Environmental Health Perspectives Vol. 57, pp. 47-68, 1984



# Teratologic Evaluation of Ethylene Glycol 

# Monobutyl Ether in Fischer 344 

# Rats and 

# New Zealand White Rabbits Following 

# Inhalation Exposure byRochelleW. 

# TyI,* Guillermo 

# Millicovsky,*t Darol E. 

# Dodd,* Irvin M. Pritts,* Kathryn 

# A. France* and Louan 

# C. Fisher*

Timed-pregnant Fischer 344 rats and New Zealand White rabbits were exposed to ethylene glycol 

### monobutyl ethervapors by inhalation ongestational days 6 through 15 (rats) or 6 through 18 (rabbits) at concentrations of0, 25, 50, 100 or 200 ppm. The animals were sacrificed ongestational day 21 (rats) or 29 

### (rabbits). Inrats, exposure to 200 or 100ppmresulted inmaternal toxicity (clinical signs, decreasedbody weight and weight gain, decreased absolute and relative organ weights, decreased food and water 

### consumption and evidence of

### anemia), embryotoxicity (increased number of totally resorbed litters and decreasednumber ofviable 

### implantations per

### litter) andfetotoxicity (reductions inskeletal ossification). Noincreaseinfetalmalformationswasobservedinanyexposuregrouprelativetocontrols. At50or25ppm, therewas no

### maternal, embryo orfetal toxicity (includingmalformations) inrats. Inrabbits, exposureto 200 ppm resulted in maternal toxicity (apparent exposure-related increases in deaths and abortions, clinical 

### signs, decreased 

### weight during exposure and reduced gravid uterine weight at sacrifice) and 

### embryotoxicity (reduced number of total and viable implantations per litter). No treatment-related fetotoxicitywas seen. Notreatment-related increases infetalmalformationsorvariationswereseenatany exposure concentration tested. There was no evidence ofmaternal, embryo, or fetal toxicity (including malformations) at 100, 50 or 25 ppm in rabbits. 

# Introduction Aspart

### of a program

### evaluatingthepotentialtoxicity of 

### ethylene glycol monobutyl ether 

### (EGBE), studies

were 

### initiated to 

### evaluate the teratogenic potential of EGBE in test 

### animals under the 

### sponsorship of 

### the Chemical Manufacturers Association 

### Glycol Ethers 

### ProgramPanel. 

### Theinitialrat

### studyemployed exposure 

### of 

### timed-pregnant Fischer 344 rats to 

### EGBE at 

### 300, 200, or

### 100 ppm or

### to

### 0 ppm

### (aircontrol) for6hrper

### day duringgestational days 6 

### through 15. Maternal clinical 

### signs, food and water 

### consumption, and 

### bodyweights

were 

### recorded. Animals were 

### sacrificed on 

### gestational day 21 

### after 

### obtainingblood 

### samples for clinical hema

### tology. Following a 

### complete ovarian and uterine 

### examination, all live 

### fetuses were 

### weighed, sexed, and examined 

### externally. Approximately one-half of the fetuses in 

### each 

### litter were 

### examined for visceral and craniofacial 

### abnormalities, and 

### the 

### remaining fetuses

were 

### examined 

### for skeletal 

### abnormalities. *Bushy Run Research Center, R. D. 4, Mellon Road, Export, PA 15632. tPresentaddress: AyerstResearchLaboratories, Chazy, NY12921. 

