Environmental Health Perspectives Vol. 57, pp. 199-206, 1984



# Further Studies on 

# the 

# Toxicology of 

# the Glycol Ethers with 

# Emphasis on 

# Rapid Screening and Hazard 

# Assessment by 

# J. E. Doe*

The discovery that ethylene glycol monomethyl ether (EGME) could affect the testis and the developing fetus in laboratory animals prompted further work to understand the effect of EGME and to examine additional glycol ethers to see if they showed EGME's reprotoxicity. Propylene glycol monomethyl ether (PGME) was shown not to cause testicular atrophy or to affect the development of rats at 600 ppm by inhalation, whereas EGME caused testicular atrophy at 300 ppm and showed teratogenic potential at 100 ppm. Diethylene glycol monomethyl ether (diEGME) was found to show no teratogenic potential when administered subcutaneously in rats at up to 1000 ,iL/kg, whereas EGME had effects at 40 ,iL/kg. EGME hasbeenshowntocauseeffects onthetestis inrats afterasingle exposure to 600 ppmorabove for 4 hr. The effects canbe seenas littleas 24 hrafterexposure. Ethylene glycol monoethyl ether (EGEE) also causes areduction intesticularweight followingasingle exposure to saturatedvapor for 3 hr (17mg/I), but ethylene glycol monoisopropyl ether (EGPE) at 15 mg/l and ethylene glycol monobutyl ether (EGBE) at 4 mg/l showed no effect on the testis.



