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2-ETH\ZHE.L4NOL .A POTESTIAL BIOLOGICAL INDICATOR

OF OCCZ'PATIOSAL EXPOSrRE TO THE PLASTICIZER

DI(2-ETH\LHEX\Z) PHTHALATE

S h a m Alfred Ellis

-4 thrsis submitted in conformity with the requirements for the degee of Master of Science,

Graduate Depanment of Community Health. University of Toronto

TS. c- Copyright b>. S h a w Alfred Ellis ( 1997)

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2-ETHYLHEXANOL .4 POTENTIAL BIOLOGICAL INDICATOR OF CCUPATIONAL

EXPOSURE TO THE PLASTICiZER DI(?-ETHYLHEXYL) PHTHALATE. Master of

Science. 1997. Shawn Alfred Ellis. Graduate Department of Cornmunit). Health.

University of Toronto.

The urinaq metabolite kthylhesanol (?-EH) was investigated as a potential biological

indicator of occupational esposure to the plasticizer di( 2-ethylhesyl ) phthalate ( DEHP).

Urine and personal air sampies wçre collrcted for 2.5 workshifis from scvrn plasticizrd

rrsin \vorkers and t ~ o controls. DEHP concentrations in workplacr air werr below 0.25

mgm' (provincial limit 5 mgm'). Airbome 2-EH. which \vas producrd from heating

DEHP. ranged from 0.92 to 4.5 mgm '. Urinan 2-EH \vas detemined by a new anal'ical

mrthod which usrd solid phase extraction of 2-EH Followed b'- esterification n i th

irichloro acrtic anhydride and analysis by GC-ECD. The limit of drtection \vas

0.0 l ug ml. Urina? 2-EH in rsposed workers genrrally increased follo\ving rach

workshili and ranged from undetected to 13.54pgmmoI creatinine. Analysis of the

correlation brt\vren DEHP esposure and urinaq ?-EH \vas inconclusir.e due to the

limiird sarnpie size of the study. Nrither DEHP nor ?-EH (airborne or urina-) were

drtected in controls

It has been said that the final product of an'. \vork symbolizing a signiticant

achievement. is always a combination of efforts. This thesis, which is the product of long

hours of toi1 by the author. nurtured to completion by the advice. suppon and

encouragement of others. is no exception. For al1 of those who assisted me. 1 offer the

fol lowing thanks:

Fint. 1 would like to thank my supervisors Jim Purdahm and Andrea Sass Kortsak

for giving me the opportuni? to design and conduct a study in a field which is of F a t

importance to me. A special thanks goes to my Father, Alfred and my brother. Macneal

who providrd kçy assistance to this projrct. without which. the cornpletion of this work

would have been more onrrous. 1 am also deeply indebted to my sister Sonia for her

suppon and to rn! Mother. Laurine. who taught. through example. that there are no

roadblocks io success. only detours. I am also vrn; grateful to the resi of rn!. farnily and

friends ivho providrd advice. lent an car at ml. tribulations. rejoiced in m!. victorirs and

cood-heartedl!. joined in jokrs on '-the process of voiding human waste fluids". c.

Finally. to my wife Karen. who unfailingly supported me through al1 aspects of

this projrct. Your understanding and patience dunng the man! long days and nights in

the lab and in the field \vas ncver taken for g~anted and your unconditional love made the

difficult times bearable. In man), ways this achievement is as much youn as i t is mine.

TABLE OF CONTESTS

ABSTRACT ......................................................................................................... II

...................................................................... ACKNOWLEDGMENTS Ill

1.0 INTRODUCTION ........................................................................................... 1

1 . 1 Chernical and Physical Properties ............................................................................................... 1

1.2 Synthesis and Trade narnes ................................... ... .................................................................. 1

1.3 l'ses and Human Exposure ........................... ... ...................................................................... 3

1.4 Health Concerns and Thesis Objective ......................................................................................... 4

TOXICOLOGY .............................................................................. DEHP 6

...................................................................................................................................... 2.1 .4 bsorption 6

2 I I Dermai 6

2 I 2 InhaIarion 7

2 I -: Oral 7

2.2 Distribution ...................................... ,. ......................................................................................... 8

2.5 Alteration of Lipid Mecabolism ........................ ..... ................. 13

2.6 Jlitochondrial Tonicin .................................................................................................................... 15

1 \

2.7 Repmduçtive Tolicity ................................... ........................................................................... 16

2 7 I Male 16

2 7 2 Femaie 2 1 - - 2 7 3 FeniIih and ErnbroytoGcity --

3.0 SELECTING A BIOLOGICAL INDICATOR AND STUDY DESIGN ............. 28

3.1 The Seed for a Biological Indicator ............................ ...... ............................................................... 28 -

3.2 Previous H ' o r k on Biological Indicators .......................................................................................... 29

..................................... 3.5 Developmenr of a 3tethod to Isolate and Detect 2-EH in Human Crine 33

. . 3 5 1 Previous Methods Ex~racting and Anatysing 2-EH J --

3 5 2 Developing a New Method for detmins ?-EH 31

3 5 3 Emacting :-EH h m Human Urine -, -, -. 1

7 5 4 Sumrna- of the method of .4naiysis used in this study 4 I

4.0 METHODS ................... ...... ...... .. 4.1 Sampling Location and Procers Description ................................................................................ 43

4.2 Subject Selection ........................................................................................................................... 45

4.3 Urine Sampling and Anaiysis .......................................................................................................... 45

4.1 DEHP and 2-EH Air Sampling and AnaJysic .................................................................................. 47

4 4 1 .Air m p l i n s 17

4 4 3 .h alysis 19

4.5 Statistical Anaiysis .......................................................................................................................... 50

5.0 RESULTS ................... .. .................................................................... 5 1

............................................................................................................... 5.1 \Torker Questionnaire - 3 1

............................ 5.2 DEHP and Airborne 2-EH exposures ... ........................................................... 52

5.4 Correlation .................................................................................................................................... 58

6.0 DISCUSSION ............... .., ....................................................................... 6 0

6.1 Air sampies ..................................................................................................................................... 60

6.2 l'rine ............................................................................................................................................... 61

...................................................................... 6.3 R-sion Analysis and 2-EH as Biological Indictor 64

7.0 SUMMARY AND CONCLUSIONS ............................................................ 66

REFERENCES ........ ........ ........ ................ ............... .... . .. .................... ......... 68

APPENDICIES ......... .. ........... .....APPENDICIES..,............................o... ........... .............. ........ ..... ..... . .... .. ....... 81

LIST OF TABLES

T.ULE 1 CHEM1C.U .-WD PHYSiC..V- PROPERTIES OF DEHP .

TABLE 2 RECOIXR)' OF ?-EH FR051 Ht+*!tIXS LW\€ C S n G COLL'ILlS ESTR\CTIO\

T.ULE 3 GC-ECD .AN.LLYSIS CONDlTIOSS

T-ABLE 4 GC-FID .\SXLYSIS COSDITlOSS

T.;\BLE 5 SL'\fif.c\RY OF WORK ASD HEALTH QLrESTIOhT\IAIRE

TABLE 6 \kDORI(SHIFT COVCESTRATIOVS (x-SD) OF DEHP A S û :-EH IX .AIR ( \ I G ! ~ I ' )

T.ABLE 7 LSD .AS.4LYSIS OF 1E.W AIRBORNE 2-EH EXPOSLRE BY CVORKTITLE

T-ABLE S C R R . 4 R ) ' ?-EH CO\CESTRATIOSS t uGISI\IOL CRE.ATISISE) XRR4hGED BI'

N'ORKTITLE

T.ABLE 9 N'ORKSHIFT CRIS.4RI- 2-EH COKCESTRATIOSS (uG/J~\IOL CRE.ATI1 I S E )

7- ABLE IO EN=-\\'IPLE OF STEPWISE XKXTIPLE REGRESSIO?\: BEW'EEN DEHP .WD POST

SHIFT LRI\.4RIi7 C 8

vii

LIST OF FIGLJRES

FIGLRX 1 STRCCTCRE OF DI(2-ETHYLHEAXL) PHTH.U.i\TE I

9 FIGLRE 2 SYNTHESIS OF DEHP - FIGLRE 3 POSTL'L.4TED \tET.aBOLIC P.ATHU'.AI' FOR 1 1

FIGURE 4 THE C.4T SYSTE'LI 14

FIGL'RE 5 THE SERTOLI CELL ADENl'L CYCL.ASE SYSTEhI: 19

FIGURE 6 DERIV.4TIZ.4TIOS REACTIOK BETU'EEN T C A 4 . G D ?-EH 36

FIGLRE 7 CHRO\I.ATOGR4PHS OF 2-EH .WD :-EH ESTER 3s

FIGCRE 8 CHROXI.ATOGR4PHS OF DERIC'XTIC'E FORZaD WITH TC.A.4 39

F I G L X 9 O\TRi'IEM- OF THE hIETHOD FOR EXTR-4CTISG ASD DETECTIhG 2-EH FROXI

HCXI.0' L'RIXE 4 I

FIGURE 10 S.UIPLIXG STRATEGI' 42

FIGURE 1 1 .&IRBOR%E DEHP .AXD :-EH ENPOSCRE 5 3

FIGURE 12 TI'PIC.-IL CHRO\l.ATOGR.4PHS OF :-EH ( .AS ?-EH ESTER) C 6

FIGURE 13 PRE- ASD POST-SHIFT LwRII\.:.4RI' :-EH PROFILES 57

LIST OF APPESDICIES

\PPE\DIS-1 S'i'\O'Kl'\ZS .ASD TR-4DE NA%fES FOR DEHP

APPELDIS-11 BLOCK DI.AGR-UI OF PVC PROCESSISG CXIT

APPEYDIS-III B-ACKGROCND INFOR\I.-lTIO?;

APPEKDIN- I i ' COKSEKT FORAI

APPEYDIS-\- HE.4LTH .WD WORK QCESTIOSNAIRE

APPE\DIN-1'1 ST.ATISTlC AL CO\SIDERATIO\S FOR REGRESSIO\ EQL.4TIOLS

.\PPESDIX-1'11 RAM' D.4T.A ASD ST.-1TISTIC.X C.4LCCLATIOSS

1 .O INTRODUCTION

1.1 Chemical and Physical Properties

Di(?-ethylhexyl) phthalate (DEHP) (CAS+: 1 17-8 1 -7), a member of a class of

chernicals hown as phthalate esters, is a colourless to yrllow oily liquid at standard

temperature and pressure. Its structure is shown Figure 1 and its properties are listed in

Table 1 ( Woodward. 1 988; PCS. 1 992: Environment and Health Canada- 1 994)

O

-CH-CH2-CH2-CH2-CH2-CH3

O Figure 1 Structure of di(2-ethylhexyl) phthalate

Table 1 Chemical and Physical Properties of DEHP

r Empirical formula: C24Hlx04 r Molecular Weight: 390.57 grnol r Vapour Pressure: 8 . 6 ~ 1 O~ Pa(at 20 '~ )

r Boiling point: 3 7 0 " ~ (at 10 1 -3 kPa)

r UV absorption region: 7 to 13pm r Density: 1.487 gmL (at 2 0 " ~ j

i log ocouioliwater coefficient: 5-9.6 1 Water Solubility: 45 p @ ( ~ ~ u ~ )

i Henry's Law Constant (KiI):3.cht 1 O" ~arn'/mol

1.2 Synthesis and Trade names

The manufacture of DEHP is shown in Figure 2 and is representative of the

production methods used for al1 phthalate esters. The rwo step estenfication process

begins with the nucleophilic alcoholysis of phthalic anhydride. which occurs rapidly

when the anhydride is dissolved in Lethyihexanol (?-EH). In the second. rate limiting

step. a reaction temperature esceeding 1 601C or a catalyst ( H:SOA or ptoluenesul fonic

acid) is usrd to complete the esterification. Until recently, acid catalysts have been

preferred for DEHP production because the' minimizr unwanted olefin. ether and isomer

formation from ?-EH. However. newer methods using autocatalysts or aprotic catalysts

such as tin osalate. have increased the p u n h of DEHP and reduced or rlirninated the

need for secondan treatments. such as catalyst rrmoval ( Weissemel and Arpe. 1993 1.

STEP 1:

STEP 2 : O

O CH2-CH3

f O- H-CHr(CHt)3-CHz -Cii2-(CH2)3-CH3 +(2-E'F'@c \

O- H-CH ?-(C Hz)3-C H

O CHrCH3 (DEHP)

Figure 2 Synthesis of DEHP

In 1992. the global annual production of al1 phthalate ester cornpounds \vas approsimaiel~~

).7s10t' tonnes. of which over 50O.a \vas produced as DEHP (IPCS, 1992). In Canada. there

are currentl! two facil irics producin- DEHP. These facilities produced 5 kilotonnes of DEHF

in 1991: equalling the total amount of DEHP imported (Environment & Health Canada.

1994).

As shotm in Appendix 1, DEHP has a plethora of synonyms and trade names

which has occasionaily led to confusion in technical Iiterature. For esample, DEHP is

routinely referred to as "dioctyl phthalate". which in tum is ofien confused with the

straight chain isomer, di-n-ocel phthalatr ( Environment & Health Canada. 1994 ).

1.3 Uses and Human Erposure

Approxirnatel!. 9.96 of global DEHP production is directed towards its use as a

plasticizer to increase the flexibility of plastics (Graham, 1 973: IPCS. 1992 ). Polyners such

as polyvinyl chloride (PVC), cellulose esters and polyvinyi acetate are commonly mised with

DEHP: \\.hile polyethylene. polypropylene, polyurethane and some rnginrenng

thennoplastics use DEHP to a lesser extent (Modem Plastics Encyclopedia, 1993). Minor

uses for DEHP include: cam-ers for pesticides and c o l o ~ n g pigments, use as a dielectnc

fluid in transfomers and use as hydraulic fluid in heaky machinely (Kluwe. 1982a).

DEHP can be found in a varieh of products including consmiction rnatclrials.

clothing, food packaging and medical tubing where the plasticizer content can range fiom

25% to 55' O (WU ) (IPCS. l99Z). Furthemore, increased world-wide consurnption of plastic

products has resulted in DEHP becoming a ubiquitous environmental contaminant. Human

esposure to DEHP cornmoni? occun from ambient air (up to 0.005 pg m'). water (from

0.1 pgL to as high as 0.1 mg'L) and food products contarninated by plastic materials (US

rstimare of 0.3 to 1 mg da-) (Thurén and Larsson. 1990: PCS. 1992).

Esposure from medical products, such as plastic transfusion bags, has been reporteci as hi@

as 1 71 mg in a 1 -5 hour transfusion pend (Peck and Albro. 1981). Occupational exposure.

which occurs during the production of phthdates and processing of plastics has ranged from

0.1 to 60 mgm' of air but is bpically below 1 m g i m ' ( ~ ~ ~ H , 1989). While the actual

number of Canadian worken processing DEHP products 1s not I n o ~ n : in 1990 there wwe

approximately 2 500 plants employing more than 100 000 worken involved in processing

plastic materials ( Society of the Plastics industry of Canada. 199 1 ).

1.4 Heaith Concerns and Thesis Otjective

While acute tosicih is low. DEHP has been show in rodent snidies to be a

reproductive toxin (Parhie. et. al., 1982: Gangolli, 1 983; Tomita et. al.. 1 982: Oishi, 1989:

Hauck et-al.. 1990: Rao et.al._ 1990). DEHP has aiso been found to cause liver cancer as well

as induce other non-specific tumours in animals (Kluwe, 198213: Rao et.& 1990 j. The

[ntemational Agency for Research on Cancer (IARC) and the National Lnstitute for

Occupational Safety and Heaith in the United States have both concluded that DEHP is

carcinogenic to rodents and ma? br carcinogenic to humans ( M C chssification 2 8 ) ( IARC.

