s' midwest resfarch institutepreface-this report war, prepared at midwest research institute,...

I

S' MIDWEST RESFARCH INSTITUTE

AD__

1~M.AMMALIAN TOXICITY OF MUNITIO1NS COMPOUNDSwIPHASE I!: Effects of Mult-C'p-e Doses

LU~ PARr II: 2,4-Dinitrotoluene

PROGRESS REPORT NO. iNovember 1978

ACotract No. DAMD 17-74-C-4073

MR! ProREct No. 3900-B~~~e Aoebr17

For

j Projec't Offiver: Dr. ,Jac. C. Dacre

, nvironmentnI Protection Research Division

U.S. Army Medical Bioengineering

Research and Development LaboratoryFort Detrick

Frvdertck, Maryland 21.70!.

I MIDWEST RESEARCH INIflTUTE A2, VOLtkKER 3CULEVARD, KANSAS CIT'i. MISSOL1II 64110 8 516 753-7600

[

Animal experimentation: Animal experiments were conducted according to the"Guide for Laboratory Animal Facilities and Care" (1972) prepared by the

Committee ort the Guide for Laborato.,. Animal Resources, Nationl Academy ofSciences, National Research Council; the regulations and standards preparedby the Department of Agriculture; and Public Law 91--570, "Laboratory AnimalWelfare Act," 1970.

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MRI - 14(0TH STAR DIVISION '10701 Hod CIrc:e Drive, Minnetonka, Minnesota 55343 • 612 933-7880

MRI WASHINGTON, D.C. 20006-- Suite 250, 1 /%0 K Street, N.W. * 202 293-3800

I

I ADMammalian Toxicity of Munitions Compounds

.• Phase II: Effects of Multiple Doses

Part !I: 2,4-T)initrotoiuene

I Progres Report No. 3

oNovember 1978

by

3I Cheng-Chun Lee

U� Hirty V. Ellis, III

Jo.,n J. Kowalski

I 1John R. HodgsonShang W. HwangRobert D. Short

* Jagdish C. RhandariJaime L. Sanyer

Thomas W. ReddigSI Jan L. Minor

Supported by

U.S. Army Medical Research and Development Coinmand

Fort Detrick, Frederick, Maryland 21701

Contract No. DAMD 17-74-C-4073

Project Officer: Dr. Jack C. DacreEnvironmental Protection Research Division

U.S. Army Medical Bioengineering Research and Development LaboratoryFort Detricl., Frederick, Maryland 21701

MlaoFt Research InstituteKansas City, Itissouri 64110

I DDC Distribution Statement

Distribution unlii..ted

3 The findings in this report are not to be construed as anofficial Department of the Army position unless so designatedby other authorized documents

MIDWEST RESEARCH INSTITUTE 425 VOLKER BOUL['- 'J KANSAS CITY, MISSOURI 64110 • 816 753-7600

'I, l••• • -. .. ..... - ..... .. ..

SECURITY CLASSIF ICATION OF TISt PAGE taMe. Dsta 101se'ee1

I I.REPRT EPORmiT DOCUMENTATIC'I PAGE UZAD00 COMPCTWO FOR

Progress Report No. 3

I Fort etIck, Frdrc,4-Marylaolu nd 2101 1/. PUMERORMN OAG.EPOTNM

I~~~R Pro1A~ 0nlasiie

"DRIE. 10ST STATMEN MO AleNT RRI.i'f

MidwstRebution unlimitued. W~hTORK UNIEAT "nal

11. SUNTRPLEINGAR NOFFIESNM N DRS

U.S. ArE y MedSCnicul onResearch a nde DefCCCvelomi enttl by block 8hnb,

SubactDetik T rexriity Disrbltind 2170-13.ni@RotOF PAGES

14.brni ToNIicity MGNYNM DDIldfeentabolim Cotroling fitroI. ECRToluee

If. DSTWB.(UT9O STTM NT aofthi erporaltu)o ~wes ee~

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I.D IS RJOU 3 EDTION STAEC of h abtrc entre I nI bSS lacETtSifretfom

j~~~~6 SUPCLEENTAY CNOTESlIWO II PE(e,

19 KEY WOD Cniu n ees ieI ecsayadIettyb lc ubr

LA.

20. Abstra'ct (concluded)

testes (depressed spermatogenesis), and the neuromuscular system (a rigid

paralysis associated with mild central nervous system lesions). The high-est no effec' levels were 5 mg/kg/day in male and female dogs, less than34 mg/kg/day n male rats and less than 38 mg/kg/day in female rats, and137 mg/kg/day in male mice and 147 mg/kg/day in female mice.

14C-2,4-DNT was wll absorbs. after oral. dosing by all species tested ex-

cept mice. The rakiiolabel was concentrated in the liver and kidney. Pri-mary biotransformat~on reactions were reduction of one or both nitros to --

amines and oxidation of the methyl to a benzyl alcohol or benzoic acid.

Secondary conjugation reactions occurred before excretion in the urine.

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v.vi TI

PREFACE

-This report war, prepared at Midwest Research Institute, 425 Volker

Boulevard, Kansas City, Missouri 64110, under U.S. Department of the Army-- Contract No. DAMD-17-74-C-4073, MR1 Project No. 3900-B, "Munition Compounds

-- Mammalian Toxicity Stu~dy." The work was supported by the U.S. Army MedicalBioengineering Research and Development Laboratory, U.S. Army Medical

Research and Development Command, Department of the Army. Cpt. John P.

Glennon, Dr. lack C. Dacre, Dr. David H. Rosenblatt and Cpt. Robert Rice,Environmental Protection Research Division, USAMBRDL, are the consecutivetechnical monitors for the project.

This work was conducted in the Biological Sciences Division, underthe direction of Dr. William B. House, between 1 August 1974 and 28 February1978. The experimental work was directed by Dr. Cheng-Chun Lee, DeputyDirector, Biological Sciences Division, for Pharmacology and Toxicology withthe asistance of Dr. Harry V. Ellis, I11, Associate Pharmacologist, andMr. John J. Kowalski, Assistant Biologist. Dr. John R. Hodgson, Head,tmocnemicai and Developmental Pharmacology Section, supervised the studieson metabolism, cytogenic and mutagenic studies. Dr. Shang W. Hwang, AssociatePharmacologist, assisted the studies on metabol'.m. Dr. Robert D. Short,Associate Pharmacologist, supervised the in vitro stud) on metabolism andin vivo study on drug metabolizing enzymes. Dr. 3. C. Ehandari and

Dr. Jaime L. Sanyer, Associate Veterinary Pathologiats, supervised the nec-ropsy and the histology preparation and performed the microscopic examination.

Mr. Thomas W. rtddig (ASCP certified M.T.), Laboratory Supervisor, super-vised the hematology and clinical laboratory tests. Mr. Jan L. Minor,Assistant Toxicologist, supervised the computer program and analysis of ex-perimental data. Technical personnel included Robert C. Byrne, Bruce S.

• "Andersen, Mary A. Kowalski, Francis H. Brown, Ellen R. Ellis, Ernesto A.Castillo, Judith D. Girvin, Patricia L. Wilkerson, Bhanu S. Gosalia, LaurelM. Halfpap, William M. Bracken, and Rita D. Freeman.

Approved for:

MIDWEST RESEARCH INSTITUTE

C. C. Lee, Deputy Director

Biological Sciences Division

November 1978

vii

:. •I~~~~AMMALIAN TOXICITY OF MUNITIONS COMPOUNDS ... ...PHASE IIz Effects of Multiple Doses m . ...............

PART II : 2,•4-Dinitrotoluone 61TIVaUrl~A•AtMLOU• CM ..

(Report No. 3) 6A it: • ••

TABLE OF CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1. Dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

A. Subacute and Subchronic Toxicities and

Reversibility .. .. .. .. .. .. .. .. . 5

1. Introduction . .. .. .. .. .. .. . .. 52. Material and Methods . • . . 53. Results . . . . . . . . . . . . . . ... . . . 7

4. Discussion and Conclusions .. .. .. . .. 10

B. Immunologic Rerponse to 2,4-DNT . .. .. . . . .

1. Introduction . .. .. .. .. .. . .. . I.

2. Material and Methods . .. .. .. .. . .. I1

3. Results and Conclusions .. .. .. .. . .. 12

C, Summary . . . . . . . . . . . . . . . . . . . . . t2

II. Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . 35


Reversibility .. .. .. .. .. .. .. . .. 35

1. Introduction . .. .. .. .. .. .. . .. 352. Material and Methods . .. .. .. .. . .. 353. Results . . . . . . . . . . . . . . . . . . . 364. Discussion and Conclusions . .. .. .. .. 40

B. Cytogenetic and Mutagenic Effects of 2,4-DNT.. 41

1. Introduction . .. .. .. .. .. .. ... 412. Material and Methods . .. .. .. .. . .. 41

3. Results . . . . . . . . . . . . . . . . . . . 434. Discussion and Conclusiona .. .. .. . .. 44

ix m

am7fhd

PASmu

TABLE OF CONTENTS (Continued)

Page

C. Imulologic Response to 2,4-DNT ... ......... ... 44

1. Introduction ..... ............... . . 44

2. Material and Methods ..... ............ ... 44

3. Result and Conclusions ... ........... .... 44

D. Summary .......... ..................... ... 45

III. Mice ........ ....... ........................... .... 83


Reversibility ...... ................. .... 83

1. Introduction ...... ............... .... 83

2. Material and Methoas ..... ............ ... 83

3. Results ....... .................. .... 83

4. Discuselon and Conclusions ..... ......... 85

B. Mutagenic Effects of 2,4-DNT .............. .... 86

1. Introduction ...... ................ ... 86


3. Results and Discussion ... ........... .... 86

4. Conclusions. ................. 86

C. Summary ........ ....... ..................... 86

IV. Disposition and Metabolism ........ ............... ... 115

A. Disposition and Metabolism of 2,4-DNT ;n

Various Species ................ 115

1. Introduction 15................15


3. Results.. . ........ .................... 1174. Discussion and Conclusions ... ......... ... 119

B. Biliary Excretion of Nitrotoluenes in Rats. . . 120

1. Introduction . .. .. .. .. .. .. . .. 1202. Material and Methods ..... ............ ... 120

3. Results. ................... 1214. Discussion and Conclusions .... ....... ... 121

xi

TABLE OF CONTENTS (Concluded)

Page

C. Metabolism of 2,4-DNT In Vitro. .. ............... 122

1. Introduction. .. ............................. 1222. Material and Methods .. ...................... 1223. Results.. .. .............. ................1234. Discussion and Conclusion. .. ................ 123

D. EFfect of 2,4-DNT on Drug Metabolizing Enzymes. . 123

1. Introduction. .. ............................. 1232. Material and Methods .. ...................... 1243. Results .. .. ................................. 1244. Conclusion. .. ............................... 125

V. General Susmmary and Conclusions .. .. ....................... 153

A. Toxic Effects. .. ................................. 153

I Toxic and Nontoxic Doses. .. .................2. Target Organs .. .. ........................... 1533. S'Oecial Toxicity Tests .. .................... 154

B. Disposition and Metabolism. .. .................... 154

1. Absorption, Distribution, Metabolism andExcretion Studies. .. ...................... 154

2. Biliary Excretion Studies. .. ................ 1543. In Vitro Metabolism Studies. .. .............. 154

4. Metabolic Interaction Studies. .. ............ 154

C. Future Research. .. ............................... 155

1. Exposure Standards .. ........................ 1552. Toxic Effects .. .. ........................... 153

References .. .........................................................

Distribution. .. ..................................................... 159

VAppendix. ............................................................ 161

xi

MAMOMALIAN TOXICITY OF MUNITION COMPOUNDSPHASE II: Effects of Multiple Dones

PART II: 2,4-binitrotoluene(Report No. 3)

EXECUTIVE SUMMARY

The effects of 2,4-DNT after oral adminisLration for up to 13 weekswere investigated in dogs, rats, and mice. A detailed study of the dis-position and metabolism was performed in rats and the metabolic pathwayswere compared in various species including the liver of human cadavers.

In dogs, daily administration of 1 or 5 mg/kg/day of 2,4-DNT for13 weeks did not cause any adverse effects. Daily treatment of 25 mg/kg/daywas toxic after 12 to 22 days and lethal after 22 or more days. There wasgreat variation in individual susceptibility. Three primary target organswere seen: the neuromuscular system, the erythrocytes and the testes. Theneuromuscular effects observed grosaly included incoordinationand rigidparalysis resulting in anorexia and weight loss. These were associated withmild demyelgnation, glioais and edema in the central nervous system. Acausal relationship is not certain, but it is significant that deuyelinationof the optic nervo was seen only in one of the two dogs that suffered fromtransient blindness. The basic erythrocyte effect was methemoglobinmamia.This led te Heinz bodies, anemia, reticulocytosis, hemosiderosis, andextramedullary hematopolesis. The testicular effect was a decrease inspermatogenesis. This effect was noticeable after treatment for 4 weekswith 25 mg/kg/day of 2,4-DNT and severe after 13 weeks. Severely affecteddogs recovered partially in 4 weeks after cessation of treatment, and com-pletely in 8 months. Serum levels of im-unoglobin E (IgE) were not affectedby 2,4-DNT treatment.

In rats, the 2,4-DNT intake of males fed the low, middle, or highlevels of 2,4-DNT in the feed averaged 34, 93, or 266 mg/kg/day, respectively.The female rats consumed an average of 38, 108, or 145 mg/kg/day, respectively.The low level caused a slight depression of weight gain. The middle levelcaused a more severe depression oi weight gain, and hemosiderosis and re-ticulocytosis, presumably due to methemoglobin, and a depression of sperma-togenesis. These effects were more severe and occurred ar'c er in rats fedthe high level. In addition, there were anemia, an unusual a4it with wide-spread and stiff hind legs, and gliosis and/or demyelination in the centralnervous system. Ten of the 16 high dosage fewales died before the end ofthe third week; whereas, 8 of the 16 high dosage males died before their schedulednecropsy. There was partial recovery 4 weeks after cessation of 2,4-DNT.Chromocomes from lymphocyte and kidney cultures from rats fed the middle

xiii

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level had increased numbers of chromatid breaks and gaps, but nio significantnumerical aberrations. Males fed the middle level for 13 wceks and thenmated produced low fertility indexes and no viable fetuses, indicatingsterility. A cy!otoxic level of 2,4-DNT had no mutagenic effect on Chinesehamster ovary cells in vitro. In rats, as in dogs, 2,4-YDNT did not affectserum IgE levels.

In mice, the 2,4-DNT intake of males fed the low, middle or highlevels of 2,4-DNT in the feed averaged 47, 137, or 413 mg/kg/day, respectively.

The female mice consumed an average of 52, 14), or 468 mg/kg/day, respectively.The low and middle levels were nontoxic. The high level caused weight loss,mild anemia, .nd a few deaths. Two of the four high dosage males terminatedafter 4 weeks had mild depression of spermatogenesis, but testicular lesions

were not seen in those terminated after 13 weeks' feeding. The mice had re-covered completely 4 weeks after cessation of treatment. In a dominantlethality test, male mice fed the 0.2% of 2,4-DNT for 13 weeks were normal,

while those fed the 0.7% level for 4 weeks had a low fertility index and anormal Implant viability index.

After oral administration (Ring-UL- 1 4 C) 2,4*DN was poorly ab-

sorbed (8 to 122 of the dose within 24 hours) by two strains of mice, butwell absorbed (752 to 85%) by rats, rabbits, dogs and monkeys. The radio-activity was concentrated in the liver and kidney in all species. Metabolismwas similar in all species studied. O(e or both niitros were reduced to

aminos. The methyl was oxidized to n benzyl alcohol or further to a reactivebenzaldehyde or a benzoic acid. All these products were conjugated to form aglucuronate or sulfate. The final products were excreted in the urine. Littleor no 2,4-DNT itself was excreted. No radioactivity from the ring carbonswas excreted in the air.

In female rats, after oral administration of radiolabeled TNT andvarious dinitrotoluenes, radioactivity appeared in the bile within 15 minutes.The time to peak height ranged from 15 minutes to over 6 hours, in the order:3,4-Dtrr < 2,3-DNT < TNT - 2,4-DNT < 2,5-DNT w 3,5-DNT < 2,6-DNT < 4-amino-2,6-Dlr. The amount of biliary excretion of radioactivity within 24 hoursof dosing ranged from 10.37. to 27.3% of the dose in the order: TNT < 2,4-DNT< 2,5-DNT - 3,4-DNT 0 3,5-DNT e 4-amino-2,6-DNT - 2,6-DNT < 2,3-DNT. Theamount of vadioactivity remaining in the GI tract plus contents and fecesranged from 3.17. to 46.87. of the dose, in the order: 2,3-DNT < 2,6-DNT <2,4-DNr - 3,5-igr < TieT - 2,5-DieT < 3,4-DNT < 4-,mino-2,6-DNT.

2,4-DNT was metabolized in vitro by liver homogenates of mice,rats, rabbits, dogs and monkeys. In these species, between 8 and 27% ofthe parent compound was metabolized at the end of 1 hour of incubation.Under aerobic conditions, the primary product was 2,4-dinitrobenzyl

I.

xiv

alcohol and the second product aminonitrotoluenes. The liver of rabbitsformed more metabolites that the other species. Under anaerobic conditions,the amount of 2,4-dinitrobensyl alcohol vas reduced while more aminonitro-toluenes were produced. More aminonitrotoluenes were formed by males thanfemales; and male rats produced the most. When male rats were fed 0.7% of2,4-DNT, as used in the toxicity studies above, for 2 weeks, neither zoxa-solamine paralyois time nor hepatic nitroanisole O-demethylase activitywere affected.

Principal Investigator

Cheng-Chun Lee, Ph.D.Deputy DirectorBiological Sciences Division

MIDWEST RESEARCH INSTITUTE425 Volker BoulevardKansas City, Missouri 64110

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MAMMALIAN TOXICITY OF MUNITION COMPOUNDiSPHASE II: Effects of Multiple Doses

PART II: 24-Dinitrotoluene(Report No. 3)

INTRODUCTION

"Under Contract No. DAMD-17.74-C-4073, entitled, "Munition Compounds

Mammalian Toxicity Study," we conducted Phase I studies on the effects of

acute exposure of various munition compounds-l During Phase II, we studiedthe effects of -ultiple exposure to selected compounds including trinitro-

. glycerin (TNG), 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DN.T)

and nitrocellulose (NC). This report summarizes the results of Phase IIstudies on 2,4-DNT. Subacute and subchronic toxicities were performed indogs, rats, and mice to determine the maximum tolerated dose and to definethe biological nature and target organ(s) of the toxic effects. Reversibil-ity of any adverse effects was determined. Mutagenicity of the compound wasassessed. Immunologic response was studied by the detection of the serum

IgE antibodies. A detailed study on the disposition and metabolism of theradiolabeled compound was performed in rats; the metabolites were isolatedand identified. Comparison of the pathways of biotransfoxmation was alsostudied in vivo in mice, rabbits, dogs and monkeys and in an in vitro systemusing tissues of the various species including the liver of human cadavers.For comparison, biliary excretion of various nitrotoluenes, including TNT,2,3-DNT, 2,5-DNT, 2,G-DNT, 3,4-DNT, 3,5-DNT and 4-amino-2,6-DNT n rats

were studied. Effetts of the test compound on drug metabolizing enzymeswere also investigated in in vivo and/or in vitro studies.

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11

I I. DOGS

TABLE OF CONTENTS

!Est

A. Subacute and Subchronic Toxicities and Reversibility ........... 5

1. Introduction ......... ............................. 5

2. Material and Methods ............................. 5

J a. Number of Dogs, Sex and Treatment ............. ..b. Experimental Procedures ....................... 5

c. Experimental Design ............ ................ 6

3. Results .......... ................................. 7

I a. General Observations and Weight Gain ..... ......... 7b. Blood Analysis ........... .................... 8

c. BSP Retention .......... ...................... 9

d. Organ Weights ............ .................... 9e. Gross and Microscopic Examination of the Tissues. . 9

I 4. Discussion and Conclusions ........ ................. 10

B. Immunologic Response to 2,4-DNT ...... ................. ... 11

1. Introduction ........ ...................... ..... 112. Material and Methods ....... .................... .... 11

3. Results and Conclusions ....... .................. ... 12

I C. Sunmnary ........... ................................. ... 12

Tables 1 -19 ............. ............................. ... 13

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5. _ A

I. DOGS

A. Subacute and Subchronic Toxicities and Reversibility

1. Introduction

These studies were perfznrmed to define the nature and extent ofeffects of 2,4-DNT on the hiological syaL-m at the biochemLcal and cellulari levels and to elucidate the dose-response relationship in dogs after admini-

stration for 4 weeks and 13 weeks. The reversibility of adverse effects

were studied after the treatment of 2,4-DNT was discontinued for 4 weeks.

2. Material and Methods

a. Number of Dogs, Sex• and Treatment

A total of 32 youug healthy beagle dogs (aztelton ResearchAnimals, Cumberland, Virginia) weighing between 7...5 and 12.5 kg were usedSfor these experiments. The dogs were conditioned and observed carefully inour animal quarters for 3 weeks after their arrival from the supplier. Theywere divided into four groups, each consisting of four males and four fe-males. The average weights of all groups were kept close.

Three groups of dogs were given 1, 5, or 25 mgikg/day of

"2,4-DNT in capsules. 2,4-DhNT was purchased from K and K Laboratories(Cleveland, Ohio), and mixed with lactose, USP, in a ball mill to give mix-tures containing 1%, 57., and 25%. of 2,4-DNT, respectively. After the weekly"weighing, appropriate amounts of the proper mixture were weighed into cap-sules, so each dog received the intended dose. The fourth group rerved asthe controls and was given empty capsules daily throughout the experimentts.Purina dog chow and water were available ad libitum, except wherever specified.

b. Experimental Procedures

All dogs were observed daily for behavioral changes and toxic

signs. Body weights of all dogs were recorded weekly. Blood samples werecollected for laboratory tests before treatment and at 4, 8, 13, and/or 17

weeks during experiment. The tests included hematology, clinical blood chem-

istry tests and serum electrolytes. For fasting blood glucose, the dogswere fasced overnight for 16 hours. At termination or when moribund, thedogs were euthanized with an overdose of sodium pentobarbital and examinedfor gross lesions. Weights of heart, liver, spleen, kidneys, adrenals, andii [gonads were recorded, and organ weight to body weight ratios were calculated.

5

Various tissues were removed, fixed, processed, sectioned and stained for

uicroscopic examination of lesions. The procedures for hematology, clinical

blood chemistry tests, and histopathology, and the normal values are given

in Appendix I.

The concentrations of ca2 +, i2+ N and k+ in serum were

determined with the itomic absorption spectrophotometer, according to thep-rce~ure u•aA, by F-hus,?./ orginally described for Ca and Mg. The resonaanclines used for the analysis of each of the elonents are: Ca, 4227 1; ma,2832 A; ea, 3302 A; and K, 7665 A. Sodiup was determined by using the 3302 Aline rather than the more sensitive 5890 A line to avoid large dilutLons ofthe serum. In thi.s way, Ca, Mg, and Na were determined after a 50-folddilution (0.2 mg serum to 10 ml) of the serum with 1,000 ppm 6trontiuw in0.1 N perchloric acid. Petassi•m was determined citer a second diiutioi:(1:1) or a total of 100-fold dilution. Plosplate interference mnd the inter-ference of sodium on potassium v3re eliminated by the addition of strontium.

Thc perchloric acid was used to remove protein interference. The serumchloride cow.entration was determined with a Buchier-Cotlove chloridometer.

Bromosulfophthalein eBSP) reteution test was performed attermination. A single dose of 5 ng/kg of the sterile test dye (Dade, Miami,Florida) was injected intravac-usly following fasting for 16 hours. Serumlevel of the dye at 15 minutes was determined and the percent of retentionin the plasma was calcultted. 2-

The results of the various parameters were compared with thereepective baseline levels and/or with those of the control groups at therespective time intervals according to the Dunnett'n multiple comparisonprocedure ./I

c. Experimental Design

At the end of 4 and again at 13 weeks of continuous treatment,one male *nd one female dog from each dosage group were euthanized for necropsy.

