CONFIDENTIAL THE UNIVERSITY OF MICHIGAN SCHOOL OF DENTISTRY Progress Report Period: July 1, 1962, to March 1, 1963 ACTION OF DETERGENTS AND FLAVOR AGENTS ON LIVING TISSUE J. K. Avery S. S. Han P. J. Martin T. C. Neumeier ORA Project 03420 under contract with: COLGATE-PALMOLIVE COMPANY NEW YORK, NEW YORK administered through: OFFICE OF RESEARCH ADMINISTRATION ANN ARBOR February 1963

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PART I TISSUE CULTURE STUDIES

RESEARCH PLAN FOR TISSUE CULTURE STUDIES ON DETERGENTS AND FLAVOR AGENTS I. VIABILITY STUDIES, GROSS A. Gross and low-power microscopic observations of the effect of dentrifrice agents on monolayer cultures in flasks and culture tubes. B. Concentrations of 2%, 1%,.6%,.4%,.2%,.1%,.06%,.04%, and.02% used. II. VIABILITY STUDIES, MICRO A. Immediate effect of low concentrations on cell viability. B. Time effect of low concentrations on cell viability. 1. To gain information concerning differences in detergent or flavor agent action after prolonged contact with cells. 2. The viability of the cells will be based on the uptake or exclusion of vital dyes. a. Dye uptake tests-methylene blue and tetrazolium stains. b. Dye exclusion tests-trypan blue, and erythrosin B. III. GROWTH AND PROLIFERATION STUDIES A. Very low concentrations of flavor agents or detergents used in combination with a seven-day monolayer of epithelial and/ or connective tissue cells. B. Cells counted at various time intervals to study relative effect of very low concentrations of detergents or flavor agents on the growth and proliferation of the cells. 2

IV. TOXICITY STUDIES A. To determine normal cellular characteristics and any changes in cell size, shape, opacity, alteration of nucleus or nucleolus by low concentrations of detergent and/or flavor agents. B. To determine normal cellular structure and any changes in the cell in relation to "specific" or "nonspecific" effects of detergents and/or flavor agents. 1. Histochemical studies 2. Time-lapse cinematography 3. Electron microscopy V. METABOLISM STUDIES A. Glucose requirements of the cells and rate of lactic acid formation with the various detergents or flavor agents. B. DNA and RNA levels determined; alteration in metabolism ascertained. VI. ORAL BACTERIA STUDIES A. The concentrations of the various detergents or flavor agents necessary to affect various oral bacteria. B. The effect of the time that various detergents or flavor agents are in contact with various oral bacteria. 5

INTRODUCTION This report is a summary of the progress on the research contract entitled "Action of Detergents and Flavor Agents on Living Tissue" during the period July 1, 1962, to March 1, 1963. During this period study has been concentrated in the area of microscopic analysis of cell viability after contact with low concentrations of the antiseptics Thiomersal, Cetylpyridinium chloride, and Hyamine 1622,and the detergent Tween 60. Relative effects of these reagents, using a method for analysis of the lethal dose for 50% of the cells, comprises Part II of the research plan. Growth and proliferation studies of HEp2 cells affected by these same reagents were done during this period. Viability studies were included here also in order to gain more accurate data than could be obtained from cell counts alone because viable and nonviable, nonlysed cells cannot accurately be differentiated by cell counting methods. In the present studies, cell counts coupled with dye exclusion tests were run to determine more accurately cell growth and viability. This is Part III of the research plan. Toxicity studies also were carried out during this period. Both histochemical studies and electron microscope studies are included in this report. No time-lapse studies are reported at this time as both the camera and the time-lapse equipment have been undergoing repairs and modification. Some data have been collected but not sufficient to report at this time. It will be included in a future report. The histochemical studies reported are in concern with the reagents Thiomersal, Cetylpyridinium chloride, Hyamine 1622, and Tween 60 at several concentrations. The May-Grunwald-Giemsa staining procedure was employed to differentiate DNA proteins and RNA proteins and the McManus Sudan Black B stain to demonstrate the presence and location of lipids. The electron microscope study includes ultrastructural changes of the HEP2 cell after introduction of 0.0075% sodium lauryl sulfate into the cell suspension. This is Part IV of the research plan. During this period Oral Bacteria Studies were continued in which the bactericidal and bacteriostatic effects of Thiomersal, Cetylpyridinium chloride, Hyamine 1622, and Tween 60 were evaluated using Lactobacillus acidophilus. These studies were a repeat of those described in the July, 1962, report and the findings are similar. This is Part VI of the research plan. 4

II. VIABILITY STUDIES, MICRO A. PURPOSE This study was initiated in order to determine the relative effects of certain reagents on HEP2 cells (human epithelial cells) over a relatively short period of time and at low concentrations. The chemical substances used in the investigation were Thiomersal, Cetylpyridinium chloride, and Hyamine 1622 (antiseptics), and Tween 60 (a detergent). B. PROCEDURE During the middle of the log phase the HEP2 cells were harvested from a suspension culture. Using the growth medium, Eagles75-tryptose phosphatel5calf serum10, the cells were diluted to a concentration of 100,000 cells per milliliter. One-tenth ml of reagent solution and 0.01 ml of 0.4% erythrosin B staining agent were added to 0.9 ml of the cell suspension five minutes before commencing the count of viable cells. The concentrations of reagent used were 0.0001%, 0.00025%, 0.0005%, 0.00075%, 0.001%, 0.0025, 0.00%, 0.0075%, 0.01%, and 0.025%. Controls were run for each concentration of each reagent. Over a period of 25 minutes the viable and nonviable cells were counted at 5-minute intervals with a hemacytometer. Using the Reed-Muench method* the lethal dose for 50% of the cells (LD50) was determined. C. RESULTS At the various low concentrations of the reagents used, Cetylpyridinium chloride appeared to be the most toxic. After 5 minutes the LD50 occurred at a concentration of 0.0074%. After 25 minutes the LD50 occurred at a concentration of 0.0043.^ Hyamine 1622 was the least toxic, producing no LD50 at 5 minutes. At 10 minutes a concentration of 0.0224% produced an LD50 and at 25 minutes the concentration required for an LD50 was 0.0216.Tween 60 showed an LD50 at a concentration of 0.0197% after 5 minutes and 0.0144% after 25 minutes, a very slight decrease. Time apparently is not a factor in the toxicity of Tween 60 or Hyamine 1622. *Reed, L. J., and Muench, H., Am. J. Hg. 27:493, 1938. 5

Thiomersal, however, showed a relatively great increase in toxicity as the time interval increased. At 5 minutes the LD50 occurred at a concentration of 0.0234* but after 25 minutes only 0.0008* was required to produce an LD50. Controls which were run concomitantly showed a viability of from 835 to 951. See tables and graphs following. TABLE I LD50* OF REAGENTS AT VARIOUS TIME INTERVALS Minutes Reagent 5 10 15 20 25 Thiomersal 0.0234 0.0135 0.0028 0.0008 0.0008 Cetylpyridinium Chloride 0.0074 0.0072 0.0072 0.0066 0.0043 Hyamine 1622 -- 0.0224 0.0208 0.0206 0.0216 Tween 60 0.0197 0.0166 0.0163 0.0164 0.0144 *Expressed in percent. 6

Detergent Number 00-101-00 5 Minutes 900 I||| 800... I II 600 ^1 i iI II I N 11 NIN I~i llllIF 500 i I:- I LI; LJ rlL~~~~rU:IL II~~~~ TrrrlII I L ~~~~L LT7~~~~~~~~~~~~~77I...... IL L II ^; ^|| I II N 1 III ^IN 11111.11I 1 500~ ~ ~~~~~~~~III ---- I|I|I I LI I I::::::::::::i:i. 200 *::::::::: r-1~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~L -1 Is I I:::: J:::: 100 IF1 I I \ FI Debtergent Cloncentration 7~~~~~~~~~~~~~~~~~~~~~~~I

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Detergent Number 00-10100 15 Minutes 800 |||||;||||^ || IIH IIIIIII Illllllllll ll llll~ llllli illl^ III^:::1 1 ]II 600 Ilil~ liillill ^ II l~ ill~ llllllllllll~ lll~ llll~ [[IIIl lilt^ 1Itllllll^ ^::^ 400~~ ~~ ~ ll^ ll I^ illlslll lt1::::::i:i::::^ ^i i i::^ ^ ^::::i:: 3 ^oo illllill1^!! i^ ^^^^^^^^^^^^^^^^^^^^^N Iiif 900 5Q.O~~~~~~~~~~~~~IIIft II I 400~ ~~~~~Ii I tll II.11 II fl 300~ ~~~~ttl il ]ill I 11 - - - - 1 1 l t l I J l a,~ ~~ ~~~~~~~~~~~~~~~~~111 Hl[ III ll P~~~~~~~~~~~~~~~~~~~~~Il ii l[ ]l a~~ ~ ~~~~~~~~~~ — ilIIII I ii Hl]IIll I rt~ ~ ~ ~ ~~~~~~~~~~itIl IlI.il ite i i rl 200~ ~~~~~~~~~_If f il9 IlI ii IIII a)~~~~~~~~~~~~~~~~~~~~~IIJl U~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Il II ll 700 llt. ll l tl.It 1 Iis till Ill I Illt a~io t i t l e I 1. I - I l l I I l

