the development of forestomach tumours in the mouse following exposure to 2-butoxyethanol by...

The development of forestomach tumours in the mousefollowing exposure to 2-butoxyethanol by inhalation: studies

on the mode of action and relevance to humans

Trevor Green *, Alison Toghill, Robert Lee, Richard Moore, John Foster

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK

Received 2 April 2002; received in revised form 8 July 2002; accepted 8 July 2002

Abstract

2-Butoxyethanol, a forestomach carcinogen in mice exposed by inhalation, has been shown to enter the forestomach

as a result of grooming and ingestion of material condensed on the skin and fur during exposure. The material entering

the stomach concentrates in the forestomach region and persists for at least 48 h post-exposure. Mice given single oral

doses of either 2-butoxyethanol or 2-butoxyacetic acid, daily for 10 days, developed a marked hyperkeratosis in the

forestomach. 2-Butoxyacetic acid was more potent than 2-butoxyethanol, the NOEL for the former being 50 mg/kg and

for the latter, 150 mg/kg. Although a dose dependent increase in cell replication was also seen with both chemicals, the

results were confounded by a high labelling rate in the controls. There was no evidence of significant binding of

radiolabelled 2-butoxyethanol to proteins in stomach tissues. 2-Butoxyethanol was metabolised in vitro in both mouse

and rat forestomach and glandular stomach fractions by alcohol dehydrogenases forming 2-butoxyacetaldehyde which

was rapidly converted by aldehyde dehydrogenases to 2-butoxyacetic acid. There was a marked species difference in

alcohol dehydrogenase activity between rats and mice with the maximum rates up to one order of magnitude greater in

mouse than rat. The alcohol and aldehyde dehydrogenases were heavily concentrated in the stratified squamous

epithelium of the forestomach of both rats and mice whereas in the glandular stomach the distribution was more

diffuse. In human stomach both enzymes were evenly distributed throughout the epithelial cells of the mucosa. It is

concluded that 2-butoxyethanol is ingested following inhalation exposure and concentrates in the forestomach where it

is metabolised to 2-butoxyacetic acid which causes cellular damage, increased cell replication and hyperkeratosis. These

changes are believed to lead to the tumours seen in mice exposed to 2-butoxyethanol for a lifetime. Differences in

structure and enzyme distribution between the rodent and human stomach suggest that the responses seen in the mouse

are unlikely to occur in humans. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: 2-Butoxyethanol; Mouse stomach tumours; Mode of action

1. Introduction

2-Butoxyethanol is used extensively as a solvent

in surface coatings, in degreasers and in a variety

of industrial and household cleaners. Its primary

* Corresponding author. Tel.: �/44-1625-515-458; fax: �/44-

1625-586-396

E-mail address: [email protected] (T. Green).

Toxicology 180 (2002) 257�/273

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acute toxicity in rodents is haemolysis of erythro-cytes leading to an anaemia and haematuria. Other

species vary markedly in their sensitivity to

butoxyethanol induced hemolysis, human erythro-

cytes being relatively less sensitive than those of

rats and mice (Ghanayem and Sullivan, 1993).

Following chronic exposure of B6C3F1 mice to 2-

butoxyethanol by inhalation at concentrations up

to 250 ppm, increased incidences of squamous cellpapillomas of the forestomach were seen in

females and hepatic haemangiosarcomas in male

mice (NTP, 1998). These tumours were not seen in

F344/N rats exposed to 125 ppm in the same

study.

2-Butoxyethanol is devoid of any genotoxic

activity (Elliott and Ashby, 1997) suggesting a

non-genotoxic mode of action for both tumourtypes. It has been proposed that the development

of the haemangiosarcomas may be related to the

haemolysis and subsequent accumulation of hae-

mosiderin in phagocytic endothelial cells resulting

in iron catalysed oxidative damage (Park et al.

2002). The development of forestomach tumours

has not been explained. Forestomach tumours in

rodents are normally associated with gavage dos-ing and it was somewhat surprising to find an

increase following exposure by inhalation. These

tumours are also uncommon in control mice of

this strain. There was, however, evidence in the

NTP 14 week study of a dose dependent inflam-

mation, necrosis, ulceration and epithelial hyper-

plasia of the forestomach of exposed mice (NTP,

1998), responses typical of irritant chemicals givenby gavage. Pre-cursor lesions of this type have also

been associated with the subsequent development

of forestomach tumours.

The haemolytic properties associated with 2-

butoxyethanol are known to be a consequence of

its metabolism by alcohol and aldehyde dehydro-

genases to 2-butoxyacetic acid, the major metabo-

lite in most species (Ghanayem, 1989; Dill et al.1998). 2-Butoxyacetic acid is also an irritant and

could, therefore, be responsible for the toxicities

seen in the mouse forestomach if it were produced

in situ from 2-butoxyethanol. The presence of

alcohol and aldehyde dehydrogenases in the gas-

tro-intestinal tract is well established (Pestalozzi et

al., 1983; Haselbeck and Duester, 1997) suggesting

that such an explanation is feasible, assuming that2-butoxyethanol enters the stomach following

inhalation exposure. There are two possible ways

in which the latter could occur; either as a result of

inhaled material entering the stomach via the

buccal cavity and oesophagus or systemically

following absorption through the lungs into blood.

