Neurotoxicity of Topically Applied Hexachlorophene in the Young Rat Robert M. Shuman, MD; Richard W. Leech, MD; Ellsworth C. Alvord, Jr., MD
Young rats 6 to 22 days of age are extremely susceptible to the neurotoxic effects of hexachlorophene given as a daily bath of undiluted antiseptic detergent containing 3% hexachlorophene (pHiso\x=req-\ Hex). At this age, most rats are clinically and histologically damaged by as few as two daily baths. Younger rats are relatively resistant, probably because they have less myelin to be affected; older rats cannot be poisoned by this route, probably because the more mature liver excretes the drug more effectively. Age-dose-response curves in rats are similar to those in humans. This experimental model is potentially useful in defining other characteristics of this drug. (Arch Neurol 32:315-319, 1975)
(HCP) Hexachlorophene antistaphylococcal agent is
an ex¬
cellent
that
commonly incorporated into preparations rang¬ ing from antiperspirant sprays to toothpastes. Its most effective use has been as a 3% suspension (pHisoHex), was
many commercial
with which newborn infants have been bathed daily in order to prevent infections in the nursery.13 Although
for publication Dec 26, 1974. From the Laboratory of Neuropathology, Department of Pathology, University of Washington School of Medicine, Seattle. Reprint requests to Division of Neuropathology, RJ-05, University of Washington School of Medicine, Seattle, WA 98195 (Dr. Alvord).
Accepted
occasional fatal poisonings have been reported by accidental ingestion of HCP or by absorption through abnor¬ mal skin,4-8 HCP was generally con¬ sidered to be innocuous when applied topically to humans with intact skin until recently, when a vacuolar en¬ cephalopathy of the brain stem retic¬ ular formation was noted in small premature infants.912 Although the Food and Drug Administration re¬ moved HCP from over-the-counter sales13 and warned of its potential danger in newborns, the complexity of the proposed etiological relation¬ ship between HCP and the vacuolar encephalopathy raised a number of questions. This complexity related to the important variables tentatively
identified, including gestational age, concentration of HCP, number of baths, area of skin bathed, and site of lesion as well as the possibility of ad¬ ditional effects of the relatively large number of diseases that brought the patients to the hospital and contrib¬ uted to their deaths. Although lesions similar to those in the premature infants have been seen in experimental animals given HCP by injection or ingestion,1417 the large number of factors tentatively impli¬ cated in the production of the lesion in humans provided the basis for
skepticism regarding not only precise role of HCP in the produc-
some
the
Downloaded From: http://archneur.jamanetwork.com/ by a UQ Library User on 06/20/2015
tion of the lesion but also the role of the lesion in producing clinically im¬ portant evidence of neurotoxicity. The present study was designed to reproduce the human situation as closely as possible under controlled experimental conditions and to ex¬ plore systematically each of the fac¬ tors tentatively identified in humans. The rat was chosen since it is nor¬ mally born so prematurely that prac¬ tically all of its brain myelinates after birth, thereby affording experi¬ mental access to viable young, which might be expected to correspond in development to the highly susceptible but poorly viable small premature hu¬ man infant. MATERIALS AND METHODS
Young Sprague-Dawley
rats
were
kept
with their mothers in steel nursing cages in the animal facilities of the University of Washington Medical School, Seattle, with regularly cycled schedules of artificial light and with free access to food and water. In general, the young rats were bathed daily in antiseptic detergent containing 3% HCP (undiluted pHisoHex) and rinsed in the manner formerly used in the human premature nursery of the University Hos¬ pital and as generally recommended.2,3 Af¬ ter washing his hands with the detergent, the experimenter applied 2 to 3 ml of the detergent to the trunk of the young rat and gently lathered it over the entire skin surface, except for the head, for about 15
