulomatous inflammation, occurred in three 150 mg/L females. Dilatation

was characterized by distention of the gallbladder lumen

up to several times normal diameter, generally with thinning of the gallbladder wall. Enlargement was characterized by a gallbladder that was not only larger than normal, but that also had a wall that was thicker than normal

and that usually had the epithelium thrown into folds.

Epithelial hyperplasia was a focal to diffuse lesion

characterized by an increase in the number of epithelial

cells and sometimes the number of cell layers with subsequent thickening of the epithelium. In more severe

cases the hyperplastic epithelium formed folds and few

to numerous papillary projections. The single carcinoma

that was observed in a female in the 60 mg/L group was

a small lesion consisting of relatively normal-appearing

cuboidal cells to anaplastic spindle-shaped epithelial

cells that had invaded the gallbladder wall.

Gallbladder (Stop-Exposure Study): The incidence of

enlargement in the 150 mg/L males was slightly

increased compared to that in the controls (Table 6).
40 Fish Studies, NTP TR 528

Kidney (Stop-Exposure Study): The incidence of mineralization in 150 mg/L males was increased compared

with controls (Table 7). Mineralization consisted of

scattered irregular bits of basophilic material within the

kidney stroma and/or tubular lumens. Casts were characterized by aggregates of amphophilic to basophilic

granular material within the lumens of scattered tubules.

Renal tubule dilatation was characterized by varying

degrees of enlargement of the renal tubular lumen up to

several times normal diameter.

Pituitary Gland (Core Study): The incidence of hyperplasia was increased in the 150 mg/L group of females as

compared with controls (0 mg/L, 3/61; 24 mg/L, 3/69;

60 mg/L, 9/58; 150 mg/L, 10/46). Pituitary gland hyperplasia was characterized by varying degrees of enlargement of the pituitary gland. The normal pituitary gland

consisted of an anterior portion and a posterior portion.

Hyperplasia was characterized by a mild to moderate,

diffuse enlargement of the anterior portion, the posterior

portion, or both. The normal architecture of the gland

was maintained and the cells of the anterior and posterior portions appeared normal.

TABLE 7

Incidences of Nonneoplastic Lesions of the Kidney in Guppies 

in the 16-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol

Trend

0 mg/L 24 mg/L 60 mg/L 150 mg/L Test

P-Valuea

Male 

Stop-Exposure Study

Number Examined Microscopically 28 22 21 24

Castsb 16 15 14 18 0.1117

Mineralization 1 4 4 8** 0.0043

Female 

Core Study

Number Examined Microscopically 61 69 58 46

Bowman’s Space, Dilatation 10 23* 12 31** <0.0001

Bowman’s Space, Hemorrhage 1 7* 2 8** 0.0048

Stop-Exposure Study

Number Examined Microscopically 35 36 28 13

Mineralization 3 6 5 4 0.0350

Renal Tubule, Dilatation 18 20 20 10 0.0279

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion
Fish Studies, NTP TR 528 41

TABLE 8

Survival of Medaka in the 14-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Animals initially in study 340 340 340 340

Died before the 9-month interim evaluation 13 11 15 10

Unaccounted fora 4 107

9-Month interim evaluation 20 20 20 20

Died after reallocationb

Core study 15 13 28 26

Stop-exposure study 13 8 7 5

Survivors

Core study 186 192 175 175

Stop-exposure study 89 95 95 97

Survival analysisc

Core study 0.1648 0.5426 0.0675 0.2983

Stop-exposure study 0.1383N 0.2738N 0.5510N 0.0772N

a The laboratory report did not account for these fish at 9 months. b At 9 months, approximately one third of the fish in each replicate was reassigned to stop-exposure aquaria; the remaining fish (core study)

were returned to their original exposure aquaria. c The result of life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparison (Cox, 1972)

between the controls and exposed animals appear in the exposure concentration columns.

14-MONTH STUDY IN MEDAKA

Survival

At the concentrations used in this study 2,2-bis(bromomethyl)-1,3-propanediol did not result in significant

reductions in survival (Table 8). 

Exposure Concentrations, Body Lengths, 

and Body Weights

Nominal exposure concentrations of 24, 60, and

150 mg/L provided actual exposure concentrations of 

19.4, 56.9, and 137.8 mg/L 2,2-bis(bromomethyl)-

1,3-propanediol, respectively. Core study animals in the

60 and 150 mg/L groups were significantly larger, in

both body lengths and weights, than control group fish.

Otherwise, there were no treatment differences determined between the control and any treatment in body

lengths or lengths in any group (Tables 9 and 10).
42 Fish Studies, NTP TR 528

TABLE 9

Body Lengths of Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediola

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 23.5 ± 0.5 25.0 ± 0.4 26.5 ± 0.3

24 mg/L 22.9 ± 0.4 25.6 ± 0.3 26.2 ± 0.3

60 mg/L 24.6 ± 0.5 26.7 ± 0.3** 26.7 ± 0.4

150 mg/L 25.2 ± 0.7 26.4 ± 0.4* 26.8 ± 0.3

* Significantly different (P#0.05) from the control group by Dunnett’s test

** P#0.01 a Data are presented as mean length ± standard error (mm).

TABLE 10

Body Weights of Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediola

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 0.203 ± 0.013 0.274 ± 0.013 0.340 ± 0.016

24 mg/L 0.186 ± 0.012 0.287 ± 0.008 0.340 ± 0.021

60 mg/L 0.238 ± 0.013 0.329 ± 0.011** 0.319 ± 0.016

150 mg/L 0.241 ± 0.016 0.318 ± 0.015* 0.322 ± 0.012

* Significantly different (P#0.05) from the control group by Dunnett’s test

** P#0.01 a Data are presented as mean weight ± standard error (mm).
Fish Studies, NTP TR 528 43

Pathology and Statistical Analyses

This section describes the statistically or biologically

noteworthy changes in incidences of neoplasms and/or

nonneoplastic lesions of the liver, gallbladder, kidney,

and thyroid tissue. Summaries of incidences of neoplasms by replicate are presented in Appendix B.

No potentially exposure-related neoplasms were

observed in male and female medaka exposed to

2,2-bis(bromomethyl)-1,3-propanediol and evaluated at

9 months.

Liver (Core Study): The incidence of hepatocellular

adenoma or carcinoma (combined) was increased in

150 mg/L males (Tables 11 and B1).

Cholangiocarcinomas occurred in a few exposed males

and females, with all but one occurring in 150 mg/L fish

(Tables 11, B1, and B3). No cholangiocarcinomas

occurred in controls. One cholangioma occurred in a

150 mg/L female, and one occurred in a control female.

Microscopically, hepatocellular adenoma and carcinoma, cholangioma, and cholangiocarcinoma appeared

similar to those previously described. The cholangiocarcinoma had highly irregular borders and invaded into the

adjacent normal parenchyma. It was composed of

numerous small, atypical, densely packed, proliferating

biliary structures within small to abundant amounts of

fibrous and/or proliferating spindle cell stroma. The biliary structures consisted of small, moderately pleomorphic, cuboidal to columnar to fusiform epithelial cells.

Incidences of bile duct dilatation were increased in

150 mg/L females compared to controls (Table 11). Bile

duct hyperplasia was generally a slight change characterized by few to several, small to moderately large,

normal-appearing bile ducts scattered within the liver.

Occasionally the hyperplastic bile ducts formed small

clusters. Bile duct dilatation was characterized by few to

several scattered bile ducts that were variably dilated,

sometimes up to several times normal diameter.

Incidences of cysts were increased in exposed groups of

males and females, and incidences of hepatocellular vacuolation in 24 and 60 mg/L females were lower than in

controls. Cysts were single or multiple clear cavities,

usually lined by flattened epithelium, scattered within

the liver. Macrophage aggregates consisted of scattered,

variably sized aggregates of small to moderate numbers

of plump, pigmented macrophages. Hepatocellular vacuolation consisted of variably sized, clear vacuoles

within the hepatocyte cytoplasm.

Liver (Stop-Exposure Study): Cholangiocarcinoma

occurred in one male and one female in the 150 mg/L

groups and in one control female (Tables 11, B2, and

B4). The incidences of bile duct hyperplasia in 60 and

150 mg/L males and the incidences of hepatocellular

vacuolation and vacuolated hepatocyte foci in all

exposed male groups were greater than those in the controls. Vacuolated hepatocyte foci blended with the surrounding parenchyma without producing compression

and consisted of round to irregular clusters of hepatocytes containing variably sized, clear, discrete vacuoles,

resembling fat droplets, within the cytoplasm. Except

for the presence of the vacuoles, cells within vacuolated

foci resembled normal hepatocytes.
44 Fish Studies, NTP TR 528

TABLE 11

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Medaka 

in the 14-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol

Trend

0 mg/L 24 mg/L 60 mg/L 150 mg/L Test

P-Valuea

Male 

Core Study

Number Examined Microscopically 47 59 56 59

Bile Duct, Hyperplasiab 1 3 0 4 0.1302

Cyst 1 10* 18** 22** <0.0001

Macrophage Aggregates 18 28 27 33 0.0481

Eosinophilic Focus 0 1 0 0

Cholangiocarcinoma 0 1 0 2 0.0788

Hepatocellular Adenoma, Multiple 0 0 0 1 0.2670

Hepatocellular Adenoma (includes multiple) 1 0 0 6 0.0008

Hepatocellular Carcinoma 0 0 0 2 0.0704

Hepatocellular Adenoma or Carcinoma 1 0 0 8* <0.0001

Stop-Exposure Study 

Number Examined Microscopically 27 21 31 29

Bile Duct, Dilatation 2 6 5 5 0.3684

Bile Duct, Hyperplasia 0 2 5* 6* 0.0120

Hepatocellular Vacuolation 1 5* 6 6 0.1212

Macrophage Aggregates 9 10 16 15 0.1263

Vacuolated Hepatocyte Focus 0 1 1 4 0.0117

Cholangiocarcinoma 0 0 0 1 0.2685

Hepatocellular Adenoma 1 0 1 3 0.0568

Hepatocellular Carcinoma 0 0 0 1 0.2685

Hepatocellular Adenoma or Carcinoma 1 0 1 4 0.0182
Fish Studies, NTP TR 528 45

TABLE 11

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Medaka 

in the 14-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

Trend

0 mg/L 24 mg/L 60 mg/L 150 mg/L Test

P-Value

Female 

Core Study

Number Examined Microscopically 59 47 50 47

Bile Duct, Dilatation 6 10 5 14* 0.0096

Cyst 22 27* 35** 30** 0.0076

Macrophage Aggregates 22 25 27 23 0.2070

Hepatocellular Vacuolation 20 4** 6** 9 0.1410

Eosinophilic Focus 0 0 1 0

Cholangioma 1 0 0 1 0.3082

Cholangiocarcinoma 0 0 0 1 0.2315

Cholangioma or Cholangiocarcinoma 1 0 0 2 0.0828

Hepatocellular Adenoma 5 3 6 3 0.4042

Hepatocellular Carcinoma 0 0 1 1 0.1673

Hepatocellular Adenoma or Carcinoma 5 3 7 4 0.4227

Ectopic Thyroid Follicular Cell Carcinoma 0 1 0 0 0.2909

Lymphosarcoma 0 1 0 0 0.2909

Stop-Exposure Study

Number Examined Microscopically 26 33 23 25

Cholangiocarcinoma 1 0 0 1 0.3084

Hepatocellular Adenoma 1 0 1 3 0.0284

Hepatocellular Carcinoma 0 0 2 0 0.4528

Hepatocellular Adenoma or Carcinoma 1 0 3 3 0.0454

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion
46 Fish Studies, NTP TR 528

TABLE 12

Incidences of Neoplasms and Nonneoplastic Lesions of the Gallbladder in Medaka 

in the 14-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

Trend

0 mg/L 24 mg/L 60 mg/L 150 mg/L Test

P-Valuea

Male 

Core Study

Number Examined Microscopically 46 53 55 57

Epithelium, Hyperplasiab 3 2 3 8 0.0249

Cystic Duct, Hyperplasia 0 1 2 4 0.0193

Stromal Polyp 0 0 1 0 0.4847

Stop-Exposure Study 

Number Examined Microscopically 25 19 30 28

Epithelium, Hyperplasia 1 1 1 6 0.0061

Stromal Polyp 0 0 0 1 0.2745

Female 

Core Study

Number Examined Microscopically 55 44 48 45

Epithelial Hyperplasia 0 1 3 2 0.1065

Stromal Polyp 0 0 0 1 0.2344

a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion

Gallbladder (Core Study): Stromal polyps occurred in

one exposed male and in one exposed female (Tables 12,

B1, and B3). Stromal polyp was a small lesion consisting of an irregular mass composed of a core of abundant

loose stroma that was covered by a single layer of tall

columnar epithelium. Epithelial hyperplasia of the gallbladder was microscopically similar to that previously

described. Cystic duct hyperplasia, consisting of hyperplasia of the cystic duct epithelium, had a similar appearance to hyperplasia within the gallbladder lumen and

was accompanied by varying degrees of enlargement of

the duct secondary to the epithelial hyperplasia.

Gallbladder (Stop-Exposure Study): A stromal polyp

occurred in one 150 mg/L male (Tables 12 and B2).
Fish Studies, NTP TR 528 47

TABLE 13

Incidences of Nonneoplastic Lesions of the Kidney in Female Medaka 

in the 14-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

Trend

0 mg/L 24 mg/L 60 mg/L 150 mg/L Test

P-Valuea

Core Study

Number Examined Microscopically 59 47 50 47

Bowman’s Space, Dilatationb 1 3 3 6* 0.0135

Casts 3 11** 12** 13** 0.0066

Mineralization 1 9** 15** 13** 0.0010

Renal Tubule, Degeneration 0 3 7** 9** 0.0003

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion

Kidney (Core Study): Compared to the control group,

incidences of casts and mineralization were increased in

all exposed female groups, and the incidences of renal

tubule degeneration were increased in the 60 and

150 mg/L female groups (Table 13). Additionally, the

incidence of dilatation of Bowman’s space was

increased in 150 mg/L females compared to that in controls. Renal tubule degeneration was characterized by

irregular shrinkage and/or varying degrees of flattening

of the tubular epithelium. Degeneration was often seen

within dilated tubules. Other kidney changes were

microscopically similar to those previously described.
48 Fish Studies, NTP TR 528

TABLE 14

Incidences of Nonneoplastic Lesions of Thyroid Tissue in Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol 

Trend

0 mg/L 24 mg/L 60 mg/L 150 mg/L Test

P-Valuea

Male

Core Study

Number Examined Microscopically 47 59 56 59

Hyperplasiab 0 1 2 11** <0.0001

Follicular Cell Hypertrophy 13 19 25 24 0.0851

Increased Basophilia 39 55 52 50 0.2957

Stop-Exposure Study 

Number Examined Microscopically 27 21 31 29

Hyperplasia 2 2 2 7 0.0177

Follicular Cell, Hypertrophy 6 5 11 15* 0.0051

Female

Core Study 

Number Examined Microscopically 59 47 50 47

Follicular Cell, Hypertrophy 14 8 19 12 0.2730

Increased Basophilia 4 4 10* 12** 0.0016

Stop-Exposure Study

Number Examined Microscopically 26 33 23 25

Follicular Cell, Hypertrophy 7 3 7 3 0.2329

Increased Basophilia 7 6 6 2 0.0600

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion

Thyroid Tissue (Core Study): Incidences of hyperplasia

in 150 mg/L males and increased basophilia of the follicular epithelium in exposed males and 60 and

150 mg/L females were greater than those in the controls

(Table 14). Microscopically, normal thyroid tissue consisted of a few follicles located just caudal to the gills

and ventral to the esophagus. Hyperplasia consisted of

a slight increase above normal in the number of thyroid

follicles as compared with normal. The follicles were

generally small, composed of small eosinophilic

cuboidal follicular cells, and contained colloid.

Hypertrophy consisted of an increase in the size of follicular epithelial cells. Increased basophilia was characterized by increased basophilic staining of follicular

cells as compared with normal cells, and may have represented a normal anatomic variation. 

Thyroid Tissue (Stop-Exposure Study): The incidence

of follicular cell hypertrophy of the thyroid tissue was

increased in the 150 mg/L male group as compared with

the control group (Table 14).
Fish Studies, NTP TR 528 49

NITROMETHANE

16-MONTH STUDY IN GUPPIES

Survival

The rapid growth of microfauna in the treatment aquaria

required the study aquaria to be brushed once and

siphoned twice each day. A number of fish deaths were

due to mechanical injury related to this activity.

Although the cause of death could not be confirmed in

many cases, mortality in the 70 mg/L treatment

appeared to indicate that this level of nitromethane

exposure was chronically toxic. This is confirmed by

the similar survival rate of guppies from all treatments

following removal from treatment aquaria and placement in stop-exposure (Table 15). When removed from

the nitromethane, survival of stop-exposure fish

improved over those fish returned to the 70 mg/L core

study aquaria. Due to the high mortality of fish in the

70 mg/L core study groups, these fish were removed

from treatment (day 396) and fixed for histological

analyses after approximately 57 weeks on study.

TABLE 15

Survival of Guppies in the 16-Month Waterborne Study of Nitromethane

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Animals initially in study 220 220 221 220

Died before the 9-month interim evaluation 9 16 7 60

Unaccounted fora 7 6 0 15

9-Month interim evaluation 20 20 20 20

Died after reallocationb

Core study 12 10 13 30

Stop-exposure study 4 4 4 6

Survivors

Core study 108 100 111 50d

Stop-exposure study 60 64 66 39

Survival analysisc

Core study <0.0001 0.3669 0.8145 <0.0001

Stop-exposure study <0.0001 0.1724 0.6177 <0.0001

a The laboratory report did not account for these fish at 9 months. b At 9 months, approximately one third of the fish in each replicate was reassigned to stop-exposure aquaria; the remaining fish (core study)

were returned to their original exposure aquaria. c The result of life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparison (Cox, 1972)

between the controls and exposed animals appear in the exposure concentration columns. d Exposure was stopped on day 396, and fish were sacrificed on day 397 after approximately 57 weeks on study.
50 Fish Studies, NTP TR 528

TABLE 16

Body Lengths of Guppies in the 16-Month Waterborne Study of Nitromethanea

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 31.8 ± 1.2 36.1 ± 1.4 33.4 ± 1.2

10 mg/L 32.6 ± 1.3 33.7 ± 1.4 35.3 ± 1.3

30 mg/L 31.8 ± 1.4 34.2 ± 1.4 35.4 ± 1.3

70 mg/L 34.2 ± 1.7 —b 38.8 ± 1.5*

* Significantly different (P#0.05) from the control group by the Dunnett’s test a Data are presented as mean length ± standard error (mm). b 70 mg/L core group terminated at 57 weeks

TABLE 17

Body Weights of Guppies in the 16-Month Waterborne Study of Nitromethanea

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 0.74 ± 0.09 1.37 ± 0.15 1.08 ± 0.12

10 mg/L 0.91 ± 0.11 1.14 ± 0.14 1.10 ± 0.10

30 mg/L 0.85 ± 0.14 1.19 ± 0.14 1.16 ± 0.12

70 mg/L 1.01 ± 0.15 —b 1.58 ± 0.16**

* Significantly different (P#0.05) from the control group by the Dunnett’s test

** P#0.01 a Data are presented as mean weight ± standard error (g). b 70 mg/L core group terminated at 57 weeks

Exposure Concentrations, Body Lengths,

and Body Weights

Nominal exposure concentrations of 10, 30, and

70 mg/L provided actual exposure concentrations of 9.9,

28.7, and 66.4 mg/L nitromethane, respectively. The

observed differences in weight and length between 

controls and 70 mg/L fish may have been an artifact of

the reduced numbers of fish in the 70 mg/L aquaria

(Tables 16 and 17).
Fish Studies, NTP TR 528 51

Pathology and Statistical Analyses

This section describes the statistically or biologically

noteworthy changes in incidences of neoplasms and/or

nonneoplastic lesions of the liver. Nonneoplastic lesions

of the pituitary gland, thyroid tissue, and kidney are also

discussed. Summaries of incidences of neoplasms by

replicate are presented in Appendix C.

No potentially treatment-related neoplasms were

observed in male or female guppies exposed to

nitromethane and evaluated at 9 months.

In the core study, a number of lesions occurred with

greater incidences in the 10 and/or 30 mg/L groups but

not in the 70 mg/L groups, as compared with controls.

This may have been due, at least in part, to the reduced

survival in the 70 mg/L groups, particularly the males. 

Liver (Core Study): The incidence of bile duct hyperplasia in the 30 mg/L male group was increased

(Tables 18 and C1). The incidence of granuloma was

greater in the 30 mg/L female group than in the controls.

Granulomas were focal to multifocal, discrete, variably

sized, nodular lesions composed of densely packed

macrophages with lightly pigmented cytoplasm and generally surrounded by a periphery of small cells with

deeply basophilic nuclei. There was a marginally

increased incidence of hepatocellular adenoma or carcinoma (combined) in 30 mg/L males, compared to the

controls.

Liver (Stop-Exposure Study): A single hepatocholangiocarcinoma, a malignant liver neoplasm consisting of

both malignant cholangiocellular (bile duct) and hepatocellular components, occurred in one female in the

10 mg/L group (Table 18).
52 Fish Studies, NTP TR 528

TABLE 18

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Guppies 

in the 16-Month Waterborne Study of Nitromethane

Trend

0 mg/L 10 mg/L 30 mg/L 70 mg/La Test

P-Valueb

Male 

Core Study

Number Examined Microscopically 60 42 60 12

Bile Duct, Hyperplasiac 3 6 13** 3 0.0046

Cholangiocarcinoma 0 1 0 0 0.3883

Hepatocellular Adenoma, Multiple 0 1 1 0 0.4285

Hepatocellular Adenoma (includes multiple) 4 2 8 0 0.0728

Hepatocellular Carcinoma 0 1 2 0 0.1561

Hepatocellular Adenoma or Carcinoma 4 3 10 0 0.0287

Stop-Exposure Study 

Number Examined Microscopically 29 29 29 14

Basophilic Focus 1 1 2 0 0.2470

Eosinophilic Focus 0 2 2 2 0.1369

Cholangioma 0 0 1 0 0.3333

Hepatocellular Adenoma (includes multiple) 5 2 1 0 0.0459N

Hepatocellular Carcinoma 0 3 0 0 0.3188

Hepatocellular Adenoma or Carcinoma 5 5 1 0 0.0423N

Female 

Core Study 

Number Examined Microscopically 47 57 50 32

Bile Duct, Hyperplasia 4 5 8 6 0.0972

Basophilic Focus 1 6 4 1 0.2046

Granuloma 13 24 23* 7 0.0470

Cholangiocarcinoma 0 1 0 0 0.3885

Hepatocellular Adenoma 1 2 2 0 0.3166

Stop-Exposure Study

Number Examined Microscopically 30 34 33 25

Hepatocellular Adenoma (includes multiple) 2 0 0 0 0.0934N

Hepatocellular Carcinoma 0 0 1 0 0.3402

Hepatocellular Adenoma or Carcinoma 2 0 1 0 0.2989

Hepatocholangiocarcinoma 0 1 0 0 0.3816

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a 70 mg/L group was not included in trend test or Fisher exact test because the fish were sacrificed earlier than the controls and other 

exposed groups b Cochran-Armitage trend test or exact permutation test (Plackett, 1981) (excludes 70 mg/L group). A negative trend or a lower incidence 

in an exposure group is indicated by N. c Number of animals with lesion
Fish Studies, NTP TR 528 53

Pituitary Gland (Core Study): The incidence of hyperplasia was increased in 30 mg/L females (0 mg/L, 1/47;

10 mg/L, 2/57; 30 mg/L, 14/50; 70 mg/L, 4/32).

Hyperplasia was characterized by a mild to moderate,

diffuse enlargement of the anterior or posterior portion

of the gland or both portions. The normal architecture of

the gland was maintained and the cells of the anterior

and posterior portions appeared normal.

Pituitary Gland (Stop-Exposure Study): The incidences

of hyperplasia were not increased in females (1/30, 3/34,

2/33, 1/25).

Thyroid Tissue (Core Study): Incidences of follicular

cell hyperplasia were greater in the 10 and 30 mg/L male

groups (0/60, 5/42, 7/60, and 0/12) and were substantially greater in all exposed female groups (4/47, 43/57,

38/50, 23/32) than those in the control groups. Thyroid

tissue hyperplasia was characterized by a diffuse

increase in the number of thyroid follicles that were

more widely scattered than in normal thyroid tissue.

Thyroid Tissue (Stop-Exposure Study): The incidence of

follicular cell hyperplasia was greater in the 30 mg/L

female group than in any other group, but the difference

was not statistically significant (0 mg/L, 4/30; 10 mg/L,

3/34; 30 mg/L, 9/33; 70 mg/L, 4/25). 

Kidney (Core Study): Renal tubule dilatation occurred

with greater incidences in 30 mg/L males (18/60, 13/42,

32/60, 6/12) and 30 mg/L females (27/47, 38/57, 38/50,

20/32) than in the controls. Renal tubule dilatation was

characterized by varying degrees of enlargement of the

renal tubular lumen up to several times normal diameter.

Mineralization occurred with greater incidences in

70 mg/L females (22/47, 33/57, 31/50, 22/32) and consisted of scattered irregular bits of basophilic material

within the kidney stroma and/or renal tubule lumens.
54 Fish Studies, NTP TR 528

TABLE 19

Survival of Medaka in the 13-Month Waterborne Study of Nitromethane

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Animals initially in study 340 340 340 340

Died before the 9-month interim evaluation 37d 28e 25f 48g

Unaccounted fora 26 29 49 28

9-Month interim evaluation 20 20 20 20

Died after reallocationb

Core study 17 19 27 14

Stop-exposure study 5 2 5 2

Survivors

Core study 143 156 136 143

Stop-exposure study 92 86 78 85

Survival analysisc

Core study 0.5414 0.2079N 0.5621 0.3430

Stop-exposure study 0.4885 0.1019 0.0627 0.3409

a The laboratory report did not account for these fish at 9 months. b At 9 months, approximately one third of the fish in each replicate was reassigned to stop-exposure aquaria; the remaining fish (core study)

were returned to their original exposure aquaria. c The result of life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparison (Cox, 1972)

between the controls and exposed animals appear in the exposure concentration columns. d Includes 3 accidental deaths e Includes 5 accidental deaths f Includes 6 accidental deaths

g Includes 19 accidental deaths

13-MONTH STUDY IN MEDAKA

Survival

To maintain acceptable water quality and treatment concentrations potentially affected by the rapid microfaunal

growth, the study aquaria were brushed once and

siphoned three times each day. Due to this frequent

activity, a number of fish probably died due to mechanical injury. Unfortunately, the cause of death could not be

confirmed in many cases; the mortality from this activity is believed to have been approximately uniform

among treatments and does not appear to have affected

the comparison of survival between treatments

(Table 19).

The effect of this aggressive maintenance can be demonstrated in the frequency of dead removed from core

study aquaria after 9 months compared to the frequency

in stop-exposure aquaria during the same period

(Table 19). The holding aquaria, which received dilution water without nitromethane, were brushed only

once each week and siphoned of debris twice per week.
Fish Studies, NTP TR 528 55

Exposure Concentrations, Body Lengths,

and Body Weights

Nominal exposure concentrations of 10, 20, and

40 mg/L resulted in actual exposure concentrations of

9.3, 20.8, and 41.7 mg/L nitromethane, respectively.

No differences between control and exposed groups

were found in body lengths or weights at the 9-month 

interim evaluation. Significant increases in length and

weight were observed in the exposed groups at

13 months. The biological significance of these changes

is unknown (Tables 20 and 21).

TABLE 20

Body Lengths of Medaka in the 13-Month Waterborne Study of Nitromethanea

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 25.0 ± 0.7 26.7 ± 0.2 26.9 ± 0.1

10 mg/L 25.9 ± 0.5 26.3 ± 0.1 27.5 ± 0.2*

20 mg/L 25.8 ± 0.5 27.5 ± 0.2** 27.8 ± 0.2**

40 mg/L 26.9 ± 0.6 27.9 ± 0.2** 27.7 ± 0.2**

* Significantly different (P#0.05) from the control group by Dunnett’s test

** P#0.01 a Data are presented as mean length ± standard error (mm).

TABLE 21

Body Weights of Medaka in the 13-Month Waterborne Study of Nitromethanea

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 0.25 ± 0.02 0.32 ± 0.01 0.30 ± 0.01

10 mg/L 0.29 ± 0.02 0.29 ± 0.01* 0.32 ± 0.01

20 mg/L 0.28 ± 0.01 0.34 ± 0.01 0.32 ± 0.01

40 mg/L 0.30 ± 0.02 0.36 ± 0.01* 0.33 ± 0.01

* Significantly different (P#0.05) from the control group by Dunnett’s test a Data are presented as mean length ± standard error (mm).
56 Fish Studies, NTP TR 528

Pathology and Statistical Analyses

This section describes the statistically or biologically

noteworthy changes in incidences of neoplasms and/or

nonneoplastic lesions of the liver, kidney, and thyroid

tissue. Summaries of incidences of neoplasms by replicate are presented in Appendix D.

Liver (9-Month Interim Evaluation): A single cholangiocarcinoma occurred in a 40 mg/L male (Tables 22

and D1). Hepatocellular adenomas occurred in two

20 mg/L males and in one 40 mg/L female (Tables 22,

D1, and D3). Otherwise, no potentially exposure-related

neoplasms were observed. 

Liver (Core Study): One cholangioma occurred in a

20 mg/L male, and cholangiocarcinomas occurred in a

few exposed males, but none occurred in control males

(Tables 22 and D1). Microscopically, the hepatocellular

adenomas, cholangioma, and cholangiocarcinomas were

similar to those previously described.

The incidences of cysts were greater in 20 mg/L males

and cystic degeneration occurred with greater incidence

in the 40 mg/L female group than in controls.

Incidences of hepatocellular vacuolization were greater

in 40 mg/L males and in 20 and 40 mg/L females than in

controls. The incidences of macrophage aggregates

were increased in 10 and 40 mg/L males. Incidences of

granuloma were decreased in all exposed groups of

males and in 10 and 40 mg/L females compared with

controls.