In this study we have sought to determine why

2-butoxyethanol targets the mouse forestomachfollowing inhalation exposure. To that end, whole

body autoradiography techniques have been used

to determine the uptake and distribution of radio-

activity following inhalation exposure to radiola-

belled 2-butoxyethanol. The stomachs of mice

concurrently exposed to radiolabelled 2-butox-

yethanol have been analysed for 2-butoxyethanol

and covalently bound radioactivity, and the effectsof 2-butoxyethanol and 2-butoxyacetic acid on

stomach morphology and cell replication rates

have been compared when these chemicals were

placed directly in the stomach by gavage dosing

for 10 days. The metabolic capacity of the rat and

mouse stomach has been compared in an attempt

to explain the lack of stomach tumours in rats

exposed to 2-butoxyethanol. Comparisons, usinghistocytochemical techniques, of the distribution

of the dehydrogenase enzymes involved in 2-

butoxyethanol metabolism have been made in

mouse, rat and human stomachs in order to assess

the potential susceptibility of the human stomach

to the effects seen in mice.

2. Materials and methods

2.1. Chemicals

2-Butoxyethanol (ethylene glycol monobutyl

ether; 99%) was obtained from Sigma�/Aldrich,

Poole, Dorset, UK. Bromodeoxyuridine (BrdU)

and nicotinamide adenine dinucleotide (NAD)

were also obtained from Sigma�/Aldrich. 2-Butox-yacetaldehyde was prepared as described by Hatch

and Nesbitt (1945) and 2-butoxyacetic acid by the

method of Rule et al. (1928). Both products were

characterised by NMR and shown to have purities

of �/99%. 2-Butoxy[1-14C]ethanol (97.6%) was

obtained from Amersham Life Sciences, Buckin-

T. Green et al. / Toxicology 180 (2002) 257�/273258

ghamshire as a custom synthesis. It had a specificactivity of 10 mCi/mmol.

2.2. Animals

Female B6C3F1 mice weighing 20�/25 g were

obtained from Harlan Olac, UK. The animals

received Rat and Mouse Number 1 (RM1) pelleted

diet from Special Diet Services, Witham, Essex,UK, and water ad libitum except during the

exposure period. The environment of the animal

room was controlled with the temperature being

maintained within a target range of 229/3 8C, a

relative humidity of 30�/70%, an artificial light

cycle of 12 h light and 12 h darkness and at least 12

air changes per hour. Animals were acclimatised

for 5 days and identified by tail marking prior tothe start of the study.

2.3. Human tissues

Human stomach tissue was obtained from the

Peterborough Hospital Human Research Tissue

Bank, Peterborough, Cambridgeshire. The tissue

was from a 68-year-old Caucasian male. It wasremoved during surgery (oesophagogastrectomy),

snap frozen, and transported to our laboratory at

�/70 8C.

2.4. Whole body autoradiography

Female mice (n�/12) were exposed whole body

in glass chambers of approximately 20 l capacity to

an atmosphere of 250 ppm 2-butoxy[1-14C]ethanol(specific activity 0.365 mCi/mmol) for 6 h. The

atmosphere, which was passed through the cham-

ber at a flow rate of 1.5 l/min, was generated by

allowing a controlled flow of 2-butoxy[1-14C]etha-

nol to evaporate into a stream of air at 90 8C. 2-

Butoxyethanol levels in the chamber were mon-

itored throughout the experiment using a Shi-

madzu GC-14BT gas chromatograph fitted with a10% Carbowax 20M column (1/8 in.�/6 ft) at

115 8C and a flame ionisation detector. 2-Butox-

yethanol had a retention time of 5.65 min. 2-

Butoxyethanol leaving the exposure chamber was

removed from the atmosphere with a cold trap at

�/70 8C.

At the end of the exposure period the animalswere removed from the chambers and placed in

wire-bottomed cages with free access to diet and

water. The mice (4 per time point) were terminated

with halothane at 5 min, 24 and 48 h post

exposure. One animal from each time point was

rapidly frozen in a mixture comprising iso-hexane

and solid carbon dioxide. The frozen carcasses

were embedded in blocks of 2% (w/v) aqueouscarboxymethylcellulose and transferred to a cryo-

stat (LEICA CM3600) at �/20 8C. Longitudinal

sagittal sections, 40 mm thick, were taken,

mounted on adhesive tape and freeze-dried for

48 h. Autoradiographs were prepared by contact

with autoradiographic film (Hyperfilm b-max,

Amersham, UK) for periods of 1, 2, 4 and 6

weeks. The film was developed and fixed beforewashing and drying.

In a second experiment, female mice (n�/12)

were given a single bodyweight dependent dose of

10 mg/kg 2-butoxy[1-14C]ethanol (850 mCi/kg) by

intravenous injection into the tail vein. The dose

was dissolved in physiological saline (5 ml/kg). The

animals were placed in wire-bottomed cages with

free access to diet and water. The mice (4 per timepoint) were terminated with halothane 4, 24 and

48 h after dosing and whole body autoradiograms

prepared from one animal per time point as

described above.

2.5. Analysis of the stomach and contents

The remaining three animals from each time

point were terminated with an overdose of ha-lothane and the stomachs removed and opened.