The treatment for one other male and female dog from each group was discon-tinued at the end of 4 and 13 weeks and they were then euthanized at the endof 8 and 17 weeks, respectively, to study the raversibility of adverse effects.

Several dogs from the high dosage group weri moribund and euthanized beforethe scheduled time. The two dogs in the high dosage group (male No. 59 andfemale No. 62) who were removed from treatment after 4 weeks were not eutha-nixed until 8 months later to test reversibility after recovery for a longer

time.

6

I3. Results

a. General Observations and Weight Gain

* The control dogs and dogs receiving I or 5 mg/kg/day of3 2,4-DNT were h6althy throughout the treatment period of 4 or 13 weeks.

Their body weights bo#fore, during and after treatment are summarized inTable 1. Some dogs consistently gained weight, and others gaitred or lost

small amounts of weight.

Iii contrast, all of the dogs trecttd with 25 mg/kg/day beganj showing toxic signs after 12 to 22 dAys of treatment. Female No. 62 fixst

had a lose of control of her hind legs on day 12 of the study. After 4weeks' ;reatment, she was removed from treatment for recovery studies. HalesNos. 59, 63, and 65 first showed similar symptoms on day 14. No. 65 waskiiled when moribund after 22 days of treatment, and No. 63 4fter 24 days.

No. 59 ias pLcced in the recovery study with female Nt-. 62 after treatmentIL for 4 weeks. Two females first showed toxic signs on day 20. No. 64 waskilled when moribund on day 36; No. 66 survived 13 waeks' treatment and wasallowed to recover thereafter for 4 weeks. The last two high dosage dogsfIrst showed symptoms on day 22. .'emale No. 60 was killed when moribund onday 48; male No. 61 war killed after 13 weeks.

The intensity of toxic signs varied between dogs and also inthe same dog from time to time. As noted above, on day 22, male No. 63 was

-moribund, while male No. 61 was Just beginning to show symptoms. Similarly,No. 65 was euthanized 8 days after he first showed symptoms, while maleNo. 59 survived 77 more days of continued treatment. Frequently, a dogshowed severe symptoms for days or weeks, apparently recovered for a while,

and then got worse again. When dogs were removed from treatment, the symptomsalways lessened. After 4 .weeks of reco'-ary, they appeared normal except for

occasional poor balance.

Toxic signs seen in virtually all affected dogs included de-creased feed cunsumption, weight loss, yellow stain on and near hiud legs,

~i 1. pale gums (sometimes blue-tinged), and a neuromuscular incoordination andparalysis. The weight loss is shown in Table 1, vith come dogs losing half

P "their initial body weight. The characteristic effects of 2,4-DNT were thosea.- on the neuromuscular system. The first manifestation was a stiffness and

incoordination of the hind legv. As this progressed, the animals had diffi-

culty maintaining their balance. Evantually, they were unable to stand, andcollapsed, lying on one siJe. Their muscles atiffened with the legs extendedand back arched. The stiffness progressed upward from hind legs to trunk,forelegs, neck, and then head. One dog with moderately severe signs was

examined by Dr. Brazil, a veterinary neurologist at the University of

Ii 7II

Missouri, Columbia. He found hyperreflexia of the extremities, intr.tspinal cord and nerves, opf.sthotonus, and a roli-ng motion to the right.

The dog showed signs similar to those of a middle ear inflammation, butno such lesion was found.

Some toxic signs were seed in only S few of the dogs. Females

woo. 62 and 66 had transient blindness. When examined by Dr. Harlan Jansen,a veterinary ophthalmologist of the University of Missouri, Columbia, Hiisouri,No. 66 was functionally blind with widely dilated pupils unresponsive tolight. Vertical or oval nystagmus was present when the dog's position was

changed or when abe was stimulated by a loud noise. The fundus appeared

normal. Several dogs had occasional tremors; male Wo. 65 had a grand Maal

type convulsion shortly before he was euthanized. At times, some dogs sali-vated excessively. A few vomited occasionally, but the time was not ob-

served.

b. Blood Analysis

Since the hematology and clinical blood chemistry results ofthe male dogs and the female dogs were not significantly different, thedata frnm both sexes from the control group and the treated groups were com-

bined and are summarized in Tables 2 through 5, respectively. In the lowand middle dosage groups, the peripheral blood elements and various clinical

chemistry parameters were not apparently alterad by 2,4-INT. However, whencompared to baseline levels within the group, or when compared to the controldogs at the same time interval, there were a number of changes. The increase

in the erythrocyte count and blood hemoglobin levels with a correspondingdecrease in cell volume and cell hemoglobin fs characteristic of maturingbeagles of the age used. The other changes were slight and inconsistent

and usually occurred in both control and treated dogs.

The high dose of 2,4-DNT (25 mg/kg/day) caused some changes

in the blood. The erythrocyte count did not increase as did the controls

and other treated groups. Hemoglobin, hematocrit and cell volume decreased.

The reticulocyte count was high, except for two female dogs (Nos. 60 and 64)tested in week 6. These two dogs were very severely affected and had almost

no reticulocytes and elevated BUN.

The treatment of one male and ottc fil& from each group wasdiscontinued after 4 weeks to study reversibility of adverse effects. As

seen in Table 6, all treatment groups are similor. A reversibility studywas also performed after 13 weeks of treatment. Only one female treated

with 25 mg/kg/day survived at the end of 13 weeks. The changes of herperipheral blood elements caused by k,4-Dk4T were reversible after the treat-

ment was discontinued for 4 weeks, although the erythrocyte count, hemato-

crit 2nd hemoglobin remained l.ower than those of the eqntrol dogs (Table 7).

8

Methemoglobin and Heinz bodies were determined after 4, 8 and,13 weeks of treatment. Results are shown in Table 8. Small amounts of

methemoglobin were seen in the surviving high dosage dogs tested at both8 and 13 iyeeks and I.n two of the four middle dosage dogs tested at 13 weeks.All levels found were minimal. Since the assay In the difference of twoabsorbance readings, some of the values may be artifact. Heinz bodies wereseen in all high dosage group dogs at all times tested. When dogs weretested after 4 weeks' recovery, none had any methemoglobin. Only one high dosagegroup female (No. 62) had 0.4% Heinz bodies at 4 weeks after 4 weeks of

2,4-DNT treatment.

Serum electrolytes were assayed before treatment and after

4, 8, and 13 weeks' treatment. Results are shown in Table 9. No changesoccurred during treatment.

c. BSP Retention

BSP retention was determined on the dogs before treatmentand the dogs euthanized after 4 or 13 weeks' treatment. The results areshown in Table 9. 2,4-DNT did not cause any retention of BSP in these dogs.

Sd. Organ Weights

I The absolute and relative organ weights of all dogs areshown in Tables 11 through 14. Changes were seen only in the high dosagedogs. Their livers were relatively large, due to decrease in body weight.

e. Gross and Microscopic Examination of the Tit'suesThe dogs in the control, low dosage and middle dosage groups

terminated at various times were in good nutritional condition vithout anyapparent gross changes. After 4 weeks, one control dog (No. 58) had mildinflammation in the lung and liver (Table 15). After 13 weeks, one controldog (No. 53) and both low dosage dogs (Nos. 77 and 78) had similar inflama-tion in their livers (Table 16). These are spontaneous lesions, riot relatedto 2,4-DNT treatment.

The high dosage dogs were In fair to poor nutritional con-dition with little or no body fat. Two dogs that were moribund during the4th week had cloudy swelling in the heart and kidney, gliosis, edema, de-

myelination in the cerebellum, brainetem and/or spinal cord (Table 15).In addition, one dog (No. 63) had subacute inflammation in the liver, tubulardegeneration in the kidney, iiucoid degeneration in the gastrointestinal tract,

inective germinal centers in the lymph node and aspermatogenesis in the testes;the other dog (No. 65) had emphysema in the lung and hemosiderosis in theliver. One dog (No. 64) was moribund during the 6th week, one dog (No. 60)

19I

was moribund during the 7th week and one dog (No. 61) was terminated at theend of the 13 weeks. These three dogs had mild cloudy swelling in the heartor kidney, severe hemosiderosis in the liver, spleen or lymph node, mild tomoderate gliosis, edema, demyelination in the cerebellum, brainste. Nr spinal

cord and/or severe aspermatogenesis in the testes (Table 16). Other occa-sional lesions included mild pneumonia and emphysema in the lung, subacuteinflmmation in the liver, tubular degeneration in the kidney and lymphoiddepletion in the spleen. The bone marrows and the myeloid/erythroid (W/E)

ratios of these dogs were normal.

The dogs treated for 4 or 13 weeks and allowed to recoverfor 4 weeks or longer were in good nutritional condition. There were a few

mild to moderate lesions in the lung, liver, and lymph nodes (Tablea 17 and18). Two high dosage dogs treated for 4 weeks and allowed to recover for8 months had only a moderate pneumonia or a mild tonsilitis. The high dosagefemale dog treated for 13 weeks and allowed to recover for 4 weeks hadmild demyelination of th, :erebrum and optic nerve.

4. Discussion nd Conclusions

Daily treatment of I or 5 mg/kg/day of 2,4-DNT for 13 weeks didnot cause any adverse effects to dogs. Daily treatment of 25 mg/kg/day wastoxic after 12 to 22 days and lethal after 22 or more days. There wasgreat variation in individual susceptibility. Three primary target organswere seen: the neuromuscular system, the erythrocytes and the testes.

The neuromuscular effects observed grossly included incoordinationand rigid paralysis resulting in anorexia and weight loss. These wereassociated with mild demyelination, gliosis and edema in the central nervoussystem. A casual relationship is not certain, but it is significant thatdemyelination of the optic nerve was seen only in one of the two dogs that

suffered from transient blindness.

The erythrocytic effects are readily explained by the well-knownmethemoglobin-producinil effects of nitro and amino compounds.-/ 2,4-DNT

or some of its metabolites oxidize hemoglobin to methemoglobin. The quantityof methemoglobin is more than the usual hemostatic systems can reduce, so itis destroyed. This process leads to the destruction of erythrocytes, de-posits of hemosiderin in liver, spleen, and/or lymph nodes, Heinz,.bodies in

the surviving erythrocytes, and a stimulation of erythropoiesis. The eryth-ropoietic effect leads to an increase in circulating reticulocytes. Most ofthese effects were observed. Blood samples were normally taken 18 to 20 f

hours after dosing, abd most methemoglobin han been removed from circulation.

The testicular effect was a decrease in spermatogene~is. Thiseffect was noticeable after treatment for 4 weeks with 23 mg/kg/day of 2,4-DNT

10

FI

and was 6'vere after 13 weeks. However, a subsequent recovery period of

j 4 weeks or longer was sufficient to restore to normal histologic appearance.

The anorexia and subsequent malnutrition complicated the body'snormal repair mechanisms by reducing the supply of exogenous protein and

calories. Therefore, the liver was unusually active in cstabolizing varioustissue constituents. Because of this increased activity, the livers of the

affected dogs did not reduce in weight, although these dogs had severe weightloss.

Morton, et ýl.•/ have recently reported significant Increases in

SCOT in workers exposed to TNT dust. We found no similar effects. Thismay be due to different prop.•irties of the two compounds or due to speciesdifference.

- There was a wide variation in individual susceptibility to the toxiceffects of 2,4-DNT: one dog was moribund when two others began to show

symptoms. Similar variability has been noted in workers exposed to 2,4-DNT

"fume4.•. Furthermore, some dogs apparently adapted to continued ingestionof the corpound and partially recovered from the toxic effects.

"When treatment with 2,4-DNT ceased, affected dogs began to recoverfrom the various effects. After 4 weeks, one dog had a minor balance problemand some demyelinatioa in the cerebrum and optic nerve. Since this dog wasfemale, we have no data on testicular recovery. Two other dogs kept for 8

months were completely recovered from all effects.

B. Immunologic Response to 2,4-DNT

1. Introduction

In humans, anaglylactic reactions were associated with high :iters

"of innumnoglobin E (IgE).-1 IgE, the allergic or hypersensitive antibody,

was determined in dogs treated with 2,4-DNT.


The immunodiffusion technique of Mancini et al.A/ was used for

"determination of serum IgE titer. Replicate samples of serum from the con-

trol dogs and dogs treated with various doses of 2,4-DNT at various intervalswere placed in wells in an imunodiffusion chamber along with suitable stan-

dards. Those dogs were used for subacute and subchronic toxicity studiesas described in Section I.A. The diffusion chamber was incubated at 37"C

for 48 hours and the diameter of the precipitin ring was measured. Since

the square root of the diameter is directly proportional to the concentration

of the antibody, the IgE concentration was quantitated with the standard

Ii antibody reagent.11

[1

3. Results and Conclusions

The results of IgE concentration of control dogs and dogs treatedwith 2,4-DNT are summarized in Table 19. Treatment of various doses of2,4-DNT for 4, 8, or 13 wQeks or treatment for 4 or 13 weeks followed byrecovery for 4 weeks did not cause any apparent changes on serum concentra-tion of IgE.

C. Suary

Doses of 1 or 5 mg/kg/day of k,4-DNT for 13 weeks caused no adverseeffects in dogs.

Doses of 25 mg/kg/diy were toxic after 12 to 22 days and lethalafter 22 or more days, with great variation in individual susceptibility.The target organs were the neuromuscalar system (incoordination and rigidparalysis), the erythrocytes (methemoglobinemia and sequelae, IncludingHeinz bodies and anemia) and the testes (decrease .n spermatogenesis).Serum IgE levels were not affected by treatment. Se'erely affected dogsrecovered partially in 4 weeks after cessation of treatment and completelyin 8 months.

12

I TABLE 1

DoM WEIGHTS OF DOGS TREATED WITH 2.4-DNT IN CAPSULES

"Dose Dog Body Wetihts (kN)

"I (m_/k/day) No. Sex Initial 4 Weeks 8 Weeks 13 Weeks 17 Weeks

0 57 M 11.0 10.80 58 F 8,6 9.01 81 H 10.6 10.4

S1 82 F 8.0 8.2

5 73 H 12.2 13.0S5 74 F 8.2 8.0

25 63 M 11.8 8.8'/25 65 H 13.2 10.80/

S0 55 M 12.4 12.2!' 12.80 56 F 8.8 9.2,k/ 9.21 79 M 12.8 1 S&b/ 13.71 80 F 7.7 .6k/ 7.85 71 M 11.2 12.4k/ 11.95 72 F 8.4 7.8k/ 8.6

25 64 F 8.0 6.2 4.4-C/25 60 F 8.2 6.8 3.8-41

I 0 53 M 12.2 12.8 14.0 15.20 54 F 8.4 9.0 9.0 10.01 77 M 10.8 11.8 12.0 13.41 78 F 8.2 7.8 8.0 9.6

5 69 M 11.0 10.6 11.8 13.35 70 F 8.0 9.8 7.8 9.0

25 61 M 13.2 13.2 10.0 9.40 51 M 10.0 11.0 11.5 12.O.b/ 12.20 52 F 9.0 9.2 9.2 9.2k/ 10.21 75 M 10.9 11.0 11.9 12.8k/ 13.41 76 F 9.0 8.6 8.9 io.o, 10.45 67 M 10,6 10.6 10.9 11.2b/ 11.45 68 F 9.4 9.8 10.5 11.2_b/ 12.0

25 66 F 9.4 8.2 8.7 9. 3k/ 10.025 59 H 12.2 9 .0k/ 9.8 11.5 12.825 62 F 8.6 6.2-b/ 7.0 8.3 8.8

a/ Terminal Leight before necropsy during week 4.A b/ Dosing discontinued thereafter.c/ Terminal weight before itecropsy during week 6.d/ Terminal weight before necropsy during week 7.

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TABLE 8 1

METHIM4GLOBIN AND HEINZ BODIES IN CONTROL DOGS ANDDOGS TREATED WITH 2 .4-DNT IMethemoglobin (L) Heinz Bodieq (7M

Dosage.Lk~Laui 4 Week.!/ 8 Weeks 13 Weeks 4 Weeks 8 Weeks 13 Weeks

0 (4./ 0 (4) 0 (4) 0 (8) 0 (4) 0 (4)1 0 (8) 0 (4) 0 (4) 0 (8) 0 (4) 0 (4)

5 0 (8) 0 (4) 1.9 (4) 0 (8) 0 (4) 0 (4)

25 0 (6) 3.5 (2) 3.8 (2) 4.1 (6) 5.5 (2) 4.9 (2)

a/ Weeks of treatment.

b/ Mean (number of dogs tested).,

21

i ' IS1II

TABLE 9

I SERUM ELECTROLYTES OF DOGS TREATD) WITH 2,4-DNIT

Dose Serum E1ectrolvtes (mea/f)

(i/aL.isAY). Va Ca C151+2 ai..IBeform- .eatment (8 dou/lroup).

Control 150 + 2/ 5.5 + 0.4 5.5 + 0.2 1.6 + 0.1 115 41151 2 5.3 + .2 : 0.1 1.5±0.1 110+45 148 + 3 5,2 + 0.2 5.6 + 0.2 1.5 + 0.1 112 + 2

25 149 + 2 5.3 + .4 + 0.1 1.5±0.1 113+3

Treatment fcr 4 Weeks (2 doogs/proup)

Contiv*4 151b/ 4.8 5.2 1.6 1091 I•13 4.7 5.3 1.4 1055 c/ 154 5.1 5.6 1.5 104

I 157 5.7 5.0 1.8 110

___ Tretmc, nt for 8 Weeks (2 dosslarouD)

I Control 154 5.1 5.5 1,.6 1081 152 4.9 5.3 1.6 1065 150 4.7 5.1 1.6 104

21/ 153 5.0 5.2 1.8 107

3 Tro-atment for 13 Weeks 4( dogularoup)

Control 148 4 2 4.4 + 0.2 5.2 + 0.2 1.5 + 0.1 104 + 11 146 + 1 4.4 + 0.2 5.1 + 0.1 1.5 + 0.1 101 + 254146 + 3 4.6 + 0.3 5.0 ± 0.3 1.5 + 0.1 102 4 2

29/ 145 4.7 4.9 1.6 101

I a/ Mean + S.E.b/ Mean.C/ Owie dog._d/ Three dogs.e/ Two dogs. t

I1I

II-I I

'1TABLE 10

BSP RETENTION OF DOGS TREATED WITH 2.4-DNT

Dome Before .Treatment After 4 Weeks After 13 Weeks£iAS/aLdayl potNo R kk gatio Dos N.~ ZRetentign PgLNo. I ZnesaIngi

Control 51 5 57 4 53 7Control 52 9 58 3 54 5Control 53 7

175 7 81 5 77 6 -

1 76 5 82 6 78 7

1 77 5

5 67 2 73 4 64 45 68 10 74 3 70 55 69 8-5 Z0 1.Q25 59 12 61 425 60 5

F 25 61 325 62

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ILTABLE 15

SUMMARY OF TIISSUE LEIONS OF DOGS TREATED

WITH 2.4-mD FO 4iO W

Done (mR ka/day)

0 1 5 25, Lee ion~a/ Doz No.: 57 5_8 8•1 8.22 3 ,4 _3 6

Heart_ •lud~y~e1ln + +

LungSubacute inflaunation +_ tnhyapa +

Liver

Subacute inflammation 4- +

Herosiderosis ++

Kidney

Cloudy swelling + +Tubular deieneratton +

Brain

Gliosis +

Edema +

Cerebellar demyelination + +

Brainstem demyelination +

Spinal cord demyelination +

Gastrointestinal TractMucoid de.gnerat ion +

Lymph NodeInactive centers +

TestesAspermat_2gnes is +

Bone Marrow_M/E 1.5 1.4 1.4 1.3 1.4 1-3 c/ c/

Tissues not listed were normal.

a/ Severity of Lesions: + - mild; ++ - moderate; +++ - severe;- very severe; + - questionable.

_/ Moribund and terminated during the 4th week.Mc Harrow smear was not prepared.

2727

Hi

|t

TABLE 16

SUMMARY OF TISSUE LESIONS OF BFAGLE DOGSTREATED WITH 2.4-DNT FO)R 13 WEEKS

Dose (g/Ikplday)0 1 5 25

Lesions-/ Dog No.: 53 54 77 78 69 70 60V 61

HeartCloudy swel lin - +

Lung -.

Pneumonia + V

Liver -

Subacute inflammation + + + +Hemosiderosis ++ +++

KidneyCloudy swelling + +Tubular dejeneration +

SpleenHemosiderosis +++ +

_ L~yyhoid depletion ++Brain

Gliosis ++ + +Edema ++ + +Cerebellar demyelination + + +Brains tern demyelination +pinkal cord djmyelination +

Lymph NodeHemos ideros is +++

Testes-_ Aspe_.ma~toseneuis 4-I-+-

Bone Marrow JiM/E ratio 1.3 1.3 1.1 1.3 1.4 1.2 1.4 1.0 0.9


a/ Severity of Lesions: + - mild; 4+-I moderate; +4- severe; ++++ = verysevere; t questionable. 17

b/ Moribund and terminated during the 6th week.cl Moribund and terminated during the 7th week.

28

I-

TABLE 17

SUMMARY OF TISSUE LESIONS OF DOGS TRIATID WITH 2.4-DNTFOR 4 WEEKS AND ALLOWED TO RECOVER

FOR 4 WEEKS OR LONGst

Dose (=fka/day)

0 1 5 25

Lesions•/ Doa No.: 55 56 759 80 71 /2 59 62

LungsPneumonia ++ ++ ++Edema +

Lymph NodeHyperplasia + +

TonsilInf lamination +

Bone Marrow1.E ratio 1 1. 1.6 1.3 1.2 1.5 _ -

Sri. . .

Tissues not listed were normal.a! Severity of Lesions: + - mild; +-I-= moderate; +++ s levere; +-I- -

4 very severe; + - questionable.b/ Allowed to recover for 8 months, the others are all for 4 weeks.S/ Marrow smear was not prepared.

I2

F'29

TABLE 18

SUM4IRY OF TISSUE LESIONS OF DOGS TREATED WITH 2.4-DNTFOR 13 WUIKS AND ALLOWED TO RECOVE FOR 4 WUKS

Dose (mg/ka/day)

0 1 5 25

LungssSubacute inf lammation + +

Li~verSubacute inflamtation + 4+Heplati~c cell necrosis +_

Brain

Cerebral dmyelination +_ pti" nekrve Am ltation _+.

Lymph Node+imnt -+

Bone Marrow- N/K ratio 1.3 1.4 1.4 1.3 1.5 1.5 _1.4

Tissues not listed were normal.Severity of Lesions: -e, - mild; +4-= moderate; +4+ = severe;

+4-4 - very severe; + - questionable.

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IT,II RATS

TABLE OF CONTENTS

Page

A. Subacute and Subchronic Toxicities and Reversibility ............ 35

1. Introduction .......... ......................... ... 352. Material and Methods ....... ..................... .... 35

a. Number of Rats, Sex and Treatment .... ........... ... 35b. Animal Husbandry ....... ... .................... 35c. Feed Preparation .......... .................... 35d. Experimental Procedure ...... ................. ... 36e. Experimental Design ........ .................. ... 36

3. Results ...... ..... ......................... .... 36

a. General Observations and Weight Gain ............. ... 36b. Feed Consumption and 2,4-DNT Intake .............. ... 37

c. Blood Analyses ......... ... ..................... 37d. Organ Weights ........ ... ..................... 39e. Gross and Microscopic Exauination of Tissues .......... 39

4. Discussion and Conclusions ....... .................. ... 40

B. Cytogenetic and Mutagenic Effects of 2,4-DNT ................ ... 41

1. Introduction .......... ......................... ... 412. Material and Methods ......... ... ..................... 41

a. Animals ........ ..... ........................ ... 41b. Cytogenetic Tests ........ ... ................... .2c. Mutagenic Effects on CHO-KI Cells Tn Vitro. .......... 42d. Dominant Lethal Mutation Tests.... . ........... ... 42

3. Results ...... ......... ........................... ... 43

a., Cytogenetic Tests ........ ................... ... 43b. Mutagenic Effect In Vitro ...... ............... ... 43c. Dominant Lethal Mutation Test ........ ............. 43

4. Discussion and Conclusions ...... .................. . 44

33

i . ..... ,_• , - IT I • ._ III

rl

TABLE OF CONTENTS (Concluded) I

C. Immunologic Response to 2,4-DNT ..... ................ .... 44

1. Introduction ......... ...................... 44.2. Material and Methods ............. .................. 44

3. Result and ConcluaLiw ...... ................. ..... 44

D. Summary ............. ........................... ... 45

Tables 20- 52 ............. .......................... ... 46Figure 1 .................................................. 79

34o

1.1

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34 ii

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!A.

