### Environmental Health Perspectives Vol. 57, pp. 69-74, 1984



# Developmental Toxicityof 

# FourGlycol Ethers Applied Cutaneously to 

# Rats

by 

# Bryan D. 

# Hardin,* Phillip 

# T. Goadt and 

# JeAnne R. Burg*

PreviousNIOSHstudiesdemonstratedtheembryo- andfetotoxicityandteratogenicityofethyleneglycol monoethyl ether (EGEE) applied to the shaved skinofpregnant rats. In thepresent studyethylene glycol monoethyl ether acetate (EGEEA), ethylene glycol monobutyl ether (EGBE), and diethylene glycol monoethyl ether (diEGEE) were tested in the same experimental model, using distilled water as the negative control and EGEE as apositive control. Water orundilutedglycols were applied fourtimes daily

ondays 7 to 16 ofgestation tothe shavedinterscapular skinwithanautomatic pipetter. Volumes ofEGEE (0.25 mL), EGEEA (0.35 mL), and diEGEE (0.35 mL) were approximately equimolar (2.6 mmole per treatment). EGBE at 0.35 mL four times daily (approximately 2.7 mmole per treatment) killed 10 of 11 treated rats, and was subsequently tested at 0.12 mL (0.9 mmole) per treatment. EGEE- and EGEEAtreated rats showed a reduction in body weight relative to water controls that was associated with completely resorbed litters and significantly fewer live fetuses per litter. Fetal body weights were also significantly reduced in those groups. Visceral malformations and skeletal variations were significantly increased in EGEE and EGEEA groups over the negative control group. No embryotoxic, fetotoxic, or teratogenic effects were detected in the EGBE- or diEGEE-treated litters.



# Introduction

The

### alkylether

### derivatives of

### ethyleneand

### diethylene gylcol are an

### important 

### class ofsolvents

### with numerous 

### applications in consumer 

### products as 

### well as 

### in indus

### trial processes. 

### Severalmembers 

### ofthischemical 

### family have 

### recently been shown to 

### be potent 

### reproductive toxins in 

### several animal 

### species, as 

### reviewed in 

### detail elsewhere 

### (1,2). Teratogenicity has 

### been 

### reported for 

### ethylene glycol monomethyl ether 

### (EGME) in mice 

### (3) and rats 

### (4); for 

### ethylene 

### glycol dimethyl ether 

### (EGdiME) in 

### mice (5); 

### for 

### ethylene glycol monoethyl ether 

### (EGEE) in 

### rabbits 

### (6) and rats 

### (6-8); and for 

### ethylene glycol monoethyl ether acetate 

### (EGEEA) in

rats 

### (4). Evidence of 

### testicular 

### toxicity has 

### been re

### ported for 

### EGME in 

### mice 

### (9-11), rats 

### (10-12), and 

### rabbits 

### (12); for 

### ethylene glycol monomethyl ether
acetate 

### (EGMEA) in 

### mice 

### (9); for 

### EGEE in 

### mice 

### (9),
rats 

### (8), and 

### dogs 

### (8); for 

### EGEEA in 

### mice 

### (9); and for 

### diethylene 

### glycol dimethyl ether 

### (diEGdiME) in 

### mice 

### and rats 

### (13). In 

### contrast, 

### ethylene glycol monobutyl ether 

### (EGBE) and 

### diethylene 

### glycol monoethyl ether 

### (diEGEE) both have been 

### tested once 

### by inhalation

exposure 

### for 

### teratogenicity (4) with 

### negative results. 

### EGBE and 

### ethylene 

### glycol monophenyl ether 

### (EGPhE)

*Department of Health and Human Services, U. S. Public Health Service, Centers for Disease Control, National Institute forOccupational Safety and Health, Division of Biomedical and Behavioral Science, 4676 Columbia Parkway, Cincinnati, OH 45226. tIntoxLaboratories, Inc. 914BarberStreet, LittleRock, AR72203. 

