Hexachlorophene Lesions in Newborn Infants John M.
Gowdy, MD, Andrew G. Ulsamer, PhD
\s=b\ Vacuolization of the white matter of the brain is produced by a number of disease entities and chemicals, including hexachlorophene. Brains of 135 stillborn infants and infants dying in the neonatal period were examined for vacuole formation in the white matter to determine if any hexachlorophene-like lesions could be found. A nonsignificant excess of vacuoles was found in infants bathed in hexachlorophene at birth compared with infants not bathed in it. Analysis of 11 brains for hexachlorophene showed that detectable levels were present in five, all of which showed vacuolization. None was detected In the remaining six, three of which also showed vacuolization. In two of these there was no hexachlorophene exposure. (Am J Dis Child 130:247-250, 1976)
Gump1 discovered In hexachlorophene septic properties the 1939
that retained its anti¬ in presence of soap, and it became a standard com¬ ponent of many topically applied drugs and cosmetics. Hexachloro¬ phene and a number of products con¬ taining it were tested extensively prior to marketing, but examination of the brains of animals involved in Received for publication Sept 30, 1974; acMarch 3, 1975. From St. Elizabeths Hospital, Washington, DC (Dr Gowdy), and the Division of Physical Sciences, Consumer Product Safety Commission
cepted
(Dr Ulsamer). The views expressed do not
necessarily agencies.
are the authors' own and reflect those of the affiliated
Reprint requests to 12408 Parkton St, Oxon Hill, MD 20022 (Dr Gowdy).
these tests was omitted.2 In experiments in China on the use of hexachlorophene for human liver fluke infection (Clonorchis sinensis), the brains of rats fed this compound were examined and reported as nor¬ mal; however, trials of the drug in man revealed severe neurotoxic reac¬ tion that precluded its systemic use.3 A neurotoxic condition is also a prom¬ inent feature of accidental hexachlo¬ rophene poisoning in humans,4 and hexachlorophene was suspected as the cause of convulsions in burn patients treated with hexachlorophene soaks.5 Kimbrough et al6 first described the central nervous system (CNS) patho¬ logical findings produced by hexa¬ chlorophene after conducting feeding studies in rats to determine its safety for use as an agricultural pesticide. Further studies in monkeys, rats,7 pigs, and tadpoles8 confirmed that hexachlorophene produced CNS le¬ sions in numerous species. By light microscopy the lesions in rats are seen as cystic spaces in the white matter of the brain and spinal cord. Electron microscopy shows that these spaces are in the wall of the myelin sheath.6 Vacuoles are most prominent in the white matter of the cerebellar folia, but are distributed throughout the white matter of the cerebrum, optic tract, pons, medulla, and cord. In rats below the age of 10 to 11 days, or above the age of 21 days, no lesions are seen when they are dosed orally with 10 mg/kg of hexachlorophene, but lesions do ap-
Downloaded From: http://archpedi.jamanetwork.com/ by a University of Manitoba User on 06/05/2015
pear at this dose in the age range of 11 to 18 days,7 which is the period of
maximum myelinization of the axons of the white matter of the rat brain.9 The lesions produced by hexachloro¬ phene in the rat brain are not diag¬ nostic for this chemical. Vacuoles are observed in both animals and man in a variety of diseases and following the administration of a number of chemical compounds. These condi¬ tions are listed in Table 1. Electron microscopy shows that hexachloro¬ phene lesions in rats are due to accu¬ mulation of some material, probably water, in the wall of the myelin sheath,10 but this distinction cannot be seen by light microscopy. Ulsamer et al7 have reported a slight decrease in the phospholipid and sterol content of the brain in rats given hexachloro¬ phene by mouth. An attractive theory is that the spaces represent accumula¬ tion of some precursor or intermedi¬ ary of myelin; however, the spaces produced by triethyltin contain only water and electrolyte.11 Biochemical studies have shown that triethyltin and similar compounds inhibit glu¬ cose metabolism and uncouple oxida¬ tive phosphorylation.12 A similar ac¬ tion has been demonstrated for hexachlorophene,13 but it is not clear that this action is related to the pro¬ duction of vacuoles in the immature myelin sheath. Webster et al8 have reported that hexachlorophene is localized in the protein fraction of myelin where it could disrupt the myelin sheath either