II. RATS

A. SubacuLe and Subchronic Toxicities and Reversibility

1. Introduction

As for the dogs, these studies were performed to define the nature

and extent of effqcts of 2,4-DhNr on the biological system at the biochemical

"and cellular levels and to elucidate the doae-resaponse relationship in the

rats fed 2,4-DNT for 4 weeks and 13 weeks. The reversibility of any adverse

effects was also studied after the feeding of 2,4-DNT was discontinued for

"4 weeks.

2. Material and Methode

a. Number of Rats, Sex and Treatment

A total of 64 male and 64 female young healthy CIO rats

(Charles River Breeding Lab.) were used for this study. They were dividedinto four groups, each consisting of 16 wales and 16 females. The averageweights of all groups were kept close. Three groups of rats were fed 0.07,

0.2, or 0.7% 2,4-DNT in their feed. The fourth group, serving as control,

was given the powdered standard rodent chow (Wayne Laboratory Meal) without

2,4-DNT.

b. Anival Husbandry

Our animal quarters have a ventilation system with 10 air

changes per hour. The room air is passed through filters to remove 99.97%of all particles larger than 0.3 P. The temperature is maintained at 75 +

50F and the relative humidity at 50 + 107.. All aniumal rooms are kept at

12-hour light cycles.

Upon arrival from the breeder, the rats were isolated andi

conditioned in our rodent quarter for at least 2 weeks before starting on

the experiment. They were housed two per plastic cage with filter tops.

Hardwood chips were used after steam-sterilization cs bedding material and

changed weekly. All cages, cover tops and water bottles were steam-

sterilized before using and once every month. Feed and watet were availableat all times.

c. Feed Preparation

.. i 2,4-DNT was purchased from K and K UIboratories (Cleveland,

Ohio) and mixed with rat feed to produce a diet contain:ing the desired 0.7%

1-1 of 2,4-DNT. The diet was placed in a wooden bon (16 x 16 x 20 in.) until

"35

U'

half full. The box was rotated about its long axis for I hour in a modifiedcement mixer at a speed of 20 rpm. Subsequently, portions of this diet weremixed with the standard chow in proper amounts to yield 0.2% and again 0.07%.of 2,4-DNT, respectively.

d. Experimental Procedure

The experimental, procedure for rats was the same as for dogs, Tdescribed in Section I.A.2.b., with the following exceptions:

(t) Feed consumption of all rats was measured throughoutthe experiment. •

(2) Blood samples were collected by cutting the tip of thetail at 0, 4, 8, 13 and/or 17 weeks for hematology tests. In additioa,termine.l blood was collected from the abdominal aorta under ether anesthesiafor clinical chemistry tests.

(3) BSP retention test was not performed.

e. Experimental D--sign [The experimental design for rats was the same as for dogs,

described in Section I.A.2.c., with the following exceptions:

(1) At the end of 4 and 13 weeks, four male and four femalerats from each group were euthenized for necropsy.

(2) Treatment was discontinued for four male and four femalerats from each group at the end of 4 weeks and 13 weeks. These rats wereobserved for 4 more weeks and then euthanized for necropsy to atudy the re-versibility of the adverse effects.

3. Results


The control rats and rats fed 0.077% or 0.2% 2,4-DNT in thefeed were heaithy throughout the experimental periods of 4 or 13 weeks.Rats fed 0.7% 2,4-DNT soon ate less, were inactive, lost weight, end died.A few animals occasionally had an unusual gait with wide spread and stiffhind legs. I.iLa resembled the gait seen in affected dogs, but the ensuingrigid paralysis was not seen.

The female rato in the high dosage group died ion aftert,:eatment began. Four died in the first week, four more in the second, and Ftwo in the third week. After 4 weeks of treatment, four survivors were

36

killed for autopsy and two survivors were given control feed without 2,4-DNTto test reversibility of the effects. Deaths in the males occurred later:the first two in week 4, after more than half of the females had died. Inaddition, there was one death in week 6, one in week 7, two in week 8, onein week 12, and one in week 13. One middle dosage group rat died in week14, just after the start of the reversibility experiment. All of the ratsdying at unscheduled times had lost wetght and had little or no body fatremaining.

The body weights of the male and female rats before, during,and after treatment are summarized in Tables 20 and 21, respectively. Theweight changes are better illustrated in Figure 1. The control rats con-

sistently gained weight throughout the experiment. The low and middledosage group rats did not gain as much weight and occasionally lost weight.

The high dosage group rats consistently lost weight. During the reversalstudy all rats gained weight; the lightest ones (high dosage group) gainingthe most.

b. Feed Consumption and 2.4-DNT Intake

Feed consumptions of rats fed various doses of 2,4-DNT aresummarized in Table 22. These results reflect the weight changes. Low andmiddle dosage group rats ate somewhat less than control rats; high dosagegroup rats ate one-third to one-half as much as controls. During the re.-covery period, all rats ate similar amounts of feed.

The 2,4-DNT intakes of the treated rats are stmnariz;•d inTable 23. Eecause of their increase in body weight, intake by the low dosagemale rats decreased from 37.6 mg/kg/day in the first 4 weeks to 31.8 mg/kg/dayin the last 5 weeks, averaging 34.3 mg/kg/day. Since the weight varied little,intake by the low dosage females fluctuated only slightly between 37.6 and39.7 mg/kg/day, averaging 38.3 mg/kg/day. In the middle dosage group rats,the males received an average of 92.8 mg/kgiday, ranging from 84.3 to 101.7mg/kg/day; the females received an average of 108.3 mg/kg/day, ranging from97.4 to 117.7 mg/kg/day. Because of relatively constant feed consumpcion

at decreasing body weight, 2,4-DNT intake by the high dosage male rats in-creased from 191.4 mg/kg/day to 292.1 mg/kg/day, averaging 265.6 mg/kg/day.The intake by the high dosage female rats was 145.2 mg/kg/day.

c. Biood Analyses

The hematology results of male control rats and male rats fedthe various amounts of 2,4-DNT are summarized in Tables 24 through 27. Therewere a number of statistically significant fluctuations in the control male

rats during the experimental period of 13 weeks (Table 24). These are within

37

normal litmits. At 17 weeks, the erythrocyte, platelet and leukocyte countsof these control males decreased with increases in various blood 1,lces.

male rats receiving 0.07% or 0.2% 2,4-DNT in the feed were substantially

similar to those of the control males (Tables 25 and 26). In addition,there was occasional and slight reticulocytosis. At fourth week, male rats re-ceiving 0.77. 2,4-DNT had decreased erythrocyte count with a compensatoryreticulocytosis, increased cell volume, and increased ceil hemoglobin

concentration (Table 27). These effects and a later decrease in hematocritand hemoglobin concentration continued throughout the treatment as the .

anemia worsened. After 13 weeks of treatment and 4 weeks of recovery, the

surviving males had returned to the same level as the controls. There were

fluctuations in platelet and leucocyte counts; but these changes were notconsistent.

"The clinical blood chemistry results from male rats terminatedaftý treatment or after treatment plus recovery for 4 weeks are shown inTables-28 and 29, respectivily. There were no apparent and consistentchanges. High values of fasting blood glucose of the control and treatedmale rats suggest that these rats were not fasted.

The hematology results of the female control rats and ratsfed various levels of 2,4-DNT are sumarized in Tables 30 through 33,

respectively. As seen in the male control rats, the control females had anumber of statistically significant fluctuations during the first 13 weeks

and a number of changes at week 17 (Table 30). Similarly, the results fromfemale rats receiving 0.07% or 0.2% 2,4-DNT in the feed were essentially

the same as those of the control females (Tables 31 and 32). Occasionaland slight reticulocytosis also occurred. The two rats receiving 0.77. of

2,4-DNT which survived for 4 weeks of treatment had decreased erythrocytecount, hematocrit and hemoglobin concentration with compensatory reticulo-

cytosis (Table 33).

The clinical blood chemistry results from female rats ter- --

minated after treatment or after treatment plus recovery for 4 weeks areshown in Tables 34 and 35, respectively. As fcr the male rats, there were

no consistent changes. High values of fasting blood glucose of the controland treated female rats suggest that these rats were not fasted.

The results of serum electrolytes of rats terminated at varioustimes are shown in Table 36. Occasionally, there was some statistical sig-

nificance. However, the values fluctuated within normal limits.

38

II

38

d. Organ Weights

The organ weights of the rats fed 2,4-DNT for 4 or 13 weeksand for 4 or 13 weeks plus 4 weeks recovery are summarized in Tables 37through 40, respectively. After 4 weeks, the livers of males fed the middlelevel and females fed the low and high levels were enlarged. The kidneysand hearts of high level males were small, but this was proportional to

their smaller body weight. After 13 weeks, based on the body weight, therelative liver, kidney and brain weights of males fed the middle dose andthe relative kidney and brain weights of females fed the middle dose wereenlarged. The relative weights of these organs of one male fed the highlevel were also enlarged. All females did not survive after 8 weeks. Based

on brain weight, the relative liver, spleen and heart of one male fed thehigh level and the relative heart of females fed the middle level were smaller

than those of their respective controls. Results for rats allowed to re-cover for 4 weeks showed partial recovery of body weight and similar variationsin various organ weights. The liver weights of treated rats after allowedto recover for 4 weeks were elevated. 'ihis presumably reflects high meta-bolic activity during the recovery process.

e. Gross and Microscopic Examination of Tissues

At autopsy, control rats and those receiving 0.07% or 0.2%

2•',..iT in the feed were in good nutritional condition with no gross lesions.Rats fed 0.7% of 2,4-DNT had little body fat, reflecting their body weightloss, but no gross lesions. Microscopic examination revealed a number of

lesions in rats from all groups. Because of the similarities between the

middle and low dosage groups, slides from the l&tter were scanned, butnot reported.

After 4 weeks of 2,4-DNT feeding, control rats had a few mildlesions in tb• lung or liver, as shown in Tables 41 and 42. The rats fed).4." •T hae ilar lesions. There was hemosiderosis in the spleen, pre-su'wably du,. to removal of methemoglobin from the circulation. In addition,

all males fed the high level of 2,4-DNT had moderate atrophy and aspermato-genesis in the testes. The bone marrows and the M/E ratios of these rats

were normal.

,.hen 2,4-DNT feeding is continued for 13 weeks, there are more

spoataneous lesions in the heart, lung, liver, spleen and kidney of controlrats, as seen in Tables 43 and 44. The treated rats had similar lesions.

However, the hemosiderosis wab more severe. The males fed 0.7% of 2,4-DNT"had very severe atrophy and aspermatogenesis in the testes. In these rats,

the testes were composed of tubules ot epithelium with practically no germcells in any stage of spermatogenesis. Rats fed the middle level had similar

39

lesions, ranging in degree from moderate to very severe. One male rat fedthe high level also had gliosis in the cerebrum. The bone marrows and theM/E ratios of these rats were normal. Hemosiderosis and the lesions in thetestes and brain were similar to those seen in the dogs treated with2,4-DNT.

lissue lesions in rats fed 2,4-DNT for 4 or 13 weeks andallowed to recover for 4 weeks are shown in Tables 45 through 48. Hemosid-erosis and testicular lesions caused by middle or high levels of 2,,4-DNTwere apparently not reversible after treatment was discontinued for 4 weeks.Gliosis and demyelination were also observed in the cerebellum of one malerat fed the middle level and one male rat fed the high level of 2,4-DNT.The M/E ratios of these rats were normal.

4. Discussion and Conclusions

The 2,4-DNT intake of the male rats fed the low, middle or highlevel of 2,4-DNT averaged 34.3, 92.8, or 265.6 mg/kg/day, respectively. Thefemale rats consumed an average of 38.3, 108.3 or at least 145.2 mg/kg/day,respectively. Since many female rats fed the high level of 2,4-DNT stoppedeating a few days before they died, the actual 2,4-DNT intake of these fe-male rats would be much higher.

The low level of 2,4-DNT was relatively nontoxic, since the onlyeffect seen was a slight depression of weight gain. This depressed effectof weight gain was accentuated in rats fed the middle level. In addition,hemosiderosis appeared in the spleen of these rats after 4 i-eks with amild reticulocytosis. The 2,4-DNT presumably produced methemoglobin by thewell-known mechanism of nitro and amino compounds.-/ These cells breakdown and the hemosiderin formed was deposited in the spleen. This resultedin a slight anemia, which was compensated by increased erythrocyte production,reflected in the reticulocytosis. At 13 weeks, there was a major decreaseor even complete cessation of spermatogenesis in the rats fed the middlelevel of 2,4-DNT.

The rats fed the high level of 2,4-DNT had more severe effectsthan the rats fed the middle level. There were severe weight losses in mostrats. Similar lesions in middle level rats appeared. The testicular lesionswere apparent by week 4; the rats were anemic by week 13, despite reticulo-,;Losis. In addition, there were some signs of a neuromuscular effect likethat seen in the dogs: wide spread and stiff hind legs, with gliosis and/ordemyelination in two rats. Finally, deaths occurred. Eight of the 16 maleson the high level and one on the middle level died before their schedulednecropsy. The high level female rats were devastated with 10 of 16 dyingin the first 3 weeks of study. These deaths occurred so rapidly that tissuelesions may not have developed enough to be visible on microscopic examina-tion. Many of these deaths occurred at night and we were unable to obtainuseful blood and tissue samples for examination.

40

, -

During the recovery studies, the rats rapidly gained weight,

particularly the high level rats which were most affected. However, 4 weekswas not sufficient for all the rats to catch up their weight with the con-trols. The erythropoietic system did return the erythrocyte levels to those

seen in controls. The tissue lesions remained. The testicular degeneration

apparently continued to worsen during the recovery period. This was pro-sumably the continuing development of a process initiated by 2,4-DNT. It

is not apparent from these studies whether the rats can recover from this

destruction of the germ cells.

To summarize, rats fed 34.3 to 38.3 mg/kg/day of 2,4-DNT had adecreased weight gain. Those fed 92.8 to 108.3 mg/kg/day had a greater de-crease in weight gain, reticulocytosis, hemosiderosis in the spleen, and

decreased spermatogenesis. Rats fed 145.2 to 265.6 mg/kg/day had severeweight loss, anemia with reticulocytosis, and a greater degree and more

rapid onset of splenic hemosiderosis and aspermatogenesis. Many of theserats died, the females after treatment for I to 3 weeks, the males after4 to 13 weeks. Four weeks' recovery sufficed for large weight gains andcorrection of the anemia, but did not eliminate the hemosiderosis and didnot lessen the aspermatogenesis. A few rats had episodes of wide spreadstiff-legged gait in the hind legs; some had mild to moderate gliosis and/ordemyelination.

B. Cytogenetic and Mutagenic Effects of 2,4-DNT

1. Introduction

The cytogenetic effect of 2,4-DNT on somatic cell chromosomes wasdetermined by examination of lymphocyte and kidney cell cultures. Thecapability of 2,4-DNT to induce single gene mutations was studied in Chinesehamster ovary cells in vitro. The mutagenic effect was studied using thedominant lethal mutation test to examine the effect of the testicular lesions

describe, above.


a. Animals

The rats used in these studies were those in the toxicity

studies described above or extra animals treated similarly. Some rats weremaintained on the diet containing 2,4-DNT for 19 weeks. Extra control fe-males were used in the dominant lethal mutation study.

41

b. Cytogetetic Tests

(1) Lymphocyte and Kidney Culture.

At the end of 5 and 13 weeks, blood samples were asepticallydrawn from the tail vein of both the control and treated rats. The lympho-

cytes were cultured by the method of Moorhead et al.12-- Kidney tissue sampleswere removed at necropsy and cultured by the trypsinization method ofFernandes.U/ All c•ltures were maintained in Eagle's medium as modified by

Vogt and Dulbecco.-

(2) Chromosome Analysis

Actively dividing kidney cultures and phytohemagglutin-stimulated lymphocytes were arrested in metaphase by short-term colchicinetreatment. The cells were trypsinized, swollen in hypotonic ,4olution, andprocessed for spreading on glass slides by the method of Moorbead and

Nowell..U/ Slides were stained with giemsa and scanned under Low power

optics. Cell polyploidy was estimated by examination of 200 cells. Slidesshowing minimrim scattering of cells in metaphase were selected for analysisunder oil immersion optics. Chromosomes were counted and morphologicdlaberrationswere examined from photographic negatives of up to 50 metaphase

cells.

c. Mutagenic Effects on CHO-Kl Cells In Vitro

Wild type Chinese hamster ovary cells (CHO-Kl)14/ capable of

growth in both a minimal and an enriched medium were exposed to selectedconcentrations of 2,4-DNT to test its ability to induce single gone mutations

in maumalian somatic cells. The concentrations used were selected from asingle cell survival curve obtained according to the method of Puck and Kao. 1_5

Potential mutants were isolated by the BUdR-visible light technique and con-

firmed by plating the cells in both media. A mutant was defined as having

the capability of growth only in the enriched medium and not in the minimalmedium. Mutagenesis was measured relative to the known mutagen ethyl methane-

sulfonate.16/

d. Dominant Lethal Mutation Test

Three groups of four or five male rats were fed standard

rodent feed or feed containing either 0.02% (low level) or 0.2% (high level)of 2,4-DNTT. The detailed procedures were the same as those described inSections II.A.2.b. and II.A.2.c. After 13 weeks of feeding, each mule was

mated with three females. The male was left overnight in a cage with twofemales. Copulation was established by morning vaginal inspections forsperm. Sperm-positive females were removed; that day is considered day 0

42

-9-

of gestation. An additional female was added to the cage with the male andthe procedure continued until each male had copulated with three females.

On day 13 of gestation, each female was killed and the uterus examined for

implantation sites, dead and viable fetuses, and resorptions. Data are

expressed as fertility index (confirmed pregnancies/sperm-positive females

x 100) and implant viability index (viable fetuses/total implants x 100).

3. Results

a. Cytogenetic Tests

The results on numerical distribution of chromosomes in culturesfrom rats fed 0.2% of 2,4-DNT are shown in Table 49. Treatment with 2,4-DNTdid not cause any significant changes in the chromosome frequency distribu-

tion or number of tetraploids in the peripheral lymphocyte cultures. Ratsfed 0.2% of 2,4-DNT in the diet for 5 or 13 weeks had increased numbers oftetraploids in the kidney cultures. However, the increases were not sta-tistically significant when compared with those before treatment.

The results on morphological aberrations of chromo somes inthese cultures are shown in Table 50. The peripheral lymphocytes sampledat week 13 did not culture properly, additional rats were tiampled after 19%eeks' treatment. The number of chromatid breaks and gaps in the lymphocytecultures were increased after treatment for 5 weeks, but this effect wasnot statistically significant until after 19 weeks. There were significant

increases in the number of chromatid breaks and gaps in the kidney culturesafter treatment for 5 or 13 weeks. The number of breaks and gaps inc,ý-easedwith the duration of treatment.

b. Mutagenic Effect In Vitro

The preliminary experiment found Lhe 1% and 5% cell survivalconcentrations to be 193 and 155 Pg/ml, respectively. Neither of theseconcentrations caused mutations, although comparable concentrations of theknown mutagen ethyl mothanesulfonate did cause mutations.

c. Dominant Lethal Mutation Test

The fertility and moplant viability indexes are sammarizedin Table 51. Rats fed 0.02% of 2,4-DNT for 13 weeks had reduced i@rtilityand implant viability indexes, but the results were not statistically sig-

nificant. Rats fed 0.27 of 2,4-DNT had significantly reduced fertilityindexes. There were no viable fetuses and the implant viability index wassero.

43


Feeding 0.2% of 2,4-DNT was toxic to chromosomes in vivo, as in-dicated by the increased chromatid breaks and gaps in the periph3ral lympho-cyte or kidney cultures. It is not known if this is the effect of 2,4-DNTitself, its meta',olite(s) or combinations of both. Rats converted part ofthe ingested compound into 2,4-diaminotoluene as discussed below. This com-pound is a suspected carcinoge 1r7 1 and likely will affect the reproductivem&terial of the cell.

The in vitro addition of 2,4-DNT at cytotoxic levels to culturesof Chinese hamster ovary cells did not induce mutations.

Rits fed 0.2% of 2,4-nNT in the diet showed an apparent dominantlethal effect. As found in Section A, toxic levels cf 2,4-DNT caused severetesticular !esions and aspermatogenesis. Thus, 2,4-DNT may have reduced thenumber of sperm and rendered the rats sterile. I. may also have producedmutations which failed to implant or caused the resorption of implants. Or2,4-DNT may have produced both of these effects resulting in a few nor-viablefertilized ova. Further testing is required to separate the toxic effect onspermatogenesis from a possible dominant lethal mutation effect.

C. Imunologic Response to 2,4-DNT

1. Introduction

Immunoglobin E (IgE), the allergic or h ersensitive antibody wasassociated with anaphylactic reactions in humans.- Serum concentrations ofIgE of rats treated with 2.4-DNT were determined.


As described for dogs in Section I.B.2., the immunodiffusion tech-nique of Mancini29/ was used to determine the serum IgE levels of rats fedvarious amounts of 2,4-DNT. The terminal blood samples from the toxicitytest, Section II.A., were used.

3. Result and Conclusion

SerwA concentrations of IgE on control rats and rats fed 0.22 or0.72 of 2,4-DNT are summarized in Table 52. There were wie variations inthe serum concentrations of IgE. 2,4-DNT even at lethal level of 0.71 in -.

the feed did not alter the serum concentration of IgE.

44

D. Summary

The low dose of 2,4-DNT (intake of 34 mg/kg/day in males and 38 mg/kg/day in females) was slightly toxic, causing only a slight depression inweight gain. The middle dose (93 and 108 mg/kg/Lay, respectively) was moretoxic and the high dose (266 and 145 mg/kg/day) was lethal to over half therats. Target organs included the neuromuscular system (abnormal gait, gli-osis and/or demyelination in the central nervous system), erythrocytes (met-hemoglobin and sequelae, including anemia and hemosiderosis) and testes (de-

pressed spermatogenesis). ?artial recovery occurred 4 weeks after cessationof treatement.

Rats fed the middle dose for 13 weeks had an increased number ofchromatid breaks and gaps, but no serious aberrations. No maitagenic effectwas seen in Chinese hamters ovary cell cultures. In a dominant lethal muta-

tion study, the males fed the middle dose for 13 weeks were sterile. Therewas no effect of 2,4--DNT on serum IgE.

45

TABLE 20

BODY WEIGHTS OF MALE RATS FED 2. *4-DNT

7. 2,4-DNT Body Weights (g•)a/

in Feed Initial 4 Weeks 8 Weeks 13 Weeks 17 Weeks

0 413 1 18 446 ± 24

0.07 418 ± 13 413 1 150.2 413 1 4 414 ± 120.1 470 m 9 ?26b/

0 395 ± 29 465 " 34R/ 528 ± 400.07 413 ± 13 455 ± 16S' 525 ±19 .