1982: 1987: NIOSH, 1989).

Given the daily human esposure to DEHP and its potential health impact. it is

important to de\elop a reliablc assessrnent of the risks associated mith DEHP esposure.

The information currently available is insuficient to make such a determination (IARC.

1987: Environment and Health Canada, 1994). To adequately assess human risk it is

neccssan; to have a yood measure of exposure. Biological marken are useful for this

purpose since they provide information on the intemal dose of a chernical received by an

exposed individual, regardless of route of ente (Bernard and Lauwexy, 1987). However,

a practical and reliable biomarker for DEHP esposure is not currentl! available ( IPCS.

1991). The objective of this thesis was to investigte the development of 2-EH as a

biological marker of DEHP exposure and to validate tbis marker in an occupational

setting using workers in the plastic industry.

2.0 TOXICOLOGY OF DEHP

2.1 Absorption

2.1.1 Dermal

Studies by ShaRer et-al.. ( 1945) (cited in Woodward 1988) and Elsisi et- al.. ( 1989)

using dermally applied radiolabelled DEKP. found radioactivity in the lungs. heart. liver.

kidney, gonads and spleen in rabbits and rats at 3 to 7 days afier administration. While the

results were not quantitative. they did indicate that DEHP can be absorbed and iveIl

distributed to interna1 organs following demal exposure.

More detailed studies of DEKP siun absorption were conducted bp Scott ct.al._

(, 1 %7), Ng et. al.. ( 1 992) and Barber e t al., ( 1 992). Treaments consisted of radiolabel led

DEHP applied in vivo and'or in \-in0 to the skins of rats, çuinea pigs and humans. Scon et-al..

( 1987) fomd that DEHP was absorbed in vitro and in vivo at rates of 23.4 jq'crn' s k i h

(human) and 5.6 pg cm' skin'hr (rat). respectively. This was in contras1 to the results of Ng.

et-al.. ( 1992) and Barber et.al., ( 1992) which indicated absorption rates of 0.42 pgcm' -

skinhr (rat). 0.27 pgcrn' sbnhr (guinea pigs) and 0.10 pgxrn' skir~lu (human). The hgher

rates obsewed by Scott et.al may be due to increased penneability as a result of the use of an

cthanol:waater solution in sample application and extraction (Barber et.al.. 1993).

Nevertheless. the results show that DEHP absorption through the sbn occurs very slowly.

2.1.2 Inhalation

Wliile a number of inhalation studies have been conducted on DEHP, they tack one

or more essential pieces of information for calculating quantitative absorption da^, such as

6

indicating the applied dose. measunng DEHP or its metabolites in escretory products.

speci-ing the use of DEHP as a vapour or aerosol or indicating the distribution of the

aerosol;vapour in inhalation charnbers (Kluwe. 1982~: Woodward. 1988). A study b!.

Gonzalez et-al.. (1978) (cited in Lawrence- 1978) found accumdation of DEHP in the liver.

kidney and hem ( 133 to 478 mgkg) in rats esposed to DEHP vapour for I hour administered

three tirnes a weck for 3 weelis (exposure dose not given). In a study rrviewd by Woodward

i 1988). investigators found that rats exposed to a single 100 mg dose of radiolabelled DEHP

vapour for 1 min escreted 9O0,{o of the applied dose in the urine and faecrs. More recent

inhalation srudies have investigated other toxicological endpoints but provide no information

for calculating pulmonary absorption (Merkle -al,, 1988: Klimisch et.al.. 1992). -4s a result

of the limited information. a rneaningtùl quantitative statement on the pulmonap absorption

of DEHP cannot be made. However. qualitatively the studies sugçest that DEHP can be well

absorbed through the rat lung.

2.1.3 Oral

In a goup of feeding studies by Daniel and Bran ( 1974). rats were @en a single

dose of radiolabelled DEHP (2.9 mgkg) dissolved in corn oil. Srven d a ~ s p s t treatrnent. it

\vas observed that gastrointestinal absorption of DEHP. as rneasured by urine and faecal

escretion. [vas greater than 9.1' O. In a similar study. Albro et-al.. ( 1987) usrd 1 to 180 mg kg

of DEHP fed in single doses or gven daily for four dap. DEHP ras found to be well

absorbed in both cases with greater than 98O0 of the applied dose recovered in excreta.

Administration of the hvo major metabolites of DEHP formed in the gui: mono(?-ethylhexyl)

pht halate < MEHP ) and 2-ethylhesanol (2-EH ) were also wel l absorbed nith recovenrs

seater than 80°-O and 96?,0. respectively (Albro. 1975: Chu et-al.. 1978). Albro rt.al._ ( 1987) c.

also found that intestinal absorption of DEKP could bt: saturated i.e. doses greater than 200

rng*kg caused large amounts of DEHP to be escreted un-metabolised.

tnformation on the intestinal absorption of DEHP in primates and humans is limited.

Radioiabelled DEHP @\-en to primates LW well absorbed. with 80°,0 king recovered in

rxcreta over four days (Astill. 1989). In a hurnan study? a single volunteer ingestrd 1 0 of

DEHP and afier 24 hours. 4.590 of the dose was recovered in the utine (Schaffer et-ai-. 1945

cited in Schmid and Schlatter. 1 985). In a similar study. Schmid and Schlatter ( 1 985) had

hvo hurnan volunteen ingest a single dose of 3Omg of DEHP. Approximateiy 1 I - 15O.o of the

dose vas escreted in the unne after 24 hours. Similar excretion patterns were found when the

volunteen ingested IOmg for 5-4 days. However. it should be noted that in the absence of

measurements of DEHP or its metabolites in the faeces. the urinary escretion levels should

bc considcred lowr limits of absorption.

The reviewved studies suggests that DEHP is well absorbed orally in rats and

primates. honever. human absorption is not clearl). detined.

2.2 Distribution

In a review of distribution studies by Woodward ( 1988), "c-DEHP (ranging fiom

1000 ppm to 12.000 ppm) orally administered in rats was found to be \ d l distributed in

blood plasma liver. lung. intestines. fat. kidney, adrenals. testes and bladder. Highest DEHP

le\& were found in the fat (3 to 9 pg). liver (0.2 to 4 pg) and adrenals (2 to 6 pg). The

lowest concentrations were found in the testes which generall y comprised less than 0.16 pg

at the hi&est administered dose. Steady state levels of DEHP were reached in hvo weeks

when rats were fed diets containing 1000 or 5000 ppm of radiolabellcd DEHP. Thex levels

rapidly decreased nith no apparent accumuiation folloning cessation of treannrnt ( Daniel

and Bratt, 1974). However. DEHP accumulation \vas observed in a female Rhesus monkey

transfused with plasma containing 37 mp of DEHP. Residual DEHP \\as found in fat (20

pg). lung ( 15 pg). spleen ( 10 pg) and liver ( 1.7 ug) 14 months pst-transfusion. Similarly. a

male rnonkev - - Sven 78.72 mg of DEHP showed 0.7. 0.4.0.8 and 3 yg of DEHP in the liver.

testis. heart and gut respectively. IF months afier transfusion (Woodward 1988). Human

adipose tissue collected From autopsies (time of procurement relative to death unspecifirdi

showed DEHP ranghg fiom 0.3 to 1 ppm (Mes et-al., 1971).

In rats administered lgkg DEHP. MEHP lrvels were found to bct approxirnately 3

times highrr in periphenl blood than DEHP and 1 . 1 ; 3.5 and 7 times higher than in the blood

circulating in the tsstis. liver and kidney. respective[!- (Oishi. 1989). Studies by Albro and

Corbrtt. ( 1978) using human blood stored in transfusion bags showed that approsimately

80°0 of the DEHP content \vas bound to lipoproteins (in the follo~\ing decreasing order of

amni'.: Low Densih. Lipoproteins. V r c Low Density Lipoprotein. High Drnsity

Lipoprotein) nith the remainder bound to albumin. In contrast. MEHP equiltbratrs in the

blood behveen plasma and albumin with no association with lipoproteins. Phamacokinetic

estimates from human cancer patients receiving blood which had leached DEHP fiom

mrdical I.V. bals (26.4 mg to 82.4 mg) showved a mono expnential disapprarance rate with

the following parameten: ha1 f l i fe of 28 minutes. an apparent volume of 1.8L m' t surface

areai and a clearance of 78 mL min-m' (Rubin and Schiffer, 1976a: Peck and Albro, 1982).

2.3 Meta bolism

The overall metabolic pathwvay for DEHP is showrn in Figure 3. In rats. ingested

DEKP is rapidly hydrolysed by pancreatic lipases to MEHP and ?-EH (Albro and Thomas.

1973; Daniel and Bratt. 1974). ûther in vitro studies indicate that non-specific lipases in the

blood as well as the lungs. kidneys and adipose tissue are also capable of similar mrtabolism

( Albro and Thomas 1973; Carter et. al., 1973: Rock et-al-, 1978). In the liver. DEHP induces

c'ochrorne P-150 JAl and is metabolized to form MEHP and ?-EH. Furthemore. cP450

-lA 1 can also be induced by MEHP and ?-EH (Pollack et al., 198% Okita and Okita 1993:

Dimen et-al.. I9Xa 1.

Followving hydrolysiç oxidation, MEHP is subjected to fûrther B and pl oxidations

wenot producin- numerous metaboiites. including phthalic acid (Albro et.al._ 1984: Lhu,

et-al.. 1985). Rats metabolise MEHP primanly through P-oxidation. but this has been found

to br a minor pathway in pnmates and humans (Albro et-ai.' 1982). Products of B-oxidation

are then metabolised via perosisomal P-osidation (Lhugenot et-al.. 1985).

In rats. ?-EH is metabolised by both alcohol and aldehyde dehydro-nase to 2-

cthylhesanoic acid (:-EH& (Albro. 1975). Studies using radiolabelled ?-EH and di(?-

rthylhes).l) adipate f DEHA) (which also produces ?-EH as a metabolite) in rats and pnmates

indicate that 2-EHA is either conjugted and eliminated directly. or is further metabolised bu

p. P-1 and Posidation to give nvo hexanoic acids. nvo heptanones. a hewnedioic acid and

CO1 ( Albro. 1975: CMA. 1985; Cornu etal., 1988).

I r ' S-@"-"

Methods for eliminating DEHP and its metabolites v a q sipificantly in rats and

rnonkeys. While both animals eliminate MEHP and its metabolites rapidl!. and

predominantly through the urine. rats o'tidize MEHP more than rnonkeys and do not rxcrete

alucuronide conjugatrs tiUbro et-al., 1981: Lhuguenot et-al.. 1985). The monkey cxcretes - DEHP metabolites as glucuronides producing the glucuronides of mono(5-hydro-2-

cthylheql lphthalate (5-OH-MEHP) as a major urina- product ( 3 3 O ) o of administered dose)

followed by the glucuronides of MEHP (79*6) ( Albro et-al.? 198 1 ). In rats, these metabolites

are low ( 13" O for 5-OH-MEHF) or absent ( Albro et.al., 198 1 ). Un-rnetabolized DEHP was

absent in rats and found in only trace amounis in monkeys (Albro etal., 1981: Peck and

Albro. 1981). Rats given oral doses of ?-EH or DEHA produced 2-ethylhexanoic acid (2-

EHA) as a major urinary metabolite (Albro, 1975: Cornu et-al.. 1988). Conversely. DEHA in

monkeys produced the glucuronides of ?-EH as the major urina- metabolite (30-lS0.~ of the

administered dose ) fol lowed by 2-€HA (approximately 1 5 O G ) . AH other compounds were

found in trace quantities.

Humans given on1 doses of DEHP. eliminate 10-1 C0/0 of the dose in 3 days (only

unne concentration reported ) ( Schrnid and Schlarter. 1 98s ). in patients receiving blood

transfwions contaminated with DEHP. more than 80°,,o of the metabolites were found

clucuronidated in the unne iPeck and Albro. 1982). Like the monkey. the major unnap C

metabolite. \vas 5-OH-MEHP (2j0,0 of the adrninistered dose) with MEHP being found at 70.0

< Peck and Albro. 1982 ). Similarlu. in poolrd urine samples of workers exposed to DEHP. 5-

OH-MEHP \vas found to be a major metabolic product (31*;0) followed by MEHP ( 2 6 O i )

( Cimen et-al., 1 993 b). In both studies. un-metabol ized DEHP \vas absent or fond in trace

amounts. which is consistent with the theop that DEHP is rapidly hydrolysed to MEHP and

?-EH (Daniel and Bran. 1974 ).

2.5 Alteration of Lipid Metabolism

It has ken established in rats that DEHP decreases cholesterol. liver glycogen.

plasma phospholipids and triglyerides as well as increasing s e m fier fa@ acids. ketons

bodies and hepatic phospholipids ( Yanagïta et-al., 1978: Sakurai et.al.? 1978: Bell et-al.?

l978a. 1 W8b 1979: Yanagita et-al.. 1979: Oishi. 1981: Winberg and Badr 1995 ). In a review

of e'tperiments by Bell (1987), male and female rats fed with 0 . j 0 o and l o i DEHP for I l

days. showed a decreax in cholesterol metabolism of -10°,0 and a decrease in the ability of

the liver to incorporate the cholesterol precunorsl acetate and mevalonate. Similar decreases

have been observed in the rat adrenal glands. testes. fetuses and in Young sucMing pups

Following matemal treatment with DEHP (Bell, 1982).The author suggested that decreased

cholesterol production ma! be due. in part. to inhibition of 3-hydro~-3-meth~~lgluta~~l CoA

rctductase ( HMGCoA j. a liver enzyme essentiai in the production of cholesterol ( Bell. 1987 ).

This conclusion appears to be supported by the experiments of Nair and i up, 1 1986) who

obsewed that male rats fed ?O8& DEHP for 30 days showed a hvo fold decrease in the specific

activih. of HMGCoA and reduced incorporation of acetate into cholesterol. In addition. the

activih of the cholesterol degradation enzyme 7a-hydro-lase increased alonç with the

apperance of cholrsteroi de-gradation products in the bile and an elevated level of bile acid

production (Nair and Kmp. 1 986 ).

, Alterations of fatty acid osidation have led

Figure 4 The CAT System

investigators to determine whether D E W at'fects

the camitine acylmsferase %stem (CATI. As

shown in Figure 4. CAT is a hvo part e q m e

systern involved in the transfer of long chain fatty

acids into the rnitochondna1 matris for osidation.

Once a faw acid is "activated through lidiage

with coenzyme A by acyl ÇoA synthetase. it is

then linked to camitine to form Acyl camitine bu

CAT 1. The actkated fa@ acid is then transferred into the rnitochondrial mams by a

translocase e-me where the acyl group is released by the CAT II erqme. The free fam

acid can then undergo osidation while carnitine can activate other fatty acids by passine

back though the translocase in eschans for acylcamitine.

Preparations of isolated mitochondria From rats fed 0.2O0 or I O o DEHP for 102

wezks. showed elevated CAT activih (Ganning et-al., 1983: 198% 199 1 ). However. MEHP

acted as a cornpetitive inhibitor of CAT 1 when CO-incubated wvith long chain fam. acids

( palmitoyl octanoyICoA) (Grolier and Elcombe, 1993). M e n CAT II was examined using

the samr methods. no inhibition \vas obsemçd. Investigations into specirs differencrs

showed that rat and mice CAT 1 are the most sensitive to MEHP inhibition. guinea pigs are

marginally sensitive and humans are the least sensitive (Grolier and Elcombe. 1993 ).

While the majority of studies have focused on MEHP. a few uçing ?-EH or its

metaboli te 2ethyl hesanoic acid haw also shom liver reductions in semm mglyceridr.

cholesterol. and ketone body production as well as inhibition of CAT activity (Woodward

1 988; Badr, et.al., 1 990: Windberg and Badr. 1 995).

