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MAMMALIAN TOXCIT OF UNITION C O S

PHASE II: Effects of Multiple Doses

I ! PART I: Trinitroglycerin

Progress Report No. 2

by

Cheng-Chun Lee<, }Harry V. Ellis, IIIJohn J. KowalskiJohn R. Hodgson

Shang W. Hwang

Robert D. ShortJadgish C. Bhandari

I Jaime L. SanyerThomas W. Reddig

Jan L. Minor ~ ~ rDanny 0. Helton

11 November 1977

Supported by

U. S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDW.ashington, D.C. 20314

I i Contract No. DAMD-17-74-C-4073MRI Project No. 3900-B

Approved for Public Release

Distribution Unlimited

The findings of this report are not to be construed as an official Departmentof the Army position unless so designated by other authorized documents.

41_ For-/o-

I, Project Officer: Dr. ,Jack C. Dacre,, / U.S. Army Medical Bioengineering Research

S.-and Development LaboratoryFore Detrick, Frederick, Maryland 21701

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19. KEYWORD (Continue on reverse. ide If necoedery and Identify by block ,marber)

The effects of or-al dose~s of trinitroglycerin (TNG) after adminis-

Itration for up to 13 weeks, were investigated in dogs, rats and mice. A de-tailed study on the disposi tion and metabolism was performed in rats and themetabolic pathways, compared in dogs, mice. rabbits, monkeys and human cadaver.1 liver.

No adverse effects were seen in dogs given 1 mg/kg/day TNG for 4

weeks followed by 5 mg/kg/day for an additional 9 weeks. Larger doses_(25 to

DI) 1473 dOITION OF NOMV 66 IS ORSOLETE

SECURITY CLA11SIFICATION OF flIS PAGE (WWe, VMSe Entered)

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S20. Abstract (concluded)

S200 mg/kg/day for 5 days) produced methemoglobinemia, peakiag about 4 hr afterosing, with the peak height and duration dose-related. Rats eating 1,400 mg/g/day of TNG showed toxic effects: decreased feed consumption, weight loss,

rough coat, testicular atrophy and degeneration and hemosiderosis. Some re-covery occurred despite zontinued dosing, before the 13th week. Mice fed 11or 100 mg/kg/day for 3 weeks followed by 60 or 580 mg/kg/day for 10 weeks hadsome extramedullary hematopoiesis, but no other effects.

Mice absorbed 50 to 70% of an oral. dose of TNG within 24 hr; the otherpecies, 72 to 95%. Most body tissues picked up the 1 4 C radiolabel. Rats andmice excreted 14%compounds in air and urine, the other species primarily in

Urine. •TNG was dnitrated to form di- and mononitrogly~erins, glycerol, other 'polar c pounds and various glucuronides. Livers from 41 species testeddenitratkd TNG in vitro.

q I

ACCUSS!;N

Nu•t ' Ie Sec _trm•c B ti Section 0

j,;S 2 •'1"

.... ........ ... ...... "

BY

"•l'.- Sb•-CIM .

- -'

""UNCLAS SIFTED.CU. ITY CLASSIFICATION OF THIS P4..(Wha.n V.et .&-w

PRE FACE

I-

This report was prepared at Midwest Research Institute, 425 Volker

Boulevard, Kansas City, Missouri 64110, under U.S. Department of the Army

Contract Nn. DAMD-17-74-C-4073, MRI Project No. 3900-B- "Munition Compounds

Mammalian Toxicity Study." The work was supported by .!- U.S. Army Medical

i lBioengineering Research and Dr-ve-opment Laboratory, USAMRDC, Department of

the Army. Cpt. John P. Glennon, Dr. Jack C. Dacres Dr. David H. RosenblattST and Cpt. Robert Rice, Environmental Research Requirement Branch, are the con-

secutive technical monitors for the project.

tThis work was conducted in the Biological Sciences Division, under

the direction of Dr. William B. House, between I March 1975 and 29 February1976. The experimental work was directed by Dr. Cheng jhun Lee, Head, Phar-mac logy and Toxicology with the assistance of Dr. Harry V. Ellis, III,"Associate Pharmacologist, and Mr. John J. Kowalski, Assistanc Biologist.

- Dr. John R. Hodgson, Senior Biochemist, supervised the studies on metabolism,cytogenesis and mutagenesis. Dr. Shang W. Hwang, Associate Pharmacologist,

assisted the studies on metafolism. Dr. Robert D. Short, Associate Pharmacologist,supervised the in vitro study on metabolism and in vivo study on drug metaboliz-

4 &ing enzymes. Dr. J. C. Bhandari and Dr. Jaime L. Sanyer, Associate VeterinaryPathologists, supervised the necropsy and the histology preparation and per-formed the microscopic examination. Mr. Thomas W. Reddig (ASCP certified M.T.),Laboratory Supervisor, supervised the hematology and clinical laboratorytests. Mr. Jan L. Minor, Assistant Toxicologist, supervised the computer pro-

gram and analysis of experimental data. Dr. Danny 0. Helton, Associate Chemist,performed the TNG assay in feed. Technical personnel included Robert C. Byrne,Bruce S. Andersen, Mary A. Kowalski, Francis H. Brown, Ellen R. Ellis, ErnestoI •A. Castillo, Judith D. Girvin, Patricia L. Wilkerson, Bhanu S. Gosalia, Laurel M.Halfpap and William M. Bracken.

Approved for:

SW. r b ec tor

Biological Sciences Division

20 February 1976

iiiA ... . . .

TABLE OF CONTENTS

Page

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

I. Dogs ............. .............................

3 IiA. Subacute and Subchronic Toxicities and Reversi-bility ....... ... .......................... 5

1. Introduction ....... ... ................. 52. Material and Methods ....... ............. 5

a. Numbev of Dogs, Sex and Treatment . . 5b. Experimental Procedures .... ........ 5c. Experimental Design ........ ........ 6

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

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

c. BSP Retention ........ ............. 8d. Urinalysis and Fecal Examination. . . 8e. Organ Weights ........ ............. 8

f. Gross and Microscopic Examination ofTissues ............ ............. 8

4. Discussion and Conclusions ...... .......... 9

B. Effect of TNG on Methemoglobin Formation .... ...... 9

1. Introduction ....... ... ................. 92. Material and Methods ..... ............. 9"3. Results .......... ..... .................. 10

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

C. Cytogenetic Effect of TNG ..... ............. .

1. Introduction ..... ................. . I.I.2. Material and Methods I.... . . . . . . . . .. 1

a. Animals .... .................. 1b. Lymphocyte and Kidney Cultures..... 12c. Chromosome Analysis .......... 12

ii

.....

TALE OF CONTIENTS (Continued)

Fane

3. Results ............ .................... .. 124. Discussion and Conclusion ............... ... 12

D. Immunologic Respcnse to 'IN ....... ........... .. 12

I. Introduction ............. ................. 122. Material and Methods ....... ............ ... 13

3. Results and Conwziusion ..... ............ .. 13

IT. Rats ................... ........................... .. 41

A. Subacute and Subchronic Toxicities and Reversibil-ity ................ ........................ .. 43

1. Introduction ......... ................. ... 43

2. Material and Methods ......... .......... .. 43

a. Number of Rats, Sex and Treatment. . . . 43

b. Animal Husbandry ..... ............ .. 43

c. Feed Preparation ..... ........... .. 43 o,

d. Stability of TNG in Feed M~ixture . . . . 44e. Experimental Procedure ............ ... 44

f. Experimeatal Design .... ........... ... 45

3. Results .......... ..................... ... 45

a. Stability of TNG iii Feed Mixture .... 45b. General Observation and Weight Gain.. 46

c. Feed Consumption and TNG Intake, . 46

d. Blood Analyses ..... ............. .. 47

e. Organ Weights ...... .............. .. 47f. Gross and Microscopic Examination of

Tissues . . ....... .............. .. 48

4. Discussion and Conclusion .... ........... ... 49 -.

B. Additional Subchronic Toxicity .... ........... .. 49

1. Introduction ............. ................. 49

2. Materieal and Methods ....... ............. 50

3. Results .......... .................... ... 50

vi

L,

°ITABLE OF CONTNTS (Continued)

a. Rats Fed 2.5% TNG ..... ............ ... 50b. Rats Fed 25% Lactose .... ......... ... 52

4. Discussion and Conclusions ......... ...... 53

C. Cytogenetic and Mutagenic Effects of TNG ........ .. 54

1. Introduction ......... ................. ... 542. Material and Methods ..... ............. .. 54

a. Cytogenetic Effects on Chromosomes . . . 54b. Mutagenic Effects on CHO-KI Cells

In Vitro ....... ............... ... 54

3. Results .......... .................... ... 55

4. Discussion and Conclusion .... ........... ... 55

D. Immunologic Response to TNG ..... ............. .. 55

1. Introduction .......... ................ ... 552. Material and Metnod ..... .............. ... 553. Results and Conclusion ....... ......... .. 55

III. Mice .................. ........................... ... 99

A. Subacute and Subchronic Toxicities and Reversibil-ity ........... ..................... .I.....0. 1

1. ). Introduction ......... ................. .. 1012. Material and Methods 1.............. 01

-. 3. Results .......... .................... .. 102

a. General Observation and Weight Gain. . 102

b. Feed Consumption and TNG Intake ... . 102

c. Blood Analysis ..... ............. .. 102d. Organ Weights. ............ 103

e. Gross and Microscopic Examination of

Tissues .......... ................ 103

E. B Discussion and Conclusions ...................... 104C. Other Studies ............... ........ 104

7 ~j m vii

TABLE OF CONTENTS (Continued)

Paae

IV. Disposition and Metabolism ...... ................ ... 123

A. Disposition and Metabolism of TNG in VariousSpecies ............ ..................... ... 125

I. Introduction ......... ............... ... 125

2. Material and Methods ................. .... 1253. Results .... .................. ........ 126

a. Distribution and Excretion ........... 126

b. Metabolites In Urine ...... ......... 127

4. Discussion and Conclusions .............. ... 128

B. Biliary Excretion of TNG, DNGs and MNGs in Rats . . 128

I. Introduction .......... ............... 128

2. Material and Methods ..... ............. ... 1283. Results ............ .................. ... 129

a. Biliary Excretion .... ........... ... 129

b. Metabolites of TNG in Bile ....... .... 130

4. Discussion and Conclusion ..... ........ .. 130

C. Metabolism of TNC In Vitro. .... ............. ... 130

1. Introduction ........ ................. ... 1302, Material and Methods ..... ............ .... 1303. Results .......... ................... ... 131

a. Metabolism by Rat Liver ........... .. 131b. Metabolism by Liver of Various

Species .......... ............... 132c. Metabolism During Development ... ..... 132

4. Discussion and Corclusions. ............. .. 132

D. Effect of ING on Drug Metabolizing Enzyme ..... 133

1. Introduction ......... .......... ..... ... 133

2. Material and Methods ..... ............. ... 133

viii

TABLE OF CONTENTS (Conci dod)

3. Results. . .... .. ... . .......................1344. Conclusion .. .. .. .. . .......................134

References .. .. .. .... .............. .. . .........................149

Appendix I -Manual f or Hematology, Clinical Blood Chemistry,Urinalysis, IHistopathology, Statistical Analysis,

arid Normal Values .................... . .. .. .. .. .. .. ......... 5.

4

Iix

I--

A BS TRACT'I

The effects of TNG after administration up to 13 weeks wereinvestigated in dogs, rats and mice. A detailed study on the disposition

and metabolism was performed in rats and the metabolic pathways were coms-pnred In vErious species Including liver of human cadavers.

In dogs, oral administration of 0.01, 0.1 or I mg/kg/day oL

TNG consecutively for 4 weeks and 0.05, 0.5 or 5 mg/kg/day for9 additional

weeks did rot cause any adverse effects, any changes in peripheral bloodelements or clinical laboratory tests, any numerical or morphologicalaberrations of chromosones in the peripheral lymphocytes or kidney cul-tures, any change in serum concentratign of igE, or any lesions. Oraladministration of 25 or 50 mg/kg/day of ING for 5 consecutive days didnot cause any apparent adverse signs; 100 or 200 mg/kg/day caused cyanosislasting a few hours 2 to 3 hours after dosing. In addition$ 200 mg/kg/daydecreased the activity without any serious effects. All these dose levelscaused formation of methemoglobin which reached peak concentration in 1 to 2hours. The height of the peaks and the duration of the methemoglobin con-centrations were dose-related. Methemoglobin disappeared from the blood in8 to 24 hours depending upon the dose. Small anounts of methemoglobinremained in some dogs ?4 hours after larger doses. AdminiLtration of3 mg/kg/day of mnthylene blue 2 hours after administration of 200 mg/kg/day

apparently prevented the methemoglobin formation to reach the high peak.However, the concentration of methemoglobin persisted for 40 hours orlonger after the administration of TNG.

In rats, the TNG intake of the males fed the low, middle ar.d highlevels of TNG in feed averaged 0.8, 6.0 or 59.0 mg/kg/day, respectively,during the first 5 weeks; and 2.6, 24.5 or 229.5 mg/kg/day, respectively,

during the additional 8 week., The TNG intake of the females averaged 0.9,6.4 or 59.3 mg/kg/day during the first 5 weeks and averaged 3.1, 26.5 or233.8 mg/kg/day during the additional 8 weeks. Both the male and femalerats fed the low or the middle level of TNG did not show any adverse signs,

any changes in peripheral olood elements or clinical laboratory tests, orlesions in any tissues related to TNG. The high level of TNG in feed

decreased the feed consumption and retarded the weight gain of rats, theeffects were more profound In the males than in the females. In addition,the high level of TNG caused elevation of SCOT in some rats without anyapparent lesions. These effects on feed consumption, growth and SOOT wo-reversible after discontinuation of TNG feeding for 4 weeks.

In another study, the rats were fed 2.5% TNG or 25% lactose infeed for 13 weeks. The TNG intake of the males and females averaged 1,176-or 1,076 mg/kg/day, respectively, during the first week and increased to

x

L .. .. ...... .-.. . .. . .. .. ..... . . . .. . ... . .. . .. . . ... .. . . . . .... .. . ..... . . .. . .. ... . .. . . ...... .. . .

ri

I 1,588 or 1,773 mg/kg/day, respectively, during the last 5 wteks. Both

the male and the female rats fed TNG consuned less feed, lost weight

I quickly, were slightly less active, and had rough hair coat through the

8th week. Thereafter, they started to gain weight and to recover from

these effects. TNG feeding caused testicular atrophy and degeneration and

aspermatogenesis in the males, and caused some changes in organ weights

in both the males and the females. In addition, both the male and female

rats fed TNG had hemosiderosis in the spleen and/or the liver. Feeding

of 25% lactose did not cause any adverse signs, any changes in peripheral Iblod elements or clinical blood chemistry tests, any effects on calcium

concentration of the blood or iron content of the liver, or any lesions

related to lactose. However, lactose feeding slightly increased the weights

of caecum of both the male and the female rats.

- An average of 59.0 to 59.3 mg/kg/day of TNG in feed for 5 weeks

and 202.4 to 206.2 mg/kg/day for additional 8 weeks did not cause any

numerical or morphological aberrations of the chromosomes in the peripheral

lymphocytes or kidney cultures. Treatment of Chinese hamster ovary cellswith TNG at concentrations which killed 657. or 99% of the population did

not appear to induce any mutations. An average of 1,406 to 1,416 mg/kg/dayof TNG in feed for 13 weeks did not alter the serum concentration of IgE.

In mice, the TNG intake of the males fed the lou, middle and high

levels of TNG in feed averaged 1.3, 11.5 or 106.7 mg/kg/day, respectively,

during the first 3 weeks; and 6.4, 60.2 or 607.2 mg/kg/day, respectively,during the subsequent 10 weeks. The TNG intake of the females averaged

1.3, 10.9 or 94.9 mg/kg/day during the first 3 weeks and averaged 6.9,58.7 or 561.2 mg/kg/day during the subsequent 10 weeks, Both the male

and the female mice fed the low, middle or high level of TNG did not

show any adverse signs, any changes in peripheral blood elements or clinc-

cal blood chemistry tests. Extramedullary hematopoiesis was seen in the

liver and spleen of mice fed TNG for 13 weeks and for 13 weeks plus re-

covery for 4 weeks.

The metabolic pathways of TNG were similar in rats, mice, rabbits,1.4K dogýs and monkeys. About 50 to 70% of an oral dose of TNG-1,3-. C was ab-

sorbed in 24 hours in mice, whereas 75 to 95% was absorbed in rats, rabbits,

dogs and monkeys. In rats and mice, the majority of the absorbed radioactiv-

ity was excreted in the urine and expired air. In rabbits, dogs and monkeys,most of the absorbed radioactivity was excreted in the urine with only

[ small amounts in the expired air. The radioactivity was highly concentrated

in the liver of all species. Other tissues, including kidney, spleen, lungand/or skeletal muscle, also contained significant amounts of radioactivity.

xiI .i

TNO' and free DNGs were ercreted o .Iy in small amounts in the urine ofmice, rat: and rabbits, and in s;.,'.tly greater amounts in the urine of

dogs and monkeys. Large amounts of unidentified polar compounds and glycerolwere found in all species. Mice excreted only small amOLnts of free MNG,MNG-glucuronides and DWG-glucuronides. Most of the urinary metabolites inrats and rabbits were free M•4G and DNG-glucuronides. Dogs and monkeys ex- "creted mostly free MNGs and MNG-glucuronides.

"14After oral administration of TNG-I,3- C to rats, the radio-

activity appeared in the bile in 15 minutes. The rate of biliary excretion

increased with time and reached a peak in 3 hours. The blood concentration

of radioactivity continued to increase through the 5th to 6th hour. Thebiliary excretion of radioactivity was high for 2-MNG, ING and 1,2-MNG and

low for I-MNG and 1,3-DNG. The amount of radioactivity remaining the gastro-intestinal tract plus contents and in the feces was small.

TNG was primarily metabolized to DNGs in vitro by livers of

various species. Livers from rats and mice produced more 1,3-DNG than 1,2-DNG; whereas, livers from rabbits, dogs, monkeys and humans produced more1,2-DNG than 1,3-DNG. Livers from mice and humans had a lcw ability tometabolize TNG. The rat liver quickly metabolized TNG in vitro to DNGs in -

15 minutes. Then the amount of DNCs decreased when the incubation was con- -

tinued for up to 2 hours. T4NG was nct appreciably metabolized by liver invitro. The addition of other tissue homogenates, including stomach andintestines, or pancreas, spleen and kidneys, did not modify the liver tometabolize TNG or MNGs in vitro. Placentas from mice, rats or humans and

mouse embryo, liver or carcass during late gestation had a poor ability,

relative to liver, to metabolize TNG in vitro. TNG metabolism in rat liversincreased with time after birth up to 7 da-s. The metabolism did not change

between 7 and 21 days. The ability of rat liver at 21 days after birth to

metabolize TNG was lower than that of the adult liver.-?1

Feeding of 0.5% TNG for 2 weeks did not affect the liver enzyme(s)to metabolize zoxa2olamine in vivo, nor affected the O-demethylase activity

in the liver in vitro.

Principal Investigator

Cheng-Chun Lee, PhDHead, Pharmacology and ToxicologyMIDWEST RESEARCH INSTITUTE

425 Volker BoulevardKansas City, Missouri 64110

xii )

INTRODUCTION

Under Contract No. DAND-17-74-C-4073, entitled "Munition

Compounds Mammalian Toxicity Study," we conducte4 Phase I styies on

It;-, effects of acute exposure of various munitioii l.ompounds,- DuringPhase II, we studied the effects of multiple exposure of selected com-

pounds including trinitzogiycerin (TNG), 2,4,.dinitrotoluene (2,4-DNT),I 2,6-dinitrotoluene (2,6-DNT) and -nitrocellulose (NC). Subacute and sub-

chronic toxicities were performed in dogs, rats and mice to determine

the maximum tolerated dose and to define the biological nature arid tar-

get organ(s) of the toxic effects. Reversibility of any adverse effects

was determined. Mutagenicity of the compound was assessed. Immunologic

response was studied by the detection of the serum IgE antibodies.Effects of the test compound on drug metabolizing enzymes were investi-

gated in in vivo and/or in vitro studies. A detailed study on the dis-

position and metabolism of the radiolabeled. compound was performed inrats; the metabolites were isolated and identified. Comparison in the

pathways of biotransformation was alao studied in vivo in micei, rabbits,dogs and monkeys and in vitro system using tissues of the various speciesincluding the liver of human cadavers. Special studies were also per-

formed to elucidate certain adverse effects. This report sunxnarize3 the

results of Phase II studies on TNG.

i"1I

IIII!I

I I. DOGS

I TABLE OF CONTENTS

Pane

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

1. Introduction ............ ........................ 52. Material and Methods .............. .................... 5

a. Number of Dogs, Sex and Treatment ...... .......... 5b. Experimental Procedures ......... ............... 5c. Experimental Design ........... ................. 6

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

a. General Observations and Weight Gain ..... ......... 7b. Blood Analysis ....... ...................... . 7c. BSP Retention. .. .. .......... ....................... 8d. Urinalysis and Fecal Examination .......... .......... 8e. Organ Weights ............. .................... 8

f. Gross and Microscopic Examination of Tissues..... 8

4. Discussion and Conclusions .......... ................. 9I 'B. Effect of TNG on Methemoglobin Formation ........ ............ 9

1. Introduction. ............. ........................... 92. Material and Methods .............. .................... 93. Results ............... ...................... . .... 104. Discussion and Conclusions ...... ................. .... 11

C. Cytogenetic Effect of ING .I. . . . .......... ................... . i

I I. Introduction ............ ........................ ... 1.12. Material and Methods ........ ................... . i..11

a. Animals ........... ....................... . I..11

b. Lymphocyte and Kidney Cultures .... ............ ... 12Ic. Chromosome Analysis .. .. .. .. .. .. .. . .. 12

3. Results ............... .......................... ... 121 4. Discussion and Conclusion ......... ................ ... 12

I3

_LBF CONTETS (Concluded)

D. Immunologic Response to TNG ....... ................... .... 12

1. Introduction .......... ...................... 122. Material and Methods. . ............. ...... 13

3. Results and Conclusion ........ .................. ... 13

Tables 1 - .5 ................... ................... ..... 14. .

Figure 1 ...................... ............................... 39

4i

1

4d

! i ~- - - - - - ---.--.---- * . ~ -

I

AI, DOGSI

A. Subacute and Subchronic Toxicities and Peversibilitv

I. Introduction

These studies were performed tc define the nature and extent ofeffects of TNG on the biological system at the biochemical and ce.lularlevels and to elucidate the dose-response relationship in the dogs afteradministration for 4 weeks and 13 weeks. The reversibility of any adverseeffects was studied after the treatment of TNG was discontinued for4 weeks. _ _ __ _I

9. Material and Methods

"a. Number of Dogs, Sex and Treatment

A total of 32 young healthy beagle dogs (ilazelton ResearchAnimals, Cumberland, VA) weighing between 6.6 and 11.8 kg were used for Ithese experiments. The dogs were conditioned and observed carefully in ouranimal quarters for 3 weeks after their arrival from the supplierý They weredivided into four groups, each consisting of four males and four females.The average weights of all groups were kept close.

Three groups of dogs were given 0.01, 0.1 or i mg/kg/day ofTNG in capsules. TNG was desensitized in lactose as 10% mixture (SDM No. 17, IAtlas Chemical Division, ICI America Inc., Wilmington, DE) and the lactosemixture was weighed twice weekly for individual dogs throughout the study.

At the end of 4 weeks, adverse effects were not observed in any dogs. Start-ing the 5th week TNG doses for all groups were increased 5-fnld to 0.05, 0.5or 5.0 mg/kg/day, respectively. The 4th group served as the controls and was Igiven empty capsules daily thoughout the experiments. Purina dog chow andwater were available ad libitum, except wherever specified.

b. Experimental Procedu, 'ýs

All dogs were observed daily for behavioral changes and toxicsigns. Body weights of all dogs were recorded weekly. Blood, urine and fecalsamples were collected for laboratory tests three times before treatment atweekly intervals and at 4, 8, 13 and/or 17 weeks during experiment. The testsincluded hematology, clinical blood chemistry tests, serum electrolytes,urinalysis, and presence of occult blood. For fasting blood glucose, the dogswere fasted overnight for 16 hours. At termination, the dogs were euthanized

!55

with an overdose of pentobarbital sodium and examined for gross lesions.Weights of liver, spleen, kidneys, adrenals and gonads were recorded, andorgan weight to body weight ratios were calculated. Various tissues wereremoved, fixed, processed, sectioned and stained for microscopic examinationof lesions. The procedures for hematology, clinical blood chemistry tests,urinalysis and histopathology, and the normal values are given in Appendix I.

2+ 2+ + +The concentrations of Ca2, Mg2, Na and K in serum were

determined with the atomý2 absorption spectrophotometer, according to theprocedure used by Pybus," originally described for Ca and Mg. The resonancelines used for the analysis of each of the elements are: Ca, 4227 •; Mg,2852 1; Na, 3302 X; and K, 7665 X. Sodium was determined by using the 3302line rather thaai the more sensitive 5890 A line to avoid large dilutions of the

serum. In this way, Ca, Mg, and Na were determined after a 50-fold dilution(0.2 mg serum to 10 ml) of the serum with 1,000 ppm strontium in 0.1 N perchloric

acid. Potassium was determined after a second dilution (1:1) or a totel of100-fold dilution. Phosphate interference and the interference of sodium onpotassium was eliminated by the addition of strontium. The perchloric. acidwas used to remove protein interference. The serum chloride concentre.tion wasdetermined with a Buchler-Cotlove chloridometer.