0.2 406 ± 30 435 ± 33S/ 496 = 500.7 439 ± 1i 289 +4S/ 396 ± 6

0 411 ± 16 475 ± 25 522 ± 30 531 ±31

0.07 394 ± 4 430 ±5 448 ± 11 401 8

0.2 419 ± 13 437 ± 16 429 ± 14 396 ± 26

0.7 414 ;- 8 269 ±12 2504/ 238-d/

o 404 k 21 468 ± 21 503 ± 24 560 , 19.S/ 583 ± 31

0.07 418 :L 3 452 3 458 ± 6 481 = 6S/ 506 ± 10

0.2 419 ± 14 442 * 16 418 ± 14 418* 17S/ 422 1 20e/

0.7 444 4. 18 334 7 292 i 151/ 305!.L&/ 3681/

a/ Mear- - .E. of faur rats, unless otherwise noted.

b/ Averagc of two rats; two other rats died in week 4.

c/ 2,4-DNT in feed discontinued thereafter.d/ 0rne raz; three othAr rats died in weeks b, 7 and 8, respectively.e/ Three rats: one other rat died in week 14.

f,' Three rats; one .0the- rat died ir week 8.

' g/ One rat; two other r..ts died In 'eeks 12 and 13.

46

TABLE 21

BODY WEIGHTS OF FEMALE RATS FED 2 .4-DNT

.% 2,4-DNT Body Weights (gm)&/

in Feed Initial 4 Weeks 8 Weeks 13 Weeks 17 Weeks

0 270 8 258 * 90.07 249 2 244 1 60.2 258 ± 8 256 * 30.7 277 9 197 ±12

0 291 1 15 292 g9h/ 323 180.07 268 * 3 271 :L 4-h/ 291 60.2 268 * 5 255 ± 12Y 282 1 90.7 284 * 4 2 0 5 .,c/ 270S/

0 281 ± 10 278 + 13 310 ±12 302 :L- 16

0.07 272 : 13 286 : 12 285 +12 265 k 150.2 264 . 4 267 t 4 266 ± 4 243 z. 50.7 281 6 d/

0 255 5 270 ±8 280 8 303 Ilk/ 307 ± 10

0.07 261 ±5 262 9 265 3 272 ±4k/ 282 t 70.2 275 ± 8 273 3 269 ± 4 274 * 4-/ 275 1 7

0.7 271 ± 8 ei e/ A/

a/ Mean + S.E. of four rats, unless otherwise noted.

b/ 2,4-DNT in feed discontinued thereafter.c/ Two rats; two other rats died in week 1 and week 2.d/ Two rats died in week 1, one in week 2, one in week 3.e/ One rat died in week 1, two in week 2, one in week 3.

47

.1

TABLE 22

AVERAGE FEED CONSUMPTION (gm/rat/day). t

OF RATS FED 2.4-DNT

7% 2,4-DNT Malesin Feed 1-4q.a 5-8 9-13 14-17-/

0 24.2 24.4' 24.4 23.3

0.07 22.8 21.7 20.3 23.20.2 21.5 18.2 19.2 25.40.7 10.2 9.9 11.6 24.7

7. 2,4-DNT Femalesin Feed 1-4 5-8 ý-13 14-17

0 15.6 19.4 16.1 14.70.07 14.2 15.6 14.6 15.0

0.2 15.6 13.1 14.4 15.40.7 5.0 -- -- -.

a/ Weeks.b/ Recovery period; all rats fed control. Feed.

148~

f 1L5

1*_ _ __._ _ _ _

1.,

TABLE 23

AVERAGE 2,4-DNT INTAKE (mR/ki/day)OF RATS DURING T.MAThENT

% 2,4-DiRT Malesin Feed 1-4l/ 5-8 9-13 1-13

0.07 37.6 34.0 3-.a 34,3

0.2 101.7 84.3 92.5 92.8

0.7 191.4 226.5 292.1 265.6r

% 2,4-DNT Femalev

in Feed 1-4 5-8 9-13 1 12

0.07 37.7 39.7 37.6 38.3

9.2 117.7 97.4 109.5 108.3

0.1 145.2 - - 145.2

a/ Weeks.

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0-53

TABLE 28

CLINICAL BLOOD CHEMISTRY DATA OF MALE CONTROL RATSAND PATS FED 2A4-DNT FOR 4 OR 13 WEEKS

Fed for 4 WeeksControlAi 0. 7% DNTIq/

Glucose (fasting), mg 7. 96.0 + 12.5 84.0 + 5.3SGOT, IU/L 146.0 + 16.5 141.5 + 12.0

SGPT, IU/L 28.8 + 0.8 51.8 + 13.1Alk. Phos., IU/L 56.0 + 5.5 60.0 + 6.5BUN, mg 7. 15.5 + 0.5 18.0 + 1.3

A A

Fed for 13 WeeksControl!-- 0.27. DNTI/ 0.77. DNTkb/

Glucose (fasting), mg 7. 120.5 + 6.7 136.0 + 5.0 145.0SGOT, IU/L 72.5 + 5.1 74.0 + 3.2 68.0SGPT, IU/L 25.8 + 2.8 33.3 + 2.3 21.0Alk. Phos., IU/L 46.8 + 4.5 68.3 + 9.0 52.0BUN, mg 7. 17.3 + 0.6 18.5 + 2.4 30.0

a/ Mean + standard error of four rats.b/ Mean of two rats.

54

TABLE 29

CLINICAL BLOOD CHEMISTRY DATA OF MALI CONTROL RATS AND RATSFED 2.4-DNT FOR 4 OR 13 WEEKS AND ALLOWED TO RECOVER 4 WEEKS

Fed for 4 Weeks andAllowed to Recover 4 Weeks

ControaiL 0.7% DN--/

Glucose (fasting), mg % 129.7 + 5.2 153.8 + 11.3

SGOT, 1U/L 97.7 + 10.7 69.5 + 3.8d/

SGPT, IU/L 26.3 + 2.3 30.0 ± 2.4Alk. Phos., UL/L 70.7 + 1.9 72.5 + 2.6

Fed for 13 Weeksard Allowed to Recov,'ur 4 Weeks

Controlk/ 0. 2% MTPA 0.77 WTfi"

Glucose (fasting), mg % 164.5 + 8.2 231.7 ±+ 11.5-d/ 210.0SGOT, IU/L 70.3 + 9.2 60.7 + 3.8 99.0SGPT, IU/L 34.0 + 2.1 42.0 + 2 .6 65.0Alk. Phos., IU/L 39.3 + 1.5 69.0 + 8.0-' 63.0BUN, mg 7. 17.5 + 1.0 21.7 + 1.7 25.0

A/ Mean + standard error of three rats.b/ Mean + standard error of four rats.c/ One rat.d/ Significantly different from the control g&rup (Student's t test).

55

m c 4 N 0 in- 0 k a a 0 499 0 in~ 0-Cc . . . S * *

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a- -r -0 - 0 0 a 04i

-so* * ltl dl 4 N I~ 99 0 ,- 0 0 in

411

566

ILI49.3

. ft In 0 ) 0 fps M a f" 0 o M 0 bi S 0

-f .e f * * . . . .

P. b, a 0 ft a

in o. -0 S4 0 00 0 a 0 aISSP 5P

ff In

log

~ Z57

.9 1 . .0 .0 0 . .0 .0 0 . *0 .0 9 0 . .060 49 I 9 9 9 . 9

SIV 3 N N A

NN M 5y CLA - ~ ~ '-9 CC * *

h .j5 . .0 4 - A N CD 0 a~ C 0 a 0 0 u

4 - 4 - N N A

22'idr ~ ~ o -a

- S S. Id

- N A N N A N - N * 0 A 11

9 9 * . ~ * . . . . *

* * N - N -- 0 0 * 58

TABLE 33

HmTOLGY DTA OF FEDLE RATS BEFUME AND DUIMN 2.4-DNT FEEDING

DOSE: 0.7 7 (BN) BASELINE(T*N) TREATMENTN a NUMBER OF RATS

WKS 0 (B, 4) WKS 4 (To 2)6 3

ERYTHROCYTES (X1O /MM ) 6.78 * .16 5.56!!

RETICULOCYTES. S 1.61 ± ..3 6.131l

HEMATOCRIT, VOL. S 47.8 ± 1.1 41.0i

HEMOGLOBIN, GM. S 16.0 9 .3 13.71/

MCV9 CUBIC MICRONS 70.6 L 2.6 74.31/

MCHBM MICRO MICROGMS. 23.7 e .7 Z4o7

MCHBC, GM S 33.6 1. .3 33.35 3/

PLATELETS (X10 /MM ) 5.9 . .3 5.53 3

LEUKOCYTES (X10 /MM • 18.5 • 2.5 16.5

NEUTROPHILS- a 8.5 t 1.7 8.0i/

LYMPHOCYTES. 9 89e5 . 2.7 91.0/

BANDS. 1 0.0 0 0.0 0.0!/

EOSINOPHILS, 1.5 0 .9 0.04/

BASOPHILS:. a 0.0 0 0.0 0.0

MONOCYTESr % .5 t .3 65

ATYPIC 0o.0 0.0 0.0

NUCLEATED Rk • 0.0 0 0.0 0.0

ENTRIES ARE MEAN + STANDARD ERROR OR MEAN.

!! Signsficcaly different from the control rts at the respective time interval(Dunnett's multiple comparison procedure-).

59F

TOBLE 34

CLINICAL BLOOD CHEMISTRY DATA OF FEMALE CONTROL RATSAND RATS FED 2.4-DNT FOR 4 OR 13 WEEKS

I

Fed for 4 WeeksControlai 0.7% in FeeWh/

Glucose (fasting), mg % 96.0 + 5.6 101.0SGOT, IU/L 93.3 + 6.2 79.5SGPT, IU/L 24.3 + 1.4 37.0AlI. Phos., WU/L 33.0 4 2.0 37.0BUY, mg 7 17.5 + 0.b 17.5

Fed for 13 %eeksControl.-! 0.22% in Feed-

Gluc-ose (fasting), mg T. 129.0 + 9.0 138.3 + 6.2iGOf, IU/L 103.,5 + 14.7 81.0 + 12.1•GPT, lU/L 35.5 + 2.9 29.3 + 2.1Alk. Phos., IU/L 23.8 + i.8 43.5 + 6.7--RBUIh, mg % 17.3 + 1.3 19.0 + 0.6

ad Mean + standard ereor of four rcts.kI Mean of two ruts.ci Significantly different from the control group (Student'n t test).

I6

60 .

TABLF 35

CLINICAL BLOOD CHEMISTRY DATA OF FEMALE CONTROL RATSAND RATS FED 2.4-DNT FOR A OR 13 WEEKS AND ALLOWED TO RECOVER 4 WEEKS


Control t ! 0.7% in Feadb/

Glucose (fasting), mg % 165.0 + 8.1 181.0SGOT, IU/L 110.5 + 10.3 74.0SGPT, IU/L 42.5 + 7.0 24.0Slk. Phos., IU/L 59.0 + 2.1 49.0BUN, mg 7. 19.5 + 0,6 19.5


Control•/ 0.27% in Feedt/

Glucose (fasting), mg % 166.0 + 5.9 181.0 + 13.6SGOT, IU/L 57.0 + 6.1 65.5 + 3.6SGPT, IU/L 29.3 + 2.1 25.3 + 1.7Alk. Phos., IU/L 30.0 + 5.5 26.5 + 6.0BUN, mg 7 20.5 + 2.0 21.0 + 1.9

a/ Mean + standard error of four rats.b/ Mean of two r ts.

61

I

LI

f, I. !I!'

1TABLE 36

SLRUK ELECTROLYTES OF RATS FED 2,4-DNT

2,4-DNT V4

in Feed Serum Electrolytes ime])A-

M Sex Na K Ca'

Fed for 4 Weeks

0 Male 152 ± 2 6.3 ± 0.2 5.2 ± 0.1 2.2 ± 0.2 100 ± 01

0 Female 148 ± 2 5.2 + 0.2 4.9 ± 0.1 2.! ± 0.2 103 ± I

0.7 Male 147 ± OP/ 6.5 ± 0.4 5.2 ± 0.2 2.7 ± 0.2 100 ± 1

0.7 Feminle-' 149 5.1 5.0 2.2 103

Fed for 13 Wcaks i

0 Male 145 ± 1 5.0 ± 0.2 5.4 ± 0.1 1.6 ± 0.1 98 ± 10 Female 146 ± 1 5.3 1 0.3 5.6 ± 0.1 1.8 ± 0.1 101± 1

0.2 Male 158 ± 1V 5.3 ± 0.), 5.2 ± 0.1 1.8 ± 0.2 101 ± 2 1

0.2 Female 152 ± 2111 4.3 ± 0.1 4.9 ± 0.1-/ 1.9 ± 0.1 96 t 2

0.7 Ma1ell 162 5..5 4.9 2.4 .o5

Fed for 4 Weeks and Allowed to Recover for 4 Wee-s

0 Malec/ 151 ± 2 6.1 t 0.3 5.5 ± 0.1 2.2 0. 2 102 1 2

0 Female 150 ± 2 5. 1 ± 0.3 5.5 ± 0.1 2.5 ± 0.2 103 ± 1 " j0.7 Male 148 ± 1 5.9 0 0.2 5.5 ± 0.1 2,0 ± 0.2 102 ± 1

b. 7 Female4 150 4,9 5.6 2.1 102

Fed for 13 Weeks and Allowed to Recover for 4 Weeks

0 Male 147 + 0 4.9 ± 0.2 5.2 ± 0.1 1.9 + 0.1 102 ± 2

k 0 Femal 144±1 46t 0.3 5.2b±0.0 2.0 0.0 102± 2

ý02 M1aec- 150 ± 1V 4.7 ± 0.2 5.8 x 0. 2.0 ± 0.1 97±

0.2 Fe-,.I 1.45 ± 2 4.2 ± 0,2 5.5 ± 0.2 1.9 ± 0.2 100 ± 3

0.7 Mle- 145 5.3 5.6 2.2 99 1a! Mean ± standard error of four rats, unlsai statod vtherwise.

b/ SignificantLy different Crom control (Student's t test).

ci Mean ± standard error ol three rats.

d/ mean of two rats.

e/ One rat.

S62 -6

TABLE 37

A•SOUT AND RELITIVE ORGAN WEIGHTS OF RATS FED 2.4-DNT ?OR. 4 WEEKS

% 24-DMT Terminal Abqolute Oraia Weigbta (na)

Sex in Feed Weigt(2 Lver Spluen Kidneys Heart

Male 0 4 4 6 ± 2 3 a/ 13.5±1.1 0.72±0.08 3.20±0.14 1.35t0.100.07 413±15 14.4±0.9 0.99±:0.08 3.06.O.17 1.17±0.08

0.2 414±12 191A±0.8gb/ 0.75±0.06 2.91±0.18 1.30t0.050.7 3 2 9 2 b-/ 12.1±0.9 0.7400.09 2.49i±0.14• 0.99±0.03k/

Female 0 258±9 7.6±0.4 0.54±0.04 1.65±0.05 0.80±0.090.07 244±6 10.77±0.5!1/ 0.54:0.05 1.7510.05 0.85±%0.17

0.2 256t3 9.2±0.6 0.42±0.08 1.62±0.14 0.79±0.040.70/ 21.2*113I/ 10 .4 -ot. 8V 0.38±0.13 1.52±0.13 0.63P'0.07

Relative Organ Welahts (glj90& Body Weight)Liverr Spleen KdneHvs Har

Male 0 3.0±0.2 0.10&O.C2 0.72±0.04 0.30:0.01

0.07 3.5±0.1 0.24±0.01-20.74±0.02 0.28±0.010.2 4.6±0.2k/ 0.1C.01 0.700.03 0.31±O.010.7 3.7±0.3 0.22±0.03 0.76±0.05 0.30.O.01

Female 0 3.0±0.1 0.21±0.01 0.64±0.04 0.31±0.030.07 4.4±0.2k/ 0.22±0.02 0.72±0.03 0.35!-0.06

0.2 3.6±0.2 0.17±0.03 0.64±0.05 0.31±0.010. 7/ 4.9±0.lk-/ 0.17±0.05 0.7310.10 0.29t0.02

_4/ Mean E standard error of four rats.b/ Significantly different from the control rats (Dunnett's multiple

coqwarison procedure-A/).c/ Two rats.

63

coo 000 00 4 0 coo 00

l. C; ; I~ C0 00

8ý C; Z0 m.0 0 g.00

000 00 - 0 0O O

.4 V 6N1 iv - 3 - '.4 4

bg - U' oO4 r44 c4,. C4 444 4 C ;

14~r .-O- ' C 14 * * * . ! . . . .

co 00000C; C; C 000 cc 00 C;

+1 tl )4 4 44

§ .41

CCe

all

41 N

::; I; C ;4;C D 0I,1

64

TABLE 39

ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF RATS FED 2.4-DNT FOR 4 WEELS ANDALLOWED TO RECOVER FOR 4 WEEKS

7. 2,4-DNT Terminal Absolute Organ Weights (gm)

Sex in Feed Weight (gm) Liver Sple Kidney Heart

Male 0 528±40-4 16.5±3.7 0.68-+0.15 3.53±0.26 1.00±0.11

0.07 525±19 21.1±1.7 0.92±0.05 3.00±0.04 1.05±0.030.2 496±50 21.7±3.1 0.55±0.16 3.10±0.20 1.27±0.210.7 396±ýk/ 20.0±0.3 0.92±0.11 3.004-0.27 1. 17±0., 6

Female 0 323±18 10.9±0.7 0.52±0.08 2.43-+0.20 0. 85*0.05

0.07 288±6 I0.6O.5 0.255+0.18 2.0.±0.06 0.82±0.03Female 0 3.4±0.7 0.3710.02 1.337±0.170 0.85-+0.050.7-S/ 267ý4 15.47±3.9 0.35±0.05 1.95±0.05 0.90±0.10

Relative Orgzan Weijth,.s (gm/lO0 xm Body Weight)Liver Spee Kidneys Hear__.t

Male 0 3.1±0.6 0.14+0.04 0.67±0.05 0.19±0.010.07 4.04-0.2 0. 18+_0.01 0.57±+0.02 0. 20+0.O000.2 4.3-.0.4 0. 11•0. 02 0.63+0.03 0.27±0.060.7 5.1±0.0k/ 0.23±0.03 0.76±0.06 0.30±0.02

Female 0 3.4±0.2 0.16±0.02 0.77±0.09 0.27±0.020.07 3.7±0,.1 0, 19±0.07 0. 70±0.03 0.29±0.010.2 3.4±0.2 0.13±0.04 0. 47±+0.05h/ 0.30±0.01 i

0.7-0 5.8t1.5-/ 0.13±0.02 0.73±0.03 0.34±0.03

aMean ± standard error of four rats.

b/Significantly diffe-,,ent from the ontrol rats (Dunnett's multiple

comparison procedure4I c/ Two rats.

• 65

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0 o NIO 0 p 0 O

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I II* I I I - I

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--- -e -I

TABLE 44

SUIM6RY OF TISSUE LESIONS OF FEMALE RATS FED 2.4-DNT FOR 13 WEKS

Rome a in feed)0 0.2

Lesions Rat No., 205 206 207 208 255 256 257 258

Heart+ + + +

_ .Myaluhod.hiprii a -a_+ +

Liver- -uka u e..fI fa -.-t-on - +

KidneyAlbumino id +

SpleenHemosiderosis +-9 + +-f + .- + .. .

+

Bone Narrow_H/ ra.Li. 1.5 1.4 1.7 1.6 1.6 1.7 1.5 1.6


f/ Severity of Lesions: + - mild; ++ - moderate; -H severe;- = very severe; + - questionable.

70

I I"II I I I I I

*-1 I I -,

IIII I I I I I

++1 + 1 ;IiS I III

CIDCIII III I I I.

I : I + I I . I

nI I p

I - II I II I

I I I II I I I

I I I II I I

I I I~ I I

I I I rII I I,, I I

III II I II

II+ I I 1 I I.I I.-

I 6Ili,,

I II

I + "II I I l ' -4

IIII +

0 Cal I I I I I I LIi.

S,,oI

4 " I rI I a a

Ail Ail m I ri I04 I I,, , , , * *

-§., a ' I %

14~ ~ IOg10 1 %4 j

I - w I S l*IW 4 pa l

Po lII S ,c1 *-01o Do L04 lo E4 NJ4

71

TABLE 46

SIIMQiAT OF TISSUE LBSIOtS IN FDWALZ RATS FED 2.4-DNT FOR 4 WEMSAND ALLOWED TO RECOVE FOR 4 WRtKS

Dose (M. i, feed)0 0.2 0.7

Lesiona- Rat No.: 209 210 211 212 259 260 261 262 297 298

Heart!Mycard iti- +

Lung_ LyMphopid_h[p-j-alsia + + + +Liver

Subacute inflaa.at ion +Hepatic cell t.*crosis + +

KidneyInterstitial nephritis +

SpleenHemosiderosin + +-- +++ ++ + + +

Bone Marrow4/.l rEt io 1.4 1.6 1.5 1.3] 1.7 1.6 1.5 1.7 1.4 1.5

Tissues not listed were nomal.a/ Severity of Lesions: + - mild; 4+ - moderate; +++ - severe; I--++ -

very severe; + - quistionable.

72

72

TABLE 47

SUMNIRY OF T-TSI LESION.S IN MALE RATS FED 2.4-DNT FOR 13 WEEKSAND ALLOWED TO RECOVER FOR 4 WEEKS

Dose (%. in foed)0 _0.2 0.7a/

Lesions- Rat No.: 101 102 103 104 151 152 153 177

Heart-Wocarditis + + ++ ++ +4-

LungsLymphoid hyperplasia + + + + +Pneumonitis + 4-4+ ++-

LiverSubacute inflausmation + + +Vacuolated hepatocytes ..+ +. . ++H 4

- _ dner at ion)

CerebellumGliosis + +

- Demyelination + _ + _

SpleenHemosiderosis + +++ ++ii ++

KidneyAlbuminoid + ++ + + +

Glomerulonephr itis ++Casts +

PancreasHemosiderotic cell inclusions + +

TestesAtrophy ++-+ H +-4+ ++++Aspermat 2oenesis -- H+-+ -- -+•--- -+

Bone Marrow-- E ratio -- 1.6 1.5 1.4 1.7 L -1-8- 1.5 - 1.4_

Tissues not listed were nurmal.Severity of Lesions: + - mild, ++ =moderate, +++ m severe; *+-+ =

very severe; + = questionable.

73l I

TABLE 48

SUMP?#RY OF TISSUE LESIONS IN FEIALE RATS FED 2.4-DNT FOR 13WEEKS AND ALLOWED TO RECOVER FOR 4 WEEKS

Dose (% in feed)0 0.2

Lesionsja- Rat No.: 201 202 203 204 251 252 253 254

Heart_ Myocareitis + - ++ ++ +

Lung- Lyph•hid hpelrpasia + + + +Liver

Subacute inflamation +- Hepatic cell nec-osis + + +Sp leen-- - - - -

Remosiderosis + + + ++++ +

KidneyTubular coagulation

necrosis +

GlomeruInonephri tLs +PancreatF

Necrosis + +

Lymph NodeHS,*3derosis +

Bone Marrow!&E ratio 1.6 1.4 1.7 1.5 1.7 1.7 1.8 1.7

Tissues noc listed were normal.q/ Scverlty of Lesions: + mild; ++ = moderate; +4+ = severe;

-4-4++ - •ory severe; + = questionable.

I7

b TABLE 49

NUMERICAL DISTRIBUTION OF CHROMOSOMESFROM RATS FED 0.2% OF 2.4-DNT

No. of Chromosome Frequency Tetraploids

Treatment- Rats '40 41 42 43 >44 per 100 Cells

Control

Lymphocyte 4 Ab/ 5 40 2 0 0.26 0.15-c/Kidney 4 4 5 39 2 0 0.48 *0.18

2,4-DNT for 5 weeks

Lymphocyte 4 2 4 42 2 0 0.25 *0.15Y.idney 3 4 4 41 1 0 0.83 *0.60

2,4-DNT for 13 weeks

Kidney 3 4 4 40 2 0 2.17 *0.72

2,4-DNT for 19 weeks

Lymphocyte 4 6 5 37 2 0 0.50 *0.35

a/ No lymphocyte cultures were obtained from rats fed the diet for 13

weeks; however, rats were bled again at 19 weeks and the lymphocytecultures were examined.

b/ Mean.

c/ Mean ± S.E.