The stomach contents were removed, pooled by

time point, and weighed. The forestomach and

glandular stomach were separated, pooled by time

point, weighed and scissor-minced. An aliquot of

each pooled sample was solubilised overnight in 1

ml Soluene. A total of 10 ml Hionic Fluor

scintillant was added and the radioactivity contentdetermined using a liquid scintillation spectro-

meter. The remainder of each sample was homo-

genised in SET buffer (250 mM sucrose, 5 mM

EDTA, 20.0 mM Tris), pH 7.4, and extracted with

2 ml ethyl acetate and assayed for radioactivity by

liquid scintillation counting. The extracts were

T. Green et al. / Toxicology 180 (2002) 257�/273 259

concentrated under a stream of nitrogen to give afinal volume of approximately 0.12 ml prior to

analysis by HPLC. A Hypersil ODS column,

operated at a flow rate of 1 ml/min, was used for

the analysis. The column was eluted with a linear

gradient changing from 100%, 10 mM aqueous

trifluoroacetic acid to 100%, 10 mM aqueous

trifluoroacetic acid:acetonitrile (1:3) over a

30 min period. 1 ml fractions were collected fromthe column and the radioactivity content deter-

mined by scintillation counting. The retention time

for 14C 2-butoxyethanol under these conditions

was 16.5 min.

The above experiment indicated that the

amount of radioactivity bound to the stomachs

of mice exposed to 2-butoxyethanol was minimal.

In order to facilitate characterising this material,maximal doses of 2-butoxyethanol were given by

the oral route. Ten female mice were each given

single doses of 500 mg/kg 2-butoxy[1-14C]ethanol

(5.5 mCi/kg), daily for 5 days. Seventeen hours

after the final dose the mice were killed with an

overdose of halothane anaesthetic and the sto-

machs removed, separated into fore- and glandu-

lar stomachs and rinsed with ice-cold 1.15%potassium chloride solution. The tissues were

pooled by type and homogenised in 10 mM

Tris�/HCl buffer, pH 7.5 containing 0.5 mM

dithiothreitol. Protein concentrations in the homo-

genates were determined by the method of Lowry

et al. (1951). Homogenates were stored at �/70 8Cuntil required.

Total radioactivity present in the tissues wasdetermined by digesting 0.1 ml aliquots of the

homogenates in 1 ml Soluene 350 at 37 8C for 3 h.

A total of 10 ml of Hionic-Fluor scintillant was

then added and the radioactivity determined by

scintillation counting.

Covalently bound radioactivity was measured as

follows. A total of 0.5 ml aliquots of the stomach

homogenates were mixed with 0.5 ml ice cold 20%trichloroacetic acid to precipitate the proteins. The

samples were placed on ice for 30 min, before

being centrifuged at 1500�/g for 15 min at 4 8C.

The supernatants were removed, neutralised with 5

M hydrochloric acid, 10 ml Ultima-Gold scintil-

lant was added, and the radioactivity content

determined by liquid scintillation counting. The

protein pellets were then repeatedly washed with4 ml ice-cold methanol. Following centrifugation

at each washing stage, 0.5 ml supernatant was

counted in 10 ml Ultima-Gold scintillant. The

protein pellets were finally air dried overnight

before being dissolved in 0.5 ml 0.1 M sodium

hydroxide. Duplicate 25 ml aliquots of each pellet

sample were counted in 10 ml Ultima-Gold

scintillant. The protein content of each pelletsample was determined by the method of Lowry

et al. (1951).

Approximately 35 mg of forestomach and 50 mg

of glandular stomach proteins were separated

using a 10�/20% linear gradient SDS gel. Follow-

ing overnight transfer onto a nitrocellulose sheet

the proteins were visualised using a phosphor-

imager.

2.6. Alcohol and aldehyde dehydrogenase activities

Tissue preparation and the dehydrogenase as-

says were based on the methods of Algar et al.

(1983) and Julia et al. (1987). Rats (typically n�/

10) and mice (typically n�/30) were sacrificed with

an overdose of halothane. The stomachs were

removed, and separated into fore and glandularsections. The tissues were pooled by species and

tissue type, thoroughly washed in ice cold 1.15%

potassium chloride, and homogenised in 10 mM

Tris�/HCl pH 7.5/0.5 mM dithiothreitol using an

Ultra Turrux homogeniser. The homogenates were

centrifuged at 41 000�/g for 30 min at 4 8C. The

supernatants were stored at �/70 8C until re-

quired. Protein content was determined as de-scribed by Lowry et al. (1951).

The alcohol dehydrogenase catalysed conver-

sion of 2-butoxyethanol to 2-butoxyacetaldehyde

was determined by measuring the reduction of

NAD in the presence of 0.5 mM semicarbazide.

The incubation mixtures contained 0.25�/100 mM

2-butoxyethanol, 0.75 mM NAD, 0.5 mM semi-

carbazide, and stomach 41 000�/g supernatent(between 0.33 and 0.75 mg protein/ml) in a final

volume of 3 ml of 100 mM sodium phosphate, pH

8.8/9 mM glycine, at 25 8C. The mixture was pre-

incubated for 2 min prior to the initiation of the

enzyme reaction by the addition of 2-butoxyetha-

nol. The conversion of NAD to NADH over


10 min, monitored spectrophotometrically at340 nm, was used as a measure of enzyme activity.

