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

by Rochelle W. 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 ether vapors by inhalation on gestational days 6 through 15 (rats) or 6 through 18 (rabbits) at

concentrations of 0, 25, 50, 100 or 200 ppm. The animals were sacrificed on gestational day 21 (rats) or 29

(rabbits). In rats, exposure to 200 or 100 ppm resulted in maternal toxicity (clinical signs, decreased body

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

decreased number of viable implantations per litter) and fetotoxicity (reductions in skeletal ossification).

No increase in fetal malformations was observed in any exposure group relative to controls. At 50 or 25 ppm,

there was no maternal, embryo or fetal toxicity (including malformations) in rats. In rabbits, exposure to

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

fetotoxicity was seen. No treatment-related increases in fetal malformations or variations were seen at any

exposure concentration tested. There was no evidence of maternal, embryo, or fetal toxicity (including

malformations) at 100, 50 or 25 ppm in rabbits.

Introduction

As part of a program evaluating the potential toxicity

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

Program Panel. The initial rat study employed exposure

of timed-pregnant Fischer 344 rats to EGBE at 300,

200, or 100 ppm or to 0 ppm (air control) for 6 hr per day

during gestational days 6 through 15. Maternal clinical

signs, food and water consumption, and body weights

were recorded. Animals were sacrificed on gestational

day 21 after obtaining blood samples for clinical hematology. 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.

tPresent address: Ayerst Research Laboratories, Chazy, NY 12921.

Exposures to 100, 200 and 300 ppm EGBE produced

statistically significant reductions in maternal body

weight gain and food consumption. Water consumption

was reduced during exposure at 300 or 200 ppm. Signs

of maternal toxicity (decrease in red blood cell count;

ocular, nasal, and urogenital discharges; hypoactivity;

etc.) were observed predominantly at 300 and 200 ppm

of EGBE. One animal exposed to 300 ppm EGBE died.

Exposures to 300 ppm EGBE resulted in an increased

incidence of embryo and fetal mortality (16 of 23 females

pregnant at sacrifice had 100% resorptions at 300 ppm).

No treatment-related fetal external or skeletal malformations 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

examination of fetuses revealed a concentration-related

incidence of enlarged lateral ventricles of the brain,
TYL ET AL.

considered a variation indicative of embryo/fetal toxicity,

at all concentrations of EGBE. Results from the skeletal examinations included concentration-related increases in the incidence of extra ribs (fourteenth,

rudimentary, considered a variation), significant only at

300 ppm, and in the incidence of reduced ossification in

those skeletal districts indicative of fetal toxicity at 200

and 300 ppm, relative to controls.

Since the exposure employed in this initial study

produced maternal and fetal toxicity at all exposure

concentrations, did not identify a "no observable effect

level" (NOEL), and excessive embryo/fetal loss was

observed at 300 ppm, another rat study and a

teratogenic evaluation of EGBE in another mammalian

species were undertaken. This report details the evaluation 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

placed in 1 gal glass bottles and blanketed with nitrogen.

The purity and stability were verified by gas chromatographic 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) equipped with a flame ionization detector was used

to monitor the EGBE vapor concentrations in the

chambers. The GC column was 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 automatic gas sampling system (station and valve programmer units and a gas sampling valve) were used

for the analyses.

Calibration of the gas chromatograph was done with

liquid injections of standard solutions of EGBE in

hexanes prepared volumetrically. The series of standard

solutions encompassed the entire range of vapor concentrations generated in the exposure chambers. A linear

calibration curve was obtained when areas (integration

counts) were plotted versus the gas equivalent concentrations of the standards. The calibration factor (KF)

was calculated as the ratio between area A and concentration C:

KF = A/C

The mean KF for all valid injections of standards was

used in subsequent microprocessor calculations. This

calibration factor was checked at least once each week

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 concentration calculated from the amount of test material

delivered and the chamber airflow during the exposure

period) were also calculated for each chamber.

Liquid EGBE was metered from a piston pump into a

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 by passage of conditioned air through

the evaporator. Chamber atmospheres containing EGBE

were filtered before leaving an exhaust stack.