## Introduction The 

### reproductive toxicology of

### glycol ethers has

### been the 

### subject of much concern in recent years. This concernwas 

### initiated

### by areport 

### (1) 

### describingtesticu

### lar 

### atrophy in mice 

### dosed with 

### ethyelene 

### glycol mono

### methyl ether 

### (EGME) and 

### ethylene 

### glycol monoethyl ether 

### (EGEE). Moreover, both 

### EGME (2) and 

### EGEE (3) have 

### been shown to cause 

### fetotoxicityand/or terato

### genicity in 

### laboratoryanimals. These 

### observations lead to a 

### general 

### reappraisal of the 

### glycol ethers as a class 

### and this paper 

### describes some of 

### the work which was 

### carried out

### within 

### Imperial Chemical Industries PLC in 

### order to 

### determine the 

### reproductive 

### toxicology of 

### several members ofthe 

### glycol ethers group

### which were 

### ofproven or

### potential commercial 

### importance. None of the 

### studies was 

### conducted tofull

### classical

### protocols, but 

### each one was 

### designed to answer a 

### specific 

### question to 

### aidineither

### productdevelopment orin

### the

### development of safe 

### handling. In retrospect, this series 

### of 

### experiments can be seen as a way in which 

### toxicology can 

### provide useful 

### data even 

### though the 

### studies are not 

### carried out to a 

### regulatory 

### protocol.
*ImperialChemicalIndustriesPLC, CentralToxicologyLaboratory, Alderley Park, Macclesfield, Cheshire, UK. 

## ComparativeReproductiveToxicology 

## by Inhalation in 

## Rats of Ethylene 

## Glycol Monomethyl 

## (EGME) and 

## Propylene 

## Glycol Monomethyl Ether 

## (PGME) This studywas carried out at a time whenthe effects 

### of 

### EGME upon

### both 

### the testes 

### by inhalation (4) and its 

### effectsofthe fetus (2) wereknown. However, therewere no 

### data available on the toxicology of PGME, and this 

### study was 

### designed to assess whether PGME was 

### capable of 

### causing the same 

### effects on 

### reproductive 

### toxicologyas 

### EGME. Itwas 

### therefore important touse 

### EGME as a 

### positive control for both the testicular 

### effects and the 

### teratogenic effects. The 

### study was 

### designed in two parts: 

### effect on testis 

### and 

### hematology and 

### determination of 

### teratogenic 

### potential. 

### Effect on 

### Testis 

### and 

### Hematology Tenmale rats ofthe 

### Alpk/Ap 

### (Wistar-derived) strain were 

### exposed to 

### the 

### following concentrations: 100 ppm 

### EGME, 300 ppm

### EGME, or200ppm

### PGME or 600

### ppm PGME for 6 

### hr/day for 10 

### consecutive 

### days in 

### 3.4 

### m3 inhalation chambers 

### (5). 

### During the exposure 

### periodJ. E. DOE



### the animals wereperiodicallyweighed and observed for 

### changes in their clinical condition or other behavioral 

### changes. Onthe

### dayafterthe 10thexposuretheanimals were 

### killed by exposure to Fluothane. Blood 

### samples were 

### taken forhematological assessment and the testis 

### weighed and 

### removed and 

### processed, along with the 

### thymus, 

### into slides for 

### subsequent histopathological 

### evaluation. Thebody

### weightgain

### andabsoluteweightsoftherats 

### exposed to 300 ppm 

### EGME were 

### significantly reduced; however, 100 ppm 

### EGME and 200 or 600 ppm PGME 

### had no 

### effect on 

### body weight. None 

### of the treatments 

### causedany

### changes in

### clinicalobservations. Atpostmortem

### there were no 

### effects at 100 ppm 

### EGME or at 200 

### and 600 ppm 

### PGME, however, the 

### thymus and 

### the testes 

### of the rats 

### exposed to 300 ppm EGME were reduced in size (Fig. 1). In the blood samples taken at postmortem 300 ppm 

### of 

### EGME was 

### shown to cause a 

### reduction in 

### total white