While the effects of DEHP. MEHP and ?-EH on lipid metabolism. arc not full'

elucidated. the mechanisms appear to be comples involvinç inhibition of CAT activity and

affecting HMGCoA and possibly other enzymes involved in the production of cholestrrol.

2.6 Mitochondrial Toxicity

Rat liver mitochondna treated wvith MEHP caused a decrease in state 3 respiration

i i.e.. ATP production in the presence of ADP) and a variable response in state 4 respiration

( resting state ATP production). Hoivever. no changes were observed in mitoc hondna treated

with DEHP (Melnick and Schiller 1985). It should be noted that the results of Melnick and

Schiller ( 1 985) did not include a stiitistical analysis. nevertheless. statistically significant

decreases in state 3 respiration werr observed in MEHP treated testicular and liver

mitochondna (Oishi. 1990: Winberg and Badr, 1995). The site of inhibition by MEHP

appears to be localised at the Cytochrome c reductase site of the respiraton. chah. MEHP

drcreased state 3 respiration obtained with succinate or malate (which provides reducing

squivalents prior to cytochrome c ) but not state 3 respiration obtained with ascorbate (which

reduces cytochrome c ). (Melnick and Schiller, 1985: Winberg and Badr 1995).

Fewv studirs have investigated the effect of 2-EH on mitochondrial tosicihp. Keller

r a (1989: 1991 ) used isolated mitochondria as well as whole livers. fiom anaesthetised

fernale rats perfused with 1 to 6 pM of ?-EH. While few statistics were used. the results

showed a marked decrease in state 3 respiration and a dose dependent increase tn state 4

respiration ( fiorn O to 6 umols of ?-EH) (Keller et-al.. 1989).

2.7 Reproductive Toxicie

2.7.1 Male

A nurnber of studies have show that rats fed a concentration of 2 gzkg or more of

DEHP or MEHP for 10 days develop testicular atrophy. decreased testosterone production.

increased zinc escretion. degeneration of spermatocytes and, in some cases. complete

cessation of spermatogenesis (Gray et.aI.. 1977: Gray and Bunenvonh. 1980: Thomas rt.al..

1981: Gangolli. 1982: Oishi and Hirag 1983: Oishi, 1989). Rats greater than 8 weeks old

were somewhat more resistant to testicular damage (Gray and Gangdi. 1986 1. Nrwthelrss.

in al1 caxs. recovem \vas lirnited following cessation of treatment. Efforts to reducr tosicity

b\ providing dietary supplernents of zinc andor testosterone (Lp. or oral) resulted in a

potentiation of DEHP's effects (Gray and Gangolli. 1986: Oishi. 1989). No testicular i n j ~ .

kvas obsrned in a feeding and in vitro sud?. using ?-EH (0.390 rngkg and 100 uL

rrspectivcly ) (Foster et-al.' 198 1 : Gangolli. 1981).

There are nvo major cell types involved in spermatogenesis: Senoli cells and Leydig

crlls. In general, Srnoli cells provide nutients to germ cells (developing spermatocytes) and

form the basis of the blood-testis barner: while Leydig celis srcrete endocrine hormones such

as testosterone which help regulate spermatogenesis (Vander. et.aI., I W O ) . Sertoli cells frorn

n t s fed 2 glig of DEHP showed estrerne vacuolation of the smooth endoplasmic reticulum

(SER) within the fint II hours and a reduction in mitochondrial respiration. By 24 hours,

severe cellular degeneration. i.e. pyknotic nuclei was evïdent and gem cells were observed

detachmg from Sertoli celis (Cos et.al., 1982). in Leydig cells, swelling of the mitochondna

was observed aiong with a disappearance or reduction in their maais granules. Also visible

\vas an increased formation of vesicles within the SER and an increased association of

macrophages wvith the surface membranes (Jones et-al.. 1 993 ).

Sertoli cells have been show in vitro to be capable of hydrolysing MEHP at a rate

comparable to hepatocytes (7.7pmoLhrmg protein vs. 9.6 pmol;hr,vmg protein,,,,) ( Albro

et al 1989): suggesting that MEHP, ?-EH andior their metabolites rnay play a greater role in ~-.?

testicular tosicity than DEHP. Sjoberr et-al.. ( 1986) co-cultured Senoli and prm cells frorn

immature rats fed either DEHP, 2-EH. MEHP or three MEHP metabolites (2 mL/kg bd.wt)

for five days. Histological examinations revealed that MEHP and MEHP metabolites caused

1 2 ° f ~ and ?OO.O. respectively. of germ cells to disassociate and degenerate fiom Sertoli cells:

consistent with earlier findinp (GangIli. 1982: Gray and Gangolli. 1986). However. neither

DEHP nor 2-EH caused any statistically significant changes.

Moss et-al.. ( 1988) postulated that MEHP ma' cause gem cell detachment by

drcreasing the production of lactate and pyruvate from Senoli cells. This uas based on the

observations thar gerrn cells are incapable of usine glucose to maintain ATP levels and are

dependant on lactate and p y v a t e produced by Senoli cells (Jutte et-al., 1983 cited in Moss

et.al.. 1988). This hypothesis ~ z i s tested by incubating Senoli cells from immature rats nith

DEHP (100 pM). ?-EH (200 PM) and MEHP (O. 1 - 200 @VI). Neither DEHP nor '-EH had

an? effect on Sertoli cells. Howevrr. MEHP produced an approsimate 2 fold decrease in

pyruvate and a 2 fold increase in lactate. Similar results were obtained by William and Foster

( 1989 1. In a related study. Siddiqui and Srivastava 11992) cultured germ cells from isolated

rats fed 1 g%g of DEHP for 115 days and found an increase in the activity of lactate

dehydrosenax (LDH) and decreases in aldose reductase (MD), and sorbitol dehyirogenax

ISODH). Taken togethcr. these results. do not necessady suggen that alteration in lactate or

pymvate are responsible for g r m ce11 detachment. While changes in the activities ALD and

SoDH suggest a perturbation in the normal use of glucose (Siddiqui and Srkastava 1992 ). the

increast: in g r m ceIl LDH activity ma? also be a compensatory mechanism to convert

elevated levels of lactate to pyuvatr.

Another possible mechanism for germ cell loss was studied b>- Thysen et-al.. ( 1990)

who investigated whether Senoli cells esposed to MEHP resulted in decreased transfemn

production. a proteio klievrd to be vital for e r m cell growth and developrnent. Senoli cçlls

isolatcd From 10 da'. old rats were cultured with MEHP in the absence or presence of folliclr

stimulating hormone (FSH), which stimulates transferrin production in Senoli cells. In

mplicate espenments. cells culnired nithout FSH showed consistent decreases in transfemn

production from 23°h to 62°G. In the presence of FSH, however. the results were more

variable nith nvo esperiments showing a decrease in transfemn production of 8'0 to 3 5 O O

n-hereas one csperiment showed an increase between 1 9 9 and 5 O . 0 .

MiIr the mechanism of germ cell detachment remains unclear. other studies have

invrstigated whethrr MEHP may affect FSH stimulated production of adenosin&'_ 5'-

monophosphate (CAMP) CAMP is a srcondan inrncellular messenor which mrdiates the

action of FSH and other hormones. in Senoli cells (Desjardins and Ewing 1993). Heindel

and Chapin (1989) cultured Senoli cells tiom 18 da!. old rats and incubated them for 24

hours uith MEHP and then treated them with FSH. MEHP treated cells showed a hcfo fold

decrease in the production of CAMP relative to controls ueated with FSH alone. To elucidate

the site of action of MEHP, the authon used a series of site specific stimulatoa and

inhibitors of CAMP production (Figure 5). In separate espenmenü. MEHP had a greater

inhibitory effect on CAMP production than adenosine or petrussis toirin: suggesting that the

molecular pathway of inhibition by MEHP 1s different from the latter ( Heindel and Chapin

1 989). S irnilarl y. the use of methylisobutyl'ranthine, which deactivates CAMP de_pdation

pathways. did not alter MEHPqs inhibitory activity. The use of cholera tosin and fonkolin.

stimulaton of CAMP production. also had no effect on MEHP's inhibitory action. This was

consistent with an earlier study (Lloyd and Foster. 1988). FinaIl!.. Senoli crlls wrrr cultured

with MEHP and isoproterenol or prostaglandin El, which act on the FSH recrptor, located on

Figure 5 The Sertoli Cell Adenyl Cyclase System (.Modified from Lloyd & Foster, 1988). ( - )= stimulators. ( - ). inhibi tors PD= Phosphodiesterase.

the outer crll membrane. to stimulate cAMP production. Interestingly. in both cases. MEHP

caused an increased production of cAMP (Heindal and Chapin. 1989 1. The authors

hyothesised that the lipophilic nature of MEHP ma! alter membrane fluidity and

di fferentiall y affect adeny l cyclase (AC ) ( Heindal and Chapin. 1989 ,. From thrse

expenments. the authors sbgested that the site of action of MEHP appears to be localisrd at

the FSH receptor membrane and that part of the rnechanism of Senoli cell toxicity ma!.

involve impaired ceIl Function due to the inhibition of FSH to initiate intracellular production

of CAMP (Heindal and Chapin. 1989).

The theory of MEHP acting at the FSH receptor membrane app*in to be supported

by the studies of Grasso et-al.. ( 1993 ). Sertoli cells frorn 18. 36 and 45 day old rats were pre

incubated for 24 hours with MEHP afier which radiolabelled FSH wvas added. In al1

expenmenrs. a clear dose related decrease in FSH binding w a s observed at MEHP

concentrations rangng from 0.0 1 pM to 100 PM. In experiments where MEHP \vas added to

the medium simultaneously wvith radiolabelled FSH. no affect on binding [vas observed. The

authors concluded that while the FSH receptor is the site of action for MEHP, MEHP does

not cornpete with FSH for binding sites (Grasso et-al.. 1993 ). The rffect of MEHP on the AC

system was studied by using Sertoii cells pre-incubated with MEHP and -miansine

mphosphatr (GTP) wvhich is involved in AC activation. At low concentrations of GTP.

binding of FSH \vas similar for the controls and MEHP treated cells. However. at higher

l e d s of GTP ( 10 and 100 mM)- FSH binding in MEHP treared cells wvas 4 Y O to 52'0 less

than controls. The authors hvpothesised that this reduction ma? be due to some MEHP

influenced action on the GTP-binding protein that couples wvith the AC system (Grasso et-al..

1993 ).

In contrast to the efforts focusrd on clucidating the effects of DEHP and iü

metabolites on Senoli cells. only a fewv studies have investigated the effects on Leydig cells.

Srivastava and Srivastava ( 199 1 ) investigated the effect of DEHP on the production of 17 P-

hydroxysteroid dehydrogenase (17 P-HSD). an enzyme invoived in the production of

testosterone. Testicular homogenates were used fiom animals given 0.15,0.5. 1 and 7 gkg of

DEHP for 15 days. While. teaicular atrophy \vas seen only at the highest dose. the levels of

17 P-HSD decreased to half of the control at 1 and 2 g k g (Srivastava and Srivastava, 199 1 ).

Jones et.al.. ( 1993). exposed isolated cultures of Lqdig cells to MEHP. Some cultures wvere

also simultaneously esposed to luteinizing hormone (LH), which stimulates testosterone

production in Leydig cells (Desjardins and Ewing. 1993). In both cases. MEHP caused dose

related decreases ( 1 O to 80940 of controls) in testosterone production (Jones et-al., 1993).

The mechanism of testicular tosicih caused by DEHP is not f'lly elucidated.

However. in vivo tosicity appears to be mocierated by age and the effects appear to be only

reproducible at very hi& concentrations. In vitro. the toxicih caused by DEHP 1s

multifactorial, and is related to MEHP's rffects in decreasing Sertoli and'or Leydig cells with

respect to energ- metabolism. s-me activity andfor hormone binding.

2.7.2 Female

Only a few studies have investigated the effects of DEHP on the ovary. Rats given 1.

10. 40. and 100 mgkg of DEHP subcutaneously esperienced dose related increases in

DNAase and RNAase. as well as related decreases in protein content, acid phosphatase and

ArPase i Arganval et. al.. 1 989 ). Histological inspection of the ovanes showed ce1 1s nith fewv

mature or developing follicles. while ma- othen had sigiificant nuclear debris: consistent

nith increased Iysosomal activity ( Arganval et-al.. 1 989). Treinen et-al.. ( 1 990 ) incubated

ovarian ganulosa cells with MEHP and observed significant decreases (40*.0 to 60°io) in the

production of CAMP. progesteronr levels and ATP levels after 30 to 48 hours. In an attempt

to elucidare the site of action of MEHP. the authon used a series of site specitic stimulators

and inhibitors of CAMP production. No rffect \ias observed in ~ m u l o s a cells CO-incubated

with MEHP in the presence of forskolin, cholera tosin and isoproterenol. However. MEHP

inhibited the maximal production of CAMP \bithout affecting the dose of FSH which yields

maximal stimulation. The authors suggested that MEHP inhibits CAMP production pnor to

the AC system: possibly acting as a non-cornpetitive inhibitor of FSH binding, or affecting

the nurnbericoupling abilih of the FSH recrptors (Treinen et-al., 1990).

The available data is too limited to draw an- meaningful conclusions. Additional

research is required to determine DEHP's mechanism of action on the ovanes.

2.7.3 Fertility and Embroytoxicity

Fenilih. assessrnenu on mature male and fernale mice injected subcutaneously with 1

to 100 mLLg of DEHP showed a dose related decreax in the incidence of pregnancy

(statistical level of sipificance not indicated) (Aganval et-al., 1989). Concentrations greater

than 10 m L k , caused stenlity (Aganval et-al., 1989). In an earlier feeding study. complete

suppression of fenilie was observed in mice at 3 gkg of DEW. while sigificant

suppression occurred at 1 g k g and no effect at 0.1 g'kg (Melnick_ 1 987). No toxic effects

were sren in a continuous breeding study using up to 7.5 g k p of DEHP (Heindel et-al..

1989 1. Similarly. an inhalation study on male rats using 0.01 m g 2 to 1 .O mgL had no affect

on fenility r Klimisch. 1992 ).

Reduced birth-rates were obsewed in pregnant mice fed 1 mLkg to 60 mLkg of

DEHP at differen~ stages of gestation (Tomita et.al.. 1982). In addition. when mice were

administered DEHP on day 7 or day 8. a higher incidence of embryo death. skeletal and goss

malformations occurred than on any other da- of gestation (259% and 6j0/0 dead embryos YS.

Ommi : < 5 * * 0 ~ \ * ~ ~ ,, ,) (Tomita et-al., 1983). Studies reviewed in IPCS ( 1 992) showed that O. 1

to 1 mLkg of MEHP @en to micr on da!. 7 of gestation caused increased rmbno deaths.

reduced body weight and increased skeleîal abnormalities. These effects were not observed

when given on da!. 8 or 9. rscept at the highest concentrations.

Mice dermally exposed to O to 3 mLkg of ?-EH for 21 days showd no si- of

rmb~tosicihteratogenicih. ( TyI. et-al.. 1992). In an oral feeding study using rats.

increased embryo death \vas observed with 7 mLl;g of 2-EH and 7ethylhesanoic acid ( 2 -

EHA) as well as 5 mLkg of DEHP (Ritter et-al.. 1987). The suniving embvos showd

levocardia and other cardiovascular defects as well as skeletal malformations of the limbs.

Ritter etal.. (1987) notrd that the most potent embroytosin and teratogen \\ris 2-EHA

followed by DEHP and 2-EH. In an interesting srudy by Hauck et-al- (1990) the racemic

mixture i= and pure entantiomenc ( R Bc S) foms of 2-EHA were investigated for their

trrtatogenic propenies. Groups of pregnant mice were given i.p. injections of 500 mgkg of

one of the different 'pes of 2-EHA four times a da! starting uith the evening of da! 7 of

gestation and continuing to day 8. R-(2-EHA) \vas the most embnotosic and teratogenic

causins 1 1 O,O r r n b ~ o draths and s9O.0 exencephal-: (=) 2-EHA caused 1040 embroytoxicity

and 3% teratogenicih. and S-(LEHA) rffected only 1 O , h of the embryos in both categories.