Cystic degeneration was characterized by clusters of

numerous small, cyst-like cavities separated by fine

cytoplasmic extensions of perisinusoidal cells. The

small cavities contained fine flocculant material and

commonly a few inflammatory cells. The other liver

lesions appeared microscopically similar to those previously described.

Liver (Stop-Exposure Study): Cysts occurred with

somewhat greater incidences in 20 and 40 mg/L females

as compared with controls (Table 22). The incidences of

granuloma were decreased in 10 and 40 mg/L males and

females.
Fish Studies, NTP TR 528 57

TABLE 22

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Medaka 

in the 13-Month Waterborne Study of Nitromethane

Trend

0 mg/L 10 mg/L 20 mg/L 40 mg/L Test

P-Valuea

Male 

9-Month Interim Evaluation

Number Examined Microscopically 12 15 14 14

Cholangiocarcinomab 0 0 0 1 0.2546

Hepatocellular Adenoma 0 0 2 0 0.4216

Core Study 

Number Examined Microscopically 72 91 90 76

Cyst 12 14 27* 19 0.0439

Hepatocellular Vacuolation 5 4 9 15* 0.0008

Macrophage Aggregates 22 45* 34 41** 0.0119

Vacuolated Hepatocyte Focus 1 1 1 4 0.0342

Granuloma 64 19** 49** 10** <0.0001N

Cholangioma 0 0 1 0 0.4293

Cholangiocarcinoma 0 1 2 1 0.2381

Cholangioma or Cholangiocarcinoma 0 1 3 1 0.2363

Hepatocellular Adenoma 1 2 1 1 0.4072

Hemangiopericytoma 0 0 0 1 0.2310

Lymphosarcoma 0 0 1 0 0.4293

Stop-Exposure Study

Number Examined Microscopically 47 35 36 41

Cystic Degeneration 32 23 25 23 0.1263

Granuloma 25 10* 23 11* 0.0330N

Female 

9-Month Interim Evaluation

Number Examined Microscopically 13 15 9 20

Hepatocellular Adenoma 0 0 0 1 0.3509

Core Study 

Number Examined Microscopically 74 69 59 70

Cystic Degeneration 50 50 44 58* 0.0168

Hepatocellular Vacuolation 7 9 15* 31** <0.0001

Macrophage Aggregates 40 42 31 46 0.1112

Vacuolated Hepatocyte Focus 12 10 7 16 0.1235

Granuloma 25 6** 14 5** 0.0005N

Eosinophilic Focus 1 0 0 0

Cholangiocarcinoma 1 0 0 0 0.1279N

Hepatocellular Adenoma 2 2 1 3 0.2957

Hepatocellular Carcinoma 0 0 0 1 0.2574

Hepatocellular Adenoma or Carcinoma 2 2 1 4 0.1542
58 Fish Studies, NTP TR 528

TABLE 22

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Medaka 

in the 13-Month Waterborne Study of Nitromethane

Trend

0 mg/L 10 mg/L 20 mg/L 40 mg/L Test

P-Value

Female (continued)

Stop-Exposure Study

Number Examined Microscopically 44 51 42 44

Cyst 20 29 28* 35** 0.0003

Granuloma 8 2* 7 1* 0.0320N

Cholangiocarcinoma 0 1 0 0 0.3115

Hepatocellular Adenoma 1 2 1 1 0.4274

Hepatocellular Carcinoma 0 1 0 0 0.3115

Hepatocellular Adenoma or Carcinoma 1 3 1 1 0.3553

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981). A negative trend or a lower incidence 

in an exposure group is indicated by N. b Number of animals with lesion
Fish Studies, NTP TR 528 59

TABLE 23

Incidences of Nonneoplastic Lesions of the Kidney in Medaka 

in the 13-Month Waterborne Study of Nitromethane

Trend

0 mg/L 10 mg/L 20 mg/L 40 mg/L Test

P-Valuea

Male 

Core Study

Number Examined Microscopically 72 91 90 76

Castsb 11 26* 30** 32** 0.0003

Mineralization 10 50** 26* 24** 0.3178

Renal Tubule, Dilatation 10 34** 31** 37** <0.0001

Renal Tubule, Degeneration 7 20* 19* 12 0.3183

Stop-Exposure Study

Number Examined Microscopically 47 35 36 41

Casts 6 2 8 11 0.0138

Mineralization 13 8 17 16 0.0635

Female 

Stop-Exposure Study

Number Examined Microscopically 44 51 42 44

Renal Tubule, Dilatation 1 6 3 6 0.0642

Mineralization 1 5 2 3 0.3279

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion

Kidney (Core Study): A number of nonneoplastic

changes occurred with greater incidences in all exposed

groups of males than in the controls (Table 23). These

changes included casts, mineralization, and renal tubule

dilatation. Renal tubule degeneration occurred with

greater incidence in 10 and 20 mg/L males than in the

controls. These changes appeared microscopically similar to those previously described.
60 Fish Studies, NTP TR 528

TABLE 24

Incidences of Nonneoplastic Lesions of the Thyroid in Male Medaka 

in the 13-Month Waterborne Study of Nitromethane

Trend

0 mg/L 10 mg/L 20 mg/L 40 mg/L Test

P-Valuea

Core Study

Number Examined Microscopically 72 91 90 76

Follicular Cells, Hypertrophyb 18 47** 51** 33* 0.0540

Follicular Cells, Vacuolation 16 71** 57** 63** <0.0001

Increased Basophilia 67 82 78 56** 0.0001N

Stop-Exposure Study

Number Examined Microscopically 47 35 36 41

Follicular Cells, Vacuolation 6 11* 10 14* 0.0214

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981). A negative trend is indicated by N. b Number of animals with lesion

Thyroid Tissue (Core Study): Incidences of hypertrophy

and vacuolation of follicular cells were substantially

greater in all exposed groups of males than those in controls (Table 24). The incidence of increased basophilia

in the 40 mg/L male group was less than in controls.

Hypertrophy consisted of slight enlargement of the follicular cells as compared with normal cells. Vacuolation

was characterized by clear vacuolation of follicular cell

cytoplasm. Increased basophilia was characterized by

increased basophilic staining of follicular cells. The significance of increased basophilia was unclear but may

have represented normal anatomic variation.

Thyroid Tissue (Stop-Exposure Study): Follicular cell

vacuolation occurred with greater incidences in 10 and

40 mg/L males as compared with controls (Table 24).
Fish Studies, NTP TR 528 61

1,2,3-TRICHLOROPROPANE

16-MONTH STUDY IN GUPPIES

Survival

The survival of exposed guppies was less than that of the

control group at 9 months. Reduced survival was evident at 6 months in the 18.0 mg/L groups, and at

7 months in the 4.5 and 9.0 mg/L groups (data not

shown). Survival was significantly reduced in the

18.0 mg/L core study group within one month of the

9-month interim evaluation (data not shown), and mortality in this group after reallocation at 9 months was

42.6% at study termination (Table 25).

Exposure Concentrations, Body Lengths, 

and Body Weights

Nominal exposure concentrations of 4.5, 9.0, and

18.0 mg/L (analyzed three times per week) resulted 

in actual exposure concentrations of 4.4, 8.8, and

18.2 mg/L 1,2,3-trichloropropane, respectively. Guppies in the 18.0 mg/L core study group were

significantly longer and weighed more than the controls.

Fish in the 18.0 mg/L stop-exposure group also weighed

more than the controls (Tables 26 and 27).

Mortality of fish during the study resulted in unequal

numbers of individuals distributed to core study and

stop-exposure aquaria at 9 months (Table 25). This

appears to have influenced the length and weight of fish

measured at study termination (i.e., the smaller tank

population allowed the fish to grow more). Observed

differences in weight and length between controls and

18.0 mg/L fish may have been an artifact of the reduced

fish numbers in the 18.0 mg/L aquaria. 

TABLE 25

Survival of Guppies in the 16-Month Waterborne Study of 1,2,3-Trichloropropane

0 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L

Animals initially in study 220 220 220 220

Died before the 9-month interim evaluation 3 14 17 32

Unaccounted fora 1 217

9-Month interim evaluation 20 20 20 20

Died after reallocationb

Core study 5 10 7 46

Stop-exposure study 3 13 3 9

Survivors

Core study 125 112 114 61

Stop-exposure study 63 49 58 45

Survival analysisc

Core study <0.0001 0.0047 0.0019 <0.0001

Stop-exposure study <0.0001 0.0015 0.0026 <0.0001

a The laboratory report did not account for these fish at 9 months. b At 9 months, approximately one third of the fish in each replicate was reassigned to stop-exposure aquaria; the remaining fish (core study)

were returned to their original exposure aquaria. c The result of life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparison (Cox, 1972)

between the controls and exposed animals appear in the exposure concentration columns.
62 Fish Studies, NTP TR 528

TABLE 26

Standard Lengths of Guppies in the 16-Month Waterborne Study of 1,2,3-Trichloropropanea

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 30.6 ± 1.0 31.1 ± 0.5 30.3 ± 0.7 

4.5 mg/L 30.2 ± 1.1 32.5 ± 0.6 32.1 ± 0.9

9.0 mg/L 29.8 ± 1.3 31.3 ± 0.7 32.5 ± 0.1 

18.0 mg/L 31.6 ± 1.6 33.9 ± 1.0* 32.8 ± 1.2

* Significantly different (P#0.05) from the control group by Dunnett’s test a Data are presented as mean length ± standard error (mm).

TABLE 27

Standard Weights of Guppies in the 16-Month Waterborne Study of 1,2,3-Trichloropropanea

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 0.69 ± 0.09 0.78 ± 0.04 0.68 ± 0.05

4.5 mg/L 0.67 ± 0.08 0.90 ± 0.05 0.86 ± 0.06

9.0 mg/L 0.67 ± 0.09 0.90 ± 0.06 0.87 ± 0.07

18.0 mg/L 0.78 ± 0.11 1.13 ± 0.09** 1.00 ± 0.09**

* Significantly different (P#0.05) from the control group by Dunnett’s test

** P#0.01 a Data are presented as mean length ± standard error (mm).
Fish Studies, NTP TR 528 63

Pathology and Statistical Analyses

This section describes the statistically or biologically

noteworthy changes in neoplasms and/or nonneoplastic

lesions of the liver, gallbladder, kidney, and pituitary

gland. Summaries of incidences of neoplasms by replicate are presented in Appendix E.

Liver (9-Month Interim Evaluation): Multiple hepatocellular adenomas occurred in one 4.5 mg/L male, and

one hepatocellular adenoma occurred in a control male

(Table 28). No other hepatocellular neoplasms were

observed; consequently, the hepatocellular adenomas

seen in the one exposed male were considered to be incidental and unrelated to exposure. Liver neoplasms were

not found in females at 9 months.

Liver (Core Study): Increased incidences of cholangiocellular (bile duct) and hepatocellular neoplasms were

observed in 18.0 mg/L males and 9.0 and 18.0 mg/L

females as compared with controls (Table 28).

Cholangiocarcinomas in 18.0 mg/L females metastasized to intestine, mesentery, and spleen. Combined

incidences of hepatocellular adenoma and multiple hepatocellular adenoma were increased in exposed groups of

males as compared with controls. In addition, hepatocellular carcinoma occurred with greater incidence in

18.0 mg/L males than in the controls. Microscopically,

these liver lesions appeared similar to those previously

described.

Incidences of basophilic focus were increased in 9.0 and

18.0 mg/L males as compared with controls. The incidences of granuloma and cystic degeneration in exposed

males, and granuloma in 18.0 mg/L females were less

than those in the controls. Mononuclear cell infiltrate

consisted of multiple, small, scattered aggregates of

mixed mononuclear inflammatory cells, generally

macrophages and lymphocytes. The microscopic

appearances of granuloma and cystic generation were

similar to those described previously. The decreased

incidences of granuloma, mononuclear cell infiltrate,

and cystic degeneration may have been secondary to the

presence of other exposure-related lesions that obscured

the presence of these lesions.

Liver (Stop-Exposure Study): Increased incidences of

cholangiocellular (bile duct) neoplasms were observed

in 18.0 mg/L females, and increased incidences of hepatocellular neoplasms occurred in 18.0 mg/L males

(Table 28).

Cholangiomas occurred in a few exposed females,

including controls. However, a few cholangiocarcinomas occurred in the 18.0 mg/L females only, and the

combined incidence of cholangioma and cholangiocarcinoma was greater in the 18.0 mg/L females than in the

controls. One of the cholangiocarcinomas in females

metastasized to the peritoneum. A single cholangiocarcinoma occurred in a 18.0 mg/L male. Incidences of bile

duct hyperplasia were slightly increased in the 9.0 mg/L

males as compared with controls. 

Hepatocellular adenoma occurred with a greater incidence in 18.0 mg/L males, and multiple hepatocellular

adenoma and hepatocellular carcinoma occurred with

greater incidence in 18.0 mg/L males compared to controls. Incidences of hepatocellular neoplasms in females

were similar across the control and exposed groups.

Basophilic focus occurred with slightly greater incidences in exposed groups of males and females as compared with controls. Eosinophilic focus occurred in a

small number of exposed males but not in control males.

Incidences in females were similar across groups.

Microscopically, eosinophilic foci appeared similar to

those previously described.
64 Fish Studies, NTP TR 528

TABLE 28

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Guppies 

in the 16-Month Waterborne Study of 1,2,3-Trichloropropane

Trend

0 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L Test

P-Valuea

Male 

9-Month Interim Evaluation

Number Examined Microscopically 9 8 12 10

Hepatocellular Adenoma, Multipleb 0 1 0 0 0.2785

Hepatocellular Adenoma 1 0 0 0 0.2308

Core Study

Number Examined Microscopically 61 47 67 27

Basophilic Focus 2 4 14** 6** 0.0011

Cystic Degeneration 43 22* 24** 9** <0.0001N

Granuloma 57 34** 44** 11** <0.0001N

Cholangioma 0 0 1 0 0.3285

Cholangiocarcinoma 0 0 0 3* 0.0022

Cholangioma or Cholangiocarcinoma 0 0 1 3* 0.0032

Hepatocellular Adenoma, Multiple 1 1 5 4** 0.0033

Hepatocellular Adenoma (includes multiple) 3 8* 17** 9** 0.0002

Hepatocellular Carcinoma 0 1 0 7** <0.0001

Hepatocellular Adenoma or Carcinoma 3 9* 17** 15** <0.0001

Hepatocholangiocarcinoma 0 0 3 2 0.0079

Stop-Exposure Study

Number Examined Microscopically 32 19 26 19

Bile Duct, Hyperplasia 3 2 8* 5 0.0299

Basophilic Focus 1 2 3 4 0.0277

Eosinophilic Focus 0 1 2 2 0.0453

Cholangiocarcinoma 0 0 0 1 0.1979

Hepatocellular Adenoma, Multiple 0 0 1 2 0.0311

Hepatocellular Adenoma (includes multiple) 3 4 5 7* 0.0113

Hepatocellular Carcinoma 0 0 1 4* 0.0018

Hepatocellular Adenoma or Carcinoma 3 4 5 10** 0.0003

Female 

Core Study

Number Examined Microscopically 64 64 47 33

Basophilic Focus 5 10 9 4 0.2266

Granuloma 29 27 16 8* 0.0147N

Bile Duct, Hyperplasia 7 9 10 6 0.1153

Mononuclear Cell, Infiltrate

Multifocal 21 23 17 5* 0.0480N

Cholangioma, Multiple 0 0 1 1 0.3846

Cholangioma (includes multiple) 0 3 4* 2 0.0509

Cholangiocarcinoma 0 0 0 6** <0.0001

Cholangioma or Cholangiocarcinoma 0 3 4* 8** <0.0001

Hepatocellular Adenoma, Multiple 0 0 1 2 0.0168

Hepatocellular Adenoma (includes multiple) 5 3 4 7 0.0125

Hepatocellular Carcinoma 0 1 0 2 0.0186

Hepatocellular Adenoma or Carcinoma 5 4 4 8* 0.0059

Hepatocholangiocarcinoma 0 0 0 2 0.0245
Fish Studies, NTP TR 528 65

TABLE 28

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Guppies 

in the 16-Month Waterborne Study of 1,2,3-Trichloropropane

Trend

0 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L Test

P-Value

Female (continued)

Stop-Exposure Study 

Number Examined Microscopically 31 31 31 27

Basophilic Focus 1 4 2 4 0.1110

Eosinophilic Focus, Multiple 0 0 0 2

Eosinophilic Focus, One 2 1 2 1

Granuloma 19 19 12 11 0.0275N

Carcinoma, Invasive 0 1 0 0 0.3194

Cholangioma 2 2 0 2 0.4942

Cholangiocarcinoma 0 0 0 5* 0.0004

Cholangioma or Cholangiocarcinoma 2 2 0 7* 0.0056

Hepatocellular Adenoma 2 0 2 3 0.1245

Hepatocellular Carcinoma 0 1 0 0 0.3194

Hepatocellular Adenoma or Carcinoma 2 1 2 3 0.1808

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981). A negative trend or a lower incidence 

in an exposure group is indicated by N. b Number of animals with lesion
66 Fish Studies, NTP TR 528

TABLE 29

Incidences of Neoplasms and Nonneoplastic Lesions of the Gallbladder in Guppies

in the 16-Month Waterborne Study of 1,2,3-Trichloropropane 

Trend

0 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L Test

P-Valuea

Males

Core Study

Number Examined Microscopically 59 44 59 24

Epithelium, Hyperplasiab 2 0 1 4 0.0064

Concretion 0 0 1 5** <0.0001

Carcinoma 0 0 0 1 0.1290

Females

Core Study

Number Examined Microscopically 61 60 44 30

Epithelium, Hyperplasia 2 2 1 3 0.0880

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion

Gallbladder (Core Study): Carcinoma occurred in one

18.0 mg/L male (Tables 29 and E1). 

Gallbladder (Stop-Exposure Study): A single carcinoma

occurred in a 4.5 mg/L female (Table E3).
Fish Studies, NTP TR 528 67

Kidney (Core Study): A number of lesions occurred

with greater incidences in exposed groups of males and

females (Table 30). These included Bowman’s capsule

hyperplasia; dilatation and hemorrhage in Bowman’s

space; glomerular dilatation, hyalinization (primarily in

males), and proliferation; mineralization; casts; and

renal tubule dilatation, degeneration, hyperplasia, and

hypertrophy. In addition, renal tubule peritubular proliferation was seen in a few exposed males and females.

The normal glomerulus was approximately twice the

diameter of a normal tubule and consisted of a few capillary loops with a small amount of mesangium composed of a few mesangial cells and scant mesangial

matrix. The glomerulus was surrounded by a narrow

clear Bowman’s space outside of which was a thin

Bowman’s capsule composed of a single layer of flattened epithelial cells lying upon a thin connective tissue

layer. Hyperplasia of Bowman’s capsule was characterized by increased numbers of epithelial cells, many of

which were cuboidal, as opposed to the normal flattened

cells, with proliferation of the underlying connective tissue leading to slight to marked thickening of the connective tissue layer. Dilatation of Bowman’s space consisted of a pronounced increase in the size of Bowman’s

space sometimes up to many times normal size.

Hemorrhage in Bowman’s space consisted of free blood

filling a usually dilated Bowman’s space. Glomerular

dilatation was characterized by prominent, mild to

marked dilatation of one or more glomerular capillary

loops producing variably sized, cyst-like spaces filled

with lightly eosinophilic, granular material (presumably

plasma) and a few blood cells, with a subsequent

enlargement of the glomerulus. Glomerular hyalinization was characterized by the filling of one or more capillary loops with homogeneous eosinophilic material that

effaced the normal structure. Glomerular proliferation

consisted of an increase in the number of glomerular

capillary loops and proliferation of mesangial cells with

a mild to marked increase in the amount of mesangial

matrix resulting in variable degrees of glomerular

enlargement. Mineralization consisted of scattered

irregular bits of basophilic material within the kidney

stroma and/or tubular lumens. Casts consisted of aggregates of amphophilic to basophilic, granular material

within the lumens of scattered tubules. Renal tubule

dilatation was characterized by varying degrees of

enlargement of the renal tubular lumen up to several

times normal diameter. Renal tubule degeneration was

characterized by irregular shrinkage and/or varying

degrees of flattening of the tubular epithelium.

Degeneration was often seen within dilated tubules.

Renal tubule hyperplasia consisted of a focal to multifocal to diffuse increase in the number of epithelial cells

in the renal tubular epithelial layer, sometimes producing

some degree of thickening of the tubular epithelium.

Renal tubule hypertrophy was characterized by an

increase in the size of renal tubular epithelial cells.

Renal tubule peritubular proliferation consisted of proliferation of the stromal elements surrounding the tubule.

Kidney (Stop-Exposure Study): Incidences of renal

tubule dilation were increased in 4.5 and 18.0 mg/L

males and in the 9.0 and 18.0 mg/L female groups

(Table 30). Incidences of mineralization and casts in

exposed females were increased compared to controls.

Pituitary Gland (Core Study): The incidences of hyperplasia were increased in 9.0 and 18.0 mg/L females

(0 mg/L, 0/64; 4.5 mg/L, 0/64; 9.0 mg/L, 7/47;

18.0 mg/L, 16/33), and the incidence was highest in the

18.0 mg/L group. Hyperplasia was not present in any

controls. A single adenoma was present in a 9.0 mg/L

female (Table E3). The normal pituitary gland was a relatively small structure consisting of an anterior portion

and a posterior portion. Hyperplasia was characterized

by a mild to moderate, diffuse enlargement of the anterior portion or the posterior portion or both. The normal

architecture of the gland was maintained and the cells of

the anterior and posterior portions appeared normal.
68 Fish Studies, NTP TR 528

TABLE 30

Incidences of Nonneoplastic Lesions of the Kidney in Guppies 

in the 16-Month Waterborne Study of 1,2,3-Trichloropropane

Trend

0 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L Test

P-Valuea

Male 

Core Study

Number Examined Microscopically 61 47 67 27

Castb 46 35 60* 24 0.0172

Mineralization 21 30** 58** 26** <0.0001

Bowman’s Capsule, Hyperplasia 1 5 10** 6** 0.0010

Bowman’s Space, Dilatation 7 11 19* 19** <0.0001

Bowman’s Space, Hemorrhage 1 0 1 7** <0.0001

Glomerulus, Dilatation 1 0 2 6** <0.0001

Glomerulus, Hyalinization 0 3 2 2 0.0678

Glomerulus, Proliferation 0 0 2 4** 0.0001

Renal Tubule, Degeneration 9 5 6 15** <0.0001

Renal Tubule, Hyperplasia 1 4 7* 4* 0.0121

Renal Tubule, Hypertrophy 0 1 5* 0 0.2179

Renal Tubule, Peritubular Proliferation 0 1 2 2 0.0197

Renal Tubule, Dilatation 32 36** 61** 26** <0.0001

Stop-Exposure Study

Number Examined Microscopically 32 19 26 19

Cast 24 16 17 18 0.1116

Mineralization 19 14 20 19** 0.0007

Bowman’s Capsule, Hyperplasia 0 0 1 2 0.0144

Bowman’s Space, Dilatation 2 5 3 6* 0.0237

Glomerulus, Dilatation 0 0 0 1 0.0435

Renal Tubule, Hyperplasia 1 1 1 4 0.0125

Renal Tubule, Dilatation 14 14* 13 14* 0.0469

Female

Core Study

Number Examined Microscopically 64 64 47 33

Cast 35 40 34* 33** <0.0001

Mineralization 30 53** 40** 30** <0.0001

Bowman’s Capsule, Hyperplasia 0 2 3 10** <0.0001

Bowman’s Space, Dilatation 20 32* 29** 25** <0.0001

Bowman’s Space, Hemorrhage 2 4 6 11** <0.0001

Glomerulus, Dilatation 0 1 5* 12** <0.0001

Glomerulus, Hyalinization 0 1 0 1 0.1212

Glomerulus, Proliferation 0 0 1 3* 0.0008

Renal Tubule, Degeneration 2 5 7* 19** <0.0001

Renal Tubule, Hyperplasia 1 2 4 3 0.0260

Renal Tubule, Hypertrophy 0 1 2 2 0.0211

Renal Tubule, Peritubular Proliferation 0 1 1 1 0.1114

Renal Tubule, Dilatation 40 45 44** 33** <0.0001
Fish Studies, NTP TR 528 69

TABLE 30

Incidences of Nonneoplastic Lesions of the Kidney in Guppies 

in the 16-Month Waterborne Study of 1,2,3-Trichloropropane

Trend

0 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L Test

P-Value

Female (continued)

Stop-Exposure Study

Number Examined Microscopically 31 31 31 27

Cast 12 17 17 19* 0.0107

Mineralization 17 23 25* 26** 0.0001

Bowman’s Capsule, Hyperplasia 0 0 0 2 0.0108

Bowman’s Space, Dilatation 5 12* 10 19** <0.0001

Glomerulus, Dilatation 0 0 0 2 0.0108

Renal Tubule, Hyperplasia 2 0 0 3 0.1258

Renal Tubule, Dilatation 15 19 27** 22** 0.0013

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion
70 Fish Studies, NTP TR 528

TABLE 31

Survival of Medaka in the 13-Month Waterborne Study of 1,2,3-Trichloropropane

0 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L

Animals initially in study 340 340 340 340

Died before the 9-month interim evaluation 11 6 18 26

Unaccounted fora 3 463

9-Month interim evaluation 20 20 20 20

Died after reallocationb

Core study 11 18 35 51

Stop-exposure study 4 11 14 18

Survivors

Core study 192 188 161 143

Stop-exposure study 99 93 86 79

Survival analysisc

Core study <0.0001 0.7867 0.0004 <0.0001

Stop-exposure study <0.0001 0.7274 0.0232 <0.0001

a The laboratory report did not account for these fish at 9 months. b At 9 months, approximately one third of the fish in each replicate was reassigned to stop-exposure aquaria; the remaining fish (core study)

were returned to their original exposure aquaria. c The result of life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparison (Cox, 1972)

between the controls and exposed animals appear in the exposure concentration columns.

13-MONTH STUDY IN MEDAKA

Survival 

Survival in the 9.0 and 18.0 mg/L groups indicated that

1,2,3-trichloropropane was chronically toxic to the

medaka; reduced survival was evident beginning at

9 months of exposure (Table 31). Mortality in the

18.0 mg/L core study group was 26.3% and mortality in

the 9.0 mg/L group was 17.3% between 9 months and

study termination at 13 months. Survival was also

reduced in stop-exposure fish from the 9.0 and

18.0 mg/L groups at the end of the study.

Exposure Concentrations, Body Lengths, and

Body Weights

Nominal exposure concentrations of 4.5, 9.0, and

18.0 mg/L resulted in actual exposure concentrations of 

4.6, 9.2, and 18.0 mg/L 1,2,3-trichloropropane, respectively. Core study medaka in the 18.0 mg/L group were

both longer and weighed more than the controls at the

end of the study (Tables 32 and 33). At 9 months, the

lengths and weights of medaka in the 9.0 and 18.0 mg/L

groups were significantly increased (Table 33).

Mortality of fish during the study resulted in unequal

numbers of individuals distributed to core study and

stop-exposure aquaria at 9 months (Table 31). This

appears to have influenced the length and weight of fish

measured at study termination (i.e., the smaller tank population allowed the fish to grow more). Observed differences in weight and/or length between controls and

9.0 or 18.0 mg/L fish may have been an artifact of the

reduced numbers of fish in these exposure aquaria.
Fish Studies, NTP TR 528 71

TABLE 32

Body Lengths of Medaka in the 13-Month Waterborne Study of 1,2,3-Trichloropropanea

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 22.9 ± 0.4 24.0 ± 0.4 26.0 ± 0.5

4.5 mg/L 23.8 ± 0.4 24.6 ± 0.4 25.4 ± 0.3

9.0 mg/L 24.5 ± 0.6* 25.2 ± 0.4 25.9 ± 0.4

18.0 mg/L 24.6 ± 0.4* 26.5 ± 0.4** 26.2 ± 0.3

* Significantly different (P#0.05) from the control group by Dunnett’s test

** P#0.01 a Data are presented as mean length ± standard error (mm).

TABLE 33

Body Weights of Medaka in the 13-Month Waterborne Study of 1,2,3-Trichloropropanea

9-Month 

Treatment Interim Evaluation Core Study Stop-Exposure Study

0 mg/L 0.183 ± 0.012 0.220 ± 0.010 0.253 ± 0.011

4.5 mg/L 0.208 ± 0.013 0.227 ± 0.013 0.229 ± 0.008

9.0 mg/L 0.248 ± 0.019* 0.257 ± 0.013 0.246 ± 0.012

18.0 mg/L 0.241 ± 0.019* 0.264 ± 0.012* 0.241 ± 0.009

* Significantly different (P#0.05) from the control group by Dunnett’s test a Data are presented as mean length ± standard error (g).
72 Fish Studies, NTP TR 528

Pathology and Statistical Analyses

This section describes the statistically or biologically

noteworthy changes in incidences of neoplasms and/or

nonneoplastic lesions of the liver, gallbladder, kidney,

and thyroid tissue. Summaries of incidences of neoplasms by replicate are presented in Appendix F.

Liver (9-Month Interim Evaluation): The incidences of

cholangiocarcinomas in 9.0 and 18.0 mg/L males were

significantly increased at 9 months (Tables 34 and F1).

Small numbers of cholangiocarcinomas occurred with

similar incidences in each of the exposed female groups,

and a cholangioma occurred in one 4.5 mg/L female

(Tables 34 and F3). A hepatocholangiocarcinoma, a

malignant liver neoplasm consisting of both neoplastic

cholangiocellular (bile duct) and hepatocellular components, occurred in one 4.5 mg/L male, an hepatocellular

adenoma occurred in one 18.0 mg/L male, and an hepatocellular carcinoma occurred in one 9.0 mg/L female.

No cholangiocellular or hepatocellular neoplasms

occurred in control males or females.

Liver (Core Study): Increased incidences of cholangiocellular and hepatocellular neoplasms occurred in

exposed groups of males and females (Tables 34, F1, and

F3).