The five chambers employed in this study were

rectangular in shape, constructed of glass and stainless

steel (Wahmann Manufacturing Company, Timonium,

MD), and measured approximately 2.1 m x 1 m x 2.1 m

(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 Magnehelic Pressure Gauge. Excellent chamber

distribution for EGBE vapors has been demonstrated.

The coefficient of variation of concentrations measured

at eight locations in the chamber in the breathing zones

of the animals was 1%.

Airflow in each chamber was approximately 1000

L/min (14 air changes per hour) with a tgg (theoretically

derived time required for the chamber to reach 99% of

the equilibrium concentration) of approximately 20 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.

Within each chamber, the animal cages were rotated

daily to compensate for any possible, but undetected,

variation in chamber exposure conditions (i.e., concentration, temperature, relative humidity).

Target concentrations were 0, 25, 50, 100 and 200

ppm. These exposure concentrations were based on a

range-finding study in pregnant rats and rabbits which

employed concentrations of 450, 200, 75 or 0 ppm, and

an initial rat teratology study with concentrations of

300, 200, 100 and 0 ppm. Maternal mortality was seen at

450 ppm (100% in both species) and maternal toxicity

was seen at all exposure concentrations employed

(except at 75 ppm for rabbits) in the two studies. Since a

48
EGBE INHALATION TERATOLOGY STUDY

no-observable effect level (NOEL) could not be ascertained from these studies, the current exposure concentrations 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 old upon 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 of selected organs and to serum

viral antibody examination for sialodacryoadenitis. Rats

were housed in stainless steel wire-mesh cages with

food (Certified Ground Rodent Chow, Ralston Purina

Co., Richmond, IN) and water (Municipal Authority of

Westmoreland County, Greensburg, PA) available ad

libitum except during exposures. Deotized Animal

Cage Board (Shepherd Specialty Papers, Inc., Kalamazoo, MI) was placed beneath the cages and changed

daily. Animals were kept on a 12-hr photoperiod and

room temperatures and humidity were recorded continuously (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 steel wire-mesh cages and the paperboard beneath

the cages was checked twice daily for dropped copulation plugs. Successfully mated (plug-positive) females

were housed singly for the duration of the study. The

day a copulation plug was found was designated gestational day (gd) 0. Thirty-six plug-positive females were

randomly assigned to each experimental group.

For exposures, plug-positive females were transferred, one per cage, to stainless steel wire-mesh cages

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 each exposure, animals were returned to 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 body weights were taken on gd 0, 6, 9,

12, 15 and 21.

At scheduled sacrifice on gd 21, rats were anesthetized by carbon dioxide inhalation for collection of blood

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. They were quarantined for 2 weeks, during which

they were examined by a veterinarian and fecal samples

taken for detection of intestinal 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 humidity were monitored.

Rabbits were bred, using in-house breeding colony

males. The date of copulation was designated gd 0.

Twenty-four mated females were randomly assigned 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). Maternal body weights were taken on gd 0, 6, 9, 12,

15, 18, 21 and 29.

At scheduled sacrifice on gd 29, blood was collected

from each doe after she was placed in a rabbit restrainer.

Following blood collection, rabbits were sacrificed by

cervical dislocation.

Thoracolaparotomy, Maternal and

Fetal Examinations

The maternal body cavities were opened by midline

thoracolaparotomy. The gravid uteri, ovaries (including

corpora lutea), cervices, vagina, and peritoneal and

thoracic cavities were examined grossly. Ovarian corpora lutea of pregnancy were counted. Maternal liver,

kidney, thymus, spleen and uterus weights were determined. The uteri were immediately ligated at their

cervical end to prevent the expulsion of conceptuses by

myometrial peristalsis. Uteri were externally examined

for signs of hemorrhage. The ligated uteri were removed from the peritoneal cavity and dissected longitudinally to expose their contents. All live and dead

fetuses, placentae, and resorption sites were noted and

recorded. Uteri from females that appeared nongravid

were placed in a 10% ammonium sulfide solution for

detection of early resorptions (2).