The control sarnples showed 6* O rmbryolrthality and malformation ( Hauck et-al. 1990 ).

At high doses. MEHP and 2-EHA have emb~otosic and teratogenic propenies.

S o m of thess rffects are speci t ic to cenain stages of gestation. however. their mechanism of

action remains to be elucidated.

2.8 Ca rcinogen icity

A number of long terni feeding studies in male and female rats have show^ that

DEKP causes hepatocellular carcinomas. neoplastic nodules and adenornas i Kluwr.

1981b: Rao et-al., 1990). However, the rnechanism of carcinogenicih is not understood.

Comprehensive revirw of the literature have show that DEHP. MEHP. and ?-EH are

neither genotoxic nor mutagenic ( Woodward. 1988. NIOSH. 1989: IPCS. 1 992).

However. it has bern suggested that DEHP's carcinogenicih ma! bt: relared to

peroxisome proliferation or its promoting activity.

Perosisornes art: single rnembraned orsanelles. predominantly found in the livcr

and kidney of rats and humans and believed to 5e present in al1 tissues cscrpt mature red

blood cells (Moser. 1993). The- perform a variety of functions. but haw bern most

studied for iheir abiiihv to P-oxidize and shonen medium and long chain fa@ acids and

substances like DEHP (Mannarns and Veldhoven. 1993 ). Whilr osidation of DEHP by

peroxisomes is similar to rnitochondna. peroxisomes have no respiratory chain and have

membrane pores instead of CAT through which fam acids can enter (ser 2.5 Alteration

of Lipid Metabolism) (Mannaerts and Veldhoven, 1993). Hence perosisornrs are not

subject to DEHP inhibition as are mitochondna.

Once a f a p acid is activated by acyl CoA synthetase. the first step in perosisomal

P-osidation imolws desaturation of the acyl -CoA and the production of hydrogen

prroside ( H 2 0 2 ) ( Mannaerts and Veldhoven. 1 993 ). It is the formation of HIOl that is

hypothesized to br critical in DEHP carcinogenesis. It is believed that constant DEHP

rxposure induces perosisornrs that contain much higher levels of H 2 0 2 fonning osidases

than catalasr. Catalasi: levels are insufficient to decompose the H-0: formed so that H:O:

escapes from the peroxisomes and causes lipid perosidation of the cell membranes and

subsequent cell injury. H z 0 2 rnay also cause DNA damage in the nuclei. mitochondria

resutting in genetic mutations ( Reddy and Rao, 1 987: Gibson. 1 993 ). Hoivever- short

t e n DEHP feedinç studies ( 2 weeks or less) show a decrease in liver catalase. whereas

long tem studies show an ovrrall increase in catalase \vithout affecting glutathione le\ els

(Ganning et-ai., 1983. 199 1 : Conway et-al., 1989: Mikalsen et-al., 1990: Tamura. 1990:

Reubsaet et-al.. 199 1 ).

Another hypothesis of carcinogenesis is that DEHP may act as a promoting agent.

It is well estabtished that DEHP. MEHP or 2-EH when administered to rats and mice

induces liver perosisome proliferation ( Moody and Reddy, 1978: Elcombe and Mitchell.

1 986: Cornu et-al.. 1992: Keith et.al.. 1992. ). It has also been demonstrated that DEHP has

the ability to stimulate replicative DNA synthesis ( Marsrnan et-al., 1988: Conway 1989).

The subsrquent increase in cell division of initiated cells could result in carcinogenesis

(IPCS. 1992). However. evidence suggesting that DEHP is a tumor promoter have bren

inconcl usiw ( Ward et-al., 1986: Woodward 1988b: Schmezer et-al.. 1988: Gerbracht

et.& 1 990 ). Furthemore. cultureci rat hepatocytes have shown are panicularly sensitive

to DEHP induccd perosisome pro1 i feration. whi le rnonke!. and human hepatocytss

appear to be unaffected ( Elcornbe and Mitchell. 1986: Dimen et.al., 19953.

The studies reviewed above s h o ~ that DEHP can cause cancer in rats: howwer,

the mechanisrn( s ) involved are unclear.

2.9 Other Effixts o f DEHP

The acute toxicity of DEHP is low, with an oral LDPl generally greater than 25 gtkg

and an intravenous LDr of 100 mg,& (Woodward 1988). Rats given I.V. transfusions of

b l d containing 250 mgkg of DEHP over a hvo week period showed pulmonq damage

called "the shock lunrr + e f f e d ( Rubin and Chang 1976b; Woodward 1988 1. The symptoms

incl uded: edema haemorrhase and infiltration of the lung vrith polynorphonuclear

Iynphocytes (PMN) which resultrd in the death of DEHP neated rabbits and dogs.

Interestinply, when the I.V. DEHP dose is given neat or as an emulsion. the required "shock

luna-' C dose increases to 500 mgkg (Woodward 1988 1.

The reaction of PMNs with DEHP was shown in vitro by Bally et-al., ( 1980) uho

isolated rabbit alveolar Pms and incubated them in blood senrrn contarninated with D E W

that had leached From I.V. transfusion bag (final concentration: 0.05 to 2.0 mg DEHP ' 100

mL of media). -4 clear dose dependent incrase of phagocytic activih and releasr of

hydrolase enzymes vas observcd.

In a review of imration studies by iPCS ( 1992). DE!+ was found to be a very weak

imtant to mammalian skin when applied topically or intradrrmaily. Most studies showed a

ncgative response and onlv one observed imitation following 10 to 14 days of repeated

application of 100 mg of DEHP to the skin of Guinea pi-. Irritant propenirs to the eye have

not ken observed. Insufficient research has been conducted to determine DEHP's potential

as a sensitizer.

2.10 Human Effects of DEHP

Studies on the effects of DEHP on humans is exirernely limited. Moderate diarrhoea

and general gastric irritation were esperienced by two adult volunteers who swallowed 10 g

of DEHP (SchaRer et-al., 1945 cited in Schmid and Schlatter. 1985). No rffects were

rspeiienced when 5 mg was inpted . DEHP leached From medical tubing (atimated ar 10-

20 mgL) caused three cases of non-specific hepatitis out of 77 patients recriving

haemodialysis for terminal renal failure. When the tubing was changed to material not

containing DEHP. the spptoms of hepatitis disappeared (Neergaard et.al., 1971 ). One case

of occupational asthrna due to DEHP esposure was reponed for a male worker al a PVC

procrssing plant. The asthmatic response was inhibited with the ux of sodium

chromoglycate pnor to DEHP esposure (Bruneni and Moscato. 1981). In an occupational

study by Nielsen et. al. ( 1985) biochernical parameters were studied in 51 men esposed to

DEHP (0.1 to 0.7 mg m ' ) at a PVC processing plant. The results showed an association

between lenkqh of crnplo-ment and a slight decrease in hacmoglobin levels. In addition.

srmm alpha-1-antitvpsin increased slightly with time of emplo-ent and semm

immunoglobulin-A was observed to rise with the parameter "rngm'.yeai' which \vas used in

the study as an index of ssposure. Nielsen et.al.. (1985) also noted that some worken

displayed symptorns and s igs of peripheral nsuropathy: however. the sample size t a s too

small to detemine the significance of these observations (Neilsen et.al.. 1985 ).

From the reviewed studies it is not possible to draw an? conclusions on the human

health effects of DEHP esposure. In particular. there is a need to inïrstigate effects on

human reproduction and carcinogenicity.

3.0 Selecting a Biological Indicator and Study Design

3.1 The Seed for a Biological Indicator

For chemicals ti.hose health effects are associated with their metabolism (1.e. not

direct acting), an accurate risk assessment of an indkidual's exposure depends on the precise

measurement of their intemal dose. While personal air sampling is commonly used to

estirnate esposure. it cannot accurately enimate the inremal dose since it does not include

modifiing factors such as metabolism or chernical exposure from skin contact, ingestion or

non-occupational sources. In addition c q i n g out a comprehensive air assessment is

expensive. requires a substantial amount of equipment and is logistically dificult to

impiement. In contrast a biologica1 indicator provides a more accurate estimate of the

internai dose by directly measuring the substance of concem, or its metabolite, in the body.

Funhmnore. it takes ïnto account al1 routes of esposure as well as the effects of metabolism

on the intemal dose. It is also relatively inexpensive and in the case of urinalysis, easy to

conduct. Hrnce. a biological indicator of exposure provides a more accurate assessment of

the intrrnal dose and assists in a more precise determination of h d t h iisks associated with

c hem ica i esposures.

.As a result of the value biological monitoring offen to expsure assessments. the

Amencan Conference of Governmental and Indusnial Hygienists ( ACGIH) has

recommended the use of t h - s e v e n Biological Exposure lndicies (BEI) for various

chemicals (ACGI)-I, 1993). Similar to airborne Threshold Lirnit Values (TLVs). BEls are

reference values that apply to an eight hour day. five da). work week and can be used to

assess a worker's occuparional exposure. However, unlilie TLVs, BEls involves collecting

either blood exhaled air or urine. While the primary use for BEIs is for estimating a worker's

occupational expoure frorn a11 routes. il has also k e n recommend for: validating air

monitoring, testing the efficacy of penonal protective equipment. as well as detennining the

contribution of non-occupationai exposures to work e.xposures ( ACGM. 1993 j.

3.2 f revious Work on Biological Indicators

Four audies have investigated the use of urina- metabolites as a biologcal indicator

of airborne exposure to DEHP. In the study by Liss et-al., (1985.). pre- and pst-shifi urine

samples of 95 employees (sex unspectfied) were analyseci for total phthalic acid

concentration. The participants worked for a minimum of six months in a phthalate

manufacturinç plant which also produced phthalic acid. Personal air samples were taken to

m a u r e individual worker exposure to DEHP and phthalic acid. The results indicated no

correlation beiween urina5 phthalate and worker exposure to DEHP. lhere \vas a weak

positive correlation between urinary and airbome phthalic acid exposure. No correlation was

found benveen airborne esposure and pre to pst-shift change.

Similady. Nielsen et.ai., ( 1985) measured total urinary phthalic acid concentration in

1 men esposed to DEHP. di-içodecyl phthalate and butyl be-l phthalate in a PVC plant.

Personal air sampling \sas carried out for the three compounds and worken wvere gwen a

medical esamination whch included a blood analysis. The minimum duration of

employnent of the worken w a s one year. The results of the urina? analysis showed a

si pi ficant increase in phthalic asid metabol ites: however, correlations benveen the urina9

metabolites and airborne concentration of phthalates were not e.mined.

Phthafic acid is clearly not a suitable biologcal indicator of DEHP exposure. Since

phthalic acid is potentially a common metabolite of al1 phthdata, it would be dificult to

detemine DEKP exposure in an industrial environment where phthalic acid itself or a varie-

of phthalates may be present.

Dirven et-al., (1993b) studied MEHP and three of its metabolites as potential

indicatoa of DEHP expsure in boot and cabie factories which both ued PVC. In the boot

factory, pre- and pst-shifi mine samples were obtainrd from nine males (length of

employment unspecified), three men each week for three weeks. Personal air sarnples for

each worker ivere taken for 2 to 1 hours on the fint and 1st day of the work week. Similady,

for sis workers in the cablr factory. pre- and pst-shift urine samples were collected on the

fint and fourth d g - of the week along wÏth personal air samples. No correlation kvas found in

cithrr factory between DEHP in air and the urinary metabolites. Statis~ically siyificant

increases in pre to pst-shiR change in the urinaq concentration for three of the metabolites

of MEHP were observed in the worken at the boot factory. No statistically significani

changes in metabolite concentration were observed in the cable factory worken. Based on

the metabolites' low interindividual vanability and their relationship with animal toxiciiy,

Diwen etal., (1993d) concluded that MEHP and its three metabolites should be used as a

biological indicator of DEHP exposure. However. gken that no correlation kvas observed

between anp of the metabolites and DEHP in air this recommendation is premature. Further.

background MEHP levels tconcentrations not stated) were obsen-ed in pooled conuol urine

samples: therefore the workers' urinary concentrations. which were not corrected for

background may have been overestimated Finally, metabolites of MEHP are dificult to

synthesize and are n~t~conimercially available, which makes routine detemination of DEHP

exposure by this method difficult and impractical.

It is clear that a more practical biological indicator for DRIP exposure is needed

When choosing a biological indicator of exposure, three main factors must be

considered: the choice of collection rnatrix (blood or wine): the specificip- of the metabolite

to the chernical of interest and the suitability of the analytical method for routine application.

Mile a biological indicator for DEHP exposure could be developed for either blood

or urine- a ,na- indicator is preferred since collection is non-invasive, simple to conduct

and creates little or no disruption in a worker's routine. Also, a urinan; biological marker

reflects. to some extent the end product of metabolism. In contrast, the concentration of a

metabolite in the b l d is dependant on the amount of the metabolized parent compound

and the amount of metabolites removed by biotransfomation and rxcretonp processes.

Hencr, for a rapidly metabolized substance like DEHP, a urin-; biologcal indicator would

be less affected by peak esposures than an indicator developed for b l d .

In reiiewing the metabolkm and elimination of DEHP (See, Sections 2.2 and 1.3

three metabolites appear to be found in suficient quanti. in the urine to malie suitable

biologïcal indicators: 5-OH-MEHP. MEHP and ?-M. As mentioned earlier. the osidation

products of MEHP are not cornmerciaily available. Therefore, even though 5-OH-MEHP is a

major metabolite in monkeys and hurnans (Peck and Albro. 1982), quantification is difficult

and ùnpractical for routine measure. Whle standards are commercial 1 y avai lable for MEHI?

Dirven et-al., ( 1993d) recommended that MEHP be quantitied along uith t h e of its

metabolites to adequately m e s s DEHP exposure. Altematively. ?-EH, which is produced in

a 1:l stoichiornetnc ratio with MEHP, is a major metabolite in monkeys and is readily

available cornmercially. Based on the sirniiarities in the metabolisrn and elimination of

DEHP in monkeys and humans, ?-EH, has the potential to be a suitable and practicd

biologtcal indicator for human exposure. While ?-EH is a metabolite of DEHP, it is also a

metabolic by-product of the plasticizers di(?-ethylhexyl) adipate (DEHA) and di(?-

ethylhe-xyl) tetrahydrophthalate (DEHT). These substances are used as alternatives to DEHP

and are not likely to be used together in the same production batch of plasticized materials.

Hence. under normal manufacninng conditions. ?-EH would be specific for DEHP.

An imponant aspect in developing a routine biological indicator for DEHP is the

method of anaiysis. While preiious attempts at developing a biological indicator wd time

consuming and labour intensive techniques, the method developed in this thesis for analysing

2-EH (discussed in detail in the Sections 3.4 and 4.3) uses mcjre efficient extraction methods

to achieve greater sample throughput and has a low detection limit. Therefore, the analflical

method would be suitable for routine ?-EH analysis.

3.4 Study Objectives and Hypothesis

The pnmary objective of this study was to assess whether 2-EH is a suitable urinas

biological indicator for estimaring human occupational exposure to DEHP. In order to meet

this objective the following tasks were completed:

1 ) Development of a method to isolate and detect 2-EH in human urine.

2) Collection and analysis of urine and air samples from workers exposed to DEHP

in an industrial environment.

3) Investigation of whether a correlation exists between urina? ?-EH excretion

and airbome exposure to DEHP in an industrial environment.

The underlying hymthesis was that 2-EH is a valid index of DEHP exposure.