### Exposures to 

### 100, 200 

### and 300 

### ppm

### EGBE 

## produced statistically significant reductions in maternal 

## body 

## weightgainand food

## consumption. Water

## consumption was 

### reduced

## duringexposure at 

### 300 or 

### 200 

### ppm. 

## Signs ofmaternal 

## toxicity (decrease in red blood cell 

### count; 

## ocular, nasal, and 

## urogenital discharges; 

## hypoactivity; etc.) were 

## observed

## predominantly at 

### 300 

### and 200 

### ppm 

### of

## EGBE. Oneanimal

## exposedto 

### 300

### ppm

### EGBE died. 

## Exposures to 

### 300 ppm

## EGBE resulted in an 

## increased incidence

## of

## embryoand

## fetal

## mortality(16of23

## females pregnantat

## sacrificehad 100%

## resorptionsat

### 300

## ppm). No 

## treatment-related fetal 

## external or 

## skeletalmalformations were 

### noted at 

### any 

### concentration of 

## EGBE. Visceral examinations 

## revealed cardiovascular 

## defects, specifically ventricular 

## septal defect in two 

### fetuses in two 

### litters 

### (out of seven 

### litters 

## examined) at 

### 300 

### ppm 

### and one 

### fetus in one 

### litter at 

### 100 

### ppm. 

## These 

## findings were not 

## statistically significant relative to 

### controls. Incidence of

## absent and/or 

## severely shortened innominate 

## artery, considered 

## variations, was 

## significantly elevated at 

### 300 

### ppm 

## relative to 

### controls. 

## Craniofacial examinationoffetusesrevealed a

## concentration-related 

## incidence of 

## enlarged lateral 

## ventricles of the 

## brain,TYL ETAL. 

### considereda

### variationindicativeof

### embryo/fetaltoxicity, at 

### all concentrations of EGBE. 

### Results from the skel

### etal 

## examinations included 

### concentration-related increases in 

### the incidence of 

### extra ribs 

### (fourteenth, 

## rudimentary, considereda

### variation), significantonlyat 300

### ppm, 

### and in

### the incidence ofreduced ossificationin 

### those

## skeletaldistricts indicative 

### offetaltoxicityat200 

## and 300 

### ppm, relative to 

### controls. 

## Since the 

### exposure 

### employed in 

### this 

### initial study 

### produced maternal and 

### fetal toxicity at 

### all exposure 

### concentrations, didnot 

### identifya 

## "no observable effect 

### level" 

## (NOEL), and excessive embryo/fetal loss was 

### observed at 300 ppm, 

### another rat study and a 

## teratogenic evaluation

## ofEGBE in

## anothermammalian 

### specieswere

### undertaken. Thisreport

### detailsthe

### evalua

### tion of EGBE for 

### teratogenic potential in 

### Fischer 344 rats and New 

### Zealand White rabbits. 

# Materials and Methods Generation of

# Atmospheres and 

# Chamber Parameters A 

### 55-gal container of 

### EGBE test material (Lot 

## Number 5804213, 

## BRRC Sample Number 45-235) was 

### received from 

## Union 

## Carbide Corporation on

### July 30, 1982. Test 

## material from the original container was 

## placedin1

## galglassbottlesand

### blanketedwith

### nitrogen. 

### The

## purityand

## stabilitywere

### verifiedbygas 

### chromato

### graphic analysis 

## both before and after the 

### study. No 

## significant compositional changes occurred while the 

## study 

## progressed. The test 

### material remained 99.6% 

### EGBE 

## throughout the study. Fisher certified hexanes was 

## used as 

## the solvent for preparation of standards. 

### The gases 

## for the chromatograph were obtained from 

## Linde 

## Corporation. The 

### nitrogen and 

## hydrogen were 

## Ultra 

## High Purity grade and the air was 

## breathing 

## quality. A 

### Perkin-Elmer Model 3920B gas 

### chromatograph 

## (GC) equippedwitha

### flameionizationdetectorwas

### used to 

### monitor 

## the EGBE vapor 

### concentrations in 

### the chambers. The 

## GC 

## columnwas 

### a

### 6-ft x 

### 4

### mm 

### (internal 

## diameter) glass column packed with 0.15% 

### Fluorad 

## FC431 (3M 

## Company, St. 

## Paul, MN) on 

### glass beads, 80-120 

### mesh. A 

## Spectra-Physics series 4000 

### central processor, a 

## data 

## interface, and a 

## Perkin-Elmer auto

### matic gas 

## sampling system 

## (station and valve pro

### grammer 

## units and a 

### gas 

## sampling 

## valve) were 

### used for the 

## analyses. Calibration of

## the gas 

## chromatographwas 

### done with 

## liquid injections of standard 

## solutions of 

## EGBE in 

### hexanes

## preparedvolumetrically. Theseries

### ofstandard 

## solutions

## encompassedtheentirerange

### ofvaporconcen

### trations 

## generated in

### the exposure 

### chambers. A

### linear 

## calibration curve was 

## obtainedwhenareas 

## (integration counts) were

## plotted versus 

## thegas 

## equivalent concen

### trations of 

## the standards. The calibration factor 

## (KF) was 

### calculatedas theratio

### betweenareaA

### andconcentration 

### C: KF = 

## A/C The mean KF for all valid 

### injections of 

### standards was 

### used in 

### subsequent microprocessor calculations. This 

### calibration factorwas checked at 

### least once 

### eachweek 

## during the exposure 

## period and did not 

### require 

### adjustment. Each chamber atmosphere was 

## analyzed for 

### EGBE 

## approximately once every

### hour during each 6-hr exposure. 

## Daily nominal concentrations (an 

### estimated con

### centration calculated from the amount oftest 

### material delivered and the 

## chamberairflow

## duringthe 

### exposure 

## period) were 

## also calculated for each 

## chamber. LiquidEGBE was

### meteredfroma

### pistonpumpintoa 

### heated glass evaporator similar in 

### design as 

### described 

## by

## Carpenter et 

### al. 

## (1). The temperature in 

### the evaporator was 

### maintained at 

### the lowest 

### level sufficient to 

### vaporize the liquid. The 

### resultant vapor was 

## carried into

### the chamber

## bypassage 

### of

## conditionedair

## through theevaporator. 

### Chamber

## atmospherescontainingEGBE were 

### filtered before 

## leaving an 

### exhaust stack. The five 

## chambers 

## employed in 

### this 

## study were 

## rectangular in

## shape, constructedof

## glass andstainless steel 

## (Wahmann Manufacturing Company, 

## Timonium, MD), 

## andmeasured

## approximately2.1m x 

### 1m x 

### 2.1m 

## (height). Total volume in 

## each chamber was 

## approximately 4350 

### L. An 

### orifice 

## plate was 

## positioned in 

### the exhaust duct of the 

### chamber and was 

## connected to a 

### Dwyer

## MagnehelicPressure

## Gauge. Excellentchamber 

## distribution for 

## EGBE vapors 

### has been

## demonstrated. The 

### coefficient ofvariation

### of

## concentrations measured at

## eightlocations in

### the

## chamberin

### the

## breathingzones 

### ofthe 

## animals was 

### 1%. 

### Airflow in 

### each 

## chamber was 

## approximately 1000 L/min 

### (14air

## changesper

## hour) witha

## tgg(theoretically derived time

## required for

## the chamber to 

### reach 

## 99% of the

## equilibriumconcentration) of

## approximately20

### min. The 

### chambers were 

## illuminated with 

## artificial room 

## light. Temperature 

## (Markson 

## Science, Inc., Model #13177) and 

## relative 

## humidity 

## (Airguide Instrument 

## Company) gauges were 

## placed inside each 

## chamber during exposures. 

## Chamber temperature, relative 

## humidity, and 

## airflow rate were 

### recorded at 

## least four times 

## during each 6-hr exposure. 

### Withineach

## chamber, the 

## animal cages were 

## rotated 

## daily to 

### compensate 

### for any

## possible, but 

## undetected, variation in 

### chamber exposure 

### conditions 

## (i.e., con

## centration, temperature, 

### relative 

## humidity). Target 

## concentrations were 

### 0, 

### 25, 50, 100 

### and 200 

### ppm. 

### These exposure 

### concentrations were 

## based on a 

## range-finding studyin

### pregnant rats 

## andrabbitswhich 

## employed concentrations of

## 450, 200, 75 or 

### 0 

### ppm, 

### and an 

### initial rat 

## teratology 

## study with 

## concentrations of 

## 300, 200, 100

## and0

### ppm. 

## Maternal

## mortalitywas seen

### at 

### 450 

### ppm 

## (100% in 

## both 

## species) and 

## maternal 

## toxicity was 

### seen at 

### all exposure 

## concentrations 

## employed (exceptat

### 75

### ppm

## for

## rabbits) in

### thetwo

## studies. 

## Sincea 48EGBE INHALATION TERATOLOGY STUDY 

### no-observable 

### effect level 

### (NOEL) could not 

### be ascer

### tainedfromthese

## studies, thecurrent

### exposureconcen

### trations were 

### selected for

## both rats 

### and rabbits in the 

### present 

### study. 

## Rats Virgin male and female 

### Fischer 344 rats 

## [NIH:(F344)/HlaBR(F141 +

## 3)] (100 

### days oldupon

## arrival) were 

## purchased from 

## Hilltop Lab 

## Animals, Inc., Scottdale, PA. 

### They were 

### quarantined for 2 

### weeks 

## during which time 

### representative animals were 

## subjected to 

### fecal 

## sampling, aerobic 

## bacteriologic cultures of

## lung

## tissue, histologic examination ofselected organs 

### and to serum 

### viral

## antibodyexaminationfor

## sialodacryoadenitis. Rats were 

### housed in 

### stainless steel wire-mesh cages 

### with food 

## (Certified Ground Rodent 

## Chow, Ralston Purina 

## Co., 

## Richmond, IN) andwater 

## (Municipal

## Authorityof 

### Westmoreland 

## County, 

## Greensburg, PA) available ad 

## libitum except 

## during exposures. 

### Deotized Animal 

## Cage Board 

## (Shepherd 

## Specialty Papers, 

## Inc., Kala

### mazoo, 

### MI) was

## placedbeneath

## the cages 

### and 

## changed daily. Animals were 

## kept on a 

### 12-hr 

## photoperiod and room 

### temperatures 

### and 

## humidity were 

### recorded con

## tinuously (Cole-Parmer 

### Hygrothermograph Seven-Day 

## Continuous 

## Recorder, Model 

## #8368-00, Cole Parmer Instrument 

## Co., 

## Chicago, IL). Rats were 

### mated 1:1 

### (one male:one 

## female) in 

### stainless 

### steelwire-meshcages and

### the

## paperboardbeneath the 

### cages was 

### checked twice 

## daily for

## dropped 

## copulation 

## plugs. 

## Successfully mated 

## (plug-positive) females were 

### housed 

## singly for the 

### duration ofthe 

## study. The 

## daya

## copulation

## plugwas 

### foundwas 

## designated gesta

### tional

## day (gd) 0. 

## Thirty-six

## plug-positive females were 

## randomly assigned to 

### each 

## experimental group. For 

### exposures, 

## plug-positive females were trans

## ferred, one

### per

### cage, to

## stainless steelwire-meshcages 

### and the cage 

## carriers, with 

## stainless steel shelves beneath 

## each row 

### of cages, were 

### moved into the 

## chambers. Food and water were 

## withheld 

## during exposures. 

## Exposures were 

### for 6 

## hr/day, gd 6 

## through 15. 

### After

## eachexposure, 

### animalswere

### returnedto

### their 

## original cages. 

### The 

## animals were 

### observed 

## daily for clinical 

## signs 

## throughout the 

## study (gd 0 

## through 21). 

## Food and water 

### consumption was 

### measured for the 

## intervals gd 

## 0-3, 3-6, 6-9, 

### 9-12, 

## 12-15, 15-18, 

### and 18-21. 

## Maternal

## bodyweightswere

## takenon

## gd0, 6, 

### 9, 12, 15 

## and 21. 

### At scheduled sacrifice on 

### gd 21, rats were 

### anesthetized

## bycarbondioxide

## inhalationfor

## collectionofblood 

## samples. 

## Following blood 

## collections, animals were 

### sacrificed 

## by carbon dioxide 

## asphyxiation. 

# Rabbits Virgin New Zealand White 

## rabbits 

## (3.0 to 

### 3.5 

## kg, approximately 5 to 

### 5.5 

## months 

## old) were 

## purchased from 

## Hazleton Dutchland 

## Laboratories, Inc., 

## Denver, PA. 

## Theywere 

## quarantined for 2 

## weeks, 

## duringwhich theywere

### examinedbyaveterinarianandfecal

### samples taken for

### detection ofintestinal parasites. The 

### rabbits were 

### individually housed in 

### stainless steel wire-mesh cages 

### with food 

### (Big Red 

### Rabbit Food, 

### Agway, Inc., 

## St. Marys, OH) and water 

### (Municipal Authority of 

### Westmoreland County, Greensburg, PA) 

### available ad 

### libitum except 

### during exposures. Paperboard was 

### placed beneath the cages, and photoperiod, temperature 

### and relative humiditywere monitored. 

### Rabbits were 

## bred, using in-house 

### breeding colony 

### males. The date of copulation was 

### designated gd 0. 

### Twenty-fourmated females were randomlyassigned to each 

### experimental group. 

### For exposures, mated 

### females were 

### transferred to 3tainless 

### steel wire-mesh cages in cage carriers 

### which were moved into the 

### chambers. Food and 

### water were 

### withheld during exposures. 

### Exposures were for 6 

### hr/day, gd 6 

### through 18, in the same 

### chambers as the rats. 

### After each exposure, 

### animals were 

### returned to their 

### original cages. 

### The animals were 

### observed daily 

### for 

## clinical signs throughout the study (gd 0 through 29). 

### Maternalbodyweights

### weretakenongd0, 6, 9, 12, 15, 18, 21 

### and 29. 

### At 

## scheduled sacrifice on 

### gd 29, 

### blood was 

### collected 

### fromeachdoe

### aftershewas

### placedinarabbit

### restrainer. 

### Following blood 

### collection, rabbits were 

### sacrificed by cervical dislocation. 

## Thoracolaparotomy, Maternal and 

## Fetal 

## Examinations The maternal 

### body cavities 

### were opened 

### bymidline 

## thoracolaparotomy. The

### graviduteri, ovaries 

### (including corpora 

## lutea), cervices, vagina, 

### and 

## peritoneal and 

## thoracic cavities were 

### examined 

## grossly. Ovarian cor

### pora 

## lutea ofpregnancywere 

### counted. Maternal 

### liver, 

## kidney, thymus, spleen anduterus

## weights were 

### deter

## mined. The uteri were 

## immediately ligated at 

### their 

### cervical end to

### prevent

### the 

### expulsionof

## conceptuses by 

## myometrialperistalsis. Uteriwere

### externally

### examined for 

## signs of 

## hemorrhage. The 

## ligated uteri 

### were re

### moved

## fromtheperitoneal

### cavityand

### dissectedlongitu

## dinally to 

### expose 

### their contents. 

### All 

### live and dead 

## fetuses, placentae, andresorptionsiteswere

### notedand 

## recorded. Uteri 

## fromfemales that appeared

### nongravid were 

## placed in 

### a 10% 

### ammonium 

### sulfide solution for 

## detection of

## earlyresorptions (2). 

### Each

### rabbit 

## fetus was 

### euthanized immediatelyupon 

### removal fromtheuterus by

### intraperitoneal injection of 

## sodium

## pentobarbital. All

## livefetuseswere

## weighedand sexed. Allfetuseswere

### examinedfor

### externalmalformations 

## including cleft 

## palate. One-half of the fetuses 

## (selected at 

## random) in 

### each litter were 

### examined for 

## thoracic and 

## peritoneal visceral 

### abnormalities 

### by 

## modificationofmethods

### described

## byStaples (3). 

### These fetuses

## were,decapitated and their

## heads were 

## fixed in 

## Bouin's 

## solution for examination of 

## craniofacial structures

## by

## sectioningmethods

## modified fromWilson (4,5) and van 

## Julsingha and 

## Bennett (6). The 

## remaining 49TYL ETAL.



### fetuses ineach

### litter were

### eviscerated, fixedin

## ethanol, and then processed 

### for 

### skeletal staining with 

### Alizarin Red 

### S 

### (7,8), and 

### examined for 

### skeletal 

### malformations. 

## Hematologic Analyses Blood 

### samples for 

### erythrocyte osmotic 

### fragility and 

### hematologic evaluation were 

### obtained fromfemale rats 

### viaretro-orbital

### bleeding

### immediatelypriorto

### sacrifice. 

### Approximately 700 

### ,uL of

### blood was 

### collected into B-D 

### MicrotainerBrandcapillarywhole

### bloodcollectiontubes 

### containing sodium 

### heparin as 

### anticoagulant (Becton

### DickinsonCo., Rutherford, NJ) for

### theosmoticfragility assay. 

### Approximately 200 

### ,uL of 

### blood was 

### collected 

### into B-D 

### Microtainer Brand Capillary whole blood collection 

### tubes 

### with EDTA as 

### anticoagulant (BectonDickinson 

### Company, Rutherford, NJ) for 

### hematologic determinations. Blood 

### samples 

### for erythrocyte osmotic and 

### hema

### tologic evaluation were 

### obtained from 

### rabbit does 

### via 

### cardiac puncture 

### immediately prior to 

## sacrifice. Approximately2.0mL

### of

### blood was

### collectedinto

### tubes withsodium

### heparin as

### anticoagulant (VacutainerBrand, 

### Becton-Dickinson Company, Rutherford, NJ) for the osmotic 

### fragility assay. 

### Approximately 2.0mL 

### ofblood

was 

### collected into tubes 

### containing EDTA as 

### anticoagulant (Vacutainer 

### Brand, Becton-Dickinson Company, 

### Rutherford, NJ) for

### hematologic determinations. A 

### 20 

### pL portion of

### blood from 

### each female rat 

### and 

### rabbit was 

### added to 

### each 

### of ten 

### Unopette Reservoirs 

### (Becton-Dickinson Co., Rutherford, NJ) containing a 

### specific concentration of 

### buffered saline. Each 

### saline 

### solution 

### containing red 

### cells was 

### incubated for 20 

### min and was 

### then 

### centrifuged for 5 

### min at 

### 2000 rpm. 

### The supernatant 

### of 

### each 

### sample was 

### decanted and the percentage of 

### hemolysis was 

### read 

### using a Trace 

### III 

### Clinical

### ChemistrySystem(Beckman

### Instruments, Inc., 

### Fullerton, CA). A 

### precision check 

### of the Trace III 

### System was 

### performed 

### immediately prior to 

### sample analysis. The 

### following hematologic parameters were mea

### sured on

### all rat 

### and

### rabbit 

### samples: 

### erythrocyte count, 

### hemoglobin, and

### hematocrit. These 

### analyses were per

### formed on a

### Coulter

### CounterModel S-Plus IV 

## (Coulter Electronics, Inc., Hialeah, 

## FL). Commerciallyavailable 

## quality control 

### samples (4C Plus II 

### Coulter 

### Counter Cell 

## Control, Coulter 

## Diagnostics, Hialeah, FL) were run 

### prior to 

### the rat 

### and rabbit samples. 

### Mean cor

### puscular volume 

### (MCV), mean 

### corpuscular 

## hemoglobin 

### (MCH) andmean

### corpuscularhemoglobinconcentra

### tions 

### (MCHC) were 

### calculated from the measured 

## hematologic values. 

## Statistical 

## Analyses Theunit ofcomparison was 

### the pregnant 

### female (9). 

### Results of the quantitative continuous 

### variables 

### (e.g., maternal 

### body weights, liver 

### weights) were 

### intercompared for 

### the four exposure groups 

### and a 

### control group 

### by use 

### of Levene's test 

### for equal variances (10), 

### analysis of variance 

### (ANOVA), and t-tests 

### with 

### Bonferroni probabilities. The t-tests were 

### used when 

### the F 

### value 

### from the ANOVA was 

### significant. When 

### Levene's test 

### indicatedhomogeneous variances andthe 

### ANOVA was 

### significant, thepooled t-test was 

### used for 

### pairwise comparisons. When 

### Levene's test 

### indicated 

### heterogeneous variances, all groups were 

### comparedby

an 

### ANOVA for unequal 

### variances (11) 

### followed, when necessary, 

### by the separate 

### variance t-test. 

### Nonparametric data obtained 

### following laparohysterectomy were 

### statistically treated by 

### using the 

### Kruskal-Wallis test 

### (12) 

### followedbythe 

### Mann-Whitney ai test 

### (12) when 

### appropriate. Incidence data were 

### compared by using Fisher's exact test 

### (12). For 

### all statistical tests, 

### the fiducial 

### limit of 0.05 

### (two-tailed)

was 

### used as 

### the criterion for 

## significance. 

# Results

Generation of

## Atmospheres and 

## Chamber 

## Parameters The means 

### of the 

### daily means 

### for EGBE 

### chamber 

### concentration, temperatureand

### humidity are

### presented in

### Table 1. AllEGBE 

### analyses were

### within + 

### 10% 

### of

### the

Table 1. 

### Summary of chamber concentrations and exposure conditions.



### Ethylene 

### glycol monobutyl ether 

### (EGBE) target concentrations 200 ppm 100 ppm 50 ppm 25 ppm 0 ppm 

### Analytical concn, ppm 201 ± 6a 98 ± 2 50 ± 1 25 ± 1 < 

### MDLb A/T 

### ratioc 1.005 0.98 1.00 1.00 Nominal conen, ppm 220 110 49 26 A/N 

### ratiod 0.92 0.89 1.01 0.94 

### Temperature, °F 79.3 ± 1.4a 78.9 ± 2.0 78.9 ± 2.0 79.0 ± 1.8 77.9 ± 2.2 Relative 

### humidity, % 41.2 ± 6.3a 41.4 ± 5.6 40.0 ± 5.8 40.7 ± 6.8 42.8 ± 6.2 aGrand mean of

### daily means ± SD. 

### b< MDL = belowminimum detection limit of 2 ppm. 

## cAnalytical/target concentration ratio. 

### dAnalytical/nominal concentration ratio. 50EGBE INHALATION TERATOLOGY STUDY



### target 

### concentrations. 

### Since 

### generation of 

### the EGBE atmosphere 

### required evaporation under heat which elevated the temperature 

### in 

### the 

### EGBE-exposure chambers, the 

### control chamber temperature was 

### also elevated to 

### keep the temperature 

### conditions 

### consistent in 

### all groups 

### (78-79°F). Relative humidity was 

### main

### tained at 

### 40 to 

### 43%.



## Rats The 

### distribution and fate ofall 

### plug-positive rats on 

### study are

### presentedin

### Table2. 

### A

### smallnumber (five or 

### six) ofrats 

### in

### each treatment group were 

### inadvertently exposed to 

### EGBE for one 

### additional day 

### (gd 16). They

were 

### subsequently removed from 

### study but all their 

### in-life, necropsy

### and

### fetalevaluationdatawere

### recorded and retained in 

### the 

### Bushy Run 

### Research Center Archives. (For 

### discussion of the 

### results from these animals, 

### please see 

### the Addendum to 

### this paper.) Pregnancy rate, as 

### determined at 

### scheduled sacrifice,

was

### equivalent across

### all groups. 

### There was a

### significant increase in

### the 

### number oftotallyresorbed 

### litters at 

### 200 ppm 

### relative to 

### controls. Clinical signs 

### observed in a 

### dose-related incidence in 

### dams during the exposure 

### period 

### (Table 3) 

### included 

### evidence of hematuria (or 

### hemoglobinuria) especially 

### early in the exposure 

### period, pale and cold extremities 

### and necrosis 

### of the tail tip. 

### Maternal toxicity was 

### also expressed as 

### significant 

### reductions in body weight during the exposure 

### period 

### (gd9, 

### 12, 15) 

### andat

### sacrifice at

### 200 ppm, as

### significantly 

### reduced weight gain during the exposure 

### period (gd 

### 6-9, 6-12, 

### and 6-15) at 

### 100 

### and 200 ppm

### andforthegd 

### 6-21 

### period for dams exposed to 

### 200 ppm 

### (Table 4). 

### Food consumption, expressed as 

### grams/dam/day, was 

### statistically reduced during the exposure 

### period (gd 

### 6-9, 9-12 

### and 6-15) 

### for dams exposed to 

### 100 

### and 200

Table 2. Distribution and fate of F-344 rats exposed to EGBE by inhalation on gestational days 6 through 15. EGBE target concentrations 200 ppm 100 ppm 50 ppm 25 ppm 0 ppm No. in study 36 36 36 36 36 No. removeda 6 6 5 5 5 No. 