blood 

### cells, the number ofred 

### blood cells and 

### the

### hemaglobin concentration ofthe red 

### bloodcells. There were no 

### effects at 100 ppm

### EGME or 

### witheitherconcentrationof

### PGME. Histological examination 

### ofthe testis 

### revealed a 

### few seminiferous 

### tubules with disordered 

### spermatogenesis with a similar inci

### dence in 

### the controls and in rats 

### exposed to 100 ppm 

### EGME, 200 ppm 

### PGME or 600 

### PGME. Pronounced 

### tubular 

### atrophywas seen in 

### the testis fromall the rats 

### exposed to 300 ppm 

### EGME, where 70 to 100% 

### of the 

### seminiferous tubules were 

### affected. 

### Determination of 

### Teratogenic 

### Potential In

### orderto

### provide a

### rapidassessment 

### oftheeffect of 

### PGME and EGME onthe

### developing embryowhenrats were 

### exposed by 

### inhalation, an 

### adaptation of 

### the procedure described by Chernoff and Kavlock (6) was 

### employed. Pregnant femalerats, 

### approximately 11 to 13 weeks 

### of age, of the 

### Alpk/Ap (Wistar-derived) strain were

### used. Groups of20 ratspergroupwere 

### exposed to 

### air, 100or300ppm

### EGME and200 or600ppm

### ofPGME for6

### hr/dayon

### days 6 to 17 (inclusiveofgestation, dayof matingbeingday0). Afterthe last exposure period the rats were removed from the exposure chamber and 

### housed singly. The rats were 

### allowed to deliver 

### their 

### litters which were observed for 3 days. The number of live and deadpups in each litterand theweight ofeach 

### litterwere

### determinedondays 1 and3postpartum. Any rats not producing 

### litters by day 24 of gestation were 

### killed, andagrosspostmortem

### examinationwas 

### carried out to determine the status 

### of

### pregnancy. All 

### the rats were pregnant: all the control rats and all the rats 

### exposed to PGME produced litters. Exposure to 300 ppm 

### EGME caused a significant 

### reduction in 

### body weight gain throughout the study, and none of these rats produced litters. Only 9 of the 20 rats exposed to 100ppmproducedlitters andintheserats thegestation 

### period was 

### significantly increased to a mean 

### value of 23.6 

### days fromacontrol value of22 days. The compara

### tive litter data are shown in 

### Thble 1. There was a 

### significant reduction in 

### the proportion of live pups 

### initiallyandtheproportionsurvivingfor3dayspostpartum 

### in 

### the litters ofdams exposed to 100 ppm EGME. 

### The weight of the pups in this group on day 1 was 

### similar to the 

### controls, although they had 

### gained 

### significantly less weight by day 3. All the pups were 

### normal externally. There was no evidence ofany 

### effect in either 

### of the groups exposed to 

### PGME.

L

.2 ,uV

L R L K 1. R K

Conlrol l(X)ppm F(iME C M[ ( )ppm (1M 2o(1)ppill l(iMI (o)(p Ip IMA,\I FIGURE 1. Effect on testis weights of the inhalation of EGME and PGME for 10 consecutive days in male rats. Asterisks denote significantly different from controls: (**) p < 0.01; (*) p < 0.05. 200SCREENING AND HAZARD ASSESSMENT

Table 1. Comparative effect of EGME and PGME by inhalation on pregnant rats: litter data. Group mean no. of Proportion of Proportion of Group mean Live pup live and dead pups live pups pups surviving per litter, weight on Treatment on day 1 (n) on day 1 to day 3 day 1 (n) day 3, g (n) Control 11.5 ± 3.0 (20) 226/229 (98.7%) 223/226 (98.7%) 6.0 ± 0.5 (20) 7.2 ± 0.8 (20)
100 ppm EGME 3.3 ± 2.4 (9)t 23/30"t (76.7%) 16/23 (69.6%) 6.1 ± 0.6 (7) 6.3 ± 1.1 (4) 300 ppm EGME
200 ppm PGME 11.7 ± 2.0 (20) 231/233 (99.1%) 226/231 (97.8%) 6.0 ± 0.4 (20) 7.1 ± 0.7 (20) 600 ppm PGME 11.7 ± 2.6 (20) 228/233 (97.9%) 227/228 (99.6%) 6.0 ± 0.4 (20) 7.0 ± 0.7 (20) tStatistically significantly different from the control group at the 1% level (Fisher's exact test, two-sided). *Statistically significantly different from the control group at the 1% level (t-test, two-sided). 