Detergent Number CO-10100 20 Minutes IIIIII I IIIIII 11 IIIIII I III lilt IIIIIIIIII lim it. lim it lim it. ] Film I I lilt l i t t l e I I I f i l l l it t l e [ [ [ [ [ [llI l i m i t- [ I lim it [little ]little LI I M T-1711171 r1% T11, CT I IliltI [if ILI in LVI-VW -1 fill 11 l i l t I I I I l i t I 1 1 t i l l Ililt I l i l t t i l l i f I I l i l t l i l t ] goo 1 1 f i l l H+ I l i l t l i l t I I f i l l l i l t I I t — +Ff+ l i l t t i l l I I 800 11 fill It I I I I L 744+ _ 1 1 1 1 1 1 1 t I I I I I I L I I I I -F-L lim it lilt 11 I f i l l i I l l l i t I l l [... f l a t [ t i l l I l l l i t 1 1 7 0 0 Ill JIF fill Ill [fill ]film f i l l I l l 1 1 1 1 11 I f f i l l f i l l I I t 1 1 I t I I l l I l l - 1 1 1 1 1 1 l i l t I I f lit tle lim it [[Ill ] f i f t h I I I I I.. I I I t I l l I I l l I.. I [ i l l I fi l l ifililli fill 11.111 I Ill - -.... I. lilt I I-I I I t I f i l l I I 11 111111. lilt II 1111 I l it I l it I [I ll I I [i ll I I, I" L -L f l i l l l i l t I l i l t I I l l I l l I [ i f 1 1 l i l t I F i l l I I L L I t i l l I L IIL 1_" _T T-I- -I- If I f i t 1 1 l i l t I H i l l I l i t t l e 1 1 1. - l i l t l i l t I t, -f i l l I i f i l l I l i l t [ ] f i l l H i l l l i t f i l l I 1 1 1 1 1 1 1 1 l i l t I f 1- 1 I I I I 1, f i l l i l l l i l t I l i l t I l i l t I I l i l t I f i l l ] t i t l e t i t l e [ l i t t l e [ l i l t 1 1 [ f i l l I ) I I I t i t l e f i l l I f i l l l i l t [ I f I l i l t l i l t l i l t 1 6 0 0' --- --- III I I.1 I Ill I III I r TL_ I I I i f I I f i t, IfI IF I III FTTTAIif II 171 IlitI I I I L I I tIIIttI 5 0 0 III ILI I I IIII I I I I II II IIIIII I II 1.1 II I I I I I I I I III l i l t I l i l t t i t l e I t i l l I l l [ f i l l I l i l t f i l l [ I l l I l i l t l i l t f i l l l l l i l t 1 1 f i l l i t l i m i t l i t t l e f i l l I f i l l 1. 1 1 f i l l I I W i t I I - I' l l I. l i l t I I f i l l f i l l I l i l t f i l l I l i [ -1 1 1 I I l i l t I I I ) I 1 1 1 1 1 I- t i t l e'' I f i l l I l i l t f i l l. l i l t H i l l I I l i l t l i l t I I I I I I I l l i f 1 1 I L - I l i l t lilt ]]if li lt -L- H ill I I I I I t + 4 0 0 I lilt I fill I I lilt ] f i l l I t i t l e I f i l l l i l t AF Jr+fill l i l t I I f i l l f i l l l i l t I f i l l [ i f f i l l 1 1 I l i l t I l i l t - - I l i l t I I l i l t I I I t i t f i l l I 11- 1 1 I l l- I 1 1 I I I i l l I 1 1 I I I I I I I I I l i t I f a i l II f i l l 1 1 ) f i l l I f [ I I f I I I I I Ar 1 I t a I IIILAII PII 3 0 0 1111 - - - - - - I A III IIII I I I I I I I l i l t I I Lpi l l I i t I I I I I A l i [ I li l t I IIIL + - + IifII I tt It t -T Fri _T TFTII IfI [ [ t i l l l e f t is t I iA II _TF I IffII I1 Tl I-I LMIII taII.tIt 200 1U Iyr-1 -dyn -F _T I L- - - - - - - 44+ -H I I I L............. - - - - - - _T I -LaRT I I.fI 10 0 II I.1f I I II I I V I I I I I I I I ( I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I El I I I I Ill lilt[ lim it A I IIIII lim it little It It + + I I A L-T I LLL LLI _LL +H+f++++

Detergent N umber 00-10100 25 Minutes 4~00~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 - - -4 l l t 9 00 a,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I ltl 800 1 1 I I -T- 1 1 I

Detergent Number 00-11217 5 Minutes 900 800 700 70:^: s o:::I: I l il l,oji |^ |i^ LI I I I I I.o I ~fo 0 0 ill7I i 100 12

Detergent Number 00-11217 10 Minutes Ilia fill 800 500 300 ~o~~~~~~~~~~~~i rH Q) 700 9oo~~~~~~i | H || I ^~~~~~~~~~~~~~~ I L HI I.1l I I: I:::::::: 6oop ^~ ~~i I 1 --------—::::::::::::: \ \ \ I ^ 600 O0 5.0025'5tergent Concentra-tion z3 9000.0(1 i l l I) I I I~ f 400 I | i i i^:: i:: i: H:: =::::::::::::::::::: - -:I::: I I I I I I:-T-::I-1 III 2 0 0::::: F: i i i::::::::;::::::::::::::: %^1^^^5''^'1^'^5','''^*^^^~~~~~~~~~~~~~~~~ LI II III ~ ~ Dtegn I olicenI atlIt ff-h I T ~f f1I

Detergent Number 00-11217 15 Minutes soo~~~~~~~~~~~~~~~~~~~~l ~~ ~ 1 I 111 i|^ f1l |I Il I|s Iii II. IIIN III I^:::::::: III::::::: 500 | \| 9 0 0 It I\ I l. \ 1 IL *: l i:t I: II: I:: i::: 3 200 ^ ^ II''ill 11 II[I I I^ ^ I I^ III ^^:: fill.N 100 \\\ \ \ |;;; n \ -! \ \ ^ ^ Illllill IIIf I fll IIII 1 1 1:::::::: )]on^ i ^ feel Ill1 a>~~~~~~~~~~Ifl i it 800 i 1.1 l t QT~~~~~~~~~~~~~ ll C)~~~~~~~~~~~~fl l I [ i l 700 I 0.1 I I S.01.02 Detergerrt Oon~~~~~~~~~~~~~~~~~enerati on ~ ~ ~ 111 fll I f l

Detergent Number 00-11217 20 Minutes oJ| \l \ u0X Jl i |I I 3 700 7 o | | | I I I I I I I I I I I1 1 1 I Il L Ild:I:: L r::::::: I I: I: T: 500 T.|..|...|T T T ||i T| ||| 1111 l l| | l | | | | |i| l1 1 | ^ 1E 400 1O0 3010 ^ I HI 1 ||^ ^| | IH\i 1 MM^ ^ ^^^1^1^^1^1 ^il^^ li~tI~l1T I Il I1 f1|[ I I ~.0025 0 0075 Detergent 0oncentration 90oho%.o25U.UI P:IT- I III= I <D::':::::::::: "' — ^ ^ i::::: =:= =::: =:::: ==::::::::: =: = =:::::::it::: ^ ^:::..'.::::; ^:: ^ E^ ^:::i:: =:::::^:: i:::: =::::::::::::::::::::4:::FT=::::=:::::: tz~:::::!^::::::::::: =j:=: =:::=:::::::::: ^::::::: =::: =::......:::: = 44=-H^ =

Detergent Number 00-11217 25 Minutes 900 800 700 I l 600 500 400 1 F L I r ~ I-I 3~ LI~I 50 |^I ||i I rl I: 11: ||^ ||i| ^'\^^:i^i^^^^^i^ ^^i^::: 300 h,-I 200 i 10O0 AT ^oo^^^ ^ii^ 0.01 4 2 Detergent Concentration 16