seconds. He then rinsed away all visible residue under running tap water with gentle massage for about five seconds and toweled the infant rat dry with cotton cloths. The entire litter was exposed, one infant after the other, once each day. The age of each litter at which the daily 3% HCP exposures began ranged from with¬ in a few hours after birth to 33 days of age. The young rats were examined at the time of washing for degrees of inactivity, decreased muscular tone, abnormal move¬ ments, and weakness. Each of these four abnormalities was graded on a scale of 0 to 2, by inverse analogy with the Apgar score used in humans. Death occurring between 12 and 24 hours after washing was arbi¬ trarily assigned a score of 8. Each litter was considered symptomatic when at least three of these four abnormalities were ap¬ parent in all of the young. In order to avoid contamination of unexposed littermates through the mother's milk (following licking of the skin of ex¬ posed infants) intralitter controls were deliberately avoided. Two other types of controls were used: (1) Young from one un¬ washed litter were killed at intervals from the first day of age through the 32nd day of age in order to establish the normal maturational pattern of the nervous sys¬ tem. (2) Two litters were blindly bathed with the suspension base of the detergent without HCP (pHisoDerm), from the first through the 16th day of age, and killed in parallel with two litters simultaneously bathed in 3% HCP under blind conditions. In order to survey histologically the cu¬ mulative effects of HCP on the brain over as broad an age range as possible, two young from each litter were killed by per¬ fusion-fixation 24 hours after every other washing. These two were chosen as being generally representative of the clinical state of the entire litter at this time. They were anesthetized with ether and killed by perfusion through the ascending aorta with cold half-strength Karnovsky fixative containing glutaraldehyde and paraformaldehyde in phosphate buffer.18 The brain and cervical spinal cord were removed, placed in phosphate-buffered 4% formalde¬ hyde solution (10% formalin) and stored at 4 C. The tissues from animals that died be¬ fore perfusion were fixed by immersion in 4% formaldehyde solution. Each specimen was identified individually; when 100 brains had accumulated, each specimen was assigned a random four-digit number by which it was subsequently identified during further processing and histological evaluation. One of each pair of dead young was processed for light microscopy, and the other initially set aside for electron mi¬ croscopy (S.M. Sumi, R.M. Shuman, unpub-
Table 1.—Number of
Young Rats Studied
Light Microscopy
Age of Animals at Which HCP Exposure Began, Days 0-4 6-22 26-33
Perfused 21 106 20
Electron Microscopy Perfused 24
Died 25 36
15
Total
Young 70 157 21
Controls 0-6 8-32 Total young *
Matched by
age at
of age (Table 2).
19* 173
perfusion
lished data), but as the clinical results were obtained, the sampling of specimens for electron microscopy was changed (by one of the investigators breaking the code) to emphasize the youngest animals (Table 1). Sections 5µ thick were stained with hema¬ toxylin-eosin, and sections 15u thick were stained for myelin with gallocyanin-Darrow red.19 The histologie slides of the animals (identified only by the random four-digit numbers) were evaluated without any knowledge of the experimental manipula¬ tion to which the rats had been subjected. Vacuolation was assessed in the reticular formation and long tracts of the brain stem and in the white matter of the cere¬ brum and cerebellum. The degree of vacu¬ olation was graded on a 0-to-5 scale, deter¬ mined by the magnification needed to see the vacuoles: 0 no vacuolation detectable at X400 magnification; 1 to 2 slight scat¬ tered vacuolation only detectable at high magnifications (x400 and xlOO, respec¬ tively); 3 frequent vacuolation easily de¬ tected at low power (x25); 4 vacuolation apparent by naked eye examination; 5 vacuolation so severe as to leave only a thin reticulum in place of the former =
=
=
=
=
neuropil.