Bromosulfophthalein (BSP) retention test was performed at ter-mination. A single dose of 5 mg/kg of the sterile test dye (Dade, Miami, ?L)was injected intravenously following fasting for 16 hours. Serum level of thedye at 15 minut 9 was determined and the percent of retention in the plasmawas calculated.l

The results of the various parameters were compared with therespective baseline levels and/or with those of the control groups at therespective4 ime interval according to the Dunnett's Multiple ComparisoniProcedure.-

c. Experimental Design

At the end of 4 and again at 13 weeks of --ontinuous treatment,one male and one female dog irom each group were to be euthanized for nerropsy.The treatment for one other male and female dog from each group was to 6e dis-continued at the end of 4 and 13 weeks and they were to be euthanized at

the end of 8 and 17 weeks, respectively, to study the reversibility of anyadverse effects.

Since adverse effects were not observed in any dogs and TNG-related lesions were not found in the dogs tha%. were euthanized for necropsyat the end of 4 weeks, the treatment *or one male and one female dog from onlythe high dose group was discontinued to study reversibility. These two dogs

6

were euthanized at the end of 8 weeks. All other dogs were continuedon treatment. At the end of 13 weeks, one male and one female dog fromIeach group were euthanized and the treatment for all other dogs wasdiscontinued. Since TNG treatment for 13 weeks did not cause any adverseeffects or lesions, the dogs that were kept for the study of reversibilitywere not euthanized for necropsy at the end of 17 weeks.

3. Results

a. General Observations and Weight Gain

I The control dogs and the dogs receivring daily various dosesof TNG were healthy throughout the treatment periods of 4 or 13 weeks,

respectively. Their body weights before, during and after treatment aresummarized in Table I. These dogs consistently gained weight.

b. Blood Analysis

Since the hematology and clinical blood chemistry results ofthe male dogs and the female dogs were not significantly different, thedata of both sexes from the control group and the groups treated with low,middle and high doses of TNG were combined for discussion and are summarizedin Tables 2 through 5, respectively. The peripheral blood elements andvarious clinical chemistry value were not apparently altered by TNG. How-ever, when compared with the baseline levels within the groups, or whencompared with control dogs at respective time intervals, there were anumber of changes. These changes were slight and inconsistent and occurredin both the control dogs and the dogs treated with TNG.

The treatment for one male and one female dog from only the

high dose group was discontinued after 4 weeks to study reversibility of 7any adverse effects. The peripheral blood elements and clinical chemistry

= Ivalues of these dogs treated with high dose of TNG for 4 weeks (Table 6)and of dogs treated with low, middle and high doses of TNG for 13 weeks(Table 7) after allowed to recover for 4 weeks were not apparently differentfrom those of the control dogs.

Methemoglobin concentration and Heinz bodies in the erythro-cytes of these dogs were negative at the end of 4, 8 and 13 weeks of treat-ment. All blood samples were taken 24 hos after t preceding doses of

TNW. Serum electrolytes including Na , K , Ca , Mg and Cl were deter-

i mined at the end of 4 weeks and again at 13 weeks. As shown in Table 8,the various electrolytes were not apparently altered during treatment.

I7I

c. BSP Retention

BSP retention was determined for the dogs terminated at theend of 4 and 13 weeks. The results are summarized in Table 9ý TNG did notcause any apparent retention of BSP in these dcgs.

d. Urinalysis and Fecal Examination

The results of urinalysis of the control dogs and dogs treated

with various doses of TNG are sunmarized in Tables 10 through 13. Smallamounts of protein, erythrocytes, leukocytes, and/or epithelial cells wereoccasionally present in the urine of both the control and the treated dogs

before and during treatment.

The results of fecal examination are summarized in Table 14.

Occult blood was occasionally found in the feces of the conLrol and thetreated dogs before atid/or during treatment.

e. Organ Weights

The absolute and relative organ weights of all dogs terminatedduring the experiment are summarized in 15 and 16, respectively. Treatmentof TNG did not cause any apparent change in the organ weights. The differencein the weights of testes was related to the age and maturity of these dogs.

f. Gross and Microscopic Examination of Tissues

The dogs terminated for necropsy at various times were in goodnutrit:ional condition without any apparent gross changes. At the end of4 weeks, one dog (No. 31) treated with 1.0 mg/kg/day had a mild subacute

inflammation in the liver and a mild thyroiditis (Table 17). These lesionswere not considered to be related to TNG. The other dogs were healthy with-out any lesions. The bone mnarrows and the myeloid/erythroid (M/E) ratios ofall dogs were normal.

At the end of 13 weeks, a number of spontaneous lesionsoccasionally occurred in both the control dogs and dogs treated with TNG.One control dog (No. 5) had severe pneuronia, a moderate tonsilitis and amild hyperplasia of the prescapular lymph node (Table 18). The other con-trol dog (No. 6) had a mild subacute inflammation of the lung. One dog(140. 11) treated with the low dose of TNG had a mild hyperplasia of themesenteric lymph node, one dog (No. 19) treated with the middle dose had a

2 8 -

mild tonsilitis, and one dog (No. 28) treated with tOn high dose had a mildpneumonia and a congenital hypoplasia of one testis. The bone marrows and:1 the M/E ratios of these dogs were normal.

T 4. Discussion and Conclusions

In dogs, daily treatment of 0.01, 0.1 or 1.0 mg/kg/day of TNG for4 weekL and 0.05, 0.5 or 5.0 mg/kg/day for 9 additional weeks did not cause

is any adverse effects. All dogs continued to gain weight during the treatmentperiod. Peripheral blood elements and various clinical chemistry tests ofthe blood, including BSP retention, did not show any apparent changes.Urinalysis and fecal examination for occult blood did not reveal any ab-normality. TNG did not cause any gross or microscopic changes in tissuesafter treatment for 4 or 13 weeks.

B. Effect of TNG on Methemoglobin Formation

1. Introduction

It is known that TNG causes methemoglobin formation in man. Thisexperiment was designed to administer larger doses of TNC, for 5 consecutive

days. Blood samples were collected at various intervals after each dose forthe measurement of methemcglobin corxcentration to determaine: first, thedose-response relationship if methemoglobin is formed; second, the d4 lappear-ance rate of methemoglobin from the blood; third, the effectrs after repeatcdadministrations. In addition, the protective effect of methylene blue on TNGinduced methemoglobinemia was investigated.


A total of 16 young adult beagle dogs (Hazelton Research Animals,Cumberland, VA) weighing between 8.8 and 14.2 kg were used and conditionedin our animal quarters for 3 weeks. They were divided into four groups, eachconsisting of two males and two females. Each group was given 25, 50, 100 or

200 mg/kg/day of TNG for 5 ccnsecutive days. TNG was desensitized as 10%mixture in lactose and was giver in capsules. Purina dog chow and water were

available ad libitum.

After each dose, blood samples from all dogs were taken at 0.5, 1,2, 4, 8, 16 and 24 hours for the determination of methemoglobirn and totalII hemoglobin concentrations. To study any protective effect on methemoglobluformation, 3 mg/kg of methylene blue (USP water solution for injection) was

I9

injected intravenously to the dogs 2 hours afte, administration of 2C0

mg/kg/day of TNG on the third day. Collection of samples was continuad for the

determination of methemoglobin and total hemoglobin cor-entrations and treat-

ment with TNG for these dogs was discontinued on the4th and 5th days.

3. Results

After treatment of 25 mg/kg/day of TNG,. methemoglobin appearedI to 2 hours after dosing each day and reached peak concentrations of 2.8

to 8.7% of total hemoglobin at 4 hours (Table 19). Methemoglobin disappearedfrom the blood 8 to 1.6 hours after daily TNG dosing. Small amounts of methemo-

globin appeared again in some dogs 24 hours after the third dose.

After treatment of 50 mg/kg/day of TNG, methemoglobin also appeared

I to 2 hours after dosing each day and also veached peak concentrations at4 hours (Table 20). The dally peak concentrations averaged 9.2 to 15.0% oftotil hemoglobin. Similarly, methemoglobin disappeared from the blood 8 to

16 hours after administration of TAG.

A•fter treatment of 100 mg/kg/day of TNG, these dogs became slightlycyanotic 2 to 3 hours after dosing each day and the cyanosis lasted fc. a

r•ouple of hours. Methemoglobin appeared In the blood earlier, i.e. 0.5 to 1hour after dosing (Tab!,- 21). Methemglobin concentrations progressively

increased with time and reached peaks of 24.7 to 42.6% c f total hemoglobin

at 4 hours. The concentrations of methemoglobin remained high at 8 hours aild

decreased thereafter. Small amounts of weathemoglobin remained in some dogs24 hours after admin-stratio, of TNG.

After treatment ot 200 mg/kg/day of TNG, tY-se dogs exhibitedcyanosis and ina,:tivitv 2 to 3 hours after dosing each Jay without any serious

effects. They retorned to nornmal in several hours. Meuhemoglobin appearedwithin 1 hour on the first day and reached peak concentration of 28.3% of

total hemoglobin qt 4 hours (Table 22). Methemoglobin disarpeared from the

blood. 8 to 16 hours attar doritxg. On the second day, methemoglobin concentra-tion quickly increased to 25.9% of total hemoglobin at 2 hours after dosing

and reached 50.7% at 4 hours. The; methemoglobin decreased slowly ana dis- 4

appeared fr r the blocd by 24 hours. Ou the third day, methemoglobin concentra-

tion increased to 3.0% at 2 hours. At this time, 3 mg/kg of methylene bluewas injected intravenously to each dog. Instead of increasing quickly to apeak, the methemoglobin concentration decreased slightly and remained rela-tively high through 16 hours after administration of TNG. Thereafter, itdecreased to 3.6% and did not disappear from the blood until 40 hours afterlast TNG dosing.

10

IMethemoglobin concentrations of these dogs after daily treatment

of various doses of TNG on the first through the 4th days are better Illus-trated in Figure 1.

4. Discussion and Conclusions

Oral administration of 25 or 50 mg/kg/day of TNG for 5 consecutivedays did not cause any apparent adverse signs; 100 or 200 mg/kg/day causedcyanosis lasting a few hours 2 to 3 hours after dosing. In addition, 200mg/kg/day decreased the activity withoutc any serious effects. All thesedoses quickly caused formation of methemoglobin. Methemoglobin concentra-tions reached peaks in 1 Lo 4 hiurs. The height of the peaks and zhe dura-tion of the methemoglobin conccntratior.s were dose-related. Methemoglobindisappeared from the blood 8 to 24 hourZ depending upon the dose. Sma--amounts of methemoglobin remained in some dogs 24 hours after larger doses,

After 25, 50 or 100 mg/kg/day of TNG for 5 consecutive days, thereappeared no tolerance in the formation of methemoglobin nor was there anyaccumulative effect on the formation of methemoglobin. However, after 200mg/kg/day of TNG, the methemoglobin concentrations on the secord day wereconsiderably higher than those on the first day and the methemoglobin at2 hours on the third day was higher t n that or, the second day. This probably in-dicates some accumulative effect of .4G on the formation of methemoglcbin.

Administration of 3 mg/kg of methylene blue 2 hours afteradministration of 200 mg/kg of TNG apparently prevented the methemoglobinformation to reach the high peak. However, the concentration of methemoglo-bin persisted for 40 hours or longer after the as'miistration of TNC.

C. Cytogenetic Effect of TNG

I. Introduction

•I The cytogenic effect. of TNG on somatic cell chromosomes wasstudied. The lymphocyte and kidney cultures from dogs treated with repeateddoses of TNG were obtained and examined for any damage.


a. Animals

Dogs treated with the high dose of TNC from the subacute andsubchronic toxicity studies were used. These dogs were administered 1.mg/kg/day of TNG for 4 weeks and 5 mg/kg/day for 9 additional weekn.

ILi-

b. Lymphocyte and Kidney Cultures

At the end of 4 and 13 weeks, blood samples were asepticallydrawn from the jugular vein cf both the control and tresed dogs. The lym-phocytes were cultured by the method of Moorhead et al.- Kidney tissuesamples were 9moved at necropsy and cultured by the trypsinization methodof Fernandes.- All ciý tures were maintained in Eagle's medium as modifiedby Vogt and Dulbecco."

c. Chromosome Analysis

Actively dividing kidney cultures and phytohemagglutin-

treatment. The cells were trypsinized, swollen in hypotonic solution, and 8/

processed for spreading on glass slides by the method of Moorhead and Nowell.-Slides were stained with giemsa and scanned under low power optics. Cellpolyploidy was estimated by examination of 200 cells. Slides showing min-imum scattering of cells in metaphase were selected for analysis underoil inmersion optics. Chromosomes were counted and morphological aberra-tionIs were examined from photographic negatives of up to 50 metaphase cells.

3. Results

The results of nume):ical . and morphological abec'rations of chrom-osomes are shown ii, Cables 23 and Z4-, respectively. Dogs treated with TNGdid not show any apparent changes in the chromosome frequency distributionor number of tetraploids or any apparent changes in the'chrotsatid breaks ortranslocations, in the peripheral lymphocytes or kidney cultures.

4, Discussion and Conclusion

Admir-Lst-ation of I mg/kg/day of TNG for 4 weeks and 5 mg/kg/dayfor 9) additiorvil weeks in dogs did not appear to cause any numerical ormorphological aberrations of chromosomes in the peripheral lymphocytes orkidney cultures. However, only a few dogs were terminated and studied at

the end of 4 and 13 weeks.

D. Immunologic Response to TNG

1. Introduction

In human, aqphylactic reactions were associated to a high immuno-globin E (IgE) titer.- IgE, the allergic or hypersensitive antibody, ofdogs treated with TNG was determined.

12

_______

III 2. Materil -and Methods

1O/

The immunodiffusion technique of Mancini et al. -- was used for

determination of serum IgE titer. Replicate I ml samples of serum from the

control dogs and dogs treated with various doses of TNG at various intervals

SI were placed in wells in an immunodiffusion chamber along with suitable stan-

dards. These dogs were used for subchronic and subchronlc toxicity studies

as described in Section I.A. The diffusion chamber was incubated at 3700 for

U 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 anti-

body reagent.

3. Results and Conclusion

The results of IgE concentration of control dogs and aogs treated

with TNG are stmmarized in Table 25. Treatment of various doses of TNG for4, 8, and 13 weeks did not cause any apparent changes on serum concentration

of IRE.

I

II~

ii'

[I__

TAB LE 1

BODY WEIGHTS OF DOGS TREATED WITH TNG IN CAPSULES

DoseS- Dog Body Weights (kg)(ma/kg/day) No. Sex Initial 4 Weeks 8 Weeks 13 Weeks 17 Weeks

0 1 F 6.6 7.30 2 M 10.7 11.1

0.01 15 F 7.2 8.40.01 16 M 9.2 10.10.1 23 F 8.6 9.5

0.1 24 M 9.6 10.81.0 31 F 9.4 10.4

1.0 32 M 10.0 11.21.0 29 F 9.0 1.t 10.4

1.0 30 M 10.3 12.0k 12.0I0 5 F 7.0 8.1 8.4 9.50 6 M 9.3 10.0 10.6 11.2

0.01-0.05 11 F 7.3 8.4 8.4 9.30.01-0.05 12 M 8.7 9.4 9.6 10.40.1-0.5 19 F 8.0 8.2 8.2 9.00.1-0.5 20 M 9.3 10.2 11.0 11.71.0-5.0 27 F 8.0 8.8 9.0 10.1I1.0-5.0 28 M 11.8 12.8 13.2 14.3

0 3 F 9.3 10.1 9.8 11.Oa/ 11.00 4 M 8.8 10.0 11.0 11.1t/ 10.80 7 F 8.3 9.2 9.8 10.8h~/ 10.60 8 M 9.0 10.2 10.8 11.6-t/ 9.4

0.01-0.05 9 F 7.8 8.9 8.0 10. 9h/ 10.40.01-0.05 10 M 10.4 12.0 12.8 14. 1!1 14.00.01-0.05 13 F 9.8 10.3 10.2 10.9-h/ 10.80.01-0.05 14 M 10.0 11.2 12.2 13.4-t/ 13.40.1-0.5 17 F 7.0 7.7 8.0 9.3t/ 8.80.1-0.5 18 M 9.8 11.4 11.8 12.4h/' 12.60.1-0.5 21 F 9.1 10.2 10.0 11.9!1 12.40.1-0.5 22 M 9.2 10.7 11.4 11. 81Y 11.41.o-5.0 25 F 7.2 R0 8.3 8.8!? 8.4

1.0-Q.0 26 m 10.6 12.0 122 13.0/' 13.4

atDoses were increased ý-fold starLing the 5th week.bj/ Dosing discontinued thereafter.

14

S-.............

ITABLE 2

•lABORATORY DATA .OF CONTROL

1 Be:N) AASCLINS

UMBER OF DOGS

-WK 1 S "S A IC- .: WKS±.3 C, 4)ERYTHROCYTES MO| IMM .98 • 25 4.7k , S.44 t .9 is1 t .:I S/

RETICULocYTES. S . T8 .0 ? t .4efi .49 .01 Ml| .09

EMATOCPIT( VOL. S 41.t1 t 12 40.3 t 1o0 44.0 1.1

HEMOOLOFtIM, 1 13.q + .4 14.0 ..'5 14.0 0 02 0S.0 04.0MCV, CUBIC MICRONS 84.4 t Z.1 87.9 4.8 74.1 t 2.5 ?1.2 1.!7

MCHAT Y]CPD MICROGAS, 20.2 t F. ?.6 1O.% 2 .8 t 0. 24.4 0.6

NCHAC* O06 1C S .33.4 t .2 34.0 . 34. . .4 34.2 0. 0

P LC T ELO T S ( XT 0 IM E ) 2 .5 - .2 2 . 1 * . 2 2 . 2 * . 3 2 . 1 .3LEUKOCYTES (X10 /MM ) I31•1091• .• I, . •1% *

LYMPHOCYTES. % 35.9 t 3.1 34.4 i 2 .1 3..• 2.S

39. S.0

BANDOS* l l . 3 . 05o t 3.0 ./ ,3 . .3 0.0 0.0 . /

BUN, MG ~ o 19.1 t .3 3 .1 ?.314 ~ 1.1 14.0 0 .0

EOSINOPHIL 2.0 t .4 4.9 t .6 7.0 3.0 .4.6 1.3

RASOPHILS, 16 0.0 t 0.0 0.0 t 0.0 ,0.0 0.0 0.0 0.0

ATYPICAL* 16 0.0 t G.0 0.0 t 0.0 0.0 0.0 0.0 0.0

NUCLEATED PRC, 'R 0.0 t 0.0 0.0 t 0,0 0.0 + 0.0 0.0 0.O0

a-CLOTTING TIME9 MItN. 4.3 .3 4.5 t .4 4.5 t 5 e.6 .9 •

GLUCOSE (FASTING). MG 1 81.7 2. 8° %.ft t P.R 89.3 t P.i 99.(l t IR [

SGUT, lU/L 32.2 t 1.8 29.5 t 2.p 25.8 I .A 21.0 ) 1.2 •

SGPT, lIJ/L 30.9 t 1.8 31.1 t 2.q 26.n ?.2 20.3 & .8 •

ALK. PHOS., IUIL 76 1. 3 6L t 4 so t 4 S/ S S •IBUN$ 146 S 15. 1 t .9 13.1 15 t• 1. 3 , 14.5 1.0

ENTRIES ARE 04FAN S TANnSRD ERROWKS -Z-O0A, 8) IS THF 4VFRAGE OF WEKIý 2. 1. ANO 0 PQIOW TO TREATMENT.

a1 Significantly different from the baseline level (Dunnett's multiple comparison procedure -/

I1I

I 'II 'I

ii _

4w en m m

.9 .1 *I .9 .1 . . . . . 1 1 9 9 9 .9 .9 1 .1 .1 .1 .9 .1

H j .'- . .9..09. .9 .1 . 9 .0 .0

1.0.

Z~t rnI a

7j I

IN, ZEr9 (D x - vn- a . . ' 4 F- C ' C n 9 . 0 .o - Int A' 0 J I

; LI o u. o 93o' 01 1

In -x 959 I n W) In In -AT ou

u o IDC W I9/ n ~ I 992 Z A

0 _j AJ LL a1 7 U 2 *

or w I v j u j -o o j. zUA LI mn w 7

LI 9. F.- ~ t2

16I

1-4

.nI .flg i0

3~~~0 .31 !;1 .3l .3 . g 4 1 3 . I . . 1 .

.2

10. C.~ . - 4 .- C 7.1- 41 # 0 f 4 1 0 . 0 1 U 1 C C1* ~ ~ ~ ~ ~ r - n - aL 0 0 0 4 7

W In.17.-t .0

if -N N C C C N li- ft

#64 . ; w

0 0 U, a. uC 0.f

4tA 0Z 0 l' (A tw

LL 0 7 -j I' 4i . . w aI-Z - M 3- 19 -1 v~* ( ' 7 4

). 2 r x 4 I.) v -, m 0 CL be

(t - Cj ~ 0 .- u j U' _j I 0 _C2

w 7 . x 7 T 2 (L J 0 D (A 0 n - w 2 7I

17

t. w

E-44

OD A90 Mn (P m In t0 m0 N 1 In V. 0 m 0 Q 0 0 c t 0 ~ i.49- *: LA lz lz Iii In N* N 0 : 0 0 p9~~4W~ In M. 4 C) n . N i .

41 .1 +1 . 1.1 +

.0

cc 4,

4 - i 0 .1 +1 .' .1 f.1 N1 i .1 01 .1 .1 0 9 qp1N 9. t

z oin~ ~ CK m9 n - N 9. 0 4

.1 . 1 1, .1 In IL .1 OIL .1 .9 19 .2 .9 . 9 9 . 1 .9 .1 9

N 0. In0 * 09M D 0 in N u LO N, in N N - NwC0 4 . n C0~~~ 7 -' U n .' . 1 an-

01 x9 x 31 1 a -. In I- c0 r w. N. . 7

U 0. 91 8 - 9. 4 *900 u

TABLE 6

LABORATORY DATA OF DOGS TREATED WITH TNG FOR 4 WEEKS AND ALLOWED TO

RECOVER FOR 4 WEEKS

(C,N) CONTROL (T,N) TREATED N NUMBER OF DOGS

DOSE: MG/KG/DAY 0 (C, 2) 1.0 (T. 2)6 3

ERYTHROCYTES (XIO /MM ) 4,36 4.44

RETICULOCYTES, % .45 957

I HEMATOCRIT, VOL. % 41.0 40.0

HEMOGLOBIN, GM. % 13.6 13.3

I MCV, CUBIC MICRONS 94.0 90.0

g MCH8, MICRO MICROGMS. 31.3 29.9

MCHBC9 GM % 33.3 33.2,•5 3

PLATELETS (X10 /MM ) 2.4 2.53 3

LEUKOCYTES (X1O /MM ) 9.8 8.2

I NEUTROPHILS4 % 54.,() 50

LYMPHOCYTES, % 42.5 36.5

BANDS9 % 0.0 0.0

MONOCYTES. % 0.0 1.5

EOSINOPHILS, % 3.5 5.0

* BASOPHILS. % 0.0 0.0

ATYPICAL, % 0.0 0.0

I NUCLEATED RBC, % .5 .5

3 CLOTTING TIME* MIN. 8.3 6.3

GLUCOSE (FASTING). MG % 108.5 102.5

3 SoOV. IU/L 24.0 24,0

SGPT9 IU/L ?8 29

I ALK. PHOS., IU/L 53 40

3BUNg MG % 13.3 14.0

ENTRIES ARE MEAN

!19

f.- u m ry

H a Na1

A. zN

abC' 0 0 ~ UN ~ ~ siN - V V 0 0 C' ' 0 U IN U

3L* .*

go a* 4N aN k op 471 .. ' 7 C ' U N 7 70 ~ ~ h 'r 0U - 2N 5 - UN t :U

~~bi

o0

EL 0ý ; o -. a ' N U N N - N A' N A 0 0 0 0 C .) C Ix UN u 1

-5~ . w 1 N 7 * A 5. 5 ' 4 N 'Q N UK - oN N . - UN '.7 N 5

NZ w 'w u u tjN w D _j _j 0 0 -1 m. z

02

I TABLE 8

SERUM ELECTROLYTES Of DOGS TREATED WITH TNG

Doses/ Serum Ele,.:trolvtes (mea/t)(mg/kg/day) Na K Ca 9_ Cl

Before Treatment (8 dogs/group)

Control 153:1-2/ 4.5+0.2 5.5±0.1 1. 6*=0.0 107±1

0.01-0.05 148*1 4.7±0.1 5.5±0.1 1.5±0.1 103±10.1-0.5 151±1 4.4±0.1 5.5..0.1 1.5*0.1 105*11.0-5.0 150±1 4.5±0.1 5.7:O.1 1.5+0.0 104±1

Treatment for 4 Weeks (2 dogs/Iroup)Control 1501/ 5.1 5.7 1.5 103

0.01-0.05 151 5.2 5.6 1.6 1050.1-0.5 151 4.9 5.5 1.4 1031 1.0-5.0 145 5.1 5.4 1.5 105

Treatment for 13 Weeks (2 dogs/group)Control 145Y- 4.9 5.4 1.7 105

0.01-0.05 148 4.9 5.4 1.5 1070.1-0.5 146 4.9 5.6 1.7 109

S1.0-5.0 146 4.8 5.6 1.6 108

a/ Doses were increased 5-fold starting the 5th week.5 b/ Mean + S.E.

c/ Mean.