One to a few cholangiomas occurred in each of the

exposed groups of males and females but not in controls

(Tables 34, F1, and F3). Incidences of cholangiocarcinomas were significantly increased in all exposed

groups of males and females; no cholangiocarcinomas

were observed in controls. Cholangiocarcinomas metastasized to a number of tissues including heart, kidney,

eye, mesentery, adipose tissue, swim bladder, spleen,

esophagus, ovary, pericardial cavity, and intestine.

Moreover, incidences of bile duct hyperplasia were substantially increased in exposed groups of females and 9.0

and 18.0 mg/L males as compared with controls.

Incidences of hepatocellular adenoma and hepatocellular

carcinoma (combined) were increased in 18.0 mg/L

males and females as compared with controls. One

18.0 mg/L female had multiple hepatocellular adenomas. One hepatocellular adenoma and one hepatocellular carcinoma occurred in control females,

otherwise there were no hepatocellular neoplasms in

control fish. A number of hepatocholangiocarcinomas

also occurred in exposed groups of males and females,

but not in controls. Hepatocholangiocarcinomas metastasized to a number of tissues including esophagus, adipose tissue, intestine, mesentery, pericardial cavity, and

spleen.

Incidences of granuloma of the liver were decreased in

4.5 and 18.0 mg/L males and 18.0 mg/L females. The

decreased incidences of granuloma may have been secondary to the presence of other exposure-related lesions

that obscured the presence of granulomas.

The incidences of cyst were lower in 18.0 mg/L males

and in all exposed groups of females than in the controls.

The decreased incidences of cyst may be secondary to

the presence of other exposure-related lesions that

obscured the presence of cysts. 

Microscopically, all of the liver lesions observed in this

study appeared similar to those previously described.

Liver (Stop-Exposure Study): Increased incidences of

cholangiocellular (bile duct) neoplasms were observed

in exposed groups of males and females as compared

with controls (Tables 34, F2, and F4). An increased incidence of hepatocellular carcinoma was observed in

18.0 mg/L females.

Cholangiocarcinoma occurred with moderately high

incidences in all groups of exposed males and females.

Cholangiocarcinoma also occurred in one control

female. Cholangiocarcinomas metastasized to a number

of tissues including esophagus, adipose tissue, eye,

heart, kidney, mesentery, pericardial cavity, peritoneum,

and spleen. Cholangioma occurred in a few exposed

males and females but not in controls. Bile duct hyperplasia occurred with greater incidences in the 9.0 and

18.0 mg/L male groups than in the control group. The

incidences of bile duct hyperplasia were similar in control and exposed groups of females. The incidences of

cystic degeneration were higher in 9.0 and 18.0 mg/L

females than in controls. Incidences of cysts were lower

in exposed groups of males and females than in controls.

The decreased incidences of cysts may be secondary to

the presence of other exposure-related lesions that

obscured the presence of cysts.
Fish Studies, NTP TR 528 73

TABLE 34

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Medaka

in the 13-Month Waterborne Study of 1,2,3-Trichloropropane 

Trend

0 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L Test

P-Valuea

Male 

9-Month Interim Evaluation

Number Examined Microscopically 10 10 11 13

Cholangiocarcinomab 0 2 5* 8** 0.0007

Hepatocellular Adenoma 0 0 0 1 0.2954

Hepatocholangiocarcinoma 0 1 0 0 0.2726

Core Study

Number Examined Microscopically 95 94 65 78

Granuloma 36 13** 18 0** <0.0001N

Cyst 16 11 10 4* 0.0158N

Cholangioma 0 6* 2 1 0.4521

Cholangiocarcinoma 0 32** 43** 45** <0.0001

Cholangioma or Cholangiocarcinoma 0 38** 45** 46** <0.0001

Hepatocellular Adenoma 0 0 1 3 0.0128

Hepatocellular Carcinoma 0 2 0 3 0.0365

Hepatocellular Adenoma or Carcinoma 0 2 1 6** 0.0011

Hepatocholangiocarcinoma 0 0 2 6** 0.0001

Stop-Exposure Study

Number Examined Microscopically 42 47 33 46

Bile Duct, Hyperplasia 3 2 13** 14** 0.0002

Cyst 8 6 6 4

Cholangioma 0 0 3 1 0.1653

Cholangiocarcinoma 0 19** 16** 26** <0.0001

Cholangioma or Cholangiocarcinoma 0 19** 19** 27** <0.0001

Hepatocellular Adenoma 0 1 0 0 0.3066

Hepatocellular Carcinoma 0 1 0 1 0.2471

Hepatocellular Adenoma or Carcinoma 0 2 0 1 0.3950

Female 

9-Month Interim Evaluation

Number Examined Microscopically 11 11 10 8

Cholangiocarcinoma 0 3 2 3 0.0366

Cholangioma 0 1 0 0 0.3400

Hepatocellular Carcinoma 0 0 1 0 0.3803

Core Study

Number Examined Microscopically 95 93 94 67

Bile Duct Hyperplasia 3 15** 30** 21** <0.0001

Eosinophilic Focus 2 1 4 5 0.0164

Granuloma 11 6 5 1* 0.0061N

Cyst 37 18** 17** 15* 0.0136N

Cholangioma 0 1 2 1 0.1642

Cholangiocarcinoma 0 29** 53** 41** <0.0001

Cholangioma or Cholangiocarcinoma 0 30** 55** 42** <0.0001

Hepatocellular Adenoma, Multiple 0 0 0 1 0.1920

Hepatocellular Adenoma (includes multiple) 1 1 5 8** 0.0001

Hepatocellular Carcinoma 1 1 2 5* 0.0043

Hepatocellular Adenoma or Carcinoma 2 2 7 13** <0.0001

Hepatocholangiocarcinoma 0 0 4 7** <0.0001
74 Fish Studies, NTP TR 528

Gallbladder (9-Month Interim Evaluation): Papillary

adenomas occurred in two 18.0 mg/L males (Tables 35

and F1). No gallbladder neoplasms were seen in the

controls or any of the other exposed groups.

Gallbladder (Core Study): Papillary adenoma occurred

with increased incidences in 9.0 and 18.0 mg/L males

and females (Tables 35, F1, and F3). In addition, carcinoma occurred in one exposed male and in one exposed

female. No gallbladder neoplasms were present in controls. Epithelial hyperplasia of the gallbladder occurred

with greater incidence in 18.0 mg/L males and females.

Hyperplasia of the cystic duct of the gallbladder

occurred with greater incidences in 9.0 and 18.0 mg/L

males and females than those in the controls. 

Papillary adenoma was generally a small- to moderatesized papillary structure composed of a branching stromal core, indicative of a complex structure, covered by

tall columnar epithelium. Some papillomas consisted of

a series of multiple branching papillary structures, while

some other papillomas were pedunculated masses composed of branching structures supported by a stalk.

Carcinoma was a moderate-sized to large lesion composed of multiple highly complex papillary structures,

some of which were covered by multiple layers of

epithelium, accompanied by localized invasion of the

neoplastic epithelium through the gallbladder wall.

Epithelial hyperplasia of the gallbladder was a focal to

diffuse lesion characterized by an increase in the number

of epithelial cells with thickening of the epithelium.

Papillary hyperplasia was characterized by thickened

epithelium that formed few to many irregular, simple,

nonbranching papillary projections composed of a small

stromal core covered by tall columnar epithelial cells,

the greater the severity of the hyperplasia the greater the

number of papillary projections. The papillary projections formed a continuous line producing a sessile, flatbased lesion. Cystic duct hyperplasia, consisting of

hyperplasia of the cystic duct epithelium, had a similar

appearance to hyperplasia within the gallbladder lumen

and was accompanied by varying degrees of enlargement of the duct secondary to the epithelial hyperplasia.

Gallbladder (Stop-Exposure Study): The incidence of

papillary adenoma in males exposed to 18.0 mg/L was

significantly increased; papillary adenoma and carcinoma were observed in most exposed groups of males

and females (Tables 35, F2, and F4). Cystic duct hyperplasia occurred with greater incidences in 18.0 mg/L

males than in controls.

TABLE 34

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Medaka

in the 13-Month Waterborne Study of 1,2,3-Trichloropropane

Trend

0 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L Test

P-Value

Female (continued)

Stop-Exposure Study

Number Examined Microscopically 57 44 49 31

Bile Duct, Hyperplasia 4 6 8 5 0.0926

Cystic Degeneration 34 30 43** 26* 0.0014

Cyst 31 18 21 10

Cholangioma 0 1 2 0 0.4016

Cholangiocarcinoma 1 15** 31** 21** <0.0001

Cholangioma or Cholangiocarcinoma 1 16** 33** 21** <0.0001

Hepatocellular Adenoma 4 2 2 1 0.2121

Hepatocellular Carcinoma 0 2 1 4* 0.0033

Hepatocellular Adenoma or Carcinoma 4 4 2 5 0.1224

Hepatocholangiocarcinoma 0 0 0 1 0.1713

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981). A negative trend is indicated by N. b Number of animals with lesion
Fish Studies, NTP TR 528 75

TABLE 35

Incidences of Neoplasms and Nonneoplastic Lesions of the Gallbladder in Medaka

in the 13-Month Waterborne Study of 1,2,3-Trichloropropane 

Trend

0 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L Test

P-Valuea

Male 

9-Month Interim Evaluation

Number Examined Microscopically 10 9 11 13

Adenoma, Papillaryb 0 0 0 2 0.0864

Core Study

Number Examined Microscopically 91 83 61 72

Epithelium, Hyperplasia 1 0 1 12** <0.0001

Hyperplasia, Papillary 0 2 2 0 0.4434

Cystic Duct, Hyperplasia 0 1 7** 19** <0.0001

Concretion 3 2 3 4 0.1760

Adenoma, Papillary 0 0 4* 9** <0.0001

Carcinoma 0 0 0 1 0.2345

Stop-Exposure Study

Number Examined Microscopically 41 42 30 46

Epithelium, Hyperplasia 1 0 3 6 0.0058

Hyperplasia, Papillary 0 0 2 1 0.1642

Cystic Duct, Hyperplasia 0 1 3 5* 0.0085

Concretion 1 0 0 6 0.0023

Adenoma, Papillary 0 0 3 4* 0.0074

Carcinoma 0 0 1 1 0.1820

Female 

Core Study

Number Examined Microscopically 85 80 88 65

Epithelium, Hyperplasia 1 0 3 10** <0.0001

Hyperplasia, Papillary 0 0 4 2 0.0338

Cystic Duct, Hyperplasia 1 3 8* 16** <0.0001

Concretion 11 6 11 10 0.2189

Adenoma, Papillary 0 1 5* 6** 0.0006

Carcinoma 0 1 0 0 0.3295

Stop-Exposure Study

Number Examined Microscopically 56 40 45 30

Cystic Duct, Hyperplasia 1 1 5 1 0.1961

Adenoma, Papillary 0 1 1 1 0.1382

Carcinoma 0 0 0 1 0.1754

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion
76 Fish Studies, NTP TR 528

Kidney (Core Study): Incidences of casts and mineralization were increased in 9.0 and 18.0 mg/L males and

in 18.0 mg/L females as compared with controls

(Table 36). The incidences of renal tubule dilitation

were increased in 18.0 mg/L males and females. The

incidences of renal tubule vacuolation in 18.0 mg/L

females and of dilatation of Bowman’s space in

18.0 mg/L males were increased relative to the controls.

Microscopically, these lesions appeared similar to those

previously described. In addition, the incidences of

clear cytoplasmic vacuolation of tubular epithelium in

some exposed groups of males and females, and the incidences of dilatation of Bowman’s space of the renal

glomerulus in some groups of exposed males were marginally greater than those in the controls.

Kidney (Stop-Exposure Study): The incidence of renal

tubule dilatation was increased in 18.0 mg/L females

(Table 36).

Thyroid Tissue (Core Study): Incidences of hyperplasia,

consisting of an increase in the number of thyroid follicles, were increased in 18.0 mg/L males (0 mg/L, 9/95;

4.5 mg/L, 12/94; 9.0 mg/L, 10/65; 18.0 mg/L, 22/78).

Incidences of increased basophilia, characterized by

increased basophilic staining of follicular cells, were

greater in 18.0 mg/L females than in the controls (6/95,

13/93, 13/94, 29/67). The significance of the increased

basophilia was questionable and may have represented a

normal anatomic variation rather than an actual lesion.

Thyroid Tissue (Stop-Exposure Study): Incidences of

increased basophilia were increased in 18.0 mg/L

females compared to the controls (2/57, 6/44, 4/49,

6/31).
Fish Studies, NTP TR 528 77

TABLE 36

Incidences of Nonneoplastic Lesions of the Kidney in Medaka

in the 13-Month Waterborne Study of 1,2,3-Trichloropropane 

Trend 

0 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L Test

P-Valuea

Male

Core Study

Number Examined Microscopically 95 94 65 78

Castsb 11 17 17* 23** 0.0012

Mineralization 14 21 20* 25** 0.0030

Renal Tubule, Dilatation 13 19 10 24** 0.0043

Renal Tubule, Vacuolation 0 0 2 3 0.0083

Bowman’s Space, Dilatation 1 3 1 8** 0.0012

Stop-Exposure Study

Number Examined Microscopically 42 47 33 46

Renal Tubule, Dilatation 7 7 6 12 0.0893

Female

Core Study

Number Examined Microscopically 95 93 94 67

Casts 5 13* 11 15** 0.0014

Mineralization 4 6 3 10* 0.0071

Renal Tubule, Dilatation 6 6 10 19** <0.0001

Renal Tubule, Vacuolation 1 1 1 6* 0.0009

Stop-Exposure Study

Number Examined Microscopically 57 44 49 31

Renal Tubule, Dilatation 4 6 8 7* 0.0193

Casts 3 3 5 5 0.0360

Mineralization 3 1 3 3 0.1553

Renal Tubule, Degeneration 0 1 3 2 0.0315

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01 a Cochran-Armitage trend test or exact permutation test (Plackett, 1981) b Number of animals with lesion
78 Fish Studies, NTP TR 528
79

One of the major efforts of the National Toxicology

Program (NTP) has been to determine the potential carcinogenic hazard from chemicals that occur in the workplace or environment (Page et al., 1977; Weisburger and

Williams, 1981; Weisburger et al., 1984; Goodman and

Wilson, 1991; Faccini et al., 1992). While rodent studies are costly and time consuming, their strengths and

weaknesses are fairly well understood (Haseman, 2000;

Bennett and Davis, 2002), and rodent studies have provided a wealth of information on chemicals that occur in

the environment and in the workplace (Gold et al., 1998;

Kielhorn et al., 2000). While long-term rodent studies

provide safety assessment for pharmaceuticals and support many regulatory policies, the search for better models continues (Festing, 1995; Alden et al., 1996;

Eastin, 1998; Law 2001, 2003). The United States

Congress has also instructed the National Institutes of

Health (NIH) to investigate the use of alternatives to animal models as well as lower phylogenetic animals

(Salem and Katz, 1998).

Assays using fish species have been reported to be more

sensitive to carcinogens (Ishikawa et al., 1984;

Zimmerer, 1984; Liu et al., 2003) and less expensive

and faster to perform than rodent studies in carcinogenicity testing (Sinnhuber et al., 1978; Ishikawa and

Takayama, 1979; Bailey et al., 1984; Simon and Lapis,

1984; Walker et al., 1985). Use of a research model

under standard test procedures may reveal limitations

that might not be obvious from limited use in a research

laboratory setting. Therefore, the NTP decided to use

two small fish models to evaluate three chemicals previously tested in NTP standard rodent studies.

While rodent studies generally involve pathogen-free

genetically-defined animals maintained on standard

rodent chow, these parameters are less well established

for fish studies (ILAR, 1974). For example, genetically

defined medaka and guppies were not available.

Therefore, laboratory-bred fish from Gulf Coast

Laboratory (University of Mississippi, Ocean Springs,

MS), where the study was conducted, were used. The

fish had been maintained as a closed colony for approximately 10 years at Gulf Coast Laboratory after they

were purchased from a commercial supplier. Since there

are no standard breeding programs to reduce genetic

drift, it may be assumed that the medaka and guppies

used in the NTP studies vary somewhat from other similar fish colonies in the United States and other countries

(ILAR, 1974; Zimmerer, 1984; Ishikawa, 2000;

Reznick et al., 2001). It has been shown that natural

populations of guppies can rapidly evolve due to predators (Reznick et al., 2001). While rodent chow has been

standardized, small fish models are fed a variety of

foods for which much less is known (ILAR, 1974). In

NTP fish studies, the fish were fed a combination of

commercial fish flake food (Ziegler Brothers) and brine

shrimp. Finally, the NTP goes to great lengths to assure

that its studies maintain pathogen-free rodents, but the

fish in this study had histological evidence of parasitic,

fungal, and bacterial infections. These issues are not

unlike those experienced when the NTP began largescale use of rodents for carcinogenicity testing.

The fish rooms were on a 16-hour light and 8-hour dark

cycle, a photoperiod used by many investigators for

long-term studies (Zimmerer, 1984; Davis et al., 2002).

The disadvantage of the photoperiod used in this study

was that it does not inhibit reproduction. Embryos were

present in female guppies, which bear live young, and

developing follicles were present in the ovaries of

female medaka. A reduced light photoperiod can be

used to inhibit reproduction in the medaka

(Koger et al., 1999; Davis et al., 2002). However, fish

exposed to restricted light are significantly smaller

(Davis et al., 2002), and the impact this may have on

carcinogenesis is not known. Therefore, the 16-hour

light, 8-hour dark was used, which is more common for

medaka studies. However, it should be assumed that

female guppies delivered live young during the study

and that the female medaka produced eggs. The aquaria

were examined daily, and immature fish were not seen

during the study. While this is standard practice with the

assumption that young born or hatched during the study

will not reach maturity (Yamamoto, 1975;

Overstreet et al., 2000), it does introduce a degree of

uncertainty about age and length of exposure for those

fish harvested at the end of the study.

DISCUSSION AND CONCLUSIONS
80 Fish Studies, NTP TR 528

One of the chemicals, nitromethane, proved especially

problematic because the chemical served as a source of

nitrogen that promoted bacterial and algal growth in the

aquaria. This effect necessitated brushing the aquaria

once and siphoning two (guppies) or three (medaka)

times each day instead of once per week. The frequent

cleaning may have caused more stress on the fish than

was experienced by fish in the 2,2-bis(bromomethyl)-

1,3-propanediol and 1,2,3-trichloropropane studies.

Despite efforts to keep the fish as healthy as possible,

microscopic granulomas related to bacteria and fungi

were seen in multiple organs in fish from all three studies. Generally, more than half of the fish in any group

had microscopic granulomas. In the nitromethane study,

there was some increase in the incidences of granulomas

with increasing dose, while in the 2,2-bis(bromomethyl)-1,3-propanediol study, the incidences of granulomas decreased with increasing dose. It was speculated

that 2,2-bis(bromomethyl)-1,3-propanediol at higher

doses inhibited bacterial growth.

In rodent studies, the dose selection is usually based on

toxicokinetic studies, clinical pathology findings, clinical signs, and histopathology using 14-day and 90-day

exposures. For the current fish studies, doses were

determined using 2-day static tests and 7-day and 28-day

flow-through range finding studies. Mortality and

lethargy were the endpoints used. The highest concentration that was predicted not to cause excessive mortality was chosen for 16-month studies. While the guppies

could tolerate the chemical exposures for 16 months,

excessive mortality occurred in the medaka. Medaka

studies were terminated at 13 months for the

1,2,3-trichloropropane study and at 14 months for the

2,2-bis(bromomethyl)-1,3-propanediol, where the doses

were the same as that for the guppy. In the studies of

guppies exposed to these same two chemicals, survival

to 16 months was adequate. In the nitromethane study,

the dose-range finding studies indicated that medaka

were more sensitive, so the doses selected were 0, 10,

20, and 40 mg/L for medaka in contrast to the 0, 10, 30,

and 70 mg/L doses selected for the guppies. Again, the

medaka had to be terminated at 13 months due to high

mortality, while the guppies survived to 16 months. This

may not be surprising since guppies are considered to

have a longer life span and to be less sensitive to

carcinogens than medaka (Hawkins et al., 2003).

The length of exposure that would be necessary for

detection of carcinogenicity of these three chemicals was

not known. The time for tumor induction in guppies has

been reported to be from 3 to 11 months (Zimmerer,

1984), while medaka exposed to potent carcinogens may

show liver tumors in 2 to 3 months (Hinton et al., 1984).

Since animals dying early are often lost for examination

in the experimental studies, an interim sacrifice was

included at 9 months for the detection of microscopic

tumors and preneoplastic lesions. Significantly

increased incidences of tumors at 9 months were seen

only in medaka and only for 1,2,3-trichloropropane.

Significantly increased incidences of tumors were not

seen for the other chemicals at 9 months including

1,2,3-trichloropropane in guppies, a study that was

clearly positive at study termination.

A hypothesis tested during these studies was that for

toxic chemicals, the fish would live longer and neoplasms might develop more readily if the chemical was

removed during the last few study months, perhaps providing a more sensitive assay than one involving continued exposure. Therefore, at 9 months, one third of the

remaining fish were placed in chemical-free water (stopexposure study) while the other two thirds were continued on exposure (core study). The 9-month interim sacrifice and the transfer of some fish to chemical-free

water (stop-exposure groups) were used to provide ancillary information. An observation period in chemicalfree water following exposure has been used in other fish

studies (Simon and Lapis, 1984).

The goal was to examine approximately 100 fish in the

continued exposure study (core study) since the sex of

the animals would not be known until histological examination. This would make the fish studies comparable to

the groups of 50 males and 50 females in rodent studies.

While large numbers of fish are easier to maintain in

many respects than rodents, they do present unique challenges. Fish counts were approximate during the study

and, since fish could be lost during the study, duplicate

aquaria containing more fish than needed were used.

The goal was to sample 20 fish per species and dose concentration at 9 months; approximately 50 per sex,

species, and dose concentration with continued exposure

(core study); and approximately 25 per sex, species, and

dose concentration for the stop-exposure study. To

accomplish these goals each group started with 220 guppies (110 per aquarium) and 340 medaka (170 per aquarium). In order to avoid bias, fish were randomly

assigned to study groups at the initiation of exposure,

and at 9 months randomly assigned for examination, 

and randomly assigned for continued exposure or 
Fish Studies, NTP TR 528 81

stop-exposure (Hawkins et al., 2003). In most groups,

enough fish survived to allow random selection of

animals for examination at terminal sacrifice.

The number of medaka examined for nitromethane and

1,2,3-trichloropropane exceeded 1,000. In the

1,2,3-trichloropropane study, the total number of

medaka examined varied from 222 fish in the high exposure group to 291 in the control group. For this study,

188 control guppies were examined, but only 106 high

exposed guppies were examined. For the nitromethane

study, more than 220 medaka were examined for each

control and exposure group, with over 160 guppies in

each group except for the high exposure where only

89 fish were available for examination. For the

2,2-bis(bromomethyl)-1,3-propanediol studies, approximately 270 medaka were examined in each group, while

approximately 115 to 190 guppies were examined for

each dose or control group.

Concentrations of each chemical were set at the highest

levels compatible with survival, and exposure lengths

were set at 13 to 16 months to maximize the possibility

of detecting a carcinogenic response. It is possible that

animals lost to mortality may have experienced more

toxicity or tumors than the survivors. However, most

early death animals were cannibalized and could not be

examined. Nitromethane was considered negative for

carcinogenicity, but less than 50% of the high exposure

guppies and approximately 70% of the high exposure

medaka were examined. Whether examination of the

early death animals would have changed this assessment

is not known. Toxicity precluded testing of these chemicals at higher concentrations.

The small size of the fish confers both advantages and

disadvantages. In contrast to rodents, there is no gross

necropsy, and most tumors are found histologically. A

whole fish fits on a histological slide, and the five longitudinal sections taken to evaluate fish include most tissues. Therefore, it is usually not possible to examine

multiple samples of tumors or take samples at different

sites within a tumor. The small size of fish also means

that many small tissues such as the thymus are easily

missed during sectioning.

Growth and size are related to the density of fish in the

aquaria (Davis et al., 2002). With increasing mortality at

higher exposure concentrations, it is nearly impossible to

maintain fish densities across exposure groups that are

comparable to those in the control aquaria.

1,2,3-Trichloropropane exposure caused increased mortality in both guppies and medaka, and in both cases

there was an increase in fish length and/or body weight.

While increased body weight might be explained by the

increased liver tumors found with 1,2,3-trichloropropane

exposure, body length is more likely related to the

reduced number of fish in the aquaria. This may introduce a confounding factor for study interpretation

because increased incidences of liver tumors are correlated with increased body weights as observed in mice

(Fullerton et al., 1992; Cohen et al., 1994;

Haseman et al., 1997; Rogers et al., 1999). However,

Rao and Crockett (2003) have pointed out that increased

body weights and liver tumors do not always correlate

for mice when housed under different conditions.

In the current studies, survival and body weight and/or

body length do not always correlate. For example, in

medaka exposed to nitromethane, there were increased

body weights, while the number of survivors was similar

across exposure concentrations. Conversely, decreased

survival was seen in guppies exposed to 2,2-bis(bromomethyl)-1,3-propanediol with little or no effect on

fish size. However, in four of the six studies, the fish in

one or more of the exposed groups were larger than in

the control group. This may be critical since body

weight gain can greatly affect tumor multiplicity, especially when occurring during the initiation and promotion stages of the cancer process (RodriguezBurford et al., 1999). Keeping aquaria concentrations

and fish size comparable across dose and control groups

will reduce uncertainty especially when a marginal

increase in tumors is found in exposed fish.

Rodents have a rich history of carcinogenicity and mechanistic studies, so much more information on classification and potential significance of rodent lesions is

available compared to the tumors that occur in fish. This

could change as fish models become more widely used

in carcinogenicity studies.

It was anticipated that evaluating the step sections from

fish would be much faster than evaluating the 10 to

12 slides usually available for each rodent in NTP studies. However, this did not prove to be the case.

Searching the whole fish and several sections for each

tissue proved to be nearly as time consuming as the evaluation for a rat or mouse. Thus, there was minimal cost

reduction for pathology effort for the fish models. Since

tumors were found essentially only in the liver, it may be

possible in the future to limit pathological evaluation to

the liver. This, of course, would limit the information

available from a fish study.
82 Fish Studies, NTP TR 528

This disadvantage might be somewhat compensated if

the overall cost of the fish studies were significantly less

than for rodents. While the fish studies were less expensive, the overall cost of evaluating three chemicals in

two fish models was still approximately half of the cost

of evaluating the three chemicals in two rodent species.

The rodent studies included a gavage study, an inhalation

study, and a feed study.

Nitromethane was selected for evaluation in small fish

models because nitromethane is a nonmutagen that gave

clear evidence of carcinogenicity in three of the four

sex/species combinations in the NTP study (NTP, 1997)

but did not primarily affect the liver. Nitromethane by

inhalation exposure was not carcinogenic for male rats

but was clearly carcinogenic for female rats with

increased mammary gland fibroadenomas and carcinomas. Nitromethane was clearly carcinogenic for male

and female mice with increased incidence of harderian

gland adenomas and carcinomas. There were increased

incidences of liver neoplasms in female mice exposed 

to nitromethane. The increased incidences of alveolar/bronchiolar adenomas and carcinomas in mice may

have been related to nitromethane exposure. Guppies

and medaka exposed to 70 mg/L of nitromethane experienced increased mortality by 9 months. There were no

increased incidences of neoplasms at any site in male or

female medaka or male or female guppies at 9 months.

At 16 months, there were no increased incidences of

neoplasms at any site in female guppies. Despite a small

increase in hepatocellular neoplasms in the 30 mg/L

core-study group, the study in male guppies was considered inadequate. In medaka, where the study was terminated at 13 months, there was equivocal evidence for

increased liver neoplasms in the male medaka for the

continued exposure but not the stop-exposure study. No

increased incidences of neoplasms at any site were found

in female medaka. Thus nitromethane, that was carcinogenic in rodents (NTP, 1997) generally at sites other than

the liver, was not predicted to be carcinogenic by these

two fish species.

2,2-Bis(bromomethyl)-1,3-propanediol was selected for

study because it is mutagenic and a multisite carcinogen

and judged to give clear evidence of carcinogenic activity in male and female rats and male and female mice

(NTP, 1996). The sites of increased neoplasm incidences in the rodents included oral cavity, skin, esophagus, and urinary bladder, sites where cancer may be predicted to occur in fish, as well as other sites including

lung, harderian gland, subcutaneous tissue, Zymbal’s

gland, and others. There was no increased mortality in

medaka exposed to 2,2-bis(bromomethyl)-1,3-propanediol, but mortality was increased in guppies exposed to

2,2-bis(bromomethyl)-1,3-propanediol. At the 9-month

interim sacrifice, there was no significantly increased

incidence of neoplasms at any site in male or female

guppies or medaka. By 16 months, the incidences of

hepatocellular adenomas and carcinomas were clearly

increased in male guppies for both the stop-exposure and

continued exposure groups. There was no significant

increase in neoplasms at any site in female guppies at

16 months. At 14 months, there was an increase in the

incidences of hepatocellular adenomas and carcinomas

in male medaka in the continued exposure group, supported by a marginal increase in the stop-exposure

group. Both the continued exposure and stop-exposure

groups were considered negative for carcinogenicity in

the female medaka. This is a more modest response than

in rodents, where neoplasms of the skin subcutaneous

tissue, mammary gland, Zymbal’s gland, oral cavity,

esophagus, forestomach, small and large intestine,

mesothelium, urinary bladder, lung, thyroid gland, seminal vesicle, and mononuclear cells were found in male

rats (NTP, 1996). Three or more sites for increased neoplasm incidences were also found in female rats and

male and female mice. 