RESULTS
Young rats proved to be very sus¬ ceptible to daily baths in 3% HCP, with clinical signs produced in all that were 22 days of age or less at their initial exposure (Fig 1). Rats less than 6 days of age required seven ex¬ posures. Between 6 and 12 days of age the sensitivity to HCP slowly in¬ creased, for the number of daily expo¬ sures necessary to produce clinical signs steadily declined from six to two. The sensitivity of rats between 13 and 22 days of age was stable and marked, with only two exposures nec¬ essary. Rats 26 days of age or more proved to be completely resistant to
Downloaded From: http://archneur.jamanetwork.com/ by a UQ Library User on 06/20/2015
49
62
with rats
exposed
to 3% HCP
beginning
11 25 284 at 6 to 22
days
this topical exposure to 3% HCP. The clinical signs of HCP-induced neurotoxicity included inactivity, de¬ creased exploratory behavior, de¬ creased feeding, and progressive weakness. A coarse tremor or quak¬ ing developed, best seen when the an¬ imal was placed in a situation of mus¬ cular tension, eg, when climbing a wire grid. Not only did 3% HCP cause clinical signs, but it killed 61 young exposed to it. This mortality (61/248 25%) is to be contrasted with that of the control population (1/36 3%, Table 1). Histological signs of neurotoxicity from 3% HCP were produced in all young rats whose exposures began between 2 and 22 days of age (Fig 2). Newborn rats bathed daily developed no vacuolar encephalopathy even af¬ ter 14 baths, but they showed very little myelination. The typical vacu¬ olar encephalopathy developed in 2day-old rats bathed daily for 12 days, and the number of exposures required dropped rapidly to only six baths by 3 days of age and two to six baths be¬ tween 6 and 22 days of age. The sen¬ sitivity of young rats between 13 and 22 days of age was remarkably stable, only two exposures being necessary. Rats 26 days old or older were resist¬ ant to topically applied 3% HCP. The histological signs of neurotoxicity consisted of vacuolation of myelin in various parts of the brain (Fig 3), as described by other investigators.14"17 The severity of the vacuolation ranged from mild focal alterations of the myelin in the reticular formation and long tracts to massive disruption of the myelin throughout the entire brain. Damage was more apparent in =
=
heavily myelinated areas (eg, long tracts) than in lightly myelinated
SN^
,
Young rats 6 to 22 days of age are extremely susceptible to the neurotoxic effects of hexachlorophene given as a daily bath of undiluted antiseptic detergent containing 3% hexachlorophene (pHiso\x=req-\ Hex). At this age, most rats are clinically and histologically damaged by as few as two daily baths. Younger rats are relatively resistant, probably because they have less myelin to be affected; older rats cannot be poisoned by this route, probably because the more mature liver excretes the drug more effectively. Age-dose-response curves in rats are similar to those in humans. This experimental model is potentially useful in defining other characteristics of this drug. (Arch Neurol 32:315-319, 1975)
(HCP) Hexachlorophene antistaphylococcal agent is
an ex¬
cellent
that
commonly incorporated into preparations rang¬ ing from antiperspirant sprays to toothpastes. Its most effective use has been as a 3% suspension (pHisoHex), was
many commercial
with which newborn infants have been bathed daily in order to prevent infections in the nursery.13 Although
for publication Dec 26, 1974. From the Laboratory of Neuropathology, Department of Pathology, University of Washington School of Medicine, Seattle. Reprint requests to Division of Neuropathology, RJ-05, University of Washington School of Medicine, Seattle, WA 98195 (Dr. Alvord).