### early deliveries 0 0 0 0 1 No. dead 0 0 0 0 0 No. nonpregnant at sacrifice 5 7 9 9 7 No. (%) pregnant at sacrifice 25 (83.3) 23 (76.7) 22 (71.0) 22 (71.0) 23 

### (76.7) No. litters totally resorbed gbt 0 ic 0 0 No. litters examined 16 23 21 22 23 

## aThese animals were removed because theywere inadvertently exposed for one additional day (gestational day 16). 

### bNine dams had 

### only resorption sites at scheduled sacrifice, seven ofwhich were found only after ammonium sulfide staining. 

## cOne damhad 

### only resorption sites at scheduled 

### sacrifice, detected after ammonium sulfide 

### staining. 

### tp < 

## 0.01 versus 

### controls, Fisher's exact test.



### Table 3. 

### Clinical observations in Fischer 344 dams 

### exposed to EGBE by inhalation on 

### gestational days 6 through 15.a EGBE concentrations 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm 

### Urogenital area stained 

### red, red black 0 0 0 + + + 

### Urogenital wetness + + + + + ++ Fur stained 

### (red) + + + + + + 

### Urogenital encrustation + + + + + ++ Fluid on tray 

### (positive for occult 

### blood) 0 0 0 + + + Red 

### fluid on tray 0 0 0 + + + Periocular wetness 0 + + + + + ++ Perinasal encrustation + + + + + + ++ Extremities cold 0 0 0 0 + + Extremities 

### pale 0 0 0 0 + ++ 

### Tail tip discolored 0 0 0 0 + + 

### Tail 

### tip ulcerated 0 0 0 0 + + 

### Tail 

### tip

### missing 0 0 0 0 + + 

## aCode: + + + = 

### majorityofdams 

### (75% ormore) exhibited the clinical 

### sign sometime 

### duringthe exposure

### period; + + = 

### approximately 50% ofthe dams 

### (30-70%) exhibited the clinical 

### sign sometime

### during the exposure

### period; + = up to 25% ofthe dams exhibited the clinical 

### sign sometime 

### during the exposure 

### period; 0 = no dams exhibited the clinical 

### sign during the exposure 

### period. 51TYL ETAL.

TIble 4. Maternal weights andweight gain of F-344 rats exposed to EGBE by inhalation.

EGBE target exposure concentrations, gestational days (gd) 6-15 200 ppm 100 ppm 50 ppm 25 ppm 0 ppm Maternal bodyweight, g 

### gd 0 194.7 ± 5.3a 195.6 ± 7.3 195.5 ± 7.2 195.7 ± 7.3 191.8 ± 6.1 

### gd 6 207.3 ± 5.6 207.1 ± 7.1 206.1 ± 7.8 206.5 ± 7.0 205.2 ± 6.7 

### gd 9 181.9 ± 6.5* 204.3 ± 10.5 208.4 ± 8.7 210.2 ± 7.5 207.8 ± 7.9 

### gd 12 191.3 ± 9.1 216.8 ± 8.1 219.0 ± 7.2 218.1 ± 15.0 218.7 ± 8.5 gd 15 203.5 ± 18.0 224.8 ± 13.1 226.7 ± 10.0 230.6 ± 11.3 230.1 ± 9.1 

### gd 21 244.5 ± 

### 30.91 277.1 ± 19.0 272.4 ± 20.2 277.6 ± 19.5 275.6 + 17.8 

### Corrected body 

### weightb 210.4 ± 13.9 215.1 ± 11.0 215.5 ± 7.9 214.5 ± 11.8 215.7 ± 8.3 

### Maternal bodyweight gain, g 

### gd 0-6 12.6 ± 4.1 11.5 ± 2.9 10.6 ± 5.4 10.8 ± 4.0 13.4 ± 3.6 

### gd 6-9

- 25.4 ± 

### 3.7*

- 2.9 ± 

### 6.5* 2.3 ± 4.0 3.7 ± 2.9 2.6 ± 4.2 

### gd 6-12

- 16.0 ± 

### 6.8* 9.7 ± 2.8t 12.9 ± 3.7 11.6 ± 12.1 13.5 ± 4.5 

### gd 6-15

- 3.8 ± 

### 17.1* 17.7 ± 

### 9.2* 20.6 ± 7.9 24.1 ± 8.0 24.9 ± 6.0 

### gd 6-21 37.2 ± 

### 30.0* 70.0 ± 15.8 66.3 ± 19.5 71.1 ± 16.1 70.4 ± 15.3 

### gd 15-21 41.0 ± 19.7 52.3 ± 14.6 45.7 ± 15.6 47.0 ± 11.5 45.5 ± 10.4 

## aValues represent mean ± standard deviation; units are in grams. 

### bCorrected bodyweight = 

### bodyweight on

### gestational day 21 minus 

### gravid uterine 

### weight. 

### *p < 0.05 compared to control (p = 0.0033 Bonferroni t-test, adjusted). 

### tp < 0.01 

### compared to control. 

### tp < 0.001 compared to control.

Table 5. Food consumption of F-344 rats 

### exposed to EGBE 

### by inhalation.



### EGBE target exposure concentrations, 

### gestational days (gd) 6-15 200 ppm 100 ppm 50 ppm 25 ppm 0 ppm Food 

### consumption, 

### g/dam/day gd 0-3 12.0 ± 

### 1.3a 11.6 ± 1.9 11.4 ± 2.4 11.6 ± 1.7 12.6 ± 1.7 

### gd 3-6 12.8 ± 1.0 12.8 ± 1.2 12.2 ± 1.9 12.8 ± 1.0 13.3 ± 1.5 

### gd 6-9 2.9 ± 1.0* 10.9 ± 1.7* 13.7 ± 1.4 14.0 ± 0.9 14.4 ± 1.0 

### gd 9-12 9.4 + 

### 2.41 15.0 ± 

### 1.2* 15.4 ± 1.2 15.3 ± 3.2 16.0 ± 1.4 

### gd 12-15 14.9 ± 3.4 15.1 ± 2.4 15.6 ± 3.3 16.0 ± 2.0 16.7 ± 1.2 

### gd 15-18 19.0 ± 2.5 18.6 ± 1.7 17.4 ± 2.0 18.3 ± 3.3 19.1 ± 2.9 

### gd 18-21 19.7 ± 2.2 19.2 ± 1.9 17.9 ± 2.8 18.2 ± 1.1 18.6 ± 1.4 

### gd 0-6 

### (prior to treatment) 12.4 ± 0.9 12.2 ± 1.3 11.8 ± 1.8 12.2 ± 1.1 12.9 ± 1.5 

### gd 6-15 

### (during treatment) 9.0 ± 2.0$ 13.7 ± 

### 1.3* 14.9 ± 1.6 15.1 ± 1.7 15.7 ± 0.9 

### gd 15-21 

### (following treatment) 19.4 ± 2.2 18.9 ± 1.6 17.6 ± 2.0 18.2 ± 2.0 18.9 ± 1.9 

## aValues represent mean ± standard deviation; units are in grams per damper 

### day. *p < 0.05 

### compared to control. 

### tp < 0.001 

### compared to control. 52EGBE INHALATION TERATOLOGY STUDY

Table 6. Water consumption of F-344 rats exposed to EGBE by inhalation.

EGBE target exposure concentrations, gestational days (gd) 6-15 200 ppm 100 ppm 50 ppm 25 ppm 0 ppm Water consumption, g/dam/day gd 0-3 16.9 ± 2.9(25)a 17.4 ± 3.3(23) 15.8 ± 4.4(21) 16.6 ± 2.0(21) 17.7 ± 2.1(22) gd 3-6 17.3 ± 2.0(25) 18.4 ± 2.4(23) 17.6 ± 2.5(21) 17.3 ± 2.5(21) 17.3 ± 1.9(22) gd 6-9 6.4 ± 2.7(25)* 18.1 ± 5.6(23) 17.9 ± 2.6(22) 18.2 ± 1.6(22) 19.1 ± 2.7(22) gd 9-12 20.1 ± 5.1(23) 25.3 ± 7.2(23) 21.9 ± 2.9(22) 22.1 ± 4.9(21) 22.0 ± 2.8(23) gd 12-15 30.1 ± 5.9(23)* 27.1 ± 7.3(23) 23.7 ± 5.3(22) 24.6 ± 4.4(21) 25.5 ± 2.9(23) gd 15-18 31.5 ± 6.5(25) 33.7 ± 3.4(22) 30.3 ± 4.8(22) 28.7 ± 6.4(22) 31.4 ± 4.0(23) gd 18-21 31.2 ± 7.7(21) 33.3 ± 

### 3.4(21)b* 30.0 ± 5.3(22) 29.7 ± 2.5(21) 30.4 ± 3.7(23) gd 0-6 (prior to treatment) 17.1 ± 2.0(25) 17.9 ± 2.4(23) 16.6 ± 2.4(20) 16.8 ± 1.6(21) 17.6 ± 1.9(21) gd 6-15 (during treatment) 19.1 ± 3.8(21)t 23.5 ± 4.0(23) 21.2 ± 2.8(22) 21.8 ± 1.9(20) 22.2 ± 2.2(23) gd 15-21 (following treatment) 31.6 ± 6.9(21) 33.6 ± 

### 3.1(21)* 30.1 ± 4.8(22) 29.1 ± 3.5(21) 30.9 ± 3.6(23) aValues represent mean standarddeviation; units are in grams perdam perday; values inparentheses indicateNforeach group. Reduced Nvalues were due to spillage. bThe statistical significance for this value was assessed using the Welch ANOVA test statistic. This value was not statistically significant when the Brown-Forsythe test (ANOVA) was employed. 

### *p < 0.05 compared to control. 

### tp < 0.01 compared to control. 

### tp < 0.001 compared to control.



### Table 7. Hematologic determinations for Fischer 344 rat dams exposed to EGBE by inhalation on gestation days (gd) 

### 6-15.a 

### Target Hemolysis, % Hematologic 

### parametersb concentration, 0.55% 0.50% 0.45% RBC Hgb, Hct, ppm saline saline saline x 

### 10-/mm3 g/dL % 200 8.2 39.5 82.4 

### 5.83* 13.8$ 

### 42.0* (7.1) (14.2) (11.2) (0.60) (1.4) (4.8) 100 4.9 33.5 76.8 

### 5.77* 11.9 35.2 (5.1) (10.4) (8.8) (0.47) (0.9) (2.9) 50 7.3 38.4 77.0 6.36 12.0 35.0 (7.5) (12.7) (10.3) (0.55) (1.0) (3.0) 25 5.1 32.4 74.4 6.37 12.0 35.0 (5.4) (9.2) (7.2) (0.34) (0.6) (1.9) 0 8.3 38.0 77.7 6.36 12.1 35.1 (6.7) (12.1) (6.5) (0.41) (0.8) (2.3) 

## aData are 

### presented as mean 

### (standard deviation). 

### bRBC = red blood cells (x 

## 106/mm3); Hgb = 

### hemoglobin (g/dL); Hct = hematocrit (%). 

### tp < 0.001 versus controls. 53TYL ETAL.



### ppm 

### (Table 5). Water 

### consumption, expressed as 

### grams/dam/day, was also 

### significantly reduced for 

### the 

### periods gd 6-9, 12-15 

### and 6-15 

### fordams at 200 

### ppm. Water

## consumptionwas

### elevatedrelativeto

### controlsfor dams 

### exposed to 100 

### ppm during the 

### postexposure 

### periods gd 18-21 

### and 15-21 

### (Table 6). 

### Hematologic determinations on dams at 

### scheduled 

### sacrifice (Tables 7 and 8) indicated no alterations in osmotic 

### fragility of

### erythrocytes but 

### significant reductions in 

### erythrocyte (RBC) 

### count, and significant 

### increases in 

### hemoglobin and hematocrit (packed 

### red 

## blood cell 

## volume) at 

### 200 

### ppm. 

### RBC count was 

### also

Table 8. Erythrocyte indices for Fischer 344 rat dams exposed to 

### EGBE by inhalation on gestation days 6-15.

Erythrocyte 

### indicesa Target 

### MCV, MCH, MCHC, concentration, ppm 

### j±m3b pgc g/dLc 200 

### 71.41 23.6k 

### 32.8k



### [1.2] (0.8) 

### (0.8) 100 

### 60.81 21.0 33.8t



## [0.9] (2.0) (0.7) 50 55.0 19.0 34.4



### [0.2] (0.4) 

### (0.6) 25 54.9 18.9 34.5



### [0.5] (0.3) (0.6) 0 55.0 18.9 34.4



### [0.6] (0.3) (0.6) 'MCV = mean 

### corpuscular volume 

# (Rm3), MCH = mean corpuscular 

### hemoglobin (pg), MCHC = mean 

### corpuscular 

### hemoglobin concentration 

### (g/dL). 

### bMedian [quartile deviation]. 

## CMean (standard deviation). 

### "O.01

- p 2 0.001 versus controls. 