### have been 

### tested for 

### testicular toxicity 

### with 

### negative results (9). The 

### glycol ethers do not have 

### especially 

### high vapor 

### pressures (14), but they

### penetrate 

### the skin

### readily and

are 

### commonlyusedin

### applications that are

### conducive to 

### skin contact. 

### Hardin et 

### al. 

### (7) 

## appliedundiluted 

### EGEE
to 

### the skin ofpregnant rats to 

## investigate its 

## potential for 

### developmental toxicity by this route 

### of exposure. 

### When 0.5 

### mL was 

## applied fourtimes 

## daily on

## days 7 to 

### 16 

### of 

### gestation, all pregnant 

### females had 

## completely resorbed 

### litters. 

## Treatment with 0.25 mL on 

### the same 

### schedule induced

### significantembryotoxic, 

## fetotoxic, and 

### teratogenic effects (7). The 

### cardiovascular system was 

### the 

### primary target for 

### visceral 

### malformations, as 

### had 

### been 

## previously 

## reported in rats 

## (4,6) and rabbits 

## (6) exposedto 

### EGEE 

### byinhalation. 

### Thepresent 

## studywas 

### conductedto

### evaluatethe

## reproductiveand

## developmental

## toxicityofthreeother

## alkylglycolethers in

### the same 

## 2utaneous exposure 

### model.



# Methods and 

# Materials

Purified 

### glycol ethers were 

### purchased from Fisher 

### Scientific: 

## EGEE, Catalog No. 

## E-180, Lot 

### 70107; EGEEA, 

### Catalog No. 

### E-181, Lot 794501; 

### EGBE, 

## Catalog No. 

## E-179, Lot 

### 703549; and 

## diEGEE, Catalog
No. 

### D-51, Lot 792796. 

### Time-mated 

## primigravida SPF 

## Sprague-Dawley rats 

### were 

## purchased from 

### Charles River 

## Breeding Laboratories, 

## Wilmington, MA. Rats

were

## shippedon

## day4

### andreceivedon

## day5

### of

## gestationHARDIN, GOAD AND BURG



### (sperm

= 

### day 1), 

### when they were

### weighed 

### andmarked for 

### individual identification by toe 

### clipping. The interscapularregion was 

### shavedwith

### electricclippers on

### day 5 or 

### 6, andrats were 

### formallyrandomizedby

### weight to

one 

### offive groups. 

### All rats were 

### singlyhousedthroughout 

### the 

### study in 

### suspended stainless steel cages 

### in a 

### ventilated cage 

### rack (Hazelton Systems, 

### Aberdeen, 

### MD) withfree access 

### to

### foodandwater. 

### These cages are 

### designed such 

### that room 

### air was 

### drawn 

### through the 

### wire mesh floors of each cage 

### and 

### into an 

### overhead 

### plenumfromwhichthe 

### exhaust air was 

### ducteddirectly
to a 

### fume hood. 

### Light was on a 

### 12-hr 

### light-dark cycle. Rats were 

### received in two separate 

### shipments in 

### consecutiveweeksandthe

### experiment was

### conductedin
two

### replicates. Tieatment 

### volumes were 

### selected based

on 

### results of the 

### earlier 

### study of 

### the teratogenicity of 

### EGEE whenapplied to

### the skinofrats 

### (7). 

### A

### volume of 

### EGEE (0.25 mL 

### four times 

### daily) that was 

### teratogenic in

### that 

### study was 

### used as 

### thepositive 

### control. Approxi

### matelyequimolarvolumes (0.35

### mL 

### fourtimes

### daily, for

a 