### Conclusions This 

### experiment demonstrated that 300 ppm of 

### EGME caused 

### hematological 

### changes, testicular atro

### phy and 

### thymic 

### atrophy in male rats and 

### complete embryo 

### lethality in pregnant female 

### rats.A 100 

### ppm level caused fetal

### mortality, 

### fetotoxicityand the 

### impairment

### of

### postnatal

### development. This 

### spectrumofeffects hasbeen

### definedas

### describing

### teratogenicpotential (6). In contrast, 

### neither 200 nor 600 ppm 

### of 

### PGME caused any 

### effects in 

### either males or pregnant females. The 

### results 

### of this 

### preliminary 

### study therefore indicate 

### clearly that 

### PGME does not 

### share the spectrum of 

### activityshown

### byEGME upon

### the

### reproductiveprocess at 

### significant dose levels. 

### The 

### studyhas beendescribed 

### previously (7). 

## Teratogenic Potential of 

## Diethylene 

## Glycol Monomethyl Ether 

## (diEGME) in the

## Postnatal DevelopmentTestin

## Rats Few or no 

### data existed at the 

### time this study was 

### carried out on 

### the effect of

### diethylene glycol ethers on the 

### developing embryo. This 

### study was 

### carried out to assess 

### the 

### effect of

### diethylene glycol monomethyl ether 

### (diEGME) on 

### the rat 

### fetus in 

### order to 

### determine whether 

### diEGME showed the same spectrum of activity as 

### EGME, whichwouldhave meant 

### reconsideration of 

### the way in 

### which the compound is 

### handled. The 

### postnatal 

### development test (6) was 

### used, as 

### EGME had 

### already been shown to 

### be positive in 

### this test 

### by 

### inhalation. As 

### diEGME is

### relativelynonvolatile, human exposure is more 

### likely to 

### be 

### by dermal contact; 

### consequently diEGME was 

### administered 

### by the subcutaneous route as this represents 

### the worst case, 

### assuming the 

### complete breakdown of 

### the barrier function of the skin. 

### Pregnant female 

### Alpk/Ap (Wistar

### derived) rats 11 to 13 weeks ofage were 

### mated 

### and the 

### day on 

### which spermatozoa were 

### detected was 

### taken as 

### day 0 of 

### pregnancy. Groups of 15 rats were 

### used and 

### theywere 

### injected subcutaneouslywith 2 mL 

### of sterile water 

### containing the 

### appropriate 

### compounds on 

### days 6 to 20 

### of

### gestation inclusive. The groups were 

### controls, 250 puL/kg diEGME, 500 

### ,LL/kg diEGME, 1000 

### ,LL/kg 

### diEGME, 40 

### ,uL/kg EGME, 250 

### p1L/kg EGME. The rats were 

### observed daily for changes in clinical conditionand

### weighed onday 1 and on each

### daybetweenday 6 and 20. 

### Each rat was 

### allowed to 

### litter and the 

### offspringweighed andthe numberofdeadand livepups were recorded on days 1 and 4 postpartum. The female rats 

### and the pups were then killed. All animals which failed to 

### produce a litter by day 24 of 

### pregnancy were 

### killed and 

### the status 

### of pregnancy determined. No 

### abnormalities in clinical condition were seen in any 

### of the rats 

### treated with 

### diEGME; however, some of the rats treated with 250 

### p1L/kg EGME showed 

### piloerectionand

### vaginal

### bleeding. The

### bodyweight gain of 

### these rats treated with 250 

### ,uL/kg EGME was 

### significantly reduced compared with controls. 

### One con

### trol animal 

### didnot

### produce a

### litterandwas 

### foundnot to be pregnant. None of the rats 

### treated with 250 

### ,uL/kg EGME produced

### litters, althoughtheywere all foundto have 

### been pregnant. The litter data are shown in

### Table 2, where it will be seen 

### that 40 

### puL/kg of EGME reduced the number of litters as well as the number of live pups per 

### litter and the 

### percentage of pups 

### surviving for 4 

### days. EGME therefore 

### clearly showed its 

### fetotoxic effect in 

### this 

### study. 

### diEGME, on the other

### hand, causednomaternal 

### toxicityand 

### little orno 

### effect on

### the

### development ofthe young at 250 or 500 

### puL/kg. There was a 

### slight, but not 

### statistically significant, decrease in the 

### survivability in 

### the 

### offspring of the rats 

### treated with 1000 

### puL/kg, and 

### this maybe 

### indicative ofa 

### slight fetotoxic effect at this 

### high dose level. 

### However, 

### diEGME certainly does not share themarked

### fetotoxicity shown

### by

### EGME, and, as the route of

### administrationwas 

### chosen tomaximize the 

### possible effects of 

### diEGME, it is 

### unlikely that dermal exposure to 

### diEGME in humans would present a 

### significant risk. 

### These data were 

### reported previously (8). 

## Effect on 

## Route of Administration on 

## Teratogenic Potential of 

## EGME EGME has been shown to cause testicular 

### atrophy and/or 

### fetotoxicity 

### by a 

### variety of routes such as 201J. E. DOE

Table 2. Effect of subcutaneous diEGME and EGME in pregnant rats: litter data. diEGME diEGME diEGME EGME EGME Control 250 ,uL/kg 500 ,L/kg 1000 ,uL/kg 40 ,uL/kg 250 ,uL/kg No. pregnant 14/15 15/15 15/15 15/15 15/15 15/15 No. of litters 14 15 15 15 7 0 No. of live pups per litter ( ± SD) 11.7 ± 2.7 12.0 ± 1.9 10.9 ± 3.1 10.5 ± 3.6 3.7 ± 4.2k Pup weight at day 1, g ( ± SD) 5.9 ± 0.6 6.0 ± 0.5 6.3* ± 0.4 6.2 ± 0.5 6.1 ± -/5 Pup weight at day 5, g ( ± SD) 8.5 ± 1.1 8.8 ± 1.2 9.3 ± 1.1 8.6 ± 1.1 10.4 ± 0.7* Weight gain per pup, g ( ± SD) 2.5 ± 0.8 2.8 ± 0.8 3.0 ± 1.0 2.4 ± 0.9 3.6 ± 0.2 No. live pups per litter day 5 ( ± SD) 11.2 ± 2.9 11.3 ± 1.9 10.6 ± 2.5 9.1 ± 3.3 2.1 ± 4.5t

- Surviving on day 5, % 95.2 91.4 89.9 85.6 46.9 No. of litters with deaths day 1-5 4/14 6/15 8/15 10/15 6/7 No. of litters with more than one death day 1-5 2/15 2/15 2/15 7/15 4/7 *Statistically significantly different from the control group means at 95% level Student t test. 'Statistically significantly different from the control group means at 99% level, Student t test, two-sided.

Table 3. Effect of route of administration on teratogenic potential of EGME.
Mean pup Mean pup Number Number of Mean litter weight, Mean litter Survival, weight, % Weight Treatment and route pregnant litters size, day 0 day 0, g size, day 4 % day 4, g gain (pups) 10 mL/kg saline IP 15 12 7.6 5.3 5.4 64 8.8 43 200 mg/kg EGME IP 15
10 mL/kg saline PO 15 15 10.7 6.1 10.7 99 9.0 48 200 mg/kg EGME PO 15 0
2 mL/kg saline SC 15 15 10.8 6.0 10.5 91 9.25 52 40 mg/kg EGME SC 15 7 4.6* 6.0 2.1* 23* 10.7 31 250 mg/kg EGME SC 15 0
2 mL/kg saline dermally 15 15 13.2 5.6 12.7 96 9.2 59 40 mg/kg EGME dermally 15 15 12.7 5.9 12.3 97 9.4 98 Control 20 20 11.5 6.0 11.5 99 7.0 17 100 ppm EGME, inhalation 20 9 3.3* 6.2 4.0* 49* 6.5

- 15* 300 ppm EGME, inhalation 20 0 *Statistically significantly different from control (p < 0.05).