75

0j0

000 00

+1+; +1 +1 +1

U)FO-4 94C

4-14 NCj'c

u.u0 4j1.. .i .1 C;0Ci2~~~+ 00+. - 54

4.1

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02.

4-4

4.1

02 41

k1 4,, 44.j U u

N5 0 002 2. C *

.~ z W- w L ~

76

MOMLAW if

TABLE 51

DOMINANT LETHAL EFFECTS IN RATS FED2,4-DNT FOR 13 WEEKS'

Implant ViabilityTreatment No. of Males Fertility Index!/ Indexb/

Control 4 92 ± 8/ 92 ± 1

2,4-DNT 4 67 ± 14 62 ±24(0.02%)

2,4-DNT 5 20 ± 13-1 V.•/(0.20%)

a/ Confirmed pregnancies/sperm positive females x 100.b/ Viable fetuses/implants x 100.c/ Mean * S.E.d/ Significantly different from the control (Student's t test).

I

J7

77

U) 'U,

+~ +1--I +1 +1U)

N4 f1% r4 Ln

+ r col ~ "I"

-4 4

4) +1 +10 +1 +1 .(-4 0 C 01

(n C LM LMn 0 4)r-4 00cn '4r' >~~

H4 -4 V-~4 0 u4.4 4)

C4 I-'

C14 4)--

E-4 0nC 1-

CV) CO -I -4

a Ln Ln to

0 r-4 -4 4

H ) a1

.~ 0 0 04)0 0 0 N 44

cu LA LA IA 4. .44)

'41

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*0

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4)(1 0 .4'

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04

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78

[II

1 600- MALES MALES

II A"

0 ///,

I'-

130 0

2001 I -- i

0 4 8 0 4 8 13 17

*----.oControl o----o Middle levelI -- ALow level A- & High level

2,4-DNT in feed discontinued

FEMALES FEMALESE

300I . 300P - .-

I ~ 200 A

0 4 8 0 4 8 13 17

WEEKS OF FEEDING

Figure 1 - Body Weights of Rats Fed Various Levels of 2,4-DNT

I7

!. IIII. MICE

j TABLE OF CONTENTS

Page

A. Subacute and Subchronic Toxicities and Reversibility.. ....... 83

1. Introduction. .. .. ........................................... 832. Material and Method. ........ ..................... .... 83

3 Results ................ ........................... ... 83

a. General Observations and Weight Gain .............. ... 83b. Feed Consumption and 2,4-DNT Intake .... .......... ... 84

I c. Blood Analysis ........... ..................... ... 84d. Organ Weights ............ ..................... ... 84e. Gross and Microscopic Examination of Tissues. ...... 85

4. Discussion and Conclusions ....... .................. ... 857

B. Mutagenic Effects of 2,4-DNT. .................................. 86

1. Introduction ............... ......................... ... 86

2. Material and Methods ......... ..................... .... 86

3. Results and Discussion ......... .................... ... 86

S4. Conclusions .............. ......................... ... 86

C. Summary .................... .............................. ... 86

Tables 53 - 76 ................. ............................. .... 87

Figure 2 ........................... ................................ lll

ie

Ai

III. MICE

A. Subacute and Subchronic Toxicities and Reversibility

1. Introduction

As for the dogs and mice, these studies were performed to definethe nature and extent of effects of 2,4-DNT on the biological system at thebiochemical and cellular levels and to elucidate the dose-response relation-ship in the mice fed 2,4-DNT for 4 and 13 weeks. The reversibility ofadverse effects was also studied in mice after the feeding of 2,4-DNT wasdiscontinued for 4 weeks.


The basic design and procedure for these experiments in mice weresimilar to those described for rats in Section iI.A.2. with the followingexceptions:

(a) A total of 64 male and 64 female young healthy albino Swissmice (National Laboratory Animals, O'Fallon, Missouri) were used for thisstudy. They were divided into four groups, each consisting of 16 malesand 16 females. The average weighte of all groups were kept close. Threegroups of mice were fed 0.07, 0.2, or 0.7% of 2,4-DNT, in powdered standardrodent chow (Wayne Laboratory Heal). The fourth group served as controls andwere given the powdered Gtandard rodent chow.

(t) Mice were kept in a separate room of our rodent quarters.They were housed four per plastic cage with filter tops.

(c) Blood samples were collected by heart puncture under etheranesthesia at termination for hematology. Clinical blood chemistry testsin mice were not performed.

3. Results


The control mice and mice fed low (0.07%) and middle (0.2%)levels of 2,4-DNT were healthy. They maintained their weight or gained

' slightly. These data are listed in Tables 53 and 54 and shown graphically

in Figure 2. Mice fed the high level (0.7%) of 2,4-DNT lost weight. Duringthe recovery period, moot of these mice regained the weight they had lost.

. I iFive deaths occurred: one low dosage male died during week 11, two high

83

dosage males died in week 5; one had Just entered the recovery period, theother was continuing on study; one high dosage female died in week 7; andone high dosage male died in week 10. None of the mice had any obvioussigns of morbidity.

b. Feed Consumption and 2,4-DNT Intake

Feed consumption of t e mice fed 2,4-DNT is summarized inTable 55. Mice fed the high level ate less, as reflected in their weightloss. The other groups consumed comparable amounts throughout the experi-ment.

Intake of 2,4-DNT is summarized in Table 56. The 2,4-DNTintake of the male mice fed 0.07, 0.02 or 0.7% fluctuated slightly and averaged47, 137, and 413 mg/kg/day, respectively. Similarly, intake of the females

averaged 52, 147, and 468 mg/kg/day, respectively.

c. Blood Analysis

The hematology resulta of the control males and males fedvarious levels of 2,4-DNT for 4 or 13 weeks, with and without re,;overy, areshown in Tables 57 through 60. Results for females are shown in Tables 61through 64.

The various peripheral blood elements of mice fed the low ormiddle level of 2,4-DNT were not apparently altered. When compared with therespective controls, there were occasional differences at the various timpintervals. However, these differences were slight and were inconsistent.Mice fed the high level of 2,4-DNT for 13 weeks had significant decreasesin hematocrit and/or hemoglobin concentrations when compared with those ofthe controls (Tables 58 and 62). In addition, there was a compensatory in-crease in reticulocyte count, although the increase was not statisticallysignificant. The middle dosage males (Table 58) had lesser effects and thechanges were not statistically significant. After allowed to 'ecover for4 weeks, these blood elements recovered and the differences disappeared

(Tables 60 and 64).

"d. Organ Weights

The various organ weights of the mire fed various levels of

2,4-DNrT for 13 weeks, or for 4 or 13 weeks and allowed to recover for 4weeks, are shown in Tables 65, 66, and 67. There were occasional increasesin kidney, liver, or brain weight, or occasional decrease or increase inspleen weight when compared with those of the respective controls. The

F differences were slight and the changes were inconsistent.

84

UT

L -- --1 1 . . . .. . . . .. . . . ...

"e. Gross and Microscopic Examination of Tissues

At necropsy, the control mice and mice fed 0.07 or 0.2% of

2,4-DNT were in good nutritional condition. Most mice fed 0.7% of 2,4-DNTfor 4 or 13 weeks had little, if any, body fat. When 2,4-DNT in feed was

discontinued for 4 voeks, the condition of most mice recovered.

After 4 weeks of feeding, there were a number of mild tissuelesions in males and females, as shown in Tables 68 and 69, respectively.Spontaneous lesions included interstitial nephritis, hematopoiesis in the

spleen, myocarditis and focal necrosis in the liver. Two of the males fed

the high level of 2,4-DWT had mild depression on spermatogenesis.

Control mice and mice fed 2,4-DNT for 13 weeks had a few

additional spontaneous lesions, including lymphoid hyperplasia and pneumoniain the lung and subacute inflanmmation in the liver and kidney a• shown in

Tables 70 and 71. There were no apparent 2,4-DNT-induced lesions in the

testes or other organs.

Control mice and mice fed 2,4-DNT for 4 or 13 weeks and allowed

to recover for 4 weeks also had a variety of spontaneous lesions, as shownin Tables 72 through 75. In addition, male and female mice in the lattergroup fed the high level of 2,4-DNT had a number of liver lesions, includinginclusion bodies, cytomegaly and pigmented Kupffer cells (Tables 74 and 75).

These lesions were mild and were not seen in mice terminated before the re-covery period.

Bone marrows and M/E ratios of all mice in all groups were

normal.


The 2,4-DNT intake for males and females, respectively, averaged47 or 52 mg/kg/day in the low level group, 137 or 147 mg/kg/day in the

middle level group, and 413 or 468 mg/kg/day in the high level group.

The low and middle levels of 2,4-DNT were nontoxic to mice. Mice

fed the high level had weight loss and a few deaths. There was mild anemiaas evidenced by decreases in hematocrit and hemoglobin concentration and

an increase in reticulocyte count. Two high dosage males terminated after

V 4 weeks had mild depresslon on spermatogenesis, but testicular lesions were

!. not caen in those terminated after 13 weeks. This may reflect tolerance

or may be incidental. After allowed to recover for 4 weeks, the mice re-

covered.

85

L.

The mice were relatively resistant to the toxic effects of 2,4-DNT.

They ingeated more 2,4-DNT related to their body weight than the rats did.

Unlike rats, mice had no apparent behavioral effects. The anemia was mild

and occurred later. There were only few deaths. Mild aspermatogenesis

was seen in two of four mice fed the high level of 2,4-DNT for 4 weeks, but

not in mice for 13 weeks. This species difference was also observed in

acute toxicity.-Y The w 5 0 s of 2,4-DNT for mice were two to four times

larger than those for rats. One possible explanation is the difference in

the absorption and metabolism of 2,4-DNT between mice and rats as discussed

below in Section IV.

B. Mutagenic Effects of 2.4-D)NT

I. Introduction

The mutagenic effect of 2,4-DNT was studied in male mice using

the dominant lethal mutation test.


The methods used were the same as in the rat study, Section II.B.2.d.,except that mating was determined by the presence of a vaginal plug and fe-

males were allowed to deliver.

3. Results and Discussion

Male mice were fed 0.2% of 2,4-DNT for 13 weeks, or 0.7% for 4

weeks. The results are shown in Table 76. The low level of 2,4-DNT pro-

duced no effects. The high level greatly reduced the fertility index but

had ao effect on the implant viability index. The reduced fertility index

was due to a majority of the females having no implants. Females bearing

young had normal numbers of implants and pups. These results suggest pre-

implant losses, possibly related to the testicular lesions discussed above.

4. Conclusions

DNT at 0.7% in thu diet for 4 weeks greatly reduced the fertility

index without effact on the implant viability index. A majority of the fe-

males mated to the treated males had no implants, whereas females bearing

young had normal numbers of implants and pups.

j C. Summary

Male mice fed up to 137 mg/kg/day of 2,4-DNT and female mice fed

up to 147 mg/kg/day for 13 weeks were unaffected. The high dose, giving

413 and 468 mg/kg/day to males and femaies, respectively, caused weight loss,

mild anemia, some decreases in spermatogenesis (decreasing fertility) and a

few deaths. Surviving mice recovered completely after cessation of t reatmenc.

86

1

I ..

TABLE 53 *01

BODY WEIGHTS OF MALE MICE FED 2.4-DNT

% 2,4-1TT Body Weights (gm)a-/in Feed Initial 4 Weeks 8 Weeks 13 Weeks 17 Weeks

0 31.8+i±.8 29.8±2.2

0.07 30.5t0.9 30.5:±2.00.2 35.5±1.0 34.5±1.00.7 32.8±2.3 26.5±2.0

0 28.5±1.7 3 3 .5:±1.7-/ 32.0±+1.5

0.07 29.0±0.4 3 0 . 8 ±O. 5P-/ 31.5±0.90.2 29.5±2.1 31.5±1.5h1/ 33.0±2.30.7 31.0±1.9 24.-3±"1.4,k/ 33.3-O.7.£/

0 31.3±0.5 33.8±0.6 38.5±0.9 35.0±2.2

0.07 30.3±0..5 31.5±1.2 33.5±1.0 33.7±0.34/

0.2 32.3±1.8 34.0±0.8 34.3±1.8 33.5±1.3

0.7 31.3±1.4 27.8+0.6 26.8+1.2 25,7±0 9M.

0 31.0±0.7 34.3±1.3 36.3±1.1 3 7 .3± 1.. / 35.0±0.9

0.07 32.8±1.8 34.8±1.5 35.3±2.0 36.0±2.1i/ 33.0±1.70.2 30.3±1.9 31.0"*:1.0 31.0±1.4 33.0±1.5 -1 30.3±2.1

0.7 29.3±1.7 28.0•0.3 28 7±1 Za/ 2 9 .0 1 .,8b-/ 3 2 . 3 ±0 . 6 S/

a/ Mean + S.E. of four mice, unless otherwise noted.b/ 2,4-DNT in feed discontinued thereafter.

c/ Three mice; one other mouse died in week 5.d/ Three mice; one other mouse died in week 11.e/ Three mice; one other mouse died in week 10.

1! 87

tl

TABLE 54

BODY WEIGHTS OF FEMALE MICE FED 2.4-DNT

% 2,4-DNT boddy Weights (gin)a/

in Feed Initial 4 Weeks 8 Weeks 1.3 Weeks 17 Weeks

O 26.0±0.4 26.0-+1.4

0.07 26.3±0.6 23.3±1.10.2 27.0-±1.2 25.3-±1.70.7 . 25.0-±1.1 22.5-±0.3

0 27.5±0.5 28.7±0.8ab/ 28.0±0.7

0.07 25.5±0.7 27.3±1.1h/ 26.3±1.10.2 25.8±0.9 28.3±1.0bb/ 26.5±0.90.7 26.3±1.0 24.0±1.1V/ 28.0±+0.9

0 26.5±0.7 29.0±1.3 28.6±1.1 2S'.3±0.80.07 26.8±0.9 27.8±0.9 30.5±1.3 29.5±1.20.2 26.3±1.1 27.8±0.9 27.5±1.2 27.8±0.8

0.7 26.3±0.9 23.3±1.3 25.7±0.9c./ 2 5 .0±1.OS/

0 26.5±0.7 27.0±0.6 27.3±0.5 29.5±1.3-- 26.8±0.6

0.07 25.8±0.8 28.8±0.8 29.5±1.0 3 1 .3±1.3k• 27.5±1.0

0.2 26.3±0.6 27.8±0.9 29.8±0.9 31.8±1.3t/ 28.5±1.0

0.7 26.0±0.3 24.0-0.4 24.8±0.6 26.7±1.0'/ 24.3±+1.7

a/ Mean + S.E. of four mice, unless otherwise noted.

b/ 2,4-DNT in feed discontinued thereafter.

c/ Three mice; one other mouse died in week 7.

88

I TABLE 55

AVERGE LEOD CONSUMPTION (G?/DAY/MUSE) OF MICE FED 2.4-DNT

% 2,4-DNT Ma 1es

In Feed I - 4- 5 -. 8 9 - 13 14 - 17h/

0 2.2 2.3 2.6 1.90.07 2.0 2.5 2.2 2,00.2 2.1 2.4 2.3 .. 10.7 1.4 2.0 2.0 2.,

Ferns lea

1 - 4 5 - 8 9 - 13 14 - 17

0 2.2 2.3 2.2 1.90.07 2.0 2.3 2.0 1.60.2 1.9 2.3 2.0 1.80.7 1.5 1.9 1.7 1.7

a/ Weeks.b/ Recovery period; all mice fed control feed.

t i. 89

If ~L

TABLE 56

AVERAGE 2.4-DNT INTAKE (MG/KG/IDAY) OF MICE DURING TREATMENT

7. 2,4-DNT 0ales540

In Feed 1- 4/ 5- 8 9 - 13 1 - 13

0.07 46.0 51.0 44.0 47.00.2 132.0 149.0 138.0 137.0

0.7 332.0 490.0 498.0 413.0

Females

1 - 4 5 - 8 9 - 13 1 - 13

0.07 53.0 54.0 47.0 52.00.2 142.0 165.0 140.0 147.00.7 434.0 547.0 458.0 468.0

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I Figure 2 - Body Weights of Mice Fed Various Levels of 2,4-DNT.

IIII 111

IV. DISPOSITION AND METABOLISM

TABLZ OF COWrNTrs

Page

A. Disposition and Metabolism of 2,4-DNT in Various Species. . . . 113

1. Introduction ... . .. . .. .. ... .... .. .. 1132. Material and Methods. . . ................. 113

a. Anim&l Species ...... ................. . . 115b. Experimental Procedure ......... ............... 115c. Methods of Identification .... ............. .... 116

3. Results .................. ..................... ... 117

a. Distribution and Excretion ................. .... 117

b. Metabolites in Urine ...... ................ ... 117

4. Discussion and Conclusions ..... ................. . 119

B. Biliary Excretion of Nitrotoluener 'n Rats .............. .... 120

1. Introduction ........... ...................... ... 120

2. Material and Methods ....... ................... ... 1203. Results .......... ... ......................... ... 1214. Discusskon and Conclusions ......... ................ 121

C. Metabolism of 2,4-DNT In Vitro. . . ......... ... ............... 122

1. Introduction ......... ........................ ..... 122

2. Material and Methods ....... ................... ... 123

3. Results ...................... ............. ....... 123

4. Discussion and ConcLusion ...... ................ ... 123

D. Effect of 2,4-DNT on Drug Metabolizing Enzymes ..... ......... 123

s 1. Introduction ....... ............... ................ 123

2. Materitl and Methods ....... ................... ... 1243. PResults .......... ... ......................... ... 124

1 4. Conclusion ........... ........................ ... 124

E. Summary ........ ......... ........................... .... 125

Tables 77 - 99 ......... ... ............................ ... 126Figure 3 ......... ........... .............................. 149

113

I

IV. DISPOSITION AND METABOLISM

A. Disposition and Metabolism of 2n4-DNT in Various Species

1. Introduction

The absorption, distribution, b otransformation, and excretion of

2,4-DNT were studied previously in rats.l/ 2,4-DNT and its metabolites

were characterized by the available conventional analytical methods and

radioassay. In the experiments reported here, similarities and differences

in the disposition and pathways of biotransformation were investigated in

mice, rabbits, dogs and monkeys.


The procedure and methods described for rats!' were generally used

to study the detailed disposition and metabolism of 2,4-DNT.

a. Animal Species

Various species used for these studies included female albino

CD-l and B6C3Fl mice (Charles River Breeding Lab.) weighing 28 to 35 gm and

18 to 26 gm, respectively, female New Zealand rabbits (Small Stock Industries,

Pea Ridge, Arkansas) weighing 1,69 to 1.87 kg, female beagle dogs (Hazelton

Research Animals) weighing 8.2 to 11.8 kg, and female rhesus monkeys (Primate

Imports, Port Washington, New York) weighing 3.0 to 3.7 kg.

b. Experimental Procedure

Each animal was fasted overnight. A single oral dose of 2,4-DNT,

approximately 10% of the acute LD50, spiked with 10 pCi of 2,4-DNT (Ring-

UL- 1 4 C, specific activity of 3.55 mCi/mM), was given via an intragastric metal

or rubber tube. The 2,4-DNT with 14 C-labeled 2,4-DNT was suspended in peanut

oil and given at a volume of 1 ml/100 gm to mice and rats, 2 ml/kg to rabbits,

or 1 ml/kg to dogs and monkeys. After dosing, the mouse or rat was placed

immediately in a "Roth-Delmar" metabolism cage.17/ The rabbit, dog ox mon-

key was placed in a stainless steel animal cage (24 in. x 24 in. x 20 in.)

All animals were given feed and water ad libitum. Feces and urine were

collected separately in the apparatus. At termination, each animal was

anesthetized with ether or pentobarbital sodium. Aortic blood was collected

and various tissues and feces were removed, weighed, and digested in 10

volumes of 2 N NaOH. Blood samples were decolorized by the addition of H0 2 .Aliquots of tissue and fecal digests, blood, plasma and urine were neutral-

ized with Beckman BBS-2, solubilized in Beckman BBS-3 and counted in the

scintillation solution using a Packard Tricarb 3375 liquid scintillation

spectrophotometer.

115

I

c. Methods of identification

Thin-laye" chromatography (TIW: Prec,'ated silica gel plates

(without fluorescent indicator, 0.25 m thickness, Brinkman Instruments,

Inc.) were used for all experiments. 11 samples were spotted 2.0 cm from

the bottom of the plate and developed at least 10 cm. The solvents usedwere (I) benzene:ethyl acetate (4:1 v/v); (II) ethyl acetate:n-heptane(9:1, v/v); and (III) n-butanol:acetic acid:water (10:1:1, v/v/v).

Gas-liquid chromatography (GL): A Hewlett-Packard model5736A gas chromatograph equipped with a flame ionization detector set at250*C was used for GLC. For the separation and identification of nitro-and aminonitrotoluenes, a stainless steel column (0.125 in. i.d. x 3 ft)packed with 10% UCW-982 on WAW-DMCS (80-100 mesh) was used. The oven tem-

perature was maintained at 1500C and nitrogen carrier gas at a flow rate of60 mi/min. For the nitro- and aminonitrobenzyl alcohols, a stainless steelcolumn (0.125 in. i.d. x 3 ft) packed with 10% OV-1 on Chromosorb P (80-100mesh) was used. The oven temperature was programed from 150*C to 180*C at2jimin, and nitrogen carrier gas 4as maintained at a flow rate of 60 ml/min.For 2,4-dinitrobenzoic acid silyl ester, a glass column (0.25 in. i.d. x 4 ft)

packed with 1.5% DC ISX-3-0295 and 1.5% GE-XE-60 on Gas Chrom Q (60-80 mesh)was used. The oven temperature was programed from 100 to 150"C at 4*/min.and nitrogen carrier gas was maintained at a flow rate of 30 ml/min. Thesilyl ester of 2,4-dinitrobenzoic acid was prepared by adding bis(trimethyl-

silyl)acetamide to an etb'• solution containing dried urine rc~idue or pure2,4-dinitrobenzoic acid.

Chemical detection tests- Aryl nitrates were detected using5% diphenylamine in absolute ethanol.EY Aryl amines were detected using

19/the Bratton-Marshall reagent.,

Enzymai% hydrolysis: Samples were prepared for enzyme treat-ment by eluting the metabolites from TLC scrapings with water. The pHwas adjusted to 5.6 by the addition of 13.6 mg/ml of sodium acetate and

treated with g-glucuronidase or aryl sulfatase (Sigma Chemical Company, St.

Louis, Miosouri). O-Glucuronidase was added at a final concentration of5,000 units/ml; aryl sulfatase was added at a final concentration of 225units/ml. All incubations were carried out for 18 hours at 37*C. After

incubation, enzyme activity was terminated by extraction with 5 volumes of

CHC13-MeOH (2:1). The resulting aqueous and nonaqueous phases were con-centrated by evaporation und the metabolites identified by TLC and GIL.

116

3. Results

a. Distribution and Excretion

The results on the disposition of 2,4-DNT in rats reportedpreviouslyl] are included here for comparison. The distribution and excre-tion of radioactivity after a single oral dose of 2,4-DNT (Ring-UL- 1 4 C) invarious species are summarized in Table 77. In mice, an average of 78.2%of the administered dose was recovered from the feces and gastrointestinaltract (GI) plus contents in 24 bours, while only 9.5% to 17.3% was recoveredI from the rats, rabbits, dogs, and monkeys duritg the same period. Both strainsof mice were similar. The total recovery from various tissues and urine was6.5% in mice and 75.8% to 83.5% in the other species. This suggests that thenet absorption of 2,4-DNT in mice approximated 17% of the administered doseand that the other species absorbed about 75% to 85% of the dose.

In all species, the majority of the absorbed radioactivitywas excreted in the urine. The urinary ey:7ction ranged 16.3% of the admin-istered dose for mice and 75.2 to 81.3% of the administered dose for rats,rabbits, dogs and monkeys in 24 hours. No radioactivity was found in theexpired air.