Detergent Number 00-11335 5 Minutes goo I 80O' 700 lO 6OO i a IlJ il li || I... - I If i l l I t t I~~~~~~~~~ ~~~~ lil [if1E[3H J3FEE 600 500 11 | \ \ \ 1^1 i| i ^ \\ | ^ I N I; I I N ^^ i^^^^^I I I I I I it] ^ ^::::::: 300 2OO <|> ~ ~ ~~ ~ I. -. - I — -, I ---- --- -- - -7 -::::::: I0I 40025'~"~000075 Detergent Ooncentrati on 17 3S 00 ^ \ \ [! i ^ \ \ \ -'.~::':::::: 200 i~~~iin \^ \ ^ \~ I I I 1 I N I I i I: 1:::: 1004::::::::::: 1 ^;^:::|;:| | 00^^l^~~~~~~~~~~~~~IitI1 i il Detergent~~~~ Concet atio 17

Detergent Number 00-11335 10 Minutes 500 H 200 a il 00.0025 0050. 00 1 l7 Detergent Concentration 18

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Detergent Number 00-11335 20 Minutes I I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9OI 800 700 900:I I IILLLTYU I. 7AA I)T ] 1I LI i 111: I: ~ ~ i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I. itI i 11 l l; I! I I! ~ I~~~~~~~~~~i i~1I 1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I II I i71 I I I I I I I IITI a~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I T I?;; I ll'l -.' J ] Ir I I. ~' ] I I A l i] ]lJ it Iil lill llif ]Z~l lJ I I N J I I lI 500 I i~~~~~~~~i I I if~~~~~~~~~~~~~~~~~~~~~ r~~~~~~~~~~~~ I I fil 1 1 li, i ~71. 1 2 0 0 I -LII~illll_ 600 1 I l l f i l l F I I O I II L1 r~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I iTII i I I~t i - Tourrrrr I~~~~ i ui~~~F I I I 1 1 I I II I1LriLT 1 i i rrr~~ ~~~~~~~~~~ It i I I I I II I I III I I ( )::) I 4 0 - II 4 I I II I II I l II i f I 3 0025 T l a i I I 0 I I I I I i.7 2~~~~~~~~~00 I I I ~~ ~ FiiF~~ff~~S~T~~r~m~j7- JJ7~ ~ ~ rITI ~ ~ nmT V I I I I I ri it I I 1. i-1rT1;:'1 I~1'Tr-~ I~~ttiF~~~-i- ~~~F~~tF;~~~3f~~ffIf I a)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.1I-H-F C) ~ ~ -c~cl~- CcC~CLCU~i Icl~-~-li~~-;-LLI-L~~-LI ~-~L~LLLLL~il.~i-ii~L~-L! ii~tt~i~S~tt1.1 L-L-t L --- it~~~~~~~~~~~~~~~~~~~~~~~~~~!Ilf

Detergent Number 00-11335 25 Mfinutes III 11 H I I II 11 II N N N 1 ~~~~IIIIIIIN.......I| || ||||i i lIlll To 1 I 1 1I:II II I1 I I IN | I00 | fil l III: i i: i: i: i! i:| N | | i 4 0 0 iiii: | il || | (| IIH IIIII II ^ H I 1 1 I ii| \ i:::::::::::::: i::::::::: it I I: I I) r I I I - -:;::::::::::::::::::. - - - - -: 800 ^ I NIIIININI~~~~~~~~hl~~~i^N \\:::::::: ^::::^;:::: ^1 N11 UN 1111 NIN~~~~~~~~~~~~~~~~~~I II I Pl 100~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ | IINN^N~NNI Iil I I IN I I III I IN I I I N I 7-00 DetergeItill 600 ml [Ill-Ili fil2 5~00 P~~~~IPI 4, 00 1J'lI fl I l i l [ i l I i l I P l T I V l I 1 i 07;~~~~~~~~~~~~~~~~~~~~~~-.002~~~~~~~~~~~~~~~~~'0,0075~~~~~~~~~~~~~~~~~~~~~~Ifl 300.111 I Dete ent Co fill Ifo 1 1 i l l [ I I I 1 1 - i l I i l 1

Detergent Number 00-11334 5 Minutes 9OO 7OO~ S0 4OO 3OO 200::i^;:;~ ~~~~~~~~~~~~~~fl [itiiiiilllllllllllllllll^Ill' 1111111111111111111 900 0.00~ 0025 0'0050 0075 0.01 o.2 o l III H I I I IIill --- lll illll ll~ l l I11 1 11 1111 1111t111 1 11 7 | llllll||Dee|rgent |on|entrati on 22 Ifo|^ ii| ti^l~l li~ lllI...ll ll^^ lllll 50(llll~ll fill [ filll ll IIllllll 400 ||||i|| lillllll~ l~ lii~ iill hiiiii^ iiii —----—::: lilit i^ i~ i i 300 II~ l l llilll lll llli ll^1 1 1 ]IIIifllll ll lli ll -::::::: lilt:::::::::::::::: SS:::: ^ Iiii I~i i i~ Ii:: t0:||;i l ll l ^ ^ ^ ^:::::::::: \ \\^::: ii^:::::; i:::::::::: ^:: ^: ^ ^ E: 1 ^::: 1 1: =:^: i: ^: ^:::^: S^ ^ ^ lil 0*0.02 *05.07 -1 - Is ~ ~ ~ Deegn toielralo - - - - - - - - - - - - - - - - - -- - - - - -

Detergent Niumber 00-11334 10 Minutes 900 I ll[ I ~~~~~t~~~~~~~oo ~ ~ ~ ~ ll 300'~~~~~~~~~~~~~~~~~litIItl -I l l ] i t I l l i l i t I l l I 1 1 I IIIIIPII i t I f l i l I l l i I 1 1 - 1 ] i l... I l i t I 1 1 i t. I a ~~~~~~~~~~~~~l l [ IIt t e ll ll [ II f l i l l =; 200~~~~~~~~~~~~~~~~~~~~~~~~~~~liteIFF I i t I l l I i t I. f i i l. I..... [ l t l 1 1 i l I i l i i... l i m i t [ l i t t e i m i t l i t l e l i t l e I tergent Ooncentra~~~litle itte on l l i t I s e f )l i t ] 1 1 1 1 f l l

Detergent Number 00-11334 15 Minute's 900 i~~~~~~~i \\\:. | |~~~~~~~~~~~ ||il |111l I| I| ||I ||I I^ I^ I^i ^^ I: TOO: ^: |||| I \ \\ \ \\\\\ \\\\ \\ ^ ^ ^[\': ^:': \\ \^ \ \ il 1I II I 500^ || ^ ~~IF 1: II I II::1 \ \ \ \1II ^ \, S~~~~~~~~~~~~~~~~~~~I I011;1 II I I I Ii N I I^ III Ii |I| I I,i 5 ~ ~~~ ~ I0o ||||; I.11:.111 I 1.::::::: 1-1111 111111: 1 III||^ |!ii i^ I lilt lDeerget Ioncenrat o II~l2 I II itIII.... iti E l I I i l I II 1 I II m l I I i l ] t.......I LIT~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I~~~I i l m l t t eI l l i l 5s~~~~~~I r(~~~~I~ I 600 Ill [I IF 1075 0. 1 062 Detergent Concentration~~~~~~~~~~~~~~~~~~~~~~~~~I 4 0 0 I I lilt I Ill I

Detergent Number 00-11334 20 Minutes 9'00 2l~~rlll~l^^lll~ l^ ii'^i^ i:::: ^ ^^ 800 700 600 500 300 I 200 r4 1O( 80 0 illllllll~ llllll if flli l 1lll lllf lll li.........^i~ i......... 700 fl|||^ | ||^ ^ ||^ ||||| ||i^ 11lfllllillll l i~~~~~~~~~~~~~~~~~~~f1 500~~~~m [il^ ll~ l l~ ililill i t ^ I Isiii I II I^ fi^^ ii 94~0025 O.0050.0075::1 O.: Detergen~t; oncentration 25Illlllli^^ ^I l 7 00 If I I --- |I ^ ^, ^ ^ |I^ 500. 02 0 0 50 0 7 mld I........ —-----—.-. ml ~ ~ ~ Dtegn Io Itato If if I I 1 I If5

Detergent Number 00-11334 25 Minutes 800 ||||;| |;:; |;| ||;| Il: 700~~~~~~~~~~~~II ||l || i |I I ||1:|||:::I::::: 900 III I I || Fi~~~~~tl 0) ~ ~~~~~~~~~~~~~ -:: --: — -. —--.......^ ^. __ ^ ^ a, 200 *: -:::::: -:::::: 100 ^ ] I ~| O.Ot06.0025 0.6050.0075 Oeo1 0.025 Detergent Oonoentratlon 26