### inhalation (4) and ingestion (2). The 

### workreportedhere 

### forms a 

### comparison of the results of 

### using EGME as a 

### positive control in 

### several studies to determine the 

### teratogenic potential ofa

### varietyof

### compounds. EGME hasbeen

### given

### bythe 

### followingroutes

### duringthe course 

### of these studies: 

### intraperitoneally, 

### orally, subcutane

### ously, dermallyand

### byinhalation. Theresultsare shown in 

### Table 3. It will be seen 

### that 200 to 250 

### mg/kg of 

### EGME caused 

### complete loss of litters 

### by the intra

### peritoneal route, 

### by the oral route, and when 

### given 

### subcutaneously; 300ppm

### of

### EGME 

### byinhalation, which 

### corresponds 

### approximately to a 

### dose of 350 

### mg/kg 

### assuming 100% 

### retention, caused a 

### similar result. At lower

### doses, EGME causes a 

### loss of

### approximatelyhalf the 

### litters, with 

### reduced litter size and 

### survivability in 

### the pups. 

### This is shown

### by40

### mg/kg EGME subcutane

### ously and 100 ppm 

### EGME 

### by inhalation 

### (which corres

### ponds to an 

### approximate dose of 115 

### mg/kg assuming 100% 

### retention). 

### Interestingly, a 

### similar dose 

### administered 

### dermally, i.e., 40 

### mg/kg 

### EGME, had no 

### effect on 

### the 

### developing fetus. This result indicates that the 

### skin has some 

### barrier function with 

### regard to 

### EGME. 

## Effect of 

## Ethylene 

## Glycol 

## Monoisopropyl Ether 

## (EGPE) on 

## the 

## Testis and Blood of Rats Groups of 10 

### male 

### Alpk/Ap 

### (Wistar-derived) rats were 

### exposed to 

### air, 300 or 1000 ppm 

### of EGPE for 6 

### hr/dayfor 5 

### days of

### week 1 and for 4 

### days of

### week2. 

### On the

### day

### followingthe lastexposure, 

### theratswere

### killed, blood

### sampleswere

### taken, andthe testeswere

### weighed. Rats in 

### the 1000 ppm 

### EGPE group 

### showed marked hematuriaonthe

### first

### dayofexposure; 

### subsequently, no more 

### hematuria was seen. 

### The skin of these rats was 

### pale in appearance up to 

### and 

### including day 4; subse

### quentlytheir 

### clinical condition 

### appeared comparable to the 

### controls. The 

### body weight gains of the rat in the 1000 ppm 

### EGPE group were 

### statistically significantly lower than the 

### controls, while the rats in 

### the 300 ppm groups

### hadweight

### gains similarto

### the controlgroup. At postmortem 

### the appearance 

### and 

### weights of the testis 

### from

### both the 1000 

### and 300 ppmgroups were 

### similar to 

### the controls. 202SCREENING AND HAZARD ASSESSMENT

At 1000 ppm, statistically significant reductions were seen in 

### hemoglobin concentration, red blood cell count and mean 

### corpuscular hemoglobin concentrations. In 

### addition, mean cell volumes and mean cell hemoglobin were 

### statistically significantly higher in the 1000 ppm group. These changes are consistent with the initial 

### hematuria observed in 

### these rats. There were no 

### changes inthewhite bloodcell counts, andapart froma 

### slight increase in mean cell volume there were no hematological effects in the rats exposed to 300 ppm 

### EGPE. This study indicates that EGPE behaves in a 

### similar manner to ethylene glycol monobutyl ether (EGBE) (9), i.e., the major effect appears to be 

### hemolysis, followed by replacement with resistant cells 

### which are younger 

### and larger. There was no suggestion 

### of the typical response seen with EGME of testicular 

### atrophy and 

### leukocytopenia. sure and throughout a subsequent 14-day observation period. On day 15 the animals were killed by over exposure to halothane and subjected to gross macroscopic postmortem examination, which included the weighingofthetestes. Theresultsare shownin

### Table 4. Both EGME and EGEE caused reductions in testis weight when compared with the controls on day 15, EGME causing an approximately 50% reduction and EGEE a 20% reduction. Neither EGPE nor EGBE caused a reduction in testes weight. Clinical observations also revealed another sign associated with the glycol ethers, hematuria in the animals treated with EGEE, EGPE or EGBE. EGME did not cause hematuria. These limited data indicate that both EGME 