In the rodents and rabbits, the various tissues includingblood contained less than 1% of the dose. The tissues of dogs and monkeyscontained an estimated 3.6% and 2.2% of the dose, respectively. The tissue/plasma radioactivity taLios after a single dose of 2,4-DNT are shown inTable 78. Since the second strain of mice (B6C3FI) were intended to checkthe unusual lack of absorption, they are omitted from the table. Radioactivitywas highly concentrated in the liver of all species. The concencration -atioof radioactivity in the liver 24 hours after dosing was 18.1 f-r rats, 17.8for monkeys and 6.3 to 8.7 for mice, rabbits and dogs. Tie concentrationratio was also high in the kidney reflecting the excretion of radioactivityin the urine. The radioactivity was also concentrated in the lung and spleenin which the concentration ratios were greater than one. In addition, the

skeletal muscle and brain of rats and monkeys contained more radioactivitythan that in the plasma.

b. Metabolites in Urine

As detailed below, 2,4-DNT is metabolized in two phases. Thefirst phase, illustrated in Figure 3, consists of reduction of the nitrogroups and/or oxidation of the methyl group. These reactions are well-known.20/ One or both nitros may be reduced to amines by the nitro reductasesystems found in liver microsomes and other tissues. The methyl group may beoxidized to a benzyl alcohol by the liver microsomal oxidation system whichoxidizes many compounds. The alcohol can then be further oxidized to a benz-aldehyde by alcohol dehydrogenase and to a benzoic acid by aldehyde dehydrog-enase. These last two enzymes are the relatively non specific soluble enzymeswhich metabolize ethanol. These oxidative and/or reductive products ra-. then

117

]

undergo the second phase of metabolism, conjugation, to form glucuronates,ethereal sulfates and perhaps other compounds, which are then excreted.

These compounds are chemically similar; their separation isdifficult. Various methods used for separation and characterization are

listed in Table 79. TLC system I was the most generally useful one. GLCwas necessary to separate some isomers, such as the aminonitrotoluenes(compounds II and I1I). We were unable to sepArate the aminonitrobenzylalcohols (compounds VI and VII), and we could not obtain authentic samplesof compounds X, XI, XII, XIV, XV, and XVI for use as standards to identifythese compounds.

Various metabolites found in the urine of mice, rats, rabbits,dogs and monkeys are listed in Tables 80 through 84, respectively. In mice

(Table 80), 57.9% of the urinary radioactivity was found to be glucuronideconjugates, primarily compounds V (19.61) and VI + VII (24.5%). Other majormetabolites included the sulfate conjugate of compound II (10.3%) and the

glucuronate of compound I11 (7.3%). The parent compound (I) was present in,iery small amounts (0.3%). A small amount (3.9%) of the oxidized benzoicacid derivative (compound XIII) was present, thus the benzaldehyde deriva-tive (compound IX) was probably produced as an intermediate, even thoughnone was detected in the urine. A total of 19.6% of the urinary radioactiv-ity in mice was not identified.

Despite the different amounts of radioactivity xcreted in theurine, the relative amounts of the various metabolites in rats, rabbits,dogs, or monkeys (Tables 81 through 84, respectively) were similar to mice.

A majority of the urinary metabolites was present as glucuronide conjugates,ranging from 54.1% for monkeys to 66.4% for rabbits. The most common metab-olites were the unreduced and partially reduced benzyl alcohols, corpounds

V, VI, and VII. Very little of the pareaLt 2,4-DNT (compound I) was present,ranging from itone for rats to 2.6% for monkeys. Although 3.2% to 9.5% ofthe urinary radiouctivity in these species was completely oxidized benzoicacid derivative (compound XIII), none of the intermediate benzaldehyde de.-rivative (compound IX) was found. A substantial amount of the urinaryradioactivity, ranging from 7.9% for rabbit to 30.7% for monkeys, was not

identified.

Total recoveries of various urinary metabolites as a percent:of the administered dose in 24 hours regardless of conjugated forms, are

shown in Teble 85. Noteworthy is the fact that 0.6% to 8.8% of the cose was

converted to 2,4-diaminotoluene (compound IV), a suspected carcinogen,-

and excreted in the urine of the variuus species studl.ed in 24 hours.

118i .... i

4. Discussicn and Conclusions

Mice absorbed about 172 of the administered radioactivity in 24hours after a single oral lose of 2,4-DNT (Ring-UL- 1 4 C). A large -mountof radioactivity was recovered in the feces and GI tract plus contents.After noting this anomalous result in our usual albino strain (CD-1), wedid a parallel experimnet in the pigmented B6C3F1 &train, but found thesame results (Table 77). One B6C3F1 mouse had very aberrant values (16.4%of dose in feces, 91.2% in urine). She was omitted from the table as an out-lier; if this datum was included, there would still be no significant differ-ences between strains of mice. This could be the result of poor absorptionafter ingestion or a rapid absorption and metabolism, excretion in the bile,and non absorption of the metabolite(s). In view of the similar pattern ofurinary metabolites in the five species studied including mice, a sufficientlydifferent metabolism and excretion of 2,4-DNT through the biliary system ofthe mice is unlikely. Furthermore, 2,4-DNT is significantly less toxic tomice than to rats or dogs. This difference in toxicity is apparently due topoor absorption through the GI tract in mice. On the other hand, most ofthe ingested 2,4-DNT was well absorbed in rats, rabbits, dogs, and monkeysand excreted in the urine within 24 hours.

After oral administration of 1 C-labeled 2,4-DNT, the radioactivitywas concentrated in the liver and kidney with less in other organs. Theliver is apparently the main organ of metabolism and site of biliary excre-tion, and the kidney is the main site of urinary excretion.

After oral admintstration, the abaorbed 2,4-DNT was extensivelymetabolized with no or only a small amount of the parent compound excretedin the urine. Metabolism of 2,4-DNT was similar in all five species studied.The most common pathway was oxidation to a benzyl alcohol, perhaps with re-duction to an aminonitrobenzyl alcohol, followed by glucuronide conjugation

and excretion. Variations included reduction of one or both nitro groupsto amines, oxidation to a benzoic acid, and excretion as aulfates or as thefree compounds.

A substantial fraction of the administered 2,4-DNT was excretedas unidentified metabolites. We did not have authentic samples of some ofthe metabolites as standards for identification. The reduced benzoic acids(compounds XIV, XV, and XVI as shown in Figure 3) are likely to be minor com-fonents, because their formation requires four or five successive enzymaticreactions and a molecule is likely to be excreted before these reactions take

place. Isolation of the authentic benzaldehydes (compound IX through XII) isunlikely because of the facile formation of stable Schiff bases, such as shownin the following reaction:

12 N , C.'G + H2N H20OH ->1 2N NH0

N----N ' NO2 -"-NO2

119

If any benzaldehyde derivatives are left in aqueous solution, it wouldreadily react with any amine. This reaction would produce a wide variety ofproducts depending on which benzaldehyde reacts with which amine. Otherpossible metabolites include other routes of conjugation. An amino compoundcould be acetylated by acetyl-CoA, as is sulfanilamide. A bensoic ac!.d de-rivative could react with coenzyme A and then with glycine to form a hippuricacid derivative. To investigat& these possibilities would be very tedious.

B. Biliary Excretion of Nitrotoluenes in Rats

I. Introduction

As discussed in the preceding Section IV.A., 2,4-DNT was highlyconcentrated in the liver of various species. Liver serves both as a sitefor metabolic biotransformation of foreign compounds and as an organ forbiliary excretion. In these experiments, biliary excretion of 2,4-DNT wasstudied. For comparison, TNT and its dinitroisomers (DNT's) including 4-amino-2,6-DNT were also studied.


Female CED rats (Charles River Breeding Lab.) wetghing 280 to 320gm, were fasted overnight before use. Under ether anesthesia, the conmonbile duct was cannulated with PE-1O plastic tubing through a midline ab-dominal incision. After the incision had been closed, a dose of TNT, DNT's,or 4-amino-2,6-DNT approximating 10% of the acute LD5 O, stiked with about10 pCi of the respective 1 4C-labeled compounds (Ring UL- C, specific activ-ity of 3.02 to 4.90 mCi/mM), was dissolved in peanut oil and administeredorally by intragastric intubation at I ml/100 gm body weight. The rats werethen confined individually in restrictive animal holders (Stoelting Company,Chicago, Illinois). Purina Rodent Chow and water were freely accessible tothe animals.

Bile was collected for the predetermined intervals and the smoun-of bile was measured by weighing. Small volumes of blood samples (200 01)were obtained periodically from the rats by cutting off the tips of theirtails and heparinized. At the end of 24 hours, the rats were removed fromthe holders and anesthetized with ether. Blood was collected from the ab-dominal aorta with heparinized syringe. Entire length of the GI tract in-cluding their contents was removed and combined with the feces which werecollected without urinary contamination.

Radioactivities in the bile, blood, plasma and the GI tract weremeasured using a Packard Tricarb 3375 liquid scintillation spectrophotometer

as described in Section IV.A.2.b. Bile was counted directly in the

120

scintillation solution. Blood, plasma and the GI tract were digested withNaOH, decolored with H2() 2 , solubilized in the scintillation solution and

countod.

3. Results

The biliary excretions of radioactivity in female rats after oral

administration of TNT and various DWr's (Ring-UL- 1 4 C) are summarized inTables 86 through 93. The DNr's included all isomers (2,3; 2,4; 2,5; 2,6;3,4; and 3,5) and 4-amino-1,6-DNT, a metabolite of TNT. Some ridioactivity

appeared in the bile within 15 minutes after oral administration of thevarious 14 C-labeled nitrotoluenes. The rate ot biliary excretion increasedwith time and reached a peak in 15 tainutes for 3,4-DNT, in I hour for 2,3-DNT,in 2 hours for TNT and 2,4-DNT, in 4 hour's for 2,5-DNT and 3,5-DNT, and in

6 hours for 2,6-DNT. Thereafter, the rate of excretion decreased. Bloodconcentration of radioactivity correlated with the rate of biliary excretion.The blood concentration and rate of biliary excretion of 4-amino-2,6-DNT itincreased slightly during the first 6 hours and continued to increase through-

out the collection period.

The total radtoactivity excreted in bile ad that remaining inthe GI tract plus contents and feces ire summarized in Table 94. After

24 hours, the total biliary excretion of radioactivity averaged 10.3% and

10.9% of the dose for TNT and 2,4-DNT; 14.4% to 14.5% for 2,5-DNT, 3,4-DiTand 3,5-DNTA 17.1% for 4-amino-2,6-DNT, and 24.87. and 27.3% for 2,6-DNTand 2,3-DNT. On the other hand, the radioactivity recovered from the GItract plus contents and faces averaged 3.17. to 8.1% of the dose fo: 2,3-DNT,2,4-DNT, 2,6-DNT, and 3,5-DNT; 14.2% to 17.9% for TNT, 2,5-DNI• and 3,4-DNT;

and 46.87. for 4-amino-2,6-DNT.

4. Discussion and Conc'usions

After oral administration of TNT and various dinitrotoluenea

(Ring-UL-1 4 C) to rats, the radioactivity appeared in the bile within 15minutes. The rate of biliary excretion reached a peak in 15 minutes for3,4-DNT, in 6 hours for 2,6-DNT and in I to 4 hours for TNT and the otherDNT's. The blood concentrations of radioactivity incraased and decreasedin close relation to the rate of biliary excretion. The blood concentrationand rate of billary excretion of 4-amino-2,6-DNT continucd to increasethroughout the collection period. The biliary exs.ration rate increased

slowly during the first 6 hours and increased only slightly during the 24thhour. The peak rate was probably reached before the 23rd hour.

__ _ _121

.... ....

The amount of biliary excretion was relatively high for 2,3-DNT

and 2,6-DNT and somewhat lover for TNT and the other dinitrotoluenes. Theamount of radioactivity remaining in the GI tract and feces 24 hours afterdosing was large for 4-auino-2,6-DN'T, small for 2,3-DNT, 2,4-DNT, 2,6-I.;Tand 3,5-DNT and intermediate for TNr and the other DNT'a.

C. Hetabolismn of 2.4-DNT In Vitro

1. Introduction

This part of the study was to describe the in vitro meL ilism of2,4-DNT by homogenates of livers from various evectes. Thowse data in con-Junction with the in vivo observations may be utilized to predict how huMidnsmetabolize 2,4-DNT.


Animals were sacrificed by devapitation (rats), cervical disloca-tion (mice), air embolism (rabbits), oa an overdose of magensium sulfate(dogs and monkeys), and the livers removed, weighed, and homogenized in threevolumes of 1.15% KC1. The homogenatj was centrifuged at 10,000 g for 30 min-utes at 4°C. The incubation medium containe..' 5 r0 magnesium chloride, 5 mMglucose-6-phosphate, 0.8 mM nicotinamide adenine dinucleotide phosphate, 1mM 14 C-2,4-DNT (0.1 pg/ml), 1.0 ml ef 0.2 M Tris-HCL pH 7.4, and 0.5 ml ofthe 1C,000 g supernatant for a final volume of 2.5 ml. All reactions were

conducted in 50 ml Erlewmeyer flasks at 37*C for 1 hour in a shaking incu-bator. Aerobic reactions were performed in room air while anaerobic reac-tions were conducted in sealed flasks which had been gassed with nitrogenfor 30 seconds before incubation. All reactions were terminated with 2.5ml of acetone. The supernatants were chromatographed with standards onsilica gel TLC plate in a solvent system of benzene and ethyl acetate (4:1).The standards were visualized with ultraviolet light, and the radioactivityassociated with the standards was quantified using liquid scintillationcounting. Protein determinations22/ were made on the 10,000 g supernatantusing bovine serum albumin as the standard. The enzymatic. formation of ineta-bolites was corrected for nonenzymatic degradation, and the reults wereexpressed as nmoles metabolite/mg protein.

A preliminary study was conducted to determine the ability of ourin vitro system to detect the difference in 2,4-DNT metabolism. Groups offour male rats were given 80 mg/kg of phenobarbital sodium intraperitoneallydaily for 4 days before sacrifice on the 5th day, or 50 mg/kg of SKF-525Aintraperitoneally for 1 hour before sacr.fice, or no treatment. The liversof these animals were removed, prepared, incubated, and analyzed as above.

31.22

3. Results

The results of the preliminary experiment involving pretreatment ofphenobarbital sodium or SKF-525A indicated that both compounds modified theability of livers from these rats to metabolize 2,4-DiJ in the in vitrosystem (Table 95). Consequently, we concluded that this system was suitableto detect species difference id 2,4-DNT metabolism.

A species comparison of 2,4-DNT metabolism under aerobic and an-aerobic conditions is presented in Tables 96 and 97, respectively. Between8 and 27% ot the parent compound was metabolized, at the end of I hour, inall of the species tested. Under aerobic conditions, 2,4-dinitrobenzylalcohol was the major metabolite in all the species tested. If incubationswere conducted under anaerobic conditions, then the amount of 2,4-dinitrobenzylalcohol was reduced while the amount of aminonitrotoluenes was increasedrelative to aerobic conditions. Differences between aerobic and anaerobic

incubations, however, were not as apparent when the quantity of unidentifiedmetabolites produced was compared. In addition, thia study suggested the sexdifferences in 2,4-DNT metabolism. Female mice and rabbits ard male dogsand rats produced more 2,4-dinitrobenzyl alcohol under aerobic conditionsthan did the opposite sex of each species. In contrast, more aminonitrotolueneswere produced under anaerobic conditions by males of all the species tested.There were no apparent sex aifferences in the amount of unidentified metab-olitet fomed under either aerobic or anaerobic conditions.

4. Discussion and Conclusion

Phenobarbital and SKF-525A pretreatment modified the ability oflivers from male rats to metabolize 2,4-DNT in an in vitro system. Similar

effects of phenobarbital and SKF-525A on drug metabolism have been reported.-L0If the disappearance of 2,4-DNT was used as a basis of comparison, then the.species tested were similar.

Under aerobic conditions, the liver of rabbits formed more metabo'.icproducts than the other species. Under anaerobic conditions, more aminonitio-toluenes were formed by males than females; and inale rats produced the most.The production of unidentified metabolites was similar for all of the speciestested and did not provide information for comparison. We tried to obtainhuman cadaver liver for parallel experiments, but were not successful.

D. Effect oi 2.,4-DNT on Drug Metabolizing Enzymes

1. Introduction

Having seen what the liver drug metabolizing enzymes do to 2,4-DNT,we then studied what 2,4-DNT did to the liver drug metabolizing enzymes.Activities of these enzymes were assayed both in vivo by the toxasolamineparalysis time and in vitro by the hepatic nitroanisole 0-demethylase activity.

123

S . .. . . . . . . . .. .... . ...


Male rats were fed diets that contained 0.7% of 2,4-DNT for 2 weeks.

Rats in the positive control group received 50 mg/kg of phenobarbital sodiumtwice daily for 3 days. At the end of treatment, the zoxazolamine paralysistime and nitroanisole O-demethylase activity were determined.

Zoxazolamine was administered i.p. to rats at a dose of 45 mg/kgin a vehicle of 0.2 N HCI. The duration of paralysis was measured in termsof the loss of the righting reflex. The values are reported as the mean +S.E., and the test of significance was the two-sample rank test.-.2 / Thelevel of significance was selected as p < 0.05.

The metabolism of nitroanisole by livers was measured in an invitro system. Rats were sacrificed by decapitation. The livers were re-moved, weighed, and homogenized in four volumes of 1.15% potassium chloride.The homogenate was centrifuged at 9,000 x g for 30 minutes. The incubationmedium contained 15 iimoles magnesium chloride, 15 wnoles glucose-6-phosphate,3 )imoles p-nitroanisole, 0.5 ml of the 9,000 x g liver supernatant, and0.5 ml of 0.5 M sodium phosphate buffer pH 7.8. Reactions were conductedfor 20 minutes in a shaking water bath at 37*C. The reaction was terminatedby the addition of 0.5 ml of 407. formalin and the color was developed with0.5 ml of 0.8 N sodium hydroxide. The product formed was measured spectro-photometrically At 420 mm. The relationship of pmoles p-nitrophenol fort;ed =Absorbance Units/10.22 was used to quantitate product formed. The Lowryprotein assay2_'/ was used to measure protein content. The activity was ex-pressed as nmoles p-nitrophenol/mg protein. The values are reported asthe mean + S.E. The test and level of significance were the same as describedabove.

3. Results

Phenobarbital pretreatment significantly decreased the duration ofzoxazolamine paralysis, as shown in Table 98. Two weeks of 0.7% of 2,4-DNT

in the feed did not significantly affect the duration.

This same diet regimen did not change the in ,4.tro ability of theliver to convert nitroanisole to nitrophenol, as shown in Table 99.

4. Conclusion

Feeding 0.77. of 2,4-DNT to male rats for 2 weeks neither affectedthe liver enzymes involved in the metabolism of zoxazolamine in vivo noraffected the in vitro liver nitroanisole O-demethylase activity.

124

I |1 ,nI,

E. Summary

Oral doses of radiolabelled 2,4-DNT were poorly absorbed (8 to 12%of the dose) by two strains of mice, but well absorbed (75 to 85%) by rats,rabbits, dogs and monkeys. Once absorbed, the compound was handled similarlyin all species. Concentrations of radiolabel were found in the liver and kid-ney. No ridiolabel was found in exhaled air, but most of that absorbed wasexcreted in the urine within 24 hours. Metabolic processes, as determinedfrom urinary metabolites, included reduction of one or both nitros to aminos,oxidation of the methyl to an alcohol or benzoic acid, and conjugation withsulfate or glucuonate.

In female rats, portions of oral doses of radiolabelled TNT or DNTisomers were excreted into the bile within 15 minutes of dosing. There werevariations between the compounds with respect to time to peak biliary excretionrats (15 minutes for 3,4-DNT to 6 hours for 4-amino-2,6-DNT), and radioactivityremaining in the GI tract, its contents, and feces (3.1% of dose for 2,3-DNTto 46.8% for 4-amino-2,6-DNT).

In vitro liver homogenates from mice, rats, rabbits, dogs and mon-keys metabolized 8 to 27% of added 2,4-DNT within 1 hour. The primary productunder aerobic conditions was 2,4-dinitrobenzyl alcohol; under anaerobic, amino-nitrotoluenes. These compounds are the first products formed in vivo, also.

Feeding 0.7% 2,4-DNT (high dose in toxicity study) to rats for 2 weeksdI.d not affect liver enzymes, as determined by zoxazolamine paralysis time andhepatic nitroanisole 0-demethylase activity.

125

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ITABLE 78

TISSUE/PLASMA IOACTVITY RAMAOS./ IN VARIOUS SPECIES OFANIMALS 24 HR AMR ORAL AMINISTRATION OF A SINGLE

iOSE OF 2,4-DWT (tIG-UL-J4C)

Mice Rate Rabbits Dojs Monkeys

Spleen 1. 7+0.3b./ -- I. 3d/ 1.14/ 2. 6d/Liver 6.3:0.8 18. 1 A.7-S/ 8.7 6.9 17.8Kidneys 3.4*0.4 7.4+0.5 7.0 4.9 6.4Brain 0.6*0.1 1.5+0.1 0.6 0.5 1.3Lungs 1.9±0.3 6.1±3.2 2.2 1.7 1.9Skeletal Muscle 0.6±0.1 1.8*-40.2 0.5 0.5 1.5

j/ Radioactivity in I ml or 9m of wet tissue per radioactivity in1 ml of plasma

b/ Mean * S.E. of four CD-1 mice.

c/ Mean + S.E. of three rats.d/ Average of two animals.

127

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I

TABLE 80

NETABOLITES OF 2.4-D•tT IN MUSE URINE COLLECTED FOR 24 HR AFTER ORALADMNISJTATION OF A SINGLE DOSE OF 2.4-DNT 0ZNG-UL-14C)

Con usa tesMetabolites Free Glucuronide Sulfate

2,4-Dinitrotoluen6 (I) 0.31/ 0 04-Amino-2-nitrotoluene (II) 1.0 1.2 10.32-Amino-4-nitrotoluene (II1) 0.1 7.3 2.12,4-Diaminotoluene (IV) 0.1 3.1 2.02,4-Dinitrobenzyl alcohnl (V) 0.3 19.6 2.72,4-Aminonitrobenzyl alcohols (VI, VII) 0.1 24.5 0.52,4-Diaminobenzyl alcohol (VIII) 0.2 1.4 02,4-I)initrobenzoic acid (XIII) 3.8 0.1 0All Identified 5.8 57.0 17.5Unidentified 19.6

_/ Percent of 14 C-radioactivity in urine. Average of four CD-1 mice.

129

TABLE 81

METABOLITES OF 2,4-DNT IN RAT URINE COLLECTED FOR 24 HR AFTER ORAL

ADMINISTRATION OF A SINGLE DOSE OF 2,4-DNT (,ING-UL-4C)

Conjuiates

Metabolites Free Glucurovide Sulfate

2,4-Dinitrotoluene (I) 0 0 0

4-Amino-2-nitrotoluene (II) 0.44/ 1,2 0.9

2-Amino-4-nitrotoluene (III) 0 0.1 0.6

2,4-Diaminotoluene (IV) 1.J 5.9 4.3

2,4-Dinitrobenzyl alcohol (V) 3.2 27.1 2.9

2,4-Aminonitrobenzyl alcohols (VI, VII) 2.2 22.5 0.7

2,4-Diaminobenzyl alcohol (VIII) 0.9 1.7 3.42,4-Dinitrobenzoic acid (XIII) 8.0 0.2 0

All identified 16.0 58.7 12.9

Unidentified 12.5

a/ Percent of C-radioactivity in urine. Average of three rats.