The aldehyde dehydrogenase catalysed conver-

sion of 2-butoxyacetaldehyde to 2-butoxyacetic

acid was determined by measuring the reduction

of NAD. The incubation mixtures contained

0.005�/1.5 mM 2-butoxyacetaldehyde, 0.75 mM

NAD, and stomach 41 000�/g supernatant (be-

tween 0.22 and 0.38 mg protein/ml) in a finalvolume of 3 ml of 100 mM sodium phosphate, pH

8.0, at 25 8C. The mixture was pre-incubated for 2

min prior to the initiation of the enzyme reaction

by the addition of 2-butoxyacetaldehyde. The

conversion of NAD to NADH, monitored spec-

trophotometrically at 340 nm over 10 min, was

used as a measure of enzyme activity.

Michaelis�/Menten constants Km and Vmax werecalculated by non-linear regression of the initial

velocity on the substrate concentration using

GraFit data fitting software (Leatherbarrow,

1998). Initial estimates were derived from Hofstee

plots.

2.7. Histocytochemistry

The distribution of alcohol dehydrogenases wasdetermined in sections of human stomach and

female rat and mouse forestomach and glandular

stomach. Cryostat sections of unfixed tissue were

incubated for 1 h at 37 8C in a solution containing

0.4 M 1-butanol, 3 mM NAD, 5 mM MgCl2,

1 mM niacinamide, 1.2 mM nitroblue tetrazolium

in 15 mM phosphate buffer, pH 7.4, then rinsed

briefly in the same buffer, followed by distilledwater. Sections were post-fixed in 10% neutral

buffered formalin for 10 min, washed, then

mounted in an aqueous mounting medium.

The distribution of aldehyde dehydrogenases

was determined as described by Bogdanffy et al.

(1986). Tissue sections were incubated in sealed

Coplin jars for 24 h at 37 8C in 40 ml of 50 mM

phosphate buffer (pH 7.4) containing 5 mMacetaldehyde, 11.3 mM sodium azide, 5.5 mM

MgCl2 �/ 6H2O, 0.34 mM nitroblue tetrazolium,

1.7 mM NAD and 1.5 mM pyrazole. Following

incubation, the slides were air dried, counter-

stained with nuclear fast red, and mounted in an

aqueous mountant.

The regions of the stomachs showing staining

were quantified as follows. The thickness of the

glandular region and of the forestomach in which

the dehydrogenases were localised was measured

using a KS400 image analysis system (Imaging

Associates, Thame, UK). About two-thirds of the

glandular region was stained and all of the stratum

corneum of the forestomach. The area of each

region was determined from photographs of dis-

sected stomachs by image analysis. The volume of

each region was then calculated.

2.8. Toxicity and cell proliferation studies

The effects of 2-butoxyethanol and 2-butoxya-

cetic acid on stomach morphology and cell repli-

cation rates were determined in mice over a period

of 10 days. Female mice, 5 per group, were given

single oral doses of either 0, 50, 150 or 500 mg/kg

2-butoxyethanol or 0, 50, 150 or 500 mg/kg 2-

butoxyacetic acid, daily for 10 days. Female mice

were used because they gave a slightly greater

carcinogenic response in the 2 year study (NTP,

1998). The doses were prepared in polyethylene

glycol at a concentration which gave a dosing

volume of 5 ml/kg. Seventeen hours after the final

dose and 1 h prior to sacrifice, each mouse

received a single bodyweight dependent subcuta-

neous injection of bromodeoxyuridine (15 mg/kg)

in physiological saline (4 ml/kg). The mice were

killed by exsanguination (cardiac puncture) under

terminal anaesthesia induced with halothane. The

stomachs were removed for histopathological ex-

amination and quantitation of cell replication

rates.

Stomachs were fixed in formol saline and

processed to paraffin wax. Sections from each

animal were stained with haematoxylin and eosin.

BrdU positive nuclei were visualised using a

histochemical technique (Soames et al., 1994).

Rates of cell replication were calculated as a unit

length labelling index (ULLI) and expressed as the

number of cells per millimetre of epithelium. Five

fields of the forestomach were observed with a

�/10 objective lens.


3. Results

3.1. Whole body autoradiography

Sections taken from mice exposed to 250 ppm 2-

butoxy[1-14C]ethanol were remarkable in three

ways. Firstly, the fur and skin of the animals

contained high concentrations of radioactivity,

even 48 h after the end of exposure (Figs. 1 and

2). Secondly, there was clear evidence of radio-

activity in the buccal cavity and oesophagus, andthirdly, there was evidence of selective accumula-

tion of radioactivity in the forestomach (Fig. 2).

The glandular stomach was conspicuously differ-

ent from the forestomach in having low back-

ground levels of radiolabelling. These observations

indicate that radiolabelled 2-butoxyethanol con-

denses on the fur and skin of mice during exposure

and suggest that subsequent grooming by theanimal results in ingestion of this material and

entry into the stomach via the buccal cavity and

oesophagus. Condensation of inhaled material in

the nasopharynx is also a potential source of

radioactivity in the oesophagus. On entering the

stomach the radioactivity selectively accumulates

in the forestomach. These processes continue for

at least 48 h following a single exposure.Following intravenous dosing, radioactivity was

also present in the buccal cavity and oesophagus,

even up to 48 h after dosing (Fig. 3). Not

surprisingly following an intravenous dose, thefur and skin were not the origin of this material.