### total 

### daily dose of 1.4 

### mL) of 

### EGEEA, EGBE, and diEGEE were 

### employed 

### initially. Because the rats 

### treated 

### with 0.35 mL 

### of 

### EGBE showed 

### marked toxic 

### effects, the treatment 

### volume was 

### reduced in 

### the second 

### replicate to 

### 0.12 mL 

### EGBE four times 

### daily. Undiluted

### glycols or

### water 

### (control) were 

### appliedto 

### the 

### shaved

### interscapularregionusing an

### automatic

### pipetter 

### (Finpipette) four

### times dailyat 

### 2.5-hrintervals on

### days 7 to 

### 16 of 

### gestation.
In

### addition to

### the 

### randomizationbodyweights (day 5 

### of 

### gestation), body weights were 

### recorded on 

### the first and 

### sixth 

### days of treatment 

### and on 

### the first 

### and fifth post-treatment 

### days (gestation days 7, 

### 12, 17 

### and 

### 21, 

### respectively). On 

### day 21, 

### females were 

### killed by CO2 asphyxiation and

### gravid uterus 

### weights were 

### recorded. 

### Fetuses were 

### weighed and examined for 

### external 

### malformations. Alternate fetuses were 

### preserved 

### in 

### Bouin's fluid or 

### 70% 

### ethanol for subsequent 

### visceral examination 

### by the Wilson (15) 

### method or 

### skeletal examination according to 

### Staples 

### and Schnell 

### (16). 

### Uterifromapparentlynonpregnant 

### females were

### placed in 10% 

### sodium sulfide to 

### reveal early termination 

### of

pregnancy 

### Allmaternal and 

### fetal

### bodyweights, maternal organ 

### weights were 

### analyzed by analysis of variance 

### tech

### niques. If 

### significant (p < 

### 0.05) 

### differences 

### between

groups were 

### detected, a 

### posteriori Waller-Duncan K-ratio t-tests were 

### conducted to 

### describe in more 

### detail the source 

### ofvariation. 

### Implantation data 

### (total andnumbers oflive and 

### dead) were 

### analyzedusingthe Kruskal-Wallis test. 

### Frequencies ofmalformations 

### and of

### variations were 

### analyzed by Fisher-Irwin exact 

### test 

### of 

### the numbers of affected litters. Because 

### repeated
contrasts were

### made with 

### the water 

### control group, 

### the level of

### significance was 

### set at p < 

### 0.01. All 

### procedures

were 

### carried out 

### using SAS 82.3 and BMDP statistical 

### packages on 

### the 

### Parklawn 

### Computer Center, Rock

## ville, MD.



# Results

Toxic signs were 

### not 

### noted 

### in 

### EGEE-, EGEEA-, or 

### diEGEE-treated rats. 

### However, red-stained urine was 

### observed 

### after only two treatments 

### with 0.35 

### mL 

### EGBE. By 

### the end of 

### the 

### first treatment 

### day, 

### EGBEtreated rats 

### produced 

### burgundy-colored urine. 

### Continued treatment 

### with 

### EGBE led to 

### ataxia

### progressing to 

### moderate to

### markedinactivityandrough

### haircoatswith dark 

### stains 

### around the 

### muzzle 

### and ano-genital area. 

### Severalrats' 

### tails

### blackened

### distallyand were

### gradually
eaten away as

### the

### apparentnecrosis

### progressed. Deaths occurred on

### thethirdto

### seventh

### days oftreatment 

### (days

Table 1. Gestational body weights (mean ± SD) of pregnant rats in various treatment groups.

Treatment group
EGEE EGEEA EGBEa diEGEE Water (0.25 mL) (0.35 mL) (0.12 mL) (0.35 mL) (control) 