Accepted
occasional fatal poisonings have been reported by accidental ingestion of HCP or by absorption through abnor¬ mal skin,4-8 HCP was generally con¬ sidered to be innocuous when applied topically to humans with intact skin until recently, when a vacuolar en¬ cephalopathy of the brain stem retic¬ ular formation was noted in small premature infants.912 Although the Food and Drug Administration re¬ moved HCP from over-the-counter sales13 and warned of its potential danger in newborns, the complexity of the proposed etiological relation¬ ship between HCP and the vacuolar encephalopathy raised a number of questions. This complexity related to the important variables tentatively
identified, including gestational age, concentration of HCP, number of baths, area of skin bathed, and site of lesion as well as the possibility of ad¬ ditional effects of the relatively large number of diseases that brought the patients to the hospital and contrib¬ uted to their deaths. Although lesions similar to those in the premature infants have been seen in experimental animals given HCP by injection or ingestion,1417 the large number of factors tentatively impli¬ cated in the production of the lesion in humans provided the basis for
skepticism regarding not only precise role of HCP in the produc-
some
the
Downloaded From: http://archneur.jamanetwork.com/ by a UQ Library User on 06/20/2015
tion of the lesion but also the role of the lesion in producing clinically im¬ portant evidence of neurotoxicity. The present study was designed to reproduce the human situation as closely as possible under controlled experimental conditions and to ex¬ plore systematically each of the fac¬ tors tentatively identified in humans. The rat was chosen since it is nor¬ mally born so prematurely that prac¬ tically all of its brain myelinates after birth, thereby affording experi¬ mental access to viable young, which might be expected to correspond in development to the highly susceptible but poorly viable small premature hu¬ man infant. MATERIALS AND METHODS
Young Sprague-Dawley
rats
were
kept
with their mothers in steel nursing cages in the animal facilities of the University of Washington Medical School, Seattle, with regularly cycled schedules of artificial light and with free access to food and water. In general, the young rats were bathed daily in antiseptic detergent containing 3% HCP (undiluted pHisoHex) and rinsed in the manner formerly used in the human premature nursery of the University Hos¬ pital and as generally recommended.2,3 Af¬ ter washing his hands with the detergent, the experimenter applied 2 to 3 ml of the detergent to the trunk of the young rat and gently lathered it over the entire skin surface, except for the head, for about 15
seconds. He then rinsed away all visible residue under running tap water with gentle massage for about five seconds and toweled the infant rat dry with cotton cloths. The entire litter was exposed, one infant after the other, once each day. The age of each litter at which the daily 3% HCP exposures began ranged from with¬ in a few hours after birth to 33 days of age. The young rats were examined at the time of washing for degrees of inactivity, decreased muscular tone, abnormal move¬ ments, and weakness. Each of these four abnormalities was graded on a scale of 0 to 2, by inverse analogy with the Apgar score used in humans. Death occurring between 12 and 24 hours after washing was arbi¬ trarily assigned a score of 8. Each litter was considered symptomatic when at least three of these four abnormalities were ap¬ parent in all of the young. In order to avoid contamination of unexposed littermates through the mother's milk (following licking of the skin of ex¬ posed infants) intralitter controls were deliberately avoided. Two other types of controls were used: (1) Young from one un¬ washed litter were killed at intervals from the first day of age through the 32nd day of age in order to establish the normal maturational pattern of the nervous sys¬ tem. (2) Two litters were blindly bathed with the suspension base of the detergent without HCP (pHisoDerm), from the first through the 16th day of age, and killed in parallel with two litters simultaneously bathed in 3% HCP under blind conditions. In order to survey histologically the cu¬ mulative effects of HCP on the brain over as broad an age range as possible, two young from each litter were killed by per¬ fusion-fixation 24 hours after every other washing. These two were chosen as being generally representative of the clinical state of the entire litter at this time. They were anesthetized with ether and killed by perfusion through the ascending aorta with cold half-strength Karnovsky fixative containing glutaraldehyde and paraformaldehyde in phosphate buffer.18 The brain and cervical spinal cord were removed, placed in phosphate-buffered 4% formalde¬ hyde solution (10% formalin) and stored at 4 C. The tissues from animals that died be¬ fore perfusion were fixed by immersion in 4% formaldehyde solution. Each specimen was identified individually; when 100 brains had accumulated, each specimen was assigned a random four-digit number by which it was subsequently identified during further processing and histological evaluation. One of each pair of dead young was processed for light microscopy, and the other initially set aside for electron mi¬ croscopy (S.M. Sumi, R.M. Shuman, unpub-
Table 1.—Number of
Young Rats Studied
Light Microscopy
Age of Animals at Which HCP Exposure Began, Days 0-4 6-22 26-33
Perfused 21 106 20
Electron Microscopy Perfused 24
Died 25 36
15
Total
Young 70 157 21
Controls 0-6 8-32 Total young *
Matched by
age at
of age (Table 2).