TABLE II VIABILITY OF CELLS AT VARIOUS TIME INTERVALS Thiomersal Cumulative Minutes Conc., % Nonviable Viable Total % Viable C ula e Nonviable Viable 5.0001 8 102 110 9 8 06.00025 10 53 63 84 18 804.0005 7 100 107 93 25 751.00075 21 77 98 79 46 651.001 19 66 85 78 65 574.0025 24 57 81 70 89 508.005 5 88 93 94 94 451.0075 19 97 116 84 113 363.01 9 152 161 94 122 266.025 9 114 123 93 131 114 10.001 10 102 112 91 10 838.00025 14 60 74 81 24 736.0005 9 98 107 92 33 676.00075 25 77 102 75 58 578.001 20 66 86 77 78 501.0025 28 59 87 68 106 435.005 89 6 95 6 195 376.0075 26 100 126 79 221 370.01 9 162 171 95 230 270.025 8 108 116 93 238 108 15.0001 11 103 114 90 11 567.00025 15 60 75 80 26 464.0005 10 97 107 91 36 404.00075 26 76 102 75 62 307.001 27 61 88 69 89 231.0025 65 18 83 22 154 170.005 102 0 102 0 256 152.0075 104 24 128 19 360 152.01 53 70 123 57 413 128.025 59 58 117 50 472 58 *27

TABLE II (Continued) Thiomersal..... C umulative Minutes Conc., t Nonviable Viable Total % Viable Cumulative Nonviable Viable 20.0001 10 102 112 91 10 376.00025 18 61 79 77 28 274.0005 10 98 108 91 38 213.00075 27 75 102 74 65 115.001 45 37 82 45 110 40.0025 82 3 85 3 192 3.005 102 0 102 0 294 0.0075 99 0 99 0 393 0.01 161 0 161 0 554 0.025 111 0 111 0 665 0 25.0001 10 102 112 91 10 333.00025 20 61 81 75 30 231.0005 10 97 107 91 40 170.00075 31 68 99 69 71 73.001 85 4 89 4 156 5.0025 84 1 85 1 240 1.005 102 0 102 0 342 0.0075 100 0 100 0 442 0.01 160 0 160 0 602 0.025 111 0 111 0 713 0 28

TABLE II (Continued) Cetylpyridinium Chloride.......Cumulative Minutes Cone., % Nonviable Viable Total % Viable Cumiulative Nonviable Viable 5.0001 11 83 94 88 11 614.00025 8 67 75 89 19 531.0005 11 100 111 90 30 464.00075 16 52 68 76 46 364.001 16 58 74 78 62 312.0025 11 75 86 87 73 254.005 11 97 108 90 84 179.0075 3 76 79 96 87 82.01 1675 172 2 254 6.025 109 1 110 1 363 1 10.0001 12 85 97 88 12 619.00025 11 64 75 85 23 534.0005 10 99 109 91 33 470.00075 18 50 68 74 51 371.001 17 60 77 78 68 321.0025 17 72 89 81 85 261.0oo5 10 100 110 91 95 189.0075 2 88 90 98 97 89.01 170 1 171 1 267 1.025 110 0 110 0 377 0 15.0001 16 80 96 83 16 601.00025 11 63 74 85 27 521.0005 11 99 110 90 38 458.00075 17 53 70 76 55 359.001 19 61 80 76 74 306.0025 15 69 84 82 89 245.005 11 83 94 88 100 176.0075 3 93 96 97 103 93.01 171 0 171 0 274 0.025 110 0 110 0 384 0 29

TABLE II (Continued) Cetylpyridinium Chloride Minutes Conc., % Nonviable Viable Total % Viable NoviblCumulative Nonviable Viable 20.0001 16 80 96 83 16 581.00025 10 63 73 86 26 501.0005 12 97 109 89 38 438.00075 17 53 70 76 55 341.001 18 60 78 77 73 288.0025 16 65 81 80 89 228.005 17 78 95 82 106 163.0075 5 85 90 94 111 85.01 171 0 171 0 282 0.025 110 0 110 0 392 0 25.0001 16 80 96 83 16 529.00025 11 63 74 85 27 449.0005 13 91 104 88 40 386.00075 19 50 69 72 59 295.001 19 62 81 76 78 245.0025 16 66 82 80 94 183.005 53 43 96 45 147 117.0075 11 74 85 87 158 74.01 171 0 171 0 329 0.025 110 0 110 0 439 0 50

TABLE II (Continued) Hyamine 1622 Minutes Conc., * Nonviable Viable Total * Viable Cumulative Nonviable Viable 5.0001 10 77 87 88 10 841.00025 11 75 86 87 21 764.0005 16 86 102 84 37 689.00075 21 58 79 73 58 603.001 13 69 82 84 71 545.0025 15 74 89 83 86 476.005 6 99 105 94 92 402.0075 14 104 118 88 106 303.01 11 78 89 88 117 199.025 1 121 122 99 118 121 10.0001 9 80 89 90 9 818.00025 8 69 77 90 17 738.0005 15 83 98 95 32 669.00075 18 61 79 77 50 586.001 17 66 83 79 67 525.0025 25 72 97 74 92 459.005 7 87 94 92 99 387.0075 22 99 121 82 121 300.01 8 78 86 91 129 201.025 7 123 130 95 136 123 15.0001 10 80 90 89 10 815.00025 10 66 76 87 20 735.0005 17 88 105 84 37 669.00075 19 60 79 76 56 581.001 16 67 83 81 72 521.0025 21 70 91 77 93 454.005 8 82 90 91 101 384.0075 20 99 119 83 121 302.01 12 89 101 88 133 203.025 5 114 119 96 138 114 31

TABLE II (Continued) Hyamine 1622 Minutes Cone., % Nonviable Viable Total % Viableulative Nonviable Viable 20.0001 10 80 90 89 10 816.00025 11 69 80 86 21 736.0005 17 89 106 84 38 667.00075 19 60 79 76 57 578.001 15 66 81 81 72 518.0025 22 69 91 76 94 452.005 8 85 93 91 102 383.0075 23 110 133 83 125 298.01 8 73 81 90 133 188.025 6 115 121 95 139 115 25.0001 10 80 90 89 10 808.00025 10 68 78 87 20 728.0005 18 89 107 83 38 660.00075 19 61 90 68 57 571.001 13 63 76 83 70 510.0025 23 68 91 75 93 447.005 7 76 83 92 100 379.0075 17 110 127 87 117 303.01 11 76 87 87 128 193.025 8 117 125 94 136 117 32

TABLE II (Continued) Tween 60..............Cumulative Minutes Cone., l Nonviable Viable Total % Viable Cumulative Nonviable Viable 5.0001 15 86 101 85 15 835.00025 11 76 87 87 26 749.0005 24 80 104 77 50 673.00075 17 73 90 81 67 593.001 18 69 87 79 85 520.0025 20 70 90 78 105 451.005 12 78 90 87 117 381.0075 17 96 113 85 134 303.01 7 93 100 93 141 207.025 9 114 123 93 150 114 10.0001 15 86 101 85 15 842.00025 17 70 87 80 32 756.0005 27 78 105 74 59 686.00075 17 73 90 81 76 608.001 19 69 88 78 95 535.0025 22 73 95 77 117 466.005 14 80 94 85 131 393.0075 19 109 128 85 150 313.01 7 96 103 93 157 204.025 8 108 116 93 165 108 15.0001 16 83 99 84 16 838.00025 16 78 94 83 32 755 0005 27 78 105 74 59 677.00075 18 71 89 80 77 599.001 21 68 89 76 98 528.0025 23 75 98 76 121 460.005 14 81 95 85 135 385.0075 20 99 119 83 155 304.01 6 94 100 94 161 205.025 9 111 120 92 170 111 33

TABLE II (Concluded) Tween 60 Minutes Cone., o Nonviable Viable Total % Viable Cumulative Nonviable Viable 20.0001 14 81 95 85 14 809.00025 16 79 95 83 50 728.0005 28 77 105 73 58 649.00075 17 72 89 81 75 572.001 19 69 88 78 94 500.0025 23 76 99 77 117 431.005 13 80 93 86 130 55.0075 15 71 86 83 145 275.01 10 98 108 91 155 204.025 10 106 116 91 165 106 25.0001 12 82 94 87 12 825.00025 15 79 94 84 27 743.0005 27 79 106 74 54 664.00075 18 71 89 80 72 585.001 18 68 86 79 90 514.0025 22 75 97 77 112 446.005 15 77 92 84 127 571.0075 25 101 126 80 152 294.01 11 97 108 90 163 195.025 9 96 105 91 172 96 54