mechanically or by interference with oxidative phosphorylation. Chemical compounds causing this lesion fall into two groups, those that are antiseptics for Gram-positive mi¬ croorganisms (hexachlorophene, tri¬ ethyltin, isoniazide, and cuprizone) and those that interfere with choles¬ terol synthesis (AY 9944 and related compounds). By light and electron mi¬ croscopy the lesions of cribriform de¬
the prophylaxis of staphylococcal in¬ fections in hospital nurseries. To eval¬ uate this possibility, material from the National Institutes of Health (NIH) perinatal study was reviewed. MATERIALS AND METHODS The scope and purpose of the NIH col¬ laborative study on cerebral palsy, mental retardation, and other neurological and sensory disorders of childhood have been detailed elsewhere.18 Some of the pregnancies in the NIH col¬ laborative study terminated in stillbirth or abortion. Other infants died after birth from various diseases. An attempt was made to secure an autopsy on every sub¬ ject who died. This included a detailed ex¬ amination of the brain with the prepara¬ tion of up to 60 tissue blocks from the cerebrum, medulla, pons, and spinal cord. Four sections were prepared from each block, one each for hematoxylin-eosin, gallocyanin, trichrome, and Luxol fast blue-
generation, triethyltin, isoniazid, cuprizone, and hexachlorophene ap¬ pear to be identical. Cuprizone and hexachlorophene are both chelating agents for copper. Copper deficiency in animals produces CNS symptoms
similar to those observed in the above experimental poisonings, and the ef¬ fects of hexachlorophene on CNS cop¬ per metabolism may merit further
study.
In rats CNS lesions appeared when the blood level of hexachlorophene reached 1.2/ig/ml.14 Investigation of blood levels following bathing of newborn infants with a commercial hexachlorophene preparation showed that values as high as 0.3jtig/ml can be attained after a single washing and up to 0.78µg/ml with repeated wash¬ ing.15 Thus, it is theoretically possible that lesions of the white matter of the CNS could result from the prac¬ tice of bathing newborn infants re¬ peatedly with hexachlorophene for Table 1.—Conditions
PAS-hematoxylin staining. A total of 76 specimens from liveborn, hexachlorophene-bathed subjects were available. Since only 18 specimens from nonhexachlorophene-bathed controls were available, 41 specimens from stillborn infants were reviewed for additional controls. Slides were studied by light mi¬ croscopy without knowledge of hexachloro¬ phene use, diagnosis, or cause of death. Be¬ cause of delays in processing, sections were available only until 1964. Slides from all areas were examined for lesions resem¬ bling those seen in the brains of hexachlorophene-treated rats and monkeys. The
Producing Vacuole Formation in the Brain
Source Magee et al" Katzman et al" Worden et al20
After reading of the slides pleted, clinical records of each
Triethyltin Triethyltin Pheniprazine hydrochloride
reviewed for maternal disease, type of de¬
Phenelzlne*
Indanylcarbethoxyhydrazlne* Heptylhydrazine*
These factors were then coordinated with the slide reading and hexachlorophene bathing at birth. Specimens representing 76 deaths fol¬ lowing live births were reviewed from hexachlorophene-using hospitals. Vacuoles were seen in 14 (18%). Eighteen specimens from live births were received from nonhexachlorophene-using institutions. Two (11%) were positive. These differences are
Isoniazid* Isoniazid mesylate AY 9944f AY 9944 AY 9944
Triparanolt Cribriform disease of mice of the Pernicious anemia
Spongy degeneration
nervous
system
not
Scrapie
CuprizoneJ *
Experimental
monamine oxidase inhibitors.
t Experimental cholesterol-lowering agents. Triparanol
was
marketed for
a
short time
as
MER 29.
Chelating agent used
in
was com¬ case were
livery, complications, birth weight, length of gestation, clinical course, autopsy diag¬ noses, and other pertinent information.
Tetrahydronaphthylhydrazine*
Green et al24 Buchanan and Davis25 Murphy and Constanzi26 Pattison and Jebbett27
.
study.