3.5 Development of a Method to Isolate and Detect 2-EH in Hurnan Urine

3.5.1 Previous Methods Extracting and Anaiysing Z E H

Prwious methods extracting and quantifjing ?-EH in urine ancbor tissues have

commonly used samples collected From animals fed radiolabelled parent compounds (2-

EH. DEHA or DEHT). The samples were then subjected to liquid-liquid extractions with

diethyl ether to separate ?-EH from the sample rnatrix. In some cases, samples were

incubated prior to extracrion with B-glu~uronidase~~arylsuphatase to de-conjugate ?-EH

from its glucuronide (See Section 2.4). Followving extraction, ?-EH w a s then analyzed by

scintillation techniques. High Pressure Liquid C hromaiography or Gas Chromatography -

Mass Spectrometry (Albro. 1975; CMA, 1985: Barber et.al.- 1994: Deisinger et-al.. 1994).

Only one of the reviewed studies used non radiolabelled ?-EH and gas chromatograph-

flame ionization detection (GC-FID) (Cornu et.&, 1988). Urine was obtained From rats

given doses of DEHA (0.6-1 -5 s'kg) for three days. The lirnit of detection (LOD) was not Y

indicated. nevertheless, the concentrations used in the experiment are much higher than

anticipated workplace exposures.

3.5.2 Developiag a 3ew Method for detecting 2-EH

Initial studies considered GC-FID to measure urinan ?-EH. However, usine a

Varian 3100 GC-FID set at m a ~ i m u m sensitivity (range 13) and a standard sipal ro noise

ratio of 5- the LOD for 7-EH was approximately 10 pgmL. Given that hpical

occupational exposures were below 1 mgm' (See Section 1.3) it was expected that

urinary ?-EH levels would be at least one order of magnitude lower than the current

LOD. Therefore. the GC-FD LOD \vas inadequate.

Chemical denvatization is ofien used to irnprove chromatogaphic properties of a

substance by converting the physical propenies of the parent compound into a structure

that can be more easily identified by a given detector (Blau, 1993). Given the low LOD

for .-EH. derivatization sermed to be rhe most suitable course of investigation. Howeïer.

a brief survey of commercial derivatizing agents such as (N-(tert-Bupldimethysily1)-N-

rnethyltrifluroacteamide t MTBSTFA) showed that. in addition to being expensive.

temperatures greater than 30°C and reaction times of up to 30 minutes were commonly

required (Zweig and Sherma, 1 972: Blau. 1993 ). In order to develop an analytical method

for routine measurement of ?-EH, it was believed that a more rapid sample preparation

method would be of greater practical value. In 1959, Fitz and Schnnl; developed a rapid

derivatization method for primary, secondar); and tenia? alcohols using acetic anhydride

vrith perchlonc acid as a catalyst. The reaction went to cornpletion at room temperature

in five minutes. The method waas originally developed for quantitative titrations.

however, it \vas believed that its use could be extended to GC-FID.

Tnitially. ?-EH was reacted with an approsimately ( 1 : 1 ) acetic anhydride:

perchloric acid mixture according to the method of Fitz and Schrink ( 1959). The reagents

and 2-EH were dissolved in ethyl acetate. Afier 5 minutes. pyridine was addrd ro

neutralize any un-reacted acidianhydride. While the resulting ?-EH ester could be

detected on GC-Fm. the chrornatographic resol ution of peaks and the limit of detection.

while improved was still poor (Le. 1 pg mL). Based on these results, it r a s believed that

a lower detection limit could be achieved by using an €CD rrith a chlorinated anhydride

derivatizing agent.

Tnchloro acrticanhydride (TCAA) was used wi th perchloric acid in ethyl acetate

and \vas successful in fonning a ?-EH ester that could be detected by ECD. Howwer.

TC.U fomsd a salt precipitate with pyridine during the neutralization reaction which

resulted in poor chrornatographic resolution. Consequentl y water \vas added to the

organic phase to dissolve the salt: afier which the water was removed. This significantlp

improved resolution and lowered the LOD to 0.05 pstrnL. Despite these changes.

derivatized samples were unstable and degraded wïthin 71 hours. The instability was

belie~ed to be aruibuted to the use of perchloric acid which, due to its water content.

l i kely reduced the activity of the moisture sensitive reagent, TCAA. In addition. the

solvent ethyl acetate. possibly due to its polarity or water content (0.001*0 1. contributed

to sample instability. When TCAA wuas used wvithout perchloric acid and cyclohexane

(water content 0.000 1 O . h ) used in place of ethyl acetate. resolution Kas enhanced. sarnple

stability improved and the detection limit lowered to 0.0 16 pgmL. Once prepared, the

derivatizing agent can be used for up to two weeh.

While additional expenmental details are gven are under Methods (Section 4.3).

the final denvatization method used in this study involved two steps. In the first step.

TCAA was added to 2-EH to fom a 3-EH ester. This was followed by neutralization of

the unreacted TCAA with pyidine. Finally the organic laver was washrd with water to

remove the pyndine-TCAA salt and the organic phase drkd nith NazSOI for CC-ECD

analysis. All reagents and samples were dissolved in cyclohexane. The stochiometry

rroverning the reaction between TCAA and 2-EH is shown in Figure 6. C

O-R

! l CH2-CH3 ?

I l

1

Where R= CH-CH-(CH2)s-CH3 !

Figure 6 Derivatintion Reaction Between TCAA and 2-EH

Studies on the rate of formation of the ?-EH ester from the reaction between ?-EH and

TCAA were conducted using Fil3 and are shown in Figure 7. Ester formation occurs

rapidly and is complete, Le. below the LOD for FID, in 2 minutes. in addition to

cyclohexane, the reaction works equally well in c&n disulfide (CS?). Once derivarized

samples remain stable at room temperame for at least one week.

A typical chromatopph of 2-EH and the intemal standard nonanol is shown in

Figure 8. It should be noted that octanol 1va.s ori-@nally chosen as the internai stanàard, but it

\vas found to contain içomeric forms of 2-EH which produced erroneous 2-EH ester peaks. It

should also be noted that derivatizations of cyclohexane alone resulted in a small peak at the

sarne retention time as ,-EH. Therefore, al1 urinaxy results had to be corrected for

background 2-EH. The correctrd LOD for this method was 0.016 pgmL. The profile

observed in the first minutes of the chromatogram was consistent in dl sarnples derivatizd

with TCAA. These peaks are rnost likely due to side reactions from irnpurities found in

the technical grade (9SoI6) TCAA.

3.53 Extracting 2-EH from Buman Urine

As reviewed in section 2.4. ?-EH is excreted in the urine of primates predominantly in its

glucuronide fom. Therefore. pnor to extraction ai1 urine sarnples were incubated with P-

elucuronidase. As previously cited liquid - liquid e.vttaction techniques were commonly used C

to cstract ?-EH from iis sarnple matrix. However, this method of exsraction is too laborious

for routine assessments so urine was applied directly to a solid phase extraction colurnn

followd by the elution of 2-EH using a suitable solvent. In order to remove and reduce the

number of unwanted compounds eluting with ?-EH, concentrated HCI was added to urine

samples prior to its application to the ex~raction colurnn. The strong acid ionized amine and

other similar groups allowing these substances to pass through colurnn while leaçing behind

2-EH. Initially. ethyl acetate was favoured in the recoven of ?-EH From the column because

of its polarity. However. a s rnentioned earlier, ethyl acetate was not a suitable solvent for

denvatization because of its moisture content. A m i - m e of cyclohexane wïth jO/o ethyl

acetate \vas therefore used. As shoum in Table 2, ihis rni'rture resulted in method recoveries

of 99.7O&34,0. as determined in spikd pooled urine samples from non DEHP esposrd

workers. Furthemore. the mixture did not sigmficantly affect the reaction rate of the

denvahzing agent nor the stabiliîy of the ester.

of the intemal standard was 0.68pgtmL.

Table 2 Recovery of 2-ER from Human Urine using Column Extraction Replicate G

-. Recovexy Concentration

of ?-EH' pgmL Area of ?-EH Pealis

Area of Interna1 Standard Peaks

3.5.4 Summary of the method of Analysis used in this study.

Figure 9 summarizes the method developed for extracthg and quanti-ing

u r i n q ?-EH. Full eqxrimental details are listed in Section 4.3. In Phase 1. ?-EH is

incubateci with J3-glucuronidase to cleave 2-EH from its glucuronide. In Phase II the fieed 2-

EH is applied to the cleanup column and eluted wîth a mixed cyclohe.we-ethyl acetate

solution. This is followed by Phase IIi, where 2-EH is estenfied with trichloro acetic

anhydride (TCAA) and analysed on GC-ECD. The LOD for this method \vas 0.0 I l pglmL

and its precison was approxirnately 2 to 3 O h .

PHASE 1 PHASE 11 PHASE III

Urine - glucuronidase lncubated overnight at 3 7 " ~

TCAA

1

SampIe transferred to cleanup column & ?-EH eluted with solvent

GC-ECD

?-EH is reacted with TCAA to form a ?-EH ester and is quantitied

Figure 9 Overvier of the method for Extracting and Detecting 2-EH from Human Crine

3.6 Sampling Strategy

To deterrnine if ?-EH can be found in the urine of DEHP exposed workers and assess if tius

correlates with airbome exposure. the sampling sh-ategy shown in Figure 10 \vas used. Ths

strategy also allowed for the separate assessrnent of changes in urinary ?-EH and DEHP

rsposure over nvo consecu<ive workshifrs. Sarnple collection bqpn with the first workday

following the weekend, therefore any urinas. ?-EH found in the Prel sarnple would be

considered background DEHP exposure. Furthemore. by collecting a total of five pre and

pst urine samples and two consecutive workshif? samples, a variety of correlations could k

e-mined such as: Workshifi l exposure vs. ( Posr !-Pre ) or Workstu fi 1 vs. Pre:.

In order ro deterrnine if airbome ?-EH is a confounder of urine sarnples. air sampling

was aiso camed out for 2-EH.

Figure 10 Sampling Strategy Air sampling occurs dunng the periods indicated by the square bracliets. Note: pre=pre-shift sampie collection: post=post-shift sample collection (assumes 8 hour workshifis).

4.0 Methods

4.1 Sampling Location and Process Description

A phthalate resin rnanufactunng plant located in South Eastern Ontario was

selected as the site for obtaining air and urine samples. The plant operated on three shifts,

24 hours a &y. seven days a week and was divided into three processing units that

produced plasticized resins according to customer requirements. The layout of the unit

where worker exposure to DEHP was monitored is show in Appendis (il). Each

processing unit was divided into two floon. On the upper floor, DEHP was pumped frorn

an outside storage tank to an indoor holding tank. From there it \vas pumped into a mixer

with PVC resin brought in by hoppen. Both the quantity of material pmped in from the

holding tanks as well as the rnixinp process itself t a s automatedi programmed at worker

operated control panels. Stabilizers and colouring agents were separately weighed and

added to each batch rnanually. Afier the resin was rnised with DEHP. the misture t a s

sent by hopper to an extruder on the first floor where it was pushed throuçh a mould and

cut into pellets. A vacuum system then carried these pellets up to the second floor to the

"cooler" where the plasticized resin was cooled to a semi-solid state. Once cooled. the

pellets dropped down a chute to a large open package container on the first floor which,

when full, was removed to the warehouse area for storage. The production unit had two

main doors. one on each floor. that were kept closed at al1 times exccpt when package

containers were being replaced or taken to the warehouse. While the extruder and the

cooler were enclosed and equipped with local evhaust ventilation. the mixer. the DEHP

holding tank and the hopper comecting the mixer to the extruder were open and had no

local euhaust ventilation. General ventilation of the hvo floors was accomplished by a

second floor fan which drew in outdoor air and shunted some of it through a duct system

to the lower floor. While in operation, the process gnerated an arnbient temperature of

30 ' '~ 'C during the month of December) and a haze could be obsemed on both floors.

Three workers were required to operate the unit. The blender worked on the

second fioor and supewised the mixing of the resin. plasticizer. stabilizer and colouring

agents. The bagger worked on the main floor and was responsible for removing resin-

filled containers to the storage area. The floor supervisor also worked on the main floor

overseeing the operation of the whole unit and providing break relief for the bagger and

t he blender.

Periodically, during production or between batches, the process was shut d o m

for approximately 1 hour and the systern was cleaned. Cleaning required ail the members

of a workshift to remove any remaining resin from the exmider and cooler. Pressurized

air hoses, hand cloths and occasionally bare hands were used to remove plasticized

pellets and unestruded resin from the cooler and extruder. respectively. Dunng the study

period. the equipment was cleaned twice dunng Workshifil. The fint involved a suitch

over from the use of DEHA (useci for 15 minutes) in the PVC resin to DEW. The second,

occurred several hours later. when a colouring agent \vas to be added to the PVC-DEHP mis.

The equipment then ran continuously for the remainder of Workshift 1 and WorkhiE.

4.2 Su bject Selection

Prior to requesting volunteen, workers at the plant were given handout sheets containing

general information about the study (See Appendix III). A total of nine male workers.

(Le. al1 three shifis), agreed to volunteer for the study. Ail pmicipants signed a consent

form (Appendis IV) and were aslied to complete a basic work and health questionnaire

(Appendix V). The questions were designed to obtain information relevant to pulmonary

absorption ( Le. age, weight, height ). as well as to identi- potential confounding factors

such as smoking, alcohol consumption or a relevant rnedical condition. In addition.

worken were reminded of proper hygîene practices (i.e. wshinç their han& afier each shft)

in order to minimize dermal exposure to DEHP. Controls were obtained from two volunteers

who worked as office personnel in a faci lity un-related to the plastic industq .

4.3 Urine Sampling and Analysis

Urine kvas collected in 150 rnL glas bottles wvith screw cap lined with aluminium

foil. To avoid an- possibility of sarnple contamination. al1 material used in the collection or

analysis of 2-EH was glas. Tefion or polypropylene. Al1 glassware was washed in soap and

\mec rinsed with deionized water. then again in methanol and dned in an oven at 150°C

The aluminium used to line the caps of the urine collection jars \vas pre-washed 14th

methanol. Boales were capped and remained unopened until sarnple collection. As indicated

in Figure 10. workers were asked to provide urine samples at the beginninç and end of their

worhhifts. Conrrols were obrained from individuals not employed the plant. Ail urine

samples were irnmediately stored between O to Sic, uniil analysis.

Following sample collection an aliquot of urine was sent to Med-Chem Laboratones

Ltd. (Scarborough, Ontario) for measurement of creatinine. In order to detenine if humans

excrete ?-EH primarily as a glucuronidr. each urine sample t a s analysed in duplicate. In the

first test tube (25 mL pyrex). a 5 mL sarnple of urine was transferred using a disposable glass

serological pipette and I mL acerate buffer ( l M pH 5 . 5 ) and 10pL (930 Units) of P-

glucuronidase type HP-I (EC 3.2. i .3 l ; Sigma Chernical Company Louis- MO, USAI is

added. The second test tube conrained oniy the urine and the acetate b u f k However, ir

shouid be noted that ?-EH \\as only found in samples containkg P-glucuronidase. The test

tubes wvere then sealed wth mbber stoppers and placed in a water bath at TC ovemgh? ( 16

IO 2 8 h m

Following this incubation. 0.2 ML of concentrated HCI and 0.1 mL of the interna1

standard 3.3 l ugmL nonanol ( WO%- Aldrich, Milwaukee. W. USA 1. were added to =ch

test tube and the tubes were vonesed. n i e samples were transferred to separate disposabi<:

Bakerbond spe' Octadecyl t C l x ) extraction columns ( VWR Scientific, Mississauga Ontario )

whîch werr previously conditionrd with 2 colurnn volumes of methanol and deionized inter,

respecrivel!.. The sarnples were applied io the column under vacuum and allowed to air @.