### and 

### EGEE can cause testicular atrophy after a single, 

### albeit, very

### high, exposurebyinhalation. However, even the saturated vapor from EGPE and EGBE did not cause testicular atrophy. 

## Effect of a 

## Single Inhalation Exposure to 

## Ethylene Glycol Monoalkyl Ethers Effect of a Single Exposure to 

## (EGME, EGEE, EGPE, EGBE) EGME on the Testis of Male Rats 

### Ethylene glycol monomethyl ether (EGME) has been shown to cause testicular 

### atrophy 

### following 90 days exposure (10) 

### and 10 

### days exposure (4,7). In 

### these 

### studies, 300 ppm 

### (= 960 

### mg/mr3) caused testicular 

### atrophy. Thework

### reportedherewas 

### designed to assess whether a 

### single very 

### high exposure to 

### EGME would also cause 

### testicular 

### atrophy. In order to determine whether there is a clear structure 

### activityrelationship, ethylene 

### glycol monoethyl (EGEE), monoisopropyl (EGPE) and 

### monobutyl (EGBE) ethers were 

### also examined in the same 

### experiment. Male 250 g

### Alpk/Ap (Wistar-derived) rats were 

### exposed for 3 or 4 hr to 

### the saturated vapor 

### generated by blowing air 

### through the relevant test 

### material contained in a sintered 

### glass bubbler. The undiluted vapor was 

### led into 1 L 

### glass exposure 

### chambers, each 

### containing five rats 

### housed 

### individually. The atmosphere concentrations were 

### estimated 

### by dividing the 

### weight loss from 

### the 

### bubbler 

### by the 

### total airflow. The rats were 

### observed 

### during exponble 4. Comparative data on effect of exposure to saturated vapor of glycol ethers on testis weights at day 15. Testis weight, % of body weight Hematuria Compound and (Present concentration 3-hr exposure Control or absent) EGME, 24mg/L 0.49 ± 0.08 1.06 ± 0.06 (7500 ppm) EGEE, 17 mg/L 0.78 ± 0.07 1.06 ± 0.06 + (4500 ppm) EGPE, 15 mg/L 0.95 0

.092 0.98 ± 0.10 + (3500 ppm) EGBE, 4 mg/L 0.95 ± 0.06 0.93 ± 0.05 + (800 ppm) '4-hr exposure. *Statistically different from control (Student's t-test, p < 0.05). The previous studyhad shown that EGME is capable ofcausing 

### testicularweight loss after a 

### single exposure to a saturated vapor. These 

### studies were 

### designed to 

### determine the 

### dose-response relationship of the effect of the 

### single exposure on 

### the testes, to 

### determine the 

### histological effect, and to determine the time course of the onset 

### of testicular 

### damage. In 

### the first 

### study, groups 

### of 20 male Alpk/AP rats were 

### exposed to 5000, 2500, 1250, 625, 300 or 150 ppm EGME. Afurthergroupofratswas exposedtoaironly. Following exposure which took place in 3.4 

### m3 whole 

### body exposure 

### chambers, they were 

### returned to their 

### holding cages for 13 days, and 14 days 

### after treatment, were killed 

### with an overdose of halothane. 

### At autopsy 

### the left and 

### right testis were 

### weighed 

### separately and stored inBouin's fixative 

### for 

### subsequentprocessingand 

### microscopic examination. The caudal

### epididymideswere 

### weighed together and 

### preserved for 

### processing and microscopic examination. The accessory 

### glands, semi

### nal vesicle and ventral prostate were 

### weighed and later examined 

### histopathologically. There was a dose-related 

### fall in 

### body weight gain as 

### shown 

### by the 

### body weight immediately before postmortem. 

### Thesewere

### accompaniedbya

### dose-related fall in testes 

### weight whichwas 

### significant down to 1250 ppm 

### (Fig. 2). There were reductions in 

### the 

### caudal 

### epididymidal weight and an 

### increase in 

### the accessory 

### gland weight at 5000 ppm of 

### EGME. 

### Histological examination of the testes 

### revealed severe 

### bilateral tubular 

### atrophy with 

### disordered 

### spermatogenesis in rats 

### exposed to 5000 ppm 

### EGME for 4 

### hr, with many 

### tubules 

### showing only stem 

### cells 

### and 

### Sertoli cells. 

### Similar, though less 

### marked 

### changes, were seen in rats 

### exposed to 2500 

### and 1250 ppm

### EGME. 

### Although testes 

### weight was not 

### reduced at 625 ppm 

### EGME, maturing spermatids showed evidence of 

### damage. 203J. E. DOE

BODY WEIGHT IN -day 13(BWG) 1101

150 12SO

Exposure level E.G.M.E. (ppm

- log scale)

TESTIS WEIGHT

- day 14 (Right)

5(1 14 ----% LbeVa l3U Exposure level E.G.M.E. (ppm

- log scale)
CAU~ EPIDIULMAL WEIGHT

- y 14 ( )

- -- '' -'-

- - - - - - - - - - - - - -- -

- - - - - - -

in -_-_-

a 625 23 Exposure level E.G.M.E. (ppm

- log scale)
ACCESSORY GLANDW WErIGHT day 14

--Ep W"

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _~

- 30 625 125 250

Exposure level E.G.M.E. (ppm

- log stale) FIGURE 2. Effect on rat testis weight of a single exposure of EGME. Significance as in Figure 1.