1,2,3-Trichloropropane was selected as a second mutagenic multisite rodent carcinogen (NTP, 1993). The

spectrum of induced incidences of neoplasms in rodents

(oral cavity, Zymbal’s gland, harderian gland, and

forestomach) was similar to 2,2-bis(bromomethyl)-

1,3-propanediol but included other sites such as uterus,

clitoral gland, intestine, and pancreas. Increased incidences of liver neoplasms were found in male medaka at

9 months with a suggestive increase in the incidences of

liver neoplasms in female medaka but were not found in

the liver of male or female guppies at 9 months. The

incidences of liver neoplasms were clearly increased in

male and female medaka at all dose concentrations at

13 months and at all dose concentrations in male guppies

at 16 months. The incidences of liver neoplasms were

clearly increased at the two highest doses in the female

guppy at 16 months. The response was greater in

medaka compared to guppies, and generally greater in

males. The incidences of neoplasms were greater in the

continued exposure groups compared to the stop-exposure groups. 1,2,3-Trichloropropane by gavage at up to

30 mg/kg in rats and 60 mg/kg in mice caused increased

cancer at multiple sites in a 15-month interim evaluation

in both rats and mice (NTP, 1993). Thus, this small
Fish Studies, NTP TR 528 83

series of three chemicals in two fish models provides

little evidence that these chemicals can be detected more

quickly or at lower concentrations than in rodents.

Stop-exposure groups after 9 months were compared to

continued exposure groups with the hypothesis that less

toxicity might enhance the growth of neoplasms,

providing greater sensitivity to detect carcinogens by

these fish species. In every instance, the incidence of

liver neoplasms was higher in groups exposed continuously when compared to the stop-exposure groups. This

would suggest that stop-exposure studies are not more

sensitive than continued exposure studies. While our

studies are limited to three chemicals, the effect was consistent for both sexes in two fish species.

It might be expected that mutagenicity or the carcinogenic response in rodents would predict the carcinogenic

response in fish. However, the traditional predictors of

toxicity in rodents fail to characterize the bioavailability

of the chemical for fish, one of the more important predictors of toxicity for aquatic species (Boudou and

Ribeyre, 1997; Parkerton et al., 2000). Hydrophobic

compounds would be expected to accumulate in the

lipid-rich tissues of fish, including the liver

(Meador et al., 1995), while hydrophilic chemicals

would remain in the water. Nitromethane and 

2,2-bis(bromomethyl)-1,3-propanediol have calculated

bioaccumulation factors of 1.0 (American Chemical

Society, 2003) and were negative or of limited carcinogenicity in our studies. 1,2,3-Trichloropropane was

clearly carcinogenic in both medaka and guppies and has

a calculated bioaccumulation factor of 18.2 (America

Chemical Society, 2003). Thus, the bioavailability

factor may be one of the most important criteria in

selecting chemicals for evaluation in small fish models.

The spectrum of neoplasms in organs and tissues in

rodents has often provided clues as to the mechanisms of

toxicity and carcinogenicity of the test chemicals.

However, most fish studies report primarily liver neoplasms (Hinton et al., 1984; Ishikawa et al., 1984;

Boorman et al., 1997; Hawkins et al., 2003). Therefore,

three chemicals were selected that caused a broad spectrum of cancers in rodents including neoplasms of the

mammary gland, oral cavity, forestomach, harderian

gland, Zymbal’s gland, lung, pancreas, kidney, and other

sites. While it may be difficult to predict a target site for

a chemical that causes increased mammary gland or

Zymbal’s gland neoplasms, it was surprising to find that

both sexes and both species of fish responded with only

neoplasms of the liver and gallbladder. The fish

response appeared less informative than the wide spectrum of neoplasms found in the rodent studies.

The loss of specimens during the study limited the information that was available for analysis. For example,

time to neoplasm and presence of preneoplastic lesions

could not be analyzed with the present study design. It

is possible that multiple interim sacrifices would be

helpful. It is worth noting that generally the 9-month

sacrifice showed few or no neoplasms, and the medaka

studies were terminated at 13 months (16 months for

guppies), providing a very small window to study neoplasm development. One concern that was difficult to

address was the possibility that fish that developed neoplasms died early and were lost to the study. For

example, all three guppy studies had markedly reduced

survival in the highest exposure group after the animals

were assigned to the core study at 9 months. As a result,

only a relatively small proportion of the animals in these

groups (ranging from 55% in the nitromethane study to

71% in the 2,2-bis(bromomethyl)-1,3-propanediol

study) were examined for liver neoplasms. It is

unknown how this early death and the resulting

possibility of underestimating the true neoplasm

incidences affected the studies sensitivity for detecting

carcinogenicity.

CONCLUSIONS

Under the conditions of these waterborne studies, 

2,2-bis(bromomethyl)-1,3-propanediol at concentrations

of up to 150 mg/L for 16 months was considered carcinogenic for male guppies based on increased incidences of hepatocellular adenomas or carcinomas. The

study in female guppies was considered inadequate

based on reduced survival. Under the conditions of

these waterborne studies, 2,2-bis(bromomethyl)-

1,3-propanediol at concentrations of up to 150 mg/L for

14 months was considered carcinogenic for male

medaka based on increased incidences of hepatocellular

adenomas or carcinomas. The study in female medaka

was considered negative.

Under the conditions of these waterborne studies, the

study of nitromethane in male guppies was considered

inadequate based on reduced survival. The study in

female guppies at concentrations up to 70 mg/L for

16 months was considered negative. The study in male

and female medaka was considered negative.
84 Fish Studies, NTP TR 528

Under the conditions of these waterborne studies,

1,2,3-trichloropropane at concentrations of up to

18 mg/L for 16 months was considered carcinogenic for

male and female guppies based on increased incidences

of a variety of liver neoplasms. Under the conditions of

these waterborne studies, 1,2,3-trichloropropane at

concentrations of up to 18 mg/L for 13 months was

considered carcinogenic for male and female medaka

based on increased incidences of a variety of liver

neoplasms and papillary adenoma of the gallbladder.
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90 Fish Studies, NTP TR 528
91

APPENDIX A

SUMMARY OF LESIONS IN GUPPIES

IN THE 16-MONTH WATERBORNE STUDY

OF 2,2-BIS(BROMOMETHYL)-1,3-PROPANEDIOL

TABLE A1 Summary of the Incidence of Neoplasms in Male Guppies 

in the 16-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

(Core Study) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

TABLE A2 Summary of the Incidence of Neoplasms in Male Guppies 

in the 16-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

(Stop-Exposure) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

TABLE A3 Summary of the Incidence of Neoplasms in Female Guppies 

in the 16-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

(Core Study) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

TABLE A4 Summary of the Incidence of Neoplasms in Female Guppies 

in the 16-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

(Stop-Exposure) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

TABLE A5 Summary of the Incidences of Granuloma in Selected Tissues of Guppies 

in the 16-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

(Core Study) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
92 Fish Studies, NTP TR 528

TABLE A1

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Groupa

A B A B A B A B

9-Month Interim Evaluation

Liverb (7) (6) (5) (8) (7) (3) (6) (5)

Hepatocellular adenomac 1 1 

Tissues Examined at 9 Months with No Neoplasms Observed

Abdominal Cavity 

Adipose Tissue 

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius 

Cranial Cavity 

Esophagus 

Eye 

Gallbladder 

Gill 

Heart 

Hematopoietic Tissue

Interrenal Tissue 

Intestine 

Kidney 

Mesentery 

Nares (Olfactory Tissue) 

Oral Cavity 

Pancreas 

Pericardial Cavity 

Peripheral Nerve 

Peritoneum 

Pharynx 

Pineal Organ 

Pituitary Gland 

Pseudobranch 

Skeletal Muscle 

Skin 

Spinal Cord 

Spleen 

Stato-acoustic Organ 

Swim Bladder 

Testis 

Thymus 

Thyroid Tissue 

Urinary Bladder
Fish Studies, NTP TR 528 93

16-Month Study

Abdominal Cavity (0) (0) (0) (0) (0) (1) (0) (0)

Adipose Tissue (15) (21) (12) (11) (4) (13) (4) (6)

Retroperitoneal, sertoli cell tumor,

invasive 1 

Blood Vessel (0) (2) (2) (0) (0) (3) (0) (1)

Bone (26) (35) (27) (23) (11) (30) (20) (18)

Brain (26) (35) (27) (23) (11) (30) (20) (18)

Chromaffin Tissue (25) (35) (25) (22) (10) (30) (18) (17)

Corpuscle of Stannius (6) (17) (12) (6) (5) (9) (10) (11)

Cranial Cavity (5) (3) (3) (5) (2) (6) (1) (2)

Esophagus (26) (35) (27) (23) (11) (30) (20) (18)

Eye (26) (35) (27) (23) (11) (30) (20) (18)

Gallbladder (24) (32) (25) (23) (11) (30) (19) (17)

Carcinoma

Gill (26) (35) (27) (23) (11) (30) (20) (18)

Heart (25) (35) (27) (23) (11) (30) (20) (18)

Ventricle, sarcoma, NOS 1 

Hematopoietic Tissue (26) (35) (27) (23) (11) (30) (20) (18)

Interrenal Tissue (25) (35) (26) (22) (10) (30) (18) (17)

Intestine (26) (35) (27) (23) (11) (30) (20) (18)

Kidney (26) (35) (27) (23) (11) (30) (20) (18)

Liver (26) (35) (27) (23) (11) (30) (20) (18)

Carcinoma, invasive

Cholangioma 1 

Hepatocellular adenoma 3 3 1 4 3 3 

Hepatocellular adenoma, multiple 2 

Hepatocellular carcinoma 1 1 1 2 2 

Mesentery (16) (17) (10) (13) (6) (14) (9) (6)

Sertoli cell tumor, invasive 1 

TABLE A1

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
94 Fish Studies, NTP TR 528

16-Month Study (continued)

Nares (Olfactory Tissue) (14) (16) (15) (16) (4) (17) (13) (12)

Oral Cavity (26) (35) (27) (23) (11) (30) (20) (18)

Pancreas (26) (35) (27) (23) (11) (30) (20) (18)

Islet cells, adenoma 1 

Pericardial Cavity (2) (5) (2) (4) (1) (4) (1) (3)

Peripheral Nerve (26) (35) (27) (23) (11) (30) (20) (18)

Peritoneum (2) (4) (5) (3) (0) (5) (1) (0)

Pharynx (6) (4) (2) (6) (0) (3) (0) (1)

Pineal Organ (19) (21) (22) (12) (8) (17) (14) (10)

Pituitary Gland (22) (32) (24) (21) (11) (28) (16) (15)

Adenoma 1 

Pseudobranch (26) (35) (27) (23) (11) (30) (20) (18)

Skeletal Muscle (26) (35) (27) (23) (11) (30) (20) (18)

Sertoli cell tumor, invasive 1 

Skin (26) (35) (27) (23) (11) (30) (20) (18)

Sertoli cell tumor, invasive 1 

Spinal Cord (25) (35) (27) (21) (11) (29) (20) (17)

Spleen (22) (34) (25) (20) (10) (26) (18) (17)

Stato-acoustic Organ (26) (35) (27) (23) (11) (30) (20) (18)

Swim Bladder (26) (35) (27) (23) (11) (30) (20) (18)

Carcinoma 1 

Testis (26) (35) (27) (23) (11) (30) (20) (18)

Sertoli cell tumor 1 

Thymus (1) (2) (0) (0) (3) (3) (0) (1)

Thyroid Tissue (26) (35) (27) (20) (11) (30) (20) (18)

Urinary Bladder (21) (35) (26) (17) (9) (29) (19) (17)

a Exposure of the 150 mg/L core group was stopped at 64 weeks and the animals were held until 69 weeks when all groups were sacrificed and examined. b Number of animals examined microscopically at the site c Number of animals with neoplasm

TABLE A1

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 95

Abdominal Cavity (0) (0) (1) (0) (0) (0) (0) (0) 

Adipose Tissue (11) (7) (5) (7) (4) (4) (6) (6)

Blood Vessel (0) (0) (0) (0) (1) (1) (1) (0)

Bonea (15) (13) (10) (12) (7) (14) (9) (15)

Vertebra, chondromab 1 

Brain (15) (13) (10) (12) (7) (14) (9) (15)

Chromaffin Tissue (15) (13) (10) (12) (7) (13) (9) (15)

Corpuscle of Stannius (3) (4) (2) (5) (2) (3) (2) (4)

Cranial Cavity (3) (4) (3) (1) (1) (0) (0) (0)

Esophagus (15) (13) (10) (12) (7) (14) (9) (15)

Eye (15) (13) (10) (12) (7) (14) (9) (15)

Gallbladder (14) (13) (9) (12) (7) (14) (8) (15)

Gill (15) (13) (10) (12) (7) (14) (9) (15)

Heart (15) (13) (10) (12) (7) (14) (9) (15)

Hematopoietic Tissue (15) (13) (10) (12) (7) (14) (9) (15)

Interrenal Tissue (15) (13) (10) (12) (7) (13) (9) (15)

Intestine (15) (13) (10) (12) (7) (14) (9) (15)

Kidney (15) (13) (10) (12) (7) (14) (9) (15)

Liver (15) (13) (10) (12) (7) (14) (9) (15)

Hepatocellular adenoma 1 2 1 1 3 1 

Hepatocellular adenoma, multiple 1 2 

Hepatocellular carcinoma 3 

Mesentery (11) (10) (7) (8) (5) (10) (5) (7)

Nares (Olfactory Tissue) (8) (7) (5) (11) (4) (3) (2) (9)

Oral Cavity (15) (13) (10) (12) (7) (14) (9) (15)

Pancreas (15) (13) (10) (12) (7) (14) (9) (15)

Islet cells, adenoma 1 

Pericardial Cavity (4) (4) (2) (3) (4) (1) (0) (1)

Peripheral Nerve (15) (13) (10) (12) (7) (14) (9) (15)

TABLE A2

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Stop-Exposure)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
96 Fish Studies, NTP TR 528

Peritoneum (2) (3) (2) (2) (1) (0) (2) (2)

Pharynx (3) (4) (4) (2) (1) (0) (0) (0)

Pineal Organ (3) (9) (8) (7) (6) (9) (6) (11)

Pituitary Gland (13) (11) (7) (10) (7) (12) (8) (15)

Adenoma 1 

Pseudobranch (15) (13) (10) (12) (7) (14) (9) (15)

Skeletal Muscle (15) (13) (10) (12) (7) (14) (9) (15)

Skin (15) (13) (10) (12) (7) (14) (9) (15)

Spinal Cord (14) (12) (10) (12) (7) (13) (9) (15)

Spleen (15) (13) (7) (12) (7) (14) (9) (15)

Stato-acoustic Organ (15) (13) (10) (12) (7) (14) (9) (15)

Swim Bladder (15) (13) (10) (12) (7) (14) (9) (15)

Testis (15) (13) (10) (12) (7) (14) (9) (15)

Thymus (0) (0) (0) (0) (0) (0) (0) (1)

Thyroid Tissue (15) (13) (9) (11) (6) (13) (9) (15)

Urinary Bladder (15) (13) (9) (11) (7) (14) (9) (15)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE A2

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Stop-Exposure) 

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 97

9-Month Interim Evaluation

Liverb (4) (4) (6) (2) (4) (7) (4) (8)

Cholangiomac 1 

Tissues Examined at 9 Months with No Neoplasms Observed

Adipose Tissue 

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius 

Cranial Cavity

Esophagus

Eye 

Gallbladder

Gill 

Heart 

Hematopoietic Tissue

Interrenal Tissue 

Intestine

Kidney

Mesentery 

Nares (Olfactory Tissue) 

Oral Cavity 

Ovary 

Pancreas 

Pericardial Cavity 

Peripheral Nerve 

Pharynx 

Pineal Organ 

Pituitary Gland 

Pseudobranch 

Skeletal Muscle 

Skin 

Spinal Cord 

Spleen 

Stato-acoustic Organ 

Swim Bladder 

Thymus 

Thyroid Tissue

Urinary Bladder 

16-Month Study

Abdominal Cavity (0) (0) (0) (0) (0) (0) (1) (0)

Adipose Tissue (16) (23) (21) (19) (11) (15) (13) (6)

Head, hemangioma 1 

Blood Vessel (1) (0) (2) (4) (0) (2) (4) (1)

Bone (32) (29) (30) (39) (31) (27) (22) (24)

TABLE A3

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Groupa

A B A B A B A B
98 Fish Studies, NTP TR 528

16-Month Study (continued)

Brain (32) (29) (30) (39) (31) (27) (22) (24)

Chromaffin Tissue (30) (28) (28) (37) (30) (25) (21) (23)

Corpuscle of Stannius (14) (6) (17) (14) (14) (11) (9) (7)

Cranial Cavity (6) (6) (1) (2) (3) (2) (1) (2)

Esophagus (32) (29) (30) (39) (31) (27) (22) (24)

Eye (32) (29) (30) (39) (31) (27) (22) (24)

Optic nerve, retinoblastoma, invasive 1 

Retinoblastoma 1 

Gallbladder (32) (29) (30) (38) (30) (27) (22) (24)

Carcinoma 1 

Gill (32) (29) (30) (39) (31) (27) (22) (24)

Hemangioma 1 

Heart (30) (29) (29) (37) (31) (26) (22) (24)

Bulbus arteriosus, hemangiosarcoma 1 

Hematopoietic Tissue (32) (29) (30) (39) (31) (27) (22) (24)

Interrenal Tissue (30) (28) (29) (38) (30) (25) (21) (23)

Intestine (32) (29) (30) (39) (31) (27) (22) (24)

Kidney (32) (29) (30) (39) (31) (27) (22) (24)

Hemangioma 1 

Liver (32) (29) (30) (39) (31) (27) (22) (24)

Carcinoma, invasive 1 

Cholangioma 2 

Hepatocellular adenoma 1 1 1 1 1 4 

Hepatocellular adenoma, multiple 1 

Mesentery (15) (24) (15) (27) (12) (14) (8) (9)

Hemangiopericytoma 1 

Nares (Olfactory Tissue) (25) (20) (25) (35) (24) (23) (18) (23)

Oral Cavity (32) (29) (30) (39) (31) (27) (22) (24)

Ovary (32) (29) (29) (38) (31) (27) (22) (24)

Pancreas (32) (29) (30) (39) (31) (27) (22) (24)

Adenoma 1 

Carcinoma 1 

TABLE A3

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 99

16-Month Study (continued)

Pericardial Cavity (2) (9) (5) (8) (3) (5) (4) (2)

Peripheral Nerve (32) (29) (30) (39) (31) (27) (22) (24)

Schwannoma 1 

Peritoneum (1) (6) (3) (5) (1) (2) (2) (1)

Pharynx (7) (5) (4) (3) (4) (2) (3) (0)

Pineal Organ (17) (11) (18) (26) (21) (16) (10) (17)

Pituitary Gland (24) (26) (28) (35) (26) (21) (22) (23)

Pseudobranch (32) (29) (29) (38) (31) (25) (22) (24)

Skeletal Muscle (32) (29) (30) (39) (31) (27) (22) (24)

Skin (32) (29) (30) (39) (31) (27) (22) (24)

Spinal Cord (30) (26) (28) (37) (29) (27) (21) (23)

Spleen (28) (27) (23) (34) (22) (15) (19) (20)

Carcinoma, invasive 1 

Hemangiosarcoma 1 

Stato-acoustic Organ (32) (29) (30) (39) (31) (27) (22) (24)

Swim Bladder (31) (29) (29) (39) (31) (27) (22) (24)

Epithelioma 1 1 

Thymus (23) (21) (22) (24) (21) (21) (13) (17)

Thyroid Tissue (30) (29) (30) (37) (30) (25) (22) (24)

Follicular cell carcinoma 1 

Urinary Bladder (27) (15) (21) (34) (24) (20) (15) (15)

a Exposure of the 150 mg/L core group was stopped at 64 weeks and the animals were held until 69 weeks when all groups were sacrificed and examined. b Number of animals examined microscopically at the site c Number of animals with neoplasm

TABLE A3

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
100 Fish Studies, NTP TR 528

Adipose Tissuea (15) (11) (17) (12) (8) (10) (3) (6)

Head hemangiomab 1

Retroparitoneal, hemangiopericytoma 1 

Blood Vessel (4) (1) (1) (0) (0) (1) (1) (2)

Bone (17) (18) (19) (17) (14) (14) (6) (7)

Brain (17) (18) (19) (17) (14) (14) (6) (7)

Chromaffin Tissue (17) (15) (17) (17) (13) (13) (5) (6)

Corpuscle of Stannius (5) (9) (10) (11) (7) (8) (4) (2)

Cranial Cavity (3) (1) (3) (2) (3) (3) (0) (0)

Esophagus (17) (18) (19) (17) (14) (14) (6) (7)

Eye (17) (18) (19) (17) (14) (14) (6) (7)

Gallbladder (17) (18) (17) (17) (14) (14) (6) (7)

Gill (17) (18) (19) (17) (14) (14) (6) (7)

Heart (17) (18) (18) (17) (14) (14) (6) (7)

Hematopoietic Tissue (17) (18) (19) (17) (14) (14) (6) (7)

Interrenal Tissue (17) (15) (18) (17) (14) (13) (5) (7)

Intestine (17) (18) (19) (17) (14) (14) (6) (7)

Hemangiosarcoma, invasive 1 

Kidney (17) (18) (19) (17) (14) (14) (6) (7)

Liver (17) (18) (19) (17) (14) (14) (6) (7)

Cholangiocarcinoma 1 

Cholangioma 1 

Hepatocellular adenoma 1 1 1 

Mesentery (12) (10) (14) (10) (9) (8) (4) (6)

Cholangiocarcinoma, invasive 1 

Hemangiosarcoma 1 

Nares (Olfactory Tissue) (15) (14) (15) (16) (10) (11) (6) (4)

Oral Cavity (17) (18) (19) (17) (14) (14) (6) (7)

Ovary (17) (18) (19) (17) (14) (14) (6) (7)

Pancreas (17) (18) (19) (17) (14) (14) (6) (7)

Adenoma 1 

TABLE A4

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Stop-Exposure)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 101

Pericardial Cavity (6) (5) (7) (3) (3) (4) (1) (1)

Peripheral Nerve (17) (18) (19) (17) (14) (14) (6) (7)

Peritoneum (3) (4) (2) (3) (1) (2) (0) (1)

Hemangiopericytoma, invasive 1 

Pharynx (4) (5) (4) (3) (1) (3) (1)

Pineal Organ (15) (11) (7) (8) (8) (10) (1) (5)

Pituitary Gland (17) (14) (10) (9) (12) (12) (6) (7)

Pseudobranch (16) (16) (16) (17) (13) (12) (4) (7)

Skeletal Muscle (17) (18) (19) (17) (14) (14) (6) (7)

Skin (17) (18) (19) (17) (14) (14) (6) (7)

Spinal Cord (17) (18) (16) (16) (12) (14) (6) (7)

Spleen (12) (11) (14) (17) (14) (13) (6) (6)

Stato-acoustic Organ (17) (18) (19) (17) (14) (14) (6) (7)

Swim Bladder (17) (18) (19) (17) (14) (14) (6) (7)

Carcinoma 1 

Epithelioma 1 

Thymus (11) (7) (13) (11) (8) (9) (4) (5)

Thyroid Tissue (17) (17) (15) (17) (14) (13) (6) (7)

Urinary Bladder (16) (15) (13) (15) (10) (10) (4) (5)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE A4

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Stop-Exposure) 

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
102 Fish Studies, NTP TR 528

TABLE A5

Summary of the Incidences of Granuloma in Selected Tissues of Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Male

Number Examined Microscopically (61) (50) (41) (38)

Adipose Tissue

Heada 17 14 8 7

Retroperitoneal 18 9 5 2

Bone

Lower Jaw 1 0 0 0

Vertebra/Perivertebral 9 6 2 2

Cranial Cavity 7 8 7 2

Gill

Arches 11 9 2 2

Branchial Wall 2 2 1 1

Heart

Atrium 15 13 5 3

Ventricle 11 5 8 1

Mesentery 20 18 12 10

Peritoneum 3 7 3 0

Pharynx 10 7 3 1

Pseudobranch 5 4 2 1

Spinal Cord

Meninges 11 6 4 3

Spleen 14 10 5 3

Testis 17 15 6 3

Female

Number Examined Microscopically (61) (69) (58) (46)

Adipose Tissue

Head 22 16 14 3

Retroperitoneal 27 25 14 6

Bone

Lower Jaw 2 2 0 0

Vertebra/Perivertebral 12 10 2 2

Cranial Cavity 12 3 4 1

Eye

Choroid Rete 1 0 1 0

Choroid 10 6 2 4

Optic Nerve 4 0 1 0

Gill

Arches 12 8 4 0

Branchial Wall 3 4 2 0

Granuloma, NOS 1 2 0 0

Heart

Atrium 16 15 9 6

Bulbus Arteriousus 5 4 1 0

Ventricle 16 13 6 6
Fish Studies, NTP TR 528 103

TABLE A5

Summary of the Incidences of Granuloma in Selected Tissues of Guppies in the 16-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Female (continued)

Number Examined Microscopically (61) (69) (58) (46)

Mesentery 25 31 19 11

Peritoneum 5 7 3 1

Pharynx 10 6 5 1

Pseudobranch 2 2 0 0

Spinal Cord

Meninges 12 8 4 0

Granuloma, NOS 1 1 0 0

Skeletal Muscle 11 8 4 2

a Number of animals with lesion
104 Fish Studies, NTP TR 528
105

APPENDIX B

SUMMARY OF LESIONS IN MEDAKA

IN THE 14-MONTH WATERBORNE STUDY

OF 2,2-BIS(BROMOMETHYL)-1,3-PROPANEDIOL

TABLE B1 Summary of the Incidence of Neoplasms in Male Medaka 

in the 14-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

(Core Study) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

TABLE B2 Summary of the Incidence of Neoplasms in Male Medaka 

in the 14-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

(Stop-Exposure) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

TABLE B3 Summary of the Incidence of Neoplasms in Female Medaka 

in the 14-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

(Core Study) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

TABLE B4 Summary of the Incidence of Neoplasms in Female Medaka 

in the 14-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

(Stop-Exposure) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

TABLE B5 Summary of the Incidences of Granuloma and Granulomatous Inflammation 

in Selected Tissues of Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
106 Fish Studies, NTP TR 528

TABLE B1

Summary of the Incidence of Neoplasms in Male Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B

9-Month Interim Evaluation

Adipose Tissuea (1) (2) (0) (0) (1) (1) (2) (0)

Retroperitoneal, follicular cell carcinoma, 

invasiveb 1 

Kidney (5) (6) (1) (6) (5) (6) (6) (7)

Follicular cell carcinoma, invasive 1 

Pericardial Cavity (0) (1) (0) (0) (1) (0) (1) (0) 

Follicular cell carcinoma, invasive 1 

Skeletal Muscle (5) (6) (1) (6) (5) (6) (6) (7)

Follicular cell carcinoma, invasive 1 

Thyroid Tissue (5) (6) (1) (6) (5) (6) (6) (7)

Follicular cell carcinoma 1 

Tissues Examined at 9 Months with No Neoplasms Observed

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius 

Esophagus 

Eye

Gallbladder 

Gill 

Heart 

Hematopoietic Tissue 

Interrenal Tissue 

Intestine 

Liver

Mesentery 

Nares (Olfactory Tissue) 

Oral Cavity 

Pancreas

Peripheral Nerve 

Peritoneum 

Pineal Organ 

Pituitary Gland 

Pseudobranch 

Skin

Spinal Cord 

Spleen 

Stato-acoustic Organ 

Swim Bladder 

Testis 

Thymus 

Urinary Bladder 
Fish Studies, NTP TR 528 107

14-Month Study

Adipose Tissue (5) (10) (6) (3) (4) (2) (4) (1)

Blood Vessel (0) (3) (1) (2) (0) (0) (0) (0)

Bone (26) (21) (35) (24) (32) (24) (32) (27)

Brain (26) (21) (35) (24) (32) (24) (32) (27)

Chromaffin Tissue (26) (21) (35) (24) (31) (24) (32) (27)

Corpuscle of Stannius (16) (12) (21) (18) (10) (14) (20) (15)

Cranial Cavity (1) (1) (0) (1) (1) (0) (0) (0)

Esophagus (26) (21) (35) (24) (32) (24) (32) (27)

Eye (26) (21) (35) (24) (32) (24) (32) (27)

Gallbladder (25) (21) (32) (21) (32) (23) (32) (25)

Stromal polyp 1 1

Gill (26) (21) (35) (24) (32) (24) (32) (27)

Heart (26) (21) (35) (24) (32) (24) (32) (27)

Hematopoietic Tissue (26) (21) (35) (24) (32) (24) (32) (27)

Interrenal Tissue (26) (21) (35) (24) (31) (24) (32) (27)

Intestine (26) (21) (35) (24) (32) (24) (32) (27)

Carcinoma, invasive 1

Kidney (26) (21) (35) (24) (32) (24) (32) (27)

Liver (26) (21) (35) (24) (32) (24) (32) (27)

Cholangiocarcinoma 1 2

Hepatocellular adenoma 1 4 1

Hepatocellular adenoma, multiple 1

Hepatocellular carcinoma 1 1

Mesentery (5) (7) (4) (5) (1) (0) (5) (5)

Nares (Olfactory Tissue) (9) (5) (16) (8) (9) (11) (13) (10)

Oral Cavity (26) (21) (35) (24) (32) (24) (32) (27)

Pancreas (26) (21) (35) (24) (32) (24) (32) (27)

Carcinoma 1

TABLE B1

Summary of the Incidence of Neoplasms in Male Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
108 Fish Studies, NTP TR 528

14-Month Study (continued)

Pericardial Cavity (3) (7) (1) (0) (1) (1) (0) (1)

Peripheral Nerve (26) (21) (35) (24) (32) (24) (32) (27)

Peritoneum (2) (5) (0) (3) (3) (2) (0) (3)

Seminoma, invasive 1

Pharynx (1) (1) (0) (3) (0) (1) (1) (0)

Pineal Organ (23) (18) (33) (19) (22) (21) (30) (22)

Pituitary Gland (24) (18) (33) (23) (32) (22) (30) (24)

Pseudobranch (26) (21) (35) (24) (32) (24) (32) (26)

Skeletal Muscle (26) (21) (35) (24) (32) (24) (32) (27)

Skin (26) (21) (35) (24) (32) (24) (32) (27)

Spinal Cord (26) (21) (35) (24) (32) (24) (32) (27)

Spleen (15) (15) (30) (14) (15) (16) (12) (9)

Stato-acoustic Organ (26) (21) (35) (24) (32) (24) (32) (27)

Swim Bladder (26) (21) (35) (24) (32) (24) (32) (27)

Testis (26) (21) (35) (24) (32) (24) (32) (27)