S21

Ii[I[I

[II ' l I _ J L

TABLE 9

BSP RETENTION OF DOGS TRE&ThD4 WITH TNG

Dose-(mg/kg/day) Dog No. 7% Retention

At the end of 4 weeks 4

Control 1 4

Control 2 7

0.01-0.05 15 70.01-0.05 16 4

0.1-0.5 23 10

0.1-0.5 24 51.0-5.0 31 4

.0-5.0 32 5

At the end of 13 weeksControl 5 3

Control 6 5

0.01-0.05 11 3

0.01-0.05 12 40.1-0.5 19 40.1-0.5 20 3

1.0-5.0 27 4

1.0-5.0 28 5 -

a/ Doses were increased 5-fold startingthe 5th week.

22

22

Ul

I

I

TABLE 10

URINALYSIS OF CONTROL DOgS .FOR TNG

Dose: Eupty Capsules

___Treatment Weeks_

0 4 8 13

Glucose: Negative 8 8 6 4< 250 mg % 0 0 0 0

>25 m&) _ 0~_ 0

Protein: Negative 6 8 6 4

< I00 mg % (4,8) 0 0 0>lOO _ _ o o _o0 _ 0_0

Microscopic 'Examination

RBC-./: Normal 8 8 6 4Moderate 0 0 0 0Excessive 0 0 0 0

WBC-": Normal 7 6 3 4

Moderate (5) (6,8) 0 (7)

Excessive 0 0 _0_ 0

Epithelium~b/: Normal 3 4 5 1

Moderate (5,6) (1,2,8) (3) (5,6,7)

I Excessive L4.,7.87. _86)_ 0 0CrystalsS/: Normal 8 8 6 4

Moderate 0 0 0 0Excessive 0

Casts : Negative- 8 8 6 4[ Positive 0 -0 - _ 0

Numbers indicate n,'mber of dogs or dog numbers (in parenthesis).a! Normal, 10 or ess cells; moderate, 10-100 cells; excessive,

> 100 cells/ ield (x 440).b/ Normal, 5 oi less cells; moderate, 5-25 cells; excessive,

> 25 cells/field (x 100).c/ Normal, none; moderate, 1-5 crystals; excessive,> 5 crystals/

field (x 100).

23

TABLE 11

URINALYSIS OF DOGS REFORE AND DURING ArI4NISTRATION OF TNQDOSE: 0.01-0.05 mg/kg/day

(Dose was increased 5-fold starting the 5th week)

Treatment Weeks

O 4 8 13

Glucose: Negative 8 8 6 4< 250 mg % 0 0 0 0> 250 0go0 0 0

Protein: Negative 8 8 6 4< 100 mg % 0 0 0 0> 1O0 m-_!o o 0 0 .o_ 0 _0_

Microscopic ExaminationRBC,/ : Normal 7 8 6 3

Moderate (14) 0 0 (11)Excessive 0 0 0 0

WBCa/: Normal 7 7 5 1Moderate (14) (12) 0 (9,10,11)Excessive 0 0 (14)_ - 0

Epteim-I ormal 4 55 1Moderate (9,13,14) (14,15,16) (14) (9,10,11)Excessive £16)_ 0 0 0 _ _

Crystalsc/. Normal 8 8 6 4Moderate 0 0 0 0Excessive 0 0 0 0 _

Casts: Negative 8 8 6 4Positive 0 _ 0-- 0 _]_ 0 ,

Numbers indicate number of dogs or dog numbers (in parenthesis). Aa/ Normal, i0 or less cells; moderate, 10-100 cells; excessive,

> 100 cells/field (x 440).b/ Normal, 5 or less cells; moderate, 5-25 cells; excessive,>25 cells

/field (x 100).c/ Normal, none; moderate, 1-5 crystals; excessive,>5 crystals/field

(x 100).

24

fTABLE 12

URINALYSIS OF DOGS BEFORE AND DURING ADMINISTRATION OTMGDOSE: 0.1-0.5 mg/kg/day

1. (Dose was increased 5-fold starting the 5th week)

_ __ Treatment Weeks

0 4 8 13

Glucose: Negative 8 8 6 4S<250 mg 7. 0 0 0 0

. . >.250 mRg. 0 0 o0 0

Protein: Negative 8 8 6 4< l00 mg % 0 0 0 0T 0mg_ 0 0 ___ 0 0

Microscopic Examination

RBCa-/: Normal 8 8 6 4Moderate 0 0 0 0Excessive 0 0 0 0

WBCI/ Normal 8 8 6 4Moderate 0 0 0 0 ý

. . .xcessive 0 0 _ - 0 ___0

Epitheliumb/: -Normal 3 4 -5 2Moderate (18,20,22) (18,20,23) (21) (19,20)

I Excessive (17,24) ,(14) 0 02Crystalsj/: N~orma 8 8 64

Moderate 0 0 0 0

- _ - -- Excessive 0 0 0 0Casts: Negative- ...... 8 . 8 6 4

Positive 0-0 0 0INumbers indicate number of dogs or dog numbers (in parenthesis).

a/ Nornal, 10 or less cells; moderate, 10-100 cells; excessive,

I > 100 cells/field (x 440).b/ Normal, 5 or less cells; moderate, 5-25 cells; excessive,> 25

cells/field (x 100).

c/ Normal, none; moderate, 1-5 crystals; excessive, >5 crystals/

field (x 100).

II!2

TABLE 13

URINALYSIS OF DOGS BEFORE '. D2URING ADMINISTRATION OF T'NG

DOSE: 1.0-5.0 m8/kg/day(Dose was increased 5-fold starting the 5th week)

Treatment Weeks0 4 8 13

Glucose: Negative 8 8 6 4S< 250 mg 7. 0 0 0 0S> 250 mrg. 0 0 0 0

Protein: Negative 6 7 6 4< 100 mg % (28,32) (27) 0 0

>OOmg 0 _0 --.--o0 o0Microscopic Examination

RBCa/: Normal 6 7 5 4

Moderate (28,31) (31) (30) 6Excessive 0 0 0 0

wW!: Normal 6 7 5 4Mod eratre (25,31) 0 (30) 0

Excessive 0 0 0

EpitheliumYb/: Normal 4 4 5 2

Moderate (28,32) (28,29,31) (30) (25,27)

Excessive (26,30) - (_3221 . 0 0

Crystals-C/: Normal 8 8 6 4

Moderate 0 0 0 0Excessive 0 0 0 0

Casts: Negative 8 8 6 4Positive 0 0 0 0

Numbers indicate n,'mber of dogs or do3 numbers (in parenthesis).a/ Normal, 10 or less cells; moderate, 10-100 cells; excessive,

> 100 cells/field (x 440).b/ Normal, 5 or less cells; moderate, 5-25 cells; excessive, >25

cells/field (.• 100).c/ Normal, noi.-ý, moderate, 1-5 crystals; excessive, >5 crystals/field

(x 100).

26

I

TABLE 14

DETECI ON 9,•_Q FECAL BLOOD IN DOOS BEFORE ANDDURING &MSTA OFIN

Doe-&/ Presence of Treatment Weeks(mg/ki/dav) Ftcal Food 0 4 8 _3

Control Negative 6/ 8 4 2

Positive (2,4) 0 (6,8) 0

0.01 - 0.05 Negative 7 6 6 2Positive (12) (10,11) 0 0

0.1 - 0.5 Negstive 5 8 6 2Positive (18,22,23) 0 0 0

1.0 - 5.0 Negative 5 7 6 2

Positive (25,30,32) (29) 0 0

a!/ Does were increased 5-fold starting the 5th week.b/ Numbers indicate number of dogs or dog numbers (in parenthesis).

27

* 27

TABLE 15

ABSOLUTE ORGAN WEIGHTS OF DOGS TREATED WITH TNG

TerminalDose4/ Dog Body Weight Absolute Organ Weights (gin)

(mg/kx/day No. (kg) Liver Spleen Kidneys Adrenals Ovaries Testes

Treated for 4 Weeks-----------------------------------

Control 1 7.3 209 54 35 0.98 0.52Control 2 11.1 291 42 46 0.83 3.670.01 15 8.4 170 31 41 0.99 0.610.01 16 10.1 241 39 41 0.95 2.690.1 23 9.5 247 58 53 0.98 0.730.1 24 10.8 325 69 45 1.01 11.001.0 31 10.4 275 68 46 0.98 0.731.0 32 11.2 325 78 62 0.85 17.00

Treated for 4 Weeks and Allowed to Recover for 4 Weeks

1.0 29 10.4 242 42 42 1.06 1.101.0 30 12.0 278 36 50 0.71 16.00

Treated for 13 Weeks

Control 5 9.0 248 60 44 1.22 0.82Control 6 11.0 294 84 44 0.85 18.00.01-0.05 11 8.6 218 68 42 1.00 0.67

0.01-0.05 12 11.1 230 114 42 0.94 12.00.1-0.5 19 9. 0 238 58 -" 1.16 0.680.1-0.5 20 11.6 282 98 50 1.10 22.01.0-5.0 27 9.6 258 30 46 0.96 0.811.0-5.0 28 13.8 300 70 66 1.34 17.4

a/ Doses were increased 5-fold starting the 5th week.

28

TABLE 16

RELATIVE ORGAN WEIGHTS OF DOGS TREATED WITH TNG

aDose/ Dog RelativeOran Weights (gm/kg Body Weight)

kIda No. Liver p Kidneys Adrenals 0 ries Testes

Treat%;d for 4 Weeks

"Control 1 28.7 7.4 4.8 0.13 G.07

Control 2 26.2 3.8 4.2 0.07 0.330.01 15 20.3 3.7 4.9 0.12 0. 0"0.01 16 23.9 3.9 4.1 0.10 0.27

0.1 23 26.0 6.1 5.6 0.10 0.08

0. i 24 30, 2 6.4 4.2 0.09 1.02 1

"" 1.0 31 26.5 6.5 4 0.09 0.07

1.0 32 29.1 7.0 5.5 0.08 1.52

Treated for 4 Weeks axu, A) lowed to Recover 4 Weeks 4

1.0 29 23.3 4.0 4.0 0.12 0.11

1.0 30 23.2 3.0 4.2 0.06 1.33

Treated for 13 Weeks

Control 5 27.6 6.6 4.9 0.14 0.09

Control 6 26.7 7.7 4.0 0.07 1.64

0.01-0.05 11 25.1 7.9 4.9 0.1? 0.08

0.01-0.05 12 20.7 10.3 3.8 0.08 1.08 I0.1-0.5 19 26.4 6.4 -0.13 0.08

-0.1-0.5 20 24.3 8.5 4.3 0.09 1.90

1.0-5.0 27 26.9 3.1 4.8 0.10 0.08

1.0-5.0 28 21.8 5.1 4.8 0.10 1.26

"a/ Doses were increased 5-fold starting the 5th week.

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TABLE 25

SERUM IE (IU/mi" OF DOGS TREATED WlITH TNG !

Dose Treatment Weeks__mgjkg/dav)-.! 0 Weekn 4 Weeks 8 Weeks 13 ,Wiks

Controls 1,673±149(8'Y 2,129±239(8) 1,250±50(4) 1,300±119(4)

0.01-0.05 1,457±135(8) 1,607±57(8)

0.1-0.5 1,354±147(8) 1,588±46(8)

1.0-5.0 1,365±103(8) 1,583±38(8) 1,651±175(4) 10068±169(4)

•/ Doses were increased 5-fold starting the 5th week.b/ Mean ± S.E. (number of dogs).

38

AIi 1

V 50-

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RTHIRD DAY FOURTH DAY

1 10\'g'

2.

0 4 6 176 24 0 4 8 16 24

HOURS AFTER DOSING

:Treated with 3mg/kg methyleg e blue nnd stopped treatmeayt with NG

Figure I -Methemoglobin Concentration in Dogs Treated With variousS---Doses of TNG.

39

• ' •0----020 mg/g/da

II. RATSn.__ .__ _S

TABLE OF CONTENTS

A. Subacute and Subchronic Toxicities and Reversibility .... ....... 43

1. Introduction ............. ........................ ... 432. Material and Methods ......... .................... ... 43

[ a. Number of Rats, Sex and Treatment. ........... ... 43b. Animal Husbandry ......... ................... ... 43c. Feed Preparation .......................... ... 43d. Stability of TNG in Feed Mixture ............... . 44

e. Experiiaental Procedure ....... ............... ... 44f. Experimental Dasigr ........ ................. ... 45

3. Results ............ ... .......................... ... 45

a. Stability of TNG in Feed Mixture.. .......... 45

b. General Observation and Weight Gain ...... ........ 46c. Feed Consumption and TNG Intake .............. ... 46

d. Blood Analyses ......... .................... ... 47e. Organ Weights ................ ............. ..... 47

f. Gross and Microscopic Examination of Tissues. . . . . 48

4. Discussion and Conclusion .......... ................ ... 49] B. Additional Subchronic Toxicity ....... .................. ... 49

1 i Introduction . . . . . . . . . . . . . . . . . . . . .. . . 49

2. Material and Methods ......... ................... ... 50

.3. Results .. ................... .......................... 50

a. Rats Fed 2.5% TNG ........ .................. ... 50b. Rats Fed 257. Lactose ......... .............. ... 52

4. Discussion and Conclusions ......... .............. ... 53

C. Cytogenetic and Mutagenic Effects of TNG. . . . . . . . . . . . . 54

I. Introduction ........... ... ........................ 542. Material and Methods ......... .................. .... 54

a. Cytogenetic Effects on Chromosomes .......... 54b. Mutagenic Effects on CHO-KI Cells In Vitro .......... 54

41

T!

TABLE OF CONTKNTS (Concluded)

3. Results ................. ........................ ... 55

4. Discutssion and Conclusion ...... ................. .... 55

D. Immunologic Response to TNG ....... ................... .... 55

1. Introduction ............ .................... . . . 552. Material and Method. . . .................. 55 .

3. Results and Conclusion .......... ................ ... 55

Tables 26- 66.............................................. . 56

Figures 2- 3 ................. ......................... ... 97

42 I

lI

* i

S4 2 I,

LHj J

I

TI. RATS

A. Subacute and Subchronic Toxicities and Reversibility

1. Introduction

As for the dogs, these studies were performed to define the natureand extent of effects of TNG on the biological system at the biochemical andcellular levels and to elucidate the dose-response relationship in the ratsfed TNG for 4 weeks and 13 weeks. The reversibility of any adverse effectswas also studied after the feeding of TNG was discontinued for 4 weeks.


a. Number of Rats, Sex and Treatment

A total of 64 male and 64 female young healthy CIP rats (CharlesRiver Breeding Lab., Wilmington, Mass.) were used for this study. They weredivided into four groups, each consisting of 16 males and 16 females. Theaverage weights of all groups were kept close. Three groups of rats werefed 0.001, 0.01 or 0.1% TNG in the feed. At the end of 5 weeks, adverseIL effects were not observed in any rats. Starting the 6th week, TNG concen-trations in the feed for all groups were increased 5-fold to 0.005, 0.05 or0.5%, respectively. The 4th group, serving as the controls, was given thepowdered standard rodent chow (Wayne Laboratory Meal) without TNG.

b. Animal Husbandry

Our animal quarters have a ventilation system with 10 air

changes per hour. The room air is passed through filters to remove 99.9%

of all particles larger than 0.3 p. The temperature i3 maintained at 75 ±56F and the relative humidity at 50 ± 10%. The light cycle in all animalrooms is kept at 12-hour on and 12-hour off with a timeT.

Upon arrival from the breeder, the rats were isolated andconditioned in our rodent quarter foi at least 2 weeks before starting on the

i I experiment. They were housed two per plastic cage with filter tops. Hardwoodchips wc o used after steam-sterilization as bedding materidl and changedweekly. All cages, cover tops and water bottles were steam-sterilized befcreii using and once every month. Feed and water were available at all times.

c. Feed Preparation

TNG was desensitized in lactose as 10% mixture (SDM No. 17,Atlas Ch~emicals DivisioA, ICI America Inc., Wilmington, Del.) and the lactosenmixture was added to powdered standard rodent chow at 1/ by weight to yield

43

a diet containing the desired 0,1% TNG. The diet was placed Lt a woodenbox (16 x 16 x 20 inches) until half full. The box was placed in it modi-fied cement mixer Sind rotated about its long axis for 1 hour at a ýtpeedof 20 rpm. Subsequently, this diet was mixed with the standard chow at10% by weight to yield 0.01% and again 0.001% of TNG, respectively. Start-ing the 6th week, the lactose mixture was added to the standard rodent chowat 5% by weight to yield a diet containing 0.5% of TNG. Accordingly, thisdiet was mixed with the standard rodent chow at 10% by weight to yielk.d0.05% and again 0.005% of TNG, respectively.

( d. Stability of TNG in Feed Mixture

The stability of TNG in feed was determined and the asaayprocedure previously reported in Appendix I on The Identification and Assayof Nitrotoluenes and Nitroglycerines-'/ was used. Two feed levels of TNGwere prepared. Duplicate samples were analyzed for TNG immediately andafter various intervals tinder various conditions.

TNG concentration in feeder under normal feeding conditionwas found to decrease with time. The question of loss of TNG by evapora-tion oi chemical reaction with rat feed components was examined by spikinga 0.5% TNU feed sample with TNG--,3- 1 4 C. The TNG-l,3-1 4 C in ethanol wasadded to 100 mg of lactose in 10 ml of ethanol. This mixture was evapor-ated to dryness under a stream of N2 and added to 125 gm of 0.5% TNG inrat feed. After it was well mixed, triplicate samples of 100 mg of thefeed mii:ture were taken and counted immediately in the scintillation so½u-tion using a Packard T icarb 3375 liquid scintillation spectrophotometerdescr-' previously The counting solution contained toluene in which

TNG was well dissolved. For safety regulations, the ING feed mixture con-taining TNG-I,3- 1 4 C was stored in an operational fume hood. Duplicate

samples were taken and counted for radioactivity at the end of 1, 4, 6S~and I0 days.

e. Experimental Procedure

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

(1) "eed consumption of all rats was measured throughoutthe experiment.

(2) Urine and fecal samples were not collected for

examination.

(3) Blood samples were collected by cutting the tip ofthe tal.] at 0, 4, 8, 13 and/or 17 weeks for hematoloy tests. In addi.lon,termi.nal blood was collected ifrom .ne abdominal aorta under ether anes-thesia for clh:ical -hemistry tests.

S~44

4 -~ I

ii - •• .. . . ... ... . .. .. - .,'...-,-,,,,-,- , •.. . .......... .~ ,'•-, • °•. -`* 4 . : •• % `W •

(4) BSP retention test was not performed.

r f. Experimental Design

Sd 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 fourt .females rats from each group were euthanized for necropsy.

(2) The treatment for four male and four female rats from

each group were discontinued at the end of 4 weeks. They were kept for ob-

servation. Since adverse effects were not observed in any rats and TNG did

not cause -'ny lesions in the rats that were euthanized at the end of 4weeks, the rats for the reversibility study were not necropsied for examin-t azion at 8 weeks as scheduled.

(3) Similarly, the treatment for four male ana four female

rr'ts from each group were discontinued at the end of 13 weeks. These rats were

euthanized for necropsy at the end of 17 weeks to study the reversibility

of any adverse effects.

3. Results

iIt a. Stability of TNG in Feed Mixture

The results on the stability of TNG in two feed mixtures aresummarized in Table 26. The extraction efficiency of TNG from rat feed

U' mixtures containing 0.01% or 1% TNG was 95 ± 4% and 99 ± 1%, respectively.TNG was stable in both tne feed mixtures when frozen for 8 days. An average

of 95% of the TNG remained at the end of 4 days when the feed mixtures wereplaced in the rat feeders under normal feeding conditions or !n a capped '

metal can stored in room temperature. When the feed mixtures were saoredin the capped metal can for 4 days plus in the rat feeder under normal feed-ing conditions for 4 additional days, an average of 82% TNG remained. TheTNG remained in the metal can after 8 days in room temperature averaged89%. When the 0.5% TNG feed mixture spiked with TNG-1,3- 1 4 C was stored in

an operoting fume hood, the radioactivity remained in the mixture at the

end of 1, 4, 6 aad 10 days averaged 81, 67, 63 and 52%, respectively. This

indicated that the loss of TNG in rat feed mixtures was due to evaporation.The larger loss,-of TNG as indicated by the loss of radioactivity was dueto larger air flow in the fume hood.

For the feeding studies in rats and mice, TNG feed mixturewas prepared weekly. The feed wap stored in room temperature. The animalfeeders were refilled after the 4th day and emptied completely after the 7thday before the freshly prepared TNG-feed mixture was used. The TNG intake

':of all animals was corrected for evaporation loss under the normal feeding3 condition. Amounts of TNG remaining in the feed mixture daily for 7 days wereobtained from the decay graph. The average was found to be 93% which was used

45

to correct the evaporation loss of TNG in all feed4,ng studies for rats andmice.

b. General Observation and Weight Gain

"The control rats and rats fed various levels of TNG in the feedwere healthy throaghout the experimental periods of 4 or 13 weeks. The body-eights of the male and female rats before, during and after treatment are.summarized in Tables 27 and 28, respectively. The weight changes of theserats are bctter illustrated in Figture 2.

The wei.ght gains of the male rats fed the lew and middle levelsof TN, were comparable to that of the controls. However, the weight gain ofthe rats receiving the high level of TNG was significatntly depressed after4 weeks. W'hen the feeding of TNG was discontinued after th- 4th or l3t'hweeks, the weight gain of these rat', increased and was greater than theweight gain of the control rats. By the end of the 8th or 17th week, thebody weights of these male rats were cowparable to, sr were only slightly3maller, than that of the controls.

As seen in the male rars, the high level of TN% In the feedalso depressed the weight gain of the female rats after 4 weeks. However,this depressed effect on the weight gain in thesE females was only slight.After TNG feeding was discontinued, the weight gain of these rate alsoincreased.

"Cc Feed Consumption ard TNG Intake

Feed consumption of the rats fed various levels of TNG )re

summarized ir Table 29. The male rats fed the low or middle level of TNG Iconsumed comparable amounts of feed as the controls; whlereas the males fedthe high level of TNC consusmed less feed between 5 and 13 weeks. In thefemale rats, the high level of TNG also decreased the feed consumption.

The TNG intak3 of the rats are summarized in Table 30. TheMaG inake was corrected for evaporat!on loss as discussed in SectionI,.A.3.a. Th.. TNG intake o• the male rats fed 0.001, 0.01 or 0.1% TNG inteed during the first 5 weeks averaged 0.8. 6.0 or 59.0 mg/kg/day, respec-tivley. !iheti the rNG conce':tratio is in the feed were inct'eased 5-foldstarting the 6th week, the TNG intake of these rats increasd to 2.5, 24.5or 229.5 mg/kgsday, respecrively. The TNG intake of the female rats fed

the low, middle or high levels of TNG was comparable to that of the malerats. The intake averaged 0.9, 6.4 or 59.3 mg/kg/day, respectively, dur-ing the first 5 weeks; and averaged 1.1, 26.5 or 233.8 mg/kg/da-,, respec-

tively, during the subsequent 8 weeks.

46

~~1 ii

d. Blood AnalyseAs

The hematology results of the control male rats and male ratsfed various levels of TNG are summaized in Tables 31 through 34. The peripher'alblood elements of these male rats were not apparently altere2d by TNG feedingfor 13 weeks. However, when comrared with the baseline level3 witnin thegroups or when compared with the control rats at the respective time tntervals,there were a number of changes. The increases In erythrocyte count, bamatocrit,hemoglobin concentration and/or the decrease in reticuloycte count in boththe control and treated rats were related to the increase in age of theserats. The other changes were slight and inconslsL':nt. The clinical blood

I chemistry values of the control male rats and male rats fed the high level ofTNG are summarized in Table 35. Two rats (Nos. 181 aivd 183) fed TNG for 1.3

weeks had elevated SGOT of 220 or 415 IU/L, respectively. After allowed torecover for 4 weeks, the SGOT level of all four rats was not significantlydifferent from that of the control u.ales.

The hematology results of the control f...male rats and the

female rats fed various levels of TNG are summarized in Tables 36 through 39.As seen in the male rats, the peripheral blood eleiiients of the female ratswere not apparently altered 1'y TNG feeding for 13 weeks. When comparedwith the baseline levels cr when compared with the control rats at therespective time Intei 'als, a number of parametera were stgnificantly altered.These changes were not related to TNG treatment. The clinical blood chemis-try values of these female rats ar. summarized in Table 40. Two rats

(Nos. 280 and 281) fed TNG for 13 weeks had elevated SGOT of 204 or 291 IU/L,respectively. As for the males, the SGOT level of all four female rats wasnot significantly different from that of the control femaleF after they were

allowed to recover for 4 weeks.

e. Organ Weights

The organ weights of the rats fed 'ar-.ous leqcls of TNG for

4 and 13 weeks and for 13 weeks plus 4 weeks of recovery are summaiized inTables 41, 42 and 43, respectively. After 4 weeks, the liver weight of the

female rats fed the middle level of TNG and the kidney w.ight of th". Pemale ratsfed the low or high levels of TNG were larger than those of the cotitrol retA(Table 41). Based on the body weight, the liver weight of these rats fedthe high level of TMG and the kidney weight of these rats fed the low level ofTNG were larger than those of the control vats.

After 13 weeks, the adrunal weight of the male rats fed thehigh level of TNG, the thyroids weight of the male rats fed all le"qls of TNG,and the liver weight of the female rats fed the high level of TNG ware smallerthan those of the respective control rats (Table 42). Based on the bodywelght, the liver weight of male rats fed the lew level of TNG, the thyroidweLght of the ua, 4' rait:s fed n I levels of "•"[,. and the liver weight of Oh.

47

female rats fed the low level of T14G were smaller than those of therespective control eats. On the other hand, the liver weight of both themale aad the female rats fed the high level of TNG, the testis jeight ofthe male raks fed the high level of TNG and the kidney weight of tnefemale rats fed the high level of TNG were larger than those of therespective control rate.