One source of the material in the upper gastro-

intestinal tract would appear to be the secretory

glands of the head region, the Harderian and

salivary glands, which contained high concentra-

tions of radioactivity. Ingestion of excreted mate-

rial may also have contributed. The mucosa of the

forestomach and glandular stomach were radiola-belled although the marked difference between the

glandular and forestomach seen following inhala-

tion exposure was no longer apparent suggesting

that the amount entering the stomach after an

intravenous dose is significantly less than that after

inhalation exposure.

3.2. Alcohol and aldehyde dehydrogenase activities

in the mouse stomach

Alcohol and aldehyde dehydrogenase activity,

measured using 2-butoxyethanol and 2-butoxya-

cetaldehyde respectively, could be detected in both

rat and mouse fore- and glandular stomachs (Fig.

4). Generally, the activities were similar in both

regions of the stomach and there were no major

species differences in aldehyde dehydrogenase

activity. There was, however, a marked speciesdifference in alcohol dehydrogenase activity be-

tween rats and mice, both in the affinity constants

(Km) and the maximal rates (Vmax) (Table 1). Km

Fig. 1. WBA section of a female mouse killed 24 h after a 6 h exposure to 250 ppm 2-butoxy[1-14C]ethanol.


values were two orders of magnitude greater in

both regions of the mouse stomach than equiva-

lent values in the rat, and maximum rates were up

to one order of magnitude greater in mouse than

rat.

3.3. Histocytochemistry

The distribution of alcohol and aldehyde dehy-drogenase activities in rat and mouse stomachs

was essentially identical. Sections of mouse sto-

mach are shown in Figs. 5 and 6. In both species,

alcohol dehydrogenase staining was highest in the

stratified squamous epithelium of the forestomach.

In the glandular stomach, activity was more

diffuse and was seen in the surface and neck cells

and to a lesser extent in scattered parietal cells.

The distribution of aldehyde dehydrogenase activ-

ity was essentially the same although the overall

staining intensity was greater and more parietal

cells in the glandular stomach had activity. Again,

the intensity of staining in the forestomach was

markedly greater than that in the glandular

stomach.

The volume of stomach tissue containing dehy-

drogenase activity in the rat was calculated to be 6

mm3 in the forestomach and 153 mm3 in the

glandular stomach, a ratio of 1:25. The distribu-

tion in the mouse was visually identical and the

ratio was assumed to be the same.

Fig. 2. WBA section of a mouse killed 48 h after a 6 h exposure to 250 ppm 2-butoxy [1-14C]ethanol.


Fig. 4. The in vitro metabolism of 2-butoxyethanol by alcohol dehydrogenase in rat and mouse glandular and fore-stomach fractions.

The in vitro metabolism of 2-butoxyacetaldehyde by aldehyde dehydrogenase in rat and mouse glandular and fore-stomach fractions.

Fig. 3. WBA section of a female mouse killed 4 h after a 10 mgkg i.. injection of 2-butoxy1-14Cethanol.


In sections of human stomach, alcohol and

aldehyde dehydrogenases were found throughout

the mucosa (Fig. 7). Alcohol dehydrogenase waspresent in the highest concentrations in the mu-

cous produced by the cells at the surface and was

noticeable in the inter-cellular spaces between

these cells. Aldehyde dehydrogenase was also

present in the highest concentrations in the surface

cells and in the pepsin secreting Chief cells towards

the base of the mucosal layer.

3.4. Toxicity and cell proliferation studies

Mice dosed with 2-butoxyethanol or 2-butox-

yacetic acid were clinically normal throughout the

study. One animal in the group dosed with 500 mg/

kg 2-butoxyacetic acid died during the study. The

death was not treatment related.

No macroscopic findings were present at post

mortem. The microscopic findings are summarised

in Table 2. The only dose dependent finding was amarked increase in hyperkeratosis, a thickening of

the keratinized layers, in the forestomach (Fig. 8).

The effect was most prominent in the mice dosed

with 500 mg/kg 2-butoxyacetic acid where all

animals in the group showed the effect. Minimal

changes were seen in three out of five animals

dosed with 150 mg/kg 2-butoxyacetic acid and 50

mg/kg was a no effect level. Keratosis was seen intwo out of five animals dosed with 500 mg/kg 2-

butoxyethanol but not in any of the mice at either

of the two lower dose levels. No effects were seen

in the glandular stomachs at any of the dose levels.

The results from the cell replication studies are

summarised in Table 3. Within the dosed groups

there was a dose dependent increase in the number

of cells in S-phase for both 2-butoxyethanol and 2-

butoxyacetic acid. However, because of a high

value for the control group, none of the changes in

the dosed groups were significantly increased.