Fibrosarcoma 1 1

Seminoma 3 1 2 5 2 3 2 7

Thymus (9) (11) (19) (14) (27) (9) (24) (21)

Thyroid Tissue (26) (21) (35) (24) (32) (24) (32) (27)

Follicular cell adenoma 1

Urinary Bladder (24) (19) (31) (22) (31) (23) (32) (23)

Multiple Organsc (26) (21) (35) (24) (32) (24) (32) (27)

a Number of animals examined microscopically at the site b Number of animals with neoplasm c Number of animals with any tissue examined microscopically

TABLE B1

Summary of the Incidence of Neoplasms in Male Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 109

TABLE B2

Summary of the Incidence of Neoplasms in Male Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Stop-Exposure)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B

Adipose Tissuea (1) (2) (0) (1) (1) (1) (3) (2) Retroperitoneal, liposarcomab 1 

Blood Vessel (0) (0) (1) (0) (0) (0) (0) (0)

Bone (16) (11) (11) (10) (16) (15) (17) (12)

Brain (16) (11) (11) (10) (16) (15) (17) (12)

Chromaffin Tissue (16) (11) (11) (10) (16) (14) (17) (12)

Corpuscle of Stannius (8) (6) (8) (10) (10) (12) (15) (7)

Esophagus (16) (11) (11) (10) (16) (15) (17) (12)

Eye (16) (11) (11) (10) (16) (15) (17) (12)

Gallbladder (14) (11) (11) (8) (16) (14) (16) (12)

Gill (16) (11) (11) (10) (16) (15) (17) (12)

Heart (16) (11) (11) (10) (16) (15) (17) (12)

Hematopoietic Tissue (16) (11) (11) (10) (16) (15) (17) (12)

Interrenal Tissue (16) (11) (11) (10) (16) (15) (17) (12)

Intestine (16) (11) (11) (10) (16) (15) (17) (12)

Kidney (16) (11) (11) (10) (16) (15) (17) (12)

Liver (16) (11) (11) (10) (16) (15) (17) (12)

Cholangiocarcinoma 1 

Hepatocellular adenoma 1 1 2 1 

Hepatocellular carcinoma 1 

Mesentery (3) (2) (2) (0) (3) (2) (4) (3)

Seminoma, invasive 1 

Seminoma, metastatic 1 

Nares (Olfactory Tissue) (6) (4) (3) (7) (8) (6) (10) (3)

Oral Cavity (16) (11) (11) (10) (16) (15) (17) (12)

Pancreas (16) (11) (11) (10) (16) (15) (17) (12)

Pericardial Cavity (2) (1) (0) (1) (0) (1) (0) (0)

Peripheral Nerve (16) (11) (11) (10) (16) (15) (17) (12)

Peritoneum (2) (0) (0) (0) (0) (0) (1) (1)

Pharynx (0) (0) (0) (0) (1) (0) (0) (0)
110 Fish Studies, NTP TR 528

Pineal Organ (11) (8) (9) (7) (14) (9) (15) (10)

Pituitary Gland (16) (11) (10) (10) (16) (14) (15) (10)

Pseudobranch (16) (11) (11) (10) (16) (15) (17) (12)

Skeletal Muscle (16) (11) (11) (10) (16) (15) (17) (12)

Skin (16) (11) (11) (10) (16) (15) (17) (12)

Spinal Cord (16) (11) (11) (10) (16) (15) (17) (12)

Spleen (9) (6) (5) (7) (8) (8) (10) (6)

Stato-acoustic Organ (16) (11) (11) (10) (16) (15) (17) (12)

Swim Bladder (16) (11) (11) (10) (16) (15) (17) (12)

Testis (16) (11) (11) (10) (16) (15) (17) (12)

Seminoma 3 1 1 4 2 2 2 

Thymus (10) (6) (8) (7) (10) (7) (10) (5)

Thyroid Tissue (16) (11) (11) (10) (16) (15) (17) (12)

Urinary Bladder (16) (11) (11) (10) (15) (15) (17) (12)

Neoplasm, NOS 1 

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE B2

Summary of the Incidence of Neoplasms in Male Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Stop-Exposure)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 111

9-Month Interim Evaluation

Pharynxa (0) (0) (2) (0) (0) (0) (0) (0)

Squamous cell carcinomab 1 

Tissues Examined at 9 Months with No Neoplasms Observed

Adipose Tissue 

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius

Esophagus 

Eye 

Gallbladder 

Gill 

Heart 

Hematopoietic Tissue 

Interrenal Tissue 

Intestine 

Kidney 

Liver 

Mesentery 

Nares (Olfactory Tissue)

Oral Cavity 

Ovary 

Pancreas 

Peripheral Nerve 

Pineal Organ 

Pituitary Gland 

Pseudobranch 

Skeletal Muscle 

Skin 

Spinal Cord 

Spleen 

Stato-acoustic Organ

Swim Bladder 

Thymus 

Thyroid Tissue 

Urinary Bladder 

14-Month Study

Adipose Tissue (3) (6) (4) (3) (0) (2) (1) (1)

Head, lymphosarcoma 1 1

Retroperitoneal, lymphosarcoma 1

Retroperitoneal, sarcoma, NOS 1

Blood Vessel (0) (1) (0) (0) (0) (0) (0) (0)

Bone (27) (32) (18) (29) (21) (29) (21) (26)

TABLE B3

Summary of the Incidence of Neoplasms in Female Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
112 Fish Studies, NTP TR 528

14-Month Study (continued)

Brain (27) (32) (18) (29) (21) (29) (21) (26)

Chromaffin Tissue (27) (31) (18) (29) (21) (28) (20) (26)

Corpuscle of Stannius (12) (19) (10) (20) (8) (14) (15) (20)

Cranial Cavity (0) (0) (0) (0) (0) (0) (1) (0)

Lymphosarcoma 1

Esophagus (27) (32) (18) (29) (21) (29) (21) (26)

Eye (27) (32) (18) (29) (21) (29) (21) (26)

Choroid rete, lymphosarcoma 1

Gallbladder (24) (31) (17) (27) (20) (28) (20) (25)

Stromal polyp 1

Gill (27) (32) (18) (29) (21) (29) (21) (26)

Hemangiosarcoma 1

Lymphosarcoma 1 1

Heart (27) (32) (18) (29) (21) (29) (21) (26)

Lymphosarcoma 1

Sarcoma, NOS 1 1

Hematopoietic Tissue (27) (32) (18) (29) (21) (29) (21) (26)

Lymphosarcoma 1 1

Interrenal Tissue (27) (31) (18) (29) (21) (28) (21) (26)

Intestine (27) (32) (18) (29) (21) (29) (21) (26)

Kidney (27) (32) (18) (29) (21) (29) (21) (26)

Lymphosarcoma 1

Liver (27) (32) (18) (29) (21) (29) (21) (26)

Cholangiocarcinoma 1

Cholangioma 1 1

Ectopic thyroid follicular cell carcinoma 1

Hepatocellular adenoma 2 3 1 2 2 4 2

Hepatocellular adenoma, multiple 1

Hepatocellular carcinoma 1 1

Lymphosarcoma 1

Mesentery (5) (10) (4) (8) (5) (5) (3) (8)

Hemangioma 1

TABLE B3

Summary of the Incidence of Neoplasms in Female Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 113

14-Month Study (continued)

Nares (Olfactory Tissue) (18) (17) (10) (10) (8) (13) (8) (6)

Oral Cavity (27) (32) (18) (29) (21) (29) (21) (26)

Submucosa, lymphosarcoma 1

Ovary (27) (32) (18) (29) (21) (29) (21) (26)

Neoplasm, NOS 1

Pancreas (27) (32) (18) (29) (21) (29) (21) (26)

Lymphosarcoma 1

Pericardial Cavity (2) (3) (0) (0) (0) (0) (1) (1)

Lymphosarcoma 1

Peripheral Nerve (27) (32) (18) (29) (21) (29) (21) (26)

Lymphosarcoma 1

Peritoneum (1) (3) (0) (3) (0) (0) (0) (0)

Pharynx (0) (2) (1) (0) (1) (0) (1) (1)

Lymphosarcoma 1

Pineal Organ (18) (25) (10) (23) (10) (19) (15) (15)

Pituitary Gland (19) (23) (17) (23) (18) (23) (16) (18)

Pseudobranch (27) (32) (18) (29) (21) (29) (21) (26)

Lymphosarcoma 1

Skeletal Muscle (27) (32) (18) (29) (21) (29) (21) (26)

Lymphosarcoma 1

Skin (27) (32) (18) (29) (21) (29) (21) (26)

Lymphosarcoma 1

Spinal Cord (26) (32) (18) (29) (21) (29) (21) (26)

Spleen (20) (16) (9) (17) (15) (19) (9) (15)

Hemangiosarcoma 1

Lymphosarcoma 1

Stato-acoustic Organ (27) (32) (18) (29) (21) (29) (21) (26)

Lymphosarcoma 1

TABLE B3

Summary of the Incidence of Neoplasms in Female Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
114 Fish Studies, NTP TR 528

14-Month Study (continued)

Swim Bladder (26) (32) (18) (29) (21) (29) (21) (26)

Thymus (9) (23) (7) (14) (19) (15) (12) (24)

Lymphosarcoma 1

Thyroid Tissue (27) (32) (18) (29) (21) (29) (21) (26)

Follicular cell adenoma

Urinary Bladder (24) (28) (15) (23) (14) (22) (18) (23)

Multiple Organsc (27) (32) (18) (29) (21) (29) (21) (26)

Lymphosarcoma 1 1

a Number of animals examined microscopically at the site b Number of animals with neoplasm c Number of animals with any tissue examined microscopically

TABLE B3

Summary of the Incidence of Neoplasms in Female Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Core Study)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 115

Adipose Tissuea (5) (6) (4) (1) (1) (2) (0) (4)

Blood Vessel (0) (0) (0) (0) (0) (1) (0) (0)

Bone (11) (15) (16) (17) (11) (12) (10) (15)

Brain (11) (15) (16) (17) (11) (12) (10) (15)

Chromaffin Tissue (11) (15) (16) (17) (11) (12) (10) (14)

Corpuscle of Stannius (5) (10) (10) (11) (8) (8) (4) (11)

Esophagus (11) (15) (16) (17) (11) (12) (10) (15)

Eye (11) (15) (16) (17) (11) (12) (10) (15)

Gallbladder (10) (11) (15) (16) (10) (11) (8) (12)

Gill (11) (15) (16) (17) (11) (12) (10) (15)

Heart (11) (15) (16) (17) (11) (12) (10) (15)

Hematopoietic Tissue (11) (15) (16) (17) (11) (12) (10) (15)

Interrenal Tissue (11) (15) (16) (17) (11) (12) (10) (14)

Intestine (11) (15) (16) (17) (11) (12) (10) (15)

Duct, carcinoma, invasiveb 1 

Kidney (11) (15) (16) (17) (11) (12) (10) (15)

Liver (11) (15) (16) (17) (11) (12) (10) (15)

Cholangiocarcinoma 1 1 

Hepatocellular adenoma 1 1 1 2 

Hepatocellular carcinoma 2 

Mesentery (2) (6) (3) (2) (4) (1) (1) (4)

Nares (Olfactory Tissue) (5) (7) (4) (8) (5) (4) (5) (7)

Oral Cavity (11) (15) (16) (17) (11) (12) (10) (15)

Ovary (11) (15) (16) (17) (11) (12) (10) (15)

Pancreas (11) (15) (16) (17) (11) (12) (10) (15)

Duct, carcinoma 1 

Pericardial Cavity (0) (1) (0) (0) (0) (0) (0) (2)

Peripheral Nerve (11) (15) (16) (17) (11) (12) (10) (15)

Neurofibrosarcoma 1 

Peritoneum (3) (0) (0) (0) (0) (0) (0) (0)

TABLE B4

Summary of the Incidence of Neoplasms in Female Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Stop-Exposure)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
116 Fish Studies, NTP TR 528

Pharynx (0) (2) (0) (1) (1) (1) (0) (1)

Pineal Organ (6) (11) (15) (12) (9) (8) (6) (11)

Pituitary Gland (10) (15) (15) (17) (9) (11) (10) (14)

Pseudobranch (11) (15) (16) (17) (11) (12) (10) (15)

Skeletal Muscle (11) (15) (16) (17) (11) (12) (10) (15)

Skin (11) (15) (16) (17) (11) (12) (10) (15)

Spinal Cord (11) (15) (16) (17) (11) (12) (9) (15)

Spleen (8) (6) (6) (13) (6) (6) (6) (10)

Stato-acoustic Organ (11) (15) (16) (17) (11) (12) (10) (15)

Swim Bladder (11) (15) (16) (17) (11) (12) (10) (15)

Gas gland, adenoma 1 

Thymus (6) (13) (11) (9) (8) (9) (6) (7)

Thyroid Tissue (11) (15) (16) (17) (11) (12) (10) (15)

Urinary Bladder (10) (14) (16) (14) (10) (11) (7) (12)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE B4

Summary of the Incidence of Neoplasms in Female Medaka in the 14-Month Waterborne Study 

of 2,2-Bis(bromomethyl)-1,3-propanediol (Stop-Exposure)

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 117

TABLE B5

Summary of the Incidences of Granuloma and Granulomatous Inflammation in Selected Tissues of Medaka 

in the 14-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Male 

Core Study

Number Examined Microscopically (47) (59) (56) (59)

Granulomaa

Adipose Tissue

Head 2 1 0 0

Heart

Atrium 14 4 2 0

Ventricle 8 2 2 0

Hematopoietic Tissue 16 11 4 0

Mesentery 8 1 1 0

Pericardial Cavity 7 1 2 0

Peritoneum 6 3 2 0

Pseudobranch 1 1 1 0

Spleen 7 1 0 0

Liver 28 12 12 0

Granulomatous Inflammation

Adipose Tissue

Head 6 4 3 0

Retroperitoneal 3 0 1 0

Mesentery 4 2 1 1

Oral Cavity Submucosa 7 7 1 2

Pericardial Cavity 3 0 0 1

Pseudobranch 12 6 0 0

Skin

Head 1 1 0 0

Lower Jaw 7 4 0 0

Upper Jaw 9 6 0 1

Stop-Exposure Study 

Number Examined Microscopically (27) (21) (31) (29)

Granuloma(s)

Hematopoietic Tissue 15 5 9 7

Heart

Atrium 8 1 4 0

Ventricle 6 0 1 0

Granulomatous Inflammation

Eye, Choroid Rete 5 0 1 0

Oral Cavity, Submucosa 11 3 4 4

Skin

Dorsum 1 0 0 0

Head 1 0 0 1

Lower Jaw 9 2 7 7

Upper Jaw 8 1 2 1
118 Fish Studies, NTP TR 528

TABLE B5

Summary of the Incidences of Granuloma and Granulomatous Inflammation in Selected Tissues of Medaka 

in the 14-Month Waterborne Study of 2,2-Bis(bromomethyl)-1,3-propanediol 

0 mg/L 24 mg/L 60 mg/L 150 mg/L

Female 

Core Study 

Number Examined Microscopically (59) (47) (50) (47)

Granulomatous Inflammation

Psuedobranch 7 1 1 0

Granuloma

Liver 9 1 7 3

Hematopoietic Tissue 4 1 1 1

Macrophage Aggregates

Heart

Stop-Exposure Study

Number Examined Microscopically (26) (33) (23) (25)

Granulomatous Inflammation

Mesentary 3 0 1 5

Granuloma

Mesentary 4 3 2 0

Liver 7 2 4 3

a Number of animals with lesion
119

APPENDIX C

SUMMARY OF LESIONS IN GUPPIES

IN THE 16-MONTH WATERBORNE STUDY

OF NITROMETHANE

TABLE C1 Summary of the Incidence of Neoplasms in Male Guppies 

in the 16-Month Waterborne Study of Nitromethane (Core Study) . . . . . . . . . . . . . . . . . . . 120

TABLE C2 Summary of the Incidence of Neoplasms in Male Guppies 

in the 16-Month Waterborne Study of Nitromethane (Stop-Exposure) . . . . . . . . . . . . . . . . . 123

TABLE C3 Summary of the Incidence of Neoplasms in Female Guppies 

in the 16-Month Waterborne Study of Nitromethane (Core Study) . . . . . . . . . . . . . . . . . . . 125

TABLE C4 Summary of the Incidence of Neoplasms in Female Guppies 

in the 16-Month Waterborne Study of Nitromethane (Stop-Exposure) . . . . . . . . . . . . . . . . . 128

TABLE C5 Summary of the Incidences of Selected Nonneoplastic Lesions in Guppies 

in the 16-Month Waterborne Study of Nitromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
120 Fish Studies, NTP TR 528

TABLE C1

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Group Group Group Groupa

A B A B A B A B

9-Month Interim Evaluation

Tissues Examined at 9 Months with No Neoplasms Observed

Adipose Tissue 

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius 

Esophagus 

Eye 

Gallbladder 

Gill 

Heart 

Hematopoietic Tissue 

Interrenal Tissue 

Intestine 

Kidney 

Liver 

Mesentery 

Nares (Olfactory Tissue)

Oral Cavity 

Pancreas 

Pericardial Cavity 

Peripheral Nerve 

Peritoneum 

Pharynx 

Pineal Organ 

Pituitary Gland 

Pseudobranch 

Skeletal Muscle 

Skin 

Spinal Cord 

Spleen 

Stato-acoustic Organ 

Swim Bladder 

Testis 

Thymus 

Thyroid Tissue 

Urinary Bladder 

16-Month Study

Adipose Tissueb (19) (11) (17) (17) (13) (17) (3) (1)

Blood Vessel (1) (0) (0) (1) (1) (0) (0) (0) 

Bone (30) (30) (22) (20) (31) (29) (8) (4)

Brain (30) (30) (22) (20) (31) (29) (8) (4)
Fish Studies, NTP TR 528 121

16-Month Study (continued)

Chromaffin Tissue (28) (28) (21) (18) (29) (28) (6) (4)

Corpuscle of Stannius (4) (8) (7) (8) (9) (10) (2) (1)

Cranial Cavity (0) (3) (3) (9) (3) (6) (0) (0) 

Esophagus (30) (30) (22) (20) (31) (29) (8) (4)

Eye (30) (30) (22) (20) (31) (29) (8) (4)

Gallbladder (28) (30) (22) (20) (28) (24) (8) (4)

Gill (30) (30) (22) (20) (31) (29) (8) (4)

Heart (30) (30) (22) (20) (31) (29) (8) (4)

Hematopoietic Tissue (30) (30) (22) (20) (31) (29) (8) (4)

Interrenal Tissue (28) (28) (21) (18) (29) (28) (6) (4)

Intestine (30) (30) (22) (20) (31) (29) (8) (4)

Kidney (30) (30) (22) (20) (31) (29) (8) (4)

Liver (30) (30) (22) (20) (31) (29) (8) (4)

Cholangiocarcinomac 1 

Hepatocellular adenoma 2 2 1 3 4 

Hepatocellular adenoma, multiple 1 1 

Hepatocellular carcinoma 1 2 

Mesentery (17) (16) (13) (18) (20) (15) (3) (0) 

Nares (Olfactory Tissue) (20) (15) (14) (12) (19) (16) (3) (3)

Oral Cavity (30) (30) (22) (20) (31) (29) (8) (4)

Pancreas (30) (30) (22) (20) (31) (29) (8) (4)

Pericardial Cavity (7) (5) (4) (7) (2) (5) (1)

Peripheral Nerve (30) (30) (22) (20) (31) (29) (8) (4)

Peritoneum (6) (0) (2) (4) (3) (2) (0) (0) 

Pharynx (5) (4) (6) (4) (4) (4) (1) (0) 

Pineal Organ (16) (16) (14) (13) (21) (19) (5) (3)

TABLE C1

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Group Group Group Group

A B A B A B A B
122 Fish Studies, NTP TR 528

16-Month Study (continued)

Pituitary Gland (27) (22) (18) (19) (22) (21) (5) (1)

Pseudobranch (30) (30) (22) (20) (31) (29) (8) (4)

Skeletal Muscle (30) (30) (22) (20) (31) (29) (8) (4)

Skin (30) (30) (22) (20) (31) (29) (8) (4)

Spinal Cord (30) (29) (21) (19) (31) (27) (8) (3)

Spleen (29) (29) (22) (19) (28) (29) (7) (3)

Stato-acoustic Organ (30) (30) (22) (20) (31) (29) (8) (4)

Swim Bladder (30) (30) (22) (20) (31) (29) (8) (4)

Epithelioma 1 

Gas gland, adenoma 1 

Testis (30) (30) (22) (20) (31) (29) (8) (4)

Thymus (0) (0) (1) (0) (1) (0) (0) (0)

Thyroid Tissue (30) (28) (22) (20) (25) (28) (8) (3)

Urinary Bladder (29) (28) (20) (20) (30) (29) (7) (4)

a The 70 mg/L group was removed and examined at 57 weeks. The control, 10, and 30 mg/L groups were removed and examined at 70 weeks. b Number of animals examined microscopically at the site c Number of animals with neoplasm

TABLE C1

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 123

Adipose Tissuea (7) (11) (12) (9) (7) (10) (4) (6)

Blood Vessel (0) (0) (0) (0) (0) (1) (0) (0)

Bone (12) (17) (16) (13) (16) (13) (7) (7)

Brain (12) (17) (16) (13) (16) (13) (7) (7)

Chromaffin Tissue (11) (17) (15) (13) (16) (12) (7) (7)

Corpuscle of Stannius (3) (3) (6) (7) (6) (4) (1) (2)

Cranial Cavity (0) (4) (3) (5) (3) (3) (1) (3)

Esophagus (12) (17) (16) (13) (16) (13) (7) (7)

Eye (12) (17) (16) (13) (16) (13) (7) (7)

Gallbladder (11) (16) (13) (12) (15) (13) (7) (6)

Adenoma, papillaryb 1 

Carcinoma 1 

Gill (12) (17) (16) (13) (16) (13) (7) (7)

Heart (12) (17) (16) (13) (16) (13) (7) (7)

Hematopoietic Tissue (12) (17) (16) (13) (16) (13) (7) (7)

Interrenal Tissue (11) (17) (15) (13) (16) (12) (7) (7)

Intestine (12) (17) (16) (13) (16) (13) (7) (7)

Kidney (12) (17) (16) (13) (16) (13) (7) (7)

Liver (12) (17) (16) (13) (16) (13) (7) (7)

Carcinoma, invasive 1 

Cholangioma 1 

Hepatocellular adenoma 2 2 1 1 1 

Hepatocellular adenoma, multiple 1 

Hepatocellular carcinoma 1 2 

Mesentery (5) (11) (12) (8) (12) (8) (2) (2)

Nares (Olfactory Tissue) (9) (10) (12) (9) (10) (8) (5) (3)

Oral Cavity (12) (17) (16) (13) (16) (13) (7) (7)

Pancreas (12) (17) (16) (13) (16) (13) (7) (7)

Pericardial Cavity (2) (2) (6) (5) (3) (7) (0) (3)

TABLE C2

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of Nitromethane (Stop-Exposure)

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Group Group Group Group

A B A B A B A B
124 Fish Studies, NTP TR 528

Peripheral Nerve (12) (17) (16) (13) (16) (13) (7) (7)

Schwannoma 1 

Peritoneum (1) (0) (0) (1) (0) (1) (0) (0)

Pharynx (3) (1) (3) (2) (2) (1) (0) (0)

Pineal Organ (8) (9) (11) (7) (13) (10) (4) (6)

Pituitary Gland (7) (9) (15) (11) (12) (13) (4) (5)

Adenoma 1 1 

Pseudobranch (12) (17) (16) (13) (16) (13) (7) (7)

Skeletal Muscle (12) (17) (16) (13) (16) (13) (7) (7)

Skin (12) (17) (16) (13) (16) (13) (7) (7)

Spinal Cord (12) (17) (15) (13) (15) (13) (7) (6)

Spleen (8) (14) (16) (13) (15) (13) (6) (7)

Stato-acoustic Organ (12) (17) (16) (13) (16) (13) (7) (7)

Swim Bladder (12) (17) (16) (13) (16) (13) (7) (7)

Epithelioma 1 

Testis (12) (17) (16) (13) (16) (13) (7) (7)

Thymus (1) (0) (0) (0) (0) (0) (2) (0)

Thyroid Tissue (8) (11) (16) (13) (16) (12) (7) (7)

Urinary Bladder (12) (15) (16) (12) (16) (13) (7) (7)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE C2

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of Nitromethane (Stop-Exposure) 

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 125

9-Month Interim Evaluation

Swim Bladdera (4) (5) (5) (6) (5) (4) (6) (6)

Epitheliomab 1 

Tissues Examined at 9 Months with No Neoplasms Observed

Adipose Tissue 

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius 

Esophagus 

Eye 

Gallbladder 

Gill 

Heart 

Hematopoietic Tissue 

Interrenal Tissue 

Intestine 

Kidney 

Liver 

Mesentery 

Nares (Olfactory Tissue)

Oral Cavity 

Ovary

Pancreas 

Pericardial Cavity 

Peripheral Nerve 

Peritoneum 

Pharynx 

Pineal Organ 

Pituitary Gland 

Pseudobranch 

Skeletal Muscle 

Skin 

Spinal Cord 

Spleen 

Stato-acoustic Organ 

Swim Bladder 

Thymus 

Thyroid Tissue 

Urinary Bladder 

TABLE C3

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Group Group Group Group

A B A B A B A B
126 Fish Studies, NTP TR 528

16-Month Study

Adipose Tissue (15) (19) (26) (22) (16) (22) (8) (14)

Blood Vessel (1) (2) (1) (0) (2) (0) (0) (1)

Bone (24) (23) (32) (25) (20) (30) (15) (17)

Brain (24) (23) (32) (25) (20) (30) (15) (17)

Chromaffin Tissue (21) (16) (23) (24) (12) (21) (10) (12)

Corpuscle of Stannius (2) (10) (8) (7) (5) (8) (1) (6)

Cranial Cavity (2) (2) (8) (11) (6) (6) (3) (2)

Esophagus (24) (23) (32) (25) (20) (30) (15) (17)

Eye (24) (23) (32) (25) (20) (30) (15) (17)

Gallbladder (20) (19) (21) (18) (16) (26) (14) (15)

Gill (24) (23) (32) (25) (20) (30) (15) (17)

Heart (24) (23) (32) (25) (20) (30) (14) (17)

Hematopoietic Tissue (24) (23) (32) (25) (20) (30) (15) (17)

Sarcoma, NOS, invasivec 1 

Interrenal Tissue (21) (17) (24) (24) (12) (21) (10) (13)

Intestine (24) (23) (32) (25) (20) (30) (15) (17)

Sarcoma, NOS 1 

Kidney (24) (23) (32) (25) (20) (30) (15) (17)

Liver (24) (23) (32) (25) (20) (30) (15) (17)

Cholangiocarcinoma 1 

Hepatocellular adenoma 1 2 2 

Sarcoma, NOS, invasive 1 

Mesentery (15) (22) (30) (25) (18) (27) (12) (15)

Nares (Olfactory Tissue) (18) (15) (28) (18) (14) (25) (10) (12)

Oral Cavity (24) (23) (32) (25) (20) (30) (15) (17)

TABLE C3

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Group Group Group Groupa

A B A B A B A B
Fish Studies, NTP TR 528 127

16-Month Study (continued)

Ovary (23) (23) (32) (25) (20) (30) (15) (17)

Sarcoma, NOS, invasive 1 

Pancreas (24) (23) (32) (25) (20) (30) (15) (17)

Pericardial Cavity (4) (6) (10) (4) (2) (7) (0) (1)

Peripheral Nerve (24) (23) (32) (25) (20) (30) (15) (17)

Schwannoma 1 

Peritoneum (2) (2) (3) (3) (3) (4) (0) (1)

Pharynx (4) (3) (4) (4) (7) (0) (1) (1)

Pineal Organ (6) (5) (9) (5) (7) (16) (5) (11)

Pituitary Gland (8) (14) (25) (18) (18) (27) (12) (15)

Pseudobranch (23) (20) (30) (25) (19) (29) (15) (16)

Skeletal Muscle (24) (23) (32) (25) (20) (30) (15) (17)

Skin (24) (23) (32) (25) (20) (30) (15) (17)

Spinal Cord (14) (21) (32) (25) (20) (30) (11) (16)

Spleen (19) (17) (28) (22) (18) (25) (9) (14)

Stato-acoustic Organ (24) (23) (32) (25) (20) (30) (15) (17)

Swim Bladder (23) (23) (31) (25) (20) (30) (15) (17)

Epithelioma 2 

Gas gland, adenoma 1 

Thymus (21) (17) (25) (20) (14) (24) (8) (14)

Thyroid Tissue (15) (22) (32) (23) (20) (30) (15) (17)

Urinary Bladder (14) (12) (15) (18) (8) (15) (9) (13)

a The 70 mg/L group was removed and examined at 57 weeks. The control, 10, and 30 mg/L groups were removed and examined at 70 weeks. b Number of animals examined microscopically at the site c Number of animals with neoplasm

TABLE C3

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Group Group Group Group

A B A B A B A B
128 Fish Studies, NTP TR 528

Adipose Tissuea (8) (11) (10) (16) (12) (13) (10) (5)

Blood Vessel (0) (1) (0) (0) (1) (0) (0) (0)

Bone (16) (14) (13) (21) (17) (16) (16) (9)

Brain (16) (14) (13) (21) (17) (16) (16) (9)

Chromaffin Tissue (12) (11) (11) (19) (13) (11) (11) (9)

Corpuscle of Stannius (6) (4) (3) (6) (4) (2) (3) (2)

Cranial Cavity (3) (6) (4) (8) (1) (5) (6) (7)

Esophagus (16) (14) (13) (21) (17) (16) (16) (9)

Eye (16) (14) (13) (21) (17) (16) (16) (9)

Gallbladder (15) (13) (12) (21) (16) (11) (15) (7)

Carcinomab 1 

Gill (16) (14) (13) (21) (17) (16) (16) (9)

Heart (16) (14) (13) (21) (17) (16) (16) (9)

Hematopoietic Tissue (16) (14) (13) (21) (17) (16) (16) (9)

Interrenal Tissue (12) (11) (11) (19) (13) (11) (11) (9)