The aqueous phases were discarded. Finaily, 4 mL of a cyclohexane solution containmg 3s 6

ethylacrtatr ( v v ) uils added to each column and the organic fractions w r e collected A 0.5

mL aliquot of each sample \vas msferred to a clean 5 rnL micro-reaction via1 (Supelco

Canada M~ssissauga Ontario ,.

The TCAA (technical gade %?/O; Aldnch M i l ~ ~ w k e e ; WI? USA) derivatizing

ragent uas made up in a 1 : 1 mi.uhire with cyclohexane. The organic phases for both samples

were then denvîtized accordmg to the following method. L'sing an Eppendorf pipette. 20pL

of TCAA \vas added to the reaction viais, the Mal bas capped, shaken and the organic phase

allowed to react for 2 minutes. Then 30pL of pyridine \\as added resultinç in the formation

of a salt precipitate. Approsimately 500pL of deionized water was added and the via1 shaken

vigorouslp mtil the organic phase became clear. The aqueous phase was removed by s ~ n g e

and the water wash repeated. The remaining o r b e c layer was dned uith NalSOl

(previously wshed wlth methanol and oven dned at 150°c), then transferred with a syinge

into a clean GC vial.

Analvsis \\as performed on a Varian 3400 gas chromatograph equipped with a N?'

detector and a Varian 8700 Autosampler (Varian L t d Mississauga Ontario) (Table 3).

Inte-ation was handled by Varian STARU Workstation Version 4 software.

4.4 DEHP and ZEH Air Sampling and Analpis

44.1 Air sampiing

Air sarnpling for DEHP was conducted accordinç to NIOSH method g5020 ( 1985).

At the bsginning of each shifi. al1 workers were equipped with a sealed filter cassene unit

containing a 0.8pm pore size. 37 mm diameter rnived cellulose ester membrane filter with

suppon pad (SKC. Intekm Environmental Burlington? Canada).The cassette was clipped to

the worker's shin collar and attached to a personal, battery operated air sampling purnp

(Gilian Hi -Flow Smpler -mode1 HFS 1 I3A or SKC Airchrck Sampler-mode1 3LWjXR)

usin2 us on" tubing. Workers were also outfïtted with air sampling equipment for 2-EH.

Charcoal tubes containine 200 mg of coconut charcoal in the front section (SKC. Intehm

Environmentai. Burlingtori, Ontario), were cut at each end and attached to either a Dupont

PlLC or Dupont Alpha -2 sampling pump with TygonR tubkg. Mer aü equiprnent was

attacheci, the pumps were turned on and the tirne documented. The worken were then Free to

conduft tbeir normal workshift duties. Occasionally a visual inspection of the pumps was

made to ensure the purnps were operating properly. At the end of the sh i f t sarnpling

equiprnent \vas remove4 the time of removal documented, and the cassettes or tubes sealed

and immediately refngerated.

Al1 pumps for DEHP sarnpling were calibrated to draw 1.5 Umin while pumps for

?-EH sampling were set to 200 mLmin using a portable SKC Cal flow meter (SKC.

integra Environmental, Burlington Ontario). with the sampling device in line. The pumps

were calibrated before sampling and the flow rate checked again afler the workshifi. The

actual Oow rate for the shifl \vas determined by taking the mean of the two values.

Table 3 GC-ECD Aoalvsis Conditions ECD and Colamn Conditions 1

ECD makeup gas ECD sensitivity Colurnn: CoIumn Carrier Gas Injector/Detector ~ernp('c)

Nitrogen (5OOkPa) range 1 SPP-1 (Supelco); 30m, 0.75 id. :flow rate 2 ml'rnin Helium 250

CoIumn Temperature Pro- 1 Initial (OC) / 120 ramp 3 'c/rnin 1 Final (OC) 150 with 2 minute hold

Column Temperahire Program 2 ramp 15 "cimin Final (OC) 200 with 5 minute hold J

4.4.2 Analysis

To avoid any possibil ity of contamination, al1 material used in the analysis of DEHP

or 2-EH was glas, Teflon or polyromlene. Ali glassware was washed in soap and mater and

nnsed with methanol and oven dned. The filten from each cassette were placed in 5 mL

glas vials and 2 mL of CS2, containing 1 1 pg;rnL of the interna1 standard diethyl phthalare.

was added. The vials were then agitated for 30 minutes in an ultrasonic bath. Similady,

charcoal tubes were alw agitated in CS2 but 100 pg'rnL of nonanol ~is used as an intemal

standard Following agitation. DEHP and 2-EH were quantified by GC-RD along with both

filter and tube blanks. GC-FID conditions are listed in Table 4. The Time Weiphted Average

(TWA) concentrations for DEKP and ?-EH were detennined from the quantity of the

matenal on the filtermhe (corrected for blank value) divided by the mean of the total

workshift air volume sampled.

-- -

1 Column: TD&S : 30m .0.25id.: flow rate 2.8 mumin 1

Table 4 CC-Fm -4nalysis Conditions Detector and Column Settings

Substance 1 DEHP (NIOSH rn~hod 5020) 1 2-EH Coiumn Temperature ec)

Initial 150 1 00; 10 minute hold rarn p I O "Ci min I O ' ~ i rn in

r

F D makeup gas FID sensitivitv

1 Final 1 250 with f 5 minute hold 1 150: with 1 minute hold

Air (6OOkPa) and Nitrogen ( SOOkPa) rame 12

1 hiector/Detector Temp ('c) 1 Iniector 1 300 1 ISO

The amount of DEHP recovered fiom mixed cellulose ester filten usine CS: uas

determined by analysing five filters spiked with 50 pg of DEHP stored overnight in sarnpling

cassettes. Similarly, 2-EH recovery wvas determined fiom the fioir sections of five charcoal

tubes spiked with O. 1 mL of ?-EH (4 mghL). Recoveiy of DEHP was greater than 960.6. In

contrasr, recoveq of ?-EH from the charcoal tube \as low at 70?,,0. In order to obtain a more

complete recovety of ?-EH fiom the charcoal, future studies should use dichlorornethane in

place of CSI to improve recoveq to 9696 (Andenson rt.al.. 1984). Breakthrough taas not

observed through the front section of the charcoal tube. Al1 results were corrected for

recoven.

4.5 Statistical Analysis

Analysis of Vanance (ANOVA) was used to test for a significant difference

between the means of the two sampled workshifts for DEHP. airborne ?-EH as well as

the pre- and post-shifi u r inap ?-EH concentrations. The Least Significant Difference

method (LSD) (Steel and Tome. 1980) was w d to test for significant differences in mean

airborne esposures and urinary concentrations among goups by workitle. Univariate

regession was used to assess the correlation of DEHP and ?-EH. Multivariate regression

analysis \ras conducted to examine the relationship behveen DEHP, urina- 3-EH and

various contriburing factors taken from the worken health questionnaires such as age. length

of employrnent.

5.0 Results

Prior to the beginninç of the study, it ws detennined that air and urine sarnples

from 50 workers would be required for a statistically valid analysis of the relationship

between DEHP exposure and urina? ?-EH (see Appendix VI). However, due to factors

outsidr of the author's conaol. it was not possible to obtain this sample size. As a resuit a

detailed stahstical analysis on a small sarnple size wodd be inappropriate. Furthemore,

many u r i n e results are near the analyticai detection limit which increases their de-me of

variability and reduces the interpretative value of the results. Therefore, the following results

and conclusions shouid be considered as illustrative of the methods and potential factors to

be considered in fiiture studies with appropriate sample sizes.

S. 1 Worker Questionnaire

Of the nine worken and two controls that ageed to pmcipate, two Operaton were

escluded fiom the stuc- due to diabetic conditions. The responses to the health questionnaire

for the remaininç nine subjects are shown in Table 5. The average age for the grooup was 34.5

years and the a\!eragr lengh of ernployment for the workers uas 7.25 years, with 4 yean

k i n g the minimum ernplo-ment period. Five of the workers were current smokers and one

( Baser-Alj, \vas a previous smoker sufferinc wîth bronchtis. One of the controls suffered

u i th rnild asthma. None of the subjects used prescription medication or consumed alcohol

during the sample collection period. Mixers A3 and A6 used d u t fibre face rnasts

approsimately half of the tirne during their shift.

Table 5 Summary of Work and Health Questionnaire. ~ - - - - - - - - - -

Work Title and ID Bagger- Al Bagger-A5 Bagger-A8 Operator-A4 Mixer&"

Suffered with Bronchitis b~uffered with mild asthma ' Does not include controls ' Used fibre face masks approsimately ';2 the time during workshiîts

5.2 DEHP and Airborne 2-EH exposures

As s h o w in (Figure I 1 ), DEHP concentrations ranged from 0.00 12 mgm' to 0.72

rngd and ?-EH exposures ranged from 0.770 mgm' to 4.50 mgm'. In general, WorkshifU

?-EH esposures apprar higher than those of Workshifi 1 : however. this pattern is reversed

wi th Mixers A6 and A7 ( F i ~ w e I 1 ). The mean airbome ?-EH concentrations were also

higher than those of DEHP b! both rvorh~-itle and ~ o u p (Table 6). Horvever. while therc was

linle change in mean exposure between Workshiftl and WorkshifU for DEHP (0.10 1 to

0.1 OJ mgm' 1. ?-EH esposure appeared ro increase ( 1 -53 to 1.94 mg mt 1. Nrither DE HP

nor ?-EH \vas found in the air samples of the controls.

The highest worhctitle DEHP exposures among workers for Workshift I was the

Operator followed by the Mixers and then the Baggers. In WorkshiE? Mixers had the

highest exposures and the Operator the lowest. With the exception of controls. no statisticall~.

significant difference in mean DEHP exposures \vas observed among the various worhitles.

Work Tite and Worker ID

Figure I I Airborne DEHP and 2-EH Exposure Profiles for Plastic lndustry

W o r ke rs.

Mean ?-EH exposures for the Operator and the Bagen were not found to be

si pi ficantl y di fferent by the LSD method. Hoivever. the remaining mean esposures bl,

~rorttitile wre sipificantly different fiom each other (Table 7). For ?-EH, the highrst mean

rsposure ~ ~ o u p for both \vorkshifis \vas the Misrn and the lowest \vas the B a g e n .

Table 6 Workshift Concentrations (xISD) of UEHP and 2-EH in Air (mg/mJ)

Bagger 1 0.093=0.069 0.080~0.0 131 0.82=0.16! 1.21=0.035 A4-Operator 1 0.232 0.051 O. 720 2.157 Mixer

I

t O. 1 3*0. 12, 0.22=0.0341 2.23i0.64 2.3710.71 gr ou^ ! 0.102=0.0981 0.104=0.0911 1.53=1.40 1.942-1.42 ND= not detected

Table 7 LSD anaiysis of Mean Airborne 2-EH Exposure by Worktitle

WorkshiM IWorkshiftl 1 WorkshiW 1

NDI NDI ND ID-WorkType Workshiftl

* Worktitle means are significantly different from rach other.

Cl A-Control l ND

!Operator

Since ?EH Mas not used as a starting matenal or as pan of the manufacturing

proccss, the relationship between DEHP and airbome ?-EH was examined. A univariate

regression analysis showed a statistically signifi cant correlation between DEHP and

airborne ?-EH for the combined worlishik (~0.700; p<0.001).

Mixers

53 Crinary 2-EH

Typical chromatopphs of urine samples collrcted from workers and controls are

show in Figure 12. In addition to ?-EH. a nurnber of other unidentified ur inq substances

Baggers 1-0.011 ,* 1.842 ,

also reacted with TCAA.

As illustrated in Figure 13, al1 of the sarnples for the Baggen, the Operator and

Mixer A7 show an increase in unkary 2-EH from pre to pst-shifi on Workshifis I and 2.

However, trends for Mixer A3 and A6, were more variable. Pre-shifi samples collected

prior to the third workshift, show ?-EH concentrations that were both Iowa (Baggen A2

and A5: Operator A4 and Mixer A l ) and higher (Bagger A8; Mixer A3 and A6) than

previous pre-shifi concentrations. Among workers. unna* ?-EH concentrations ranged

from undetected to 1 3.54ugirnmol creatinine (Figure 1 3, Table 8). The Mixers had the highest

rnean urinary 2-EH concentrations followed by the Operator and then the Baggen (Table 8).

However. LSD analysis showed that this trend was not statistically significant. Excluding

controls. there was a significant increase in the pst-shift mean from the pre shifi mran

for Workshifil(Tab1e 9 ). No change was observed in WorkshiW.

Table 8 C'rinary 2-EH Concentrations (pg/mmol creatinke) Arranged by W'orktitle

Table 9 Workshift Crinary 2-EH Concentrations (pg/mmol creatinine)

Mean Standard deviat ion range

Controls n=10

O O O

Pre-S hift 1.93 3.39

(O- 10.28)

Baggers n=15 1.98 3.74

(O- 1 1.16)

* Pre- and p s t concentrations sipificantly different excluding controls (F=6.64: de1 ,l3 ; fl.074)

%

n=9 ( a11 groups) Mean Standard Deviation R a n ~ e

Pre-Shi ft 1.31 1 -6 1

(0-4.89)

Operator n=5 3.84 3.66

( 1 -20-9.79)

Post-Shifi 2.77 3.28

(0-9.79)

Post-Shi fi 4.84 4.67

(0-1 3.54)

Mixers n=15 4.56 4.29

(O- 13.54)

Pte-S hi ft 2.18 3.90

(0-10.43)

O ' I 4

Ç ' O , 0'01 ç ' 0: 0 ' 0 , , I

ç ' O ! I

9, , 1 0 0 ; - 5 1 = 8ul;;puq urrrr 000 '03XTi Pu3 .. ' - 060'0 autJ Fu3 000 '0

9Ievj = uoi;anua:ay uyu/un e p 1 ;:enueaatl rrrrrtpx~ g 9 - i : totaunue2ay u~ui /un e p - 1

Concentration (uglmmol creatinine

5.4 Correlation

As a result of the small sarnple size and analytical variability, the following

regession analyses in Table 10 should be considered as illutrative of the processes and

potential factors to be considered in future regession anaiyses with more cornplete data sets.

The dependant variable used \vas minary ?-EH and the independent variables, listed

in Table 1 O, included airborne rneasurements as well as other factors taken fiom the worker

questionnaire (Table 5 ). Log transformations of DEHP and airborne 2-EH werr included in

the analysis since occupational exposures are generally lognormally disaibuted (Rappapon

and Selvin. 1987). Mass. hzight and age were selected based on their relationship to an

individuais lipid content. thoracic voiurne, pulmonary ventilation and creatinine excretion

(Miller et-al., 1987: Boeniger et-al., 1993). Finally. smoking \vas included to investigatr its

potential affect on urina- ?-EH excretion.

Table 10 Esample of Stepwise .Multipie Regession Between DEHP and Post Shifi Urinary ?-EH for WorkshifE

As shown in Table 10. the ANOVA for the regession equation as well as several of

the variables are not significant. Therefore, a stepwise analysis \ras conducteci, i-e., the least

.&VOVA ( regression) correiation coefficient Statistic In terceut

F=2 8 1 ; df====, 1 1 ;p=0.06 -. f= 0.605

Coefficient / P-value 40.039 1 0.025

F=4.X;dw, 13; p=0.02 ?= O 573

d

CoefGcient ( P-value 34.3 15 1 0.026

F=3.49;df%, 12, p=û O3 . f = 0.592

Coefficient 34.440

P-value 0.029

sipificant variables were removed until the remaining variables becornr significant. This is

show in colurnns B and C. In this example, the stepwise analysis mas terminated at the point

when the variables in the regression equation w a s closest to being statistically significant.