### tp < 0.001 versus controls. 

### reduced at 

### 100 ppm. 

### The size 

### of the red blood cell 

### (MCV) was 

### significantly enlarged at 

### 200 

### and 100 ppm, 

### and the 

### hemoglobin per 

### cell (MCH) was 

### significantly increased. The mean

### corpuscular

### hemoglobinconcentra

### tion 

### (MCHC) was 

### significantly increased. The mean 

### corpuscular hemoglobin concentration (MCHC) was 

### significantly reduced at 

### 100 

### and 200 ppm 

### relative to 

### controls. Atscheduledsacrifice,

### maternalgraviduterine

### weight

was 

### significantly reduced, and absolute and 

### relative spleenweight and

### relativekidneyweight were 

### elevated 

### compared to 

### controls at 

### 200 ppm 

### (Table 9). Reproductive toxicity was 

### also evident at 

### 200 ppm 

### (Table 10). 

### The number of viable implants and the percent 

### live fetuses per 

### litter were 

### reduced relative to 

### controls andthe

### numberofnonviable implants, primar

### ily due to 

### the elevated number 

### of early embryonic 

### resorptions, was

### elevatedat

### 200 ppm

### relativeto

### controls. Preimplantation loss was 

### also elevated, but the difference was 

### not 

### statistically significant. Sex ratio 

### (% 

### males) and the 

### bodyweight ofmale or 

### female fetuses per 

### litter were 

### unaffected by treatment 

### (Table 10). 

### The 

### results of fetal 

### evaluations are 

### presented in 

### Tables 11 

### and 12. 

### There were no 

### statistically significant 

### increases in

### the incidence 

### ofexternal, visceral, 

### skeletal

or

### total

### malformations due to 

### treatment 

### (Table 11). 

### The incidence 

### of 

### variations 

### observed in rat 

### fetuses in 

### the present 

### study is 

### presented in 

### Table 12. 

### Evidence for 

### retarded 

### skeletal ossification was seen 

### at 

### 100 

### and 200 ppm. 

### At200 ppm, 

### there was a

### significantincreasein

### the number of 

### litters 

### with one or more 

### fetuses exhibiting unossified 

### skeletal 

### elements, e.g., 

### the anterior 

### arch of the 

### atlas, cervical centra 

### 5 

### and 6, 

### and poorly ossified skeletalelements, e.g., 

### cervicalarches, sternebrae 1, 3, 4

### and

### 6and

### proximalphalanges in

### the

### forelimbs. At 100 ppm, 

### there was a

### significant increasein

### theincidenceof

Table 9. Maternal organ 

### weights of F-344 rats 

### exposed to 

### EGBE 

### by inhalation.

EGBE target exposure 

### concentrations, 

### gestational days 

### (gd) 6-15a 200 ppm 100 ppm 50 ppm 25 ppm 0 ppm 

### Maternal bodyweight 

### (gd21), g 244.5 ± 30.9 277.1 ± 19.0 272.4 ± 20.2 277.6 ± 19.5 275.6 ± 17.8 

### Organ

### weights, g Uterus 34.09 ± 28.62t 62.04 ± 17.91 56.94 ± 20.44 63.10 ± 11.76 59.90 ± 17.56 Liver 10.42 ± 2.09 11.30 ± 1.04 10.67 ± 1.06 11.19 ± 1.22 11.17 ± 0.88 

### Relative 

### liver, %b 4.95 ± 0.97 5.25 ± 0.39 4.96 ± 0.48 5.20 ± 0.41 5.18 ± 0.38 

### Thymus 0.25 ± 0.10 0.26 ± 0.06 0.25 ± 0.08 0.27 ± 0.08 0.26 ± 0.07 Relative 

### thymus, %b 0.12 ± 0.04 0.12 ± 0.03 0.12 ± 0.03 0.12 ± 0.04 0.12 ± 0.03 

### Spleen 0.65 ± 0.09* 0.57 ± 0.06 0.60 ± 0.26 0.53 ± 0.07 0.54 ± 0.05 Relative 

### spleen %b 0.31 ± 0.04k 0.27 ± 0.03 0.28 ± 0.12 0.25 ± 0.03 0.25 ± 0.02 

### Kidney 1.54 ± 0.17 1.44 ± 0.08 1.39 ± 0.10 1.42 ± 0.10 1.46 ± 0.14 Relative 

### kidney, %b 0.74 ± 0.09* 0.68 ± 0.04 0.65 ± 0.04 0.66 ± 0.05 0.68 ± 0.05 

## aValues represent mean ± 

### standard deviation. 

### bRelative organ

### weight = organ

### weight 

### expressed as a

### percent of corrected

### bodyweight. *p < 0.05 as 

### compared to 

### control. 

### tp < 

## 0.01 as 

### compared to 

### control. 

### tp < 0.001 as 

### compared to control. 54EGBE INHALATION TERATOLOGY STUDY

Table 10. Reproductive toxicity evaluation of F-344 rats exposed to EGBE by inhalation.

EGBE target exposure concentrations, gestational days (gd) 6-15a 200 ppm 100 ppm 50 ppm 25 ppm 0 ppm 

### Corpora lutea 15.7 ± 5.4 13.2 ± 1.2 13.2 ± 5.3 13.0 ± 1.7 12.9 ± 1.8 Total 

### implants 8.7 ± 3.8 10.3 ± 3.2 9.1 ± 3.6 10.3 ± 2.2 10.0 ± 3.5 

### Viable implants 4.7 ± 

### 4.5k 9.9 ± 3.3 8.9 ± 3.7 10.0 ± 2.2 9.4 ± 3.2 Nonviable implants 4.0 ± 

### 4.6t 0.4 ± 1.1 0.2 ± 0.4 0.3 ± 0.5 0.5 ± 0.7 Embryonic resorptions 2.8 ± 

### 4.7bt 0.0 ± 0.0 0.1 ± 0.3c 0.0 ± 0.0 0.0 ± 0.0 

### Embryonic resorptions with placenta 1.2 ± 2.2 0.3 ± 0.9 0.1 ± 0.4 0.2 ± 0.4 0.4 ± 0.7 Dead and macerated fetuses 0.0 ± 0.2 0.1 ± 0.3 0.0 ± 0.0 0.0 ± 0.2 0.1 ± 0.3 Live 

### fetuses, % 52.7 ± 

### 45.1t 94.4 ± 12.9 93.3 ± 21.4 97.5 ± 4.3 95.7 ± 5.9 

### Preimplantation loss, % 39.7 ± 30.6 22.2 ± 22.7 25.2 ± 29.2 19.6 ± 18.0 23.7 ± 26.2 Sexratio 43.8 ± 17.2 51.8 ± 20.0 51.2 ± 15.9 50.6 ± 19.8 50.5 ± 23.2 Mean litter weight, g Male 

### fetus weight 4.4 ± 0.4 4.5 ± 0.2 4.6 ± 0.4 4.5 ± 

### 0.ld 4.5 ± 0.2 Female fetus weight 4.0 ± 0.5 4.2 ± 0.2 4.3 ± 0.4 4.2 ± 0.2 4.3 ± 0.2e 

## aValues represent mean ± standard deviation on a per litter basis. 

### bNine of

### twenty-five dams had embryonic resorptions, seven ofwhich were found to be gravid by ammonium sulfide 

### staining

## only. cOne of twenty-two dams had 

### embryonic resorptions, detected 

### onlyby ammonium sulfide 

### staining. 

### dN = 

## 22, 20, 21, and 22 for exposure groups 100, 50, 25 and 0 ppm, 

## respectively. One litter from each group had only female fetuses. eN = 22; one

## litterhad onlymale fetuses. 

## tp < 0.01 versus controls. 

### tp < 0.001 versus controls.

Table 11. 

### Malformations observed in Fischer 344 rat 

### fetuses exposed to 

### EGBE in utero.a



### Fetusesb Littersc 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm 0 ppm 25 ppm 50 ppm 100 ppni 200 ppm 

### EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE 

### EGBE EGBE Number 

### exanmined 

### externallyd Umbilical hernia No. Number examined 

### viscerallye 

### Right aortic arch No. Ventricular 

### septal defect No. Situs inversus No. 

### Left carotid artery emergesfromarch on

### right No. Dorsal 

### deflected aortic arch No. 

### Retroesophageal aortic 

### arch No. 

### Microphthalmia, right No. 217 221 196 228 117 23 22 21 23

0 1 1 0 0 0 1 1 0 (0.0) (0.5) (0.5) (0.0) (0.0) (0.0) (4.5) (4.8) (0.0) 112 115 104 119 64 23 22 21 23 0 0 0 0 1 0 0 0 0 (0.0) (0.0) (0.0) (0.0) (1.6) (0.0) (0.0) (0.0) (0.0) 16 0 (0.0) 16 1 

### (6.2)

0 1 0 0 1 0 1 0 0. 1 (0.0) (0.9) (0.0) (0.0) (1.6) (0.0) (4.5) (0.0) (0.0) 

### (6.2) 0 0 0 0 1 0 0 0 0 1 (0.0) (0.0) (0.0) (0.0) (1.6) (0.0) (0.0) (0.0) (0.0) (6.2)

0 0 1 0 0 0 0 1 0 0 (0.0) (0.0) (1.0) (0.0) (0.0) (0.0) (0.0) (4.8) (0.0) (0.0)

0 0 0 0 1 0 0 0 0 (0.0) (0.0) (0.0) (0.0) (1.6) (0.0) (0.0) (0.0) (0.0) 1 

### (6.2)

0 0 1 0 0 0 0 1 0 0 

### (0.0) (0.0) 

### (1.0) (0.0) (0.0) 

### (0.0) (0.0) 

### (4.8) 

### (0.0) 

### (0.0)

1 0 0 1 1 1 0 0 1 1 

### (0.9) (0.0) 

### (0.0) (0.8) 

### (1.6) (4.3) 

### (0.0) (0.0) 

### (4.3) 

### (6.2) 55TYL ETAL.



### Table 11. (continued) 

### Fetuses' Littersc 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE Microphthalmia, left No. Incomplete septation ofaorta and pulmonary artery No. 0 0 1 1 0

(0.0) (0.0) (1.0) (0.8) (0.0)

0 0 0 0 1 (0.0) (0.0) (0.0) (0.0) (1.6) 0 0 1 1 0 (0.0) (0.0) (4.8) (4.3) (0.0)

0 0 0 0 1 (0.0) (0.0) (0.0) (0.0) (6.2) Number examined 

### skeletally' Thoracic vertebrae curved No. Unossified 

### rib, unilateral No. Sternebrae 

### misshapen No. 

### Digit, curved and 

### abnormal size No. 

### Tympanic annuli abnormal No. Microagnathia No. 

### Total malformations 

### Externald No. Viscerale No. 

### Skeletalf No. Total No. 105 106 92 109 49 23 22 209 229 

### 15h

0 1 0 0 0 (0.0) (0.9) (0.0) (0.0) (0.0)

0 0 0 0 1 (0.0) (0.0) (0.0) (0.0) (2.0)

0 1 0 0 0 (0.0) (0.9) (0.0) (0.0) (0.0)

0 1 0 0 0 (0.0) (0.9) (0.0) (0.0) (0.0)

0 1 0 0 0 (0.0) (0.9) (0.0) (0.0) (0.0) 0 1 0 0 0 (0.0) (0.9) (0.0) (0.0) (0.0)

0 1 1 0 0 (0.0) (0.5) (0.5) (0.0) (0.0) 1 1 2 2 3 

### (0.9) (0.9) (1.9) (1.7) (4.7) 0 3 0 0 1 (0.0) (2.8) (0.0) (0.0) (2.0) 1 5 3 2 4 

### (0.5) (2.3) (1.5) (0.9) (3.4) 0 1 0 0 0 (0.0) (4.5) (0.0) (0.0) (0.0)

0 0 0 0 1 (0.0) (0.0) (0.0) (0.0) (6.7)

0 1 0 0 0 (0.0) (4.5) (0.0) (0.0) 

### (0.0)

0 1 0 0 0 (0.0) (4.5) (0.0) (0.0) 

### (0.0)

0 1 0 0 0 (0.0) (4.5) (0.0) (0.0) (0.0) 0 1 0 0 0 (0.0) (4.5) (0.0) (0.0) (0.0)

0 1 1 0 0 (0.0) (4.5) (4.8) (0.0) (0.0) 1 1 2 2 3 (4.3) (4.5) (9.5) (8.7) (18.7) 0 3 0 0 1 (0.0) (13.6) (0.0) (0.0) 

### (6.7) 1 5 2 2 4 (4.3) (22.7) (9.5) 

### (8.7) (25.0) 'A 

### single fetus maybe 

### represented more than once in 

### listing individual defects. 

## bOnly live fetuses were examined formalformations. 

## CIncludes litters with one ormore 

### malformed fetuses. 

### dAll fetuses were examined 

### externally. 

## eApproximately 50% of each litter were

## examined viscerally 

## (3) and for soft tissue craniofacial malformations 

### (4). 

### fApproximately 50% of each litter were examined for skeletal malformations after 

### stainingwith Alizarin Red S. 

## NOne litter contained 

### only one live fetus. 

### By convention this fetus was 

### subjected to visceral and craniofacial examination. 

## hThe skeletal 

### preparations from one litter were macerated and could not be evaluated.



### unossified 

## cervical centrum 6. 

### At 100 and 200 

## ppm, At 200 

## ppm there was also a 

### decreased 

## incidence of 

## there was a 

### decreased incidence of 

## bilobed cervical 

## bilobed thoracic centra 9 

### and 13 

### and of

## poorly ossified centrum

### 5 

## (primarilybecauseat

## these

## exposureconcen- 

## proximal

## phalangesofthehindlimbrelativeto

## controls. trations 

### this 

## skeletal element was 

## largely unossified). 

## Rudimentaryriband/orbone 

### islandat 

### the first 

### lumbar 56EGBE INHALATION TERATOLOGY STUDY

Table 12. Variations observed in 

### Fischer 344 rat fetuses 

### exposed to EGBE in utero.a

Fetusesb 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm EGBE 

### EGBE EGBE EGBE EGBE Number examined externallyc Ecchymoses, trunk No. Ecehymoses, head No. Ecchymoses, extremitie No.