III. GROWTH AND PROLIFERATION STUDIES A. PURPOSE The purpose of the study during this period of investigation was to determine and compare the effects of low concentrations of Thiomersal, Cetylpyridinium chloride, and Hyamine 1622 (antiseptics), and Tween 60 (a detergent) on the growth and viability of human epithelial cells. In the past, growth of HEp2 cells has been determined by counting on a Coulter counter the total number of cells. Since viable and nonviable, nonlysed cells cannot be accurately differentiated by this method, stains indicating viability were included in the present study because little or no cytolysis was expected with the low concentrations of reagents used. Also, this method of figuring percent of viable cells is a more accurate indication of reagent toxicity than counts of the total numberuof remaining cells in a growing culture. B. PROCEDURE Fifty-milliliter suspension cultures of HEp2 cells were planted at concentrations ranging from 1.2 x 105 to 4.5 x 105 cells per ml and allowed to enter early log phase of growth. The growth medium was similar to that used previously-Eagles75, tryptose phosphate15, and calf seruml0, with 0.1% methyl cellulose and 0.1% sodium citrate added. Log phase was entered at intervals ranging from 21.5 to 45.5 hours when the cell count ranged from 2.2 x 105 to 8 x 105 and at that time one ml of one of the antiseptic and detergent solutions was introduced. The concentrations of the solutions used were 0.001%, 0.00075%, 0.0005%, and 0.0001%. All of these concentrations were used with the cultures of lower cell count and in addition the 0.001% solutions were used with cultures of the highest cell count in order to demonstrate any varied effects due to a different density of the cell population. The cultures were placed on a rotary shaker for constant agitation. Every 24 hours two one-ml samples were withdrawn from the cultures for counting and viability determinations. The total cell count was obtained by use of the Coulter counter. The percentage of viable cells was determined by staining with 0.4% erythrosin B and counting the stained (nonviable) and unstained (viable) cells with a hemacytometer. The inclusion of the viability determination demonstrates more clearly the toxic effects of the antiseptic and detergent solutions. In previous studies the inhibition of growth was difficult to differentiate from death of the cells when lysis of the dead cells did not occur. Also, any error introduced by fractionation of the cells, a possibility that exists because frac35

tionation may increase the total cell count as determined by the Coulter counter, is eliminated. C. RESULTS 1. Effects of 0.001* Solutions At this concentration a large variance was made in the initial cell population density. Examinations were made using an initial inoculum of 1.2 x 105 cells per ml and 4.5 x 105 cells per ml. The solutions were added when the cell count reached approximately 2,2 x 105 cells per ml and 8 x 105 cells per ml respectively. A definite variation in toxic reaction was noticed between the different cell population densities. For example at the higher level the population appears to be unaffected by Tween 60. At the lower level there is a reduction in growth rate and the plateau phase occurs at the same time as in the control but at a lower cell number. At the lower cell population Cetylpyridinium chloride seems to be only slightly more toxic than Tween 60 when the total cell count is considered. However, at a higher cell population Cetylpyridinium chloride is the most toxic of the four chemicals tested. This could be attributed to an increase in total cell lysis by Cetylpyridinium chloride in a higher cell population as compared to that by other chemicals. On the basis of viable cell counts, the cells in the series of tests at the lower cell population have been affected to such an extent that Thiomersal, Cetylpyridinium chloride, and Hyamine 1622 caused almost immediate death and the viable cell count was so low that any practical comparison of these three antiseptics was impossible. Tween 60 showed only a slight effect upon the viability. At the higher population levels there is a more distinct variation in viability. Cetylpyridinium chloride is the most toxic, Thiomersal is slightly more toxic than Hyamine 1622, and Tween 60 is almost identical to the control. An interesting finding that was observed at both high and low population levels is that Thiomersal produced an immediate reduction in the number of viable cells but withCetylpyridinium chloride and Hyamine 1622 the reduction did not occur for at least 24 hours. However, Thiomersal did not prove to be ultimately the most toxic. 2. Effects of 0.00075% Solutions This concentration level seems to divide the four compounds into two groups. Tween 60 and Hyamine 1622 had a slight effect on the cells while Cetylpyridinium chloride and Thiomersal showed absolute toxicity. The former group seems only to 36

inhibit mitosis without any toxic effect on the cell proper. 3. Effects of 0.0005% Solutions The results at this concentration are similar to those at 0.00075%. The decrease in viable cells again points out the toxic action of Thiomersal and Cetylpyridinium chloride. One difference between the two concentrations, however, is that the time required before reduction in the number of viable cells by Thiomersal and Cetylpyridinium chloride has been prolonged. At 0.00075% the reaction time varies from zero to 24 hours. At the concentration of 0.0005% this lag is increased to an interval ranging from 65 to 89 hours. 4. Effects of 0.0001% Solutions At this concentration Thiomersal, in addition to Tween 60 and Hyamine 1622, appears to be almost nontoxic. With Hyamine 1622 the growth rate is even greater than in the control. Thiomersal did show a toxic reaction after approximately 76 hours. Cetylpyridinium chloride caused a reduced rate of growth of the cells 26 hours after its introduction but the percentage of viable cells did not begin to decrease until after 47 hours. D. SUMMARY Tween 60 has been shown to be the least toxic throughout the concentration range examined. Hyamine 1622 is the second least toxic over all but is less toxic than Tween 60 at the lowest concentration tested. Thiomersal rates third in degree of nontoxicity and Cetylpyridinium chloride is the most toxic. A decrease in the degree of toxicity of the three antiseptic reagents in a more dense cell population has been shown. This may be due to a number of factors. Possibly a more rapidly growing culture shows more resistance to the toxicity of these reagents or perhaps the increased amount of protein inactivates the antiseptics. It is also possible that a given amount of these reagents can react only with an optimum number of cells. If that be the case then it would explain why a dense and continuously growing cell population would appear to be less affected than a more sparse cell population. As has been reported previously, the concentration of the reagents is a definite factor in the degree of toxicity. The present study has shown also that the time required before a decrease in the number of viable cells becomes evident is dependent upon the concentration of the reagents. A reduction in concentration will increase the length of time preceding a reaction. These studies showed that a relatively small difference in concentration 37

of the three antiseptics produced a great difference in toxicity. At 0.001*, 0.00075$, and 0.0005* they were quite toxic but at 0.0001% they were, with the possible exception of Cetylpyridinium chloride, nontoxic. In previous reports on higher concentrations viability tests were not necessary because total lysis of the cells usually occurred immediately after introduction of the reagent. However, at the lower concentrations tested in the present study significant cytolysis was not expected and it was essential to determine the viability of the cells. While the total cell count often showed little change the number of viable cells dropped sharply. 38

F-I CH O) 0 O wd O r-l m\ JOO n cH *H * Cr- c 0 H ~ 0 H tH C O 0 0 0 0 XI ~ r -d- t- \o n \KN o 0 *l Ha OCN c \ 0\ COH c.! U *H o O -0.H a.H OH U) K a CO n CcON r-^ Hi n Cr nCO O 40 \ a + 3 ro 0...... a E-i 0 x _____ o r- I c \ct jco -:- nr\ 0 \oj co Co d r- n \ j cO rH- _icto fc\ o 0 0 H H.O Ca ~ Ol H co 04 ~rl I X s O lA O O.1 Fn C O Q 0 0O O ^.- 0 c > c H - _ _ _ i C_\j r- O _O C Xj o o 0 co E- j H \ raU IP\OOJHK^ CU cO n 0 co COJ -- 1 N co P o - E- 0 x W ^ -0 rQ'-i^ OJOJr-I r O _:J- _:J- n-rl \ 0 0\ E-1 1 H H Q>U HHHHO o \ 4-ooo -L Q-.ozto o EH ~.~ r (O> 0 H n n O0 \0 H0 LCA r\Q - _-L\ j t-.O..O. O\ t-. r HO I o +P co -d\ 0-\ t1\ ON C; CC -~ oC \ G\ 0\ co \" -=;( ~ *.-l (I O 0 CC - n - -- O 0 O \LN 0 0 0 0O H — o! {NW\ 0O H 0\ zf 00\ tr -AN o > 0 U).0 -C-(N-Ol>lN.O 0Lr\LL n foNLL 0U O *rHr*oc*jt- - o*H*-*r**oo CjW - CO C\Jin n.L N C\j O i C- O n E 0 XH H -p4 o,.c; co \ U o-,\ o,,c,:o cN,,\ n n t> L rO cOH O a > O iO p C W 0 O co- C t KC I O O\ r0 e A- -o 39

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17 0.%0. 001%-HIGH CELL POPULATION 0 4i _- _ _. - - ------ - 1 i i A A I...llllllllll.. +-__ _+ _ __ __ -- - Ti=m=e 1 =o=ursI 8 _= =| ||=|==_ EE|___ =|E_|_ EEE_ E||||II I I I I I I IE1E1-11 I =_==== =====Time in or s Z I____ ____,___ ___1____= _== IX|I 44 2 t Tll)ilT