The final regession mode1 that best represents the relationship between urinan ?-EH, and

the independant variables is showm below:

(Eq. A) y = lL83870.6 1 3 ~ ~ - 0 . 0 6 2 9 ~ ~ - 6 . 9 9 ~ : (r'4.542)

where: y = ?-EH ( pg mmol creatinine)

s, = log[?-EHaÎr (mgmi)] (pQ.046)

x2 = Mass (kg) ( ~ 0 . 0 6 9 )

s. = Height (m) (p=0.067)

The completr regression analysis is shown in Appendis VII.

6.0 Discussion

6.1 Air samples

Worker exposure to DEHP was well below the Ontario Time Weighted Exposure

Value of 5 mgmi which corresponds to the ACGM TLV-TWA' (ACGLH. 1993: Ontano

Ministry of Labour, 1992). In cornpanson 14th previous studies? exposures were slightiy

hgher than those observed in a phthalate manufacturing plant (0.038 mg m; - 8 hour TWA)

but were generally similar or within the range of other PVC processing plants (0.130 to 0.760

m g rn' : 0.0 1 to 5 mgm' 7-hour TWA) (Liss et.& 1985: Vainiotaio and PfafNi. 1990: Dirven

et-al., 1 993 b ). -

In cornpanson to DEHP. worker e-uposure to ?-EH was substantidly higher. Since 7-

EH was not used in the processing of PVC resin. it is believed rhat heat generated fkom the

mising process either cleavrd the ethylhexyl moiety from DEHP, or volatilized unreacted 7-

EH in the DEHP. This hypthesis is supponed bp the strong univariate comlation brtween

airborne DEHP and ?-EH Ievels.

There are currently no occupational exposure standards for ?-EH or occupational

hpjrneepidemiolog. studies on its health effects. However? animal studies suggest that 7-

EH ma? play a role in mitochondnal toxicity: alteration of lipid metabolism, carcinogenesis

and reproductiw rRects ( Section 2.0 ). Therefore. to assess the extent of such exposures and

determine what concentrations of ?-EH pose human health rislis (if any). the evaluation of 2-

EH exposure should be included. in addition to DEHP. in future occupational hygiene and#or

epidemiology studies of the plasticized resin industry. If worker 3-EH exposure is a concern

in faciliries with sirnilar plant Iayouts to the one studied exposures could be reduced by

adding a local e,xhaust ventilation system at the mixer, and near the hopper connecting the

mixer to the main floor.

The fact that the mean airbome DEHP concentrations were not significantly different

among worktities (excluding controls) kvas not unexpected, @ven the hi& variability in

workshifi exposures (Table 6). In conhast, the significant difference in airbome ?-EH

esposures according to worh?itle appeared to be a good reflection of anticipated exposures.

e.g. Baggers who spent less time in the processing area had lower exposures than Mixers

(Table 7).

6.2 Urine

Given that ?-EH was undetected in urine samples not incubated with P-çlucuronidase

s h o ~ that humans. sirnilar to primates, excrete ?-EH primarily as glucuronide conjugates

(CMA. 1985: Cornu 1988). Furthemore, the low pre-shift, high pst-shift urina? profile

obxned for most of the worken (Figure 13). suggests that occupational esposure is not

accumulated in the body but is rliminated to nearhelow pre-shifi levels after 13 houn. For

the workers in which this trend was not obsen-ed, certain work conditions may have affected

the ir urinan excretion profiles.

The uncharacteristically hi& pst-shift urinary ?-EH for Bagger A8, ma' be due to

his activity levrl during equipment cleaning. At the time of the second equipment shutdown.

Bagser A8 tvas responsible for cleaning the cmler and assisted in cleaning the mixer. During

normal operations. the door to the cooler is kept closed and the local exhaust ventilation is

lefi on. However. during cleaning. the ventilation [vas off and B=er A8 was observed

actively cleaning the cooler. Since DEKP and possibly 2-EH are readily soluble in the

blood (Section 2.2)- and blood uprake of soluble substances fiom the lung increases with

the level of activity (Bergerova. 1987; Brugone. 1987). it is possible that an increased

level of activity in an area with high exposures. resulted in Bagger A83 elevatrd urina?

?-EH concentration.

For Mixer A6, high urinary 2-EH may have been related to changes in his

workshift. On the second da- of the study Worker A6 exchanged his moming shifi for

Worker A 3 3 night shifi. With less than 8 hours beîween Workshifil and WorkshiftZ,

there may have been insufficient time for Workers A6's body to eliminate the

occupational cxposure to pre-shift levels. It is possible this resulted in some

accumulation, which is reflected in the hi& prr-shifi ?-EH concentrations observed in

Workshift 2 and 3 (Figure 13).

In contrast mith Worker A6, Worker A3 had more than 12 hours between

~vorkshifts. During that time urinary ?-EH would have been rspected to return to pre-

shift levels. Therefore, the shifi exchange with worker A6, cannot account for the pre-

shifi. post-shift profile obsewed during Workshift- (Figure 13). One possible explanation

is that the "pre-shifi" sample provided by Worker A3 \vas actually collected 1-3 hours

into the shifi because he was unable to provide an radier sample. While this seems to

explain the high pre-shift concentration. it remains unclear why 2-EH could not be

detected in the post-shifi urine.

As a result of the high pre-shifi ?-EH concentrations obsewed in Mixers A3 and

A6. the mean pre-shifi value for WorkshiW (Table 9 ) n-as skewed upwards. This

explains why the pre- to pst-shifi change in urinary 2-M l a s not sipificant for

Workshifts. In fact, if the data is re-analyzed excluding Mixer A3 and A6, the pre to post

shifi change becornes more significant (F=5.5: df- 1,9: p-0.046 vs. p=0.66). This result

along with the exposure patterns show in Figure 13, sugests that urinary ?-EH generally

reflects occupationa.i exposure.

The lack of significance of urinary ?-EH arnong the different work titles is a result

of the large variation in exposures and a smail sample size. However the significant

difference observed between the Controls and the Operator and Mixers (Table 8) is

consistent with earlier findinp suggesting that u r h q ?-EH reflects occupational exposure.

In reviewing Figure 13, it is interesthg to observe that most \vorkers had a

Workshiftl pre-shift concentration of urinary ?-EH. Since this study ~d on a Monda! and

none of the worken were occupationally eliposed on the weekend, the presence of urinary 2-

EH must be amiuted to background sources. There are four possible sources which could

account for the pre-workweek &EH: non-occupational airborne DEHl?Q-EH exposure;

d e t e intake of DEHP. ci-grette smoking or carryover of occupational exposure from the

previous worhweek.

It is unlikely that urinary 2-M is related to non-occupational airborne espures

since arnbient DEHP and 2-EH levels are in the jqyrn" range or lower (Andersson et-al.,

1984: Thurén and Lanson, 1990; Vitali et. al., 1993). As observed in Figure 13. dl of the

workers \+ho srnolied, with the exception of Worker A5, also had a Workshiftl pre-shifi 7-

EH level. It is also interesting to note that urinary ?-EH was not detected in either of the

controls (both were non-smokers). While cigarette smohnç is unlikely to pve off DEHP, it

ma' produce quantities of 2-EH or another chemical that could be metabolised to 2-EH in

the body hence it is possible that ?-EH reflects worker smoking habits. Indeed, it is more

likely that pre-shift 7-RI is related to dieîaxy intake of, or preiious worhweek exposure to

DEHP. This is supported by the fact that DEHP is lipid soluble and has k e n found in the

adipose tissue of primates several months after a single DEHP esposure (Woodward 1988a).

Furthemore, it has been estimated that the average die= internai dose for DEHP ranges

From 0.3 to 1 rng@erson/day (IPCS, 1992).

Nevertheless, the results do suggen rhat non-occupational esposures. whatever the

source, can influence urinary2-EH. Therefore, to understand how these factors affect urinary

?-EH profiles. future studies should record the number of cigarettes smoked per da>- as

well as the diet for each subject. In addition, the sarnpling p e n d should be estended to

observe changes in urinan ?-EH for a full work week. Depending on the source and

influence of non-occupational exposures, it may be necessan to subtract pre-workshift 2-

EH from post shifts values in order to avoid overestirnating occupational exposure.

6 3 Regression Analysis and 2-EH as Biological Indictor

One of the main purposes of a biologcal marker of exposure is to relate a person's

interna1 dose of a substance to the external chernical exposure they received. For the intemal

dose rstimate to k rnraningfid, a relationship must exist betwen the extemal exposure and

the biological rnarker. It has already ken discussed how unna? ?-EH may reflect

occupational esposure. This information could be used to calculate a person-s internai dose

b'. assurning a 1 : 1 rnolar relationship behveen DEHP and 2-EH. However, the lirnited sarnple

size of the sru& prevents a correlation analyses from determining if u i n q 2-EH is related

65

to either DEHP or airborne ?-EH exposure. Ir also prevents identi-ng factors that may

moderate urinary 2-EH excretion, such as mass. To illustrate, the associated confildence

intervai for the correlation coeficient in quation A (Section 5.4). is unacceptably 1-e

( 1.61, 0.59). As a result, the relationship expresseci in the regession mode1 is meaningless.

Thetefore- future studies should collect samples from a minimum of 50 workers in order to

obtain suirable confidence intervals for the correlation coefficient and establish statistically

valid regession models. Correction of results with pre-workshifi concenaations as well as

investigatinç other metabolites of 2-EH, such as kthylhexanoic acici, should also be

considered in order to detemine which exposure period and;or metabolite best reflecü

DEHP exposure. Until these additional studies c m be conducted, 2-EH should only be

considered a potential biological indicator of DEHP exposure.

7.0 Summary and Conclusions

1. Solid phase extraction. in combination with the derivatizing agent TCAA and

GC-ECD, is a suitable method for routine analysis of 2-EH in human urine.

Incubation of urine samples with f3-glucuronidase is an important step in the

process. since ?-EH is rscreted as a giucuronide.

3 -. The results fiom this study showed that worker DEHP exposures were wel1 below

the Ontario and ACGM level of 5 rngfm-'. While no similar standards esist for

?-EH, exposures were approsimaetly one order of magnitude higher than DEHP and

were most likely produced by heat degidation of the plasticizer during mi'ting. Hi$

?-EH exposures can be reduced in facilities with similar plant layouts to the one

studied by improving general ventilation or adding local rxhaust systems at the mixer

and ar the hopper connecting the mixer ro the main Roor. In pneral, airborne ?-EH

reflected rxposures espected with pamcular activities (Le. Controls Baggen <

Operator < Miser).

3. Like DEHP. ?-EH exposure has been shown to have health effëcts in anirnals.

Therefore. future occupational hygiene and epidemiolog? studies should srwly both

DEHP and ?-EH esposures in the plasticized resin indusm in order to detemine

their potential human health risks. In addition. dichloromethane. ivhich is bener

67

than carbon disulfide at recovering ?-EH from charcoal tubes. should be used to

more accuratel y assess ?-EH exposure.

4. DietandsmohngarepotentialsourcesofDEHP~2-EHthatmayintluence~n-

?-EH. In order to assess how non-occupational factors may affect urinary 2-EH

profiles. future studies should extend the sample collection pen'od to one hl1 work

week: recordinç the quantity of cigarettes smoked and dietary habits as related to

plastic umppedjmkaged foods. It ma?; also be neces- to correct occupational

exposures with non-occupational exposures in order to accurately estimate worker

e.psure.

C -. It has k e n shown that u r i n q 2-EH may reflect occupational exposure in plastic

indus. workers. However. due to the study's limited sample size. it is unclear

whether urina- ?-EH correlates with DEHP or airborne ?-EH exposure. Future

studies should include a minimum sarnple size of 45 workers. so that statinical ly

valid models describing the relationshp behveen urinary ?-EH and DEHP/airborne

2-EH can be developed

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Williams. J. and Foster, P.M.D., ( 1989). The Effects of I .3-Dinitrobemene and Mono-t 7- ethvlhesvl )phthalale on Horrnonallv StimuIated Lactate and Pvruvate Production bv Rat Sertoli Cr11 Cultures. Tosicology Letters. 17: 249-257.

Winbag. L.D. and Badr, M. W., (1995). Mechanisim of phthdate-lnduced Inhibition of Hewtic Mitochondrial O-oxidation. Toxicolog Lettea.76: 63-69.

Woodward. K.N., (1988). Phthaiate Esters: Toxicin. and Metabolimi Volume 1. CRC Press Inc. Boca Raton. Florida.

Yanagita. T.. Kobayashi, K. and Endomoto; N.. ( 1978). Accumulation Hepatic Phosoholipids in Rats Fed Di-(?-ethvlhenp1)ohthalate. Biochemical Pharmacolo~. 27: 3283-2387-

Yanagita. T.. Knihm S., Enomoto,N.. Shimad, T. and S u p o , M., ( 1979). Effrcts of Di(?- ethvlhewt)~hthalate on the Content of Hepatic Mitochondriai and Mrcrosornal Phos~holiptds in the Rat. Biochemical Pharrnacology. 18: 3 1 15-3 12 1.

Zweig, G. and Shema J., (1972). CRC Handbook of Chromatographv. Volume U. CRC Press inc. Cleveland_ Ohio. pp 7 1 M X .

Appendix-1 Synonyms and Trade Names for DERP

1 ; bis(kthylhexy1) ester (CAS name) i r di(ethylhexy1) phthalate 1

Common Synonyms for DEEIP (TPCS,1992)

i phthalic acid bis(2-ethylhexyl) ester ( iUPAC) i d i m i phthalate

r BEHP 1----------------- - IloP

1,2-benrenedicaboxylic acid i di(3ethylhe4xyl ) orthephthalate

I i bis(?-ethylhexyl) mer of phthalic acid / i octyl phthalate

I 1.2-~ed.icaboxylic acid biiethylhexyl) mer r ethyl hex~'1 phthalate l

I I bis(?-ethyl hexyl) phthaiate 1 i phthalic acid dioctyl ester

r bis(kthyIhexy1) 12 benzenedicaboxyiate

Common trade names for DEHP

r kthylhexyl phthaiate

Bisoflex 8 1 I i Goodrite GP 264 1 i PX-138

I - Bisonex l / > Reomol D 79P I I

i Compound 889

fl DAF 68

r Ergoplast FDO

r Eviplast 80

i Eviplast 8 1

i Kodaflex DOP / 150 v Mollan O

r Nuoplaz DOP

h Octoil

> Palatin01 AH

I Staflex DOP

i Tniflex DOP

r Vestinol AH

i Vinicizer 80

Appendix-II Block Diagram of PVC Processing C'nit vent I ControI Panel 1

O Extruder

Hopper from. Mixer

nm - Stairs (up)

Chute from Cooling Room t-

Container

t- Estruder esaust to cooling room 2nd floor /'

Sliding door

YIAIS FLOOR

vent

*[

R - Stairs

Weighing Station

DEHP

Hopper brining PVC Hopper to resin from outdoor Main F'oor storage tanks

I l

SECOSD FLOOR

Appendix-III Background Information

What is DEHP?

DEHP or Di(2-Ethylhexy1)phthalate is a chernical which is commonly mixed with

plastics resins like PVC to make them more flexible.

Cao DEHP cause health problems?

There is no evidence that DEHP causes health problems in humans. Studies in animals

have s h o w that DEHP can cause m o r s and interfere with reproduction.

What will this study be trying to accomplish?

Normdly DEHP exposure is measured by air sampling which can be expensive

and difficult to cany out. We are trying to develop an easier method of

monitoring exposure by measuring a breakdown product of DEHP in the unne.

How will this be done?

This study wiIl be lookins at 2 areas:

1 We will measure how much DEHP is in the air around the workers. This

will be done using srnall. lightweight air sampling pumps.

7 - h ' e will c o l k t urine sarnples and measure 2-Ethylhesanol (?-EH) in the

urine.

By douig both of these things we hope to find out if the amount of DEHP in air is

related to the amount o f 2-EH.

Who can take part in the study and what is involved?