Each rabbit fetus was euthanized immediately upon

removal from the uterus by intraperitoneal injection of

sodium pentobarbital. All live fetuses were weighed and

sexed. All fetuses were examined for external malformations including cleft palate. One-half of the fetuses

(selected at random) in each litter were examined

for thoracic and peritoneal visceral abnormalities by

modification of methods described by Staples (3). These

fetuses were,decapitated and their heads were fixed in

Bouin's solution for examination of craniofacial structures by sectioning methods modified from Wilson (4,5)

and van Julsingha and Bennett (6). The remaining

49
TYL ET AL.

fetuses in each litter were eviscerated, fixed in 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 from female rats

via retro-orbital bleeding immediately prior to sacrifice.

Approximately 700 ,uL of blood was collected into B-D

Microtainer Brand capillary whole blood collection tubes

containing sodium heparin as anticoagulant (BectonDickinson Co., Rutherford, NJ) for the osmotic fragility

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 hematologic evaluation were obtained from rabbit does

via cardiac puncture immediately prior to sacrifice.

Approximately 2.0 mL of blood was collected into tubes

with sodium heparin as anticoagulant (Vacutainer Brand,

Becton-Dickinson Company, Rutherford, NJ) for the

osmotic fragility assay. Approximately 2.0 mL of blood

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 Chemistry System (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 measured on all rat and rabbit samples: erythrocyte count,

hemoglobin, and hematocrit. These analyses were performed on a Coulter Counter Model S-Plus IV (Coulter

Electronics, Inc., Hialeah, FL). Commercially available

quality control samples (4C Plus II Coulter Counter

Cell Control, Coulter Diagnostics, Hialeah, FL) were

run prior to the rat and rabbit samples. Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentrations (MCHC) were calculated from the measured

hematologic values.

Statistical Analyses

The unit of comparison 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 indicated homogeneous variances and the

ANOVA was significant, the pooled t-test was used for

pairwise comparisons. When Levene's test indicated

heterogeneous variances, all groups were compared by

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) followed by the 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, temperature and humidity are presented

in Table 1. All EGBE 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 = below minimum detection limit of 2 ppm. cAnalytical/target concentration ratio.

dAnalytical/nominal concentration ratio.

50
EGBE 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 maintained at 40 to 43%.

Rats

The distribution and fate of all plug-positive rats on

study are presented in Table 2. A small number (five or

six) of rats 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 fetal evaluation data were 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 of totally resorbed 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

(gd 9, 12, 15) and at 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 and for the gd

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 they were inadvertently exposed for one additional day (gestational day 16).

bNine dams had only resorption sites at scheduled sacrifice, seven of which were found only after ammonium sulfide staining.

cOne dam had 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: + + + = majority of dams (75% or more) exhibited the clinical sign sometime during the exposure period; + + = approximately

50% of the dams (30-70%) exhibited the clinical sign sometime during the exposure period; + = up to 25% of the dams exhibited the clinical

sign sometime during the exposure period; 0 = no dams exhibited the clinical sign during the exposure period.

51
TYL ET AL.

TIble 4. Maternal weights and weight 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 body weight, 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 body weight 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 body weight = body weight 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 dam per day.

*p < 0.05 compared to control.

tp < 0.001 compared to control.

52
EGBE 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 standard deviation; units are in grams per dam per day; values in parentheses indicate N for each group. Reduced

N values 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.

53
TYL ET AL.

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 for dams at 200 ppm.

Water consumption was elevated relative to controls for

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 hemoglobin concentration (MCHC) was significantly increased. The mean

corpuscular hemoglobin concentration (MCHC) was

significantly reduced at 100 and 200 ppm relative to

controls.

At scheduled sacrifice, maternal gravid uterine weight

was significantly reduced, and absolute and relative

spleen weight and relative kidney weight 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 and the number of nonviable implants, primarily due to the elevated number of early embryonic

resorptions, was elevated at 200 ppm relative to controls.

Preimplantation loss was also elevated, but the difference was not statistically significant. Sex ratio (%

males) and the body weight of male 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 of external, 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. At 200 ppm, there was a significant increase in 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

skeletal elements, e.g., cervical arches, sternebrae 1, 3,

4 and 6 and proximal phalanges in the forelimbs. At 100

ppm, there was a significant increase in the incidence of

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 body weight

(gd 21), 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 body weight.