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IV. TOXICITY STUDIES Phase 1. Histochemical Studies A. PURPOSE The purpose of this aspect of the investigation was to determine the cytochemical effects of Thiomersal, Cetylpyridinium chloride, and Hyamine 1622 (antiseptics), and Tween 60 (a detergent) on HEP2 cells. Ribonucleoproteins (RNAProtein) and deoxyribonucleoprotens (DNA-Protein) were demonstrated by the MayGrunwald-Giemsa method and lipids by a modified Sudan Black B method. B. PROCEDURE HEP2 cells derived from our stock line were transplanted to Leighton tubes and incubated for 24 hours in a growth media composed of Eagle's media75, tryptose phosphate15, and calf seruml0. After the incubation period the coverslips, with the cellsattached, were removed from the Leighton tubes and exposed to concentrations of 0.1%, 0.01%, 0.001%, and 0.0001% of the reagents dissolved in Hanks balanced salt solution. For the May-Grunwald-Giemsa method 0.1% solutions were omitted. The cells were allowed to incubate in the solution containing the antiseptic or detergent for one hour. 1. Ribonucleoproteins and Deoxyribonucleoproteins Three runs of each concentration of each reagent were carried out for this stain. In two of the runs there were approximately 10 x 105 cells per ml and in the third approximately 3 x 105 cells per ml. The purpose was to determine any variations due to differences in cell population. After the one-hour exposure to the reagent the cells were rinsed in three changes of Hanks balanced salt solution, fixed in absolute methanol and stained by the May-Grunwald-Giemsa method.* This staining procedure is excellent for the differential demonstration of ribonucleoproteins (RNA-Protein) and deoxyribonucleoproteins (DNA-Protein) in monolayer cultures. The RNA-Protein stains blue and the DNA-Protein stains red-purple. With this method cytological characteristics of the cells are retained but lipids are dissolved. *Merchant, D. J., Kahn, R. H., and Murphy, W. H. Handbook of Cell and Organ Culture. Minneapolis: Burgess Pub. Co., 1960, p. 154. 54

2. Lipids Two runs for each concentration of each reagent were carried out. The initial incubation medium for each run contained 2 x 105 cells per ml. After exposure to the reagent the coverslips with attached cells were removed from the Leighton tubes, rinsed in Hanks balanced salt solution and fixed in neutral 10% formalin. Lipids were stained by a modification of the McManus method.* The cells were chromated in 5% potassium dichromate, washed, stained in 0.7% Sudan Black B in 70% ethanol and counterstained in 0.5% aqueous carmine. After rinsing, the coverslips with cells were mounted in an aqueous medium of Arlex and gelatin. The lipids stained black or blue and the nuclei stained red. C. RESULTS HEP2 cells affected by various concentrations of Thiomersal, Cetylpyridinium chloride, Hyamine 1622, and Tween 60 were stained for ribo- and deoxyribonucleoproteins and for lipids. The cytotoxic effects as revealed by these methods were compared but evaluation of the comparative toxicity of the reagents was difficult. In some instances DNA Protein was lost from the nucleoplasm and in others vacuolation of the cytoplasm occurred. Occasionally these reactions were observed in the same cell but not always and because either reaction is indicative of a toxic environment the relative significance of each could not be determined. Of the four reagents tested Hyamine 1622 caused the most severe toxic reaction. Thiomersal and Cetylpyridinium chloride caused similar reactions but Thiomersal was perhaps slightly more toxic. Tween 60 appeared to be the least toxic. 1. Ribonucleoproteins and Deoxyribonucleoproteins In cells stained for RNA- and DNA-Proteins many phenomena were noted, most of which indicated cellular reaction to injury. A number of pyknotic cells in various stages of degeneration were observed. Also of interest was the generalized increase in size and number of cytoplasmic vacuoles which were most apparent at the lower concentrations of the reagents. This would indicate an active transport of the reagent and was most noticeable with Thiomersal and Cetylpyridinium chloride. Alteration of cytoplasmic protein structure probably has occurred and this certainly would alter cell metabolism and function. With all reagents there *Pearse, A.G.E. Histochemistry, Theoretical and Applied. Boston: Little, Brown and Co., 1960, p. 850. 55

seemed to be some loss of RNA-Protein from the cytoplasm but with 0.01% Cetylpyridinium chloride the loss seemed to be complete. Changes in the cell periphery also were indicative of cytotoxicity. Tween 60 caused little change but with Hyamine 1622 the periphery became distinct because of cytoplasmic condensation. Both Thiomersal and Cetylpyridinium chloride caused spindle-like extensions of the cytoplasm as well as cytoplasmic condensation. The extensions are a common reaction to insult and could be outgrowths of the cytoplasm or merely the result of portions of the cytoplasm adhering to the glass while the rest condensed. Nuclear reactions were most interesting and were somewhat similar to those described in previous reports of toluidine blue staining of the nucleoproteins in cells affected by sodium lauryl sulfate and sodium-N-lauroyl sarcosinate (May 1 to December 1, 1960). The nucleoplasm had become hyalinized and the intensity of staining of the nucleoli decreased. Both reactions indicate serious and probably irreversible damage to the cells. All four of the reagents tested in the present study caused enlargement of the nucleus, the greatest increase being seen with Tween 60. Cetylpyridinium chloride 0.01* apparently was responsible for a loss of RNA-Protein in the nucleoli, nuclear granules and nuclear membrane. At the lower concentrations tested Thiomersal caused a slight reduction in the size of the nucleoli and was the only reagent to do so. The small, blue nuclear granules which are distinct in normal HEp2 cells were affected to some extent by all compounds. Their number was reduced by Thiomersal and Hyamine 1622 and they were completely removed by the higher concentrations of Tween 60. Cetylpyridinium chloride 0.01* caused a change in staining which indicated loss of RNA-Protein. The nucleoplasm was affected to some degree by the four reagents. At concentrations of 0.01* and 0.001* all compounds removed a significant amount of DNA-Protein. At 0.0001%, however, Thiomersal was the only reagent to remove DNA-Protein from the nucleoplasm. This finding is consistent with those of other areas of this study in that Thiomersal is the most toxic of the four reagents and that the others become relatively nontoxic between 0.01* and 0.001*. The nuclear membrane showed a reaction to all the reagents. It either became thickened or indistinct. Such a change was difficult to compare or evaluate. 2. Lipids The results of the Sudan BlackB stain were quite similar for 0.1%, 0.01%, and 0.001% of all four reagents. For that reason the findings of only 0.01% and 0.0001% will be illustrated and described. The results of this staining 56

procedure were somewhat disappointing in that the cells undergoing degeneration were heavily stained and cytologic features were masked. Sudan Black B is dissolved in mammalian lipids, including phospholipids and neutral fats. Phospholipids form a layer of all membranes in the cell. Uptake of the dye was heaviest in pyknotic cells and least in normal cells. Apparently the metabolism of the reagent-affected cells was so altered that the enzymes which normally hydrolyze the lipids were not available. Thus there was an accumulation of lipids in the cell accounting for the intense and generalized staining. There is some evidence that a complex was formed by the phospholipids and cellular proteins. This complex also stained black. Cells affected by Tween 60 showed the least uptake of the dye. Hyamine 1622 again seemed to cause the most toxic reaction as noted by the heavy staining of the sparse, pyknotic cells. It was of interest to note that Thiomersal produced a specific reaction manifested by a concentration of lipid material around the nucleus. 57

Control May-Grunwald-Giemsa Mag. 830x The normal characteristics found in HEp2 cells are seen. The round nucleus is large and prominent. It is often in the center of the cell but may be near the periphery. The nucleoli are prominent and several in number. The shape of the nucleoli varies, either' round with a smooth outline or round with numerous small projections from the periphery. The nucleoli stain blue which indicates the presence of RNA-Protein. Numerous diffuse blue granules having no regular distribution are present in the nucleus. The nucleoplasm is very diffuse and stains red- purple indicating the presence of DNA-Protein. The nuclear membrane is distinct and blue indicating large amounts of RNA-Protein. An occasional granule of RNA-Protein may be seen in close proximity to the membrane. The cytoplasm stains blue indicating RNA-Protein. It is diffuse with large granules and no vacuoles. The periphery of the cell often is not well defined due to cellular activity in these areas. 58

Thiomersal 0.01 May-Grunwald-Giemsa Mag. 830x The nucleus is quite large in relation to total cell size. Several nuclei are present which are larger than those found in a normal environment. Nucleoli have not been affected by the antiseptic. The nucleoplasm no longer stains red-purple, indicating a loss of DNA-Protein from the nucleus. An increase in thickness of the nuclear membrane is apparent in a few cells. The blue color of the cytoplasm has been replaced by a redpurple color. This change may be due to migration of DNA-Protein from the nucleus to the cytoplasm. Many vacuoles are present in the cytoplasm. The outline of some cells cannot be established, and in some cells the periphery is refractive, indicating that it has become thickened. 59

Thiomersal 0. 001% May-Grunwald-Giemsa Mag. 830x The DNA-Protein (red-purple) of the nucleoplasm is absent. There also has been a loss of the RNA-Protein granules of the nucleus, which indicates a loss of both RNA-Protein and DNA-Protein from the nucleus. Little cytological effect is seen in the nucleus proper. The nuclear wall has become less distinct when compared to that of the normal cell. The cytoplasm has retained its blue color indicating that RNA-Proteins are present. Vacuolation of the cytoplasm has occurred, accompanied by a loss of cytoplasm in some cells. 60

Thiomersal 0.0001l May-Grunwald-Giemsa Mag. 830x The size of the nucleus is similar to the normal nucleus. The size of the nucleoli, however, has diminished. The red-purple color of the nucleoplasm has been lost as have most of the blue RNA-Protein granules of the nucleus. The nuclear membrane has lost much of its distinct characteristics and the membrane is not so intensely stained. A large reduction of cytoplasm has taken place and several large vacuoles are present. The cell periphery is quite distinct with many spindle-like extensions, as contrasted to the polygonal shape of normal epithelial cells.