Intestine (16) (14) (13) (21) (17) (16) (16) (9)

Kidney (16) (14) (13) (21) (17) (16) (16) (9)

Liver (16) (14) (13) (21) (17) (16) (16) (9)

Carcinoma, invasive 1 

Hepatocellular adenoma 1 

Hepatocellular adenoma, multiple 1 

Hepatocellular carcinoma 1 

Hepatocholangiocarcinoma 1 

Mesentery (8) (11) (11) (16) (17) (14) (15) (7)

Nares (Olfactory Tissue) (12) (11) (12) (16) (11) (8) (11) (4)

Oral Cavity (16) (14) (13) (21) (17) (16) (16) (9)

Ovary (16) (14) (13) (21) (17) (16) (16) (9)

Pancreas (16) (14) (13) (21) (17) (16) (16) (9)

Pericardial Cavity (2) (4) (2) (6) (4) (4) (1) (1)

TABLE C4

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of Nitromethane (Stop-Exposure)

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 129

Peripheral Nerve (16) (14) (13) (21) (17) (16) (16) (9)

Sarcoma, NOS 1 

Peritoneum (0) (1) (1) (4) (0) (3) (0) (0)

Pharynx (1) (3) (2) (3) (3) (3) (2) (2)

Pineal Organ (13) (10) (0) (5) (6) (4) (10) (6)

Pituitary Gland (15) (12) (7) (15) (10) (7) (12) (6)

Pseudobranch (14) (12) (13) (18) (16) (15) (16) (8)

Skeletal Muscle (16) (14) (13) (21) (17) (16) (16) (9)

Skin (16) (14) (13) (21) (17) (16) (16) (9)

Spinal Cord (16) (14) (12) (20) (17) (13) (15) (8)

Spleen (12) (11) (10) (21) (17) (16) (16) (8)

Hemangiosarcoma 1 

Stato-acoustic Organ (16) (14) (13) (21) (17) (16) (16) (9)

Swim Bladder (16) (14) (13) (21) (17) (16) (16) (9)

Thymus (15) (9) (12) (14) (17) (13) (11) (7)

Thyroid Tissue (16) (14) (13) (21) (17) (16) (16) (9)

Urinary Bladder (9) (7) (8) (15) (12) (11) (12) (1)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE C4

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of Nitromethane (Stop-Exposure)

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Group Group Group Group

A B A B A B A B
130 Fish Studies, NTP TR 528

TABLE C5

Summary of the Incidences of Selected Nonneoplastic Lesions in Guppies in the 16-Month Waterborne Study 

of Nitromethane

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Male 

Core Study

Number Examined Microscopically (60) (42) (60) (12)

Granuloma

Adipose Tissue

Heada 11 16 16 1

Retroperitoneal 22 14 14 2

Cranial Cavity 1 9 5 0

Eye

Choroid Rete 3 2 7 0

Choroid 10 8 4 1

Cornea 1 0 0 0

Iris 2 0 0 0

Retina 1 0 1 0

Swim Bladder

Gas Gland 3 5 8 1

Gill 1 0 0 0

Arches 3 12 8 1

Branchial Wall 3 3 5 0

Heart

Atrium 15 11 20 1

Bulbus Arteriosus 3 0 2 0

Ventricle 13 8 10 1

Hematopoietic Tissue 42 35 56 5

Skeletal Muscles 11 10 13 1

Stop-Exposure Study 

Number Examined Microscopically (29) (29) (29) (14)

Granuloma

Pericardial Cavity 3 10 8 3

Mononuclear Cell Infiltrate

Urinary Bladder 1 9 7 3

Female 

Core Study 

Number Examined Microscopically (47) (57) (50) (32)

Granuloma

Adipose Tissue

Head 16 35 26 9

Retroperitoneal 20 28 25 12

Cranial Cavity 0 12 8 4

Eye

Choroid Rete 4 5 13 1

Choroid 1 16 3 4

Cornea 0 0 1 0

Iris 0 0 1 0

Retina 0 1 2 0
Fish Studies, NTP TR 528 131

TABLE C5

Summary of the Incidences of Selected Nonneoplastic Lesions in Guppies in the 16-Month Waterborne Study 

of Nitromethane

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Female (continued)

Core Study (continued)

Number Examined Microscopically (47) (57) (50) (32)

Granuloma (continued)

Swim Bladder

Gas Gland 10 19 15 5

Gill 0 2 2 1

Arches 5 20 16 2

Branchial Wall 1 8 7 2

Heart

Atrium 8 17 25 2

Bulbus Arteriosus 1 6 5 2

Ventricle 9 16 20 1

Hematopoietic Tissue 31 45 42 25

Intestine

Serosa 0 1 0 0

Submucosa 4 20 10 2

Ovary 18 39 30 5

Embryo 0 1 0 0

Oviduct 2 6 5 2

Peripheral Nerve 3 6 10 1

Skeletal Muscle 7 22 13 5

Spinal Cord 0 1 0 0

Meninges 3 12 15 2

Spleen 13 28 17 2

Liver 13 24 23 7

Macrophage Aggregates

Adipose Tissue

Head 2 3 2 0

Retroperitoneal 16 29 27 15

Cranial Cavity 1 10 6 1

Heart

Atrium 29 43 40 23

Bulbus Arteriosus 0 1 0 0

Ventricle 6 11 12 0

Ovary 0 23 11 2

Skeletal Muscle 1 0 0 0

Swim Bladder

Gas Gland 4 4 0 1

Wall 4 13 4 1

Mononuclear Cell Infiltrate

Adipose Tissue

Head 6 3 1 0

Perivertebral 1 0 0 0

Retroperitoneal 3 0 0 0

Cranial Cavity 2 1 0 0

Gill

Arches 31 53 40 17

Branchial Wall 5 12 7 6

Infiltrate Cellular 4 4 3 1
132 Fish Studies, NTP TR 528

TABLE C5

Summary of the Incidences of Selected Nonneoplastic Lesions in Guppies in the 16-Month Waterborne Study 

of Nitromethane

0 mg/L 10 mg/L 30 mg/L 70 mg/L

Female (continued)

Core Study (continued)

Number Examined Microscopically (47) (57) (50) (32)

Mononuclear Cell Infiltrate (continued)

Heart

Atrium, Focal 1 1 2 1

Atrium, Multifocal 11 7 2 3

Bulbus Arteriosus 0 4 1 2

Ventricle, Focal 1 1 3 0

Ventricle, Multifocal 12 6 10 1

Intestine 2 10 8 2

Ovary 0 1 1 1

Skeletal Muscle

Focal 1 2 6 1

Multifocal 1 12 5 2

Swim Bladder

Gas Gland 3 4 2 3

Stop-Exposure Study

Number Examined Microscopically (30) (34) (33) (25)

Granuloma

Ovary 12 20 15 12

Oviduct 4 3 7 4

Macrophage Aggregates

Ovary 10 22 11 11

a Number of animals with lesion
133

APPENDIX D

SUMMARY OF LESIONS IN MEDAKA

IN THE 13-MONTH WATERBORNE STUDY

OF NITROMETHANE

TABLE D1 Summary of the Incidence of Neoplasms in Male Medaka 

in the 13-Month Waterborne Study of Nitromethane (Core Study) . . . . . . . . . . . . . . . . . . . 134

TABLE D2 Summary of the Incidence of Neoplasms in Male Medaka 

in the 13-Month Waterborne Study of Nitromethane (Stop-Exposure) . . . . . . . . . . . . . . . . . 138

TABLE D3 Summary of the Incidence of Neoplasms in Female Medaka 

in the 13-Month Waterborne Study of Nitromethane (Core Study) . . . . . . . . . . . . . . . . . . . 140

TABLE D4 Summary of the Incidence of Neoplasms in Female Medaka 

in the 13-Month Waterborne Study of Nitromethane (Stop-Exposure) . . . . . . . . . . . . . . . . . 144

TABLE D5 Summary of the Incidences of Selected Nonneoplastic Lesions in Medaka 

in the 13-Month Waterborne Study of Nitromethane (Core Study) . . . . . . . . . . . . . . . . . . . 146
134 Fish Studies, NTP TR 528

9-Month Interim Evaluation

Livera (5) (7) (6) (9) (7) (7) (7) (7)

Cholangiocarcinomab 1 

Hepatocellular adenoma 1 1 

Pseudobranch (5) (7) (6) (9) (7) (7) (7) (7)

Hemangioma 1 

Swim Bladder (5) (7) (6) (9) (7) (7) (7) (7)

Epithelioma 1 

Tissues Examined at 9 Months with No Neoplasms Observed

Adipose Tissue 

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius 

Esophagus 

Eye 

Gallbladder 

Gill 

Heart 

Hematopoietic Tissue 

Interrenal Tissue 

Intestine 

Kidney 

Mesentery 

Nares (Olfactory Tissue)

Oral Cavity 

Pancreas 

Pericardial Cavity 

Peripheral Nerve 

Peritoneum 

Pharynx 

Pineal Organ 

Pituitary Gland 

Skeletal Muscle 

Skin 

Spinal Cord 

Spleen 

Stato-acoustic Organ 

Testis 

Thymus 

Thyroid Tissue 

Urinary Bladder 

TABLE D1

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 135

13-Month Study

Adipose Tissue (15) (12) (6) (9) (9) (14) (7) (6)

Head, lymphosarcoma 1 

Retroperitoneal, lymphosarcoma 1 

Blood Vessel (0) (0) (1) (0) (1) (5) (1) (1)

Bone (41) (31) (48) (43) (46) (44) (36) (40)

Brain (41) (31) (48) (43) (46) (44) (36) (40)

Chromaffin Tissue (41) (30) (48) (42) (45) (44) (36) (40)

Corpuscle of Stannius (15) (15) (28) (18) (24) (22) (21) (23)

Cranial Cavity (0) (0) (0) (1) (0) (1) (0) (0)

Esophagus (41) (31) (48) (43) (46) (44) (36) (40)

Eye (41) (31) (48) (43) (46) (44) (36) (40)

Lymphosarcoma 1 1 

Gallbladder (40) (31) (48) (42) (45) (43) (34) (38)

Gill (41) (31) (48) (43) (46) (44) (36) (40)

Lymphosarcoma 1 

Heart (41) (31) (48) (43) (46) (44) (36) (40)

Lymphosarcoma 1 1 

Hematopoietic Tissue (41) (31) (48) (43) (46) (44) (36) (40)

Lymphosarcoma 1 1 

Interrenal Tissue (41) (30) (48) (42) (45) (44) (36) (40)

Intestine (41) (31) (48) (43) (46) (44) (36) (40)

Lymphosarcoma 1 

Kidney (41) (31) (48) (43) (46) (44) (36) (40)

Lymphosarcoma 1 

Liver (41) (31) (48) (43) (46) (44) (36) (40)

Cholangiocarcinoma 1 1 1 1 

Cholangioma 1 

Hemangiopericytoma 1 

Hepatocellular adenoma 1 1 1 1 

Hepatocellular adenoma, multiple 1 

Lymphosarcoma 1 

TABLE D1

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of Nitromethane (Core Study) 

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
136 Fish Studies, NTP TR 528

13-Month Study (continued)

Mesentery (13) (8) (4) (7) (7) (11) (9) (7)

Lymphosarcoma 1 1 

Nares (Olfactory Tissue) (25) (10) (7) (14) (23) (16) (22) (24)

Oral Cavity (41) (31) (48) (43) (46) (44) (36) (40)

Pancreas (41) (31) (48) (43) (46) (44) (36) (40)

Lymphosarcoma 1 

Pericardial Cavity (7) (7) (1) (1) (1) (1) (3) (0)

Carcinoma, invasive 1 

Peripheral Nerve (41) (31) (48) (43) (46) (44) (36) (40)

Peritoneum (3) (1) (2) (1) (1) (1) (1) (1)

Pharynx (0) (2) (1) (0) (0) (3) (2) (0)

Pineal Organ (28) (23) (40) (22) (31) (31) (26) (23)

Pituitary Gland (31) (26) (43) (31) (27) (31) (26) (32)

Pseudobranch (41) (31) (48) (43) (46) (44) (36) (40)

Lymphosarcoma 1 

Skeletal Muscle (41) (31) (48) (43) (46) (44) (36) (40)

Carcinoma, invasive 1 

Lymphosarcoma 1 

Skin (41) (31) (48) (43) (46) (44) (36) (40)

Lymphosarcoma 1 

Spinal Cord (40) (30) (48) (43) (45) (44) (36) (38)

Spleen (37) (25) (17) (36) (40) (35) (31) (27)

Lymphosarcoma 1 1 

Seminoma, metastatic

Stato-acoustic Organ (41) (31) (48) (43) (46) (44) (36) (40)

Swim Bladder (41) (31) (48) (43) (46) (44) (36) (40)

Testis (41) (31) (48) (43) (46) (44) (36) (40)

Hemangioma 1 

Lymphosarcoma 1 

Seminoma 1 2 2 1 1 3 

Thymus (18) (13) (21) (11) (28) (14) (9) (15)

Lymphosarcoma 1 

TABLE D1

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of Nitromethane (Core Study) 

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 137

13-Month Study (continued)

Thyroid Tissue (41) (31) (48) (43) (46) (44) (36) (40)

Carcinoma 1 1 

Urinary Bladder (31) (26) (46) (36) (38) (35) (33) (38)

Multiple Organsc (41) (31) (48) (43) (46) (44) (36) (40)

Lymphosarcoma 1 1 

a Number of animals examined microscopically at the site b Number of animals with neoplasm c Number of animals with any tissue examined microscopically

TABLE D1

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of Nitromethane (Core Study) 

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
138 Fish Studies, NTP TR 528

Adipose Tissuea (4) (7) (1) (1) (4) (4) (3) (5)

Retroperitoneal, schwannoma, malignant, 

invasiveb 1 

Blood Vessel (0) (1) (0) (0) (1) (0) (0) (0)

Bone (27) (20) (20) (15) (20) (16) (22) (19)

Brain (27) (20) (20) (15) (20) (16) (22) (19)

Chromaffin Tissue (27) (20) (20) (15) (19) (16) (21) (19)

Corpuscle of Stannius (11) (7) (12) (5) (11) (8) (11) (8)

Cranial Cavity (0) (0) (0) (0) (0) (1) (0) (0)

Esophagus (27) (20) (20) (15) (20) (16) (22) (19)

Schwannoma, malignant, invasive 1 

Eye (27) (20) (20) (15) (20) (16) (22) (19)

Choroid rete, seminoma, metastatic 1 

Gallbladder (25) (20) (19) (15) (20) (15) (21) (19)

Gill (27) (20) (20) (15) (20) (16) (22) (19)

Heart (27) (19) (20) (15) (20) (16) (22) (19)

Hematopoietic Tissue (27) (20) (20) (15) (20) (16) (22) (19)

Interrenal Tissue (27) (20) (20) (15) (19) (16) (21) (19)

Intestine (27) (20) (20) (15) (20) (16) (22) (19)

Sarcoma, NOS, invasive 1 

Kidney (27) (20) (20) (15) (20) (16) (22) (19)

Schwannoma, malignant, invasive 1 

Liver (27) (20) (20) (15) (20) (16) (22) (19)

Mesentery (5) (10) (6) (5) (6) (5) (6) (4)

Sarcoma, NOS 1 

Schwannoma, malignant, invasive 1 

Nares (Olfactory Tissue) (10) (5) (6) (4) (2) (4) (9) (5)

Oral Cavity (27) (20) (20) (15) (20) (16) (22) (19)

Pancreas (27) (19) (20) (15) (20) (16) (22) (19)

Pericardial Cavity (1) (3) (0) (1) (4) (2) (0) (0)

TABLE D2

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of Nitromethane (Stop-Exposure) 

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 139

Peripheral Nerve (27) (20) (20) (15) (20) (16) (22) (19)

Peritoneum (2) (2) (0) (0) (4) (0) (0) (1)

Schwannoma, malignant 1 

Pharynx (0) (0) (0) (0) (0) (1) (2) (0)

Pineal Organ (19) (18) (13) (8) (15) (11) (17) (15)

Pituitary Gland (19) (15) (10) (8) (17) (10) (14) (17)

Pseudobranch (27) (20) (20) (15) (20) (16) (22) (19)

Skeletal Muscle (27) (20) (20) (15) (20) (16) (22) (19)

Schwannoma, malignant, invasive 1 

Skin (27) (20) (20) (15) (20) (16) (22) (19)

Ventrum, sarcoma, NOS, invasive 1 

Spinal Cord (26) (20) (20) (14) (20) (16) (21) (19)

Spleen (15) (17) (13) (9) (6) (12) (10) (5)

Stato-acoustic Organ (27) (20) (20) (15) (20) (16) (22) (19)

Swim Bladder (27) (20) (20) (15) (19) (16) (22) (19)

Testis (27) (20) (20) (15) (20) (16) (22) (19)

Schwannoma, malignant, invasive 1 

Seminoma 1 1 1 2 2 

Thymus (12) (10) (4) (8) (16) (7) (8) (14)

Thyroid Tissue (27) (20) (20) (15) (20) (16) (22) (19)

Urinary Bladder (24) (15) (20) (15) (19) (15) (19) (14)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE D2

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of Nitromethane (Stop-Exposure) 

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
140 Fish Studies, NTP TR 528

9-Month Interim Evaluation

Adipose Tissuea (0) (0) (0) (0) (1) (3) (1) (1)

Head, lymphosarcomab 1 1 

Retroperitoneal, lymphosarcoma 1 1 

Cranial Cavity (0) (0) (0) (0) (0) (0) (1) (0)

Lymphosarcoma 1 

Esophagus (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma 1 

Eye (7) (6) (10) (5) (4) (5) (10) (10)

Choroid rete, lymphosarcoma 1 

Lymphosarcoma 1 

Gill (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma 1 1 

Heart (7) (6) (10) (4) (4) (5) (10) (10)

Lymphosarcoma 1 1 

Hematopoietic Tissue (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma - 1 1 

Intestine (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma 1 

Liver (7) (6) (10) (5) (4) (5) (10) (10)

Hepatocellular adenoma 1 

Lymphosarcoma 1 

Mesentery (1) (0) (0) (0) (0) (2) (2) (1)

Lymphosarcoma 1 

Seminoma, metastatic 1 

Oral Cavity (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma 1 

Ovary (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma 1 

Seminoma 1 

Peripheral Nerve (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma 1 

Pharynx (0) (0) (0) (0) (1) (1) (2) (0)

Lymphosarcoma 1 1 

TABLE D3

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 141

9-Month Interim Evaluation (continued)

Pseudobranch (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma 1 

Skeletal Muscle (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma 1 1 

Skin (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma 1 1 

Spleen (6) (5) (9) (4) (4) (5) (6) (7)

Lymphosarcoma 1 

Stato-acoustic Organ (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma 1 

Swim Bladder (7) (6) (10) (5) (4) (5) (10) (10)

Epithelioma 1 

Thymus (2) (2) (5) (3) (3) (2) (6) (5)

Lymphosarcoma 1 1 

Multiple Organs (7) (6) (10) (5) (4) (5) (10) (10)

Lymphosarcoma 1 1 

Tissues Examined at 9 Months with No Neoplasms Observed

Blood Vessel 

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius 

Gallbladder 

Interrenal Tissue 

Kidney 

Nares (Olfactory Tissue) 

Pancreas 

Pineal Organ 

Pituitary Gland 

Spinal Cord 

Thyroid Tissue 

Urinary Bladder 

TABLE D3

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
142 Fish Studies, NTP TR 528

13-Month Study 

Adipose Tissue (6) (13) (8) (7) (6) (9) (4) (3)

Retroperitoneal, dysgerminoma, metastatic 1 

Blood Vessel (0) (0) (3) (1) (0) (0) (1) (0)

Bone (33) (41) (31) (38) (35) (24) (35) (35)

Brain (33) (41) (31) (38) (35) (24) (35) (35)

Chromaffin Tissue (33) (41) (31) (37) (35) (24) (34) (35)

Corpuscle of Stannius (11) (16) (13) (16) (20) (11) (17) (19)

Cranial Cavity (1) (0) (0) (0) (0) (0) (0) (0)

Esophagus (33) (41) (31) (38) (35) (24) (35) (35)

Eye (33) (41) (31) (38) (35) (24) (35) (35)

Choroid rete, seminoma, metastatic 1 

Gallbladder (32) (40) (31) (37) (32) (22) (32) (34)

Gill (33) (41) (31) (38) (35) (24) (35) (35)

Lymphosarcoma

Heart (33) (41) (31) (38) (35) (24) (35) (34)

Lymphosarcoma

Hematopoietic Tissue (33) (41) (31) (38) (35) (24) (35) (35)

Dysgerminoma, metastatic 1 

Interrenal Tissue (33) (41) (31) (37) (35) (24) (34) (35)

Intestine (33) (41) (31) (38) (35) (24) (35) (35)

Schwannoma, malignant, Invasive 1 

Kidney (33) (41) (31) (38) (35) (24) (35) (35)

Liver (33) (41) (31) (38) (35) (24) (35) (35)

Cholangiocarcinoma 1 

Hepatocellular adenoma 1 1 2 1 1 2 

Hepatocellular carcinoma 1 

Seminoma, metastatic 1 

Mesentery (7) (8) (9) (18) (11) (9) (11) (5)

Dysgerminoma, metastatic 1 

Schwannoma, malignant, invasive 1 

Seminoma, metastatic 1 

TABLE D3

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 143

13-Month Study (continued)

Nares (Olfactory Tissue) (26) (20) (11) (17) (21) (11) (20) (26)

Oral Cavity (33) (41) (31) (38) (35) (24) (35) (35)

Ovary (33) (41) (31) (38) (35) (24) (35) (35)

Dysgerminoma 1 1 

Schwannoma, malignant, invasive 1 

Seminoma 1 1 1 1 

Pancreas (33) (41) (31) (38) (35) (24) (35) (35)

Pericardial Cavity (3) (2) (0) (1) (1) (2) (1) (0)

Carcinoma, invasive

Peripheral Nerve (33) (41) (31) (38) (35) (24) (35) (35)

Peritoneum (0) (0) (0) (1) (0) (1) (0) (1)

Schwannoma, malignant 1 

Seminoma, metastatic 1 

Pharynx (5) (5) (2) (2) (3) (2) (0) (0)

Pineal Organ (22) (28) (21) (22) (20) (16) (24) (23)

Pituitary Gland (23) (31) (23) (22) (22) (12) (23) (23)

Pseudobranch (33) (41) (31) (38) (35) (24) (35) (35)

Skeletal Muscle (33) (41) (31) (38) (35) (24) (35) (35)

Skin (33) (41) (31) (38) (35) (24) (35) (35)

Spinal Cord (32) (41) (31) (36) (35) (24) (35) (33)

Spleen (29) (30) (15) (30) (26) (19) (31) (21)

Dysgerminoma, metastatic 1 

Seminoma, metastatic 1 

Stato-acoustic Organ (33) (41) (31) (38) (35) (24) (35) (35)

Swim Bladder (33) (41) (31) (38) (35) (24) (35) (35)

Thymus (19) (18) (9) (15) (19) (6) (11) (20)

Thyroid Tissue (33) (41) (31) (38) (35) (24) (35) (35)

Urinary Bladder (17) (28) (24) (23) (27) (16) (23) (23)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE D3

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
144 Fish Studies, NTP TR 528

Adipose Tissuea (8) (6) (11) (6) (5) (8) (5) (3)

Retroperitoneal, seminoma, metastaticb 1 

Blood Vessel (0) (0) (0) (2) (0) (0) (1) (1)

Bone (20) (24) (23) (28) (21) (21) (25) (19)

Brain (20) (24) (23) (28) (21) (21) (25) (19)

Chromaffin Tissue (20) (24) (20) (26) (20) (19) (25) (19)

Corpuscle of Stannius (7) (7) (9) (13) (9) (10) (12) (10)

Cranial Cavity (0) (0) (0) (0) (0) (0) (0) (1)

Esophagus (20) (24) (23) (28) (21) (21) (25) (19)

Eye (20) (24) (23) (28) (21) (21) (25) (19)

Gallbladder (17) (24) (22) (28) (21) (21) (22) (19)

Gill (20) (24) (23) (28) (21) (21) (25) (19)

Heart (20) (24) (23) (28) (21) (21) (25) (19)

Hematopoietic Tissue (20) (24) (23) (28) (21) (21) (25) (19)

Interrenal Tissue (20) (24) (20) (26) (20) (19) (25) (19)

Intestine (20) (24) (23) (28) (21) (21) (25) (19)

Mesothelioma 1 

Kidney (20) (24) (23) (28) (21) (21) (25) (19)

Liver (20) (24) (23) (28) (21) (21) (25) (19)

Cholangiocarcinoma 1 

Hepatocellular adenoma 1 2 1 1 

Hepatocellular carcinoma 1 

Mesentery (11) (11) (8) (12) (8) (5) (10) (5)

Hemangioma 1 

Sarcoma, NOS

Seminoma, metastatic 1 1 

Nares (Olfactory Tissue) (9) (16) (15) (21) (10) (15) (12) (10)

Oral Cavity (20) (24) (23) (28) (21) (21) (25) (19)

Ovary (20) (24) (23) (28) (21) (21) (25) (19)

Sarcoma, NOS 1 

Seminoma 1 2 

TABLE D4

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of Nitromethane (Stop-Exposure) 

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 145

Pancreas (20) (24) (23) (28) (21) (21) (25) (19)

Pericardial Cavity (1) (0) (1) (1) (0) (0) (1) (0) 

Seminoma, metastatic 1 

Peripheral Nerve (20) (24) (23) (28) (21) (21) (25) (19)

Peritoneum (0) (0) (1) (2) (0) (0) (0) (0) 

Seminoma, metastatic 1 1 

Pharynx (1) (0) (0) (0) (1) (1) (1) (1)

Pineal Organ (12) (14) (6) (9) (16) (10) (15) (14)

Pituitary Gland (8) (12) (5) (14) (20) (8) (18) (12)

Pseudobranch (20) (24) (23) (28) (21) (21) (25) (19)

Skeletal Muscle (20) (24) (23) (28) (21) (21) (25) (19)

Seminoma, invasive 1 

Skin (20) (24) (23) (28) (21) (21) (25) (19)

Spinal Cord (19) (24) (23) (22) (20) (21) (24) (17)

Spleen (12) (18) (14) (24) (8) (16) (15) (5)

Seminoma, metastatic 1 

Stato-acoustic Organ (20) (24) (23) (28) (21) (21) (25) (19)

Swim Bladder (20) (24) (23) (28) (21) (21) (25) (19)

Gas gland, adenoma 1 

Thymus (6) (11) (9) (15) (16) (10) (14) (12)

Thyroid Tissue (20) (24) (23) (28) (21) (21) (25) (19)

Urinary Bladder (14) (12) (10) (21) (18) (16) (17) (13)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE D4

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of Nitromethane (Stop-Exposure) 

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Group Group Group Group

A B A B A B A B
146 Fish Studies, NTP TR 528

TABLE D5

Summary of the Incidences of Selected Nonneoplastic Lesions in Medaka in the 13-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Male 

Number Examined Microscopically (72) (91) (90) (76)

Granuloma 

Adipose Tissuea

Head 0 0 1 0

Retroperitoneal 2 0 1 1

Heart

Atrium 47 8 20 6

Ventricle 25 2 10 0

Hematopoietic Tissue 48 10 39 13

Pericardial Cavity 11 1 0 1

Liver 64 19 49 10

Granulomatous Inflammation

Adipose Tissue

Head 21 3 14 3

Eye

Choroid Rete 16 1 6 3

Optic Nerve 1 0 1 0

Right, Inflammation 1 0 0 0

Granuloma, NOS 1 0 1 0

Oral Cavity Submucosa 31 9 18 2

Pericardial Cavity 2 1 2 1

Pseudobranch 35 7 22 6

Skeletal Muscle

Focal 8 1 5 2

Multifocal 18 1 12 4

Skin

Fin 1 0 0 0

Head 1 0 0 0

Multifocal 1 0 0 0

Lower Jaw 61 15 51 15

Upper Jaw 48 17 42 6

Mononuclear Cell Infiltrate

Oral Cavity Mucosa 29 19 17 7
Fish Studies, NTP TR 528 147

TABLE D5

Summary of the Incidences of Selected Nonneoplastic Lesions in Medaka in the 13-Month Waterborne Study 

of Nitromethane (Core Study)

0 mg/L 10 mg/L 20 mg/L 40 mg/L

Female 

Number Examined Microscopically (74) (69) (59) (70)

Granuloma

Adipose Tissue

Head 1 1 0 0

Retroperitoneal 2 0 2 2

Heart

Atrium 16 1 7 2

Ventricle 7 0 3 0

Hematopoietic Tissue 16 2 12 4

Ovary 5 3 1 6

Pericardial Cavity 2 0 1 0

Liver 25 6 14 5

Granulomatous Inflammation

Adipose Tissue

Head 9 2 7 3

Eye

Choroid Rete 7 1 3 0

Oral Cavity Submucosa 2 1 0 0

Ovary 5 5 3 0

Pericardial Cavity 2 1 2 0

Pseudobranch 15 5 6 4

Skeletal Muscle

Focal 2 1 1 1

Multifocal 6 0 4 0

Skin

Lower Jaw 3 1 4 2

Upper Jaw 1 1 2 0

Mononuclear Cell Infiltrate

Oral Cavity Mucosa 6 3 3 2

a Number of animals with lesion
148 Fish Studies, NTP TR 528
149

APPENDIX E

SUMMARY OF LESIONS IN GUPPIES

IN THE 16-MONTH WATERBORNE STUDY

OF 1,2,3-TRICHLOROPROPANE

TABLE E1 Summary of the Incidence of Neoplasms in Male Guppies 

in the 16-Month Waterborne Study of 1,2,3-Trichloropropane (Core Study) . . . . . . . . . . . 150

TABLE E2 Summary of the Incidence of Neoplasms in Male Guppies 

in the 16-Month Waterborne Study of 1,2,3-Trichloropropane (Stop-Exposure) . . . . . . . . 153

TABLE E3 Summary of the Incidence of Neoplasms in Female Guppies 

in the 16-Month Waterborne Study of 1,2,3-Trichloropropane (Core Study) . . . . . . . . . . . 155