19* 173
perfusion
lished data), but as the clinical results were obtained, the sampling of specimens for electron microscopy was changed (by one of the investigators breaking the code) to emphasize the youngest animals (Table 1). Sections 5µ thick were stained with hema¬ toxylin-eosin, and sections 15u thick were stained for myelin with gallocyanin-Darrow red.19 The histologie slides of the animals (identified only by the random four-digit numbers) were evaluated without any knowledge of the experimental manipula¬ tion to which the rats had been subjected. Vacuolation was assessed in the reticular formation and long tracts of the brain stem and in the white matter of the cere¬ brum and cerebellum. The degree of vacu¬ olation was graded on a 0-to-5 scale, deter¬ mined by the magnification needed to see the vacuoles: 0 no vacuolation detectable at X400 magnification; 1 to 2 slight scat¬ tered vacuolation only detectable at high magnifications (x400 and xlOO, respec¬ tively); 3 frequent vacuolation easily de¬ tected at low power (x25); 4 vacuolation apparent by naked eye examination; 5 vacuolation so severe as to leave only a thin reticulum in place of the former =
=
=
=
=
neuropil.
RESULTS
Young rats proved to be very sus¬ ceptible to daily baths in 3% HCP, with clinical signs produced in all that were 22 days of age or less at their initial exposure (Fig 1). Rats less than 6 days of age required seven ex¬ posures. Between 6 and 12 days of age the sensitivity to HCP slowly in¬ creased, for the number of daily expo¬ sures necessary to produce clinical signs steadily declined from six to two. The sensitivity of rats between 13 and 22 days of age was stable and marked, with only two exposures nec¬ essary. Rats 26 days of age or more proved to be completely resistant to
Downloaded From: http://archneur.jamanetwork.com/ by a UQ Library User on 06/20/2015
49
62
with rats
exposed
to 3% HCP
beginning
11 25 284 at 6 to 22
days
this topical exposure to 3% HCP. The clinical signs of HCP-induced neurotoxicity included inactivity, de¬ creased exploratory behavior, de¬ creased feeding, and progressive weakness. A coarse tremor or quak¬ ing developed, best seen when the an¬ imal was placed in a situation of mus¬ cular tension, eg, when climbing a wire grid. Not only did 3% HCP cause clinical signs, but it killed 61 young exposed to it. This mortality (61/248 25%) is to be contrasted with that of the control population (1/36 3%, Table 1). Histological signs of neurotoxicity from 3% HCP were produced in all young rats whose exposures began between 2 and 22 days of age (Fig 2). Newborn rats bathed daily developed no vacuolar encephalopathy even af¬ ter 14 baths, but they showed very little myelination. The typical vacu¬ olar encephalopathy developed in 2day-old rats bathed daily for 12 days, and the number of exposures required dropped rapidly to only six baths by 3 days of age and two to six baths be¬ tween 6 and 22 days of age. The sen¬ sitivity of young rats between 13 and 22 days of age was remarkably stable, only two exposures being necessary. Rats 26 days old or older were resist¬ ant to topically applied 3% HCP. The histological signs of neurotoxicity consisted of vacuolation of myelin in various parts of the brain (Fig 3), as described by other investigators.14"17 The severity of the vacuolation ranged from mild focal alterations of the myelin in the reticular formation and long tracts to massive disruption of the myelin throughout the entire brain. Damage was more apparent in =
=
heavily myelinated areas (eg, long tracts) than in lightly myelinated
SN^
,