Number examined 

### viscerally' Thymus, hemorrhagic No. Shortened innominate a No. 217 221

21 22 

### (9.7) (10.0) 5 3 (2.3) (1.4) 4 5 

### (1.8) (2.3)

112 115 3 6 (2.7) (5.2) rtery 0 2 (0.0) (1.7) Missing innominate artery No. 0 % (0.0) White nodule, caudate lobe of liver No. 1 % (0.9) Liver discoloration No. 0 % (0.0) Liver hemorrhagic No. 0 % (0.0) Red foci, right kidney No. 1 % (0.9) Red foci, both kidneys No. 0 % (0.0) Dark red coagulation in abdominal No. 0 % (0.0) Lungs hemorrhagic No. 0 % (0.0) Fetal atelectasis 0 (0.0) 2 (1.7)

1 (0.9)

0 (0.0) 2 (1.7)

0 (0.0) cavity

0 (0.0)

0 (0.0) No. 2 4 % (1.8) (3.5) Fourth cusp on aortic semilunar valve No. 1 0 % (0.9) (0.0) Red foci on right atrium No. 0 0 % (0.0) (0.0) Lateral ventricles dilated (cerebrum) No. 2 1 % (1.8) (0.9) Third ventricles dilated (diencephalon) No. 1 0 % (0.9) (0.0) Enlarged fourth ventricle (cerebellum) No. 21 14 % (18.7) (12.2) Nasal septum or passage distorted No. 2 8 % (1.8) (7.0) 196 27 (13.8) 3 (1.5) 5 (2.6)

104 6 (5.8) 2 (1.9)

0 (0.0) 2 (1.9) 1 (1.0)

1 (1.0)

0 (0.0) 1 (1.0)

1 (1.0)

1 (1.0) 3 (2.9)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0) 19 (18.3) 8 (7.7) 228

29 (12.7) 3 (1.3) 8 (3.5)

119 6 (5.0)

0 (0.0)

0 (0.0) 2 (1.7)

0 (0.0)

0 (0.0) 2 (1.7)

0 (0.0)

0 (0.0)

0 (0.0) 2 (1.7)

0 (0.0)

1 (0.8)

1 (0.8)

0 (0.0) 14 (11.8) 7 (5.9) 117

16 (13.8) 4 (3.4) 4 (3.4)

64 4 (6.2) 2 (3.1)

1 (1.6) 2 (3.1)

0 (0.0)

0 (0.0)

1 (1.6)

0 (0.0)

0 (0.0)

0 (0.0) 8 (12.5)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0) 8 (12.5) 1 (1.6) Littersc 0 ppm EGBE 23 15 (65.2) 4 (17.4) 3 (13.0)

23 3 (13.0)

0 (0.0)

0 (0.0)

1 (4.3)

0 (0.0)

0 (0.0)

1 (4.3)

0 (0.0)

0 (0.0)

0 (0.0)

1 (4.3)

1 (4.3)

0 (0.0) 2 (8.7)

1 (4.3) 13 (56.5) 2 (8.7) 25 ppm EGBE

22 12 (54.5) 3 (13.6) 5 (22.7)

22 6 (27.3) 2 (9.1)

0 (0.0)

1 (4.5)

1 (4.5)

0 (0.0) 2 (9.1)

0 (0.0)

0 (0.0)

0 (0.0) 2 (9.1)

0 (0.0)

0 (0.0)

1 (4.5)

0 (0.0) 9 (40.9) 5 (22.7) 50 ppm EGBE

21 12 (57.1) 3 (14.3) 5 (23.8)

21 5 (23.8) 2 (9.5)

0 (0.0) 2 (9.5)

1 (4.8)

1 (4.8)

0 (0.0)

1 (4.8)

1 (4.8)

1 (4.8) 2 (9.5)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0) 11 (52.4) 5 (23.8) 100 ppm 200 ppm 

### EGBE EGBE 23 16 16 8 (69.6) (50.0) 3 3 (13.0) (18.7) 5 3 

### (21.7) (18.7)

23 16 5 4 (21.7) (25.0) 0 2 (0.0) (12.5) 0 1 (0.0) (6.2) 2 1 (8.7) (6.2) 0 0 (0.0) 

### (0.0) 0 0 (0.0) (0.0) 2 2 (8.7) (6.2) 0 0 

### (0.0) 

### (0.0) 0 0 

### (0.0) 

### (0.0) 0 0 

### (0.0) 

### (0.0) 2 4 (8.7) (25.0) 0 0 

### (0.0) 

### (0.0) 1 0 (4.3) (0.0) 1 0 (4.3) (0.0) 0 0 

### (0.0) (0.0) 9 7 

### (39.1) (43.7) 4 1 (17.4) (6.2) 57TYL ETAL.



### Table 12. (continued) Fetusesb Littersc 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE Gel-like material between skin and nasal bone No. 0 0 1 % (0.0) (0.0) (1.0) Number examined 

### skeletallyf106 106 92 Anterior arch ofAtlas, unossified No. 1 5 2 % (0.9) (4.7) (2.2) Cervical centra 1-3 and/or 4, poorly ossified No. 57 73 58 % (53.8) (68.9) (63.0) Cervical centra #5, unossified No. 13 17 11 %o (12.3) (16.0) (12.0) Cervical centra #5, bilobed No. 19 11 13 % (17.9) (10.4) (14.1) Cervical centra #6, unossified No. 2 4 5 % (1.9) (3.8) (5.4) Cervical centra #6, bilobed No. 40 33 27 % (37.7) (31.1) 

### (29.3) Cervical arches 1-6 and/or 7, 

### poorly ossified No. 2 4 7 % (1.9) (3.8) (7.6) 

### Throacic centra #1, bilobed No. 47 30 46 % (44.3) (28.3) (50.0) Thoracic centra #9, bilobed No. 15 16 10 % (14.2) (15.1) (10.9) Thoracic centra #13, bilobed No. 31 24 22 % (29.2) (22.6) (23.9) Sternebra #1, 

### poorly ossified No. 4 12 5 % (3.8) (11.3) (5.4) 

### Sternebra 

### #3, 

### poorly ossified No. 4 11 4 % (3.8) (10.4) (4.3) Sternebra #4, 

### poorly ossified No. 13 23 14 % (12.3) (21.7) (15.2) Sternebra #6, 

### poorly ossified No. 28 24 19 % (26.4) (22.6) (20.7) 

### Sternebra #5, 

### bilobed No. 14 34 18 % (13.2) (32.1) (19.6) Proximal 

### phalanges poorly ossified 

### (hindlimb) No. 66 53 35 % 

### (62.3) (50.0) (38.0) Proximal 

### phalanges unossified 

### (forelimb) No. 12 18 14 % (11.3) (17.0) (15.2) 

### Rudimentary rib 

### lumbar-1, unilateral No. 3 5 3 % 

### (2.8) (4.7) (3.3) 

### Rudimentary rib 

### lumbar-1, bilateral No. 0 1 2 % (0.0) (0.9) (2.2) Extra 

### rib, unilateral No. 0 0 0 % (0.0) (0.0) (0.0) Bone island at lumbar-1 No. 2 2 5 0 0 0 (0.0) (0.0) (0.0) 109 50 23 3 10 1 (2.8) (20.0) (4.3) 62 9 22 (56.9) (18.0) (95.7) 22 24 9 (20.2) (48.0) (39.1) 6 1 14 

### (5.5) (2.0) (60.9) 11 12 2 (10.1) (24.0) (8.7) 20 6 19 (18.3) (12.0) (82.6) 2 7 2 (1.8) (14.0) (8.7) 34 22 20 (31.2) (44.0) (87.0) 10 4 11 (9.2) (8.0) (47.8) 17 6 18 (15.6) (12.0) (78.3) 5 12 3 (4.6) (24.0) (13.0) 11 10 3 (10.1) (20.0) (13.0) 25 16 7 (22.9) (32.0) (30.4) 28 35 11 (25.7) (70.0) (47.8) 16 9 9 (14.7) (18.0) (39.1) 56 10 22 (51.4) (20.0) (95.7) 23 21 7 (21.1) (42.0) (30.4) 5 4 3 (4.6) (8.0) (13.0) 1 3 0 (0.9) (6.0) (0.0) 0 2 0 (0.0) (4.0) (0.0) 0 (0.0) 22 4 (18.2) 22 (100.0) 10 

### (45.5) 9 

### (40.9) 4 (18.2) 17 

### (77.3) 4 (18.2) 

### 12* 

### (54.5) 10 

### (45.5) 16 (72.7) 8 (36.4) 8 (36.4) 13 

### (59.1) 14 (63.6) 

### 17* 

### (77.3) 20 

### (90.9) 12 

### (54.5) 5 (22.7) 1 

### (4.5) 0 

### (0.0) 1 

### (4.8) 209 2 (10.0) 20 (100.0) 9 

### (45.0) 11 

### (55.0) 4 (20.0) 13 (65.0) 4 (20.0) 18 (90.0) 9 

### (45.0) 13 (65.0) 3 (15.0) 4 (20.0) 9 

### (45.0) 12 (60.0) 14 (70.0) 15 

### (75.0) 8 (40.0) 3 (15.0) 2 (10.0) 0 (0.0) 0

(0.0) 229 2 

### (9.1) 22 (100.0) 14 (63.6) 

### 4* (18.2) 10* 

### (45.5) 13 

### (59.1) 2 

### (9.1) 16 (72.7) 7 (31.8) 11 (50.0) 5 (22.7) 6 

### (27.3) 13 

### (59.1) 14 (63.6) 11 (50.0) 22 

### (100.0) 10 

### (45.5) 4 (18.2) 1 

### (4.5) 0 

### (0.0) 0

(0.0) 15h 

### 7* (46.7) 

### 7* (46.7) 

### 12* (80.0) 

### 1* (6.7) 10l (66.7) 

### 5* 

### (33.3) 7* (46.7) 12 (80.0) 

### 2* 

### (13.3) 3* (20.0) 

### 7* (46.7) 

### 7* (46.7) ll* 

### (73.3) 14* 

### (93.3) 9 (60.0) 

### 8* 

### (53.3) 10* (66.7) 4 (26.7) 3 (20.0) 2 

### (13.3) 7 4 2 2 5 7 4 

### (1.9) (1.9) (5.4) 

### (6.4) (8.0) (8.7) 

### (9.1) 

### (25.0) (31.8) 

### (26.7) 58EGBE INHALATION TERATOLOGY STUDY

Table 12. (continued) 

### Fetusesb Littersc 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE 

### Rudimentary rib, thoracic area No. 0 0 0 0 1 0 0 0 0 1 % (0.0) (0.0) (0.0) (0.0) (2.0) (0.0) (0.0) (0.0) (0.0) (6.7) 'A single fetus may be represented more than once in listing individual defects. 

### bOnly live fetuses were examined for defects. 

## cIncludes litters with one or more affected fetuses. 

### dAll fetuses were examined externally 

## eApproximjately50% of each litterwere examined viscerally (3) and for soft tissue craniofacial defects (4). 

### 'Approximately 50% of each litter were examined for skeletal defects after staining with Alizarin Red S. Only those parameters whose incidence differed significantly from that of controls at p < 0.05, Fisher's exact test (two-tailed) and related parameters are indicated. 90ne litter contained 

### only one live fetus. 

### By convention this fetus was 

### subjected to visceral and craniofacial examination. 

### hThe skeletal preparations from one litter were macerated and could not be evaluated. 

### *p < 0.05 versus controls, Fisher's exact test (two-tailed). Table 13. Distribution and fate of NZW rabbits exposed to EGBE by inhalation on gestational days 6 through 18. EGBE target 

### concentrations 200 ppm 100 ppm 50 ppm 25 ppm 0 ppm No. in study 24 24 24 24 24 No. removed 0 0 la 0 0 No. abortions 

### 4b 1 0 0 0 No. dead 4c 0 0 0 0 No. sacrificed (gd 29) 16 23 23 24 24 No. nonpregnant at sacrifice 1 3 1 1 4 No. (%) pregnant at sacrifice 15 (93.8) 20 

### (87.0) 22 (95.7) 23 (95.8) 20 (83.3) No. litters examined 15 19d 22 22e 20 aAnimal removed from study because it died immediately following cardiac puncture; all fetuses were dead when cesarean section was performed. 

### bThe number of 

### abortions in this group was not 

### significantly elevated relative to that in 

### controls by 

### Fisher's exact test. 

### CAll females were confirmed pregnant at postmortem

## necropsy. The number of deaths was not significantly elevated relative to controls. 

### dOne rabbit was found gravid at cesarean 

### section with only one 

### embryonic resorption with placenta. 

### eOne rabbit was found gravid at cesarean section with 10 dead and macerated fetuses 

## only.

Table 14. Clinical observations in NZW rabbit does exposed to EGBE on gestational days 6 through 18.' EGBE concentration 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm Deaths By gd 8 

### 2b By gd 10 1 Sacrificed in extremis (gd 9) 1 Behavior Hypoactive 1 Listless 1 Lethargic 1 Alopecia + + + + + 

### Urogenital area stained + + + + + + Fur stained + + + + + + Periocular wetness + + + + + + + Periocular encrustation + + + + + Perinasal wetness + + 0 0 + + Perinasal 

### discharge 0 0 0 0 + Red fluid on tray or paperboard 0 0 0 + + Abortion 

### (placental/fetal tissue on 

### paperboard; gd 21-25) la 4a 

## aCode: + + = approximately 50% of the does (30-70%) exhibited the clinical sign sometime during the exposure 

### period; + = up to 

### 25% of the does exhibited the clinical sign 

### sometime during the 

### exposure period; 0 = no does 

### exhibited the clinical sign 

### during the exposure period. 

### bNumber of does exhibiting the clinical sign sometime during the exposure or postexposure period. 59TYL ETAL.

Table 15. Maternal weights andweight gain ofNZW rabbits exposed to EGBE by inhalation.

EGBE target exposure concentrations, gestational days (gd) 6-15 200 ppm 100 ppm 50 ppm 25 ppm 0 ppm 

### Maternal bodyweight, g 

### gd 0 3892.1 ± 520.6a 3910.5 ± 517.0 3748.3 ± 563.5 4086.6 ± 529.7 4077.8 ± 612.2 

### gd 6 4047.9 ± 535.2 4071.0 ± 505.8 3958.2 ± 641.5 4249.1 ± 530.3 4242.2 ± 599.1 

### gd 9 3844.5 ± 

### 449.6b 4007.6 ± 477.7 3877.7 ± 596.9 4190.4 ± 525.7 4164.3 ± 583.9 

### gd 12 3786.5 ± 393.6c 4009.7 ± 451.1 3889.1 ± 586.5 4195.7 ± 526.0 4196.7 ± 536.5 gd 15 3811.3 ± 

### 406.2c* 4022.7 ± 436.1 3945.0 ± 590.3 4227.7 ± 506.1 4221.8 ± 540.8 gd 21 3878.1 ± 422.2c 4099.0 ± 443.1 4050.6 ± 593.7 4316.9 ± 500.7 4290.9 ± 

### 383.1d gd 29 3994.4 ± 426.0e 4163.5 ± 

### 445.3' 4047.7 ± 514.9 4314.7 ± 

### 502.7h 4363.1 455.0 

### Corrected 

### body weightg 3553.5 ± 380.6 3692.1 ± 413.7 3507.5 ± 456.6 3784.8 

### 474.oh 3795.2 ± 434.0 

### Maternal bodyweight gain, g 

### gd 0-6 155.8 ± 101.4 160.5 ± 72.2 209.9 ± 145.3 162.5 ± 104.7 164.4 ± 126.2 

### gd 6-9 -134.2 ± 

### 144.1b -63.4 ± 66.5 -80.4 ± 82.3 -58.7 ± 60.4 -77.9 ± 80.8 

### gd 6-12 -102.6 ± 

### 58.0c -61.3 ± 89.9 -69.1 ± 90.5 -53.4 ± 94.1 -45.6 ± 99.9 

### gd 6-15 -77.7 ± 70.0c -48.3 ± 119.5 -13.1 ± 113.7 -21.4 ± 133.5 -20.4 ± 129.5 

### gd 6-21 -11.0 ± 152.3c 28.0 ± 247.9 92.4 ± 146.4 67.8 ± 202.5 74.2 ± 233.4 

### gd 6-29 85.7 ± 162.7e 66.3 ± 

### 347.3f 89.6 ± 275.7 55.3 ± 

### 199.4h 120.9 ± 246.9 

### gd 21-29 33.1 ± 123.8e 28.9 ± 

### 204.7f -2.8 ± 190.1 -3.5 

### 135.6h 41.9 ± 

### 177.8d 

## aValues represent mean ± standard deviation; units are in grams. 

### bN = 21

-Two rabbits dead prior to gestational day 9. 