Compound/Condition
Smith et al2' Suzuki and DePaul22 Rawlins and Uzman23 Cremer'2
overall extent of involvement was esti¬ mated subjectively and recorded as nega¬ tive to 4 + Four plus represents vacu¬ olization of 50% or more of the white matter of the cerebellum; 3+ represents involvement of 25% to 50%; 2+ is involve¬ ment of 10% to 25%; and 1 + is involvement of less than 10%. Reliability of the diag¬ nosis varies with the percent of involve¬ ment. In the studies of Ulsamer et al,7 1 + areas of vacuolization were seen in some control animals. Sections were considered negative if there was no cerebellar involve¬ ment or if involvement was not confined to the white matter. In general, lesions in positive cases were seen in all areas of white matter in the brain and cord, but were most pronounced in the white matter of the cerebellum. The incidence of brain vacuoles in sub¬ jects washed with hexachlorophene at birth was compared to the incidence in those who were not washed in it. The use of hexachlorophene bathing in the new¬ born nurseries was determined by inquiry of the hospital involved. Two institutions used 3% hexachlorophene daily (pHisoHex) and were classified as high-level users. Most hospitals used dilute 3% daily and were considered medium-level users. Two institutions used dilute 3% hexachloro¬ phene at birth only and were classified as low-level users. The definition of dilute is somewhat vague. One formula is 15 ml of 3% hexachlorophene in 1 liter of water. In practice it is likely that the preparation was added to the bath water without ac¬ tually measuring the amount. Hexachloro¬ phene bathing of newborns was not used during the period covered by this study in two hospitals. One hospital did not use hex¬ achlorophene during the first year of the
analysis for copper.
Downloaded From: http://archpedi.jamanetwork.com/ by a University of Manitoba User on 06/05/2015
statistically significant.
A total of 41 specimens from nonlive births was reviewed. Of 30 from hexachlorophene-using institutions, vacuoles were seen in six (20%). No vacuoles were seen among the 11 cases from nonuser hos-
pitáis. Table 2 gives these results quantita¬ tively. In an attempt to account for the lesions in stillborn infants, an analysis was made of the use of hexachlorophene as the anti¬ septic for sterilization of the perineum prior to delivery. Hexachlorophene is read¬ ily absorbed from this area in man and crosses the placenta to produce congenital defects in animals.17 Twelve percent of the hexachlorophene group showed lesions compared to 13.7% of the control group. There is a tendency for the percentage of lesions to increase with increasing birth weight among both liveborn and stillborn, suggesting that the phenomenon is corre¬ lated with increased maturity. As men¬ tioned above, lesions do not appear in rats until myelinization of the cerebral white matter tracts begins. In humans this is around the second month of life. In infants surviving 30 days or longer all of the posi¬ tive findings are in the hexachlorophenebathed group. However, only one infant from the control group survived 30 days. Brains, fixed in an aqueous solution of formaldehyde, from which the slides were prepared were available at NIH, and as a further check, several specimens were ana¬ lyzed for hexachlorophene content. This was determined by gas-liquid chromatog¬ raphy, using a radioactive nickel electroncapture detector as described previously by Ulsamer.18 Results are shown in Table 3. The halflife of hexachlorophene in man is given variously as 8 to 24 hours,2·10 and no hexa¬ chlorophene is found in blood 15 days after exposure stops, even though in rats the brain lesions persist for at least three months after exposure. During this period, lesions decline from 4+ to 1 + or 2+ in ex¬ tent and also decrease in size.6·10 In cases 2, 4, and 5, hexachlorophene levels and slide readings agree, and all infants were hexachlorophene-bathed. Tissue levels in case 1 could be due to postnatal contact with hexachlorophene-containing soaps, lotions, and other preparations. Although this in¬ fant was delivered in a hexachloropheneusing institution, hexachlorophene could not remain in the brain for 18 months without continued exposure. While other contact with hexachlorophene, such as pre¬ natal use of soap and cosmetics containing the antiseptic, may be a factor, case 6 could be due to the use of hexachlorophene solution for predelivery washing of the mother. The use of hexachlorophene-containing talc on infants has been implicated in hexachlorophene poisoning and death.15 The positive reading in case 3 may be due to prenatal exposure to isoniazid. Case 7 could be from maternal use of hexachloro¬ phene soaps and cosmetics or to unknown
factors not related to use of the antiseptic. In case 8 hexachlorophene levels would have fallen to zero by three months after exposure. In cases 9 through 11, levels and readings agree. It should be remembered
Table 2.—CNS Lesions in
that this tissue
fixed with formalde¬
was
hyde prior to hexachlorophene level deter¬ mination, and the effects of this on the quantitation of the chemical are not known.