After treatment for 13 weeks and recovery for 4 weeks, theheart weight of the male rats fed the low or middle level of TNC werelarger than that of the control rats, and the liver and kiduey weightsof the male rats fed high level of TNG were smaller than those cf thecontrol rats (Table 43). Based on the body weight, the heart weights ofthe male rats fed the low or middle level of TNG, the liver weight of themale rats fed the middle level of TNG, and the heart weight of the femalerats fed the middle level of TNG weze larger than those of the respectivecontrol rats.

f. Gross and Microscopic Examination of Tissues

The rats fed various levels of TfG were in good nitritionalcondition at various times of necropsy. Microscopic examination of tissuesrevealed a number of lesions in both the control rats and rats fed TNG.

After TNG feeding for 4 weeks, spontaneous lesions occurred inthE control rats and rats fed the high level of TNG in both sexes (Tables 44 and45). These lesions included lymphoid hyperplasia and/or pneumonia In thelung, portal inflammation or necrosis of the hepatic cells in the liver, orextramedullary hematopoiesis in the spleen. The bone marrows and the M/Eratios of these rats were normal.

After feeding for 13 weeks, a number of spontaveous lesions

alco occurred in the heart, lung, liver and spleen of control rat& and ratsfed high level of TNG (Tables 46 end 47). One treated male rat also had mildacute inflammation in the pancreas. The bone marrowa and the M/E ratios ofthese rats were noriwal.

Tissue lesions of inale and female rats fed TNG for 13 weeksand allowed to recover for 4 weeks are summarized in Tables 48 and 49,reapectively. As seen in the control rats and rats fed TNG for 4 or 13 weeks,n ni.itber of occasional lesions were also seen in the hea:rt, lung, liver,

Stciictn and/or kJdney pf. bot'i tht control rat-s and the rats Ced the highlevel olp TN(G for 13 weeks and allowed to recover foc 4 weeks. These lesions

w ere tiaturally-occu.-rIng alid were noc related to TNG. The bone martows andthe M/E 1 iatlos of these rats were normal.

48 -.

I!

1i 4. Discussion and Conclusion

The TNG intake of the male rats fed the low, middle or highlevel of TNG in feed averaged 0.8, 6.0 or 59.0 mg/kg/day, respectively,during the first 5 weeks; and 2.6, 24.5 cr 229.5 mg/kg/day, respectively,

during the additional 8 weeks. The TNG intake of the female ratsaveraged 0.9, 6.4 or 59.3 mg/kg/day during the first 5 weeks and 3.1,26.5 or 233.8 mg/kg/day during the additional 8 weeks.

1 3ý-th the male and the female rats fed the low or the middle].'.el of TNG did not show any adverse signs, any changes in peripheralblood elements or clinic,,l laboratory testis, or lesions in any tissues

related to TNG. The high level of TNG in :Ieed decreased the feed con-sumption and retarded the weight gain of rats. These effects were moreprofound in the males than in the females and were reversible after

discontinuation of TNG feeding for 4 weeks., High level TNG for 13 weeksalso caused alevation of SGOT in some rats. However, these rats did not haveany lesions in any organs. After allowed to recover for 4 weeks, the SGOTlevel of all rats was not significantly different from that of the control rats.

The absolute and/or relative weights of heart, liver, kidney, thyroidand/or testis of the male and/or female rats fed TNG for 4 or 13 weeks weresignificantly different from those of the respective control rats. However,

V these changes were not related to TNG feeding and were not considered to beL, clinically significant. First, the changes were slight and inconsistent.

Second, these organs did not have any consistent lesions related to TNGfeeding.

I.

B. Additional Subchronic Toxicity

1. Introduction

As discussed in Section II.A.4., feeding of 0.5% TNG for 13 weeksdepressed feea consumption and weight gain in rats. it is imperative toestablish a dose-response for the adverse effects of TNG. In addition,the 0.5% TNG diet contained 4.5% lactose. The question arose whetherlactose in the diet had any adverse effects. The objectives of these

U" experiments were 2-fold. First, the TNG concentration in the diet wasincreased to 2.5% and the effects of feeding this diet for 13 weeks werestudied. Second, the effects of feeding 25% laptose for 13 weeks were

I: compared.

4

1 4


In the first experiment, a total of 7 male and 7 female young healthyCDO rats (Charles River Breeding Lab.) were used. Four males and fourfemales, serving as controls, were fed a powdered standard rodent chow(Wayne Laboratory Meal); three males and three females were fed 2.5% TI4Gin the feed. The preparation of the diet containing 2.5% TNG was similar

to that for rats described in Section II.A.2.c. The 10% TNIG lactose mixturewas mixed with powdered standard chow at 25% by weight to yield the diet con-taining the desired 2.5% TNG every week. Feed consumption of all rats weremeasured throughout the experiments and body weights were recorded weekly.At the end of 13 weeks of feeding, arterial blood (abdominal aorta) ofeach rat was collected under ether anesthesia for hematolugy, clinicalblood chemistry and measurement of serum electrolytes. Heart, liver, spleenand kidneys were weighed; various tissues were removed for histopathological

examination.

In the second experiment, a total of 10 males and 10 females wereused. The experimental design and procedure were similar to those describedf or the first experiment. Five males and five females, serving as controls,were fed the powdered s andard chow; five males and five females were fed 25%of lactose in the feed. Lactose (USP) was mixed with the powdered standard .

chow at 25% by weight. In addition to the parameters studied in the firstIi experiment, calcium content in the femur bone and iron content in the liverwere determined at termination.

The femur was weighed, digested in concentrated nitric acid andanalyzed for calcium with a Varian Model 6 atomic absorption spectrophotom-eter using the resonance line 2399 X. A nitrous oxide-acetylene flame wasused to eliminate any interference from phosphate. The liver was weighed,digested in concentrated nitric acid with the addition of 70% perchloric -

acid and analyzed for iron with the same spectrophotometer. An air-acetylene flame was used.

3. Results

a. Rats Fed 2.5% TNG

General observations, body weight, feed consumption andTNG intake: The body weights, feed consumption and TNG intake of the controlrats and rats fed 2.5% TNG are summarized in Table 50. Both the male and thefemale rats fed TNG lost weight rapidly during the first 4 weeks and continuedto lose weight throuigh the 8th week. During this period, these rats wereslightly less active, appeared in poor nutritional condition and had roughhair coat without any other adverse signs. Thereafter, the condition ofthese rats improved and they started to gain weight. By the 13th week, they

50

II

regained che lost weight. On the other hand, the male and female controlsappeared heaithy and persistently gained weight throughout the experiment.

I The weight changes of the control and treated rats are better illustrated inFigure 3.

Both the male and female rats fed 2.5% TNG consumed about

one-half or less of the feed as the control group during the first 8 weeks.

Thereafter, their feed consumption increased. This decrease and change in

feed conswmption of these rats correlated with their weight loss or the re-gained weight. The TNG 1Lit-ko :of the male rats averaged 1,406 mg/kg/dayand of the female ratb ,,vei:,•Y 1,416 mg/kg/day. As discussed in SectionlI.A.3.a., the TNG incake- corrected by a factor of 93X for the evapora-

tion loss in the fd.er during the feeding week.

Blood analysis: The results of hematology and clinical

blood chemistry tests are summarized in Table 51. Both the male and the

female rats fed TNG had increases in erythrocyte count, hematocrit and hemo-globin concentration. In addition, the fasting blood glucose of these ratsdecreased and the alkaline phosphatase increased as compared with those ofthe respective controls. In the males, the leukocyte count also decreased.I However, the decrease in leukocyte counts were not considered clinicallysignificant. Methemoglobin was not detected in the blood of any male or

•iIr female rats. iiSerum electrolytes of the control rats and rats fed TNG

.are summarized in Table 52. Concentrations of Na, K, Ca, Mg and Cl of ratsfed 2.5% TNG for 13 weeks were not significantly altered.

Oren weights: The absolute and relative weights ofa, various organs are summarized in Table 53. The absolute weights of brain,

heart, liver aud kidneys of the male rats fed 2.5% TNG for 13 weeks weresmaller than those of the control males. The relative weights of brain,kidneys and spleen based on the body weight were larger than those of thecontrols, and the relative heart weight based on the brain weight was

i •osmaller than those of the controls.

The absolute weights of spleen and kidneys ef the femalerats fed 2.5% TNG were larger than those of control females. The relative

weights of both organs, based on -he body -,eight or ti- brain weight, werealso larger than those of the controlR.

Gross and microscopic examination of tisnoesl Thecontrol rats and rats fed 2.5% TNG were in good nutritional condition at timeof necropsy. Tissue lesions in male and female rats are summarized inTables 54 and 55. respectively. The males fed 2.5% TNG had modecate to•svere tosticuhir atrophy, mild to moderate testicular depeneration, and1 severe aspermatogenesiq In the teste•s. In addition, there were hemoalderosls

51

in the spleen and liver. A nunber oO spontaneous lesions also occasionallyoccurred in the male co.itrols and/or the TNG treated males. These lesionsincluded myocarditis, lymphoid hyperplasia and pneumonia in the lung, sub-acute inflammation in the liver, glomervlonaphritis or pyelonephritis inthe kidney, and/or vacuolar degeneration in the adrenal gland. The bonemarrows and the M/E ratios of these males were normal.

The female rats fed 2.5% TNG had mild to severe hemosid-erosis in the spleen and mild hemositerosis or bile duct proliferationin the liver. As seer in the males, a number of spontaneous lesions occasionallyoccurred in the female controls and TNG treated females. These lessons in-eluded lymphoid hyperplasia, pneumonia and/or emphysema in vhe lang, subacuteinflammation in the liver, interotitial nephritis in the kidney and/orkeratosis, edema and Inflammation of the stomach. The bone marrows and theMWE ratios of these females were normal.

b. Rats Fed 25% Lactose

General observation, body weight and feed consumption: Thecontrol rats and rats fed 25% lactose for 13 weeks appeared healthy throughout -,

the experimcnt. The body weights and feed consumption of the rats are summarizedin Table 56. Both the male and the female rats fed lactose persistently gainedweight. Their weight gains, as better illustrated in Figure 3, were comparableto the male and female controls,, respectively. Feed consumption of the ratsfed lactose was also comparable to that of the controls.

Blood analysis: The resulte of hemdtology and clinicalblood chemistry tests are sunmiarized in Table 57. The peripheral blood ele-mentr and various clinical chemistry values of both the male and female ratsfed 25% lactose for 13 weeks were not apparently altered as compared withthose of the respective control rats. Methemoglobin was not found in any rats.The mean corpusular hemoglobin concentration of the female rats fed lactosewao slightly but significantly less tVan that of the female controls. Thischange was not considered clinically significant.

Serum electrolytes of these rats are summarized inTable 58. Serum levels of Na, K, Ca, 1ig and Cl of rats fed 25% lactosefor 13 weeks were not significantly 41tered.

Calcium content in the bone and iron content in theliver of these rats are summarized in Table 59. These levels in the ratsfed lactose were not significantly different from those of the control zats.

52

IIOrgan weih~ts: Tie absolute and relative weightu, of

varc.,s organs are summarized in Table 60. The absolute and relativeweights of zaecum, based on body weight or brain weight, of rats fed 25%lactose for 13 weeks were significantly larger than those of the controlrarp. The other organ weights of rats fed lac..tose were not apparently

* altered.

Gross ard microscopic examination of tissues: At ter-3 umination, both the control rats and the rats fed 25% lactose were in goodnutritional conditicn with normal deposits of tody fat. Tissue lesions inmale and female rats are summarized in Tables 6i and 62, respectively. Anurwber of lesions occasionally occurreJ in these control rats and rats fed

lacttse. These lesions included focal myocarditis of the heart, lymphoidbyperplasia or pneumonia of the lung, inflammation or focal nucroeis of the

liver, and/or interstitial nephritls or mononuclear cell infiltration of thekidney. In addition, one female control had hemosiderosis in the spleen and

iI mesinteric lymph node, and one female rot fed lactose had a fibroadenoma ofthe mammary gland. These lesions were mild to moderate; they were spontan-eoi's and not related to lactose.


i _The male and female rats fed 2.5% ThG for 13 weeks coasunted anaverage of 1,406 or 1,416 mg/kg/day of TNC, respectively. Both the maleIand the fewale rats fed ThG consumed less feed and lost weight quirkly.They continued to lose weight, were siightly less active, and had Loughhair coat through the 8th week. Thereafter, these rats started to gain

weight and their conditions tmprovcd.

These rats fed TNG had some changes in the blood and organ weightaand lesions in the testis, liver and spleen Erythrocyte count, hematocrit,hemoglobin concentration, and serum level of alkaline phosphatase in'creasedand fasting blood glucose decreased in both the male and female rats. TheIerythrocyte count of both the male and the female contrtls were re)atively lowbefore treatment; however, -he 7,alues were within normal limits for rats observed

7 in our laboratory as shown in rabje L in Appendix I. In the males, TNG feedingincreased the relative weights of spleen, kidney and brain based on the body

weight and decreased the relative heart weight based on the brain weight. Inthe females, TNG feeding increased the relative weights of spleen and kidneybased on the body weight and 'ncreaaed the relatIve kidney weight basedon the brain weight. TNG caused testicular atrophy and degeneeration, andaspermatogenesis in the males. Both the males and the females fed TNG hadhemosiderosis in the spleen and/or the liver. Hemosideroass in these organsis often seen in rodents. However, it was not seen in these control tatsor mice used for these studies.

53

The male and female rnts fed 25% lactose for 13 weeks did notshow any adverse signs, any ihanges in the peripheral blood elements orclinical blood chemistry values. any effects on calcium content of the boneor iron :ontent of the liver, or any lesions related to lactose. However,lactose feeding increased the weights of caecum of both the ,ale and thefemale rats.

C. Cytogenezic and Mutagenic Effects of TNG

I introduction

The cytogenetic effect of TNC or) somatic cell chromosomes was studied.The lynwhocyte and kidney cultures from rats fed TNG were obtained and examinedfor any damages. In addition, the capability of TNG to induce single genemutations was studied in Thinese hamster ovary cells in vitro. The Chinesehamster ovary system is capable of detecting mutations induced in nine dif-ferent specific locL simutaneously.


a. Cytogenetic Effects on Chromosomes

The procedure on the cytogenetic study described for dogs inSection I.C.2. was used. Peripheral lymphocyte and kidney cultures wereobtained from rats fed the nigh level TWG for 4 and 13 weeks. The male ratswere fed an average of 59.0 mg/kg/day TNG during the first 5 weeks and 229.5mg/kg/day during the additional 8 weeks; the female rats were fed an averageof 59.3 and 233.8 mg/kg/day for the respective periods. Accordingly, thelymphocytes or kidney cultures were activated, arrested, processed, stainedand examined for both numerical and morphological aberrations of the chromo-somes.

b. Mutagenic Effects on CHO-KL Cells In Vitro

Wild type Chinese Hamster Ovary (CHO-KI) cells!-/ capable ofgrowth In both a minimal and an enriched medium were exposed to selectedconcentrations of TNG to test its ability to induce single gene nmutations inmammalian somatic cells. The concentrations used were selected from a singlecell survival curve obtained according to the method of Puck and Kao..12/ Po-tential 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 thecapability of growth only in the enriched medium and net in the minimal medium.Mutagenesis was measured relative to the known mutagen ethyl methanesulfonate.-3/

54

3 3. Results

The results on numerical ana morphological aberrations of chromo-somes are shown in Tables 63 and 64, resp%;tctively. Rats fed TNC for 4 and

ii 13 weeks did not show any changes in the chromosome frequency distributionor number of tetraploids, or any chanes in the chromatid breaks or trans-

SI locations, in the peripheral lymphocytes or kidney cultures.

The results of the in vitro single gene mutation study is shownin Table 65. No mutants were found in the cultures treated with TNG at

,l concentrations which killed 65 and 99% of the cell population, respectively.Ethyl methanesulfonate, on the other hand, induced mutants at the frequency3of 28.0 x 10-6 which is similar to that reported by Kao and Puck.--/

4. Discussion and Conclusion

SI An average of 59.0 to 59.3 mg/kg/day of TNG in feed for 5 weeksand 229.5 to 233.8 mg/kg/day for an additional 8 weeks to rats did not causeany numerical or morphological aberrations of chromosomes in the peripherallymphocytes or kidney cultures.

Treatment of Chinese hamster ovary cells with TNG at concentrationswhich killed 65% and 99% of the population did not appear to induce anymutations. However, since this test system does not incorporate a metabolicactivating system, no information on the mutagenicity of metabolites of TNGwas obtained.

D. Immunologic Response to TNC

1. Introduction

Immunoglobin E (IgE), the allergic or hypersensitive antibody, washssociated with anaphylactic reactions in h~unan.-2 Serum concentration of IgE

of rats treated with TNG was determined.

2. Material and Method

As described for the dogs in Section I.D.2., the immunodiffusiontechnique of ManciniP.0 was used to determine tbe serum IgE of rats fed 2.5%TNG for 13 weeks. These rats were used for the subchroniL toxicity studydescribed in Section II.B.

3. Results and Conclusion

3 Serum concentrations of IgE of control rats and rats fed 2.5% TNGfor 13 weeks are summarized in Table 66. TNG did not apparently alter theserum concentration of !gE. The TNG intake of the male rats averaged 1,406mg/kg/day and in the female rat averaged 1,416 mg/kg/day.

55

TABLE 26

STABILITY OF TNG IN FEED MIXTURE FOR RAT?

Feed Mixture No, 1

Sample %. NG %. Remaining

1. Time zero-fresh 0.l' 100

2. Time zero-frozen 0.41 100

plus 8 daysI3. 4 days in feeder 0.39 954. 4 days in capped can 0.41 1005. Sample No. 4 after 0.36 88

4 days in feeder

6. 8 days in capped can 0.38 93

Feed Mixture No. 2

1. Time zero-fresh 0.042 100

2. Time zero-frozen plus 0.042 100

* 8 days

3. 4 days in feeder 0.040 95I4. 4 days in capped can 0.038 905. Sample No. 4 after 0.031 744

4 days in feeder

6. 8 days in capped can 0.036 86

a/ Average of duplicate samples.

56

TA BLF 27

BODY WEIGHTS OF MALE RATS FEL TNG

%/. TNG Body Weights (gm)I

in FeedcO Initiai 4 ',ý.eks 8 Weeks 13 Weeks 17 Weeks

0 278+9 441+10

0.001-0.005 285+7 458+230.01-0.05 278+9 423+210.1-0.5 279+6 417+1

0 276+10 434+9-S 114+16I 0.001-0.005 289+6 458+16C 553+21

0.01-0.05 267+7 4 107 6 -S-, 495+10- 422- lC,

0.1-0.5 290+6 422+U 544+15

0 270+7 446+20 532+27 555+220.001-0.005 275+5 428+11 500+_15 560+16

0.01-0.05 276+9 429+10 509+18 552+280.1-0.5 267+12 394+14 434+18 440+18

0 288+9 462+10 542+18 603+20 642+210.001-0.005 274+9 441+17 522+21 571+27S- 617+32

I0.01-0.05 281+111 427.+14 501+20 564T18.c/ 621+32

10.1-0.5 276+3 406+6 443+5 4 76+ 3 -Y 576+7

a/ TNG concentrations in the feed were increased 5-fold starting the6th week.

b/ Mean + S.E. of four rats.

c/ TNG in feed discontinued thereafter.

-5

1 57

TABT E 28

BODY WEIGHTCS OF FEMALE RATS FED TNG

% TNG Body Weights (gm)in Feede/ Initial 4 WeeKs 8 Weeks 13 Weeks 17 Weeks

0 188+4b-/ 244+70.001-0.005 185+6 242+7

0.01-0.05 199+5 267+40,1-0.5 191+6 226+1

0 185+3 243+7C• 272+70.001-0.005 186+6 248+4 279+4

0.01-0.05 198+4 298+10-/ 283+120.1-0.5 204+9 251+11-C 316+5

0 195+7 268+9 266+18 299+110.001-0.005 198+3 268+7 298+8 316+70.01-0.05 193+6 252+6 27544 291+50.1-0.5 196+6 238+7 257+10 251+9

c/0 190+4 257+9 291+9 308+9--, 328+16

0.001-0.005 186+4 251+7 281+6 305+8 i/ 342+130.01-0.05 187+4 253+10 273+9 294+12-, 329+130.1-0.5 191+7 249+9 261+12 272+12-- 315+12

a/ TNG concentrations it the feed were increased 5-fold startingthe 6th week.

b/ Mean + S.E. of four rats.c/ TNG in feed discontinued thereafter.

58

I

TABLE 29

AVERAGE FEED CONSUMPTION (gm/day/rat) OF RATS FED TNGI% TNG Males

I In Feed-S/ 1- Z7_ 5-8 9-13

0 28.4 26.9 26.50.001-0.005 28.0 26.5 27.50.01-0.05 26.4 26.9 26.30.1-0.5 27.1 22.6 22.6

%• TNG Females

In Feeda/ 1- 5-8 9-13

0 19.4 19.4 17.61

0.001-0.005 18.0 17.4 17.6

0.01-0.05 18.3 16.0 1.5.7

0.1-0.5 17.5 13.7 14.3

a/ TNG concentrations in the feed were increased 5-fold starting the

S~6th week.

i b/ Weeks.

I.

ill

TA BBLE 30

AVERAGE TNG INTAKE (mg/1kg/day) OF RATS DURING TREATMENT

% TNG Masles .

In Feed Lb/ 4 8 13

0.001-0.005 0.9 0.7 2.7 2.5

O.G1-0.05 6.5 5.4 22.6 20.6

0.1-0.5 64.1 54.0 207.9 196.8

% TNG FemalesIn FeedA-/ 1 4 8 13

0.001-0.005 0.9 0.8 3.2 2.9

0.01-0.05 6.7 6.0 23.1 23.60.1-0.7 56.1 62.5 228.9 183.5

a/ TNO concentrations in the feed were increased 3-foldstarting the 6th week.

b/ Weeks.

60

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63

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644

- TABLE 35

THE CLINICAL BLOOD CRE4I.STRY DATA OF CONTROLMALE RATS AND MALE RATS FED HIG.H LEVEL TNG

II Alkaline% TNG Fasting Glucose SOOT SGPT Phosphatase BUN

in Feed/ MR %) L,,LL (IU/L (IUL)mg

Fed for 13 Weeks

Control 144 4 6h/ 126 3- 10 36 6 53 k 2 110.1 - 0.5 92 ± 21 262 4: 79 48 ± 9 75 + 17 18 ± 3

Fed for 13 Weeks and Allowed to Recover for 4 Week!

Control 172 d: 27 96 6 28:± 1 44 3 18 20.1 - 0.5 138 A: 8 131 37 41 ± 11 44 1 44 1

I a/ TNG concentration was increased 5-fold starting the 6th week.b/ Mean t S.E.

0I1I •

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7 ., CUi OD m v 0 10 01 C C) c.-I

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Lnz1~ n

40) +1 + 1 + 1A

rDU4 en r-

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c; c9. ;

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~ 006

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4.L

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ul 0 6.01LIN .- 0 7 4 4 7 A C C A

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x I U 1- j 7' m- 2 7 'm 0 4. 0Ij r m03 O 0urid * * * * * * * S * * * S 67

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- 14 * 0 'X b9' ~ 4 . 4 1 4 1

TABLE 40

THE CLINICAL BLOOD CHEMISTRY DATA OF CONTROL FLEM

RATS AND FEMALE RATS FED HIGH LEVEL_ TN

Alkaline

7% TNG Fasting Glucose SGOT SGPT Phosphata~e BUN

ina Feeda-/ (Talg %/. (IU/L) (I jL (mg %.

Fed for 13 Weeks

Control 107 ± 3 / 155 31 51 ±20 29 ±1 10 ±30.1 - 0.5 87 4 248 44 36 ±2 29 -t 2 14 2

Fed for 13 Weeks and Allowed to Recover for 4 Weeks

Control 163 16 111 19 29 1 38 h 2 16± 20.1 - 0.5 131 + 4 104 ± 13 30 1 1 32 + 2 18 1 1

a/ TNG concentration was increased 5-fold starting the 6th week.

b/ Mean t S.E.

70L -~- *~~ -~ -

T ABLE 41

ABSOLUTE AND RELATIVE 01AWI WEIGHTSOF RATS FMI TNG FOR 4 WEWS

Ift, TNG Absolute Organ Weight

Sex In Feed Terminal Live Kidney S•leen

I Male Control 441 + lOA/ 18.2 + 1.6 1.74 + 1.05 0.77 + 0.040.001 458 + 23 19.4 + 1.4 1.88 + 0.11 0.80 + 0.100.01 423 + 22 19.0 + 1.8 1.72 _+ 0.11 0.78 + 0.090.1 408 + 9 19.0 + 1.7 1.53 + 0.13 0.95 + 0.11

- Female Contcol 244 + 7 8.6 + 0.3 0.78 + 0.05 0.54 + 0.040.001 242 + 7 8.8 + 0.3 0.98 + 0.05b/ 0.51 + 0.030.01 267 + 4 9.9 + 0.L/ 1.05 + 0.05b/ 0.59 ± 0.04

- 0.1 226 + 1 9.3 + 0.t 0.80 + 0.03 0.51 + 0.03

7. TNG Relative Organ Weights (m/Ilggm Body Weight)

Sex In Feed Liver Kidney Spleen

Male Control 4.1 + 0.3 0.40 + 0.01 0.17 + 0.010.001 4.2 + 0.2 0.41 + 0.01 0.18 + 0.020.01 4.5 + 0.3 0.41 + 0.03 0.18 + 0.020.1 4.6 + 0.3 0.37 + 0.02 0.23 + 0.02

Female Control 3.5 + 0.2 0.32 0.03 0.22 + 0.010.001 3.6 ± 0.3 0.41 + 0.02./ 0.21 + 0.020.01 3.7 + 0.6 0.39 + 0.01 0.22 + 0.020.1 4.1 + 0.06k/ 0.35 + 0.01 0.23 + 0.01

I a/ Mean + S.E.