3.5. Protein binding

In mice killed immediately after the end of a 6 h

inhalation exposure, most (�/70%) of the

radioactivity present in the stomach and its

contents was extractable into ethyl acetate and

analysis of the extracts by hplc detected a single

component which was identified as 2-

butoxyethanol. The remaining radioactivity, which

was more polar in nature than 2-butoxyethanol,

could not be identified because of the low levels of

material present at any given time and difficulties

in separation and isolation of this material. At the

24 and 48 h time points, the residual radioactivity

present in the stomach tissues was largely (�/80%)

covalently bound to protein. There was no

significant difference between the glandular and

forestomach and the absolute concentrations of

bound material were very low, B/0.4 nmol

equivalents/mg stomach tissue. When washed

forestomachs and glandular stomachs taken from

mice given daily oral doses of radiolabelled 2-

butoxyethanol for 5 days were homogenised, the

homogenate supernatents were found to contain

1074 and 1823 nmol equivalents of non-covalently

bound radioactivity respectively. The concentrat-

ions which were covalently bound, 8.5 and

5.9 nmol equivalents/mg protein in forestomach

Table 1

Alcohol and aldehyde dehydrogenase activities in rat and mouse stomach fractions

Species/tissue Alcohol dehydrogenase Aldehyde dehydrogenase

Km (mM) Vmax (nmol/min per mg) Km (mM) Vmax (nmol/min per mg)

Rat forestomach 0.29 1.627 0.037 3.738

Rat glandular stomach 0.73 2.170 0.029 5.624

Mouse forestomach 46.59 17.094 0.056 8.576

Mouse glandular stomach 87.01 13.986 0.135 8.950

Dehydrogenase activities were measured in stomach 41 000�/g supernatant fractions. Alcohol dehydrogenase was measured using

2-butoxyethanol as the substrate and aldehyde dehydrogenase with 2-butoxyacetaldehyde as the substrate.


and glandular stomach respectively, were again

very low and precluded any structural identifica-

tion of the bound material. Consistent with this,

SDS-PAGE analysis failed to detect any radio-

activity that was specifically bound to individual

proteins. These results suggest that covalent bind-

ing is not a major feature of the toxicity of 2-

butoxyethanol.

Fig. 5. The distribution of alcohol dehydrogenase in forestomach and glandular stomach of the mouse.


4. Discussion

The finding of forestomach papilloma following

exposure by inhalation is rare and implies that the

chemical either enters the stomach through the

buccal cavity and oesophagus or that it targets the

stomach systemically following absorption

through the lungs. These studies demonstrated

Fig. 6. The distribution of aldehyde dehydrogenase in forestomach and glandular stomach of the mouse.


that radioactivity entered the stomach via the

buccal cavity and the oesophagus after inhalation

exposure. Analysis of the stomach contents identi-

fied the radioactivity in the stomach as 2-butox-

yethanol itself. The major source of the ingested

material appears to be the skin and fur of the

animals where 2-butoxyethanol was found to

condense during exposure, the condensate then

Fig. 7. The distribution of alcohol and aldehyde dehydrogenases in human stomach.


entering the upper gastro-intestinal tract during

grooming. It also seems probable that 2-butox-

yethanol condensed in the nasopharynx during

inhalation exposure and was subsequently swal-

lowed (muco-ciliary clearance). The fact that 2-

butoxyethanol behaves in this way is not too

surprising since it is a high boiling (171 8C), fully

water miscible, relatively non-volatile material

which forms a vapour which is prone to conden-

sing onto any surface. 250 ppm is a relatively high

concentration for such a low volatility material

and corresponds to about 20% of the theoretical

saturated vapour concentration. Consistent with

this, an atmosphere of 250 ppm 2-butoxyethanol

was found to readily condense onto most surfaces

at room temperature.

Although there is evidence for ingestion of test

material, inevitably a large proportion of the

inhaled material will enter the systemic circulation

via the lungs. The stomach lesion noted in this

study, and in the NTP studies (NTP, 1998), was of

Table 2

Incidence of microscopy findings in female mice given single oral doses of either 2-butoxyethanol or 2-butoxyacetic acid daily for 10

days

Control 2-Butoxyethanol (mg/kg) 2-Butoxyacetic acid (mg/kg)

0 50 150 500 50 150 500

No. examined 5 5 5 5 5 5 4

No abnormalities detected 5 5 5 5 5 5 0

Hyperkeratosis*/total 0 0 0 2 0 3 4

Minimal 0 0 0 2 0 3 0

Slight 0 0 0 0 0 0 3

Moderate 0 0 0 0 0 0 1

Fig. 8. Sections of forestomach from control mice and from

mice given single oral doses of 500 mg/kg 2-butoxyacetic acid,

daily for 10 days.

Table 3

Frequency of S-phase in the forestomach of female B6C3F1

mice dosed for 10 days with either 2-butoxyethanol or 2-

butoxyacetic acid

Chemical Dose (mg/kg) ULLI (mm�1)

Control 0 12.069/2.41 (4)

2-butoxyethanol 50 7.719/2.50*

150 9.339/2.55 (4)

500 12.889/2.60

2-butoxyacetic acid 50 8.729/4.97

150 9.019/2.32

500 16.229/5.61 (4)

Values are given as mean9/S.D. (number per group). Five

animals per group except where indicated.