Hexachlorophene-Bathed and Control Subjects Slide Reading
3+
Treatment
High Medium Low Total bathed with hexachlorophene Controls Stillborn Liveborn Total
Table 3.—Tissue
Sex
34 24
13 27
76 59 41 18 135
62 51 35 16 113
14 15 11 16
Hexachlorophene
13
Levels
Compared With Slide Readings* Hexachlor¬ Hexachlor¬
Weight,
Gestation,
Slide
gm
Weeks 42 32 30 38 35 39 40 39 39 30 40
Reading
3,360 1,310 1,414 2,545 1,225 2,980 2,955
3t
3,220 10 11
M
2,875 1,280 2,810
3+ 3+ 3+ 2+ 2+ 2+ 2+ 2+
Use
Table 4.—Association of Lesions With Various Slide Anoxia L* Anoxia Sf Premature L Premature S Atelectasis L Pneumonia L Hyaline membrane disease L Congenital defect L Congenital defect S Septicemia L
Septicemia S Digitalis L L signifies liveborn. t S signifies stillborn. One not bathed in hexachlorophene. § Two not bathed in hexachlorophene. *
j
Downloaded From: http://archpedi.jamanetwork.com/ by a University of Manitoba User on 06/05/2015
M|
0.70 0.40 0.0 0.34 0.20 0.10 0.0 0.0 0.0 0.0 0.0
Correlation of slide readings against tissue levels gives omitted, r = 0.71 and < .025. t M signifies medium level hexachlorophene bathing. Ì Isoniazid exposure. § H signifies high-level hexachlorophene bathing. [| signifies maternal preparation only.
3+
ophene
ophene,
*
Condition
Negative Positive
39 33
Birth Case
%
1+
2+
2+
3Í 1Í 4t
r
M
None
H§ None H None M :
0.57 and
Survival 18 mo 5 days
Stillborn 102 25 5 33 3 33 29 8
< .1 ; if
hr hr
days hr mo
hr hr weeks case
3 is
Autopsy Diagnoses
Reading
%
Negative 8 10 29 10 15 15 20 11
2§ 11
Positive
29
25 25 13 41 29 43 15
The
of death and other autopsy compared with the inci¬ dence of vacuolization. This is shown in Table 4. Among live births anoxia was not related to the lesion, being recorded in 18% of both the negative and positive groups. However, among stillbirths, anoxia was present in half the subjects in whom le¬ sions were seen. An excess of cardio¬ vascular malformations was found in the positive group, occurring in 32% compared with 7% in the negative group. Pneumonia was less common in the positive group, being 9% compared with 36% in the nega¬ tive group. Atelectasis was recorded in 27% of the positive group and 15% of the nega¬ tive group. Finally, septicemia was seen in 14% of the positive group and 7% of the negative group. If anoxia and atelectasis are omitted, the figures in Table 2 become nine out of 48 positive for the bathed group and four out of 44 for the nonbathed group, which is not statistically signifi¬ cause
diagnoses
were
cant.
Prescription drug intake of the mothers during pregnancy was reviewed for isonia¬ zid and triparanol, the only two drugs in clinical use at that time implicated in vacu¬ ole production. One mother of a stillborn with 3+ vacuolization received isoniazid during her pregnancy. Aside from this, cardiac glycosides, and the small amounts of vitamin A contained in prenatal supple¬ ments, the subjects of the study were not exposed to any of the agents listed in Table 1. Most of the agents producing vacuoliza-
tion are laboratory curiosities or industrial chemicals that need not be considered in the differential diagnosis in the present cases.
COMMENT
Comparison of hexachlorophene brain tissue levels and slide readings shows one false positive or a 10% er¬ ror in interpretation. No hexachloro¬ phene was detected in the brains of infants not washed with this sub¬ stance. Correlation of slide readings and tissue levels gives a correlation coefficient of .57, which is not signifi¬ cant (P < .1). Although some lesions appear to be associated with hexa¬ chlorophene exposure, all such lesions are not due to the practice of bathing newborns in hexachlorophene, since vacuoles were found in subjects who were not bathed with this material. Prenatal use of hexachlorophene preparations by the mother and the use of lotions and other products con¬ taining this antiseptic on infants may have produced lesions in some cases. Detectable, albeit minute, amounts of hexachlorophene have been found in the cord blood of unbathed newborns.28 During the period of the perinatal study (1960 to 1970),
hexachlorophene was widely used, and both prenatal and postnatal ex-
posure to it
might well have occurred. Under the conditions of this study there were no noteworthy differences in the incidence of CNS vacuolization between hexachlorophene-bathed and control subjects. The occurrence of vacuolization and hexachlorophene levels in the same subjects is sugges¬ tive, but does not prove a causal rela¬
tionship.