### Group size 18 18 9 13 17 

### Body weight, g Day 5 188 ± 8 192 ± 9 192 ± 10 193 ± 7 189 ± 7 

### Day 7 210 ± 7 212 ± 12 219 ± 12 213 ± 10 210 ± 7 

### Day 12 239 ± 10 240 ± 14 239 ± 13 242 ± 12 246 ± 11 

### Day 17 262 ± 15 256 ± 17 268 ± 16 269 ± 17 278 ± 14 Day 21 284 ± 32 268 ± 29 336 ± 30 323 ± 31 331 ± 22 

### Bodyweight gain, g Day 5-7 22 ± 8 20 ± 7 27 ± 6 19 ± 8 21 ± 5 

### Day 

### 5-12* 51 ± 9xy 48 ± 

### 9Y 48 ± 12Y 49 ± 7x,y 57 ± lOx Day 5-17t 74 ± 13Y 65 ± 12z 77 ± 18Y 76 ± 13Y 89 ± 11x Day 

### 5-21t 97 ± 29Y 76 ± 

### 27Z 144 ± 31x 130 ± 27x 142 ± 19x Gravid uterus 

### weight, 

### gt 23 ± 21Y 9 ± 12z 59 ± 22X 65 ± 20X 60 ± 15x Extragestational 

### body

### weight, gb 261 ± 18 259 ± 24 276 ± 18 258 ± 18 271 ± 15 

### Extragestational 

### bodyweight gain, 

### g* 74 ± 15xy 67 ± 20v 85 ± 

### 18X 65 + 16Y 82 ± 15x 

## aReplicate 2 

### only. 

## All other groups are combined data for 

### replicates 1 and 2. 

## bDay 21 

### bodyweight minus 

### gravid uterus 

### weight. 

## cExtragestational bodyweight minus 

### day 5 

### body

### weight. 

### *Significant difference 

### (p < 

### 0.05). 

### Groups with the same 

### superscript do not differ from each other 

### by a

## posteriori test. 

### tSignificant difference (p < 0.001). 

### Groups with the same 

### superscript do not differ from each other 

### by a

### posteriori test. 70DEVELOPMENTAL TOXICITY OF CUTANEOUSLYAPPLIED GLYCOLS



### 9-13 

### of gestation). In 

### replicate 1, 

### only one 

### of 11 rats 

### treated with 0.35 

### mL 

### EGBE 

### survived. In 

### the 

### second replicate, the EGBE treatment 

### volume was 

### reduced to 

### 0.12 mL 

### four 

### times 

### daily (0.48 

### mL 

### EGBE/day) and no 

### toxic 

### signs were 

### noted. Bodyweights 

### ofpregnant 

### females are 

### summarized in 

### Table 1, 

### along

### with gravid uterus 

### and 

### extragestational body 

### weight (day 21 

### body weight minus 

### gravid uterus 

### weight). Day 5 

### (randomization) body weights 

### did not 

### differ 

### between 

### replicates and 

### body weight data were 

### therefore

### combined across

### replicatesfor

### analysis. Analysis ofvariance 

### confirmed that

### bodyweights on

### day5

### did

not 

### differ across 

### treatment groups. 

### Body 

### weights on 

### days 7, 12, 17, 

### and 21 were 

### analyzed by multivariate analysis ofthe 

### change relative to

### day5. Treatment

### with 

### EGEE and 

### EGEEA

### profoundlyreduced

### maternalbody weight gain, and at 

### days 17 

### and 21 

### bodyweight gain in the 

### EGEEA group was 

### significantly lower than in 

### the 

### EGEE positive controls. 

### Gravid uterus 

### weights were 

### also 

### markedly reduced in 

### those groups, 

### accounting for 

### muchofthedifference in

### body

### weight. 

### Extragestational body 

### weights did not 

### differ 

### significantly (p < 

### 0.10), but 

### extragestational bodyweight gain was 

### significantly different across groups 

### (p < 

### 0.05). 

### Extragestational weight gain in EGEEA 

### and diEGEE groups was 

### significantly less than in 

### the water 

### controls 

### by a 

### posteriori test. 