Cetylpyridinium Chloride 0.01% May-Gruinwald-Giemsa Mag. 630x The nucleoli which normally are blue, indicating the presence of RNA-Protein, have become red-purple indicating DNA-Protein. This change in staining properties of the nucleoli may be due to a loss of RNA-Protein and the possible unmasking the DNA-Protein normally present. In addition to the nucleoli reversing color, the diffuse granules of the nucleus and the nuclear membrane have also changed from blue to red-purple. The nuclear membrane is more granular than normal. The cytoplasm has become condensed and there are no vacuoles. The cell outline is distinct with spindle-like projections. 62

Cetylpyridinium Chloride 0.001, May-Grunwald-Giemsa Mag. 830x An increase in the size of the nucleus, as compared with the normal cell, is apparent. The nucleoli have remained similar in size and color. Most of the blue RNA-Protein granules usually found in the nucleus are absent. The red-purple DNA-Protein of the nucleoplasm is also absent. The cytoplasm contains numerous small vacuoles, and is no longer diffuse. The cell periphery is well defined because of a condensation or loss of cytoplasm. Many of the cells have spindlelike processes, the result of cell insult. 63

Cetylpyridinium Chloride 0.0001% May-Grunwald-Giemsa Mag. 830x The nucleus has many normal characteristics at this low concentration. The nucleoplasm is red-purple denoting the presence of DNA-Protein which is normal. The number of blue-staining RNA-Protein granules present in the nucleus is, however, less than in a normal cell. A few large vacuoles and many smaller ones are present in the cytoplasm. As in the normal cells the periphery of many cells cannot definitely be established. 64

Hyamine 1622 0.01% May-Grunwald-Giemsa Mag. 830x The nucleus and nucleoli appear cytologically normal. The diffuse blue-stained granules of the nucleus are present. The redpurple stain of the nucleoplasm is absent indicating that DNA-Protein is lost from the nucleus. A thickening of the nuclear membrane has occurred in some cells. The cytoplasm is condensed as compared with normal HEP2 cells, and contains a large number of small vacuoles. Due to the condensation of the cytoplasm the cell periphery is more clearly defined. 65

Hyamine 1622 0.001% May-Grunwald-Giemsa Mag. 830x The nuclei of many cells appear larger than those normally found but the nucleoli are normal in size and number. Blue-stained granules (RNA-Protein) are reduced in number. The nucleoplasm stains a red-purple color indicating the presence of a small amount of DNA-Protein in the nucleus. Except for the large number of vacuoles present in the cytoplasm, it appears fairly normal. 66

Hyamine 1622 0.0001% May-Grunwald-Giemsa Mag. 830x The nucleus is larger than that found in a normal epithelial cell. The nucleoli and nuclear granules appear normal and the nucleoplasm stains a definite red-purple indicating the normal presence of DNA-Protein. The nuclear membrane is less distinct than in the normal cell. The cytoplasm contains many vacuoles but otherwise it is normal. 67

Tween 60 0.01% May-Grunwald-Giemsa Mag. 830x The nuclei of several cells are much larger than normal and the majority of nuclei show some increase in size. The nucleoli remain normal in size and stain blue as in the normal cell. The small, blue (RNA-Protein) granules of the normal nucleus are absent and the nucleoplasm which normally is red-purple now is a light blue, indicating a loss of DNA-Protein. The nuclear membrane is interrupted in many cells. A reduction in the amount of cytoplasm has occurred with the inclusion of a few vacuoles. The periphery of the cell remains normal. 68

Tween 60 0.001% May-Grunwald-Giemsa Mag. 830x The nuclei appear slightly larger than those of the normal cell. The nucleoli appear normal. As seen with the higher concentration of Tween 60 the small, blue (RNA-Protein) granules of the nucleus are absent and the red-purple color of the nucleoplasm is lost. The nuclear membrane has lost its distinct blue color, and often is difficult to distinguish from the cytoplasm. A reduction in the amount of cytoplasm has occurred with the inclusion of a few small vacuoles. 69

Tween 60 0.0001% May-Griunwald-Giemsa Mag. 830x Many cells show an enlarged nucleus. The nucleoli appear normal in size and color (blue). There are fewer diffuse, bluestaining granules in nucleoplasm than are found in a normal cell. The nucleoplasmas is normal, stains red-purple denoting the presence of DNA-Protein. The cytoplasmic portion of the cell appears similar to that of the normal cell except for a few large, scattered vacuoles. Included in some of the large vacuoles is a red-purple (DNA-Protein) body. This was observed only with this compound and this concentration. 70

Control Sudan Black B Mag. 680x A prominent, round nucleus with a well defined membrane is seen. The large nucleoli which normally are present do not appear distinctly with this stain. The nucleoplasm is a homogenous grey. Many indistinct lipid granules are present. Numerous dark-staining granules are present in the cytoplasm. These granules are fairly well distributed throughout the cell although, with a slight affinity for the periphery parts of the cell. A few large, isolated vacuoles are present. The cytoplasm appears rather homogenously stained with the carmine counterstain. The cell boundary is not always distinct. 71

Thiomersal 0.01% Sudan Black B Mag. 680x Thiomersal at this high concentration has little effect upon the nuclear size but there is a concentration of granules around the nuclear membrane. The nuclear granules are indistinct and the nucleoli are absent. The cytoplasm contains many lipid granules located at the center of the cell and near the nucleus. Unstained vacuoles are seen at the periphery of each cell. The cellular borders are well defined due to cytoplasmic condensation. 72

Thiomersal 0.0001% Sudan Black B Mag. 680x The cytoplasmic and nuclear concentration of lipids does not appear to vary significantly from the control. The few cells containing large amounts of lipids apparently are undergoing degenerative changes. In most cells small granules of lipid are scattered throughout the cytoplasm. The cytoplasm is diffusely vacuolated. 73

Cetylpyridinium Chloride 0.01% Sudan Black B Mag. 680x The size of the nucleus has been reduced in the degenerating cells. Some vacuoles can be seen in the nuclei which indicates extensive degeneration. A heavy concentration of lipid is apparent in the reduced cytoplasm. There is a general opacity of cells as well as a clumping arrangement. A few thin, cytoplasmic extensions denote a toxic reaction. 74

Cetylpyridinium Chloride 0.0001% Sudan Black B Mag. 680x The nuclei are indistinct with interruption of the nuclear membrane. Granulation or vacuolation of the nuclear area has not occurred. Three common reactions to cell injury are present: numerous cytoplasmic villae on the outer cell membrane, long spindle-like cytoplasmic extensions and pyknosis. Each stage of cellular degeneration shows a corresponding increase in lipid concentration, i.e., the more normal cells show the least amount of lipid, and the pyknotic cells show the largest amount. 75

Hyamine 1622 O.01% Sudan Black B Mag. 680x The cells are extremely pyknotic with a large amount of lipid material present throughout the cytoplasm and nuclei. The cell number has been reduced because of loss of attachment to the coverslip. The cells are round and appear pyknotic. Vacuolation is apparent in all cells. 76

Hyamine 1622 0.0001% Sudan Black B Mag. 680x The effects on the cells of the lower concentration of Hyamine 1622 are similar to those of the higher concentration. The cells are round and pyknotic and contain large amounts of lipids. Vacuoles are present in all cells. 77

Tween 60 0.01 Sudan Black B Mag. 680x The nucleus is intact but poorly differentiated from the cytoplasm. The nucleoli are apparent and the nucleoplasm is homogenous. The cytoplasm is homogenous with lipid granules at the periphery of the cell. The cells appear to be least affected by Tween 60 than by any of the other reagents at this concentration. 78

r Tween 60 0.0001% Sudan Black B Mag. 680x The nucleus is more distinct than at the higher concentration of Tween 60. The amount of lipids in the cells is similar to that seen with the higher concentration but cytologically the cells appear more normal. A few degenerating cells containing large amounts of lipid are seen. 79