.L/ Signflficant ly dtiferent from t-he (-.oitrol rat *. (i)uinett's multipleI comparisoni provedure-/)

71

U,

0o 1 n' ffc4 -i --t C' C'J r-4w- +1 +1+1 +1 +1 +i+1 +1

U, ) -'- 000 '-40000 "

411++ p11 + +1+ 1 +1+ 1+

0 14(Iu ,t~ 0% tor-4 to Lnc 00HL 000 to U) (4e

0) 4+1+1 +1 +1 +1 +1+1 +1 O:TH rý 00-i m ~ *J ON 0, 0OI l l 41; 0 699a

CA HcJn (A,- V

tma ~ ~~ ~ ~ ~ 0 -L)m C4 4Nr4- 0 H- 04 0 0 04 0ý

44) cn 0 % D ( ) 1+ 1 +1 +1 +1 +12

0 +1 +1 +1 +1 +1 +1 +1 +1 H0 s2 H ' N '

Pd0 . . . 00 0)00

4J~~C 0 0 - C C OCHH00 0 00 0 r4J *

0o +21 +1 +1+ +1++ 00001 00001U00,11~~~21++ +12++ +1+ 1 + + 1+ t0 f n'Zrr

- ' r4 0 A J kC1 ~ r-- cn Co a)

000 00 0 C) 0 4

C ~ 5- 4 1- 1r 0 2-

HO, C> H 000 0 ) - 0IT C4 (%2 I - - %- -r! *

:3 1 +1 +1 +1 +1+1 +21 +t > - r4 1414141 -41 14 14] %DV2-." 0 00 m0 00 -r00 r) 0I0 000n

in- He' OD r, 00 r- J-%J m O ; ;C 8

H-4CO) ,- 000 C11 C1 2 11-41 H ~ ~ 4 ' H % -

:4>O 000 14C0 C-4 CD c04 C, r-A H4 0 0 0 0 0 0 0 C 4-a

0) +1 +, +1 +'J H1 +1 +14 H1 co ifl 0.$a 2 2 2 - 2 24>- -ti +2 +2 C+ 4 +: +2 , + D +2 4 +1 +1 +1 +.0 +1' +0 +1 +1H ~

,'1~ 0) LI'OH , -4 'U 04 4 I .a-c C'c C'4C' P(ýH o00a00 000 %22 ia-W

0~~ 0w r

-- "-a

TABLE 43

I ABSOLUTE AND REUATIVE ORGAN WEIGHTS OF RATS FED TNCFOR 13 WEEKS AND ALLOWFD TO RECOVER FOR 4 WEKS

I Terminal% TN(G Body Absolute Oragn Weight (gPi)

sex In Feed/ Weight Heart Liver Spleen Kidneys

Male Control 642+21-b/ 0.96+0.06 18.3040.98 0.724+0.08 4.43+0.300.001-0.005 616+25 1.36+0.06-S/ 19.47+0.87 1.01+0.11 3.95+0.32"0.01-0.05 615+31 1.29+0.05R/ 20.4640.90 1.07+0.03 4.06+0.040.1-0.5 576+6 0.85+0.11 1 4 . 2 9+0. 6 3 cS/0 . 8 1. 0 . 1 5 2.91+0.26-./

Female Control 328+16 0.90+0.04 8.30_0.98 0.90Q0.05 1.84+0.110.001-0.005 341+13 1.00+0.02 10.23+0.95 0.86+0.09 2.04+0.41

0.01-0.05 319+18 1.02+0.05 8.75+2.13 0.86+0.08 1.86+0.20"0.1-0.5 315+12 0.96+0.07 8.33_+0.34 0.89+0.06 1.66+0.18

% ING Relative Organ Weights (gm/lOOgm Body Weight)Sex In Feed Heart Liver Splee Kidneys

SMale Control 0.15+0.01 2.85+0.13 0.1140.01 0.70+0.070.001-0.005 0.22+0.00]/ 3.16+0.07, 0.1640.02 0.65+0.07

0.01-0.05 0.21+0.01S/ 3.34O+0.15-/ 0.1840.01 0.66+0.030.1-0.5 0.15+0.02 2.65+0.06 0.14-+0.03 0.75+0.05

Female Control 0.27+0.00 2.77+0.12 0.274-0.01 0.57+0.050.001-0.005 0.30+0.01 2.99+0.17 0.25+0.02 0.59+0.130.01-0.05 0.32+0.01./ 3.21+0.27 0.27+0.02 0.5840.060.1-0.5 0.30_,u.01 2.65+0.06 0.28+0.04 0.53+0.05

_8 a/ TNG concentrations were increased 5-fold starting the 6th week.

b/ Mean + S.E.c/ Significantly differeut from the control rats (Dunnett's multiple

compirison procedure4/).

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79

TABLE 50

BODY WEIGHTS (GM) OF RATS FED 2.5% TNG

Treatment Weeks

Sex Treatmenta/ 0 4 8 13

Male Control 316+9kb/ 454±10 525±7 584±5 dTNC 306±7 233±3S/ 212±8_d 322,3

Female Control 185±5 226±6 246±8 252±10TNG 202±7 148±5!/ 134±5-dd 202±9A/

FEED CONSUMPTION (GM/RAT/DAY) OF RATS FED 2.5% TNG

Treatment Weeks0-1 2-4 5-8 9-13

Male Control 28.8c/ 28.7 31.0 30.5TNG 12.4 12.8 12.2 22.0

Female Control 14.7 14.8 15.2 14.7

TNG 7.5 7.4 7.1 15.4

TNG INTAKE (MG/KG/DAY) OF RATS FED 2.5% TNG

Treatment. Weeks

0-1_ 2-4 5-8 9-13 Ave-rage

Male TNG 1176S/ 1277 1339 1588 1406

Female 171(11 1076 1163 1252 1773 1416

a/ Four rats for control groups and three rats for TNG groups,

b/ Mean ± S.E.c/ Meand/ Sign [f icant ly different from tho (cont rols (student "t" tst)

80 JAq

I

IZ

IL

a00

• -6

t,

- " -a

+31

C~~~~~ N ' 9 9-

a -

.1N81

TABLE 52

SERUM ELECTROLYTES OF RATS FED 2.57. TNG FOR 13 WEEKS

Serum Electrolytes (m.eq/L)Sex Treatment!/ Na K Ca Cl

Male Control 14 2+,1/ 4.9+0.1 4.8+0.0 1.74+0.0 102+1

TNG 149+5 5.5±0.5 5.0+0.2 2.2+0.3 100+3

Female Control 146+1 5.6+0.1 5.2+0,1 2.0+0.1 106+2

TNG 147±-1 5.9+0.3 5.14+0.1 2.340.1 101+2

a/ Four rats for control groups and three rats for TNG groups.b/ Mean + S.E. (number of rats).

4

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85

TABLE 56

BODY WEIGHTS (GM) OF RATS FED 25% LACTOSE

Treatment Week ___

Sex Tre2atment-l 0 4 8 13

Male Control 2 62+17b/ 403+17 486+20 524+24

Lactose 270+17 401+10 480+18 510+19

Female Control 210+5 257+5 284+8 294+6Lactose 204+6 269+5 295+9 299+7

FEED CONSUMPTION (GM/RAT/DWY) OF RALTS FED 25% LACTOSE

Treatment WeekSex Treatmenta/ 1 4 8 13

Male Control 22.9c/ 26.1 27.2 24.2Lactose 17.2 28.3 25.9 21.7

Female Control 16.8 18.1 18.0 16.7Lactose 13.9 20.3 18.1 16.3

a/ Fi..e rats per group.b/ Mean + S.E.c!/ ean.

I;I -0

............... .I ... .... .

.1 . 0 01 .1 41 . 1 0 . 1 0 01 01 01 N1 .1 -1 4 1

4~~ N-

on .0 .A .0 .0 .0 0 . 0 . 0 . 0 . 1 .0 *0 1 .0 .1 .0 .1 .1

0 M - 400

f,- tII

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0- N - 0 7 *~

m1 .01

0 u I 0 0 4~

W -I N a a 9 -1 9 n U'A C- M 9

L 0 . 0 . 0 * 0 . 0 . 0 . 0 . 0 . 0 . 0 . 0 .

4.-. 87

TABLE 58

SERUJM ELECTROLYTES OF IW.TS FED 25% LACTOSE FOR 13 WEEKS

Serum Electrolytes (meg/L)Sex Treatmenta/ Na K Ca Cl

Male Control 142-2k/ 6.2+0.1 5.3+0.1 2.5+0.1 97+1Lactose 153+1 5.6+0.1 5.6+0.1 2.4+0.2 101+1

Female Control 151+1 5.2+0.3 5.3+0.1 2.2+0.1 103+1Lactose 149+1 5.2+0.4 5.4+0.1 2.3+0.1 96+1

a/ Five rats per group.bi Mean + S.E.

8I

88

- -.-.--- --- ,-_~ ~~ij

'tf 01!SOM

UII

I TABLE 59

i BONE CALCIUM AND LIVER IRON OF RATS FED 257. LACTOSE

I Bone Ca Liver Fe*Sex Treaulmenta-f (°/.•). (iuaL

Male Control 9.24 + 1.14h/ 151 + 18

Lactose 9.41 T 0.91 158 ± 18

i Female Control 9.06 + 0.64 390 + 28LactLose 8.87 + 0.89 446 + 47

II a/ Five rats per group.

A5b Mean + S.E.

I 89

ll>

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0 -0 14- r-f N u-' P -4~ Q%D 4m

00 0

J ý m C*4 0 00 0 0 00

+_ +14~ 1 1c1 1 + 1+1+ 1+04C

,.S14 CV)u.134 554 V-4 . * .

4.) 0 ; C; 0 0 0)0 4 00 00w. +1 +1 +1 +1 ca > +1 +1 +1 +1 f:~ +1 +1 +19+1 -

r-4 U 00 b C H t- C %D' 00 car 00om c0 0 Itq

r 4 04

to 41'a'a N CNJ~ ,--4e'J .- 0% 400

00 00 0 o 0 C4U4 u ,-r4~ S-P4 0 . .)4

C) 00 00 4. 0 00> w 00 00 rP- ++1 99 . +1+ +19+1 > +1 + +19+9

11 IA %D -4 en t 0% *- 0,-4 '-H4 ,-'a - CI) Uw a O 0ONT% 0 I re) en c'4 Ln mr. 0 c

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0)0 C*4 -4 01%ON en %D %D' 4. -F-4 'N(' *n t4

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to tj. r. uci rIU u uU to 0U u i IU0 mC 0 c 4 0 0( o W 0 toC 0C to kv -

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TABLE t6

SERUM IIE OF RATS FED 2.5%TNG FOR 13 WEEIZ

Sex Treatment-- IjWE (U/mMI)

Males Control 1250 + 4611/WCG 1333 + 44

Females Control 1263 + 24TNG 135(0 + 44

a/ Five rats for control groups and three ratsfor TNO group.

b/ Mean + S.E.

96

650 MALES MA LES

550 A -

E /' * /ES 450 -

350 X

25 / I I . ,

0 4 8 0 4 8 13 17

*- *Control o---o Middle levelA----A Low level &-A High level

t TNG in feed discontinued

350 FEMALES FEMALES A

CE

0~0

co15 - - .. I

250

0 4 8 0 4 8 13 17

I VWEEKS OF FEEDING

Figure 2 - liody Weights of Rats Fed Various Level-s of TNC

~1 97

560-

480 /

0 •E

Male -TNG /Male - Lactosej400

0

320(

\\ //

240 // /_\ /

I " ! I I0 4 8 13 0 4 8 13

o--O Control .----- Treated

320

E0

_240-1• •el

I- /

SNG Female- Lactose

0 r

0 4 8 13 0 4 8 13

WEEKS OF FEEDING ,

IFl glirv - IhBdy WeIghts oF Rnts F'ed 2.5% 'I'NG or- 25% 1aetose J,

98__i

I ii

III. MICE

TABLE OF CONTENTS

SOe£A. Subacute and Subchronic Toxicities and Reversibility ........... 101

1. Introduction .I................. ....................... 101

"2. Material and Methods .......... .................. . I.. 101

3. Results ................... .......................... 102

a. General Observation and Weight Gain ........... .... 102

b. Feed Consumption and TNG Intake .................. 102

Sc. Blood Analysis .......... ................. ... 102

d. Organ Weights ......... ................... .... 103

e. Gross and Microscopic Examination of Tissues ..... ... 103

B. Discussion and Conclusions ........ ................... ... 104

C. Other Studies ............... ......................... ... 104

Tables 67 -84 .............. ........................... .... 105

6~ IRit ,

.V

99I

1 99

III. MICEt:i

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 TNG on the biological system at thebiochemical and cellular levels and to elucidate the dose-response relation-ship in the mice fed TNG for 4 and 13 weeks. The reversibility of any ad-verse effects was also studied in mice after the feeding of TNG was dis-conttnuted for 4 weeks.


The basic design and procedure for these experiments in micewere similar to those described for rats in Section II.A.2. with thefollowing exceptions:

a. A total of 64 male and 64 female young healthy albino swissmice (National Laboratory Animlals, O'Fallon, Missouri) were used for thisstudy. They were divided into fcur gvoups, each consisting of 16 malesand 16 females. The average weights of all, groups were kept close. Threegroups of mice were fed 0.001, 0.01, or 0.1% of TNG in powdered standardrodent chow (Wayne Laboratory Meal). At the end of 3 weeks, adverse effects

• were not observed in any mice. Starting the 4th week, TNG concentrations

in feed for all groups were increased 5-fold to 0.005, 0.05 or 0.5%, re-spectively. The 4tb group served as controls and was given the powered standardrodent chow.

b. 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.

d. Since adverse effects were not observed in any mice and TNGdid not cause any lesions in any mice that were terminated at the end of4 weeks, the mice for the reversibility study were not necropsied forexamination at 8 weeks as scheduled.

."•101

. 7

3. Resul ts

a. General Observation and Weight Gain

The control mice and mice fed various levels of TNG were

healthy throughout the experiment. The body weights of the male and female

mice before, during and after treatment are summarized in Tables 67 and 68,

respectively. The weight gains of both the male and the female mice fed -,

the low, middle or high level of TNG were comparable to those of the controls.

b. Feed Consumption and TNG Intake

Feed consumption of the mice fed TNG are summarized int Table

69. Both the male and female mice fed various levels of TNG consumed com-

parable amounts of feed as the controls throughout the experiment.

TNG intake of these mice are summarized in Table 70. The

ING intake of the male mice fed 0.001, 0.01 or v,.1% TNG during the first

3 weeks averaged 1.3, 11.5 or 106.7 mg/kg/day, respectively. When the TNGconcentrations in the feed were increased 5-fold starting the 4th week, the

TNG intake of these mice increased and averaged 6.4, 60.2 or 607.6 mg/kg/

day, respectively. The ING intake of the female mice fed the low, middle

or high level was comparable and averaged 1.3, 10.9 or 94.9 mg/kg/day during

the first 3 weeks, and averaged 6.9, 58.7, or 561.2 mg/kg/day, respectively,during the subsequent 10 weeks.

c. Blood Analysis

The hematology results of the control mice and male mice fed

variois levels of TNG for 4 or 13 weeks, or for 13 weeks and allowed to re-

cover for 4 weeks, are summarized in Tables 71, 72, or 73, respectively.

The peripheral blood elements were not apparently altered by TNG. When com-

pared with the control males, however, there were a few occasional differences

at the various time intervals. These differences were slight and were incon-

sistent.

The hematology results of the control female mice and female

mice fed various levels of IG are summarized in Tables 74 through 76. As

for the males, the peripheral blood elements of the females were not ap-

parently altered by TNG. When compared with the control females, there were

a few occasional differences at the various time intervals. The differences

were slight and were not consistent.

I

1i

3

I d. Orga., Weithts

The organ weights of the mice fed various levels of TNG for& 13 weeks are summarized in Table 77. The absolute and relative spleen

weights, based on the body weight, of the female mice fed TNG were largerI than thoce of the control females. However, due to large individual varia-

tion, the increase in spleen weights was not statistically significant. Ther absolute and relative weights .of other organs of mice fed TNG were not ap-

ST parently altered as compared with those of the control mice.

After feeding for 13 weeks and allowed to recover for 4 weeks,the absolute kidney weight of the female mice fed the middle level of TNG

IL was significantly larger than that of the control mice (Table 78). Basedon the body weight, this difference was not statistically significant.Since absolute kidney weight was not increased in the female mice fed thelow or high level if TNG or in the male mice fed any levels of TNG andsince no lesion was found in the kidneys of these mice, the increase of theabsolute kidney weight was not considered clinically significant and wasnot related to TNG. The slight but not significant increases in the absoluteand relative spleen weight, seen in the female mice fed TNG for 13 weeks,was not apparent in the mice after they were allowed to recover for 4 weeks.

e. Gross and Microscopic Examination of Tissues

At necropsy, the control mice and mice fed various levelsof TNC were in good nutritional condition at various Lime periods. Micro-scopic examination of tissues revealed a number of tissues in both the"control mice and mice fed TNG for 4 weeks. In the males, 3 of 4 controlmice had microfoci of subacute inflammation i'i the liver (Table 79). Onemouse also had a focal chronic perivasculitis of the kidney. Two of Lhe4 male mice fed the high level of TNG had focal chronic interstitialnephritis. in the females, 2 control mice had microfoci of subacuteinflammation in the liver (Table 80). Another control had a chronicinterstitial nephritis and tubular basophilia of the kidney. Three of the4 female mice led the high level of TNG had focal chronic murine pneumonia,microfoci of subacute inflammation in the liver, and/or chronic interstitialnephritis. These occasional lesions were spontaneous and are commonly seenin the mice.

After TNG feeding for 13 weeks, a number of spontaneouslesions also occurred in the control mice and mice fed TNG. In the males,

~ there were microfoci of subacute inflammation in the liver and/or inter-stitial nephritis (Table 81). In the females, there were murine pneumoniawith tzacheitis, microfoci of subacute inflammation and/or pinworms in thelarge intestine (Table 82). In addition, extramedullary hematopoiesis occurredih the liver and spleer) of the male and female mice fed the high and middle

_W levels TN( and in thu spleen of one mate and one female moijse fed the low

S~103

leve) TNG. Bone marrows of the control mice and mice fed high levels of

TNG were normal; the M/E ratios were within normal ranges.

After TNG feeding for 13 weeks and allowed to recover for

4 weeks, a number of spontaneous lesions were also seen in these mice(Tables 83 and 84). These lesions included lymphoid hyperplasia or focal

pnetimonia in the lung, foci of subacute inflamnation in the liver, inter-

stitial nephritis in the kidney, and/or fatty degeneration in the adrenal.

These lesions occurred in the control mice and/or in the mice fed the TNGand allowed to recover for 4 weeks. As seen in the mice fed TNG for 13

weeks, extramedullary hematopoiesis occurred in the liver and spleen of

these male and female mice. The bone marrows of t.he control mice and mice

fed the high level, of TNG, were normal; the M/E ratios were within normal

ranges.

B. Discussion and Conclusions

Both the male and female mice fed the low, middle or high

level of ING did not show any adverse signs, any changes in hematologyor clinical blood chemistry tesLs. The TNG i-taks of the male mice fedthe low, middle and high levels in the feed averaged 1.3, 11.5, or 106.7

mg/kg/day, respectively, during the first 3 weeks; and 6.4, 60.2 or 607.2mg/kg/day, respectively, during the subsequent 10 weeks. The TNG intake

of the female mice averaged 1.3, 10.9 or 94.9 mg/kg/day, respectively,

during the first 3 weeks; and averaged 6.9, 58.7 or 561.2 mg/kg/day,

respectively, during the subseqiient 10 weeks.

The absolute and relative spleen weights of female mice fed TNGfor 13 weeks were larger than those of che control mice. However, the

incceases were not statistically significant. After feeding for 13 weeks

and allowed to recover for 4 weeks, the slight iicreases in the absolute and

relative spleen weights were not apparent. Extramedullary hematopoiesis was

seen in the liver and spleen of mice fed TNG for 13 weeks and for 13 weeksplus recovery for 4 weeks. The significance of this extranmedullary hema-

topoiesis was not clear. First, extramedullary hematopoiesis is often seen

in rodents. Second, the extramedullary hematopoiesis in the liver andspleeii of these mice was mild. Third, there was no dose-relationship inII1h e~xtr inwedmg I I 1t, u op i 's hh i i livs•t, mice i1ed lit, high, nmiddle or low

levels of TNC.

C. Other Studies

Metabolic studies are described in Section IV. Mutagenic and

immunologic studies were not performed in mice.

104

S1i

TABLE 67

BODY WEIGHTS OF MALE MICE FED TNG

7 TNG Body Weights (gin)in FeedaV/ Initial 4 Weeks 8 Weeks 13 Weeks 17 Weeks

i 0 29.3+1.4- 34.0+0.60.001-0.005 26.8+1.5 30.0+1.60.01-0.05 27.5+1.5 31.0+1.60.1-0.5 28.0+1.3 30.340.8

0 26.8+0.8 32.0+0.4--C 33.8+1.00.001-0.005 27.5+0.6 31.5+1.3- 33.3+1.7

0.01-0.05 23.3+1.4 29.371.5S// 28.7+0.90.1-0.5 27.5+0.7 31.8+O. 9-- 33.5+1.2

0 26.0+1.4 31.5+1.6 33.5+1.8 28.8+1.6

0.001-0.005 27.3+1.3 32.0+1.0 33.3+1.7 29.0+1.70.0i-0.03 235.+ 1.0 30.5+1.6 32.. A+0.9 29.3+0.90.1-0.5 25.0+1.2 28.5+0.7 31.0+0.7 28.3+0,8

0 27.8+0.6 29.8+1.0 32.8±1.1 40.5+0.6 36.8+0.3

0.001-0.005 29.0+1.5 32.0+2.0 35.0+2.1 36.3+1.3-- 38.8+1.30.01-0.05 26.5+1.2 27.8+1.8 33.5+1.9 35.81.1-S/ 38.0+1.40.1-0.5 28.0+0.8 30.3+0.5 31.8+0.5 36.3i0.6-S/ 35.5+1.0

I


b/ Mean + S.E. of four mice.

c/ TNG in feed discontinued thereafter.

il 105

S. . .. _ . ... -- - --... ,- .,

TABLE 68

BODY WEIGHTS OF FEMALE MICE FED TNG

7 TNGin Feed!/ Initial 4 Weeks 8 Weeks 13 Weeks 17 Weeks

0 25.8+1.1- 27.3+1.30.001-0.005 23.0+0.4 25,5+0.90.01-0.05 22.87+0.8 21.87+1.00.1-0.5 23.1 1.3 25.3+1.1

0 24.3+0.5 26.8+0.3$ 27.0+0.40.001-0.005 23.5-+1.3 25.5+1.9c5/ 28.5-+.

25.5+1.3 28S 5+1.70.0-0.05 23.3+1.0 25.5+0.C/ 30.5+0.90.i-0.5 22.8+1.2 26.0+0.7- 28.0+1.8

0 23.5+0.7 26.8+0.3 28.5+0.7 26.3+0.60.001-0.005 22.0+0.6 23.3+1.6 27.3+0.8 25.3+1.00.01-0.05 23.3+1.0 25.3+1.J 29.0+2.2 28.0+1.10.1-0.5 25.3+0.8 26,5+0.3 29,5+0.3 27.0+2.0

0 22.5+0.5 24.3+0.6 25.8+0.3 30.3+0.5 27.8+0.90.001-0.005 21.5+0._1 24.3+1.1 26.5+1.0 28.5+0.7cS/ 29.5+1.30.01-0.05 23.8+0.5 25.0+0.7 28.5+1.2 32.0+1.2S/ 31.3+1.4

0.1-0.5 24.5+0.9 26.0+1.1 28.54-0.3 32.3+0.9cS/ 32.0+0.9


b/ Mean + S.E. of four mice.c/ TNG in feed discontinued thereafter.

1 0

106 1

STABLE 69

AVERAGE FEED CONSUMPTION (im/day/mouse) OF MICE FED TNG

7% TNG MalesIn Feed!'/ 1-4 5 5-8 9-13 17th

0 3.8 4.5 4.4 4.10.001-0.005 3.7 4.8 3.7 4.8

0.01-0.05 3.7 4.4 4.3 4.3

0.1-0.5 3.7 4.5 4.1 4.1

In Feede! 1-4 5-8 9-13 17th

0 3.5 3.5 3.5 3.2

0.001-0.005 3.6 4.1 3.3 2.90.01-0.05 3.8 4.6 3.9 4.00.1-U.5 3.4 4.6 3.6 3.6

a/ TNG concentrations in the feed were increased 5-fold startingthe 4th week.

b/ Weeks.

-10

S..