### Data in 

### Table 2 

### reveal that, relative to 

### the water 

### controls, both EGEE 

### and 

### EGEEA treatments were 

### stronglyembryotoxic as

### reflectedin

### significantlyhigher frequenciesof

### completelyresorbedlittersand

### increased numbers 

### of 

### dead implants per 

### litter. The number 

### of dead 

### implants per 

### litter was 

### also 

### significantlyhigher in 

### EGEEA- than in 

### the positive control 

### EGEE-treated litters. 

### Considering only 

### litters with at 

### least one 

### live fetus, there was a

### significantreductioninthe

### numberof live 

### fetuses per 

### litter in 

### the EGEE- and EGEEAtreated groups. 

### Similarly, the 

### body 

### weights of live fetuses in 

### these two groups were 

### significantly reduced relative to 

### the 

### water-treated controls. No 

### evidence of 

### embryo- or 

### fetotoxicity was 

### noted in 

### the 

### EGBE and 

### diEGEE groups. 

### On gross 

### examination of 

### fetuses, the 

### only 

### malformations noted were 

### three fetuses from 

### EGEE-treated litters with 

### acaudia and 

### imperforate

anus 

### (Table 2).



# Discussion

The 

### results of this study 

### confirmed the 

### previously reported embryo- and 

### fetotoxicityandteratogenicity of 

### EGEE 

### applied to 

### the skin of pregnant rats. 

### As was 

### previously reported, 

### cardiovascular defects were 

### the 

### predominantvisceralmalformation. A

### wide spectrum

### of 

### skeletal variants was 

### also seen

### both in 

### the 

### present and 

### the 

### earlier skin 

### teratology evaluation of 

### EGEE. Verte

### bral malformations and reduced skeletal 

### ossification 

### also occurred with 

### increased 

### frequency in 

### EGEE

### treatedlitters

### but were 

### not 

### statistically

### significant. The 

### only gross

### malformations noted in

### the 

### study were 

### three 

### EGEE-treated fetuses 

### with acaudia and 

### imperforate

anus, a 

### defect 

### also seen 

### in 

### the 

### previous evaluation of 

### cutaneously 

### applied EGEE. 

### The spectrum 

### and frequency of malformations and 

### variations in 

### the 

### EGEEA-treated litters was 

### remarka

### bly similar to 

### that described for 

### the 

### EGEE-treated

group. 

### Statisticallysignificant increases in

### cardiovascu

### lar

### malformationsand

### skeletalvariations were

### detected. Similar responses 

### to 

### EGEE and 

### EGEEA are 

### not 

### unexpected since 

### EGEEAshouldbe

### rapidly

### hydrolyzed in

### vivo to 

### EGEE and acetate. 

### At an 

### equimolardose, in 

### fact, 

### EGEEA in 

### this 

### study caused even more severe 

### maternal, embryo, and 

### fetal 

### toxicity than 

### did 

### EGEE. 

### This was 

### reflected in 

### significantly reduced in 

### body weight gain (Table 1) and 

### increased dead 

### implants per 

### litter 

### (Table 2) in

### EGEEA-treated litters relative to 

### the 

### EGEE litters. 

### Neither 

### EGBE nor 

### diEGEE was 

### associated with 

### embryo- or 

### fetotoxicity, and the incidence of visceral 

### and skeletal defects was 

### not 

### increased in 

### either group.

Table 2. Observations at necropsy.