Participation in the study is open to anyone who has been working at the Company

for 6 months or more in areas where DEHP is presmt. Participation is completely

voluntary. Each worker will be asked to 1 ) give 5 samples of urine: one at the beginning

and end of the work shifl for 2 days and the fi% sarnple the following day 3) Wear a

Iightweight air sarnpling purnp attached to a sampling device for two work shifts 3 )

provide information on their heaith and work history.

Q. Will you be looking for anything else in the urine? Absolufely not! Our research agreement with Healih Canada allows us to look

for breakdown products of DEHP &. We have no interest in any other materials and lwlcing for a else, without your written consent, would be unehical.

Will health information and urine results be kept confidentid?

Absolutely! Information will be reponed in such a way that no indikdual or

Company will be idenhfied. Information you provide to us will not be shared with anyone elr. Once the study is finished al1 idenciQing information such as narnes

will be destroyed.

Can 1 find out my personal results after the study is completed?

Yes!. After the study is completed we will be happy to send your resulu to you if

you wish.

Who will be conducting this study?

Jim Purdham, Andrea Sas-Kortsak and Shawn Ellis fiom the University of Toronto will be conducung this snidy. Shawn Ellis will be responsible for analyzing al1 urine and air samples.

,

Why should 1 participate? These are some of the ways that your participation will help other people: e It rnay identify work areas where DEHP levels are high, dlowing

- workers and management to take action to d u c e DEHP levels. 4 The new sarnpling method may help future researchers to determine if

there are human health effecü due to DEHPexposure. GP I t may provide information on how the hurnan body processes DEHP.

DEHP can aiso be found in many places outside the workplace. The new

sampling methods from this study may be used to find out how much other people are exposed to DEHP.

Appeadix- 1C' Consent Form

1 have been invited to participate in a study being camed out by Dr. Jim Purdham.

Dr. Andrea Sass-Konsak and Mr. Shawn Ellis from the University of Toronto airned at

detennining whether a urinav metabolite can be used as an indicator of exposure to di-

(2-ethylhesyl) phthalate (DEHP). My participation in this study \vil1 involve the

following procedures being camed out.

1 . 1 \vil 1 be asked some questions about rn'. hralth and about my work history ivhich \vil1

takr approxirnately I O minutes.

1. I \vil1 give a sample of urine at the beqinning and end of the work shifi for two da+

and a fifth sample the following da).

3. 1 \ d l wear on rny belt. lightweight sampling pumps attached to sampling devices.

which w i l l be clipped to rny collar. in order to measure my airbome esposure to

DEHP and other solvents over the two shifis.

The procedures involved are al1 safe. My involvement in this study is cornpletel!.

i olunrary and whrther or not 1 panicipate wili have no effect on rny relationship with rny

smplo-er. Al1 of the health information and urine results collected from me \vil1 be krpt

contidential by the University of Toronto and the investigators. No information will be

relcased to anyone nithout m. s i ~ e d consent, excrpt the airborne DEHP esposure levels

which will bs provided to rny emplo!.er. I can withdraw from the study at an>- time.

DATE:

S A M E :

Appendix-\' Health and Work Questionnaire

Amount of Alcohol Consumed i! II

/ Type of alcohol 1 / Beer (canubottles) I

Wine (glasses) l a

t 1 1

Liquor (shots) I , 1 -

Section A: persona1 Information

First name

1 ) Name:

Last name

2 ) Address:

3 ) Phone:

4 ) Ses: Male Female

5 ) Date of Birth: (

Da' Month Year

6)Height: ( f t )(cm)

7)Weight: ( Ib)( l ig)

Section B: Emplovment Information

1 i Company name:

2 i Length of employment: From ( ' H o ( j

D M Y D M Y

3 ) What is your current job title'?

4 ) Job Description:

5 ) Houv long have you been ernployed in the job title indicaied in question = 3?

From ( 10 ( 1

M Y Y

6) What was your previous job title?

7) Job Description: .

8) How long have you k e n empioyed in the job Me indicated in

question #6? From ( I ) to ( 1 )

M Y M Y

7 ) In the past two weeks. has Four normal work routine changed

(example: rqui pment shutdown, took vacation etc.. )?

No O Yes O ; if yes please describe

8) How often do you were respirators?

Never O Sometimes O Most of the time 0 Always

Section C: Health Information

1 ) Have -ou ever smoked a tobacco product'?

Yes Cl No O

2 ) If !.ou answred Yrs complete the following table

1 1

a pipe t 1 1

1 ! l 1 ii

il i Ii

Age first started 1 Age finally 1 srnokins I smoking stopped 1

1 Average n rr of years

;i e

Y Filter cigarettes 1 1

Chetring tobacco 1 l l 1

g smoked:times

I 1 1

- .

5 i Are you currently having your period?

smoked

b Non filter cigarettes 1 ! I l

6 1 if the answer to 25 was Yes what phase of your period are ?ou in:

In the beggining 01 In the middle O At the end

1 perday 1

7) Are vou currentlv taking an'. prescription medication?

Yes Cl No Ul 8) If you answered Yes in question =7 what was the name of the medication?

If ?ou cannot rernember the name( s ). you ma! \\Tite donn what condition the

medication \\.as supposed to treat.

9 ) In the last week ha\ r !ou taken an!. prescription medication''

Yes O No 811

10) If o u ansrvered Yes in question -5 what was the name of the medication?

If !.ou cannot remember the namet s). !ou ma- \\rite d o m what condition the

medication was supposed to treat.

1 1 ) In the last 2 years has your doctor diagnosed !ou \fith asthma? Yes a No

12) Do !ou have bronchitis? Yes O No Ci

13 ) In the Past I I hours have !ou consumed any alcoholic bewxagss'' ( pg 1 table )

Appendix-\l Statistical Considerations for Regression Equa tions

In order for a simple linear regession mode1 to reduce the average prediction

variance below that of the simple mean. the squared correlation coe6cient must bt: greater

than 0.5. A squared correlation coefficient of less than 0.75 would be of little practical value

for predicting cxposure to DEHP on the ba i s of a biological index. The confidence in the

dope incrrases ivith srnaller residual variance, larger sarnple size and a p a t e r range in the

predictor variable ( x ). However. for the correlation coeficient r a unitlcss measure ). Fisher

derived a transformation for which the probability is approxirnately nonnally distributeci,

with vanance dependant only on sample size (Snedecor and Cochran. 1976). Using this. a

sarnple size of at least 45 irould readily reject a squared correlation of 0.5 when the mir

squared correlation was 0.75 at a signifkance leïel of 0.05 and for a statistical power of 0.8.

With a sample size of 15 and a squared correlation coefficient of 0.75 we would rspect a

confidence intenal for the R' of 0.69 and 0.81 based on Fisher's transformation. an

acceptably narron- range.

Calculation of I-rinaw Workshift 2-EH From Plastic Industn: U'orkers r

blank A3A

GC-.=A

4.47SE-04 1.551E-05

b h k c ~ m ~ ~ d

1.103EtOS

~ - E H ( ~ M o L C S A T [ N I N E )

1.489E-00

2-EH c o r w e d b!' std clIrne (drnl)

2.114E-02

CREATLNINE ( M M O m L )

1.420E-03

Summary Airborne Workshift DEHP and 2 I H concentrations (mglm3)

Least Sianificant Difference Analvsis For Airborne DEHP Arranqed bv Worktitle

ID-WorkType

Anova: Single Factor

SUMMARY

DEHP

- -

Groups Count Sum Average Variance

Workshift 1

Bagger Operator Mixer

Workshiftî 0.095 O. 244 0.05

0.0727 O. 177 O. 224 0.0719

O O

A2-Bagger 1 0.0333 A3-Mixer i 0.00116

ANOVA Source of SS d f MS F P-value F crit Variation

Between Groups 0.02352 2 0.01 176 1.970867 0.185 3.98 Within Groups 0.065635 11 0.005967 Total 0.0891 55 13

A4-Operator A5-Bagger AG-Mixer A7-Mixer A8-Bagger C 1 A-Control

0.232 O. 1692 0.208 O. 194 0.0769

O CMA-Control i O

Least sig nifican t difference IBagger [Operator !Mixer 1

1 Bagger i I ;Operator I r

0.0545; 0.08821 0.0337j

Least Significant Difference Analysis For Airborne2-EH Arranged by Worktitle


SUMMARY Groups Count Sum Average Variance

Bagger 6 8.699 1 A49833 O. 1 1221 9 Operator 2 2.877 1.4385 1.032485 Mixer 6 19.7 3.283333 0.761 822


Between Groups 11.56175 2 5,780874 1 1.76999 0.001 849 3.982308 Within Groups 5.402691 11 0.491154 Total 1 6.96444 13

Least significant difference 1 IBaoaer io~erator [Mixer

p p p p p

l Bagger l 1 -0.01 11'1.842 [Operator

I

I 1 1'1 -853 I

ANOVA Analvsis of the Chancie in Airborne DEHP Concentrations from Dav 1 to Dav 2 Amonq different worktitles


SUMMARY Grou~s Count Sum Averaae Variance

Day 1 7 0.91456 O. 130651 0.008481 Day2 7 0.9346 O. 133514 0.006373


Between Groups 2.87E-05 1 2.87E-05 0.003862 0.9514 4.747 Within Groups 0.0891 26 12 0.007427 Total 0.0891 55 13

ANOVA Analvsis of the Chanqe in Airborne 2-EH Concentrations from Dav 1 to Dav 2 Amonq different worktitles



Day 1 7 13.80j43 1.971633 1.613417


Between Groups 0.964388 1 0.964388 0.72334 0.41 17 4.747 Within Groups 15.99891 12 1.333242 Total 16.9633 13

Urinary Workshift 2-EH concentrations (pgimmol creatinine) IPRE-SHlFT. IPST-SHIFT. 1 PR€-SHlFT: ( PST-SHIFT: PRE-SHIFT

Least Siqnificant Difference Analvsis For Urinarv 2-EH Arrancied bv Worktitle Anova: Single Factor

SUMMARY - - -

Groups Count Sum Average Variance Bagger 15 28.931 1 -928733 7.679063 Operator 5 17.986 3.5972 75.29225 Mixer 15 68.394 4.5596 18.43207


Between Groups 52.44509 2 26.22254 1.966422 O. 156507 3.29453 1 Within Groups 426.7249 32 13.33515 Total 479.17 34

Least significant difference i Bagger IOperatorlMixer

i ! 1

: Bagger ! ! 1.67/ 2.631 [Operator I

I I 1 O. 9621

f32df =2.03; (0.05) IS~=~&MSE*(I /ni)}'-" MSE=13.33515 lsd=1.69{13.3351 5'(1/15+1115+1/5)}~~~

lsd=3.59

ANOVA Analvsis of the Chanae in Workshift Urinary 2-EH Concentrations


SUMMARY Grou~s Count Surn Averaae Variance

ANOVA Source of SS df MS F P-value F crit Variation

Between Groups 72.1 0882 1 72.1 0882 6.639026 0.024248 4.747221 Within Groups 130.3363 12 10.86136 Total 202.445 1 13




Between Groups 3.03 1 988 1 3.031988 0.201003 0.661 9 4.747221 WithinGroups 181.0113 12 15.08427 Total 184.0433 13

Re-analvsis of Shift 2 excludina Mixers A6 and A3




Between Groups 33.4421 7 Within Groups 48.1663 Total 81 -5878

Regression Analysis of Workshifts 1 and 2 (1,2) for Airborne DEHP vs Post-Shift Urinary 2-EH

Worker # 2-EH (1,2) DEHP(1,2) masslheight len empl. mass(kg) height(m) age Smoker Post-

A2 A2 A3 A3 A4 A4 A5 A5 A6 A6 A7 A7 A8 A8 C l A CIA CMA CMA

SUMMARY OUTPUT

---- Regression ---- Statistics Multiple R O. 77829884 R Square 0.60574909 Adjusted R Square O. 390703 14 Standard Error 3.16781469 Observations 18

ANOVA d f

---. .- ----- SS MS F Significance F Regression 6 169.6025149 28.26708582 2816836 0,0651 32664 Residual Total

Coefficients Standard t Stat P-value Lower 95% Upper 95% Error

ln tercept 40.03981 16 15.54141 899 2.576329204 0.025758 5.833361 747 74.24626149 log(DEHP1.2) -0.9991 881 1.655668619 -0.603495226 0.558418 -4.64329201 2.64491 5795 Iog(2EHI ,2) 1.41642233 1.358599134 1 .O42560895 0.31 9523 -1.573835715 4.406680374 height(m) -1 5.51 8887 8.308761 451 -1.867773861 0.088646 -33.80635736 2.768582445 age 0.10620145 0.101161516 1,049820660.316318 -0,116453659 0.328856558 mass(kg) -0.1540327 0.077120446 -1.997300625 0.071 132 -0.323773757 0.01 5708328 Smoker -1.853559 2.155498495 -0.859921 283 0.4081 83 -6.597781633 2.890663568

SUMMARY OUTPUT

Regression Statistics ---- - -- Multiple R 0.769867% R Square O. 59269557 Adjusted R 0.42298539 Square Standard Error 3.08275282 Observations 18

ANOVA -- p-p-p-p

d f SS MS F Sianificance F Regression 5 165.9476846 33.18953693 3.492398 0.035233351 Residual Total

Coefkien ts Standard t Stat P-value Lower 95% Upper 95% Error

lntercept 37.2082816 14.41 842701 2.580606161 0.024071 5.793228359 68.62333479 log(2EH 1,2) 0.62287234 0.332537799 1.873087338 0.08561 2 -0.101 66527 1.347409952 height(m) -1 3.2621 83 7,220501 346 1.836739907 0.091 13 -28.99430367 2.469937727 age O. II277206 0,09787331 4 1.152224832 0.271667 -0.100475566 0,32601 9692 mass(kg) -0.7568532 0.07491 1678 2.093841 735 0.0581 77 -0.320071721 0.006365325 Smoker -1.5580976 2.042795582 0.762728082 0.460357 -6.008966702 2.892771 589

SUMMARY OUTPUT

Rearession Statistics Multiple R 0.75693438 R Square O. 57294965 Adjusted R 0.441 54954 Square Standard Error 3.03275707 Observations 18

ANOVA d f SS MS F Sianificance F

Regression Residual Tota I

Coefficients Standard t Stat P-value Lower 95% Upper 95% Error

lntercept 34.31 52833 13.68498837 2.50751 2781 0.02621 5 4,75066901 2 63.87989749 tog(2Et-i 1.2) 0.51 749228 0.297571 959 1.739049234 O. 105634 -0.125372728 1.160357291 heig ht(rn) -1 2.91 8269 7.0895361 14 -1.8223 59935 0.091 508 -28.23427732 2.397739989 age 0.08766994 0.0906791 72 0.966814545 0.351 28 -0.108230461 0.283570346 mass(kg) -0.1274568 0,0631 94807 -2.01 6886952 0.064847 -0.263980835 0.009067272

SUMMARY OUTPUT

. -. - Regression --------- Statistics Multiple R 0.73637201 R Square O. 54224373 Adjusted R 0.4441531 Square Standard Error 3.02567928 Observations 18

ANOVA d f SS MS F Sianificance F

Regression 3 151.821 7724 50.60725747 5.527987 0.01 023702 Residual Total

Coefficients Standard t Stat P-value Lower 95% Upper 95% ..-- --

Error lntercept 38.81 58269 12.8389088 3.023296408 0.00912 1 1.2790816866.35257203 log(2EH 1.2) 0.61 288122 0.280084303 2.188202675 0,0461 08 0,OI 21 59602 1,213602838 heig ht(m) -1 3.921 142 6.996872836 -1.98962341 7 0.066534 -28.9279551 3 1.085671 022 rnass(kg) -0.1238825 0.062939347 -1.968284296 0,0691 64 -0.2588741 23 0.01 1 IO9065

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