I1 107

I1

TABLE 70

AVERAGE TNG INTAKE (mg/kg/day) of MICEDURING TREATMENT

7. TNG Ms leaIn Feed!l 0-3ŽI 4-8 9-13

0.001-0.005 1.3 7.0 5.70.01-0.05 11.5 54.2 66.20.1-0.5 106.7 580.5 634.7

7% TNGIn Feed!-/ 0-3 4-8 9-13

0.001-G.005 l. 7.6 6.20.01-0.05 19.9 64.7 52.6

M.1-0.5 94.9 642.3 480.1

a/ TNG concentrations in the feci were increased 5-fold starting the

4th week. •b/ Weeks.

i1

" I

i l

108Ai

I ~HEM4TOLMG DATI, OF MLE IMICE FED TNG FOR 4 WEEKSI

IC*NI CONTR(OL (T9NI THEATFL) 4 a NUU4UW. Of MICE

-- DOSE TWG in Fe.d 0.0 (Co 41 0.1-0.5 (1. 4 5

E4IH'14S ~i M 4090 6 41 4.13 .3

RETICULOCYIES9 I .54 00 .5j4j

I .EIATOCRIr. VOL* S 44.5 ±. 3 41.0 1.11

HEMOGLONINs GM. 140 i.e. 1 66

I4CV. CWIC MICRONS '1~4 A.L1 101.7 1).h

"MCHO$ MICR4O MICROGiMSO 30.t9 ?.rP .'* . f,'

MCHUC, 6,M I 33.3 ±. .3 b/. .4

PLATELETS (XIO /MM ) .7 'm 6~ .5 . I

LEUKOLYTES( 1X0 MH 1 9% S : Y.4 1.I

I vMPHOCYTPS. % 98 4. Ih0 .

HANDSo It 0.0 ±.0.0 U1.0 +. 0.0

MUNOCYTF'Sq % 1~ ... 3 * 3

FUINIMH1159 1.% * 3 .3 j.

HASIJPHILS, 0.0 ±.0.0 (1000 0.00

?.IYPICAL. 1& 0.0 0.0 0.0 0.0

-. NUCLEATED RHtLO t 0.0 j0.0 0.0 0.1)

k .. ENTRIES AHL MEAN t SIANUAND ERROR

a/ TNG concentratiý,% was increased 5-fold startin~g the 4th wweko. 4hiSignificantly different from the control mnice (Dunniett's multiple comparison procedure

109

Nn ED CU4

00

*1 o4 *1 *1 *1 *1 .1 of of . . . .

0D 0 Nu -D n ND ca

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V- u i2 4

P. - . 0. -p. .c . a a A j

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a i .9 .j0 . 9 . 0 . 0 . 9 . 9 . 9 . 0-

TABLE 74

K•&TOLOC Y DATA OF FEI&LE MICE FED TNG FOR 4 WEEKS

1CeN) CUMIROL (TvN) INHA1lt. N X NUMbIA 14 )VICE

005E; % TNG in Feed!l 0.0 (C, 41 0.1-0.5 (T. 41)

t.RYIH RUCYTtS (XIO / MM )4 .4 A4 .13 b 3 P :

METICULOCYTES, S 070 1 .04 1.34 .. .J6

HEMATOCRIT9 VOL. 1 4b.0 L- 1.2 4h,. .. i."

HEMOGLOSIN, 6M. 4 15.4 t .4 lb.v ±, ,•

MCV* CUNIC MICHONS 102.8 !. 4.1 t(7,! _. 3.1

MCHNW MICIHU MICHObMS. 34.4 1 1.4 .-*

MCHHCO GM % 33.4 .J .34.0 ± .4

PLAIELFTS (XI0 /MM ) S.H ±. 8 7 S.• ±_ o3 3

LE.UKOCYT'ES 1X10 /NN)MM . ~ 904 .9

NLUTI)OPHILS. 4 13.3 t* 4.r 1i.0 t 4.9

LYMPHOCYTFS. 4 H2.' 2.,M ± *41.O

HANIDS, S 0.0 . 0.0 0.0 • 0.0

SONO.YTES9 .3 . ..s . _. •b/

FOSINOPHILS9 4 1.3 t. .S (,.0 * n.W--

HASOIIPHILS, o 0.0 :t 0.0 0.0 _ 0.0

ATYPICAL. - 0.0 . 0.0 0.0 _ 0.0

NUCLEATFD WHC, 4 0.0 ± 0.0 0.0 _ 0. 0

ENIHILS ARE MEAN I !3IANUAHD EHRoH

a/ 'ING concentrations were increased 5-fold starting the 4th weel.

b/ Significantly different from the control mice (Dunnett's multiple comparison procedure

112

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LV Ir 1' '0 z r I m. Z Lii fol9.09

114

TABLE 77

ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF MICE FEDTNG FOR 13 WEEKS

Terminal% TNG Body Absolute Weight (gin)

Sex In Feeda/ Weight Heart Liver Kidneys Spleen

Male Control 28.8+1.7Al 0.16+0.01 1.25+0.03 0.55+-0.02 0.14+0.010.001-0.005 29.5+1.3 0.17+0.02 1.21+0.08 0.55+_0.06 0.15+0.040.01-0.05 29.3+0.9 0.15+0.01 1.28+0.07 0.58+0.03 0.12+0.010.1-0.5 28.3+0.8 0.15+0.01 1.39-10.10 0.564-0.04 0.104-0.01

Female Control 26.3+0.6 0.13+-0.01 1.19+0.09 0.33+-0.01 0.07+0.01S0.01-0.05 28.0-I._ 0.15+0.01 1.36+0.09 0.42+0.03 0.14+0.01

0.1-0.5 30.0+42.0 0.17+0.01 1.54+0.22 0.42+0.03 0.18+0.06

% 7o TNG Relative Organ Weights (gIm/lO0 gim body weight)Sex In Feed Heart Liver Kidney Spleen

Male Control 0.55+0.02 4.37+0.20 1.95+0.16 0.49+0.040.001-0.005 0.58+0.04 4.16+0.13 1.90+-0.2 0.53+0.140.01-0.05 0.52+0.02 4.38+0.18 1.98+0.03 0.40+_0.040.1-0.5 0.54+0.04 4.92+0.34 1.98+0.18 0.36+0.02

Female Control 0.51+0.05 4.67+0.45 1.31+0.05 0.27+0.030.01-0.05 0.52+0.03 4.86+-0.35 1.50+0.13 0.50+0.04

0.1-0.5 0.55+0.03 5.21+0.83 1.434-0.16 0.62+0.22

a/ TNG concentrations were increased 5-fold starting the 4th week.b/ Mean + S.E.

[

I 115

i1

TABLE 78

ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF MICE FED TNG FOR

13 WEEKS AND ALLOWED TO RECOVER FOR 4 WEEKS

Terminal%° TNG Body Absolute Weight (gin)

Sex In Feeds/ Weight Heart Liver Kidneys Spleen

Male Control 31.1+0.3T/ 0.17+0.01 1.37+0.13 0.64+0.01 0.15+0.07

0.001-0.005 35.0+_-1.9 0.21+0.03 1.57+0.13 0.63+0.01 0.11+0.01

0.01-0.05 33.0+1.6 0.22+0.03 1.454-0.05 0.73+0.06 0.13+0.02

0.1-0.5 31.0+_0.7 0.19+_0.01 1.38+-0.05 0.56+_0.02 0.12+0.01

Female Control 24.3+0.6 0.14+0.01 1.05+0.06 0.36-0.01 0.08+0.01

0.001-0.005 24.8+1.3 0.14+0.01 1.24+0.06 0.40+0.02 0.14+0.02

0.01-0.05 26.5+1.6 0.16+0.01 1.24+0.08 0.45+0.04S/0.12+0.02

0.1-0.5 29.0*1.1-/ 0.14+0.01 1.26-0.04 0.38+0.01 0.12+0.02

•J

7% TNG Relative Organ Weights (Rm/100 gm body weight)

Sex In Feed!/ Heart Liver Kidneys Spleen

Male Control 0.56+0.0.3 4.40+_0.43 2.07+0.06 0.47+0.22

0.001-0.005 0.60+0.07 4.50+0.36 1.81+0.10 0.32+0.02

0.01-0.05 0.65+_0.07 4.42+0.19 2.21+0.15 0.39+0.05

0.1-0.5 0.62+0.03 4.46+0.13 1.79+-0.02 0.40+0.02

Female Control 0.58+0.05 4.32+0.20 1.48+0.06 0.31+0.03

0.001-0.005 0.59+0.03 5.05+0.28 1.61+0.06 0.56+0.09

0.01-0.05 0.60+0.04 4.69+0.27 1.70+0.06 0.47+0.07

0.1-0.5 0.51+0.06 4.40+0.31 1.34+0.08 0.41+0.10

a/ TNG concentrations were increased 5-fold starting the 4th week.

b/ Mean + S.E.

c/ Significantly different from the contrL .,Aice (Dunnett's multiple

comparison procedure-4/).

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I

IV. DISPOSITION AND MEM JISM

3 TABLE OF CONTE4TS

A. Disposition and Metabolism of TNC in Various Species...... . 125

3 1. Introduction.............. ........ .. . . . . . . 1252. Material and Methods ....... ..................... . 125

3. Results ...................................... . 126

a. Distribution and Excretion .... .............. .... 126b. Metabolites in Urine ....... ............... .... 127

4. Discussion and Conclusions ...... ................. .... 128

I B. Biliary Excretion of TNG, DNGs and MNGs in Rats ........... ... 128

1. Introduction ................................ .... 128f 2. Material and Methods ........ ................... .... 128

3. Results ...................................... .... 129

a. Biliary Excretion ...... ................. ..... 129b. Metabolites of TNG in Bile ..... ............. .... 130

[4. Discussion and Conclusion ....... ................ .... 130

g C. Metabolism of TNG In Vitro........ .................... . 130

1. Introduction .......... ........................ ... 130

2. Material and Methods ....... .................... .... 130

3. Results ...................................... .... 131

a. Metabolism by Rat Liver ..... ............... .... 131b. Metabolism by Liver of Various Species ..... ........ 132c. Metabolism During Development ........ ........... 132

14. Discussion and Conclusio'q. ...... ................. .... 132

1I1 123

I

TABLE OF CON-TENTS (Concluded)

D. Effect of ING on Drug Metabolizing Enzyme ..... ........... ... 133

1. Introduction ............. ....................... ... 133

2. Material and Methods ......... .................... ... 133

3. Results .......... ....................... ......... 1344. Conclusion ............... ........................ .. 134

Tables 85 98 ................................................. 135

12i

IIV. DISPOSITION AND METABOLISM

A. Disposition and Metabolism of TNG in Various .Slcies I1. Introduction,

7 Absorption distribution, biotransformation, and excretion of TNG

were studied in rats--/. TNG and its metabolites were identified by the avail-

able conventional analytical methods and radioassay. In these experiments, isimilarities and differences in the disposition and pathways of blotransforma-

-" dton were investigated in mice, rabbits, dogs and monkeys.


11The procedure and methods described for rats-/ were generally used

to study the detailed disposition and metabolism of TNG in female albino Swiss Imice (National Laboratory Animals, O'Fallan, MO) weighing 20 to 25 gm,

male and female New Zealand rabbits (Small Stock Ind., Pea Ridge, AR)

weighing 2.30 to 2.37 kg, male and female beagle dogs (Haselton Research

Animals, Cumberland, VA) weighing 9.0 to 9.2 kg, and male and famalerhesus

monkeys (Primate Imports, Port Washington, NY) weighing 3.8 to 3.9 kg.

Each animal was fasted overnight. A single oral dose of TNG approx-imately 10% of the acute LDj0, spiked with 10 ptCi of TNG-1,3-14 C (specific

activity of 53.25 mCi/rH) was given via an intragastric metal or rubber tube.The TNG with TNG-l,3- 1 4 C was suspended in peanut oil and given at a volume of

0.2 ml/lO gm for mice, 1 mi/lO0 gm for rats, or I ml/kg for rabbits, dogs and

monkeys. After dosing, thmouse or rat was placed immediately in a "Roth-

Delmar" metabolism cage.- The rabbit, dog or monkey was placed in a stainless

steel animal cage (24 in. x 24 in. x 20 in.) inside of a closed air-tight

Plexiglass box. All animals were given food and water ad libitum. Tihe chamberswere vented continuously with CO2 -free air and the expired CO2 was collected

and absorbed in 5% NaOH for rodents and in LiOH for the larger animals. Feces

and urine were collected separately in the apparatus. At termiuation, each

animal was anesthetized with ether or pentobarbital sodium. Aortic blood wascollected and various tissues including feces were removed, weighed, and

digested. Aliquots of tissue digests, filtrates, blood, plasma and urine

were decolored with H202 and counted in the scintillation solution usin3 aPackard Tricarb 3375 liquid scintillation spectrophotometer. 14C02 samples

from the air trap were spotted on filter paper, dried and counted. Various

125I

Lechniques including TLC, GLC, chemical detection 1 sts, B-glucuronidase

treatment and tissue extracts described previously- were used to identify

TNG and its metabolites.

3. Results

a. Distribution and Excretion

The y7sults on the disposition and metabolism of TNG in rats

reported previously- are included for discussion in the present report.

i..e distribution and excretion of radioactivity after a single oral dose

of TNG in various species are suamnarized in Table 85. In mice, an average

of 30.7%/ of the administered dose was recovered in the gastrointestinal

tract plus contents and feces in 24 hours, whereas in rats, rabbits,

dogs and monkeys less than 127/ was recovered.

The total recovery of radioactivity in tissues and excreta

averaged from 78.5 to 83.3% in mice, rats and monkeys, 91.4/. in rabbits

and 99.2% in dogs. This suggests that the absorption of TNG in mice aver-

aged 50 to 707/ of the administered dose in 24 hours, whereas the absorp-

tion in other species was somewhat larger, averaging 75 to 95% of the dose.

In rats and mice, the majority of the absorbed radioactivity was excreted

in the urine and expired air. In 24 hours, mice Lxcreted 19.2 and 18.8%

of the dose in the urine and expired air, respectively, whereas rats ex-

creted 25.5 or 39.8% of the dose in the urine and expired air, respectively.

In monkeys, dogs and rabbits, most of the absorbed radioactivity was ex-

creted in the urine, averaging 45.0 to 72.0% of the dose in 24 hours. Only

2.5 to 7.8% of the administered radioactivity were recovered in the ex-

pired air during this period in these species. At the end of 24 hours,

the liver of all species contained significant amounts of radioactivity,

rangintg from 4.3 to 6.8% of the dose. Dogs and monkeys retained an average,

of 9.5 or 13.0% of the administered radioactivity in the skeletal muscle,whereas only 2.1 to 2.8% of the dose were recovered in the skeletal

muscle of rabbits, rats or mice. Kidneys, spleen, lung and brain of all

species contained only small amounts of radioactivity.

The tissue/plasma radioactivity ratios after a single oraldose of 'ING-I,3- 14 C in various species are sunmiarized in Table 86. Radio-

activiLy was highly concentrated in the liver of all species. The tissue/

plasma concentration ratio of radioactivity in the liver of rabbit ormouse was 21.0 or 30.1, respectively. The concentration ratios in theliver of rats, dogs and monkeys ranged from 7.4 to 7.8. The concentration

ratio of radioactivity was also relatively high in the kidney, probably

reflecting the excretion of radioactivity in the urine. The radioactivitywas also concentrated in the spleen and lung in which the concentration

t 126

"f~

ratios were greater than one. The concentration ratios in the brain of

mice, rats and rabbits were greater than one and of dogs and monkeys were

less than one; the concentration ratio in the skeletal muscle of rabbitswas greater than one and of the other species were less than one.

b. Metabolites in Urine

Metabolites of TNG In the 24-hour urine of various speciesare summarized in Table 8V. The parent compound, TNG, was detected onlyin small amounts, averaging less than 0.1% of the dose in the 24-hoururine of mice, rats and rabbits, and 0.3 to 0.8% in dogs and monkeys. Thefree dinitroglycerines (DNGs) were also excreted in small amounts in theurine, averaging less that 0.1% in mice, 0.3% in rabbits, 1.1% in rats,I • 2.5% in monkeys, and 3.6% in dogs.

Most of the uri ary metabolites in mice were unidentifiedpolar compounds, averaging 12.0% of the administered dose. One component

moved in the ethyl acetate and petroleum ether solvent system and accountedfor ii.2% of the dose. Other urinary metabolites included 2.0% of the doseas free mononitroglycerines (MNGs) 2.4% as DNG-glucuronides, 0.2% as MNG-glucuronides and 2.1% as glycerol.

Most of the urinary metabolites in rats were free MNGs(10.6% of the administreed dose) and 1,2-DNG-glucuronides (10.0%). Otherurinary metabolites included 3.5% of the dose as 1,3-DNG-glucuronide and1.5% of the dose as MNG-glucuronide. Glycerol accounted for 6.9% of thedose and the unidentified polar components accounted for 6.2% of the dose.

_ I The unidentified polar compounds in the urine of rabbits

accounted for most of the urinary radioactivity, averaging 44.8% of theadministered dose. The major component moved in the ethyl acetate andpertroleum ether solvent system. Rabb.ts also excreted large amounts of1,3-DNG-glucuronide and glycerol, averaging 11.2% and 8.8% of the dose,respectively. Free MNGs, 1,2-DNG-glucuronide and MNG-glucuronide accountedfor 2.4%, 2.8% and 1.6% of the dose, respectively.

Dogs and monkeys excreted more MNGs and more MNG-glucuronidesthan o-her species. Free MNGs in the urine of dogs and monkeys accountedfor 18.2% and 15.8% of the dose, respectively; and MNG-glucuronides accountedfor 5.2% and 7.5%, respectively. Dogs also excreted 8.9% of the dose asDNG-giucuronides; aud monkeys excreted only 0.9% as the DNG-glucuronides.1 IGlycerol accounted for 8.8% i.nd 4.8% of the dose in dogs and monkeys,respectively; whereas the unidentified polar compounds accounted for 22.0%and 12.6% in dogs and monkeys, respectively.

- - 127

4. Discussion and Conclusions,

The metabolic pathways were similar in rats, mice, rabbits,dogs and monkeys. About 50 to 70% of an oral dose of TNG-l,3- 1 4C wasabsorbed in 24 hours in mice, whereas 75 to 95% was absorbed in rats,rabbits, dogs and monkeys. In rats and mice, the majority of the absorbedradioactivity was excreted in the urine and expired air. In rabbits, dogsand monkeys, most of the absorbed radioactivity was excreted in the urinewith only small amounts in the expired air. The radioactivity was highlyconcentrated in the liver of all species. Other tissues, including kidney,spleen, lung and/or skeletal muscle, also contained significant amountsof radioactivity.

TNG and free DNGs were excreted only in small amounts in theurine of mice, rats and rabbits, and in slight amounts greater in theurine of dogs and monkeys. Large amounts of unidentified polar compoundsand glycerol were found in all species. Mice excreted only small amountsof free MNG, MNG-glucuronides and DNG-glucuronides, indicating relativelycomplete biotransformation of ThG. Most of the urinary metabolites in ratsand rabbits were free MNG and DNG-glucuronides. Dogs and monkeys excretedmostly free MNGs and MNG-glucuronides.

The lesser amount of absorption and ccmplete metabolism of TNGin mice reflects the relative toxicity of TNG in this species. As reportedpreviously, the acute oral toxicity of TNG was less in mice than in rats.In the present studies, the subacute and subchronic toxicities of TNGwere more in dogs and rats than in mice as described in Sections I, II,and III.

B. Biliary E2cretion of TNG. DNGs and MN~s in Rats

I. Introduction

As discussed in the preceding Section IV.A., TNG was highly con-centrated in the liver of various species. Liver serves both as a site formetabolic biotransformation of foreign compounds and as an organ for excre-tion. In these experiments, biliary excretion of TNG, its dinitroisomers(DNGs) and mononitro-isomers (MNGs) in rats were compared.

2. Mal erfal and Methods

Female CUR rats (Charles River Breeding Laboratories, Wilmington,MA), weighing 280 to 320 gm, were fasted overnight before use. Under

ether anesthesia, the common bile duct was cannulated with PE 10 plas-tic tubing through a midline abdominal incision. After the incision

128

I had been closed, a dose of TNG, DNGs or MNGs approximating 10% of the acute

LD5 0 , spiked with about 10 ;Ci of the respective 14C-labeled compoundsI (specific activity of 36.67 to 53.25 mCi/mM), was dissolved in peanut

oil and administered orally by intragastric intubation. The rats werethen confined individually in restrictive animal holders (Stoelting

Y Company, Chicago, IL). Purina Rodent Chow and water were freelyaccessible to the animals.

Bile was collected for the predetermined interwls and theamount of bile was measured by weighing. Small volume (200 pl) bloodsamples were obtained periodically from the rats by cutting off the tipsof their tails and heparinized. At the end of 24 hours, the rats wereremoved from the holders. and anesthetized with ether. Blood was collectedfrom the abdominal aorta with heparinized syringe. Entire length ofgastrointestinal tract includ'ng their contents was removed and combinedwith the feces which were collected without urinary contamination.

Radioactivities in the bile, blood, plasma and the GI tractwe-! measured using a Packard Tricarb 3375 liquid scintillation spectro-photometer as described in Section IV.A.2. Bile was counted directly in

the scintillation solution. Blood, plasma and the GI tract were digestedwith NaOH, decolored with HA20, solubilized in the scintillation solu-

tion and counted.

1 3. Results

a. Biliary Excretion

The biliary excraion of radioactivity in female rats afteroral administration of TNG-1,3-- C is suamitrized in Table 88. The radio-activity appeared in the bile within 15 min.-tes after dosing. The rate

of biliary excretion increased with time and reached a peak in 3 hours.Thereafter, the rate of excretion decreased. The total biliary excretionaveraged 13.50% of the dose in 24 hours. The blood concentrition ofradioactivity continued to increase through the 5th to 6th hour and de-3 creased thereafter.

The biliary excretions of radioactivity Wter oral admin-istration of I-MNG, 2-MNG, 1,2-DNG and 1,3-DNG (NG-1,3- C) are summar-Ized in Table 89. The biliary excretion of radioactivity was high for2-MNG and l,2-DNG, averaging 18.90% and 11.20% of the administered dosein 24 hours, respectively. The biliary excretion was low for 1-MNG andI,3-DNG, averagfng 3.81% and 1.63% of the dose, respectively. The re-

cuvery of radioactivity in the gastrointestinal tract plus contents andfeces was 4.64% of the dose for l-MNG and less than 1% for 2-MNG, 1,2-DNG or 1, 3-DNG.

129

.,, D,, or

b. Metabolites of TNG in Bile

Metabolites of TNG in the 24-hour bile of rats are summr-ized in Table 90. As described in the urine in Section IV.A.2.b., TNGwas detected only in small amounts in the 24-hour bile of rats, less than

0.1% pf the administered dose. The frae DNGs were also excreted in smallamounts in the bile, averaging 0.3% of the dose or less. In addition to1,2-DNG-glucuronide (3.9% of the dose), 1,3-DNG-glucuronide (2.5%), glycerol(2.8%) and unidentified polar compounds (2.4%), other major metabolitesin the bile were MNG-glucuronides (2.0%). Free MNGs accounted for 1.0%of the dose.

Separate experiments showed that the concentration of 1,3-DNG in the first-hour bile was higher than that in the 5th-hour bile,whereas the concentration of MNG was higher in the 5th-hour bile than inthe first-hour bile. This indicates the progressive denitration of theDNG to MNG.

4. Discussion and Conclusion

14After oral administration of TN. ,.I- C to rats, the radio-

activity appeared in the bile in 15 minutes. The rate of biliary ex-cretion increased with time and reached a peak in 3 hours. Thc bloodconcentration of radioactivity continued to increase through the 5th Ito 6th hour. The biliary excretion of radioactivity was high for 2-MNG,TNG and 1,2-MNG and low for 1-MNG and 1,3-DNG. The amount of radioactiv-ity remained in the gastrointestinal tract plus contents and in the fereswas small. This suggests relatively complete absorption after oral ad-ministration of these compounds.

C. Metabolism of TNG In Vitro

1. Introducti.on

This phase of the study was to describe the in vitro metabolismof TNG by homogenates of livers from various species. These data in con-junction with the in vivo observations mvy be utilized to predict howhumans metabolize TNG. In addition, metabolism of TNG by placentas andLissues of several s[3cies during development was investigated.


Animals were sacrificed by decapitation (rats), cervical dis-

location (mice), air embolism (rabbits), or an overdose of magnesiumsulfate (dogs and monkeys) and the livers removed, weighed, and homogen-ized in three volumes of 1.15% K1l. Human liver was obtained from autopsy

130

It

samples and similarly processed. The i vitro sys t em , as modified fromN ~ ~Needleman and Krantz,-!/contained 1 mM TNG-1,3-'4C or 1-MNG-1,3-1C

8 mM reduced gluthathione, 8 mM potassium cyanide, phosphate buffer andliver homogenate at a final pH of 7.4. The incubations were conducted at370c for 10 minutes, except where specified, and terminated with mercuric

chloride and absolute ethanol. Metabolites were resolved by silica gelthin-layer chromatography in a solvent system of benzene and ethyl acetate(4:1). In this system, TNG, 1,3-DNG, and 1,2-DNG are resolved on the platewhile MNGs, glycerol and glucuronides remain at the origin. The Lowryi/protein assay was used to measure protein assay. The ability of livers tometabolize TNG was expressed as nanomoles metabolites per milligram pro-tein. Since there were no sex differences in TNG metabolism, the valuesfor both males and females were combined.

[ 3. Results

a. Metabolism by Rat Liver

The in vitro system metabolized TNG primarily to 1,3-DN3Gand 1,2-DNG after a 15-minutes' incubation. The results are shown in

Table 91. If the reactions were continued for up to 2 hours, then theamount of DNGs decreased and the amount of polar components at the originincreased as a function of time. The material at the origin probably repre-sented MNGs since 94% of l-MNG incubated in the same system was presentas the parent compound after a 90-minute incubation. These observationssuggest that the liver, in vitro was not able to completely denitrate TNGas the in vivo system.