130

130

I"

TABLE 82

METABOLITES OF 2.4-DNT IN RABBIT URINE COLLECTED F)OR 24 HR AFTER ORALADMINISTRATION OF A SINGLE DOSE OF 2.4-DNT (RING-UL-14C)

ConiugatesMetabolites Free Glucuronide Sulfate

2,4-Dinitrotoluene (I) 0.3-/ 0 0

4-Amino-2-nitrotoluene (II) 0.4 0.5 7.92-Amino-4-nitrotoluene (III) 0.6 4.2 1.82 ,4-Diaminotoluene (IV) 0.9 4.0 0.72,4-Dinitrobenzyl alcohol (V) 0.5 40.3 3.0

2,4-Aminonitrobenzyl alcohols (VI, VII) 0.3 9.4 0.52,4-Diaminobenzyl alcohol (VIII) 1.0 5.9 0.62,4-Dinitrobenzoic acid (XIII) 7.5 2 0All identified 11.4 66.4 14.5Unidentified 7.9

a/ Percent of 14C-radioactivity in urine. Average of two rabbits.

I

131

jiII~

TABLE 83

METABOLITES OF 2.4-DNT IN DOG URINE COLLECTED FOR 24 HR AFTER ORAL --

ADMINISTRATION OF A SINGLE DOSE OF 2.4-DNT (RING-UL-' 4 C)

Conj ugates •

Metabolites Free Glucuronide Sulfate

2,4-Dinitrotoluene (1) 0.22/ 0.5 0 _

4-Amino-2-nitrotoluene ýII) 0.1 4.6 3.72-Amino-/-.-nitrotoluene (111) 0.2 2.9 o.4"2,4-Diaminotoluene (IV) 0.2 4.8 0.42,4-Dlnitrobenzyl aicohol (V) 0.1 33.1 1. 62,4-Aminonitrobenzyl alcohols (VI, "vii) 0.4 17.9 0. 22,4-D.'.3minobenzyl alcohol (VIII) 0.5 1.4 1.5 .2,4-Dinitrobenzoic acid (XIII) 5.7 0_!_QQAll identified 7.4 66.2 7.8-

Unidentified 18.6

a/ Percent of 1 4 C-radionctivltv in urine. Average of two dogs.

13.2I -i

132

I!

TABLE 84

METADOLITES OF 2.4-DNT IN MONKEY URINE COLLECTED FOR 24 HR AFTER ORALi ~ ~ADMINISTRATION OF A SINGLE DOSE OF 2,4-DKT fRI'NG UL-1C

Coniugates

Metaboliter Free Glucuronide Sulfate

2,4-DInitrotoluene (I) 0.41/ 2.2 04-Amiao-2-nitiotoluene (II) 1 0 0.7 0.42-Amino-4-nitrutoluene (III) 0.3 6.8 1.62,4-Diaminotoluene (IV) 0.4 3.3 1.12,4-Dinitrobenzyl alcohol (V) 1.5 21.5 2.3

2,4-Aminonitrobenzyl alcohols (VI, VII) 0 17.9 0.22,4-Diaminobenzyl alcohol (VIII) 0.5 1.3 1.42,4-Dinitrobenzoic acid (XIII) 4.5 0.4 0.11 identified 8.2 54.1 6.9Unidentified 30.7

a/ Percent of 14 C-radioactivity in urine. Avere e of two monkeys.

133

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TABLE 98

DURATION OF ZOXAZOIAMINE PARALYSIS IN MALE RATS PRETRE&TE) WIThPHENOBARBITAL OR F]) A CONTROL DIET OR A DIET CONTAINING 0.7% OF 2.4-DNT

Duration of Paralysis

TreaUnt, (minutes)

Experiment I

Control 98 + 11 (10)-9/

Phenobarbital 34 + 3 7)h/

Experiment II

Control 134 + 14 (8)2,4-DNT 105 + 6 (6)

_q/ Mean + 4tandard error (number of rats)b/ Significantly different from control-I'

147

Ii

TABLE 99

NITROANISOLE O-DEMETHYIASE ACTIVITY IN LIVERS OF RATS FED A CONTROL DIET

OR A DIET CONTAINING 0.77 OF 2.4-DNT

Treatment ActivitVA/

Control 1.32 ± 0.17 (3)bý/

2,4-DNT 1.14 0.01 (3) .

a/ nmoles g-nitrophenol/mg prctein in 9,000 x gsupernatant.

b/ Mean + standard error (number of rats).

1484

CH3 C142 0H CHO COOHN02r NO2~~N02 N02N0

NO 2 NO 2 NO 2 NO 2

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NH2 NH2 NH 2 NH2

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NO 2 NO 2 NO 2 NO 2

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NH2 NH2 NH2 NH2

*....IV V III X ll XVI

ii.

"Figure 3 - Pousibli Early Steps (Phase I) ofMotAbolic Pathways of 2,4-DNT IM Harals

i49

I!

1j V. GENERAL SUNKARY AND CONCLUSIONS

TABLE OF CONTvNTS

A. To•icc Effects ........................ 153

1. Toxic and Nontoxic Doses ..... ............... .... 1532. Target Organs .. .. .. .. .. . ... ... .. . 153

3. Special Toxicity Testc ....... ................ ... 154

* B. Disposition and Metabolism ....... ................. ... 154

1. Absorption, Distribution, Metabolism and ExcretionStudi . . . ................... 154

2. Biliary E:cretiort Studies .... ............. .. 1543. In Vitro Matzbollsrn Studies .... ............. .... 1544. Metabolic Interaction Studies ............. 154

C. Fucura Research ...................... 155

?... Exptoure Standardr!.................. 155

2, Toxic Effoca... . ....... ................. .... 155

1I

ii

I~Ii

1.51

S.. .

I V '=

V. GENERAL SUMMARY AND CONCLUSIONS

A. Toxic .rf(f4eIR

1. Toxic and Nontoxic Doses

Dogs were the most sensitive species tested in these subchronicdose studies, with 5 mg/kg/day having no effects and 25 mg/kg/day being

t'rtic to all and lethal to some. In rats, a dose In feed giving 34 mg/kg/'. males and 38 mg/kg/day to females was slightly toxic; no nontoxic

dose was found in this study. Mice were much less affected, since intakesof 137 mg/kg/day by males and 147 mg/kg/day be fema.es had no adverse ef-fects, and intakes of the high dose of 415 mg/kg/day by males and 468 mg/kg/day were not as toxic as much lower doses given dogs or rats.

The cause of the relative nontoxicity of 2,4-DNT to mice is appar-ent in the absorption studies. Mice only absorbed 8 to 12% of an oral dose,while rats, rabbits, dogs and monkeys absorbed 75 to 85% of the dose. Whenthis effect was seen li the albino strain used in the toxicity study (CD-11,we considered the possibility of a peculiarity in that strain. However, vir-tually identical results were seen in the pigmented B6C3Fl strain (Fl hybrids

' of C57 BL/6 black mice and C3H agouti mice), the standard strain of the NCIS• Carcinogenesis Bioassay Program. Therefore, the low absorption of 2,4-DNT

is a peculiarity of the species Mus musculus.

A noteworthy phenomenon is the extreme individual variation in sus-ceptibility to 2,4-DNT toxicity. A good example is the dogs given 25 mg/kg/day. On day 22, one died, but two others were finally having minimal toxicsigns.

2. Target Organs

Typical non-specific toxic effects (decreased weight gain or evenweight loss) were seen in the rodents. This was the only effect seen in the

].ow-done rats.

All species had methemoglobinemia and its sequelae (Heinz bodies,S .anemia, reticulocytosis, hemosiderosis, etc.) in varying degrees.

Dogs and, to a lesser extent, rats had neuromuscular symptoms ofl- incoordination and rigid paralysis. Some gliosis and demyeliniation was

seen, but the connection between lesions and symptoms is not clear.

Males of all species tested had decreased spermatogen, is. Insufficient dose, this could produce functional sterility as seen in the

11.! dominant lethal mutation study in rats.

153

3. Special Toxicity Tests

No increases of immunoglobulin E were seen in dogs and rats.

No unequivocal mutagenic effect was seen in the cytogenetics, dom-

inant lethal mutation, and cell culture studies. The toxic effect of 2,4-DNT on spermatogenesis confounded the dominant lethal mutation study. Thechromatid breaks and gaps seen in the cytogenetics study have dubious pre-

dictive value.

B. Disposition and Metabolism

1. Absorption, Distribution, Metaboli.r -,Ad Fxcretion Studies

Oral doses of radiolabelled 2,4-DNT were poorly absorbed (8 to 12%of the dose) by two strains of mice, but well absorbed (75 to 857) by rats,rabbits, dogs and monkeys. Once absorbed, the compound was handled similarly

in all species. Concentrations of radiolabel were found in the liver andkidney. No radiolabel was found in exhaled air, but most of that absorbedwas excreted in the urine within 24 hours. Metabolic processes, as determined

from urinary metabolites, included reduction of one or both nitros to aminos,oxidation of the methyl to an alkohol or benzoic acid, and conjugation with

sulfate or glucuronate.

2. Biliary Excretion Studies

In the female rats, portions of oral doses of radiolabelled TNT orDNT isomers were excreted into the bile within 15 minutes of dosing. There

were variations between the compounds with respect to time to peak biliaryexcretion rate (15 minutes for 3,4-DNT to 6 hours for 4-amino-2,6-DNT),total biliary excretion in 24 hours (10.3% of dose for TNT to 27.3% for 2,3-

DNT), and radioactivity remaining in the GI tract, its contents, and feces

(3.1% of dose for 2,3-DNT to 46.8% for 4-amino-2,6-DNT).

3. In Vitro Metabolism Studies

In vitro liver homogenates from mice, rats, rabbits, dogs and mon-keys metabolized 8 to 27% of added 2,4-DNT within 1 hour. The primary prod-uct under aerobic conditions was 2,4-dinitrobenzyl alcohol; under anaerobic,

aminonitrotoluenes. These compounds are the first products form in vivo, also.

4. Metabolic Interaction Studies

Feeding 0.7% 2,4-DIT (high dose in toxicity study) to rats for 2weeks did not affect liver enzymes, as determined by zoxazolamine paralysistime and hepatic nitroanisole 0-demethylase activity.

154

r

C. Future Research

1. Exposure Standards

j •2,4-DNT is obviously toxic to mammals. Setting a reliable exposurestandard will require a lifetime exposure to evaluate carcinogenicity. Be-cause of their aberrant absorption, mice would be of relatively little usein standard setting.

2. To, , c c ts

Additl[.: i,.,ork will be necessary to fully understand some of thetoxic effec¢t of 2,ý.- NT.

Methemoglobinemia is a well known effect.5/ The only remaining quest-ion is what exposure level in humans produces no effect, a routine industrialhygiene problem. Heinz bodies seem to be a useful diagnostic tool.

The neuromuscular effect has not been reported previously. Itsmechanism is unknown. It may be related to some neurologic complaints of2,4-DNT workeri/ but that is a guess. Because of this lack of knowledge,extrapolation to humans is less certain than for better-studied effects, suchas methemoglobinemia.

The depressed spermatogenesis is also a new effect for these com-pounds. The most serious unanswered question is its reversibility--how greata depression can the victim recover from? The accompanying toxicity is sogreat that it is unlikely that 2,4-DNT would be a useful lead compound fordevelopment of a male contraceptive.

I1

S1551

REFERENCES

1. Lee, C. C., J. V. Dilley, J. R. Hodgson, D. N. Roberts, D. 0. Helton,

and W. J. Wiegand: Mammalian Toxicity of Munition Compounds:

Phase I. Acute Oral Toxicity, Primary Skin and Eye Irritation,

Dermal Sensitization and Disposition and Metabolism. USAMRDC Con-

tract No. DAMD-17-74-C-4073. Report No. 1: 1-100, 22 July 1975.

2. Pybus, J.: Determination of Calcium and Magnesium in Serum and Urine

by Atomic Absorption Spectrophotometry. Clin. Chim. Acts., 23: 309,

1969.

3. Seligson, D., J. Marino, and E. Dodson: Determination of Sulfobromo-

phthaleun in Serum. Clin. Chem., 3: 638, 1957.

4. Dunnett, C. W.: A Multiple Comparison Procedure for Comparing Siveral

Treatments with a Control. J. Am. Stat. Assoc., 50: 1096, 1955.

5. Harris, J. W., and R. W. Kellerman: The Red Cell. Harvard UniversityPress, C&mbridge, Massachusetts, 1970.

6. Morton, A. R., M. V. Ranadive, and J. A. Hathaway: Biological Effects

of Trinitrotoluene from Exposure Below the Threshold Limit Value.

Amer. Ind. Hyg. Assoc. J., 37: 56, 1976.

7. McGee, L. C., A. McCausland, C. A. Plume, and N. C. Marlett: Metabolic

* -Disturbances in Workers Exposed to Diiiitrotoluene. Amer. J. Dig.

Dis. 9: 329, 1942.

8. Reisman, R. E., and C. E. A'besman: Systemic Allergic Reactions Dueto Inhalation of Penivillin. J. Am. Med. Assoc. 203: 986, 1968.

9. Mancini, G., A. 0. Carbonara, and J. F. Heremans: Immunochemical

Quantitation cf Antigens by Single Radial Immunodiffusion. Iimuno-chemistry, 2: 235, 1964.

10. Moorhead, P. S., P. C. Nowell, W. J. mellman, D. M, Battips, and D. A.

Hungerford: Chromosome Preparations of Leukocytes Cultured from"Human Peripheral Blood. Expt. Cell Res., 20: 613, 1960.

11. Fernandes, M. V.: The Development of a Human Ammion Strain of Cells.fl Texas Repts. Biol. Med., 16: 48, 1958.

12. Vogt, M., and R. Dulbecco: Virus-Cell intera-14 on with Tumor ProducingVirus. Proc. Nat. Acad. Sci., 46: 365, 1960.

1[57 .

REFERENCES (Concluded)

13. Moorhead, P. S., and P. C. Nowell: Chromosome Cytology. In Methods

in Medical Research (H. N. Eisen, Editor). Year Book Medical Publ.Inc., Chicago, 10: 310, 1964.

14. Kao, F. T., and T. T. Puck: Genetics of Mammalian Cells. VII. Induction

and Isolation of Nutritional Mutants in Chinese Hamster Cells. Proc.

Nat. Acad. Sci., 60: 1275, 1968.

15. Puck, T. T., and F. T. Kao: Genetics of Somatic Mammalian Cells. V.

Treatment with 5-Bromodeoxyuridine and Visible Light for Isolation

of Nutritionally Deficient Mutants. Proc. Nat. Acad. Sci., 58: 1227,

1967.

16. Kao, F. T., and T. T. Puck: Genetics of Somatic Mamalian Cells. IX.

Quantitation of Mutagenesis by Physical and Chemical Agents. J. Cell

Physiol., 74: 245, 1969.

17. Roth, L. J., E. Leifer, J. R. Hogness and W. H. Langham: Studies on

the Metabolism of Radioactive Nicotinic Acid and Nicotinamide in Mice.

J. Biol. Chem., 176: 249, 1948.

18. Feigl, F., V. Anger and R. E. Oesper: Seot Tests in Organic Analysis,

Seventh Edition, Elsevier Publishing Company, New York, p. 300, 1966.

19. Bratton, A. C., E. K. Marshall, Jr., D. Babbitt and A. R. Hendriekson:

A New Coupling Component for Sulfanilamide Determination. J. Biol.Chem., 128: 537, 1939.

20. Goldstein, A., L. Aronow and S. M. Kalman: Principles of Dru& Action.

Second Edition. John Wiley and Sons, New York, 1974.

21. Christensen, H. E., T. T. Luginbyhl, and B. S. Carroll: Suspected

Carcinogens. A Subfile of the NIOSH Toxic Su•bstances List. NationalInstitute for Occupational Safety and Health, Rockville, Maryland,1975.

22. Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall: Protein

Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193:265, 1951.

23. Mann, H. B., and D. R. Whitney: On Test of Whether One of Two

Random Variables is Stochasticaliy Larger than the Other. Ann. Meth.Stat., 18: 50, 1947.

158

l

PISTRIBUTION LIST

No. of

4 HQDA (SGRD-AJ)

Ft. Detrick

Frederick, MD 21701

25 U.S. Army Medical BioengineeringResearch and Development Laboratorv

Attn: SGRD-UBG-RFort Detrick

Frederick, MD 21701

12 Defense Documentation Center (DPC)Attn: DDC-TCA

Cameron Station

Alexandria, VA 22314

SuperintendentAcademy of Health Sciences, U.S. ArmyAttn: AHS-COM

Fort Sam Houston, TX 78234

Dean

School of MedicineUniformed Services University of the

Health SciencesOffice of the Secretary of Defense6917 Arlington Road

Bethesda, MD 20014

11 159

.. . .... . .......

APPENDIX II

V 161

I'

Ii :I'I

II

HIEDATOLOGY, CLINICAL LABORATORY TESTS, HISTOPATHOLOGY,STATISTICAL ANALYSIS, AND NORNAL VALUES

Cheng-Chun LosChuen-Bin Hang

Jagdish C. Bhandari

Judith D. GirvinJohn J. Kowalski

"Midwest Research Institute

-. January 1977

L

11*

TABLE OF CONTENTS

1. Hematology and Clinical Laboratory Tests...........1

A. Hematology. .. .. .......................... .. .......... 1B. Clinical Blood Chemistry .. .. .......... ................2C. Urinalysis. .. .. ......................................3D. Occult Blood in Feces. .. .......... ....................4

E. Precision of Hematology and Clinical Blood ChemistryTests .. .. ........................ ..................4

1. Reproducibility. .. ............ ..................42. Reproducibility Within a Test Day........ 43. Proficiency Test Service. .. .. ....................5

II. Histopat'hology. .. .. ...................... ..................

A. Necropsy and Gross Examination. .. .. ....................5B. Organ Weights .. .. ........................ ............5C. Tissues for Microscopic Examination .. .. ................6D. Fixation and Staining of Tissues. .. .. ..................6

111. Statistical Aralysis .. .. ............ ........................6

IV. Normal Values .. .. ........................ ..................7

A. Hematology, Clinical L.aboratory Tests and Bone Marrow 7B. Absolute and Relative Organ Weights .. .. ................7C. Presence of Varivue Substances in the Urine. .. .......... 7D. Occult Blood in Feces. .. ............ ..................8

V. References. .. .. ...................... ......................8

Tables A 0 0.. .. ...................... .......................10 -24

1_ .-- 1 I- '- ", . n."t.,.v 'v" I.'.n , r -. n-W -f~. 17' -_________

H•I-TOLOGY, CLIHICAL LADOCLTOICY TESTSO HISTOPATHOLSGY,,STATISTICAL ANALYSIS, ANY) NORMAL 7ALUES

.• 1. HEMATOLOGY AND CLINICAL LAJBORATORY TESTS

The usual blood sample from dogs is 8 wl, from monkeys 4 ml, and

from rats 0.3 =1 for hematology 3nd about 8 ml for fill analysis tat termin-

at ion.

.. Hematology

The following hemarological analyses ai'e performed on all blood

j samples from rats, dogs and monk3"ya.

1. Erythrocyte and leukocyte counts: A Coulter ElectL'onic

I Particle Counter with 100 g aperture is u6ed.I' Particle-free diluents

(Isoton for RBC, Zap-Ogkobin in Isotou for WBC, Coulter Electronics, inc.)

ore counted to establish the background. Each blood sample is counted in

auplicate. For each test day, twG control blood samples (Diagnostic

Technology, Inc.) are counted separately in duplicate.

2. Hematocr!t: Ilematocrit is determined in capillary tubes

using a microcapillary centrifuge (International Equipment Compeny, Model

MB). 'wo control blood samples (Diagnostic Technology, Inc.) are measuredI> separately duplicate.

3. Hemoglobin: hemoglrbii. is measured as cyanomc themoglobin-.-

Si Each blood sample is measured in dxplicate. Cyanomethemoglobin (Coulter

Electronics, Inc.) is used as the standard. For each assay, twý, levels of

the standard are used and two control blood samples (Diagnostic Technology,

IInt.) are measured in auplicate.

4. Me~hemoglobin (Met-Hb): Met-Hb is measured by the method of

SJ Dubowski.-/ A positive control is made by adding potassium ferrinyanide

to control blood.

5. 4e'nz bodies: Heinz bodies are stained with methyl violet and

the percent of Heinz bodies 1s calculated.

i 6. Mean corpusculaT volume (MCV): MCV is calculated as follows:

MCV 3) Hematne-it x 10

Ezythr...:yt" in millions 7mm•SEyha1 rye

I'• , • ., • • •,," . . .,'::- . - . ",'-"---- ' "•-- ."t •'' - -- ".?: •- ,. .. "-- .- - T- . ... ...

7. Mean corpuscular hemoglobin (MCHb): MCHb is calculated asfollows:

MCHb (M•g) -M Hemoglobin (gin %) x 10Erythrocytes in millions/mm3

8. Mean corpuscular hemoglobin concentration (MCHbC): MCHbCis calculated as follows:

MCHbC (Sm %) . Hemoglobin "ý m ) x 100Hematocrit *1

9. Differential leukocyte counts: Wright's stain is used to

stain the leukocytes for examination.

10. Reticulocyte count, Reticulocytes are counted by the methy-lene bMae method using the Miller disc.4/

11. Platelet count: A Coulter Electronic Particle Counter with70 g aperture is used.•5-- Particle-free Isoton is used as diluent andconted to establish the background. At weekly intervals, platelets are

also visually counted in a hemocytometer with a phase microscopa forcomparison6 .6

12. Clotting time (dog and monkey): Clotting time to determinedby the capillary tube procedure using two capillary tubes.7/ The time

elapsed from the appearanci of the blood from the animal and coagulationin either tube iC measured.

B. Clinical Blood Tests

The following clinical blood chemistry tests are performed onall blood samples from dogs and monkeys and on blood samples from ratsat termination.

1. Llood glucose: Fasting blood glucose is deterwined by Stein's phexokinase method-.8 Stai.dard glucose solution (Dade) is usea to establish

a standard curve. For each assay, one level of the standard and two con-trols (Reference Serum, Worthington; and Validate, General Diagnostics) aremeasured.

2. Serum glutamic-oxaloacetic transamirase (SGOT): SGOT is

measured by the method of Amador and Wacker.'9_/ Validate and ReferenceSerum are used as the enzyme reference for each assay.

2

Fwr'

3. Serum gletamlc-pyruvic transaminase (SGPT): SGPT is measured

by titmethod of Henry ei al.LP01 Validate and Reference Serum are uted as

thE ,-azyyme reference for ealch nssay.

4. Alkalinc phomphatase: Alkaline phosphatase is marured by

the meth•d of Bowers and McComb.li/ Validate and Reference Serum are usedas the enzyme reference for each assay.

1 5. BUN4: BUN is measured using the BUN Strate Kit (Genercl DWag-

noatlc) wNhich lis based on tha urease met.hod. ill Three levels of Calibrate(General DiagrsostiL.s) are used to establish a standard curve. For each

"I assay two controls (Calibrate I and Validate) are usee an the referen4e.

-s6. Creptinine: Creatinire is measuied by a modified kineticI alkaline picrate procedure.l-3/ Creatinine S.andard Solutions (Sigma Chemi-

cal Company) are used to establish a standard curve. For each assay, two

I levels of the standard and two controls (Calib'rate I and Validate) areused as reference.

7. Lactate dehydrogenase (LDH): WDH is measured by the method

of Wacker at ali21- Precinorm E end Precipath E (Boehringer, MannheimCorporation) are used as the enzyme controls for each assay.

j 8. a-Hydroxybutyrate dehydrogebt ue .(a-HtDH): a HJIDM is measuredby the method of Rosalki and Wilkinson.A-' Precinorm E and Precipath E are

used as the enzyme controls ior each assay.

9. Cueatine prtosphokinase (CPK): CPK to measured by the improved

procedure of Rosalkltlk based on the methods of Oliver.171 Preciuorm E and

I Precipath E are used as the enzyme coutrols for each assay.

C. U-tinalysis

* Urine samples are collected from animals before and durin treat-waent as are tk bloud samples. The urine from rat& is collected by slIght

manipulation of their body, and samples within each group are pooled. The

I nonkeys and dogs a.e placed individually in metabolism cagep, and urine is

collected in the stairile's steel pa:,. The urine from each dog and the pooled

urine from rats are tested and examined for the following:

1, Protein: Urinary pritain is deterviaed with Labstix (AmesCompany, Elkhart, Indiana).