*p < 0.05 as compared to control.

tp < 0.01 as compared to control.

tp < 0.001 as compared to control.

54
EGBE 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

Sex ratio 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 of which were found to be gravid by ammonium sulfide staining only.

cOne of twenty-two dams had embryonic resorptions, detected only by 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 litter had only male 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

emerges from arch

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)

55
TYL ET AL.

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 of aorta

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 may be represented more than once in listing individual defects.

bOnly live fetuses were examined for malformations.

CIncludes litters with one or more 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 staining with 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 (primarily because at these exposure concen- proximal phalanges of the hindlimb relative to controls.

trations this skeletal element was largely unossified). Rudimentary rib and/or bone island at the first lumbar

56
EGBE 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)

57
TYL ET AL.

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 skeletallyf 106 106 92

Anterior arch of Atlas, 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)

58
EGBE 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 eApproximjately 50% of each litter were 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.

59
TYL ET AL.

Table 15. Maternal weights and weight gain of NZW 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 body weight, 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 body weight 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 body weight = body weight on gd 29 minus gravid uterine weight.

hN = 22- One rabbit died immediately following cardiac puncture on gestational day 29; a final body weight 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.

vertebra was observed in fetuses from all groups with

no treatment-related changes in incidence.

Rabbits

The distribution and fate of all mated New Zealand

White rabbits on study are presented in Table 13. There

was an apparent, but not statistically significant increase in spontaneous abortions and deaths 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 periocular wetness, perinasal

wetness and discharge, red fluid on tray, and stained fur

as possible treatment-related clinical signs.

Reduced maternal 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) indicated no apparent treatment-related changes in any

parameter evaluated. Statistically significant increases

in hemoglobin content and hematocrit were seen at 100

ppm but not at 200 ppm.

60
EGBE INHALATION TERATOLOGY STUDY

The only treatment-related change in maternal organ

weights was a reduction in gravid uterine weight at 200

ppm (Table 18). Evaluation of reproductive parameters

(Table 19) indicated a significant reduction in number of

total implants and viable implants per litter at 200 ppm

relative to controls. There were no treatment-related

effects on the number of nonviable implants, preimplantation loss, percent live fetuses, sex ratio or male

and female fetal body weight per litter.

The type and incidence of malformations and variations observed in rabbit fetuses are presented in Tables

20 and 21, respectively. There was no statistically

significant increase in the number of fetuses or of litters

with one or more affected fetuses with pooled external,

visceral, skeletal or total malformations in any treatment group relative to controls. For individual malformations, there was an increase in the number of

Table 17. Erythrocyte indices for NZW 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 papillary muscles 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

Table 21. There was a significant reduction in unossified

sternebra 6 and in rudimentary rib 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

variations found 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 toxicity and embryotoxicity but no fetal toxicity 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 of anemia confirms and extends the work done

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 in rats at 100 and 200 ppm are consistent with

destruction of mature erythrocytes and release of

immature and/or young erythrocytes into the peripheral

circulation (13,15). Increased absolute and relative

Table 18. Maternal organ weights of NZW 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 body weight

(gd 29), g 3994.4 ± 426.0a 4163.5 ± 445.3 4047.7 ± 154.9 4314.7 ± 502.7 4363.1 t 455.0

Organ weights, 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 body weight.

*p < 0.05 compared to control.

61
TYL ET AL.

Table 19. Reproductive toxicity evaluation of NZW 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

Dead and macerated

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 only nonviable 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 with placenta 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 only male 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)

Severely shortened tail

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

62
EGBE 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 carotid branches 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 constricted pulmonary artery, channel to aorta from rt. ventricle, extra branches off aorta, vestigal rt. ventricle, absent tricuspid

valve and associated papillary muscles

No. 0

% (0.0)

Excessive fluid within pericardium

No. 0

% (0.0)

Ventricular septal defect

No. 1

% (1. 1)

Ventricular walls of heart thin

No. 0

% (0.0)

Missing papillary muscle(s)

No. 1

% (1. 1)

Two urinary bladders

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 centra malaligned

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)

0O

(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)

63
TYL ET AL.

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 of clavicle

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 may be represented more than once in listing individual defects.

bOnly live fetuses were examined for malformations.