TABLE E4 Summary of the Incidence of Neoplasms in Female Guppies 

in the 16-Month Waterborne Study of 1,2,3-Trichloropropane (Stop-Exposure) . . . . . . . . 158

TABLE E5 Summary of Incidences of Granuloma in Selected Tissues of Guppies 

in the 16-Month Waterborne Study of 1,2,3-Trichloropropane . . . . . . . . . . . . . . . . . . . . . . . 160
150 Fish Studies, NTP TR 528

TABLE E1

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B

9-Month Interim Evaluation

Hematopoietic Tissuea (4) (5) (3) (5) (5) (7) (4) (6)

Lymphosarcomab 1 

Liver (4) (5) (3) (5) (5) (7) (4) (6)

Hepatocellular adenoma 1 

Hepatocellular adenoma, multiple 1 

Multiple Organsc (4) (5) (3) (5) (5) (7) (4) (6)

Lymphosarcoma 1 

Tissues Examined at 9 Months with No Neoplasms Observed

Adipose Tissue 

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius 

Esophagus 

Eye 

Gallbladder 

Gill 

Heart 

Interrenal Tissue 

Intestine 

Kidney 

Mesentery 

Nares (Olfactory Tissue)

Oral Cavity 

Pancreas 

Pericardial Cavity 

Peripheral Nerve 

Peritoneum 

Pharynx 

Pineal Organ 

Pituitary Gland 

Pseudobranch 

Skeletal Muscle 

Skin 

Spinal Cord 

Spleen 

Stato-acoustic Organ 

Swim Bladder 

Testis 

Thymus 

Thyroid Tissue 

Urinary Bladder 
Fish Studies, NTP TR 528 151

16-Month Study

Adipose Tissue (19) (25) (11) (16) (23) (23) (6) (12)

Blood Vessel (0) (0) (0) (0) (1) (4) (0) (0)

Bone (27) (34) (20) (27) (33) (34) (9) (18)

Vertebra, osteofibroma 1 

Brain (27) (34) (20) (27) (33) (34) (9) (18)

Chromaffin Tissue (26) (32) (20) (26) (31) (30) (8) (18)

Corpuscle of Stannius (7) (12) (4) (6) (7) (10) (5) (8)

Cranial Cavity (3) (4) (4) (6) (6) (12) (3) (1)

Esophagus (27) (34) (20) (27) (33) (34) (9) (18)

Eye (27) (34) (20) (27) (33) (34) (9) (18)

Gallbladder (25) (34) (19) (25) (30) (29) (7) (17)

Carcinoma 1 

Gill (27) (34) (20) (27) (33) (34) (9) (18)

Heart (27) (34) (20) (27) (32) (34) (9) (18)

Hematopoietic Tissue (27) (34) (20) (27) (33) (34) (9) (18)

Interrenal Tissue (26) (32) (20) (26) (31) (30) (8) (18)

Intestine (27) (34) (20) (27) (33) (34) (9) (18)

Carcinoma, invasive 1 

Kidney (27) (34) (20) (27) (33) (34) (9) (18)

Liver (27) (34) (20) (27) (33) (34) (9) (18)

Cholangiocarcinoma 3 

Cholangioma 1 

Hepatocellular adenoma 2 2 5 8 4 1 4 

Hepatocellular adenoma, multiple 1 1 2 3 4 

Hepatocellular carcinoma 1 2 5 

Hepatocholangiocarcinoma 2 1 1 1 

Mesentery (18) (19) (9) (17) (17) (20) (3) (5)

Nares (Olfactory Tissue) (12) (17) (5) (15) (19) (25) (6) (10)

TABLE E1

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
152 Fish Studies, NTP TR 528

16-Month Study (continued)

Oral Cavity (27) (34) (20) (27) (33) (34) (9) (18)

Pancreas (27) (34) (20) (27) (33) (34) (9) (18)

Carcinoma 1 

Pericardial Cavity (4) (6) (1) (3) (4) (7) (2) (1)

Peripheral Nerve (27) (34) (20) (27) (33) (34) (9) (18)

Peritoneum (5) (7) (2) (1) (4) (3) (1) (1)

Pharynx (7) (5) (5) (8) (3) (7) (1) (1)

Pineal Organ (16) (25) (14) (20) (21) (26) (6) (14)

Pituitary Gland (21) (26) (15) (23) (28) (28) (9) (13)

Pseudobranch (26) (34) (20) (27) (33) (34) (9) (18)

Skeletal Muscle (27) (34) (20) (27) (33) (34) (9) (18)

Skin (27) (34) (20) (27) (33) (34) (9) (18)

Spinal Cord (27) (34) (20) (27) (31) (31) (9) (18)

Spleen (26) (34) (19) (26) (30) (33) (8) (18)

Stato-acoustic Organ (27) (34) (20) (27) (33) (34) (9) (18)

Swim Bladder (27) (34) (20) (27) (33) (34) (9) (18)

Carcinoma 1 

Testis (27) (34) (20) (27) (33) (34) (9) (18)

Thymus (2) (2) (2) (5) (1) (2) (1)

Thyroid Tissue (27) (33) (20) (27) (33) (34) (9) (18)

Urinary Bladder (25) (33) (20) (26) (31) (32) (9) (15)

a Number of animals examined microscopically at the site b Number of animals with neoplasm c Number of animals with any tissue examined microscopically

TABLE E1

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 153

Adipose Tissuea (12) (8) (7) (6) (10) (7) (3) (6)

Bone (17) (15) (10) (9) (15) (11) (9) (10)

Brain (17) (15) (10) (9) (15) (11) (9) (10)

Chromaffin Tissue (17) (14) (9) (9) (14) (11) (9) (8)

Corpuscle of Stannius (10) (6) (5) (3) (7) (3) (1) -

Cranial Cavity (5) (3) (2) (2) (5) (2) (2) (3)

Esophagus (17) (15) (10) (9) (15) (11) (9) (10)

Eye (17) (15) (10) (9) (15) (11) (9) (10)

Gallbladder (17) (15) (8) (9) (15) (9) (9) (9)

Gill (17) (15) (10) (9) (15) (11) (9) (10)

Heart (17) (15) (10) (9) (15) (11) (9) (10)

Hematopoietic Tissue (17) (15) (10) (9) (15) (11) (9) (10)

Interrenal Tissue (17) (14) (9) (9) (14) (11) (9) (8)

Intestine (17) (15) (10) (9) (15) (11) (9) (10)

Kidney (17) (15) (10) (9) (15) (11) (9) (10)

Liver (17) (15) (10) (9) (15) (11) (9) (10)

Cholangiocarcinomab 1 

Hepatocellular adenoma 3 3 1 2 2 2 3 

Hepatocellular adenoma, multiple 1 1 1 

Hepatocellular carcinoma 1 4 

Mesentery (15) (9) (7) (8) (12) (8) (8) (7)

Nares (Olfactory Tissue) (6) (5) (4) (6) (6) (4) (2) (3)

Oral Cavity (17) (15) (10) (9) (15) (11) (9) (10)

Pancreas (17) (15) (10) (9) (15) (11) (9) (10)

Pericardial Cavity (5) (2) (3) (3) (0) (3) (1) (1)

Peripheral Nerve (17) (15) (10) (9) (15) (11) (9) (10)

Peritoneum (4) (4) (2) (2) (2) (0) (0) (0)

Pharynx (12) (2) (6) (7) (5) (2) (2) (1)

TABLE E2

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane (Stop-Exposure)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
154 Fish Studies, NTP TR 528

Pineal Organ (10) (9) (8) (5) (12) (8) (8) (8)

Pituitary Gland (10) (11) (7) (6) (14) (7) (8) (7)

Pseudobranch (17) (15) (10) (9) (15) (11) (9) (10)

Skeletal Muscle (17) (15) (10) (9) (15) (11) (9) (10)

Fibrosarcoma 1 

Skin (17) (15) (10) (9) (15) (11) (9) (10)

Spinal Cord (17) (14) (10) (9) (15) (10) (9) (9)

Spleen (17) (15) (9) (8) (15) (11) (9) (10)

Hemangiosarcoma 1 

Stato-acoustic Organ (17) (15) (10) (9) (15) (11) (9) (10)

Swim Bladder (17) (15) (10) (9) (15) (11) (9) (10)

Carcinoma 1 

Testis (17) (15) (10) (9) (15) (11) (9) (10)

Thymus (0) (0) (0) (0) (1) (0) (1) (2)

Thyroid Tissue (17) (15) (10) (9) (15) (11) (9) (8)

Urinary Bladder (17) (14) (10) (9) (15) (11) (9) (10)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE E2

Summary of the Incidence of Neoplasms in Male Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane (Stop-Exposure)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 155

9-Month Interim Evaluation

Tissues Examined at 9 Months with No Neoplasms Observed

Adipose Tissue 

Blood Vessel 

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius 

Cranial Cavity 

Esophagus 

Eye 

Gallbladder 

Gill 

Heart 

Hematopoietic Tissue 

Interrenal Tissue 

Intestine 

Kidney 

Liver 

Mesentery 

Nares (Olfactory Tissue) 

Oral Cavity 

Ovary 

Pancreas 

Pericardial Cavity 

Peripheral Nerve 

Peritoneum 

Pharynx 

Pineal Organ 

Pituitary Gland 

Pseudobranch 

Skeletal Muscle 

Skin 

Spinal Cord 

Spleen 

Stato-acoustic Organ 

Swim Bladder 

Thymus 

Thyroid Tissue 

Urinary Bladder 

TABLE E3

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
156 Fish Studies, NTP TR 528

16-Month Study

Adipose Tissuea (22) (20) (20) (23) (17) (16) (13) (9)

Blood Vessel (1) (0) (1) (1) (3) (3) (2) (0)

Bone (36) (28) (33) (31) (25) (22) (19) (14)

Brain (36) (28) (33) (30) (25) (22) (19) (14)

Chromaffin Tissue (33) (18) (28) (27) (17) (15) (14) (13)

Corpuscle of Stannius (5) (3) (7) (3) (5) (2) (7) (1)

Cranial Cavity (4) (8) (8) (7) (1) (3) (1) (2)

Esophagus (36) (28) (33) (30) (25) (22) (19) (14)

Eye (36) (28) (33) (31) (25) (22) (19) (14)

Gallbladder (35) (26) (31) (29) (22) (22) (17) (13)

Gill (36) (28) (33) (31) (25) (22) (19) (14)

Heart (35) (28) (29) (29) (25) (20) (19) (14)

Hematopoietic Tissue (36) (28) (33) (31) (25) (22) (19) (14)

Interrenal Tissue (33) (19) (28) (27) (17) (15) (14) (13)

Intestine (36) (28) (33) (31) (25) (22) (19) (14)

Cholangiocarcinoma, invasiveb 1 

Kidney (36) (28) (33) (31) (25) (22) (19) (14)

Liver (36) (28) (33) (31) (25) (22) (19) (14)

Cholangiocarcinoma 4 2 

Cholangioma 3 2 1 1 

Cholangioma, multiple 1 1 

Hepatocellular adenoma 3 2 1 2 2 1 2 3 

Hepatocellular adenoma, multiple 1 2 

Hepatocellular carcinoma 1 2 

Hepatocholangiocarcinoma 2 

Mesentery (22) (19) (21) (20) (18) (17) (11) (11)

Cholangiocarcinoma, metastatic 1 

Nares (Olfactory Tissue) (20) (16) (21) (20) (20) (15) (12) (8)

Oral Cavity (35) (28) (33) (30) (25) (22) (19) (14)

Ovary (36) (28) (33) (30) (24) (22) (19) (14)

TABLE E3

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 157

16-Month Study (continued)

Pancreas (36) (28) (33) (31) (25) (22) (19) (14)

Carcinoma 1 1 

Islet cells, adenoma 1 1 

Pericardial Cavity (10) (4) (3) (6) (3) (6) (2) (3)

Peripheral Nerve (36) (28) (33) (31) (25) (22) (19) (14)

Peritoneum (7) (6) (3) (2) (0) (4) (2) (1)

Pharynx (11) (7) (11) (11) (6) (14) (1) (2)

Pineal Organ (23) (9) (11) (9) (16) (7) (12) (2)

Pituitary Gland (33) (22) (19) (17) (22) (15) (19) (12)

Adenoma 1 

Pseudobranch (34) (21) (31) (29) (23) (18) (13) (13)

Skeletal Muscle (36) (28) (33) (31) (25) (22) (19) (14)

Skin (36) (28) (33) (31) (25) (22) (19) (14)

Spinal Cord (32) (19) (21) (19) (18) (15) (16) (5)

Spleen (25) (16) (22) (23) (16) (12) (14) (11)

Cholangiocarcinoma, invasive 1 

Stato-acoustic Organ (36) (28) (33) (31) (25) (22) (19) (14)

Swim Bladder (36) (28) (33) (30) (25) (22) (19) (14)

Carcinoma 1 1 

Epithelioma 2 1 1 

Thymus (23) (15) (26) (24) (19) (17) (12) (7)

Thyroid Tissue (34) (28) (30) (29) (25) (18) (19) (14)

Transverse Septum (0) (0) (0) (0) (1) (0) (0) (0)

Urinary Bladder (21) (16) (22) (14) (16) (12) (15) (6)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE E3

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
158 Fish Studies, NTP TR 528

Adipose Tissuea (7) (14) (11) (12) (9) (12) (11) (8)

Blood Vessel (1) (0) (0) (1) (1) (0) (2) (2)

Bone (14) (17) (16) (15) (14) (17) (15) (12)

Brain (14) (17) (16) (15) (14) (17) (15) (12)

Chromaffin Tissue (11) (16) (14) (13) (12) (13) (11) (11)

Corpuscle of Stannius (2) (3) (5) (2) (1) (3) (4) (2)

Cranial Cavity (3) (2) (3) (3) (3) (4) (6) (4)

Esophagus (14) (17) (16) (15) (14) (17) (15) (12)

Eye (14) (17) (16) (15) (14) (17) (15) (12)

Gallbladder (13) (17) (16) (15) (13) (17) (15) (12)

Carcinomab 1 

Gill (14) (17) (16) (15) (14) (17) (15) (12)

Heart (13) (17) (16) (14) (14) (16) (15) (12)

Hematopoietic Tissue (14) (17) (16) (15) (14) (17) (15) (12)

Interrenal Tissue (11) (17) (15) (14) (12) (14) (11) (11)

Intestine (14) (17) (16) (15) (14) (17) (15) (12)

Kidney (14) (17) (16) (15) (14) (17) (15) (12)

Adenoma, tubular 1 

Liver (14) (17) (16) (15) (14) (17) (15) (12)

Carcinoma, invasive 1

Cholangiocarcinoma 3 2 

Cholangioma 1 1 2 1 

Cholangioma, multiple 1 

Hepatocellular adenoma 2 1 1 2 

Hepatocellular adenoma, multiple 1 

Hepatocellular carcinoma 1 

Mesentery (11) (14) (11) (14) (11) (15) (10) (12)

Nares (Olfactory Tissue) (7) (7) (7) (8) (11) (10) (10) (10)

Oral Cavity (14) (17) (16) (15) (14) (17) (15) (12)

Ovary (14) (17) (16) (15) (14) (17) (15) (12)

Pancreas (14) (17) (16) (15) (14) (17) (15) (12)

TABLE E4

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane (Stop-Exposure)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 159

Pericardial Cavity (2) (6) (5) (4) (5) (4) (4) (2)

Peripheral Nerve (14) (17) (16) (15) (14) (17) (15) (12)

Peritoneum (2) (4) (2) (2) (1) (4) (3) (0)

Cholangiocarcinoma, invasive 1 

Pharynx (8) (11) (9) (9) (4) (12) (6) (6)

Pineal Organ (8) (10) (9) (9) (10) (7) (11) (3)

Pituitary Gland (12) (12) (14) (12) (13) (10) (15) (10)

Pseudobranch (10) (17) (16) (14) (12) (16) (12) (10)

Skeletal Muscle (14) (17) (16) (15) (14) (17) (15) (12)

Skin (14) (17) (16) (15) (14) (17) (15) (12)

Spinal Cord (11) (14) (13) (10) (11) (11) (15) (10)

Spleen (6) (17) (12) (8) (9) (9) (11) (5)

Stato-acoustic Organ (14) (17) (16) (15) (14) (17) (15) (12)

Swim Bladder (14) (17) (16) (15) (14) (17) (15) (12)

Epithelioma 2 

Thymus (8) (12) (13) (11) (11) (15) (9) (7)

Thyroid Tissue (14) (15) (16) (12) (14) (15) (15) (12)

Urinary Bladder (10) (10) (14) (9) (8) (8) (7) (3)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE E4

Summary of the Incidence of Neoplasms in Female Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane (Stop-Exposure)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
160 Fish Studies, NTP TR 528

TABLE E5

Summary of Incidences of Granuloma in Selected Tissues of Guppies in the 16-Month Waterborne Study 

of 1,2,3-Trichloropropane

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Male

Core Study

Number Examined Microscopically (61) (47) (67) (27)

Hearta

Atrium 21 10 16 3

Testis

Granuloma, NOS 35 19 24 9

Sperm 30 24 41 18

Stop Exposure

Number Examined Microscopically (32) (19) (26) (19)

Adipose Tissue

Head 14 10 9 3

Vertebra and Perivertebral Area 8 8 9 1

Eye, Choroid 11 5 7 4

Heart, Atrium 10 5 6 6

Mesentery 18 8 15 8

Skeletal Muscle 8 5 7 2

Testis 11 10 11 11

Female

Core Study

Number Examined Microscopically (64) (64) (47) (33)

Ovary

Granuloma, NOS 46 24 20 12

a Number of animals with lesion
161

APPENDIX F

SUMMARY OF LESIONS IN MEDAKA

IN THE 13-MONTH WATERBORNE STUDY

OF 1,2,3-TRICHLOROPROPANE

TABLE F1 Summary of the Incidence of Neoplasms in Male Medaka 

in the 13-Month Waterborne Study of 1,2,3-Trichloropropane (Core Study) . . . . . . . . . . . 162

TABLE F2 Summary of the Incidence of Neoplasms in Male Medaka 

in the 13-Month Waterborne Study of 1,2,3-Trichloropropane (Stop-Exposure) . . . . . . . . 166

TABLE F3 Summary of the Incidence of Neoplasms in Female Medaka 

in the 13-Month Waterborne Study of 1,2,3-Trichloropropane (Core Study) . . . . . . . . . . . 168

TABLE F4 Summary of the Incidence of Neoplasms in Female Medaka 

in the 13-Month Waterborne Study of 1,2,3-Trichloropropane (Stop-Exposure) . . . . . . . . 172
162 Fish Studies, NTP TR 528

TABLE F1

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B

9-Month Interim Evaluation

Livera (5) (5) (5) (5) (6) (5) (7) (6)

Cholangiocarcinomab 1 1 3 2 6 2 

Hepatocellular adenoma 1 

Hepatocholangiocarcinoma 1 

Gallbladder (5) (5) (5) (4) (6) (5) (7) (6)

Adenoma, papillary 1 1 

Tissues Examined at 9 Months with No Neoplasms Observed

Adipose Tissue 

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius 

Esophagus 

Eye 

Gill 

Heart 

Hematopoietic Tissue 

Interrenal Tissue 

Intestine 

Kidney 

Mesentery 

Nares (Olfactory Tissue) 

Oral Cavity 

Pancreas 

Peripheral Nerve 

Pineal Organ 

Pituitary Gland 

Pseudobranch 

Skeletal Muscle 

Skin 

Spinal Cord 

Spleen 

Stato-acoustic Organ 

Swim Bladder 

Testis 

Thymus 

Thyroid Tissue 

Urinary Bladder 
Fish Studies, NTP TR 528 163

13-Month Study

Adipose Tissue (7) (7) (8) (3) (4) (10) (6) (4)

Head, lymphosarcoma 1 

Retroperitoneal, cholangiocarcinoma, invasive 1 2 

Retroperitoneal, hepatocholangiocarcinoma,

invasive 1 

Retroperitoneal, lymphosarcoma 1 1 

Bone (52) (43) (44) (50) (38) (27) (36) (42)

Brain (52) (43) (44) (50) (38) (27) (36) (42)

Chromaffin Tissue (50) (43) (44) (49) (38) (27) (36) (42)

Corpuscle of Stannius (34) (19) (15) (30) (21) (12) (16) (24)

Cranial Cavity (0) (1) (0) (0) (0) (2) (1) (0)

Lymphosarcoma 1 

Esophagus (52) (43) (44) (50) (38) (27) (36) (42)

Cholangiocarcinoma, invasive 1 

Eye (52) (43) (44) (50) (38) (27) (36) (42)

Choroid rete, cholangiocarcinoma, metastatic 1 

Choroid rete, lymphosarcoma 1 

Lymphosarcoma 1 

Gallbladder (49) (42) (39) (44) (35) (26) (35) (37)

Adenoma, papillary 2 2 6 3 

Carcinoma 1 

Gill (52) (43) (44) (50) (38) (27) (36) (42)

Carcinoma, scirrhous, invasive 1 

Lymphosarcoma 1 

Heart (52) (43) (43) (50) (38) (27) (36) (42)

Lymphosarcoma 1 

Sinus venosus, cholangiocarcinoma, invasive 1 1 1 

Hematopoietic Tissue (52) (43) (44) (50) (38) (27) (36) (42)

Lymphosarcoma 1 1 1 

Interrenal Tissue (50) (43) (44) (49) (38) (27) (36) (42)

Intestine (52) (43) (44) (50) (38) (27) (36) (42)

Adenomatous polyp 1 

Cholangiocarcinoma, invasive 1 

Lymphosarcoma 1 

Wall, sarcoma, NOS, invasive 1 

TABLE F1

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
164 Fish Studies, NTP TR 528

13-Month Study (continued)

Kidney (52) (43) (44) (50) (38) (27) (36) (42)

Cholangiocarcinoma, metastatic 1 1 

Lymphosarcoma 1 

Renal tubules, adenoma 1 

Renal tubules, carcinoma 1 

Liver (52) (43) (44) (50) (38) (27) (36) (42)

Cholangiocarcinoma 17 15 26 17 19 26 

Cholangioma 5 1 2 1 

Hepatocellular adenoma 1 3 

Hepatocellular carcinoma 2 3 

Hepatocholangiocarcinoma 1 1 3 3 

Lymphosarcoma 1 

Mesentery (10) (14) (6) (10) (6) (14) (3) (5)

Lymphosarcoma 1 1 

Nares (Olfactory Tissue) (24) (19) (20) (18) (12) (11) (15) (18)

Lymphosarcoma 1 

Oral Cavity (52) (43) (44) (50) (38) (27) (36) (42)

Pancreas (52) (43) (44) (50) (38) (27) (36) (42)

Lymphosarcoma 1 

Pericardial Cavity (4) (2) (2) (2) (1) (4) (2) (1)

Lymphosarcoma 1 

Peripheral Nerve (52) (43) (44) (50) (38) (27) (36) (42)

Carcinoma, scirrhous, invasive 1 

Lymphosarcoma 1 

Peritoneum (1) (2) (1) (0) (0) (1) (1) (1)

Sarcoma, NOS 1 

Pharynx (4) (1) (2) (1) (1) (1) (1) (0)

Carcinoma, scirrhous 1 

Pineal Organ (27) (29) (29) (40) (26) (13) (29) (37)

Pituitary Gland (48) (31) (35) (48) (34) (21) (35) (39)

Pseudobranch (52) (43) (44) (50) (38) (27) (36) (42)

Skeletal Muscle (52) (43) (44) (50) (38) (27) (36) (42)

Carcinoma, scirrhous, invasive 1 

Lymphosarcoma 1 

TABLE F1

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 165

13-Month Study (continued)

Skin (52) (43) (44) (50) (38) (27) (36) (42)

Lymphosarcoma 1 

Spinal Cord (50) (43) (44) (49) (38) (27) (36) (42)

Spleen (32) (34) (35) (30) (23) (21) (12) (27)

Cholangiocarcinoma, metastatic 4 2 

Hepatocholangiocarcinoma, metastatic 1 

Lymphosarcoma 1 

Stato-acoustic Organ (52) (43) (44) (50) (38) (27) (36) (42)

Lymphosarcoma 1 

Swim Bladder (52) (43) (44) (50) (38) (27) (36) (42)

Testis (52) (43) (44) (50) (38) (27) (36) (42)

Germ cell neoplasm 1 1 

Lymphosarcoma 1 

Sarcoma, NOS, invasive 1 

Seminoma 1 3 1 2 

Thymus (19) (20) (15) (25) (16) (11) (22) (20)

Lymphosarcoma 1 

Thyroid Tissue (52) (43) (44) (50) (38) (27) (36) (42)

Carcinoma, scirrhous, invasive 1 

Urinary Bladder (46) (42) (40) (43) (32) (21) (36) (41)

Multiple Organsc (52) (43) (44) (50) (38) (27) (36) (42)

Lymphosarcoma 1 1 1 

a Number of animals examined microscopically at the site b Number of animals with neoplasm c Number of animals with any tissue examined microscopically

TABLE F1

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
166 Fish Studies, NTP TR 528

Adipose Tissuea (4) (4) (3) (1) (0) (3) (0) (2)

Head, lymphosarcomab 1 

Retroperitoneal, lymphosarcoma 1 

Bone (24) (18) (25) (22) (15) (18) (21) (25)

Brain (24) (18) (25) (22) (15) (18) (21) (25)

Chromaffin Tissue (24) (18) (25) (22) (15) (18) (21) (24)

Corpuscle of Stannius (9) (11) (9) (11) (10) (8) (13) (11)

Esophagus (24) (18) (25) (22) (15) (18) (21) (25)

Eye (24) (18) (25) (22) (15) (18) (21) (25)

Choroid rete, cholangiocarcinoma, metastatic 1 

Gallbladder (23) (18) (22) (20) (12) (18) (21) (25)

Adenoma, papillary 3 1 3 

Carcinoma 1 1 

Gill (24) (18) (25) (22) (15) (18) (21) (25)

Heart (24) (18) (25) (22) (15) (18) (21) (25)

Atrium, cholangiocarcinoma, metastatic 1 

Lymphosarcoma 1 

Hematopoietic Tissue (24) (18) (25) (22) (15) (18) (21) (25)

Lymphosarcoma 1 

Interrenal Tissue (24) (18) (25) (22) (15) (18) (21) (24)

Intestine (24) (18) (25) (22) (15) (18) (21) (25)

Kidney (24) (18) (25) (22) (15) (18) (21) (25)

Liver (24) (18) (25) (22) (15) (18) (21) (25)

Cholangiocarcinoma 8 11 9 7 11 15 

Cholangioma 1 2 1 

Hepatocellular adenoma 1 

Hepatocellular carcinoma 1 1 

Mesentery (6) (7) (8) (5) (4) (3) (2) (9)

Cholangiocarcinoma, invasive 1 

Nares (Olfactory Tissue) (11) (5) (13) (9) (6) (6) (3) (4)

Oral Cavity (24) (18) (25) (22) (15) (18) (21) (25)

Lymphosarcoma 1 

Pancreas (24) (18) (25) (22) (15) (18) (21) (25)

TABLE F2

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Stop-Exposure)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 167

Pericardial Cavity (1) (2) (1) (1) (0) (2) (0) (0)

Peripheral Nerve (24) (18) (25) (22) (15) (18) (21) (25)

Peritoneum (1) (2) (4) (0) (0) (0) (0) (0)

Cholangiocarcinoma, metastatic 1 

Pharynx (0) (1) (0) (0) (0) (0) (0) (1)

Lymphosarcoma 1 

Pineal Organ (19) (17) (21) (18) (10) (15) (19) (16)

Pituitary Gland (23) (15) (24) (20) (15) (16) (19) (24)

Pseudobranch (24) (18) (25) (22) (15) (18) (21) (25)

Skeletal Muscle (24) (18) (25) (22) (15) (18) (21) (25)

Lymphosarcoma 1 

Skin (24) (18) (25) (22) (15) (18) (21) (25)

Lymphosarcoma 1 

Spinal Cord (24) (18) (25) (22) (15) (18) (21) (25)

Spleen (16) (13) (19) (10) (9) (13) (13) (9)

Cholangiocarcinoma, metastatic 1 

Stato-acoustic Organ (24) (18) (25) (22) (15) (18) (21) (25)

Lymphosarcoma 1 

Swim Bladder (24) (18) (25) (22) (14) (18) (21) (25)

Testis (24) (18) (25) (22) (15) (17) (21) (24)

Seminoma 1 1 1 

Thymus (8) (13) (13) (11) (2) (13) (18) (13)

Thyroid Tissue (24) (18) (25) (22) (15) (18) (21) (25)

Urinary Bladder (16) (14) (19) (19) (11) (16) (17) (24)

Multiple Organsc (24) (18) (25) (22) (15) (18) (21) (25)

Lymphosarcoma 1 

a Number of animals examined microscopically at the site b Number of animals with neoplasm c Number of animals with any tissue examined microscopically

TABLE F2

Summary of the Incidence of Neoplasms in Male Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Stop-Exposure)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
168 Fish Studies, NTP TR 528

9-Month Interim Evaluation

Liver (6) (5) (6) (5) (5) (5) (4) (4)

Cholangiocarcinomaa 1 2 2 2 1 

Cholangioma 1 

Hepatocellular carcinoma 1 

Pancreas (6) (5) (6) (5) (5) (5) (4) (4)

Duct, carcinoma 1 

Tissues Examined at 9 Months with No Neoplasms Observed

Adipose Tissue 

Bone 

Brain 

Chromaffin Tissue 

Corpuscle of Stannius 

Esophagus 

Eye 

Gallbladder

Gill 

Heart 

Hematopoietic Tissue 

Interrenal Tissue 

Intestine 

Kidney 

Mesentery 

Nares (Olfactory Tissue) 

Oral Cavity 

Peripheral Nerve 

Pineal Organ 

Pituitary Gland 

Pseudobranch 

Skeletal Muscle 

Skin 

Spinal Cord 

Spleen 

Stato-acoustic Organ 

Swim Bladder 

Ovary 

Thymus 

Thyroid Tissue 

Urinary Bladder 

TABLE F3

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 169

13-Month Study

Adipose Tissue (6) (8) (11) (6) (11) (13) (8) (7)