These results regarding the ability of livers to metabolizeTNG are in general agreement with the rports of others 1 5, 1 7 , 1 8 • Since

1Cto14liver homogenates metabolized glycerol- C, but not TNG-4C, to 002,they concluded that TNC was not denitrated to glycerol in the liver. Inaddition, the conversion of TNG- 14 C to 14CO2 which occurred in the intactanimal was blocked when rats were eviscerated. Since the removal of lir,stomach, intestines, pancreas, spleen and kidneys interferred with themetabolism of TNG- 4 C,it was possible that these organs acted in conjunctionwith the liver to metabolize TNG. This proposal was tested in the in vitrosystem by incubating homogenates of the liver and various organs with TNG-1,3-14C and I-MNG-I,3 C. As shown in Tables 92 and 93, the addition ofnone of the tissues which were removed during evisceration modified the

ability of the liver to metabolize either substrate. The results of thisexperiment still leave In question the role of the liver and various otherorgans in the complete denitration of TNG to glycerol and ultimately to

CO •2

1 131

b. Metabolism by Liver of Various Species

Comparative studies on TNG metabolism were ccnductfl by in-cubating hmogenates of livers from various species with TNG-l,3- C or

114I-MNG-I,3- "C for 10 minutes.4Livers from rats, dogs, rabbits, and monkeys

were not able to metabolize C-l-MNG to glycerol. As shown in Table 94,tie primary metabolites of TNG were 1,3-DNG and 1,2-DNG. Polar components

at the origin represented 5% or less of the total radioactivity and wereexcluded from the comparison. No sex difference in TNG metabolism was ob-

served for any of the animal species tested and the results for males and

females were combinedi. Livers from rats and mice produced more 1,3-DNG than

1,2-DNG. In contrast, more 1,2-DNG was produced by livers from rabbits,

dogs, monkeys and humans. Livers from mice and humans had a low abilityto metabolize TNG. In addition, the human livers had a lower ability to form

1,3-DNG than any of the other species. The formation of 1,2-DNG by livers

from rats and humans was equivalent.

c. Metabolism During Development

Since the metabolism of many compounds changes during develop-.

ment, studies were conducted to determine the ability of various developing

tissues to metabolize TNG. Placenta from mice, rats, and humans had a poor

ability, relative to the liver, to produce 1,3-DNG and 1,2-DNG. The results

are shown in Table 95. Low levels of TNG metabolism were found in mouse

embryos on day 12 of gestation and in mouse livers and carcasses on day 18 of

gestation. The results are shown in Table 96. TNG metabolism in rat livers

increased between I and 7 days after birth and did not change between 7 and

21 days after birth. Livers from rats at 3 weeks of age produced less DNGsthan the adult liver and the ratio of 1,3-DNG to 1,2-DNG was 1.2. These

results suggest that at 3 weeks of age rats do not produce the adult pattern

of TNG metabolites.


TNG was primarily metabilized to DNGs in vitr~o by livers of various

species. Livers from rats and mice produced more 1,3-DNG than 1,2-DNG; where-

as, livers from rabbits, dogs, monkeys and humans produced more 12-DNG than1,3-DNG. Livers from mice and humans had a low ability to metabolize TNC.

The rat liver quickly metabolized TNG in vitro to DNGs in 15 minutes.

Then the amount uf DNGs decreased when the incubation was continued for up

to 2 hours. MNC was not appreciably metabolized by liver In vitro. These

results suggested ihal the liver jjj xILro was not able to compleLel-y deruirrate

"TNG as in vivo system. The addition of oiher tissue homogenates, including

stomach and intestines, or pancreas, spleen and kidneys, did not modify theability of the liver to metabolize TN(m or MNCs in vitro.

t32

MI

Placentas from micep rats or humans and mouse embryo,

liver or carcass during late gestation had a poor ability, relative

to liver, to metabolize TNG in v . TNG metabolism in rat livers in-

creased with time after birth up to 7 days. The metabolism did not change

between 7 and 21 days. The ability of rat liver at 21 days after birth

to metabolize TNG was lower than that of the adult liver.

DoD. Effect of TNG on Drue Metabolizin2 Enzyme

1. Introduction

The effect of TNG on the ability of rats to metabolize test

compounds was studied. The test compounds selected for this study were

zoxazolamine and nitroanisole. The in vivo metabolism of zoxazolaminewas followed by the duration of the loss of the righting reflex following

treatment. The in vitro metabolism of nitroanisole in liver was mea-

sured in terms of the formation of p-nitrophenol.


Male rats were fed diets that contained 0.5% TNG for 2 weeks.

Rats in the positive control group received 50 mg/kg of phenobarbital

sodium twice daily for 3 days. At the end of treatment, the zoxazolamine

paralysis time and nitroanisole 0-demethylase activity were determined.

I Zoxazolamine was administered i.p. to three rats at a dose

of 45 mg/kg in a vehicle of 0.2 N HCl. The duration of paralysis was

measured in terms of the loss of the righting reflex. The values are

reported as the mean ± S.E. and the test of significance was the two-

sample rankl/ 9Jtest. The level of significance was selected at P <0.05.

TLe metabolism of nitroanisole by livers was measured in ann r system. Rats were sacrificed by decapitation and the livers

were removed, weighed, and homogenized in 4 volumes of 1.15% potassium

chloride. The homogenate was centrifuged at 9,000 x g for 30 minutes.

The incubation medium contained 15 pmoles magnessium chloride, 15 pmoles

glucose-6-phosphate, 3 pmoles p-nitroanisole, 0.5 ml of the 9,000e x g

liver supernatant, and 0.5 ml of 0.5 M sodium phosphate buffer pH 7.8.

Reactions were conducted for 20 minutes in a shaking water bath at 37 0 C.

The reaction was terminated by the addition of 0.5 ml of 40% fortoalin

311d tie color was developed with 0.5 ml of 0.8 N sodium hydroxide. The

product formed was measured specirophotometrically at 420 nut. The

1 133

!I

relationship of pmoles p-nitrophenol formed - Absorbance Units/10.22was used to quantitate product foired. The Lowry protein assaymi/was used to measure protein content. The activity was expressed as Inanomoles p-nitrophenol/mg protein. The values are ruported as themean ± SoE, The test and level of significance were the same as de-scribed above.

3. Resu~lts i

The results on the zoxazolamine paralysis in rats are summarizedin Table 97. Pretreatment of phenobarbital sodium significantly simulatedthe metabolism of zoxazolmiine and reduced the duration of paralysis

as compared to control rats. On the other hand, feeding of 0.5. TNG for2 weeks did not affect the duration of zoxazolamine paralysis. This re-sult indicates that the liver enzyme(s) to metabolize zoxazolamine wasnot affected by TNG.

The results of nitroanisole 0-demethylase activity in rat liversare summarized in Table 98. The nitroanisole O-demethylase activity inlivers of rats fed 0,5% TNG for 2 weeks was not significantly differentfrom that of the control rats.

4. Conclusion J

Feeding of 0*5% TNG for 2 weeks did not affect the liver enzyme(s)to .LeL.bolize zoxazolanine in 3ivo. nor affected the 0-demethylase activityin -- liver `n vitro

I 'lit!

I1 'ABLE %

DISTRIBUTION AND EXCRETION OF RADIOACTIVITY IN VARIOUS SPECIES4 OF ANIMALS 24 HR AFTER ORAL AII4NISTRATIDN OF A

SINGLE DOSE OF TNG-1,3- 14 C

7. of Administered DoseSMi.•ce R~at•_._•s Rabbits 2 Monkeys

GI plus Con-

tents 2.2 + 0.i. 3.0 + 0.2-c/ 3.74/ 4.8A/ 8.4+ 3.5e./Feces 28.5 + 4.2 6.3 + 1.0 0.1 1.8 3.2 + 1.6Whole Blooda/ 0.3 + 0.0 0.7 + 0.0 0.6 1.6 2.1 + 0.1Expired Air 19.2 + 1.2 25.5 + 1.5 7.8 2.5 3.6 + 0.6

Urine 18.8 + 7.5 39.8 + 2.6 72.0 71.6 45.0 + 0.9Liver 6.3 + 0.6 4.3 + 0.9 4,8 6.4 6.8 + 2.2Kidneys 0.2 + 0.0 0.3 + 0,0 0.2 0.4 0.3 + 0.0Spleen < 0.1 -- <0.1 0.1 <0.1Lungs 0.1 + 0.0 0.1 + 0.0 0.1 0.3 0.2 + 0.0Brain b/ 0.1 + 0.0 0.1 +_ 0.0 <0.1 0.2 0.7 + 0.1Musce I- 2.5 + 0.7 2.8 + 0.2 2.1 9.5 13.0 + 1.0

Recovery 78.5 + 9.7 82.9 + 6.4 91.4 99.2 83.3 + 3.8

a/ Based on 7% of the body weight.b/ Based on 407. of the body weight.

c/ Mean + S.E. of four animals.d/ Mean of two animals.e/ Mean + S.E. of three animals.

135

[

TABLE 86

TISSUE/PLASMA RADIOACTIVITY RATIOS!/ IN VARIOUS SPECIES OFANIMALS 24 HR AFTER ORAL ADMINISTRATION OF A

SINGLE DOSE OF TNG-1.3- 14 C

Tissue Mice Rats Rabbits Dogs Monkeys

Liver 30.1 + 5.6h/ 7.8 + 1.711/ 21.OOF/ 7.5/ 7.4 + 2.81/Kidney 4.1 + 1.1 2.8 + 0.3 4.3 2.7 2.2 + 0.2Spleen 2.6 + 0.7 -- 3.3 1.8 1.3 + 0.1Lungs 2.8 + 0.7 1.7 4- 0.1 2.8 I.A 1.3 + 0.1Brain 1.2 + 0.2 1.0 + 0.1 1.5 0.7 0.9 + 0.3Muscle 1.8 + 0.6 0.6 + 0.1 0.6 0.8 0.8 + 0.1

a/ Radioactivity in 1 gm of wet tissue per radio-

activity in 1 ml of plasma.b/ Mean + S.E. of four animals.c/ Mean of two animals.d/ Mean + S.E. of three animals.

t3

.4

TABLE 87

METABOLITES OF TNG IN URTNE OF VARIOUS SPECIES COLLECTED FOR 24 HR

AFTER ORAL ADMINISTRATION OF A SINGLE DOSE- OF TNG-,3-14C

% of Administered Dose

Metabolite Mice Rats Rabbits Do2s Monkeys

TNG <~! <0.11- o a/ o.11Y- 0. 8k/ 0.31/

1,3-DNG <0.1 0.4 0.2 2.8 1.3

1,2-DNG <0.1 0.7 0.1 0.7 1.2

MNGs 2.0 1C.6 2.4 18.2 15.8

1,3-DNG-glucuronide 1.5 3.5 11.2 5.5 0.4

1,2-DNG-glucuronide 0.9 10.0 2.8 3.4 0.5

MNG-glucuronides 0.2 1.5 1.6 5.2 7.5

Glycerol 2.1 6.9 8.8 8.8 4.8

Polar Compounds

Rf 0.0 in Solvent C 0.8 4.9 5.4 18.6 12.4

Rf 0.6 in Solvent C 11.2 1.3 39.4 3.4 0.2

a/ Mean cf three animals.b/ Mean of two animals.

A

0

(

1 137

iI

144

0 V- ,4 -l

0 0w

%D %0O c ,- C I 0

o 0go

--4 r4 +I+I4+I+I 4ir-4i+I+ +-

0e~~~ cn enC0lea 0 0004C- %D000004

0- C 4- g 4 04 -4 4 C;4

H 0

04 0 0 r- N Cv0 -- 4

+t-4++>+II+I+II+I+

1- H00 0v-4 4,-

0Qr 0n .0-4COe'Nr.4 M M -0

o4 N

'-"4

0 ~00C 00-4-4 10 -4 4C4Aý0

444-0 r.4- M%0 M 4O L .4

8 3] 000000000- r-ý%D00 0.

0b 000--4 000M0 r-

0 l0J0ri4r- 7 141-4 C C; C'J o 0C

r-4w

, 0 %o % -0S.4-4C 1

44 C4C4 kDD Lf r4 1380

I 4-4

010

~O0 0

1-4 0 - -zI0-4

IL0 % C'J '0

in 0 +1 4 +1 +1 +

04 CCu00 c 'o' - 41

0 'ý4od 0) .. 0 Go~ 0 0% r

0 . C0 U .4 C)ac

1-04 0 04 0

00 04UD CC., a, ~ '

0 C1 4) M

0 C) 0

1- 1 +1 1 +

4.391p z JD 0 0 %D0 4Ii:u

TABLE 90

METABOLITES OF TNG IN RAT BILE COLLECTED FOR

24 HOURS AFTER ORAL ADMINISTRATION OF AS SINGL2E .DO& •"TNc- ."- 1 4 c

Mtb t Radioactivity in Bile

Metabolites 7. in Bile % of Dose

TNG < 0.1 < 0.1

1,3-DNG 1.7 ± 0.3 0,3 + 0.1

1,2-DNG 0.4 + 0.1 0.1 + 0.0

MN~s 6.4 + 1.6 1.0 + 0.2

1,3-DNG glucuronide 16.3 + 2.6 2.5 + 0.41,2-DNG glucuronide 25.9 + 3.8 3.9 + 0.6

MNG glucuronides 13.2 + 4.7 2.0 + 0.7Glycerol 18.9 + 4.2 2.8 + 0.6

Polar compounds 17.2 + 4.3 2.6 + 0.6

a/ Mean + S.E. of three rats.

140

I IIi

14.

METABOLISM OF TNG-I,3- C IN RAT LIVER

AS A FUNCTION OF TIME

I Time _ Moles/mg Protein A(minutes) l,3-DNG 12-DNG Orgi

15 36.8a-/ .9 2.8+ 2.6 + 1.9 + 0.4

30 29.8 20.2 6.4 4* 1.8 + 1.4 + 0.8

160 19.4 8.6 13.3+1.2 + 0.8 + 1.6 4

120 7.1 5.7 21.7+ 0.5 + 0.7 + 2.4

al Mean + S.E. for three livers from male rats.

IL

,1 4'

4,.

II 141

I • •i I -, : : i

TABLE 92

METABOLISM OF TNG-I,3-14C IN LIVERS OFFEMALE RATS IN THE PRESENCE AND

ABSENCE OF VARIOUS TISSUEPREPARATIONS

nMoles/mg Protein

Tissue 1,3 DNG I."D NG

Liver- 44.3-/ 29.6+ 2.2 + L.8

Liver + 31.3 23.5PreparaLion 1b/ + 0.7 + 0.1

Liver 1 ci 49.5 33.6Preparation 2 + 5.3 A- 3.4

&a! One volume liver hcmogenate.b/ 0.5 volume liver homogenate and 0.5 volume

homogenate of stomach and intestines.c/ 0.5 volume liver homogenate and 0.5 voolume

homogenate of pancreas, spleen, and kidneys.

d/ Mearn +5, •, for three observations.

142

.. j .

=r

I;1INI

TABLE 93

METABOLISM OF 1-MNC- 13- C IN LIVERSFROM FEMALE RATS IN THE PRESENCE AND

ABSENCE OF VARIOUS TISSUEPREPARATIONS

noles/rng Protein-- Tissue l-MG Crigin

Liverd/ 93A/ 1.6

+5 + 0.3

Liver + 93 1.3Preparation -b/ + 5 + 0.2

if Liver 4 93 1.31. Preparation 2 ±1 _+ 5 + 0.2

a/ One volume liver homogenate.b/ 0.5 volume liver homogenate and

0.5 volume homogenate of stomach

and intestines.c/ 0.5 volume liver n)mogenate and

0.5 volume homogenate of pancreas,spleen, and kidnevs.

S/ Mean + S.E. for three observations.

Ii

I 143

.!- h

TABLE 94

14METABOLISM OF TNG-l.3- C IN LIVERS FROM

DTFFERENT SPECIES

Numberof nMoles/mg Protein

Species Determinations 1,3-DNG 1,2-DN. G 1,3-DNG/I,2-DNG

Rat 7 40.6 + 1. 9 y 28.0 + 1.3 1-4

Mouse 6 16.7 + 0.9 7.0 + 0.3 2.4

Rabbit 6 18.3 + 0.8 51.3 + 2.2 0.36

Dog 7 21.1 + 0.7 46.8 + 2.0 0.45

Monkey 5 16.3 + 2.1 52.7 + 5.2 0.31

Humana/ 4 12.5 ± 2.9 32.5 + 4.9 0.38

S/ Autopsy samples frow a 65-year old NegLo male died 3f a dissectinganeur,ýem of the •orta, a 21-year old white male died of a gunshotwound, a 67-year old white female died with CNS seizures, and a

60-year old female died with a lung carcinonia.3/ Mean + SeE, or mean of the inadicated numbcr of determinations. Values

for males and females were combined.

144

IT

[TABLF 95

METABOLISM OF TNG-1,3-1 4 C INPLACENTAS FROM VARIOUS SPECIES

nM'Iles/mg ProteinSpecies 1 3-DNG i,2-DNG

Mouse-- Day 12 1.5 + 0.V-a/ 3.9 + 0.9

Day 18 3.1 + 0.1 5.2 + 0.4

Day 19 2.5 + 0.6 3.8 + 0.2

"HumanTerm 0.7 + 0,4 4.7 + 1.3

a/ Mean + S.E. fvr three determinations.

T.4

Ire

1 '4-I.I

I 1£i

I

li

TABLE 5-6

METABOLISM OF TNG-1 3- 1 4 C DURING DEVELOPMENT IN MICE AND RATS

DaysAfter nMoles/mg Protein

Snecies Tissue Birth 1,3-DNG I,2-DNG

Embryo -7 0 3.8 + 0.9 •1

Liver -1 7.4 + 0.6-a/ 5.9 + 0.2Carcass -1 3.4 + 1.2 4.9 + 0.4 .

Rat

Liver -2 12.2 + 0.8 9.4 + 1.0.iver 1 14.6 + 0.4 9.1 + 0.1

Liver 7 27.0 + 1.0 23.1 + 0.6Liver 14 26.9 + 1.2 21.4 + 2.0"Giver 21 29.1 + 1.0 24.8 + 1.8 .

a/ Mean + S.E. for three determinations.

1

*11

I'1

• ~146

!I TABLE 97 1DURATION OF Z0XAZ0LAM4INE PARALYSIS IN MALE RATS PRETREATED

WITH PHENOBARBITAL SODIUM OR FED A CONTROLDIET OR A DIET CONTAINING 0.5% TNGI

Duration of ParalysisTreatment (Minutes) j

Expeciment IControl 98 + 11 (10)-cPhenobarbitala/ 34 4 - (7)A/

Experiment IIlo Control 134 + 14 (8)

0.5% TNGbk/ 149 + 11 (8)

"S a/ 50 mg/kg, I.P., twice daily for 3 days.b/ Incorporated into diet and fed for 2 weeks.c/ Mean 4- S.E. (number of observations).d/ Signilicantly different from control (two rank testli9/).

A

1I4

III

II

I 147

!

TABLE 98

NITROANISOLE O-DEMETHYLASE ACTIVITY IN

LIVERS OF RATS FED A CONTROL DIETOR A DIET CONTAINING 0.5% TNG

Treatment Actiltyi--/

Control 1.32 + 0.17 (3)c/0.5% TNU'/ 1.14 + 0.01 (3)

a!TInncporateo into diet and fed for2 weeks.

b/ nMo1e3 p-nicrophenol/mg protein in9,000 x g supernatant.

c/ Mean ± S.E. (number of observations).

'i

148 J

-j$~

I

REFERENCES

1. Lee, C. C., J. V. Dilley, J. R. Hodgson, D. N. Roberts, D. 0. Helton

and W. J. Wlegand. Mammalian Toxicity of Munition Compounds: PhaseI. Acute Oral Toxicity, Primary Skin and Eye Irritation., Dermal Sen-sitization and Disposition and Metabolism. USAMRDC Contract No. DAMD-17-74-C-4073. Report No. I: 1-100, 22 July 1975.

2. ?ybus, J.: Determination of Calcium and Magnesium in Serum and Urineby Atomic Absorption Spectrophometry. Clin. Chem. Acts, 23: 309, 1969.

3. Seligson, D., J. Marino, and E. Dodson: Determination of Sulfobromoph-thalein in Serum. ClGn. Chem., 3" 638, 1957.

4. Dunnett, C. W.: A Multiple Comparison Procedure for Comparing SeveralTreatments with a Couttol. J. Am. Stat. Assoc., 50: 1096, 1955.

5. Moorhead, P. S,, P. C. Nowell, W. J. Mellman, D. M. Battips, and D. A.Hungerford: Chromosome Preparations of Leukocytes Cultured fromHuman Peripheral Blood. Expt. Cell Res., 20: 613, 1960.

6. Fernande.3, M. V.: The Development of a Human Ammion Strain of Cells.Texas Repts. Biol. Med., 16: 48, 1958.

7. Vogt, M., and R. Dulbecco: Virus-Cell Interactiovi with Tumor ProducingVirus. Proc. Nat. Acad. Sci., 46: 365, 1960,

8. Moorhead, 2. S., and P. C. Nowell: Chromosome Cytology. In Methods inMedical Research (H. N. Eisen, Editor). Year Book Medical Publ. Inc-,Chicago, 10: 310, 1964.

9. Reisman, R. E., and C. F. Arbesman: Systemic Allergic Reactions Due ToInhalation of Penicillin. J. Am. Med, Assoc. 203: 986, 1968.

t 10. Mancini, G., A. 0. Carbonara, and J. P. Heremans: Inmnunochemical

Quantitation of Antigens by Single Radial Inmnunodiffusion. Immuno-chemistry, 2: 235, 1964.

11. Kao. F. T., and T. T. Puck: Genetics of Somatic Mammalian Cells: Vi1.Inducl ioni and Isolal Ilon ot Nuiri I olial MuiiatS I,-in Chinese Hamster

GeLls. 1'roc. Nat. Acad. Sel., 60: 1275, 11t8.

1?. Pi -;k, I%'I" .. a•'id F. Tr. Kao: CAnets IISOf SomatLI c Mammal Ian Ce Ils. V.I Treatment wi'h 5-Bromodeoxyui [dine and Visible Light for Isolation of

= Nutritionally Deficient Mutants. Proc. Nat. &cad. Sri., 58: 1227, 1967.

149

13. Kao, F. T., and T. T. Puck: Genetics of Somatic Manvuallan Cells. IX.

Quantitation of Mutagenesis by Physical and Chemical Agents. J. Cell.Physiol., 74: 245, 1969.

14. Roth, L. J., E. Leifer, J. R. Hogness and W. H. Langham: Studies on theMetabolism of Radioactive Nicotinic Acid and Nicotinamide in Mice.J. Biol. Chem., 176: 249, 1948.

15. Needleman, P., and J. C. Kirantz: The Biotransformation of Nitroglycerin,

Biochea. Pharmacol., 14: 1225, 1965.

16. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall: Protein

Measurement with the Folin Phenol Reagent. J. B3.ol. Chem., 193: 265-275,1951.

17. Needleman, P. and F. E. Hunter: The Transformation of Glyceryl Trinitrateand Other Nitrates by Glutathione Organic Nitrate Reductase. Nolec.Pharmacol.,l: 77, 1965.

18. Needleman, P., D. J. Blehm, A. B. Harkey, E. M, Johnson, and S. Lang:The Metabolic Degradation of Glyceryl Trinitrate. J. Pharmacol. Exp.Ther., 179: 347, 1971.

19. Mann, H. B. and D. R. Whitney: On a Test of Whether One of Two Random.Variables is Stochastically larger than the Other. Ann. Math. Stat.,18: 50, 1947.

'1

1.5o 1

150

I

III

APPENDIX 1II

I MANUAL FOR

HEMATOLOGY. CLINICAL BLOOD CHEMISTRY. URINALYSIS.HISTOPATHOLOGY, STATISTICAL AWALYSIS. AND NORMAL VALUES

Cheng-Chun LeeLoren D. KintnerThomas W. ReddigJohn J. Kowalski

I

- Midwest Research Institute

May 1975

I

l 151

I,, , Il:

I

II

S APPENDIX I

MANUAL FOR

HEMATOLOGY, CLINICAL LABORATORY TESTS, HISTOPATHOLOGY,STATISTICAL ANALYSIS. AND NORMAL VALUES

Cheng-Chun LeeChumn-Bin Hong

Jagdigih C. BhandariJudith D. GirvinJohn J. Kowalski

"Midwest Research Institute

January 1977

'S

tl

TABLE OF CONTENTS

?Mge

I. Hematology and Clinical Laboratory Tests. . . . . . . . . . . . 1

A. Hematology . . . . . .1... . . . . . . . . 1B. Clinical Blood Chemistry ........ ................. 2

C. Urinalysis. . . . . . . . . . . . ......... . . 3D. Occult Blood in Feces ..... ............. 4E. Precision of Hematology and Clinical Blood Chemistry

Tests ....... ................. . . . . . . ... 4

1. Reproducibility ................... 42. Reproducibility Within a Test Day ......... 43. Proficiency Test Service .... ........... . . 5

II. Histopathology ..... ......................... 5

A. Necropsy and Gross Examination ..... .............. . 5B. Organ Weights . . . ......... . . . . . . . . . 5

. Tissues for Microscopic Examination . . . ........ 6-- D, Fixation and Staining of Tissues ......... . 6

III. Statis2ical Analysis .......... ...................... 6

IV. Normal Valu,:.s ........ . ........................... 7

"A. Hematology, I'linical Laboratory Tests and Bone Marrow . . 7B. Absolute and RL"ntive Organ Weights ........... 7C. Presence of Variouc Substances in the Urine .... ....... 7D. Occult Blood in Feces . ................. 8

V. References...... . . . .................. 8

Tables A - 0 .i.................... ................. ......... 10 - 24

zIi

It

HEMATOLOGY, CLINICAL LAB.ORATORY TESTS, HISTOPATHOL(__.