* Statistically significantly lower than the control group:

Student’s t -test, 2-sided (P B/0.05).


the lining of the forestomach and was identical tothat seen after gavage dosing suggesting that the

test material reached the target tissue in exactly the

same way. The lesion appears to develop as a

result of irritant material present in the lumen of

the forestomach coming into contact with the

lining. The question arises as to whether 2-

butoxyethanol present in the systemic circulation

could give rise to a similar response. An attempt totest this hypothesis by dosing 2-butoxyethanol

intravenously was confounded when radioactivity

was found in the buccal cavity, oesophagus and

stomach. The source of this material was appar-

ently the Harderian and salivary glands. Thus, the

fact that radioactivity entered the stomach in this

way after intravenous dosing made it impossible to

demonstrate systemic uptake into the stomach. Itcannot, therefore, be entirely discounted that 2-

butoxyethanol enters the stomach from the circu-

lation even though there is no known precedent for

this. However, there can be little doubt that 2-

butoxyethanol does enter the stomach via the

buccal cavity and the oesophagus following in-

halation exposure.

The responses seen in the forestomach in thecurrent study, and in the NTP study, which

include irritation, hyperkeratosis, ulceration and

hyperplasia of the forestomach lining, are well

characterised responses, seen with a wide range of

chemicals, which are known to lead to papilloma

and squamous cell carcinoma of the forestomach.

Many of these chemicals, including 2-butoxyetha-

nol, are devoid of any genotoxic activity, cancerdeveloping as a result of cellular damage and

reparative hyperplasia (Kroes and Webster, 1986).

Attempts to measure increased cell division in the

forestomach at the end of the 10-day study were

only partially successful. Although a clear dose

response was seen for both 2-butoxyethanol and 2-

butoxyacetic acid, the significance of the dose

dependent increase was confounded by the highcontrol value. The reason for this is not fully

understood but the cell turnover rate in the

stomach is known to be high and variable and is

influenced by food intake and the entry of material

into the stomach. Consequently, an accurate

assessment in mice fed on laboratory diet and

given gavage doses of test material is difficult to

achieve. It is, however, very apparent from the

hyperkeratosis that considerable cell proliferation

had occurred in the treated animals.

It appears from the protein binding results that

binding is either not a major factor in the

development of the lesion in the forestomach of

mice or that its detection is difficult due to the

rapid replacement of damaged cells. The concen-

trations of bound radioactivity were low, B/10

nmol equivalents/mg protein, which is not too

surprising since the proposed toxic metabolite, 2-

butoxyacetic acid, is not a reactive electrophile and

its covalent interaction with protein would be

expected to be minimal. It is clear from the very

marked hyperkeratosis that cells damaged by the

irritant action of 2-butoxyacetic acid are rapidly

replaced by new cells. Thus, cells which may have

contained bound material are rapidly shed to be

replaced by new cells which do not. This would not

have been the case in the glandular stomach where

cell damage did not occur. Consequently, compar-

isons of protein binding between the two regions

of the stomach is not meaningful since the bulk of

bound material would have been lost in the

forestomach due to the high cell turnover rates.

Characterisation of the remaining low levels of

bound material proved to be impossible.

It was evident from the 10 day gavage study that

whilst both chemicals induced the same response,

2-butoxyacetic acid was significantly more potent

than 2-butoxyethanol. These results imply that 2-

butoxyacetic acid is the toxic entity. Consistent

with this, 2-butoxyacetic acid is known to be an

irritant and it was also responsible for the haema-

tological changes seen in the study reported by

Ghanayem (1989). These conclusions imply that

the stomach has the capacity to metabolise 2-

butoxyethanol to 2-butoxyacetic acid. Assays of

alcohol and aldehyde dehydrogenase activities

with the relevant substrates in mouse fore- and

glandular stomach fractions demonstrated that

both enzymes were present in both regions of the

stomach. Thus, although 2-butoxyacetic acid was

not detected in the forestomach following inhala-

tion exposure due to methodological limitations, it

is clear that stomach tissues are able to form this

metabolite.


The dehydrogenase activities were similar inboth regions of the stomach and failed to explain

the specificity for the forestomach seen in the acute

and long-term studies. The histocytochemical

studies localising these enzymes did, however,

provide an explanation for the difference in

sensitivity between the two regions. In the forest-

omach, the dehydrogenase activity was heavily

localised in the lining of the stomach whereas inthe glandular region it was more evenly distributed

throughout the stomach wall. When homogenates

were prepared using the whole tissue from each

region the highly localised activity in the forest-

omach was diluted by none active tissue, whereas

the glandular stomach homogenates more accu-

rately reflected the more even distribution of

activity in this region. Assessment of the areaand volume of each stomach compartment con-

taining enzyme activity found a ratio of approxi-

mately 1:25 between forestomach and glandular

stomach. Thus, the enzyme activity in the forest-

omach is highly concentrated in a much smaller

volume of tissue than that in the glandular

stomach. When this is taken into account the

enzyme activity per unit volume is considerablyhigher in the forestomach than in the glandular

stomach.

A further factor which also makes a major

contribution to the specificity for the forestomach

is the nature and function of the forestomach

itself. Internally the forestomach is separated from

the glandular stomach by a prominent limiting

ridge. Material entering the rodent forestomach isstored in this region prior to entry to the glandular

stomach where initial digestion occurs. The resi-

dence time in the forestomach is considerable, in

fact it is proposed that the forestomach is rarely

empty whereas the glandular stomach empties

relatively quickly. Consequently, exposure of the

forestomach to potentially toxic chemicals is

significantly greater than that of the glandularstomach. This combination of concentrated en-

zyme activity and prolonged contact with the

substrate is the fundamental reason why the

forestomach is the target.