Retroperitoneal, cholangiocarcinoma, invasive 1 1 1 

Bone (48) (47) (49) (44) (44) (50) (39) (28)

Brain (48) (47) (49) (44) (44) (50) (39) (28)

Chromaffin Tissue (46) (47) (49) (44) (44) (50) (39) (28)

Corpuscle of Stannius (29) (25) (19) (26) (19) (26) (18) (16)

Cranial Cavity (0) (0) (0) (0) (2) (0) (1) (0)

Esophagus (48) (47) (49) (44) (44) (50) (39) (28)

Cholangiocarcinoma, invasive 1 1 

Hepatocholangiocarcinoma, invasive 1 

Eye (48) (47) (49) (44) (44) (50) (39) (28)

Gallbladder (42) (43) (45) (35) (40) (48) (39) (26)

Adenoma, papillary 1 2 3 3 3 

Carcinoma 1 

Gill (48) (47) (49) (44) (44) (50) (39) (28)

Heart (48) (47) (49) (43) (44) (50) (39) (28)

Atrium, cholangiocarcinoma, invasive 1 

Sinus venosus, cholangiocarcinoma, invasive 1 1 1 

Ventricle, cholangiocarcinoma, metastatic 1 

Hematopoietic Tissue (48) (47) (49) (44) (44) (50) (39) (28)

Interrenal Tissue (46) (47) (49) (44) (44) (50) (39) (28)

Intestine (48) (47) (49) (44) (44) (50) (39) (28)

Adenomatous polyp 1 

Hepatocholangiocarcinoma, metastatic 1 

Wall, sarcoma, NOS, invasive 1 

Kidney (48) (47) (49) (44) (44) (50) (39) (28)

Liver (48) (47) (49) (44) (44) (50) (39) (28)

Cholangiocarcinoma 14 15 23 30 21 20 

Cholangioma 1 1 1 1 

Hepatocellular adenoma 1 1 2 3 3 4 

Hepatocellular adenoma, multiple 1 

Hepatocellular carcinoma 1 1 2 4 1 

Hepatocholangiocarcinoma 3 1 6 1 

TABLE F3

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
170 Fish Studies, NTP TR 528

13-Month Study (continued)

Mesentery (16) (12) (23) (12) (19) (19) (9) (8)

Hepatocholangiocarcinoma, metastatic 1 

Nares (Olfactory Tissue) (21) (19) (35) (26) (20) (34) (22) (16)

Oral Cavity (48) (47) (49) (44) (44) (50) (39) (28)

Ovary (48) (47) (49) (44) (44) (50) (39) (28)

Cholangiocarcinoma, metastatic 1 

Dysgerminoma 1 

Germ cell neoplasm 1 

Leiomyosarcoma 1 

Sarcoma, NOS, invasive 1 

Pancreas (48) (47) (49) (44) (44) (50) (39) (28)

Pericardial Cavity (4) (2) (0) (0) (4) (3) (3) (0)

Cholangiocarcinoma, invasive 1 1 

Hepatocholangiocarcinoma, invasive 1 1 

Sarcoma, NOS, invasive 1 

Peripheral Nerve (48) (47) (49) (44) (44) (50) (39) (28)

Peritoneum (0) (0) (1) (0) (3) (1) (0) (0)

Sarcoma, NOS 1 

Pharynx (1) (3) (2) (0) (0) (1) (0) (0)

Pineal Organ (30) (31) (29) (26) (27) (34) (26) (18)

Pituitary Gland (44) (44) (45) (38) (40) (41) (34) (26)

Pseudobranch (48) (47) (49) (44) (44) (50) (39) (28)

Skeletal Muscle (48) (47) (49) (44) (44) (50) (39) (28)

Skin (48) (47) (49) (44) (44) (50) (39) (28)

Spinal Cord (46) (47) (49) (44) (44) (50) (38) (28)

Spleen (36) (32) (43) (30) (30) (34) (23) (19)

Hepatocholangiocarcinoma, metastatic 1 

Lipoma 1 

Stato-acoustic Organ (48) (47) (49) (44) (44) (50) (39) (28)

Swim Bladder (48) (47) (49) (44) (44) (50) (39) (28)

Cholangiocarcinoma, invasive 1 

TABLE F3

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 171

13-Month Study (continued)

Thymus (11) (16) (15) (15) (16) (16) (22) (13)

Thyroid Tissue (48) (47) (49) (44) (44) (50) (39) (28)

Urinary Bladder (40) (41) (37) (36) (40) (39) (33) (22)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE F3

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Core Study)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
172 Fish Studies, NTP TR 528

Adipose Tissuea (3) (9) (4) (4) (6) (5) (5) (4)

Retroperitoneal, cholangiocarcinoma, invasiveb 1 1 

Bone (28) (29) (21) (23) (26) (23) (15) (16)

Brain (28) (29) (21) (23) (26) (23) (15) (16)

Chromaffin Tissue (27) (29) (21) (23) (26) (23) (14) (16)

Corpuscle of Stannius (13) (17) (7) (11) (11) (14) (11) (10)

Esophagus (28) (29) (21) (23) (26) (23) (15) (16)

Cholangiocarcinoma, invasive 1 

Eye (28) (29) (21) (23) (26) (23) (15) (16)

Gallbladder (27) (29) (18) (22) (23) (22) (15) (15)

Adenoma 1 

Adenoma, papillary 1 1 

Carcinoma 1 

Gill (28) (29) (21) (23) (26) (23) (15) (16)

Hemangiosarcoma 1 

Heart (28) (29) (20) (23) (26) (23) (15) (16)

Atrium, cholangiocarcinoma, invasive 1 

Sinus venosus, cholangiocarcinoma, invasive 1 1 

Hematopoietic Tissue (28) (29) (21) (23) (26) (23) (15) (16)

Interrenal Tissue (27) (29) (21) (23) (26) (23) (14) (16)

Intestine (28) (29) (21) (23) (26) (23) (15) (16)

Adenomatous polyp 1 1 

Wall, hemangiosarcoma 1 

Kidney (28) (29) (21) (23) (26) (23) (15) (16)

Cholangiocarcinoma, metastatic 1 

Liver (28) (29) (21) (23) (26) (23) (15) (16)

Cholangiocarcinoma 1 8 7 18 13 11 10 

Cholangioma 1 2 

Hepatocellular adenoma 1 3 2 2 1 

Hepatocellular carcinoma 1 1 1 3 1 

Hepatocholangiocarcinoma 1 

Mesentery (5) (11) (12) (9) (15) (11) (6) (3)

Cholangiocarcinoma, invasive 2 1 

Cholangiocarcinoma, metastatic 2 

Nares (Olfactory Tissue) (16) (6) (13) (7) (16) (5) (7) (8)

TABLE F4

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Stop-Exposure)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
Fish Studies, NTP TR 528 173

Oral Cavity (28) (29) (21) (23) (26) (23) (15) (16)

Ovary (28) (29) (21) (23) (26) (23) (15) (16)

Dysgerminoma 1 

Pancreas (28) (29) (21) (23) (26) (23) (15) (16)

Pericardial Cavity (0) (1) (0) (1) (1) (1) (0) (1)

Cholangiocarcinoma, invasive 1 

Peripheral Nerve (28) (29) (21) (23) (26) (23) (15) (16)

Peritoneum (0) (0) (1) (0) (0) (1) (0) (1)

Pineal Organ (23) (20) (13) (17) (16) (16) (12) (11)

Pituitary Gland (27) (23) (20) (23) (26) (21) (15) (15)

Pseudobranch (28) (29) (21) (23) (26) (23) (15) (16)

Skeletal Muscle (28) (29) (21) (23) (26) (23) (15) (16)

Skin (28) (29) (21) (23) (26) (23) (15) (16)

Spinal Cord (28) (29) (21) (23) (26) (23) (15) (16)

Spleen (22) (18) (15) (11) (19) (16) (4) (10)

Stato-acoustic Organ (28) (29) (21) (23) (26) (23) (15) (16)

Swim Bladder (28) (29) (21) (23) (26) (23) (15) (16)

Thymus (16) (22) (11) (20) (9) (17) (13) (11)

Thyroid Tissue (28) (29) (21) (23) (26) (23) (15) (16)

Urinary Bladder (25) (18) (17) (18) (20) (20) (14) (13)

a Number of animals examined microscopically at the site b Number of animals with neoplasm

TABLE F4

Summary of the Incidence of Neoplasms in Female Medaka in the 13-Month Waterborne Study 

of 1,2,3-Trichloropropane (Stop-Exposure)

0 mg/L 4.5 mg/L 9 mg/L 18 mg/L

Group Group Group Group

A B A B A B A B
174 Fish Studies, NTP TR 528
175

APPENDIX G

CHEMICAL CHARACTERIZATION

AND GENERATION OF AQUARIA CONCENTRATIONS

PROCUREMENT AND CHARACTERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

EXPOSURE SOLUTION GENERATION AND EXPOSURE SYSTEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

EXPOSURE SOLUTION CONCENTRATION MONITORING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

EXPOSURE SOLUTION CHARACTERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

FIGURE G1 Infrared Absorption Spectrum of 2,2-Bis(bromomethyl)-1,3-propanediol . . . . . . . . . . . . . . 180

FIGURE G2 Infrared Absorption Spectrum of Nitromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

FIGURE G3 Infrared Absorption Spectrum of 1,2,3-Trichloropropane . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

TABLE G1 Gas Chromatography Systems Used in the Waterborne Studies

of 2,2-Bis(bromomethyl)-1,3-propanediol, Nitromethane, and 1,2,3-Trichloropropane . . . 183

FIGURE G4 Exposure Chambers Used in the Waterborne Studies 

of 2,2-Bis(bromomethyl)-1,3-propanediol, Nitromethane, and 1,2,3-Trichloropropane . . . 184

FIGURE G5 Schematic of the Exposure Solution Delivery System in the Waterborne Studies 

of 2,2-Bis(bromomethyl)-1,3-propanediol, Nitromethane, and 1,2,3-Trichloropropane . . . 185

TABLE G2 Summary of Exposure Aquaria Concentrations Administered to Medaka and Guppies 

in the Waterborne Studies of 2,2-Bis(bromomethyl)-1,3-propanediol . . . . . . . . . . . . . . . . . . 186

TABLE G3 Summary of Exposure Aquaria Concentrations Administered to Medaka and Guppies 

in the Waterborne Studies of Nitromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

TABLE G4 Summary of Exposure Aquaria Concentrations Administered to Medaka and Guppies 

in the Waterborne Studies of 1,2,3-Trichloropropane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
176 Fish Studies, NTP TR 528

CHEMICAL CHARACTERIZATION

AND EXPOSURE SOLUTION STUDIES

PROCUREMENT AND CHARACTERIZATION

2,2-Bis(bromomethyl)-1,3-propanediol

2,2-Bis(bromomethyl)-1,3-propanediol was obtained from Aldrich Chemical Company (Milwaukee, WI) in three

lots (16728PG, 11215BQ, and 00713BQ) that were used in the 14- and 16-month studies. Lots 11215BQ and

00713BQ were manufactured in the same batch but were assigned different lot numbers by the supplier because

they were received on different dates. Identity, purity, and stability analyses were conducted by the study

laboratory.

Lots 16728PG and 11215BQ were identified as 2,2-bis(bromomethyl)-1,3-propanediol by the University of

Mississippi (Oxford, MS) (lot 16728PG) and by the University of Southern Mississippi (Hattiesburg, MS)

(lot 11215BQ) using infrared spectroscopy. The spectra were consistent with the structure of

2,2-bis(bromomethyl)-1,3-propanediol. The infrared spectrum is presented in Figure G1.

The purities of lots 16728PG and 11215BQ were determined by the study laboratory using gas chromatography

(GC) by system A (Table G1). GC indicated no contaminants in either lot. Information provided by the supplier

indicated that the purity of lot 16728PG was greater than 99%. The overall purity of lots 16728PG and 11215BQ

was determined to be greater than 99%.

The bulk chemical was stored at approximately 4° C (lot 16728PG) or at room temperature (lots 11215BQ and

00713BQ). The stability of bulk 2,2-bis(bromomethyl)-1,3-propanediol was monitored monthly during the

14- and 16-month studies using GC by system A. No contaminants were found during the course of the studies.

Nitromethane

Nitromethane was obtained from Aldrich Chemical Company in one lot (CG02304HF) that was used in the

13- and 16-month studies. Identity, purity, and stability analyses were conducted by the study laboratory.

Lot CG02304HF, a colorless liquid, was identified as nitromethane by the University of Mississippi using infrared

spectroscopy. The spectrum was consistent with the structure of nitromethane and is presented in Figure G2.

The purity of nitromethane was determined by the study laboratory using GC by system B. GC indicated that

lot CG02304HF contained no contaminants other than water. Information provided by the supplier indicated that

the purity of lot CG02304HF was greater than 99%. The overall purity of lot CG02304HF was determined to be

greater than 99%.

The bulk chemical was stored at approximately 4° C. The stability of bulk nitromethane was monitored monthly

during the 13- and 16-month studies using GC by system B. No contaminants were found during the course of the

studies.

1,2,3-Trichloropropane

1,2,3-Trichloropropane was obtained from Aldrich Chemical Company in one lot (01628HG) that was used in the

13- and 16-month studies. Identity, purity, and stability analyses were conducted by the study laboratory.

Lot 01628HG, a colorless to straw-colored liquid, was identified as 1,2,3-trichloropropane by the University of

Southern Mississippi and by the supplier using infrared spectroscopy. The spectra were consistent with a reference

spectrum (Aldrich, 1985). The infrared spectrum is presented in Figure G3.
Fish Studies, NTP TR 528 177

The purity of lot 01628HG was determined by the University of Southern Mississippi using GC by system C, the

chemical supplier using GC by system E, and the study laboratory using GC by system E. GC by system C

indicated that lot 01628HG was highly pure; GC by system D indicated a purity of 99%; GC by system E indicated

the absence of significant contamination. The overall purity of lot 01628HG was determined to be 99%.

The bulk chemical was stored at approximately 4° C.

EXPOSURE SOLUTION GENERATION AND EXPOSURE SYSTEM

Exposure was by intermittent flow through and was conducted in a closed chamber similar to that described by

Walker et al. (1985) (Figure G4). A schematic of the exposure solution delivery systems used in the 13-, 14-, and

16-month studies is shown in Figure G5. Stock solutions of 2,2-bis(bromomethyl)-1,3-propanediol, nitromethane,

or 1,2,3-trichloropropane were prepared by adding neat chemical to diluent well water in glass carboys 

(2,2-bis(bromomethyl)-1,3-propanediol, 19 L; nitromethane, 49 L; 1,2,3-trichloropropane, 19 L) to produce

concentrations sufficient to provide the required concentrations in exposure aquaria. A water partitioner was 

timer-regulated to perform at least five volume additions per day to each exposure aquarium. Diluent well water

entered the water partitioner through a solenoid-controlled valve immediately after filtration and ultraviolet light

sterilization. A float switch within the water partitioner activated liquid dispensing pumps (Hamilton Company,

Reno, NV) that were preset to remove the appropriate volume of stock solution from the carboy containers. The

dispensing pumps injected the stock solution through Teflon® microbore tubing into glass mixing\splitter boxes

immediately prior to delivery of two liters of diluent water. Diluent water alone was delivered to the control

aquaria mixing\splitter boxes. Exposure solutions were delivered from mixing\splitter boxes to duplicate 35 L

exposure aquaria through calibrated glass delivery lines. Aquaria volumes were maintained by an overflow drain

siphon designed to remove water from near the bottom of each aquarium. 

The resin-coated plywood exposure chamber measured 333 cm long, 118 cm wide, and 244 cm high. Access to

the chamber was through sliding polycarbonate doors on each side of the chamber. Aquaria were placed at random

in a central waterbath maintained at 26° ± 1° C. Timer-controlled fluorescent lights with dimmer ballasts provided

16 hours of light per 24 hours with 30-minute transitions simulating dawn and dusk. The chamber was maintained

at a slightly negative pressure.

Individual test aquaria measured 48 cm long, 35.5 cm wide, and 24 cm high. A water depth of 20.5 cm was

maintained. Aquaria were individually covered and covers of adjacent tanks were not raised simultaneously.

EXPOSURE SOLUTION CONCENTRATION MONITORING

Concentrations of 2,2-bis(bromomethyl)-1,3 propanediol, nitromethane, and 1,2,3-trichloropropane in the exposure

aquaria were monitored using GC by systems A, B, and D, respectively, approximately 3 times each week.

Duplicate samples were analyzed from each aquarium. Summaries of the aquaria solution concentrations are given

in Tables G2 through G4.

EXPOSURE SOLUTION CHARACTERIZATION

2,2-Bis(bromomethyl)-1,3-propanediol

Exposure solution characterization was performed prior to the start of the 14- and 16-month studies. The

generation of target concentrations of 2,2-bis(bromomethyl)-1,3-propanediol exposure solution in exposure aquaria

by intermittent flow through was determined with and without fish present. Duplicate aquaria at target

concentrations of 10, 35, and 100 mg/L were sampled prior to the first injection and 3, 6, 9, 12, 14, and 24 hours

after the initial injection. Samples were analyzed using GC by system A. All aquaria reached their approximate

target 2,2-bis(bromomethyl)-1,3-propanediol concentrations within 24 hours of the initial injection.
178 Fish Studies, NTP TR 528

The uniformity of distribution of 2,2-bis(bromomethyl)-1,3-propanediol in single 10 and 100 mg/L aquaria was

determined following 6-day intermittent flow-through generation and stability studies. Samples were collected

from nine locations 2 cm below the surface of each aquarium solution and from nine locations within 2 cm of the

bottom of each aquarium. Samples were analyzed using GC by system A. Uniformity of distribution was

established.

The persistence of 2,2-bis(bromomethyl)-1,3-propanediol in the exposure aquaria was determined by monitoring

the exposure solution concentration with intermittent flow through of diluent water only after stable target

concentrations were reached. Samples were collected every 3 hours for 12 hours and analyzed using GC by system

A. The measured concentration was below the limit of detection after 6 hours in the 10 mg/L aquaria, after

12 hours in the 35 mg/L aquaria, and was less than 6 mg/L after 12 hours in the 100 mg/L aquaria.

Stability of 2,2-bis(bromomethyl)-1,3-propanediol in the intermittent flow-through exposure solution was

monitored with and without fish present for 132 hours after approximate target concentrations were reached in 10,

35, and 100 mg/L exposure aquaria. Samples were collected approximately every 4 hours and analyzed using GC

by system A. Stability was confirmed for at least 132 hours. The stability of 2,2-bis(bromomethyl)-1,3-

propanediol in exposure solution without intermittent flow through was monitored with and without fish present

for 24 hours. The 2,2-bis(bromomethyl)-1,3-propanediol exposure solution was stable for up to 24 hours in the

absence of intermittent flow through.

Nitromethane

Exposure solution characterization was performed prior to the start of the 13- and 16-month studies. The

generation of target concentrations of nitromethane exposure solution in exposure aquaria by intermittent flow

through was determined with and without fish present. Duplicate aquaria at target concentrations of 8.6, 24.5, and

70.0 mg/L were sampled prior to the first injection and every 2 hours for 14 hours after the initial injection.

Samples were analyzed using GC by system B. All aquaria reached their approximate target nitromethane

concentrations within 14 hours of the initial injection.

The uniformity of distribution of nitromethane in a single 70.0 mg/L aquarium was determined during 6-day

intermittent flow-through generation and stability studies. Samples were collected from nine locations 2 cm below

the surface of the aquarium solution and from nine locations within 2 cm of the bottom of the aquarium. Samples

were analyzed using GC by system B. Uniformity of distribution was established.

The persistence of nitromethane in the exposure aquaria was determined by monitoring the exposure solution

concentration of a 100 mg/L exposure solution prepared by injection of neat nitromethane into the mixing/splitter

box. Intermittent flow through contained diluent water only after stable target concentrations were reached.

Samples were collected every hour for 13 hours and analyzed using GC by system B. The nitromethane

concentration was below the limit of detection after 13 hours.

Stability of nitromethane in the intermittent flow-through exposure solution was monitored with and without fish

present for 132 hours after approximate target concentrations were reached in 8.6, 24.5, and 70.0 mg/L exposure

aquaria. Samples were collected approximately every 4 hours and analyzed using GC by system B. Stability was

confirmed for at least 132 hours. The stability of nitromethane stock carboy solutions was monitored for

144 hours. Samples were collected approximately every 24 hours and analyzed using GC by system B. The

results indicated that nitromethane solution was stable in stock carboys for at least 144 hours. The stability of

nitromethane in exposure solution without intermittent flow through was monitored for approximately 70 hours

with and without fish present in 100 mg/L exposure solutions prepared by injection of neat nitromethane into the

mixing\splitter box. Nitromethane concentrations decreased to approximately 60% of initial target concentrations

after 3.5 hours.
Fish Studies, NTP TR 528 179

1,2,3-Trichloropropane

Exposure solution characterization was performed prior to the start of the 13- and 16-month studies. The

generation of target concentrations of 1,2,3-trichloropropane exposure solution in exposure aquaria by intermittent

flow through was determined with and without fish present. Duplicate aquaria at target concentrations of 10, 35,

and 100 mg/L were sampled prior to the first injection and 3, 6, 9, 12, 14, and 24 hours after the initial injection.

Samples were analyzed using GC by system D. All aquaria reached their approximate target 

1,2,3-trichloropropane concentrations within 24 hours of the initial injection.

The uniformity of distribution of 1,2,3-trichloropropane in single 10 and 100 mg/L aquaria was determined during

6-day intermittent flow-through generation and stability studies. Samples were collected from nine locations 2 cm

below the surface of the aquarium solution and from nine locations within 2 cm of the bottom of the aquarium.

Samples were analyzed using GC by system D. Uniformity of distribution was established.

The persistence of 1,2,3-trichloropropane in the exposure aquaria was determined by monitoring the exposure

solution concentration with intermittent flow through of diluent water only after stable target concentrations were

reached. Samples were collected every 3 hours for 12 hours and analyzed using GC by system D. After 12 hours,

the measured concentrations were 0.96 mg/L in the 10 mg/L aquaria, 3.63 mg/L in the 35 mg/L aquaria, and

9.16 mg/L in the 100 mg/L aquaria.

Stability of 1,2,3-trichloropropane in the intermittent flow-through exposure solution was monitored with and

without fish present for 120 hours after approximate target concentrations were reached in 10, 35, and 100 mg/L

exposure aquaria. Samples were collected approximately every 4 hours and analyzed using GC by system D.

Stability was confirmed for at least 120 hours. The stability of 1,2,3-trichloropropane in exposure solution without

intermittent flow through was monitored with and without fish present for 24 hours. After 24 hours, the average

1,2,3-trichloropropane exposure solution concentrations in all aquaria declined to approximately 54% of the

measured concentrations before flow through was terminated.
180 Fish Studies, NTP TR 528

FIGURE G1

Infrared Absorption Spectrum of 2,2-Bis(bromomethyl)-1,3-propanediol
Fish Studies, NTP TR 528 181

FIGURE G2

Infrared Absorption Spectrum of Nitromethane
182 Fish Studies, NTP TR 528

FIGURE G3

Infrared Absorption Spectrum of 1,2,3-Trichloropropane
TABLE G1

Gas Chromatography Systems Used in the Waterborne Studies

of 2,2-Bis(bromomethyl)-1,3-propanediol, Nitromethane, and 1,2,3-Trichloropropanea

Detection System Column Carrier Gas Oven Temperature 

Program

System A

Flame ionization

System B

Flame ionization

System C

Flame ionization

System D

Electron capture

System E

Flame ionization

DB-5, 15 m × 0.32 mm,

0.25-μm film thickness (J&W

Scientific, Folsom, CA)

DB-Wax, 15 m × 0.32 mm,

0.5-μm film thickness (J&W

Scientific)

Not available

20% FFAP on Chromosorb W

HP, 6 ft × 3.5 mm

D8-1701, 30 m × 0.25 mm

(J&W Scientific)

Helium at 2 mL/minute

Helium at 2 mL/minute

Not available

Nitrogen at 5 mL/minute

Helium

150° C for 3 minutes, then

10° C/minute to 200° C, then

held for 1 minute

80° C for 4 minutes, then

20° C/minute to 120° C, then

held for 4 minutes

100° C for 2 minutes, then

10° C/minute to 195° C

120° C isothermal

80° C to 230° C at 10° C/minute,

then held for 5 minutes

a Gas chromatographs were manufactured by Perkin Elmer (Torrance, CA) (systems A and B), Tracor (Austin, TX) (system D), 

and Varian, Inc. (Palo Alto, CA) (system E).

Fish Studies, NTP TR 528 183
184 Fish Studies, NTP TR 528

FIGURE G4

Exposure Chambers Used in the Waterborne Studies 

of 2,2-Bis(bromomethyl)-1,3-propanediol, Nitromethane, and 1,2,3-Trichloropropane
Fish Studies, NTP TR 528 185

FIGURE G5

Schematic of the Exposure Solution Delivery System in the Waterborne Studies 

of 2,2-Bis(bromomethyl)-1,3-propanediol, Nitromethane, and 1,2,3-Trichloropropane INJ INJ INJ INJ INJ RES

WP

SB SB

TA TA TA TA

WP Water PartitionerINJ Syringe InjectorRES Reservoir

(Common to all Injectors)

SB Splitter Box/Mixing ChamberTA Treatment Aquarium
186 Fish Studies, NTP TR 528

TABLE G2

Summary of Exposure Aquaria Concentrations Administered to Medaka and Guppies 

in the Waterborne Studies of 2,2-Bis(bromomethyl)-1,3-propanediol 

Target Concentration Average Concentrationa Coefficient of

(mg/L) (mg/L) Variation

14-Month Study (Medaka) 

24 19.4 ± 3.9 19.9

60 56.9 ± 11.0 19.2

150 137.8 ± 27.3 19.8

16-Month Study (Guppy)

24 20.0 ± 6.3 31.3

60 53.5 ± 10.1 19.9

150 139.0 ± 16.6 12.0

a Mean ± standard deviation

TABLE G3

Summary of Exposure Aquaria Concentrations Administered to Medaka and Guppies 

in the Waterborne Studies of Nitromethane

Target Concentration Average Concentrationa Coefficient of

(mg/L) (mg/L) Variation

13-Month Study (Medaka)

10 9.3 ± 2.4 26.2

20 20.8 ± 3.2 15.6

40 41.7 ± 5.9 14.2

16-Month Study (Guppy)

10 9.9 ± 2.5 24.8

30 28.7 ± 3.5 12.1

70 66.9 ± 6.9 10.4

a Mean ± standard deviation
Fish Studies, NTP TR 528 187

TABLE G4

Summary of Exposure Aquaria Concentrations Administered to Medaka and Guppies 

in the Waterborne Studies of 1,2,3-Trichloropropane

Target Concentration Average Concentrationa Coefficient of

(mg/L) (mg/L) Variation

13-Month Study (Medaka)

4.5 4.6 ± 0.3 7.7

9.0 9.2 ± 0.6 6.6

18.0 18.0 ± 1.9 10.5

16-Month Study (Guppy)

4.5 4.4 ± 0.4 9.3

9.0 8.8 ± 0.7 8.4

18.0 18.2 ± 1.6 8.9

a Mean ± standard deviation
188 Fish Studies, NTP TR 528
189

APPENDIX H

NUTRIENT COMPOSITION

AND CONTAMINANT LEVELS IN

FISH FEED AND AQUARIA WATER

TABLE H1 Nutrient Profile of Aqua-Tox (Specialized Formula) Flakes . . . . . . . . . . . . . . . . . . . . . . . . . . 190

TABLE H2 Contaminants in Brine Shrimp Larvae, Aqua-Tox (Specialized Formula) Flakes, 

and Well Water used in the Fish Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
190 Fish Studies, NTP TR 528

TABLE H1

Nutrient Profile of Aqua-Tox (Specialized Formula) Flakesa

Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45% 

Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10%

Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3%

Ash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9%

Moisture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7%

a Ziegler Brothers, Inc (Gardners, PA)

TABLE H2

Contaminants in Brine Shrimp Larvae, Aqua-Tox (Specialized Formula) Flakes,

and Well Water used in the Fish Studies

Brine Shrimp Larvae Aqua-Tox Flakes Well Water

Compound Value LDLa Value LDL Value LDL

(μg/kg) (μg/kg) (μg/kg) (μg/kg) (μg/kg) (μg/kg)

Toxaphene NDb 700 ND 255 ND 0.1

"-BHC ND 9 ND 3.2 ND 0.005

$-BHC ND 18 ND 6.4 ND 0.005

*-BHC ND 26 ND 9.6 ND 0.005

Heptachlor ND 9 ND 3.2 ND 0.005

Aldrin ND 12 ND 4.25 ND 0.005

Heptachlor epoxide ND 240 ND 88 ND 0.005

Endosulfan I ND 41 ND 15 ND 0.03

Dieldrin ND 6 ND 2.1 ND 0.01

p,pN-DDE ND 12 ND 4.25 ND 0.01

Endrin ND 18 ND 6.4 ND 0.01

Endosulfan II ND 12 ND 4.25 ND 0.01

p,pN-DDD ND 32 ND 11.5 ND 0.03

Endrin aldehyde 102 67 ND 25 ND 0.005

Endosulfan sulfate ----c ---- ND 70 ND 0.03

Lindane ND 12 ND 4.25 ND 0.02

p,pN-DDT ND 35 ND 13 ND 0.03

Methoxychlor ---- ---- ND 185 ND 0.1

Arochlor 1016 ND 300 ND 210 ND 0.1

Arochlor 1221 ND 300 ND 210 ND 0.1

Arochlor 1232 ND 300 ND 210 ND 0.1

Arochlor 1242 ND 300 ND 210 ND 0.1

Arochlor 1248 ND 300 ND 210 ND 0.1

Arochlor 1254 ND 300 ND 210 ND 0.1

Arochlor 1260 ND 300 ND 210 ND 0.1

Malathion ---- ---- ND 105 ND 0.1

Methyl parathion ---- ---- ND 105 ND 0.1

Parathion ---- ---- ND 105 ND 0.1

a LDL = lower detection limit; b ND = not detected; c Dashed line indicates no analysis