Inthe secondstudy, groups of90, 8-week-oldAlpk/AP rats were exposed to either 2500 or 1000 ppm EGME for a 

### single 4-hrperiod. A further group of 90 rats was 

### sham-exposed to air. Ten rats per group were killed on each of 

### days 1, 2, 3, 4, 5, 8, 10, 15 and 19 

### following exposure. Individual 

### body weights were recorded on the 

### daybefore exposure and 

### subsequently ondays 4, 8, 15 and 19. 

### Testes, epididymides and accessory gland weights were recorded at autopsy. 

### Samples of these tissues were preserved in Bouin's fixative prior to examination. The results are shown in 

### Figure 3. In the second 

### study, testis 

### weight reduction was seen in 

### exposed rats 48 hr after exposure to both 2500 and 1000 ppm, and testes 

### weights were 

### lower than control 

### values in 

### both the 

### exposed groups at 

### subsequent time 

### points. Histological examination of the testes revealed 

### damage to the

### germinal epithelium24 hrafter exposure with 

### primary spermatocytes as the target cells for 

### EGME. 

### Recovery was not evident at 

### day 19 

### following exposure to 

### 2500 ppm

### EGME, and the germinal 

### epithelium remained 

### disordered. Electron 

### microscopy revealed 

### changes in the 

### Sertoli 

### cells, with 

### cytoplasmic retractionand

### swollen

### mitochondriaevident 4

### daysafter exposure to 

### EGME. There was a biphasic response in BUG

Cont,' I o means

110o 2500



## Ioo

- 3000

Ut. 90

X 0 Cont'l means 70

6C

2500

110
Ut. oo

Cont'I means
70

Ut. I
cont'1 mans 10 0

- - :p

Eoh,ft ah%f%^ I 1n - -nv204

JW 625

uW 3USCREENING AND HAZARD ASSESSMENT

TESTIS WEIGHT

- (Right) (Values for right and left testes were similar)

8 10 DAYS POST EXPOSURE

CAUDA EPIDIDYMAL WEIGHT

- (Paired)

8 10 DAYS POST EXPOSURE

ACCESSORY GLAND WEIGHT

*

Nb-~ ** ** 1 2 3 4 5 8 10

DAYS POST EXPOSURE

FIGURE 3. Time course of the effect of a single exposure of EGME in male rats. Significance as in Figure 1. exposed animals withregard to epididymal weight. The increased weight at day 4 probablyreflects the passage through the epididymides at thispoint oflarge numbers of immature germ cells shed from the testes following the initial insult. Subsequently, epididymal weights

were reduced when compared to control values, again reflecting the situation in the testes where there was a marked reduction in the number of spermatozoa produced following exposure. EGME would appear to be exerting its effect primarily on rapidly dividing cells, with primary spermatocytes being affected initially. Subsequently, Sertoli cells showed signs ofdamage and this may account for the resulting marked loss of immature germ cells. These studies indicate that even a relatively short exposure to EGME vapor can causedmarked testicular atrophy although at high concentrations. Even at concentrations comparable with those causing testicular atrophy over a 90-day period (i.e., 300 ppm versus 625 ppm), there were subtle effects on spermatogenesis highlighting the need for further study of the effect of EGME on the testes. The results were reported previously (11). 

## General 

## Conclusion The work reported in this paper demonstrates very clearlythe role oftoxicology examinations which do not 110
100 Wt. 90

8C Cont'l 70

means 6o 2 3 4 5 15 19

** 130
120 Wt. 110 ^,100 ,0 Cont'l 90

means 80 70 **

2 3 4 5 15 **

110 19

Wt.

Contr'

means 100

90

80

15 19 205206 J. E. DOE



### fall clearly into the type of testing currently done for regulatorypurposes. BoththetesticulareffectofEGME and 

### the effect on the developing fetus can be seen in relatively short term and/or inexpensive experiments. The effect of EGME on the testis has been shown after 10days exposure orindeedaftera

### single exposure. This has 

### enabled the effect of other glycol ethers to be 

### examined very 

### quickly. These 

### data were in many cases 

### generatedbefore the effects oftheother

### glycol ethers in regulatory type studies were 

### available and where 

### this has 

### occurred these studies have 

### accurately predicted the 

### effect in the 

### regulatory studies. 

### Thus, EGME has been 

### shownto

### affect the testis (10). 

### EGBE onthe other hand, does not appear to affect the testis in a similar manner (9). It is therefore reasonable to assume that EGPE which did not affect the testes 