## CN = 19- Four rabbits dead prior to gestational day 12. 

### dN = 16- Four rabbits were

## accidentally not weighed. eN = 15

- Four 

### rabbits dead prior to gestational 

### day 12 and four rabbits aborted prior to gestational day 29. 

### fN = 20- One rabbit 

### aborted

### prior to gestational day 29. gCorrected

### bodyweight = 

### body

### weight on

### gd 29 minus 

### gravid uterine 

### weight. 

### hN = 22- 

### One 

### rabbit died immediately 

### following 

### cardiac puncture on gestational day 29; a final bodyweight was not obtained. 

### *p < 0.05 versus 

### controls.



### Table 16. 

### Hematologic determinations for NZW rabbit does exposed to EGBE by inhalation on gestational days 6-18.' 

### Target Hemolysis, % Hematologic 

### parametersb 

### concentration, 0.55% 0.50% 0.45% 

### RBC 

### Hgb, 

### Hct, ppm saline saline saline x 

### 1076/mm3 g/dL t 200 20.2 54.2 88.1 5.87 13.1 40.6 

### (14.0) (19.8) (10.7) 

### (0.40) (0.8) 

### (2.7) 100 23.8 56.1 87.3 6.13 

### 13.5t 

### 41.5t (19.4) 

### (21.4) (12.5) 

### (0.62) (1.2) (4.0) 50 19.2 47.7 81.4 5.94 13.1 40.0 (13.2) (18.8) (14.0) (0.43) (0.9) (2.8) 25 22.5 53.0 82.2 5.71 12.6 38.5 (12.3) (16.7) (11.9) (0.70) (1.5) (4.4) 0 25.4 57.0 86.0 5.66 12.4 38.1 (11.6) (13.5) (8.2) (0.52) 

### (1.1) (3.3) 

## aData expressed as mean 

### (standard 

### deviation). 

## bRBC = red blood cells (x 

## 106/mm3); Hgb = 

### hemoglobin (g/dL); 

## Hct = hematocrit (%). 

## tO.01 > p > 0.001 versus 

### controls.



## vertebrawas observed in 

## fetuses fromall 

## groups with no 

## treatment-related changes in 

### incidence. 

# Rabbits The distribution 

## and fate 

## ofall

## mated New Zealand Whiterabbitson

## studyare

## presentedin

### Table 13. 

### There was 

### an 

## apparent, but not 

## statistically significant increase 

### in

## spontaneous abortions anddeaths at 200

## ppm 

## relative to 

## controls. All 

## dead females were 

## pregnant. 

## Pregnancy rate at 

## scheduled 

## sacrifice on 

## gd 29 

### was 

## equivalent across treatment 

## groups. 

## Clinical observations 

## (Table 14) included

## periocularwetness, perinasal wetness

### and

## discharge, red

## fluidon

## tray, andstainedfur as 

## possible treatment-related clinical 

## signs. Reducedmaternal

## body

## weight at

## gd 15 was seen at 

### 200 

## ppm, but 

## there were 

### no 

## significant 

## weight gain 

## depressions in 

## any

## exposure group relative to 

## controls 

## (Table 15). Hematologic 

## determinations (Tables 16 

### and 

## 17) indi

## cated no 

## apparent treatment-related 

## changes in 

## any 

## parameter 

## evaluated. 

## Statistically significant increases 

### in

## hemoglobin content

## andhematocritwere seen

### at 100 

## ppm

## but not at 

### 200 

## ppm. 60EGBE INHALATION TERATOLOGY STUDY



### Theonlytreatment-related change inmaternal organ 

### weights was a

### reductionin

### graviduterineweight at 

### 200 ppm 

### (Table 18). 

### Evaluation ofreproductive parameters 

### (Table 19) 

### indicated a

### significantreductioninnumberof 

### total

### implants andviable implants per

### litterat 

### 200 ppm 

### relative to 

### controls. There were no 

### treatment-related 

### effects on 

### the number 

### of nonviable implants, pre

### implantationloss, percent

### livefetuses, sex

### ratio or

### male 

### and 

### female fetal body weight per 

### litter. 

### The type 

### and incidence of

### malformations and varia

### tions 

### observedin

### rabbit 

### fetuses are

### presented in

### Tables 20 

### and 21, 

### respectively. 

### There was no 

### statistically 

### significantincreasein

### thenumberof

### fetuses or

### of

### litters with one or more

### affected

### fetuseswith

### pooledexternal, 

### visceral, skeletal or 

### total malformations in any 

### treatment group 

### relative to 

### controls. For 

### individual malfor

### mations, there was an 

### increase in 

### the 

### number of

Table 17. Erythrocyte indices forNZW rabbit does exposed to EGBE by inhalation on gestational days 6-18. Tar-get concentration,

ppm 200 100 50 25

0 

## Am3s

68.6 [0.9] 67.8 [2.0] 67.1 [1.0] 67.4 [1.2] 67.3 [1.1] Erythrocyte indicesa

MCH,

pg 22.4 (0.8) 22.1 (0.9) 22.0 (0.8) 22.1 (0.7) 22.0 (0.6) MCHC,

g/dLc 32.4 (0.4) 32.5 (0.6) 32.7 (0.6) 32.7 (0.6) 32.6 (0.4) 'MCV

= mean corpuscular volume (pLm3); MCH

= mean corpuscularhemoglobin(pg);MCHC = meancorpuscularhemoglobinconcentration (g/dL). bMedian [quartile deviation]. CMean (standard deviation). 

### litters at 

### 100 ppm

### with one or more 

### fetuses exhibiting fusion

### of

## papillarymuscles in 

### the left

### ventricle relative to 

### controls. Five 

### fetuses in 4 

### litters out 

### of 19 

### litters examined exhibited this 

### malformation at 

### 100 ppm. 

### There was no 

### concentration-response, i.e., no 

### other fetuses at any 

### other exposure group 

### exhibited this 

### malformation (Table 20). 

### This malformation was 

### therefore not 

### considered treatment-related. 

### Variations seen 

### in rabbit 

### fetuses are 

### presented in Table21. 

### There was a

### significantreductioninunossified sternebra 6 

### and in 

### rudimentaryrib at 

### the first 

### lumbar 

### vertebra, bilaterally, both at 

### 200 ppm 

### relative to 

### controls. 

### Except for these two 

### findings, there were no 

### treatment-related changes in 

### the incidence or 

### type 

### of 

### variationsfound across

### treatment groups. 

### Rudimentary rib or 

### extra

### rib was seen across 

### all groups 

### at

### relatively high 

### frequency with no 

### exposure-related incidence. 

# Discussion The present 

### study, 

### evaluating the effects of 

### EGBE administered by inhalation 

### during organogenesis, indicates 

### that EGBE 

### produces maternal and 

### embryo/fetal 

### toxicity in 

### Fischer 344 rats at 

### 100 

### and 200 ppm, 

### and maternal

### toxicityand

### embryotoxicitybut no 

### fetaltoxic

### ity in 

### New 

### Zealand White rabbits at 

### 200 ppm. 

### No 

### teratogenicity was seen 

### in 

### either 

### species at any 

### of the exposure 

### concentrations 

### employed. In rats 

### at 

### 100 

### and 200 ppm, 

### the 

### finding of maternal 

### hematuria and/or 

### hemoglobinuria seen 

### early in 

### the exposure 

### period and evidence 

### ofanemia

### confirms andextendstheworkdone 

### previously in rats 

### (13,14). The 

### reduced red blood cell count, 

### elevated 

### hemoglobin and hematocrit, increased cell size and 

### increased 

### hemoglobin per 

### cell found at 

### sacrifice inrats at 

### 100 

### and 200 ppm are 

### consistentwith 

### destruction of mature 

### erythrocytes and 

### release of 

### immatureand/oryoung

### erythrocytesintothe

### peripheral circulation 

### (13,15). Increased absolute and relative

Table 18. Maternal organ

### weights ofNZW rabbits 

### exposed to 

### EGBE 

### by inhalation.

EGBE target exposure 

### concentrations, gestational 

### days (gd) 6-18 200 ppm 100 ppm 50 ppm 25 ppm 0 ppm Maternal 

### bodyweight 

### (gd29), g 3994.4 ± 426.0a 4163.5 ± 445.3 4047.7 ± 154.9 4314.7 ± 502.7 4363.1 t 455.0 

### Organweights, g Uterus 440.83 ± 

### 99.84* 417.43 ± 187.89 540.20 ± 120.40 526.62 ± 103.07 567.87 ± 157.00 Liver 83.92 ± 11.94 97.52 ± 21.99 88.12 ± 16.80 93.21 ± 17.25 95.53 ± 21.25 

### Relative 

### liver, %b 2.37 ± 0.32 2.64 ± 0.54 2.51 ± 0.36 2.45 t 0.42 2.52 t 0.48 

### Thymus 3.57 ± 1.96 2.82 ± 1.10 3.17 ± 1.60 3.30 ± 1.47 3.89 ± 2.21 Relative 

### thymus, %b 0.10 ± 0.05 0.08 ± 0.03 0.09 ± 0.04 0.09 ± 0.04 0.10 ± 0.05 

### Spleen 1.45 ± 0.30 1.66 ± 0.91 1.66 ± 0.70 1.51 ± 0.42 1.72 ± 0.75 Relative 

### spleen, %b 0.04 ± 0.01 0.04 ± 0.02 0.05 ± 0.02 0.04 ± 0.01 0.04 ± 0.02 

### Kidney 17.13 ± 2.03 19.19 ± 3.13 17.24 ± 2.45 18.20 ± 2.17 18.96 ± 2.96 

### Relative 

### kidney, %b 0.48 ± 0.06 0.52 ± 0.07 0.49 ± 0.05 0.48 ± 0.06 0.50 ± 0.05 

## aValues represent mean ± 

### standard deviation. 

## bRelative organ

### weight = organ

### weight expressed as percent of corrected

### bodyweight. *p < 0.05 

### compared to 

### control. 61TYL ETAL.

Table 19. Reproductive toxicity evaluation ofNZW rabbits exposed to EGBE by inhalation.

EGBE target exposure concentrations, gestational days (gd) 6-18

Corpora lutea Tbtal implants Viable implants Nonviable implants Embryonic resorptions Embryonic resorptions with placenta Deadandmacerated fetuses Live fetuses, % Preimplantation loss, % Sex ratio Fetal body weights, g Male

Female 200 ppm 10.1 + 2.1a 8.4 ± 1.9* 7.2 ± 2.4* 1.2 ± 1.7 0.1 ± 0.4

0.9 ± 1.7 0.2 ± 0.6 85.8 ± 20.0 16.0 ± 11.8 58.4 ± 13.1

39.7 ± 6.6 39.2 ± 6.0 100 ppm

11.3 t 2.7 9.0 t 3.3 7.6 t 3.4 1.4 t 2.7 0.0 ± 0.0

0.8 ± 2.7 0.6 ± 0.9 83.5 ± 28.7 22.2 ± 27.0 58.8 ± 21.9b

39.6 ± 

### 64d 38.7 ± 7.1g 50 ppm

10.5 ± 2.5 9.2 ± 2.7 8.5 ± 2.2 0.7 ± 1.6 0.0 ± 0.0

0.3 ± 1.1 0.5 ± 0.9 94.0 ± 11.8 13.2 ± 15.2 48.9 ± 17.4

41.4 ± 6.1l 41.6 ± 6.7 25 ppm

10.8 + 2.9 9.5 ± 2.3 8.4 ± 3.0 1.1 ± 2.1 0.0 ± 0.0

0.3 ± 0.5 0.8 ± 2.1 88.3 ± 20.9 12.4 ± 14.1 52.5 ± 16.1c

40.6 ± 

### 4.9' 40.5 ± 

### 6.4' aValues represent mean + standard deviation. 

### bN

= 19- One pregnant female had onlynonviable uterine implantation sites. CN

= 22

- One pregnant female had only nonviable uterine implantation sites. dOne rabbit was found gravid at C-section with one embryonic resorption withplacenta only (N

= 19). eOne rabbit had only female fetuses (N

= 21). 

### 'One rabbit was found gravid at C-section with 10 dead and macerated fetuses only (N

= 22). gTwo rabbits had onlymale fetuses and one rabbit had an embryonic resorption with placenta only (N = 17). hOne rabbit had only male fetuses (N = 19). 