### TIeatment group
EGEE EGEEA EGBEa diEGEE Water (0.25 mL) (0.35 mL) (0.12 mL) (0.35 mL) 

### (control)


### Group size 18 18 9 13 17
No. (%) litters totally resorbed 

### 7*(38.9) 13*(72.2) 0 0 0
No. implants per 

### litterb 11.6 ± 2.9 11.7 ± 2.7 11.2 ± 4.3 11.8 ± 1.7 11.6 ± 2.5
No. dead implants per 

### litter"t 7.7 ± 5.1y 10.6 ± 3.3x 1.2 ± 1.0Z 1.2 ± 1.2z 1.2 ± 1.2z
No. live fetuses per litter with live 

### fetuses"t 6.5 ± 4.1y 4.2 ± 

### 3.4Y 10.0 ± 3.8x 10.6 ± 2.3x 10.4 ± 2.6X (no. litters) (11) (5) (9) (13) (17) Fetal bodyweight, 

### gbt 3.0 ± 0.6y 2.6 ± 1.0v 4.2 ± 0.7x 3.9 ± 0.8x 3.9 ± 0.6x Gross malformations litters 

### (fetuses) 

### 3(3)C 0 0 0 0 

### aReplicate 2

## only. All other groups are combined data for replicates 1 and 2. 

### bMean ± SD. 

## cAcaudia and imperforate anus. 

### *Differs significantly (p < 0.01) from water controls. 

### tSignificant differences (p < 0.001). 

### Groups with the same 

### superscript do not differ from each other (p > 0.05) 

### by a

### posteriori test. 71HARDIN, GOAD AND BURG



### EGBE is a 

### potent 

### hemolytic agent 

### in 

### rodents, as 

### reflected in 

### the 

### rapid onset 

### ofred-stainedurine 

### (within 5 hr 

### of 

### first 

### dosing) and 

### subsequent death of rats 

### receiving0.35-mLdoses. 

### Equimolarvolumes of

### EGEE, EGEEA and 

### diEGEE caused no 

### mortality and no 

### clinical 

### signs of 

### toxicity. The 

### extragestational 

### body weight gain (Table 1) of 

### diEGEE-treated rats was 

### significantly reduced, to a 

### degree comparable to 

### the reductionthe

### EGEEA-treatedrats. 

### Thus, while

### diEGEE

was 

### not 

### embryo- or 

### fetotoxic, it was 

### maternally toxic under 

### these test conditions.
In summary, 

### the results of 

### this 

### study confirm that 

### EGEE can 

### be 

### absorbed in 

### embryotoxic, fetotoxic, and 

### teratogenic quantities 

### through the intact skin 

### of preg

### nant

### rats. 

### EGEEAwas 

### shownto

### sharethese

### properties 

### and to 

### be at 

### least as 

### active as 

### EGEE by this route. 

### It 

### has been previously reported 

### that 

### inhalation exposure 

### ofpregnant 

### rats to 

### EGBE resultedin no

### developmental 

### toxicity, and none was 

### detected here at 

### approximately 

### one-third the

### molardoseusedfor 

### EGEE and 

### EGEEA. A treatment 

### volume of

### EGBE equimolar to 

### the 

### EGEE 

### and 

## EGEEA volume was 

### strongly 

### hemolytic and ulti

### mately lethal, demonstrating that 

### EGBE also pene

### trates 

### the skin readily. There also was no 

### measurable 

### developmental toxicity in rats 

### treated with 

### diEGEE, 

### despiteadministrationat a

### dose causing

### slight

### maternal toxicity. 

### These data

### demonstrate that

### reproductive and 

### developmental toxicity, like 

### other 

### manifestations 

### of glycol ether toxicity, are 

### possible 

### following cutaneous

exposure.

Table 3. Visceral malformations and variations: number of litters (fetuses) affected.