2. Sugir: Urinary glucose and reducing substance are determined

with Labstix (Ames Company).

3( I

3.' Microscopic examination: Urine samples are centrifuged andthe supernatant discarded. The residue Is resuspended and examined micro-scopically for the presence of erythrocytes, leukocytes, epithelial cells,and crystaJ.s under high power field arud for casts under low power field.

A positive urine control prepared with known amounts of proteinand glucose in salini adjusted to pH 6.0 is run with each assay to c'heckthe reliability of the Labstix.

D. Occult Blood in Feces

Fecal samples are collected from animals beforc and during

treatment as are the blood and urine samples. Occult blood in the fecesis determined with Hematest Reagent Tablets (Ames Company, Elkhart, Indiana).A positive control (whole blood) and a negative control (distilled water)

are included with each assay to check the reliability of the Hematesttablets.

E. Precision of Hematology and Clinical Blood Chemistry Tests

1. Reproducibility

For erythrocyte and leukocyte counts, hewatocrit, hemoglobin, andthe various clinical blood chemistry tests, the same cortrol bl.od samplesor control standards are used for day-to-day assays. The replication ofresults are excellent and are summarized in Table A.

The determinatlon of differential leukocyte counts and retf.&ulocytecounts are performed by experienced personnel. At weekly intervals, a bloodsample is counted by two or more personnel to confirm the accuracy of thecounting. Also at weekly intervals, the platelet counts obtuined from aCoulter Electronic Particle Counter are compared with the direct visualcounts in a hemocytometer using a phase microscope.

2. Reproducibility Within a Test Day

At monthly intervals, a blond sample is taken from a control dogand six or more determinations for erythrocyLe, leukocyte, reticulocyte,and plapeleý counts, hemoglobin, and varloue clinical blood chemistry testsare performed to establish cha reprcducibility within s assay. The resultsare sunmarized in Table B.

4I

3. Proficiency Test Service

We subscribe to the Proficiency Test Service of the Institutefor Clinical Science, Hahnemann Medical College, Philadelphia, Pennsylvania(F. Wm. Sunderman, M.D., Director). On the first day of each month, thisservice sende two samples containing two different sera or solutions to all

Ssubscribers for measurements of one or more of the parameters usually analyzedin clinical laboratories. Participants report their results on a form fur-ii nished by the service. On the 15th day of the munth, each participant re-

* •ceives a report from the service which includes: the results of a statistical3 analysis of the values reportcd by all the participating laboratories; a

current review of pertinent methodology; a comprehensive bibliography; and3 validation of the results which the participating laboratory reported. This5• service enables each participating laboratory to obtain an unbiased and criti-

cal assessment of its proficiency in relation to that of 1,000 or so otherclinical laboratories throughout the country. The service has been in con-

I tinuous operation since 1949 and was given endorsement by the American Societyaf Clinical Pathologists in 1952 and by the Association of Clinical Scientists

* iin 1957 and 1968. Our results have been found to be satisfactory and are5 |summarized in Table C.

' I II. HISTOPATHOLOGY

* A. Necropsy and Gross Examination

At termiration or prior to imminent death, rats are killed withI ether, and dogs and monkeys with an overdose of sodium pentobarbital. Animalsthat die on tests are.kept refrigerated but not frozen until necropsy. The

I general physical condition and nutritional status of Each animal at the time5 of death or termination are observed and recorded. Necropsy is performed as

Bsoon as possible after death. Gross changes of all tissues are carefullyexauined and recorded.

Ii B. OrMn Weights

The brain, liver, spleen, kidneys, adrenals, thyroids and gonadsare trimmed free from surrounding tissues and weighed. The organ weight tobody weight and/or brain weight ratios are then calculated.

5!-. 1 5

..2- •'.

"iI

C. Tissues for Microscopic Examination

Tissues to be examined include the eye, skin (breast), trachea,lung, tongue (except rat), salivary gland, liver, gallbladder (except rats),pancreas, esophagus, fundic and pyloric stomach, duodenum, Jejunum, ileum,cecum, colon, kidneys, urinary bladder, gonads, and accessory organs,diaphragm and gracilis muscle, anterior pituitary, Ihyroids/parathyroids,adrenals, tonsil (except rat), thymus, spleen, prescapular (except ratp)and mesenteric lymph nodes, rib bone with bone marrow, brain (sasgttalsection for rats; coronal sections of cerebral cortex, cerebellum, andbrain stem for dog and monkey), spinal cord (lumbosacral plexus, dog and

monkey), sciatic nerve and any other structures not mentioned which showabnormal gross changes.

D. Fixation and Staining of Tissues

All tissues are cut not to exceed 1 cm in thickness for fixation.For most tissues, neutral buffered 10% formalin is used. Sufficient volumeof fixing solution is used and the tissues are changed to a fresh solutionafter 24 hours. The fixed tissues are processed in an Autotechnicon fordehydration, clearing, and infiltration and then embedded in paraffin-Routine H & E staining is used to stain the sectioned tissues for micro-scopic examination.

Supplementary tissue fixatives and staining techniques may beemployed for more positive identification of special lesions such as calci-fication, pigments, fat deposition and other abnormal changes.

III. STATISTICAL ANALYSIS

Data are analyzed statistically using the Dunnett's multiple com-parison procedure following an analysis of variancet181 or our modificationof this procedure for uneven numbers among groups. The chosen criterionsignificance is p < 0.05. 7he means of each group at various intervalsduring treatment are compared with pretreatment levels. For most experi-ments in-beagles, three baseline (pretreatment) levels are obtained. Thebaseline levels for each animal are averaged and the mean is used in theanalysis. In addition, the means of the various treated groups are comparedwith that of the control group at the respective time intervals.

6

IV. NORMAL VALUES

A. Hematology, Clinical Laboratory Tests and Bone Marrow

SI Since June 1971, we have used about 180 rhesus monkeys (WoodardResearch Corporation, Herndon, Virginia, Primate Imports, Port Washington,

- New York, and PrimeLabs, Inc., Farmingdale, New Jersey) for various studies.

SThe peripheral blood elements and clinical blood chemistry values of thesemonkeys before treatment and the myeloid/erythroid (M/E) ratio of the bonemarrow of the monkeys used as normal controls varied among individual ani-mals. The mean ± S.D. and the range of the various parameters for the malesand females are summarized in lables D and E, respectively.

5 Since September 1971, we have used about 525, 5 to 9 months old,beagles dogs (AKC registered, Hazelton Research Animals, Inc.). The periph-eral blood elements, clinical blood chemistry values and the M/E ratio ofthe bone marrow varied considerably among individual dogs. The mean + S.D.and the ranges of the various parameters for the males and females aresummarized in Tables H and I, respectively.

During the same period, we have used about 500, 7 to 10 weeks old,male albino rats (CD® Strain, Charles River Breeding Laboratories). As

I for the dogs, the individual variations of the peripheral blood elements,clinical blood chemistry values and the M/E ratio of the bone marrow werelarge. The mean ± S.D. and the ranges of the various parameters for these

I male rats are summarized in Table L.

I B. Absolute and Relative Organ Weights

Organ weights, both absolute and relative to body weight, ofrhesus monkeys, beagle dogs, and albino rats are summarized in Tables Fand G, J and K, and M, respectively. These were control animals used be-

SI tween June 1971 and December 1976.

C. Presence of Various Substances in the Urine

Various substances occasionally occurred in the urine of monkeys,

dogs and rats. The results are susmarized in Table N. Large percentage ofurine samples from monkeys contained epithelial cells, i.e., 34.7% to 52.0%.Other substances occurred in 8.1% or less of the urine samples.

In dogs, protein, erythrocytes, leukocytes and epithelial cells

were present in 19.1 to 21.6%, 16.5 to 19.8%, 22.6 to 24.6% or 24.7 to 25.7%,respectively, of the samples from dogs collected for analysis. Glucose,

7B _ _ _ _ _ _ _ _ _

crystals, and casts occurred in less than 2% of these samples. Some dogshad been bled and returned to the metabolism cages before the urine wasremoved for analysis. The high incidence of some of these substances inthe urine of these dogs might be due to contamination with the fecal materialand traces of blood dropped in the cage. Special care to avoid contaminationhas been undertaken.

In rats, large percentage of urine samples contained protein, i.e.,29.8 to 36.0%. A few samples contained erythrocytes, leukocytes, epithelialcells and crystals.

D. Occult Blood in the Feces

Less than 10% of the feces samples from monkeys or dogs was positivewith the Hematest for occult blood. The results are summarized in Table 0.

V. REFERENCES

1. Brecher, G., M. Schneiderman, and C. Z. William: Evaluation of theElectronic Red Cell Counter. Am. J. Clin. Path., Zt: 1439, 1956.

2. Selegson, D.: Standard Methods of Clinical Chemistry, Academic ?ress,Inc , New York, Vol. 2, p. 52, 1958.

3. Dubowski, K. M.: Measurement of Hemoglobin Derivatives in Hemoglobin,Its Precusors and Metabolites. (F. W. Sunderman and F. W. Sunderman,Jrs., eds.), J. B. Lippincott Company, Philadelphia. p. 29, 1964.

4. Brecher, G., and M. Schneiderman: A Time-Saving Device for the Count-ing of Reticulocytes. Am. J. Clin. Path., 20: 1079, 1950.

5. Bull, B. S., M. A. Schneiderman, and G. Brecher: Platelet Counts Withthe Coulter Counter. Am. J. Clin. Path., 44: 678-688, 1965.

6. Brecher, G., M. Schneiderman, and E. P. Cronkite: The Reproducibilityand Constancy of the Platelet Count. Am. J. Clin. Path., 23: 15,1953.

7. Hepler, 0. E.: Manual of Clinical Laboratory Methods, p. 83, Charles C.Thomas, Springfield, Illinois, 1935.

8. Slein, M. W.: Methods of Enzymatic Analysis (Bergmeyer, 1. U., ed.),p. 117, Academic Press, New York, 1963.

8

____ ___ ___ _ _____

II

9. Amador, E., and W. E. C. Wacker: Serum Glutamic-Oxaloacetic Trans-aminase Activity: A New Modification and an Analytical Assessmentof Current Assay Technics. Clin. Chem., 8: 343, 1962.

10. Henry, R. J., N. Chiamori, 0. J. Golub, and S. Berkman: RevisedSpectrophotnmetric Methods for the Determintation of Glutamic-IOaloacetic Transaminase, Glutamic-Pyruvic Transaminase, andLactic Dehydrogevase. Am. J. Cdin. Path., 34: 381, 1960.

11. Bowers, G. N., Jr., and R. B. McComb: A Continuous SpectrophotometricMethod 7or Measuring the Activity of Serum Alkaline Phosphitase.Clin. Chem., 12: 70, 1966.

I 12. Chaney, A. L., and E. P. Manback: Modified Reagents for Determinationof Urea and Ammonia. Clin. Chem., 8: 130, i962.

1 13. Lustgarten, J. A.: A Simple, Rapid, Kinetic Method for CreatinineConcentration. Clin. Chem., 18: 1419, 1972.

14. Wacker, W. E. C., D. D. Ulmeir, and B. L. Vallee: Metallors.zymes andMyocardial Infarction. II. Malic and Lactic Dehydiogenaie Activrities

I and Ziuc Concentrations in Serum. ,N1ew Eng. J. Mad., 2 449, 19j6.

.5. R)snlli, S. B., and J. H. Wilkinson: Reduction of a-Ketobutyrate byI Human Serum. Nature (London), 1& 1110, 1960.

16. Rosalki, 3. B.: An Improved Procedure for Setum Creatine PhouphokinaseDetermination. J. Lab. Clin. Med., 69 696, 1967.

17. Oliver, I. T.: A Spectrophotometric Method for the Determination of

Creatine Phosphokinase and Myokinase. Biochem. J., 61, 116, 1955.

18. Dunnett, C. W.: A Multiple Comparison Procedure for Comparing Several3 ~Treatments with a Control. J. Am. Stat. Aos.oc., 50; 1096-11"U,1 1955.

9

LI!9

! .- |

TABLE A

REPRODUCIBILITY AMONG TEST DAYS ON THESAME CONTROL SAMPLES OR STiNDARDS!-

No. of "

Deteri•iastions Mean :t S.D. Ranze

Erythrocytes (x 10 6 /m- 3 )

Normal level 20 4.51 ± 0.07 4.36 - 4.67Abnormal level 20 2.32 ± 0.04 2.25 - 2.40

Hematocrit (vol %)Normal leval 20 44.3 * 0.40 44 - 45Abnormal level 20 22.8 ± 0.60 22 - 24

Hemoglobin (gm %)Normal level 20 14.2 * 0.20 13.6 - 14.5Abnormal level 20 7.4 ± 0.20 6.9 - 7.8

Leukocyte Counts (x 10 3 /n, 3 )Normal level 20 7.3 ± 0.50 6.8 - 8.7Abnormal level 20 17.6 * 0.80 16.3 - 18.7

Fasting Blood Glucose (mg %) 20 163.0 + 7.5 10I - 178SGOT (iU/1) 23 61.7± 3.9 55 - 68SGPT (IU/I) 23 51.3 ±2.6 46 - 55Creatinine (mug 2) 18 2.2 ± 0.3 1.6 - 2.6BUN (mag %) 19 9.8 ± 0.2 9.5 - 10.2Bilirubin (rag 2) 11 0.8 ± 0.1 0.8 - 1.0Alkaline Phosphatase (IU/1) 22 71.6 ± 5.4 62 - 80CPK 11 153.0 ± 7.7 139 - 161LDH 8 98,0 ±2.4 95 - 101HBDH 8 226.0 ± 7.2 214 - 238

a/ Performed in December 1976.

10

'iI

____________________________________O

I TAfiLE B

REPRODUCIBILITY WITHIN A TEST DAYON THE SAll SPECrImwfI

Mean s.D./

Erythrocytes (x 10 6 /m 3 ) 5.90 ± 0.14 5.73 - 6.08Reticulocytes (t) 0.63 ± 0.12 0.44 - 0.79

Hematocrit (vol %) 46.8 ± 0.6 46.0 - 47.5

Hemoglobin (gm, %) 16.1 i, 0.2 15.8 - 16.1

I Platelets (x 105/mm3 ) 1.56 ± 0.07 1.49 - 1.66

Leukocytes (x 10 3 /mm3 ) 10.8 * 0.4 10.2 - 11.3

Bands (0) 0 0 - 0Neutrophils (2) 64.3 3.1 61 - 69Lymphocytes (2) 29.0 * 4.9 23 - 35Eosinophils (Z) 3.2 ± 0.8 2 - 4Basophils (M) 0 0 0 - 0Monocytes (2) 3.4 * 0.9 3 - 5

Atypital (1) 0 0 0- 0Nucleated RBC () 0 0 0- 0Methemoglob.n (gmZ) 0 0 0- 0Pasting Glucose (tug %) 96.7 + 3.0 32 - 101SGOT (IU/M) 23.2 * 2.8 21 - 28SGPT (IU/I) 25.3 ± 2.1 24 - 28Creatinine (mag %) 0.6 ± 0.1 0.5 - 0.6

BUN (mg 2) 9.0 ± 0.0 9 - 9

Alkaline Pho~phatase (lUll) 63.5 + 1.1 62 - 65

CPK 44.0 1.6 43 - 46

LUH 38.5 1.6 37 - 40

HBDH 42.0 1.6 40 - 43

ai Performed in October 1976.b/ Six determinations from an adult beagle blood sample.

I 11

TABLE C

P,3FICIENICY TEST SFRVICH (FTS) REPORTS (1975-1976)A/

ParticipatingLaboratories

W-1 PiS (10-90 Percentiles) AcceptableUnkniowns Results Results Median Mean Performance-b/

Hemoglobin 13.8 gm % 13.8 13.8 13.8 13.6 - 14.018.1 "• % 17.9 17.9 17.8 17.6 - 18.2

Serum Protein 6.6 mg X 7,1 7.0 7.0 6.7 - 7.3

Fasting Glucose 272.0 pig % 264.5 266.0 263.0 240 - 290229.0 mg Z 221.4 220.5 222.5 200 - 240

BUN 12.1 mg % 12.0 12.0 12.2 11.0 - 13.038.4 mg Z 40.1 40.3 39.2 36.0 - 44.0

Creatinine 1.0 mg % 1.0 1.0 1.0 0.8 - 1.34.3 mg % 4.4 4.5 4.4 3.9 - 4.9

Bilirubin 3.9 mg % 4.16 4.15 4.14 3.5 - 4.61.3 mg % 1.78 1.80 1.77 1.5 - 2.1

Cholesterol 175.0 mg % 161.4 161.0 162.0 145 - 175100.0 Mg % 109.8 109.4 111.0 98 - 120

Ca 15.1 meq/l 15.4 15.4 15.3 14.1 - 16.49.5 meq/i 9.8 9.8 9.8 9.2 - 10.3

Na 156.0 meq/f 155.8 156.0 155.5 153 - 158

K 7.3 meq/f 7.5 7.5 7.5 7.3 - 7.7

Cl 96.0 meq/1 97.8 98.0 97.5 96 - 10178.0 meq/1 79.4 79.0 80.0 77 - 83

Hg 1.0 meq/f 1.1 1.1 1.2 0.9 - 141.9 meq/1 2.0 2.0 2.1 1.8 - 2.3

A/ To date, we have received u!nknowns for phosphorus, uric acid, and seruriron. We do not routinely perform these determinations.

b/ Based on values submitted by participants by 10th of month.

12

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TABLZ F

ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF MALE RHESUS MONKEYSa'

AbsoluteOrgan We'-ght Mean ± S.D. Range

1.±ver (gin) 82 * 17 64 - 122Fp-e-'tn (gin) 4.6 ± 1.8 2.0 - 9.3Kidneys (gmj 15.1 ± 3.8 8.0 - 22.0Adrenals (gmn) 0.73 ± 0.15 0.45 - 0.86Thyroids (gin) 0.57 ± 1.30 0.37 - 0.81Teetes (go) 1.29 ± 0.67 0.53 - 3.30

Relative (per kg body weight)Mean ± S.D. Range

Liv'rr (gin) 23.4 ± 2.5 18.8 - 33.4

Spleen (gmu 1.25 ± 0.47 0.57 - 2.38Kiireys (gm) 4.13 ± 0.92 2.20 - 6.43

A&dnals (zig) 201 ± 44 129 - 254Thyroids (ag) 154 + 42 86 - 250Testes (gin) 0.34 ± 0.11 0.18 - 0.53

a/ Data collected between September 1971 and December 1976 from 17

monkeys weighing 3.71 + 0.48 kg, used as co, trol animals.

15

S15

TABLE G

ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF FEMALE RHESUS MONKEYSa-

AbsoluteOrgan Weight Mean - S.D. Range

Liver (gm) 83 ± 17 64 - 122

Spleen (gin) 3.8 ± 1.4 2.0 - 6.0

Kidneys (gin) 14.5 - 2.8 11.0 - 20.0Adrenals (gin) 0.68 + 0.16 0.53 - 1.14

Thyroids (gin) 0.60 + 0.20 0.37 - 1.11Ovaries (gin) 0.28 _ 0.10 0.14 - 0.4r

Relative (per kg body weight)

Mean ± S.D. Range

Liver (gin) 25.4 ± 5.8 19.2 - 37.4

Spleen (gm) 1.16 ± 0.49 0.60 - 1.89Kidneys (gm) 4.40 ± 0.86 3.20 - 6.25

Adrenals (mg) 212 ± 80 138 - 438Thyroids (mag) 173 ± 66 97 - 346

Ovaries (mg) 82 ± 28 43 - 140

a/ Data collected between September 1971 and December 1976 from 11monkeys weighing 3.39 ± 0.58 kg, used a,: controls.

16

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18

I 6.

TABLE J

ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF MALE BEAGLE DGGSO-/

Absolute

Organ Weight Mean S.D. Range

Liver (gm) 264- 51 166 - 384

Spleen (gm) 58 ± 25 22 - 167

Kidneys (gm) 53 - 10 32 - 71

Adrenals (gm) 1.12 - 0.26 0.74 - 1.75

Thyroids (gm) 1.03 ± 0.32 0.55 - 2.50

Testes (gm) 6.60 ± 4.56 1.32 - 18.00

Relative (per kg body weight)Mean ± S.D. Range

Liver (gm) 27.9 ± 4.2 19.6 - 42.3

Spleen (gm) 6.0 ± 2.0 2.8 - 12.5

Kidneys (gm) 5.6 ± 0.8 4.0 - 7.7

Adrenals (mg) 117 ± 25 70 - 165

Thyroids (mg) 108 ± 34 56 - 211

Testes (gm) 0.67 ± 0.39 0.13 - 1.67

a/ Data collected between September 1971 and December 1976 from 51 dogs,

weighing 9.3 ± 1.8 kg, used as control animals.

19

TABLE K

ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF FEMALE BEAGLE DOGS.l

___ ___ _________ Absolute___

Organ Weight Mean ±S.D. Range

Liver (gmi) 218 ±51 106 - 322Spleen (gui) 48 ±21 16 -103Kidneys (gum) 43 ± 9 24 - 71Adrenals (gin) 1.04 ± 0.26 0.49 - 1.65Thyroids (gin) 0.88 ± 0.25 0.55 - 1.91Ovaries (gin) 0.74 ± 0.24 0.38 - 1.27

Relative (per kg body weight)Mean ±S.D. Range

Liver (gin) 28.2 ±5.0 20.7 - 38.8Spleen (gmi) 6.0 ±2.3 3.1 - 10.9Kidneys (g-.a) 5.5 ±0.~9 3.7 - 7.9Adrenals (mng) 135 ~.35 67 - 215Thyroids (tag) 1.2 ±31 75 - 219Ovaries (mng) ±33 54 - 222

a/ Data collected between September 1971 and December 3976 from 49 dogs,weighing 7.7 ±1.5 kg, used as control animals.

20

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TABLE M

ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF MALE ALBINO RATSa

AbsoluteOrgan Weight Mean ±S.D. Range

Liver (gin) 10.89 ±2.87 7.18 - 15.09Spleen (gmn) 0.65 ±0.11 0.34 - 0.89Kidneys (gmn) 2.64 ± 0.37 1.84 - 3.58Adrenals (ing) 63.6 ± 9.5 21.9 - 73.5Thyroids (mug) 26.3 ± 5.8 14.3 - 37.7Testes (gmn) 2.98 t 0.51 1.76 - 3.81

Relative (per 100 gin body weight)Mean ± S.D. Range

Liver (gin) 2.96 ± 0.42 2.09 - 4.01Spleen (gin) 0.19 ± 0.08 0.10 - 0.30Kidneys (gin) 0.76 ± 0.10 0.22 - 0.88Adrenals (mug) 18.6 ± 5.8 5.8 - 22.4Thyroids (mng) 7.6 ± 2.7 4.2 - 12.7Testes (gin) 0.87 ± 0.15 0.23 - 1.09

a/ Data collected between September 1971 and December 1976 from 139 rats,weighing 352 ±59 gin, used as control animals.

22 J

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TABLE 0

PRESENCE OF OCCULT BLOOD IN THE FECES OF MALEAND FEMALE MONKEYS AND DOGS

Species: Monkeys DozeNo. of Animals: 4 a 8 I18l! 30

No. of Collections: 44 481/ 118 156-/-

Occult Blood: Negative 9C.9 (40)S/ 95.8 (46) 94.1 (II1) 91.7 (143)

Positive 9.1 (4) 4.2 (2) 5.9 (7) 8.3 (13)

a/ Baseline data collected from all animals employed between July 1974 andDecember 1976.

b/ Data collected at weekly intervAle for 4-7 collections from controlsemployed between July 1974 and December 1976.

c/ Percent of total (number of samples).

J

24

I7

s' midwest resfarch institutepreface-this report war, prepared at midwest research institute,...

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