IV. TOXICITY STUDIES Phase 3. Electron Microscopy A. PURPOSE The purpose of the present study was to continue observations on the early effects of detergents on the HEp2 cell. The high resolution of the electron microscope makes it possible to detect and characterize the earliest structural modifications which cannot be ascertained through optical microscopy. The report describes ultrastructural cytoplasmic changes produced minutes after introduction of 0.0075% sodium lauryl sulfate into the cell suspension. B. PROCEDURE HEP2 cells, grown in suspension in a modified Eagle's medium, were harvested by slow centrifugation and then resuspended in 0.0075% sodium lauryl sulfate for two minutes. The cells were centrifuged again and fixed in acetate-buffered 2% osmic acid at pH 7.5. Following dehydration in ascending percentages of ethanol, the cells were embedded in a mixture of epoxy resin, cured at 60~C, sectioned on a Porter-Blum microtome, and observed in a Hitachi HU-11 electron microscope. C. RESULTS All membranes of cells exposed to sodium lauryl sulfate appeared to be affected to varying degrees. Instead of the fingerlike projections seen in the normal cells and described in the previous report, the surface plasma membrane of the detergent-affected cells showed numerous blunt projections which appeared as cytoplasmic blebs ready to separate from the cell by pinching off. Other portions of the cytoplasm were segregated by the endoplasmic reticulum and contained numerous ribosomes (Figure 1). The Golgi apparatus appeared to have shrunken to a much smaller size than in normal cells and was composed only of numerous small vesicles and short bilaminar membranes (Figure 2). The dense globular bodies containing minute vesicles, which seem to be associated with the Golgi apparatus in normal cells, were isolated in various parts of the cytoplasm (Figure 3). The most prominent changes observed were in the mitochondria where there werelocalized swelling of the limiting membranes and displacement of cristae 80

mitochondriales. At the same time the matrix became somewhat less dense than normal (Figures 4, 5, and 6). Along the displaced and often broken cristae were small particles which were similar in diameter to ribosomes (Figure 5). With further swelling large vacuoles appeared which could be identified as mitochondria only by the double membrane rather than as normal vacuoles which have a single membrane. Cristae mitochondriales have disappeared (Figure 7). The diameter of those "mitochondrial vacuoles" reached 2p. or larger. Since formation of vacuoles within the cytoplasm frequently was observed in timelapse cinematographic studies, it might be reasonable to assume that degenerating mitochondria were producing some of these vacuoles. Another structural alteration often seen with the electron microscope was disruption or total disappearance of the nuclear membrane. 81

Figure 1 A peripheral portion of the cytoplasm of a HEp cell subjected to 0.0075% sodium lauryl sulfate for 2 minutes prior to fixation. The formation of cytoplasmic bleb is seen on the right. Notice the segregation of portions of the cytoplasm by the endoplasmic reticulum (arrows). The segregated cytoplasm contains an increased number of ribosomes. Mitochondria (M) have lost most of their internal cristae. 82

Figure 2 A portion of the cytoplasm of a HEP2 cell subjected to 0.00754 sodium lauryl sulfate for 2 minutes prior to fixation. The Golgi apparatus (G) is smaller than in normal cells. Laminated membranes are very short and only small vacuoles and vesicles are present.

Figure 3 A portion of the cytoplasm of a HEp2 cell subjected to 0.0075% sodium lauryl sulfate for 2 minutes prior to fixation. Note the round dense bodies with small vesicles in them (arrows). In normal cells these bodies were associated with the Golgi apparatus. 84

Figure 4 A portion of the cytoplasm of a HEP2 cell subjected to 0.0075% sodium lauryl sulfate for 2 minutes prior to fixation. The initial swelling of 2 mitochondrion (M) is shown. Only a few cristae mitochondriales are visible. 85

Figure 5 Two mitochondria of a HEp2 cell subjected to 0.0075% sodium lauryl sulfate for 2 minutes prior to fixation. Cristae mitochondriales appear to be broken and displaced and have small granular materials along their surface. There is also a break in the limiting membrane (arrow). 86

Figure 6 Mitochondria of a HEp2 cell subjected to 0.0075% sodium lauryl sulfate for 2 minutes prior to fixation. Variations in the structural damage to mitochondria are shown. A and B might be profiles of partially swollen mitochondria sectioned through less dense areas as seen in C and D. 87

Figure 7 Two large vacuoles found in the cytoplasm of a HEP cell subjected to 0.0075% sodium lauryl sulfate for 2 minutes prior to fixation. The smaller of the two shows a double limiting membrane which resembles a normal mitochondrial limiting membrane, indicating that the vacuole might have been produced by extreme swelling of a mitochondrion. 88

Figure 8 The nucleus of a HEp2 cell subjected to 0.0075% sodium lauryl sulfate for 2 minutes prior to fixation. The nucleoplasm shows many small aggregates of dense granular material and apparently empty areas. The nucleolus has become quite round. 89

Figure 9 A portion of the nucleus of a HEP2 cell subjected to 0.0075% sodium lauryl sulfate for 2 minutes prior to fixation. The dense, round nucleolus is packed with granules and the nuclear membrane (arrows) appears to be damaged. 90

VI. ORAL BACTERIA STUDIES A. PURPOSE The purpose of the study during this period of investigation was to repeat the analyses of the bactericidal and bacteriostatic effects on Lactobacillus acidophilus of Thiomersal, Cetylpyridinium chloride, and Hyamine 1622 (antiseptics), and Tween 60 (a detergent). B. PROCEDURE Pure cultures of Lactobacillus acidophilus were prepared and tested daily for purity by using Gram's stain. Growth was determined daily by plating 0.1 ml of the culture which previously had been checked for turbidity on a Bausch and Lomb colorimeter. The number of bacteria were correlated with the turbidity reading so that when the culture was added to reagent solutions the known number of bacteria in each inoculum could be kept constant by determining only the turbidity. The reagents were dissolved in Earle's balanced salt solution and diluted to ten times the desired ultimate concentration. One ml of each solution was added to nine ml of peptonized milk for final reagent concentrations of 0.01%, 0.001%, and 0.0001% by weight. The stock culture again was checked for purity by Gram's stain and 0.1 ml of it added to each tube. After incubation periods of 24 and 48 hours the reagent-affected cultures and controls were plated on tomato juice agar. The standard tube dilution method was used to reduce the number of organisms in each series of plates. C. RESULTS Thiomersal and Hyamine 1622 were found to be bactericidal at a concentration of 0.001%. Cetylpyridinium chloride was found to be bactericidal at all concentrations tested. Tween 60 had little or no effect on the growth of organisms, 91

TABLE IV LACTOBACILLUS PLATE COUNTS (1) (2) (3) (4).0ooo0 Thiomersal 24 hr 972 87 6 0 48 hr 1,790 170 47 7 Cetylpyridinium Chloride 24 hr 0 0 0 0 48 hr 0 0 O Tween 60 24 hr NP NP 13,560 1,056 48 hr NP NP 21,426 2,352 Hyamine 1622 24 hr NP NP 10,630 960 48 hr NP NP NP 1,906 Control 24 hr NP NP NP 1,175 48 hr NP NP NP 2,500 (1) = 0.1 ml sample of detergent-affected culture per plate. (2) = 1 ml of (1) in 9 ml B.S.S. - 1:10 dilution (0.1 ml plated). (3) = 1 ml of (2) into 9 ml B.S.S. - 1:100 dilution (0.1 ml plated). (4) = 1 ml of (3) into 9 ml B.S.S. - 1:1000 dilution (0.1 ml plated). NP = not plated because of abundance of bacteria. 92

TABLE IV (Concluded) (1) (2) (3) (4).001% Thiomersal 24 hr 00 0 0 48 hr 00 0 0 Cetylpyridinium Chloride 24 hr 00 0 0 48 hr 00 0 0 Tween 60 24 hr NP NP 11,065 873 48 hr NP NP NP 1,864 Hyamine 1622 24 hr 2,240 0 0 0 48 hr 3,165 0 0 0 Control 24 hr NP NP NP 1,065 48 hr NP NP NP 2,340.01% Thiomersal 24 hr 00 0 0 48 hr 00 0 0 Cetylpyridinium Chloride 24 hr 00 0 0 48 hr 00 0 0 Tween 60 24 hr NP NP 10,032 765 48 hr NP NP NP 1,742 Hyamine 1622 24 hr 0 0 0 0 48 hr 00 0 0 Control 24 hr NP NP NP 1,105 48 hr NP NP NP 2,462 93

UNIVERSITY OF MICHIGAN 3 9015 02499 5246