### *p < 0.05 versus controls.



### Table 20. Malformations observed in NZW rabbit fetuses exposed to 

### EGBE in 

### utero.a

Fetusesb Littersc 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE 

### EGBE EGBE Number examined 

### externallyd 181 195 Imperforate anus No. 0 0 So (0.0) (0.0) Severelyshortenedtail No. 0 0 % (0.0) (0.0) Gastroschisis No. 0 0 % (0.0) (0.0) Malrotated limb No. 0 0 % (0.0) (0.0) Midfacial hypoplasia No. 0 1 So (0.0) (0.5) Shortened limbs No. 0 1 % (0.0) (0.5) Number examined viscerallye 94 102 Papillary muscles in left ventricle fused No. 0 0 % (0.0) (0.0) Papillary muscles in right ventricle fused No. 1 0 % (1.1) (0.0) Right atrioventricular valve not closed No. 0 0 % (0.0) (0.0) 187 152 108 20 22 22 19 15

0 0 1 0 0 0 0 1 (0.0) (0.0) (0.9) (0.0) (0.0) (0.0) (0.0) (6.7) 0 0 2 0 0 0 0 2 (0.0) (0.0) (1.9) (0.0) (0.0) (0.0) (0.0) (13.3)

0 0 1 0 0 0 0 1 (0.0) (0.0) (0.9) (0.0) (0.0) (0.0) (0.0) (6.7) 1 0 0 0 0 1 0 0 (0.5) (0.0) (0.0) (0.0) (0.0) (4.5) (0.0) (0.0)

0 0 0 0 1 0 0 0 (0.0) (0.0) (0.0) (0.0) (4.5) (0.0) (0.0) (0.0)

0 0 0 0 1 0 0 0 (0.0) (0.0) (0.0) (0.0) (4.5) (0.0) (0.0) (0.0) 100 81 59 20 22 22 19 0 5 0 0 0 0 

### 4* (0.0) (6.2) (0.0) (0.0) (0.0) (0.0) (21.1) 2 2 2 1 0 2 2 (2.0) (2.5) (3.4) (5.0) (0.0) (9.1) (10.5) 0 1 0 0 0 0 1 (0.0) (1.2) (0.0) (0.0) (0.0) (0.0) (5.3) 15

0 (0.0) 2 (13.3)

0 (0.0) 0 ppm 11.7 ± 2.1 9.8 ± 3.2 9.0 ± 2.9 0.8 ± 1.2 0.1 ± 0.4

0.3 ± 0.8 0.4 ± 0.9 93.4 ± 9.6 17.3 ± 24.1 58.2 ± 15.0

41.8 ± 6.3 40.1 ± 

### 6.0h 62EGBE INHALATION TERATOLOGY STUDY

Table 20. (continued) Fetusesb Littersc 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE Left carotidbranches and 

### off innominate No. 0 1 0 0 0 0 1 0 0 0 % (0.0) (1.0) (0.0) (0.0) (0.0) (0.0) (4.5) 

### (0.0) 

### (0.0) (0.0) 

### Small constrictedpulmonary 

### artery, channel to aorta fromrt. 

### ventricle, extrabranches offaorta, 

### vestigal rt. 

### ventricle, absent 

### tricuspid valve and associated papillary muscles No. 0 % (0.0) Excessive fluid withinpericardium No. 0 % (0.0) Ventricular septal defect No. 1 % (1.1) Ventricular walls ofheart thin No. 0 % (0.0) Missingpapillarymuscle(s) No. 1 % (1.1) Two urinarybladders No. 0 % (0.0) Fused ureters No. 0 % (0.0) Horseshoe kidney (same fetus No. 0 % (0.0) Hydronephrosis, right No. 0 % (0.0) Hydronephosis, bilateral No. 0 % (0.0) Liver misshapen No. 0 % (0.0) Liver necrotic (same fetus as a No. 0 % (0.0) Midfacial hypoplasia (repeat) No. 0 % (0.0) Number examined 

### skeletallyf 87 Cervical centra fused No. 0 % (0.0) as a

abov 0 (0.0) 0

(0.0) 0 1 (0.0) (1.0) 0 0 (0.0) (0.0) 0 0 (0.0) (0.0) 0 0 (0.0) (0.0) 0 0 (0.0) (0.0) 0 0 (0.0) (0.0) 

### tbove) 0 0 (0.0) (0.0) 0 0 (0.0) (0.0) 4 0 (3.9) (0.0) 0 0 

### (0.0) (0.0) ,e) 0 0 (0.0) (0.0) 1 0 (1.0) (0.0) 93 87 0 0 (0.0) (0.0) 0 1 0

(0.0) (1.7) (0.0) 0 (0.0) 0 (0.0) 3 

### (3.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.2) 1 

### (1.2) 0 (0.0) 0 (0.0) 0 (0.0) 71 0 (0.0) Cervical centramalaligned No. 0 0 0 0 % (0.0) (0.0) (0.0) (0.0) Cervical centra irregularly shaped (same fetus as above) No. 0 0 0 0 % (0.0) (0.0) (0.0) (0.0) Cervical arches irregularly shaped (same fetus as above) No. 0 0 0 0 % (0.0) (0.0) (0.0) (0.0) Cervical vertebrae malaligned (same fetus as above) No 0 0 0 0 % (0.0) (0.0) (0.0) (0.0) Caudal vertebrae fused (same fetus as above) No. 0 0 0 0 % (0.0) (0.0) (0.0) (0.0) Cervical rib (bilateral) No. 0 0 0 0 % (0.0) (0.0) (0.0) (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.7) 1 (1.7) 1 (1.7) 0 (0.0) 1 

### (1.7) 1 

### (1.7) 1 

### (1.7) 0 

### (0.0) 49 1 (2.0) 1 (2.0) 1 (2.0) 1 (2.0) 1 

### (2.0) 1 

### (2.0) 1 

### (2.0) 0 (0.0) 1 

### (5.0) 0 (0.0) 1 

### (5.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 20 0 

### (0.0) 0 

### (0.0) 0 

### (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 0 (0.0) (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 

### (9.1) 0 (0.0) 0 (0.0) 1 

### (4.5) 22 0 (0.0) 0 

### (0.0) 0 

### (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 

### (0.0) 1 

### (4.5) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 22 0 (0.0) 0 (0.0) 0 

### (0.0) 0 (0.0) 0 (0.0) 0 

### (0.0) 0 

### (0.0) 0 1 (0.0) (6.7) 0 (0.0) 0 (0.0) 2 (10.5) 0 (0.0) 0 

### (0.0) 0 (0.0) 0 (0.0) 1 (5.3) 1 

### (5.3) 0 

### (0.0) 0 (0.0) 0 (0.0) 189 0 (0.0) 0 

### (0.0) 0 

### (0.0) 0 

### (0.0) 0 O 

### (0.0) 0 

### (0.0) 0 

### (0.0) 0 

### (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (6.7) 1 

### (6.7) 1 

### (6.7) 0 (0.0) 1 (6.7) 1 

### (6.7) 1 

### (6.7) 0 (0.0) 15 1 (6.7) 1 (6.7) 1 (6.7) 1 

### (6.7) 1 

### (6.7) 1 

### (6.7) 1 

### (6.7) 63TYL ETAL.

Table 20. (continued) Fetusesb Littersc 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE Duplication ofclavicle No. 0 0 0 1 0 0 0 0 1 0 So (0.0) (0.0) (0.0) (1.4) (0.0) (0.0) (0.0) (0.0) (5.6) (0.0) Missing cervical centra No. 0 0 0 1 0 0 0 0 1 0 % (0.0) (0.0) (0.0) (1.4) (0.0) (0.0) (0.0) (0.0) (5.6) (0.0) Thoracic centra malaligned No. 0 0 1 0 0 0 0 1 0 0 % (0.0) (0.0) (1.1) (0.0) (0.0) (0.0) (0.0) (4.5) (0.0) (0.0) Thoracic centra fused (same as fetus above) No. 0 0 1 0 0 0 0 1 0 0 % (0.0) (0.0) (1.1) (0.0) (0.0) (0.0) (0.0) (4.5) (0.0) (0.0) Thoracic arches malaligned (same as fetus above) No. 0 0 1 0 0 0 0 1 0 0 So (0.0) (0.0) (1.1) (0.0) (0.0) (0.0) (0.0) (4.5) (0.0) (0.0) Thoracic-12 fused to lumbar-1 centra and arch No. 0 1 0 0 0 0 1 0 0 0 % (0.0) (1.1) (0.0) (0.0) (0.0) (0.0) (4.5) (0.0) (0.0) (0.0) Rib branched No. 1 0 0 0 0 1 0 0 0 0 % (1.1) (0.0) (0.0) (0.0) (0.0) (5.0) (0.0) (0.0) (0.0) (0.0) All ribs poorly developed No. 0 0 0 0 1 0 0 0 0 1 % (0.0) (0.0) (0.0) (0.0) (2.0) (0.0) (0.0) (0.0) (0.0) (6.7) 

### Total malformations 

### Externald No. 0 1 1 0 3 0 1 1 0 2 % (0.0) (0.5) (0.5) (0.0) (2.8) (0.0) (4.5) (4.5) (0.0) (13.3) Viscerale No. 2 6 3 11 6 2 4 3 7 5 % (2.1) (5.9) (3.0) (13.6) (10.2) (10.0) (18.2) (13.6) (36.8) (33.3) 

### Skeletalf No. 1 1 1 2 4 1 1 1 2 3 % (1.1) (1.1) (1.1) (2.8) (8.2) (5.0) (4.5) (4.5) (11.1) (20.0) 

### Ibtal No. 3 7 5 13 10 3 5 5 7 7 % (1.7) (3.6) (2.7) (8.6) (9.3) (15.0) (22.7) (22.7) (36.8) (46.7) 

## aA single fetus maybe 

### represented more than once in 

### listing individual defects. 

## bOnly live fetuses were examined formalformations. 

## CIncludes litters with one ormore malformed fetuses. 

### dAll fetuses were examined 

### externally. 

### 'Approximately 50% of each litter were examined 

### viscerally (3), and for soft tissue craniofacial malformations. 

### 'Approximately 50% of each litter were examined for skeletal malformations after 

### staining with Alizarin Red S. 

### 90ne litter contained 

### only one live fetus. 

### By convention this fetus was 

### subjected to visceral and craniofacial examination. p < 0.05 versus 

### controls, Fisher's exact test 

### (two-tailed).

Table 21. Variations observed in NZW rabbit fetuses exposed to EGBE in 

### utero.a

Fetusesb Littersc 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE Number examined 

### externallyd 181 195 187 152 108 20 22 22 19 15 

### Ecchymoses, limbs No. 10 2 13 5 15 3 2 6 4 3 % (5.5) (1.0) (7.0) (3.3) (13.9) (15.0) (9.1) (27.3) (21.1) (20.0) 

### Ecchymoses, head No. 11 6 20 14 6 4 3 9 7 4 % (6.1) (3.1) (10.7) (9.2) (5.6) (20.0) (13.6) (40.9) (36.8) (26.7) 

### Ecchymoses, trunk No. 3 3 18 4 8 3 3 9 3 4 % (1.7) (1.6) (9.6) (2.6) (7.4) (15.0) (13.6) (40.9) (15.8) (26.7) 64EGBE INHALATION TERATOLOGY STUDY

Table 21. (continued) 

### Fetusesb Littersc 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE Placental membrane 

### filled with red fluid No. 0 0 0 % (0.0) (0.0) (0.0) Placental membrane filled withyellow fluid No. 0 0 0 % (0.0) (0.0) (0.0) Right hind leg extended No. 0 1 2 % (0.0) (0.5) (1.1) 

### Right hind 

### leg inverted No. 0 0 1 % (0.0) (0.0) (0.5) Umbilical 

### cord split No. 0 1 0 % (0.0) (0.5) (0.0) Number examined 

### viscerally 94 102 100 

### Spleenmisshapen No. 0 1 0 % (0.0) (1.0) (0.0) Accessory spleen No. 1 3 1 % (1.1) (2.9) (1.0) 

### Spleen short No. 0 0 0 % 

### (0.0) (0.0) (0.0) Redmasses attached to gall 

### bladder membrane No. 3 6 2 % (3.2) (5.9) (2.0) Liver discolored No. 1 0 0 % (1.1) (0.0) (0.0) Liver nodule No. 1 0 0 % (1.1) (0.0) (0.0) 

### Liver, reticular pattern No. 1 0 0 % (1.1) (0.0) (0.0) 

### Stomach

### wall, red in color No. 0 0 0 % (0.0) 

### (0.0) (0.0) Excessive fat No. 1 0 0 % (1.

### 1) (0.0) 

### (0.0) Thymus hemorrhagic No. 0 2 0 % (0.0) (2.0) (0.0) 

### Body cavity 

### filled with

### milkywhite fluid No. 1 0 0 % (1.

### 1) (0.0) (0.0) 

### Kidney

### discoloration, left

### milkywhite fluid No. 0 0 1 % 

### (0.0) (0.0) 

### (1.0) Renal 

### pelvis dilation No. 0 0 1 % 

### (0.0) (0.0) (1.0) Heart indented and/or darkened at apex No. 0 1 % (0.0) (1.0) Papillarymuscle(s) in right ventricle shortened No. 3 1 % (3.2) (1.0) Papillarymuscle(s) in left ventricle shortened No. 0 0 % (0.0) (0.0) 1 

### (1.0) 0 

### (0.0) 0 

### (0.0) 2 0 0 (1.3) (0.0) (0.0) 1 0 0 (0.7) (0.0) (0.0) 0 0 0 (0.0) (0.0) (0.0) 0 0 0 (0.0) (0.0) (0.0) 0 0 0 (0.0) (0.0) (0.0)

81 59 20 0 1 0 (0.0) (1.7) (0.0) 1 0 1 (1.2) (0.0) (5.0) 1 0 0 (1.2) (0.0) (0.0) 1 2 3 (1.2) (3.4) (15.0) 0 0 1 (0.0) (0.0) (5.0) 0 0 1 (0.0) (0.0) (5.0) 0 0 1 (0.0) (0.0) (5.0) 0 1 0 (0.0) (1.7) (0.0) 0 0 1 (0.0) (0.0) (5.0) 2 0 0 (2.5) (0.0) (0.0) 0 0 1 

### (0.0) 

### (0.0) (5.0) 1 0 0 (1.2) (0.0) 

### (0.0) 0 0 0 

### (0.0) (0.0) (0.0) 1 2 0 

### (1.2) (3.4) (0.0) 2 1 3 (2.5) (1.7) (15.0) 3 0 0 

### (3.7) (0.0) (0.0) 0 (0.0) 0 (0.0) 1 

### (4.5) 0 (0.0) 1 

### (4.5)

22 1 

### (4.5) 2 

### (9.1) 0 (0.0) 5 (22.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 

### (9.1) 0 

### (0.0) 0 

### (0.0) 0 (0.0) 1 (4.5) 1 (4.5) 0 

### (0.0) 0 1 0 (0.0) 0 (0.0) 1 (4.5) 1 

### (4.5) 0 (0.0)

22 0 (0.0) 1 

### (4.5) 0 (0.0) 2 

### (9.1) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 

### (0.0) 1 (4.5) 1 (4.5) 1 

### (4.5) 0 

### (0.0) 0 

### (0.0) 

### (5.3)

1 

### (5.3) 0 

### (0.0) 0 

### (0.0) 0 

### (0.0)

19 0 

### (0.0) 1 

### (5.3) 1 

### (5.3) 1 

### (5.3) 0 

### (0.0) 0 

### (0.0) 0 

### (0.0) 0 

### (0.0) 0 

### (0.0) 1 

### (5.3) 0 

### (0.0) 1 

### (5.3) 0 

### (0.0) 1 

### (5.3) 2 

### (10.5) 2 

### (10.5) (0.0)

0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

15 1 

### (6.7) 0 (0.0) 0 

### (0.0) 2 

### (13.3) 0 (0.0) 0 (0.0) 0 (0.0) 1 

### (6.7) 0 (0.0) 0 

### (0.0) 0 

### (0.0) 0 

### (0.0) 0 

### (0.0) 2 

### (13.3) 1 

### (6.7) 0 

### (0.0) 65TYL ETAL.



### Table 21. 

### (continued) 

### Fetuses' Littersc 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm 0 ppm 25 ppm 50 ppm 100 ppm 200 ppm EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE EGBE Red 

### markings on heart No. Atria engorged No. Atria discolored No. %I Fetal atelectasis No. 

### Pulmonary artery 

### largei No. %I Granulation in nasal pas No. % White substance in nasa No. % Cochlea dark No. % Indentations on soft an No. 