### weights after 

### either 10 

### days exposure at 1000 ppmor 4hrexposure to a saturated vaporwould not cause 

### testicular

### atrophy in a 

### longer regulatory study. The effect of EGME upon the developing embryo is 

### dramatically revealed 

### using the test for 

### teratogenic potential (6). Athigherlevels EGME causes complete resorption

### of all 

### embryos and at 

### lower levels 

### all three of 

### the factors suggested by Chernoff and Kavlock (6) 

### affected, i.e., numbers 

### oflitters, numbers 

### ofpups, 

### their 

### survivability to

### day4. Again, 

### theseresults are inagreementwith

### the results of full teratology studies which have been carried out with EGME. EGME has been shown to be fetotoxic and teratogenic in the rabbit (10) and in the mouse (2). This activity of EGME gives added confidence to the results found with 

### PGME and 

### with diEGME in 

### this test. Even though EGME has such a dramatic 

### effect, neither PGME nor 

### diEGME caused any significant changes in the parameters listed by 

### Chernoffand 

### Kavlock (6). It is 

### important to 

### remember, however, that inboth the studies reportedhere the dose 

### oftest 

### compound was not so

### high that 

### there was severe 

### maternal 

### toxicity. It is important when trying to interpret the results ofthis type of study 

### that extrava

### gantlyhighdoses arenotused. These studies

### give a

### fair amount ofconfidence in

### predictingthat

### neither

### diEGME nor 

### PGME will 

### be significantly teratogenic. Taken overall, these studies indicate that evenwith a limited resource in terms of time 

### and availability of 

### experimental facilities, significant 

### knowledge can 

### be gained on the 

### toxicology of a group of compounds, provided that agoodpositive control 

### substance, suchas EGME, is available. The studies reported here were carried out as a collaborative effort by I. S. Chart, D. Hart, L. K. Head, S. Millward, L. Pinto, D. M. Samuels, D. J. Tinston, G. A. Wickramaratne and D. R. Woodcock. Thanks are also due to Miss L. Pritchard for typing this manuscript.

REFERENCES
1. Nagano, K., Nakayama, E., Oobayashi, H., Yamada, T., Adachi, H., Nishizawa, T., Ozana, H., Nakaichi, M., Okuda, H., Minami, K., and Yamazaki, K. Embryotoxic effects of ethylene glycol monomethyl ether in mice. Toxicology 39: 335-343 (1981). 2. Nagano, K., Nakayama, E., Hoyano, M., Dobayski, H., Adachi, H. andYamada, T. Testicularatrophyofmice inducedbyethylene glycol monoalkyl ethers. Japan. J. Ind. Health 21: 29-25 (1979). 3. Andrew, F D., Buschbom, R. L., Cannon, W C., Miller, R. A., Montgomery, L. F., Phelps, D. W, and Sikov, M. R. Teratologic assessment of ethylbenzene and 2-ethoxyethanol. Batelle Pacific Northwest Laboratories, report to NIOSH, 1981. 4. Miller, R. R., Ayres, J. A., Calhoun, L. L., Young, J. T., and McKenna, J. M. Comparative short-term inhalation toxicity of ethylene glycol monomethyl ether and propylene glycol monomethyl ether in rats and mice. Toxicol. Appl. Pharmacol. 61: 368-377 (1981). 5. Doe, J. E., and Tinston, D. J. Novel chambers for long term inhalation studies. In: Proceedings of the Inhalation Toxicology and Technology Symposium (B. K. J. Leong, Ed.), Ann Arbor Science Publishers, Ann Arbor, MI, 1981. 6. Chernoff, N. and Kavlock, R. J. A potential in vivo screen for the determination of teratogenic effects in mammals. Teratology 21 (2): 33A (1980). 7. Doe, J. E., Samuels, D. M., Tinston, D. J., and Wickramaratne, G. A. Comparative aspects of the reproductive toxicology by inhalation in rats of ethylene glycol monomethyl ether and propyleneglycolmonomethyl ether. Toxicol. Appl. Pharmacol. 69: 43-47 (1983). 8. Doe, J. E., Hart, D., andWickramaratne, G. A. The teratogenic potential of diethylene glycol monomethyl ether (DGME) as assayed in the post natal development test by the subcutaneous route in rats. Toxicologist 3: 70 (1983). 9. Dodd, D. E., Snellings, W M., and Ballantyne, B. Ethylene glycolmonobutyl ether (EGBE) vapor 9dayand 90dayinhalation studies in Fischer 344 rats. Toxicologist 2: 159 (1982). 10. Miller, R. R., Ayres, J. A., Young, J. T. and McKenna, M. J. Ethyleneglycolmonomethyl ether. I. Subchronicvaporinhalation study with rats and rabbits. Fundam. Appl. Toxicol. 3: 49-54 (1983). 11. Samuels, D. M., Doe, J. E., andTinston, D. J. The effects on the rat testis of single inhalation exposures to ethylene glycol alkyl ethers, in particular ethylene glycol monomethyl ethers. Paper presented at European Society of Toxicology Meeting, Rome, March 1983.