CIncludes litters with one or more 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)

64
EGBE 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 with yellow 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

Spleen misshapen

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)

Red masses 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 milky white fluid

No. 1 0 0

% (1. 1) (0.0) (0.0)

Kidney discoloration, left milky white 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)

Papillary muscle(s) in right ventricle shortened

No. 3 1

% (3.2) (1.0)

Papillary muscle(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)

65
TYL ET AL.

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

Fetal

No.

%I

atelectasis

No.

Pulmonary artery largei

Granulation

No.

%I

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, poorly o:

No.

Sternebra #6, poorly o:

Sternebra

No.

%I

#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)

d

11 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 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. eApproximately 50% of each 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 of controls (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).

66
EGBE INHALATION TERATOLOGY STUDY 67

spleen weights and relative kidney weight at 200 ppm in

rats also indicate stimulation of hematopoiesis. That the

homeostatic mechanisms of compensation were not

entirely successful is indicated by the significant reduction in mean corpuscular hemoglobin concentration

seen at 100 and 200 ppm.

The early rat conceptus appeared vulnerable to insult

by EGBE since 9 of 25 dams pregnant at sacrifice at 200

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,

and occurred in the presence of maternal toxicity. Fetal

body weights were unaffected by treatment but fetotoxicity was indicated by the increased incidence of

poorly ossified or unossified skeletal elements seen in gd

21 fetuses. These skeletal districts were those examined

by Aliverti et al. (16), who suggested that retardation of

skeletal ossification, especially on gd 21 in rats, provided a reliable index of delayed development in teratogenicity studies. 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 supernumerary ribs as a fetal variation, a designation also

followed in this study which demonstrated this finding

across all groups. Heart and great 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 briefly by Nelson

et al. (18).

New Zealand White rabbit does exhibited toxicity to

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 indicated by reduced gravid uterine weight and a concomitant reduction in total and viable fetuses at scheduled

sacrifice only at 200 ppm. No evidence of fetal toxicity

was observed at any exposure concentration (i.e., no

treatment-related reduction in fetal body weight, no

increase in malformations or variations, including reduced ossification in 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

The present study indicates that inhalation exposure

to EGBE at 100 ppm (rats) or 200 ppm (rats and

rabbits) during organogenesis results in maternal toxicity and toxicity to the conceptus (embryotoxicity in rats

and rabbits; fetotoxicity in rats), but no teratogenicity.

Exposure to 100 ppm (rabbits), 50, or 25 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 per exposure group exposed one additional day (gd

16) to EGBE indicated the same profile of maternal

toxicity: clinical signs, decreased weight and weight

gains during exposure, elevated relative spleen weights,

decreased food and water consumption and evidence of

anemia. With the small number of litters in each group,

no alterations in status of implantation sites were seen.

Only one fetal malformation was observed: missing

median lung lobe at 25 ppm. Fetal variations seen were

similar to those reported herein, i.e., treatment-related

reduced ossification of skeletal districts, and rudimentary rib and/or bone island at the first lumbar vertebra

distributed across all treatment groups.

This work was sponsored by the Chemical Manufacturers Association, 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 of the following Bushy Run Research

Center personnel who provided expert technical assistance are

gratefully acknowledged. Teratology Personnel: K. F. D'Aurora, D. L.

Eisler, G. J. Haines, L. A. Meckley, T. A. Savine, C. E. Baylock, P J.

Guzzie; Inhalation Personnel: Dr. W M. Snellings, W J. Kintigh, F L.

Howard, M. L. Steel, D. W Fait, D. J. Nachreiner; Clinical

Chemistry Personnel: Dr. C. Troup, S. J. Kozbelt, P J. Brown, D. V

lurley, J. L. Beskitt, C. M. Anuszkiewicz, M. Tallant, W C. Hengler;

Quality Assurance Personnel: L. J. Calisti, J. R. Bernard and W J.

Conroy; Animal Care Personnel: M. J. Cardella, Supervisor.

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1. Carpenter, C. P, Kinkead, E. R., Geary, D. L., Jr., Sullivan L. J.,

and King, J. M. Petroleum hydrocarbon toxicity studies 1.

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