2-Butoxyethanol is metabolised to 2-butoxyace-

tic acid via 2-butoxyacetaldehyde, the first step

being catalysed by alcohol dehydrogenases and the

latter by aldehyde dehydrogenases (Ghanayem et

al., 1987). The toxicities associated with these

chemicals are irritancy and haemolysis, there being

neither structural alerts for, nor evidence of

genotoxicity (Chiewchanwit and Au, 1995; Elliott

and Ashby, 1997). Although the evidence to date

has identified 2-butoxyacetic acid as the toxic

metabolite formed from 2-butoxyethanol (Gha-

nayem, 1989), a question arises about the potential

role of the intermediary metabolite 2-butoxyace-

taldehyde. The metabolism of alcohols to alde-

hydes is known to be a reversible reaction whereas

the oxidation of aldehydes to acids is irreversible.

Typically, the rate of the latter reaction is fast

enough to maintain negligible concentrations of

the aldehyde in the body, the metabolism of

ethanol to acetaldehyde and acetic acid being a

common example of this (Testa and Jenner, 1976).

In this study the rate of metabolism of 2-butox-

yacetaldehyde to 2-butoxyacetic acid was signifi-

cantly faster than the conversion of 2-

butoxyethanol to the aldehyde. Consequently, 2-

butoxyacetaldehyde would be rapidly metabolised

to 2-butoxyacetic acid as soon as it was formed

from the alcohol and would not therefore accu-

mulate in the stomach. Finally, the observation

that 2-butoxyacetic acid, which is not metabolised

to 2-butoxyacetaldehyde, was far more toxic than

2-butoxyethanol, which is, confirms that 2-butox-

yacetaldehyde does not play a significant role in

the observed toxicity.

Although forestomach tumours were not seen in

rats exposed to 2-butoxyethanol, the precursor

lesions were observed in the NTP (1998) study,

albeit to a lesser extent than in the mouse. This is

entirely consistent with the dehydrogenase data

which showed that the alcohol dehydrogenase

activity (Vmax) was an order of magnitude lower

in the rat than the mouse forestomach. Further-

more, the affinity constant, Km, was 160-fold

higher in the mouse than the rat. Bearing in

mind that the dose is held in the forestomach,

the mouse has the capacity to metabolise large

amounts of butoxyethanol without saturation

occurring. By contrast, metabolism in the rat

would be limited by saturation at relatively low

doses.


In addition to the metabolic differences betweenmice and rats it is also worth noting that the

maximum doses used in the NTP study were

different, 250 ppm in mice but only 125 ppm in

rats. It is possible that this contributed to a higher

skin/fur burden of condensed material in mice

than rats. The inhaled dose is also considerably

greater in mice than rats. Assuming 100% absorp-

tion and a ventilation rate of 7.9 l/h in rats and 2.1l/h in mice (Brown et al., 1997), the dose in mg/kg

over a 6 h inhalation period in rats would have

been 114 mg/kg, and in mice 508 mg/kg. Again,

this would facilitate condensation occurring in the

nasopharynx of mice to a greater extent than in

rats. Overall, the enzyme activities and differences

in dose are consistent with more test material

entering the stomach and being activated in micethan in rats.

Human stomach contrasts markedly with that

of mice and rats, both in structure and in enzyme

distribution. The forestomach is a structure which

is found only in rodents and hence humans do not

store ingested material in the same way as the rat

and mouse. Both dehydrogenases were distributed

throughout the stomach mucosa. Alcohol dehy-drogenase was particularly concentrated in the

mucous found on the surface of the stomach and

between the mucous secreting cells lining the

stomach. This distribution is consistent with that

reported previously by Pestalozzi et al. (1983)

using an immunohistochemical technique. It seems

reasonable to assume that metabolism of 2-butox-

yethanol by this enzyme in the mucous layer willbe of less toxicological consequence than that

which occurs intra-cellularly in rodents. The high-

est concentrations of aldehyde dehydrogenase

were found in and around the mucous producing

cells and in the pepsin secreting Chief cells. Over-

all, the distribution of both enzymes in the human

stomach is more comparable with that in the

glandular region of the rodent stomach, which isnot a target for butoxyethanol or for the large

number of rodent forestomach toxins and carcino-

gens.

In conclusion, the circumstances which led to

the uptake of 2-butoxyethanol into the gastro-

intestinal tract of the mouse following exposure by

inhalation do not occur in humans. The lack of a

forestomach in humans means that ingested ma-terial would not be retained as it is in the mouse

and, finally, the specific localisation of the dehy-

drogenases which activate 2-butoxyethanol in

the forestomach lining is unique to rodents. It is

highly unlikely, therefore, that the acute and

chronic responses seen in the forestomachs of

mice exposed to 2-butoxyethanol will occur in

humans.

Acknowledgements

These studies were sponsored by member com-panies of the Oxygenated Solvent Producers

Association, a CEFIC Sector group.

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