Treatment Group

EGEE EGEEA EGBEa diEGEE Water (0.25 mL) (0.35 mL) (0.12 

### mL) (0.35 

### mL) (control) 

### Tbtal examined 10(34) 3(10) 8(45) 13(70) 17(87) Malformations 

### Cardiovascular: total 

### 8(9)t 3(5)t 0 0 0 

### Right aorta 

### 4(4)b 2(2) 0 0 0 Double aorta 1(1) 1(1) 0 0 0 

### Right ductus 

### 3(3)b 1(1) 0 0 0 Ventricular 

### septal defect 5(5) 3(5) 0 0 0 Abnormal 

### subelavian 

### 1(1) 0 0 0 0

Renal: total 

### 7(8)* 1(2) 0 

### 2(3) 3(3) Hydronephrosis 4(4) 1(1) 0 1(2) 0 

### Hydroureter 4(4) 0 0 

### 1(2) 3(3) Ectopic 

### kidney 1(2) 1(1) 0 1(1) 0 Hypoplastic kidney 0 1(1) 0 0 0 Fused 

### kidneys 1(1) 0 0 0 0

Brain: total 

### 4(4)* 1(2) 0 2(3) 0 Hydrocephalus 2(2) 1(2) 0 2(3) 0 

### Hemorrhage 2(2) 0 0 0 0



### Eye: total 2(2) 2(2) 0 0 0 

### Microophthalmia 1(1) 0 0 0 0 Abnormal optic nerve 1(1) 0 0 0 0 Folded retina 2(2) 2(2) 0 0 0



### Tobtal malformations 

### 10(21)t 3(8) 

### 0(0) 3(5) 3(3) Variations Cardiovascular 

### Missing innominate 2(2) 1(1) 0 0 0

Renal: total 5(7) 0 

### 4(5) 6(13) 

### 10(15) Dilated renal 

### pelvis 2(3) 0 0 

### 3(6) 

### 6(7) Dilated ureter 4(5) 0 

### 4(5) 4(9) 

### 7(10)



### Tbtal variations 5(8) 1(1) 4(5) 6(13) 

### 10(15)



### Testicular defects Males examined 21 6 23 40 39 Testicular: total 3(3) 1(1) 1(1) 4(4) 1(1) 

### Ectopic (slight) 2(2) 1(1) 1(1) 4(4) 

### 1(1) 

### Undescended 1(1) 0 0 0 0 

### Agenesis 1(1) 0 0 0 0 

## aReplicate 2

## only. All other groups are combined data for 

### replicates 1 and 2. 

### bIncludes one fetus with situs inversus. 

### *Differs significantly fromwater controls (p < 0.01) 

### by two-tail Fisher-Irwin exact test of litters affected. 

## tDiffers significantly from water controls (p < 0.001) 

### by

## two-tail Fisher-Irwin test of litters affected. 72DEVELOPMENTAL TOXICITY OF CUTANEOUSLYAPPLIED GLYCOLS 73

Table 4. Skeletal malformations and variations: number of litters (fetuses) affected.

Treatment group
EGEE EGEEA EGBEa diEGEE Water (0.25 mL) (0.35 mL) (0.12 mL) (0.35 mL) (control)


### Tbtal examined 11(36) 5(11) 9(45) 13(67) 17(90) Malformations Ribs: total 2(2) 2(2) 1(1) 2(4) 1(1) Missing ribs 

### 2(2)b 0 0 2(3) 1(1) Fused, malformed 

### 1(1)b 2(2) 0 1(2) 0 Cervical 0 0 1(1) 0 0

Vertebrae: total 4(5) 0 0 2(4) 0 Fused arches 

### 2(2)b 0 0 1(1) 0 Extra lumbar 2(2) 0 0 1(3) 0 Reduced lumbar 

### 2(3)b 0 0 0 0 Reduced thoracic 

### 1(1)b 0 0 1(1) 0 Absent arch 0 0 0 1(1) 0

Scoliosis 0 1(1) 0 0 0 Missing tympanic ring 0 0 0 1(1) 0 Total malformations 4(5) 2(2) 1(1) 3(5) 1(1)

Variations Ribs: total 

### 6(13)* 5(6). 3(4) 5(12) 1(1) Extra thoracic 2(6) 0 0 0 0 Extra lumbar 

### 5(7)b 5(6) 3(4) 5(12) 1(1)

Vertebrae: total 

### 10(32)t 5(11) 1(1) 3(6) 0 Reduced sacral (< 6) 

### 2(2)b 0 0 0 0 Missing lumbo-sacral arch 2(2) 1(1) 1(1) 0 0 Increased thoracic 1(3) 0 0 0 0 Centra: 

### misshaped 

### 7(16)b 5(10) 0 2(3) 0 Centra: absent, incomplete 7(23) 5(11) 0 2(5) 0

Sternebrae: total 11(32) 4(9) 6(13) 7(18) 13(28) Misaligned 6(8) 0 0 1(3) 2(2) 

### Split 0 1(1) 0 0 0 Decreased number (< 6) 9(29) 4(9) 6(13) 7(17) 13(27)

Reduced ossification: total 5(14) 

### 5(11) 3(3) 3(3) 1(1) Skull 

### 2(8) 5(11) 3(3) 

### 2(2) 0 Pelvic 

### girdle 

### 4(8)b 4(6) 0 0 0 Decreased metatarsals (< 4) 

### 2(3)b 4(7) 0 1(1) 0 Decreased 

### metacarpals (< 3) 2(5) 2(5) 1(1) 0 1(1) 

### Tbtal variations 11(34) 5(11) 7(16) 9(22) 13(29) 

## aReplicate 2

## only. All other groups are combined data for replicates 1 and 2. 

### bIndicates two fetuses with acaudia and imperforate anus. 

### *Differs significantly from water controls (p < 0.01) by two-tail Fisher-Irwin exact test of litters affected. 

### tDiffers significantly fromwater controls (p < 0.001) by two-tail Fisher-Irwin exact test of litters affected.

WewishtothankPeggyL. Johnson, GarthE. Peterson, RonaldL. SchulerandTerryW Thrnerfortheirassistancewithtreatments and necropsies. We are also grateful to Sandra L. Clark for her help in preparing the manuscript. Mention of a company name or product does not constitute endorsement by the National Institute for Occupational Safety and Health.

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