### Gap between skull 

### plate No. Lateral ventricles dilate No. Number examined 

## skeletally' Rudimentary rib 

### lumba: No. 

### Rudimentary rib lumba No. Extra rib 

### lumbar-1, uni No. Extra rib 

### lumbar-1, bik No. 

### Sternebra #5, 

### poorlyo: No. 

### Sternebra 

### #6, 

### poorly 

### o: No. %I 

### Sternebra 

### #5, unossifie No. % 

### Sternebra 

### #6, unossifie No. surface

1 0 0 0 0 1 0 (1.1) (0.0) (0.0) (0.0) (0.0) (5.0) (0.0) 0 0 0 0 1 0 0 (0.0) (0.0) (0.0) (0.0) (1.7) (0.0) 

### (0.0) 0 0 0 0 1 0 0 (0.0) (0.0) (0.0) (0.0) (1.7) 

### (0.0) (0.0) 19 17 13 5 9 10 13 '20.2) (16.7) (13.0) (6.2) (15.3) (50.0) 

### (59.1) r than normal 2 0 1 0 1 1 0 (2.1) (0.0) (1.0) (0.0) (1.7) (5.0) (0.0) 3sage and/or on 

### palate 2 6 9 6 3 2 4 (2.1) (5.9) (9.0) (7.4) (5.1) 

### (10.0) 

### (18.2) 1 passage 4 1 2 0 0 2 1 (4.3) (1.0) (2.0) (0.0) 

### (0.0) (10.0) 

### (4.5) 4 3 3 11 0 2 2 (4.3) 

### (2.9) (3.0) (13.6) 

### (0.0) (10.0) 

### (9.1) d/or hard 

### palate 0 1 0 0 0 0 1 (0.0) (1.0) 

### (0.0) (0.0) (0.0) 

### (0.0) (4.5) Ls and skin 0 6 0 1 0 0 1 

### (0.0) (5.9) (0.0) 

### (1.2) (0.0) 

### (0.0) (4.5) d 

### (cerebrum) 13 5 7 12 5 8 4 

### (13.8) (4.9) (7.0) (14.8) (8.5) (40.0) 

### (18.2) 87 93 87 71 49 20 22 

### r-1, unilateral 18 22 12 11 6 14 15 

### (20.7) (23.7) (13.8) 

### (15.5) 

### (12.2) (70.0) (68.2) 

### r-1, bilateral 9 10 4 3 0 6 8 

### (10.3) (10.8) 

### (4.6) 

### (4.2) (0.0) (30.0) 

### (36.4) ilateral 10 16 11 6 7 10 10 (11.5) 

### (17.2) (12.6) 

### (8.5) (14.3) 

### (50.0) (45.5) ateral 36 37 49 35 34 16 16 (41.4) 

### (39.8) (56.3) 

### (49.3) 

### (69.4) (80.0) (72.7) ssified 32 49 42 30 30 15 21 

### (36.8) 

### (52.7) 

### (48.3) 

### (42.3) 

### (61.2) (75.0) 

### (95.5) ssified 35 26 28 17 14 15 15 

### (40.2) 

### (28.0) 

### (32.2) 

### (23.9) (28.6) (75.0) 

### (68.2) d11 6 6 10 7 6 3 

### (12.6) (6.5)( 

### 6.9) 

### (14.1) (14.3) 

### (30.0) (13.6) ,d 7 9 9 3 0 6 5 

### (8.0) 

### (9.7) 

### (10.3) 

### (4.2) (0.0) (30.0) 

### (22.7) 0 0 0 

### (0.0) 0 

### (0.0) 0 

### (0.0) 8 

### (36.4) 1 

### (4.5) 3 

### (13.6) 2 

### (9.1) 2 

### (9.1) 0 

### (0.0) 0 

### (0.0) 4 

### (18.2) 22 11 

### (50.0) 3 

### (13.6) 10 

### (45.5) 19 

### (86.4) 19 

### (86.4) 14 

### (63.6) 4 

### (18.2) 5 

### (22.7) 

### (0.0)

0 

### (0.0) 0 

### (0.0) 4 

### (21.1) 0 

### (0.0) 4 

### (21.1) 0 

### (0.0) 3 

### (15.8) 0 

### (0.0) 1 

### (5.3) 9 

### (47.4) 189 7 

### (38.9) 3 

### (16.7) 6 

### (33.3) 17 

### (94.4) 14 

### (77.8) 9 

### (50.0) 6 

### (33.3) 2 

### ( 1.

### 1) 

### (0.0)

1 

### (6.7) 1 

### (6.7) 5 

### (33.3) 1 (6.7) 1 

### (6.7) 0 

### (0.0) 0 

### (0.0) 0 

### (0.0) 0 

### (0.0) 2 

### (13.3) 15 6 

### (40.0) 0* 

### (0.0) 7 

### (46.7) 14 

### (93.3) 14 

### (93.3) 9 

### (53.3) 4 

### (26.7) 0* 

### (0.0) 

## aA single fetus maybe 

### represented more than once in 

### listing individual defects. 

### bOnly

### live fetuses were examined for defects. 

## CIncludes litters with one or more affected fetuses. 

## dAll fetuses were 

### examined

## externally. 

## eApproximately 50% ofeach litter were examined 

### viscerally (3), and for soft tissue craniofacial defects 

### (4,6). 

### fApproximately 50% of each litter were examined for skeletal defects after 

### staining with Alizarin Red S. 

### Only those 

### parameters whose incidence was 

### significantly different from that ofcontrols 

### (p < 

### 0.05, Fisher's exact 

### test) and related 

### parameters are indicated. 

### 90ne litter 

### contained only one live fetus. 

### By convention this fetus was 

### subjected to visceral 

### and craniofacial examination. 

### *p < 0.05 versus 

### controls, Fisher's exact test 

### (two-tailed). 66EGBE INHALATION TERATOLOGY STUDY 67 

### spleenweightsand

### relativekidneyweightat200ppmin rats

### alsoindicate

### stimulationof

### hematopoiesis. Thatthe 

### homeostatic mechanisms of compensation were not 

### entirelysuccessful is

### indicatedbythe significantreduction in mean 

### corpuscular hemoglobin concentration seen 

### at 100 

### and 200 

### ppm. The

### earlyratconceptus appeared

### vulnerabletoinsult 

### byEGBE 

### since 9

### of25damspregnantat

### sacrificeat200 ppm 

### had totally 

### resorbed litters, 7 of 9 

### detected only after 

### staining of the 

### uteri. This embryotoxic effect is 

### responsible for the 

### significant elevations 

### in nonviable 

### implants and resorptions and the 

### reduction in viable (i.e., 

### live) fetuses at 

### scheduled necropsy 

### at 200 ppm, 

### andoccurredin

### thepresence

## ofmaternal toxicity. Fetal 

## body 

## weights were 

### unaffected 

## by treatment 

### but 

## fetotoxicity was 

### indicated by the increased incidence of poorly

### ossifiedor

### unossified

### skeletalelementsseenin

### gd 21 

### fetuses. 

### Theseskeletaldistrictswere

### thoseexamined 

## byAlivertiet

### al. (1

### 6), who

## suggestedthatretardationof skeletal 

### ossification, especially on gd 21 in 

### rats, provided

### areliable indexof

### delayeddevelopment interato

### genicitystudies. Khera 

### (17) has interpreted 

### reductions in ossification as 

### indications of toxicity and suggested 

### that the 

## gd 21 

### fetal 

## skeleton is the most 

### uniform and 

### therefore the most 

### appropriate to 

### examine. Khera (17) 

## included as 

## fetal "aberrations" 

## retardations, variations 

### and deviations. He 

### includes supernu

### merary 

### ribs as a 

### fetal 

## variation, a 

## designation also followed in 

### this 

## studywhich demonstrated this 

## finding across

### allgroups. Heart

### andgreat

### vessel

## malformations, seen 

### with 

### increased 

## frequency after exposure of rats to 

## ethylene glycol 

## monomethyl and 

## monoethyl ether 

### (18,19) were not 

### seen 

### with 

### increased 

## frequency in 

### this 

## study. The lack of 

## teratogenicity in 

### the presence 

### of

## maternal 

## toxicity in rats 

## after exposure to 

## EGBE, seen 

### in

### this 

## study, was 

## also 

## reported

## brieflybyNelson et 

### al. (18). New

### ZealandWhite

### rabbit does 

### exhibitedtoxicityto 

### EGBE at 200 

### ppm. 

## Treatment-related increases in 

### maternal deaths and spontaneous 

## abortions, 

## depressed 

## body weight during the exposure 

## period and some 

## relatively nonspecific clinical 

## signs were 

### observed at 

## this exposure concentration. 

## Embryotoxicity was 

### indi

## cated

## byreduced

## graviduterine

## weight and a 

### concomitant 

### reduction in 

### total and viable fetuses at 

### scheduled 

## sacrifice 

## onlyat 

### 200 

### ppm. No 

### evidence offetal 

## toxicity was 

## observed at 

### any 

### exposure 

## concentration 

## (i.e., no 

### treatment-related reduction in 

### fetal 

## body 

## weight, no 

### increase in 

## malformations or 

## variations, 

## including re

### ducedossificationin

### skeletal

## elements). 

## Supernumerary ribs, 

## presenting as 

## rudimentary or extra 

## ribs, and 

### observed across 

### all groups, is the most 

## commonly occurring skeletal variation in New 

### Zealand White 

### rabbits 

## (20). 

# Conclusions Thepresent 

## studyindicates that inhalation exposure to 

## EGBE at 

### 100 

### ppm 

## (rats) or 

### 200 ppm 

## (rats and 

## rabbits) 

## duringorganogenesis

### resultsin

### maternaltoxicity

### andtoxicitytotheconceptus 

### (embryotoxicityinrats 

### and

### rabbits; 

### fetotoxicity inrats), 

### but no 

## teratogenicity. Exposure to 100ppm (rabbits), 50, or25

### ppm (rats and 

### rabbits) results in 

### no observable 

### maternal, embryo or 

## fetal 

## toxicity. 

# Addendum Analysis of the 

### data generated 

### from the five to six rats

### perexposure

### groupexposedone

### additionalday 

### (gd 16) 

### to EGBE 

### indicated the same 

### profile of 

### maternal 

### toxicity: clinical signs, decreased 

### weight and 

### weight 

### gains

## duringexposure, elevatedrelativespleen

### weights, 

## decreased food andwater

### consumptionand evidence of anemia. 

### Withthe 

### smallnumberof

### litters in

### each

### group, no

### alterations in

### status ofimplantationsiteswere 

### seen. 

## Only one 

## fetal malformation was 

### observed: 

## missing medianlunglobeat 

### 25ppm. Fetal

### variations seen

### were 

## similarto

### thosereportedherein, i.e., 

### treatment-related reduced ossification of skeletal 

### districts, and 

### rudimentaryrib

### and/orbone 

### islandat the 

### first lumbarvertebra 

### distributed across 

### all treatment 

### groups. This work was sponsored by the Chemical Manufacturers Associ

### ation, 

### Glycol Ethers Program Panel. The companies participating in the program are Dow Chemical, USA; ICI Americas, 

### Inc.; Olin 

### Corporation; PPG Industries; Shell Chemical 

### Company; Tennessee 

### Eastman Company; Texaco Chemical Company; and Union Carbide 

### Corporation. The contributions ofthe followingBushyRunResearch Center personnel who provided expert technical assistance are gratefullyacknowledged. TeratologyPersonnel: K. F. D'Aurora, D. L. Eisler, G. J. Haines, L. A. 

### Meckley, T. A. Savine, C. E. Baylock, PJ. 

### Guzzie; InhalationPersonnel: Dr. W M. Snellings, WJ. Kintigh, F L. Howard, M. L. Steel, D. W Fait, D. J. Nachreiner; Clinical 

### ChemistryPersonnel: Dr. C. Troup, S. J. Kozbelt, P J. Brown, D. V lurley, J. L. Beskitt, C. M. Anuszkiewicz, M. Tallant, W C. Hengler; 

### QualityAssurance Personnel: L. J. Calisti, J. R. Bernard and W J. 

### Conroy; Animal 

### Care Personnel: M. J. Cardella, Supervisor.

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