STATISTICAL ANALYSIS. AND NORMAL VALUES

I. HEMATOLOGY AND CLINICAL LABORATORY TESTS

The usutal blood sample from dogs is 8 ml, from monkeys 4 ml, andI from racs 0.3 -l for hematology and about P ml for full analysis at termin-

at ion.

I A. Hematology

The following hematological analyees are performed on all blood

samples from rats, dogs and monkeys.

1. Erythrucyte and leukocyte counts: A Coulter ElectronicParticle Counter with 100 A aperture is used.'/ Particle-free diluents(Isoton for RBC, Zap-Oglobin in Isoton for WBC, Coulter Electronics, Inc.)

I are counted to establish the background. Each blood sample is counted induplicate. For each test day, two control blood samples (DiagnosticTechnology, Inc.) are counted separately in duplicate.I _sdee__edi

2. Hematocrit: Hematocrit is determined In capillary tubesusing a microcapillary centrifuge (International Equipment Company, ModelMB). Two control blood samples (Diagnostic Technology, Inc.) are measuredseparately in duplicate.

T3. Hemoglobin: Hemoglobin -s measured as cyanomethemoglobia.2/4 Each blood sample is measured in duplicate. Cyanomethemoglobin (CoulterElectronics, Inc.) is used as the standard. For each assay, two levels ofthe standard are used and two control blood samples (Diagnostic Technology,

Inc.) are measured in duplicate.

4. Methemoalobin (Met-Hb): Met-Hb is measured by the method ofDubowski.3/ A positive control is made by adding potassium ferricyanideto control blood.

1 5. Heinz bodies: Heinz bodies are stained with methyl violet andthe percent of Heinz bodies is %alculated.

6. Mean corpuscular volume (MCV): MCV is calculated as follows:

MCV (M3 ) - Hematocrit x 10Erythrocytes in millions/mm3

1 1ia im ~ ,. . . .. . . .... . . . . . . .. . . . .... _ . - ' . .. l

.-- ";7.

7. Mean corpuscular hemoglobin (MCHb): MCHb is calculated asfollows:

MCHb (gig) = Hemoglobin (gin m) x 10Erythrocytes in millions/fm3

8. Mean corpuscular hemoglobin concentration (MCHbC): MCHbCis calculated as follows:

MCHbC (gin Z) = Hemoglobin (gin %) x 100Hematocrit

9. Differential leukocyte counts: Wright's stain is used tostain the leukocytes for examination.

10. Reticulocyte count: Reticulocytes are counted by the methy-lene blue method using the Miller disc.,

11. Platelet count: A Coulter Electronic Pe_ýticle Countei with70 g aperture is used.-!/ Particle-free Isoton is used as diluent andcounted to establish the background. At weekly intervals, platelets arealso vislally counted in a hemocytometer with a phase microscope forcomparison-1

12. Clotting time (dog and monkey): Clotting time is determined

by the capillary tube procedure uping two capillary tubes.!1 The timeelapsed from the appearance of the blood from the animal and zoagulationin eithier tube is r-asured.

B. Clinical Blood Tests

The following clinical blood chemistry tests are performed onall blood samples from dogs and monkeys and on blood samples frcm ratsat tertination.

1. Blood glucose: Fast_!ng blood glucose is deternined by Stein'shexokinase method. Standard glucooe solution (Dane) is used to establisha standard curve. For each assay, one level of the standard and two con-trols (Reference Serum, Worthington- and Validate, General Diagnos.'ics) aremeasured. "

2. Serui gluta ic-oxaloacetic trans~minsse _: SGCT i&meas,_red by the method of Amador and Wacker..9 Validate and ReferencvSerum are used as the enzyme reference for tach assay.

2

UIJ

I..

3. Serum glutamic-pyruvic transam-.nase (SGPT): SGPT is measuredby the method of Henry et al.LO! Validate and Reference Serum are used as

the enzyme reference for e:ch assay.

4. Alkaline phosphatase: Alkaline phosphatase is measured bythe method of Bowers and McComb.Ail Validate and Reference Serum are usedas the enzyme reference for each assay.

5. BUN: BUN is measured using the BUN Strate Kit (General Diag-nostic) which is bused on the urease method.12/ Three levels of Calibrate(General Diagnostics) are used to establish a standard curve. For each

-. assay, two controls (Calibrate I and Validate) are used as the reference.

6. Creatinine: Creatinine is measured by a modified kineticalkaline picrate procedure.13/ Creatinine Standard Solutions (Sigma Chemi-cal Company) are used to establish a standard curve. For each assay, twolevels of the standard and two controls (Calibrate I and Validate) are

-.. used as reference.

7. Lactate dehydrogenase (LDH): LDH is measured by the methodof Wacker et al.1i4 1 Precinorm E and Precipath E (Boehringer, MannheimCorporation) are used as the enzyme controls for each assay.

8. a-Hydroxybutyrate dehydrogenase (a-HBDH): a HBDH is measuredby the method of Rosalki and Wilkinson.:-/ Precinorm E and Precipath E areused as the enzyme controls for each assay.

9. Creatine phosphokinase (CPK): CPK is measured by the improvedprocedure of RosalkiL6 / based on the methods of Oliver.17-/ Precinorm E andPrecipath E are used as the enzyme controls for each assay.

C. Urinalysis

Urine samples ire collected from animals before and during treat-ment as are the blood samples. Thr urine from rats is collected by slightmanipulation of their body, and samples within each group are pooled. Themonkeys and dogs are placed individually in metabolism cages, and urine iscollected in the stainless steel pan. The urine from each dog and the pooledurine from rats are tested and examined for the following:

1. Protein: Urinary protein is determined with Labstix (AmesCompany, Elkhart, Indiana).

2. Sugar: Urinary glucose nd reducing substance are determinedwith Labstix (Ames Company).

3

.',. .

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 crystals under high power field and for casts under low power field.

A positive urine control prepared with known amounts of proteinand glucose in saline adjusted to pH 6.0 is run wvih each assay to checkthe reliability of the Labstix.

D. Occult Blood in Feces

Fecal samples are collected from animals before and duringtreatment as are the blood and urine samples. Occult blood in the fecesis determined with Ilematest 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

E. Reproducibility

Fo- erythrocyte and leukocyte counts, hematocrit, hemoglobin, and Athe various clinizal blood chewitry tests, the same control blood samplesor control standards are used for day-to-day assays. The replication ofresults are excellent and are summarized in Table A.

The determination of differential leukocyte counts aud reticulocytecounts are performed by experienced personnil. At weekly intervals, a bloodsimp1F is counted by two or more personnal to confirm the accuracy of thecounting. Alao at weekly intervals, the platelet counts obtained from aCoulter Electrouic Particle Counter are compared witn the direct visual

counts in a bemocytometer using a nhase microscope.

2. Reproducibility Withir. a lest Day

At monthly intervals, a blood sample is taken from a control dogand eix or more determinations for erythrocyte, leukocyte, reticulocyte,and platelet counts, hemoglobin, and various clinica] blood chemiatry testsare pecformed to establish the reprnduclbil:ty within an aawiy. The resu,tsare summarized in Table B.

r r

4

3. Proficiency Test Service

We si7scribe to the Prof 4 ciency Test Service of the Institute *

for ClInical Science, Hahnemann Meotcal College, Philadelphia, Pennsylvania(F. Wm. Sundirman, M.D., Director). On the first day of each month, thisservice sends t,'o samples containing two different sera or solutions to alloubscviberg for measurements of one or more of the parameters usually analyzedin clinical laboratories. Participants report their results on a form fur-nished by the service. On the 15th day of the month, each participant re-ceives a report from the service which includes: the results of a statisticalanalysis ot the values reported by all the participating laboratories; acurrent review o0 pertinent methodology; a comprehensive bibliography; andvalidntion o. the results which the participating laboratory reported. Thisservice enables each partizipating laboratory to obtain an unbiased and criti-cal assessment of %ts proficiency in relation to that of 1,000 or so otherclinitcal laboratories throughout the country. The service has been in con-tinuous operation since 1949 and was given endorsement by the American Societyof Clirical Patl-,'ogists fn 1952 and by the Association of Clinical Scientistsin 1957 and 1968. Our results have been found to be satisfactory and aresummarized in Table C.

JTr II. HISTOPATHOLOGY

A. Necropsy and Gross Examination 'I

At termination or prior to imminent death, rats are killed withether, and dogs and monkeys with an overdose of sodium pentobarbital. Animalsthat die on tests are.kept refrigerated but not frozen until necropsy. Thegeneral physical condition and nutritional status of each animal at the timeol death or termination are observed and recorded. Necropsy is performed assoon as possible after death. Gross changes of all tissues are carefullyexamined and recorded.

B. Organ Welghts

The brain, liver, spleen, kidneys, adrenals, thyroids and gonads iare trimmed free from surrounding tissues and weighed. The organ weight tobody weight and/or brain weig•ht ratios are then calculated.

I 5II! ,,'.- •--.

C. Tissues for MicrosLopic Examination

Tissues to be examined include the eye, skin (breast), trachea,lung, tongue (except rat), salivary gland, liver, gallbladder (except rats),pancreac, esophagus, fundic and pyloric stomach, duodeoum, joJunumn ileum,cecum, tolon, kidneys, urinary bladder, gonads, and accessory organs,diaphragm and gracilis muscla, anterior pituitary, thyroids/parathyroids,adrenals, tonsil (except rat), thymus, spleen, prescapular (except rats)and mesenteric lymph nodes, rib bone with bone marrow, brain (sagittalsectiov for rats; coronal sections of cerebral cortex, cerebellum, and:braitn stem for dog and monkey), spinal cord (lumbosacral plexus, dog andmonkey), sciatic nerve and any other structures not mentioned which showabnormal gross changev.

D. Finetion and Staining of Tissues

AK1 . tissues are cut not to exceed 1 um in thickness for fixation.For nzot tissues, neutral buffered 10% formalin is used. Sufficient volumeof Yixing solution is used and the tiszues are changed to a fresh solutionafter 24 hours. ITe iixed tissues are processed in an Autotechnicon fordehydration, clearing, and infiltration and then embedded in paraffin.Roucine H & 7 staining ia used to 6uain the sectioned tissues for micro-scopic examination.

Supplementary tissue fixatives and staining techniciues may beemployed for more positive ident!ficatiun of special lesions such as calci-fication, pigments, !Pt depost.itio and other abnormal changes.

III. STATISTICAL ANALYSIS

Data are analyzed statisticeilly usina the Duqnstt's multiple com-pVrisoo nrocedure following an analysis of variance,18-/ or our modificationof tnis procodure for uneven numbe. s among groups. The chosen criterionoigniffeance ig p C 0.0•. The means of each group at various intervalsduring treatmerit are comnpared with pretreatment levels. For most experi-ments in-beagles, three baseline (pretreatuent) levels are obtained. Thebaseline levels for each animzl are averaged and the mean is used ip theanalysis. In addition, the means of the various treated groups are comparedwith that of the co:trol group at the respective time iutervals.

6

I ~IV. NORMkL VALUES

A.Hematology, Clinical Laboratory Tests and Bone Marrow

Since June 1971, we have used about 180 rhesus monkeys (WoodardR~esearch Corporation, Herndon, Virginia, Primate Imports, Port Washington,New York, and PrimeLabs, Inc., Farmingdale, New Jersey) f or various studies.The peripheral blood elements and clinical blood chemistry values of theseI monkeys before treatment and the myeloid/erythroid (M/E) ratio of the bone

t marrow of the monkeys used as normal controls varied among individual ani-

t mals. The mean *j S.D. and the range of the various parameters for the malesaud females are summarized in Tables D and E, respectively.

Since September 1971, we have used about 525, 5 to 9 months old,II 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.

x ~During the same period, we have used about 500, 7 to 10 weeks old,male albino rats (CD® Strain, Charles River Breeding Laboratories). Asfor the dogs, the individual variations of the peripheral blood elements,clinical blood chemistry values and the M/ E ratio of the bone marrow were

large. The mean ± S.D. and the ranges of the various parameters for thesemale rats are summarized in Table L.

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 F'I and G, J and K, and M, respectively. These were control animals used be-tween June 1971 and December 1976.

C. Presence of Various Substances in the Urine

I Various substances occasionally occurred in the urine of monkeys,dogs and rats. The results are summarized in Table N. Large percentage ofurine samples from monkeys contained epithelial cells, i.e., 34.7% to 52.0%.I Other substances occurred in 8.1% or less of the urine samples.

Tn dogs, protein, erythrocytes, leukoc.ytes and epithelial cellswere 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,

7

crystals, and casts occurred in less than 2% of these samples. Some dogshad been bled and returned to the maetabolism 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: Evnluation of theElectronic Red Cell Counter. Am. J. Clin. Path., &: 1439, 1956.

2. Selegson, D.: Standard Methods of Clinical Chemistry, Academic Press,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, H. U., ed.),p. 117, Academic Press, New York, 1963.

8

_ _ _ ____ ____ _ _j

. ... . .................

9. Amador, E., and W. E. C. Wacker: Serum Glutamic-Oxaloacetic Trans-

aminase Activity: A New Modification and an Analytical Assessment3 of Current Assay Technics. Clin. Chem., 8: 343, 1962.

10. Henry, R. J., N. Chiamori, 0. J. Golub, and S. Berkman: RevisedSSpectrophotometric Methods for the Determination of Glutamic-

Oxaloacetic Transaminase, Glutamic-Pyruvic Transaminase, andLactic Dehydrogenase. Am. J. Clin. Path., 34: 381, 1960.

11. Bowers, G. N., Jr., and R. B. McComb: A Continuous SpectrophotometricMethod for Measuring the Activity of Serum Alkaline Phosphatase.3 Clin. Chem., 12: 70, 1966.

S12. Chaney, A. L., and E. P. Manback: Modified Reagents for Determination3 of Urea and Ammonia. Clin. Chem., 8: 130, 1962.

* 13. Lustgarten, J. A.: A Simple, Rapid, Kinetic Method for Creatinine

. Concentration. Clin. Chem., 18: 1419, 1972.

- 14. Wacker, W. E. C., D. D. Ulmer, and B. L. Vallee: Metilloenzymes and' Myocardial Infarction. II. Malic and Lactic Dehydrogenase Activities

and Zinc Concentrations in Serum. New Eng. J. Med., 225. 449, 1956.

S15. Rosalki, S. B., and J. H. Wilkinson: Reduction of a-Ketobutyrate byHuman Serum. Nature (London), IM 1i10, 1960.

* 1 16. Rosalki, J. B.: An Improved Procedure for Serum Creatine Phosphokinase- Determination. J. Lab. Clin. Med., 69 696, 1967.

1 N 17. Oliver, I. T.: A Spectrophotometric Method for the Determination ofm Creatine Phosphokinase and Myokinase. Biochem.-J., 61, 116, 1955.

18. Dunnett, C. W.: A Multiple Comparison Procedure for Comparing SeveralTreatments with a Control. J. ka. Stat. Assoc., 50: 1096-1121,1955.

I 9

TAELE A

REPRODUCIBILITY AMONG TEST DAYS ON THE -R

SAME CONTROL SAMPLES OR STANDARDST-a

No. ofDeterminations Mean t S.D. Range

Erythrocyces (x 10 6 /-u- 3 )

Normal level 20 4.51 ± 0.07 4.36 - 4.67Abnormal level 20 2.32 1 0.04 2.25 - 2.40

Hematocrit (vol Z)Normal level 20 44.3 * 0.40 44 - 45Abnormal level 20 22.8 ± 0.60 22 - 24

Hemoglobin (gmi %)Normal level 20 14.2 * 0.20 13.6 - 14.5Abnormal level 20 7.4 f 0.20 6.9 - 7.8

Leukocyte Counts (x 10 3 /mn3 )

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 + 7.5 151 - 178SGOT (lU/1) 23 61.7 ±3.9 55 - 68SGPT (IU/1) 23 51.3 ±2.6 46 - 55Creatinine (, 18 2.2 ±0.3 1.6 - 2.6BUN (mg %) 19 9.8 ± 0.2 9.5 - 10.2

Bilirubin (mg 2) 11 0.8 _ 0.1 0.8 - 1.0Alkaline Phosphatase (lU/i) 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 ir, December 1976.

10

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3 TABLE B

REPRODUCIBILITY WITHIN A TEST DAY

I ON THE SAME SPECIMM6-!

Mean * S.D._/ Raige

Erythrocytes (x 10 6 /m"3 ) 5.9C ± 0.14 5.73 - 6.08

Reticulocytes (M) 0.63 ± 0.12 0.44 - 0.79

I Hematocrit (vol X) 46.8 ± 0.6 46.0 - 47.5

Hemoglobin (gm %) 16.1 k 0.2 15.8 - 16.1

Platelets (x l051Mn 3 ) 1.56 ± 0.07 1.49 - 1.66

Leukocytes (x 10 3/mm 3) 10.8 ± 0.4 10.2 - 11.3

Bands (M) 0 0 0-0 iNeutrophils (%) 64.3 * 3.1 61 - 69

I Lymphocytes (M) 29.0 * 4.9 23 - 35

Eosinophils (M) 3.2 t 0.8 2- 4

Basophils (M) 0 0 0- 0

Monocytes (M) 3.4 ± 0.9 3 - 5SAtypical (%) 0 0 0 -0

Nucleated RBC 0 0 0 0

SI Methemoglobin (gin %) 0 * 0 0 - (

Pasting Glucose (mg %) 96.7 + 3.0 32 - 101

GOT (IU,) 23.2 •2.8 21 - 28SSGPT (IU/I ) 25.3 ± 2.1 24 - 28

Creatinine (mag 2) 0.6 ± 0.1 0.5 - 0.6

BUN (mag %) 9.0 ±0.0 9-9

Alkaline Phosphatase (IU/i) 63.5 ± 1.1 62 - 65

CPK 44.0 1.6 43 - 46

LDH 38.5 ±1.6 37 - 40

HBDH 42.0 1.6 40 - 43

a/ Performed in October 1976.b/ Six determinations from an adult beagle blood sample.

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TABLE C 1PROFICIENCY TEST SERVICE (PTS) REPORTS (1975-1976)a!

Participating__Laboratories

MRI PTS (10-90 Percentiles) AcceptableUnknowns Results Results Median Mean Performanceb/

Hemoglobin 13.8 gm % 13.8 13.8 13.8 13.6 - 14.018.1 gm % 17.9 17.9 17.8 17,.6 - 18.2

Serum Protein 6.6 mg % 7.1 7.0 7.0 6.7 - 7.3

Fasting Glu ose 272.0 mg % 264.5 266.0 263.0 240 - 290

229.0 mg % 221.4 220.5 222.5 200 - 240

BUN 12.1 mg % 12.0 12.0 12.2 11.0 - 13.038.4 mg % 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 - 4.6

1.3 mg % 1.78 1.80 1.77 1.5 - 2.1

Cholesterol 175.0 mg % 161.4 161,0 162.0 1-6 - 175100.0 ms % 109.8 109.4 111.0 98 120

Ca 15.7 meq/1 15.4 15.4 15.3 14.1 - 16.4

9.5 meq/f 9.8 9.8 9.8 9.2 - 10.3

Na 156.0 meq/j 155.8 156.0 155.5 153 - 158

7.3 mneq/1 7.5 7.5 7.5 7.3 - 7.7

Cl Q6.0 meq/1 97.8 98.0 97.5 96 - 10178.0 meqe 79.4 79.0 80.0 77 - 83

M8 1.0 meq/i 1.1 1.1 1.2 0.9 - 1.41.9 mtq/P 2.0 2.0 2.1 1.8 - 2.3

_/ To date, w have received unknowns ýor phosphorus, uric acid, and serum

iron. We do not routinely perform these deteiminations.b/ Basad on vclues submitted. by participants by 10th of month.

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3 TABLE F

ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF MALE RHESUS MONKEYS-a/

AbsoluteI Organ Weight Mean ± S.D.

Liver (gm) 82 ± 17 64 - 122Spleen (gn) 4.6 ± 1.8 2.0 - 9.3Kidneys (gm) 15.1 ± 3.8 8.0 - 22.0Adrenals (gin) 0.73 ± 0.15 0.45 - 0.86Thyroids (gm) 0.57 ± 1.30 0.37 - 0.81Testes (gin) 1.29 ± 0.57 0.53 - 3.30

Relative (per kg body weight)Mean ± S.D. Range

Liver (gin) 23.4 ± 2.5 18.8 - 30.4Spleen (gm) 1.25 ± 0.47 0.57 - 2.38Kidneys (gm) 4.13 ± 0.92 2.20 - 6.43Adrenals (mng) 201 ± 44 129 - 254

Thyroids (mg) 154 ± 42 86 - 250Testes (gin) 0.34 ± 0.11 0.18 - 0.53

a/ Data collected between September 1971 and December 1976 from 17monkeys weighing 3.71 _ 0.48 kg, used as control animals.

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TABLE G

ALSOLt"E AND R'thATIVF ,..,GA- WTrHTS OF FEMALE RHESUS MOmNKEYS!

. AbsoluteOrgan WeiAffx. Mean t S.D. Range

Liver 8 8 17 64 - 122

Spleen (n') A±F 1.4 2.0 - 6.0

Kidneys (gin) 14.5 ± 2.8 11.0 - 20.0Adrenuls (gmi) 0.68 ± C.16 0.53 - 1.14Thyroids (gm) 0.6J ± 0.20 0.37 - 1.11Ovariev (gin) 0.28- ± O.1O 0.14 - 0.45

Relative (per kg body weight)Mean ± S.D. Range

Liver (gin) 25.4 t 5.8 19.2 - 37.43p3een (gm) 1.16 t 0.49 0.60 - 1.89Kidnevs (gm) 4.40 ± 0.86 3.20 - 6.25Adrenals (mag) 212 ± 80 138 - 438Thyroids (mag) 173 ± 66 97 - 346 jOvaries (mg) 82 ± 28 43 - 140

a/ Data coLlected between September 1971 and December 1976 from 11monkeys weighing 3.39 ± 0.58 kg, used as controls.

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i I ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF MALE BEAGLE DOGS'/

Absolute

Organ Weight Mean S.D. Range

Liver (gm) 264 _ 51 166 - 384

Fpleen (gm) 58 25 22 - 167

Kidneys (gm) 53 1 10 32 - 71

Adrenalo (gm) 1.12 ± 0.26 0.74 - 1.75

1 Thyroids (gm) 1.03 ± 0.32 0.55 - 2,50

STestes (gm) 6.60 ± 4.56 1.32 - 18.00

Relative (per kg body weight)

Mean ± S.D. Range

I Liver (gm) 27.9 ± 4.2 19.6 - 42.3

t 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 (ing) 108 ± 34 56 - 211STestes (gm) 0.67 ± 0.39 0.13 - i.67

a/ Data collected between September 1971 and December 1976 from 51 dogs,

weighing 9.3 ± 1.8 kg, used as control animals.

191

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TABLE K

ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF FEMALE BUGLE DOGSS/

AbeouluteOrgan Weight Mean t S.D. Range

Liver (gui) 218 ± 51 106 - 322Spleen (gmi) 48 ± 21 16 - 103Kidneys (gm) 43 ± 9 24 - 71Adrenals (go) 1.04 ± 0.26 0.49 - 1.65Thyroids (gu) 0.88 ± 0.25 0.55 - 1.91Ovaries (gm) 0.74 ± 0.24 0.38 - 1.27

Relative (per kg body weight)Mean t S.D. Range

Liver (gu) 28.2 ± 5.0 20.7 - 38.8Spleen (gmi) 6.0 ± 2.3 3.1 - 10.9Kidneys (gui) 5.5 t 0.9 3.7 - 7.9Adrenals (mg) 135 ± 35 67 - 215Thyroids (mg) 112 ± 31 75 - 219Ovaries (mg) 96 ± 33 54 - 222

a/ Data collected bet~een September 1971 and December 1976 from 49 dogs,weighing 7.7 ± 1.5 kg, used as control animals.

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TABLE M

ABSOLUTE AND RELATIVE ORGAN WEIGHTS OF MALE ALBINO RATS-!/

Absolute .organ W,.eight Mean t S.D. Rz

Liver (gi) 10.89 ± 2.87 7.18 - 15.09Spleen (gin) 0.65 ± 0.11 0.34 - 0.89Kidneys (Sm) 2.64 ± 0.37 1.84 - 3.58Adrenals (mag) 63.u+ ± 9.5 21.9 - 73.5Thyroids (mg) 26.3 ± 5.8 14.3 - 37.7

Testes (gin) 2.98 ± 0.51 1.76 - 3.81

Realative (per 100 am body weight)Mean - S.D. Range

Liver (gm) 2.96 ± 0.42 2.09 - 4.01Spleen (gin) 0.19 ± 0.08 0.10 - 0.30Kidneys (gin) 0.76 t 0.10 0.22 - 0.88

Adrenals (mag) 18.6 ± 5.8 5.8 - 22.4Thyroids (mg) 7.6 -± 2.7 4.2 - 12.7

Testes (gia) 0.87 t 0.15 0.23 - 1.09

a/ Data collected between September 1971 and December 1976 from 139 rats,weighing 352 ± 59 gm, used as control animals.

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TABLE 0

PRESENCE OF OCCULT BLOOD fIN THE FECES OF VALEAND FEMALE NM S AND DOGS

3pecies: Monkeys Dos-No. of Animals: 44. 8 11-87! 30

No. of Collections: 44 48k/ 116i 156b/

Occult Blood: Negative 90.9 (40)S/ 95.8 (k6) 94.1 (111) 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 intervals for 4-7 collections from controlsemployed between July 1974 and December 1976.

c/ Percent of total (number of samples).

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