It would
seem
to avoid the
preparations
prudent to continue hexachlorophene
use of on infants
and children until a definitive answer to the ques¬ tion of toxicity can be found. Joseph S. Drage gave permission to examine clinical records of the Perinatal Research Branch of the National Institute of Neurological Diseases and Strokes. Toshio Fujikura, MD, National Institute of Neurological Diseases and Strokes, gave permission to study slides and provided advice on interpretation of slides. William Cockran, MD, Robert O. Fisch, MD, William Clark, Jr, MD, Carl Zelson, MD, Marguerite Yates, MD, Raymond Vande Weile, MD, Brigette de la Burde, MD, Janet Hardy, MD, Harry M. Beine, MD, Sheldon Korones, MD, Adolph Sellman, MD, and Harmon Jordan supplied information on hexachlorophene use in the various hospitals.
Nonproprietary Names and Trademarks of Drugs Hexachlorophene—pHisoHex. Pheniprazine-Cainm.
References 1. Gump WS: Development of a germicidal soap. Soap and Sanitary Chemicals, 1945, pp 36\x=req-\
39, 50-85.
2. Gump WS: Toxicological properties of hexachlorophene. J Soc Cosmet Chem 20:173-184,1969. 3. Chung HL, Ts'ao WC, Hu HC, et al: Hexachlorophene as a new specific drug against Clonorchis sinensis. Chin Med J 82:691-701, 1963. 4. Herter WB: Hexachlorophene poisoning. Kaiser Foundation Med Bull 7:228, 1959. 5. Larson DL: Studies show hexachlorophene causes burn syndrome. Hospitals 42:63-64, 1968. 6. Kimbrough RD, Gaines TB: Hexachlorophene effects on the rat brain. Arch Environ Health 23:114-118, 1971. 7. Ulsamer AG, Yoder PD, Kimbrough RD, et al: Toxic effects of hexachlorophene in neonatal rats: Morphology, tissue levels and biochemistry. Food Cosmet Toxicol, to be published. 8. Webster H deF, Ulsamer AG, O'Connel MF: Hexachlorophene induced myelin lesions in the developing nervous system of Xenopus tadpoles: Morphological and biochemical observations. J Neuropathol Exp Neurol 33:144-163, 1974. 9. Cragie EH: Neuroanatomy of the Rat. New York, Academic Press, 1963. 10. Kimbrough RD: Review of the toxicity of hexachlorophene. Arch Environ Health 23:119\x=req-\ 121, 1971. 11. Magee PN, Stover HB, Barnes JM: The experimental production of edema in the central
nervous
system of the rat by triethyltin
pounds. J Pathol 73:107-124,
com-
1957.
12. Cremer JE: Biochemical changes associated with neurotoxicity. Proc Eur Soc Study Drug Toxicity 8:169-176, 1967. 13. Caldwell RS, Nakaue HS, Buhler DR: Biochemical lesion in rat liver mitochondria induced by hexachlorophene. Biochem Pharmacol 21: 2425-2441, 1972. 14. Curley A, Hawk RE: Hexachlorophene: I. Analysis in body fluids and tissues of experimental animals. Read before the 161st national meeting of the American Chemical Society, Los Angeles, 1971. 15. Gluck L, cited by Lockhart JD: How toxic is hexachlorophene? Pediatrics 50:229-235, 1972. 16. Berendes H: Structure and scope of the collaborative study: Present status. Proc AMA Natl Conf Infant Mortality 1966, pp 95-99. 17. Kimel CA, Moore W Jr, Stora JF: Hexachlorophene teratogenicity in rats. Lancet 2:765, 1972. 18. Ulsamer AG: The determination of hexachlorophene in mammalian tissues by gas liquid chromatography. J Am Osteopath Assoc 55:1294, 1972. 19. Katzman R, Aleu F, Wilson C: Further observations on triethyltin edema. Arch Neurol 9:178-187, 1963. 20. Worde. AN, Palmer AC, Noel PRB, et al: Lesions in the brain of the dog induced by pro-
Downloaded From: http://archpedi.jamanetwork.com/ by a University of Manitoba User on 06/05/2015
longed administration of monoamine oxidase in-
hibitors and isoniazid. Proc Eur Soc Study Drug Toxicity 8:149-161, 1967. 21. Smith ME, Hasinoff CM, Fumagalli R: Inhibitors of cholesterol synthesis and myelin formation. Lipids 5:665-671, 1970. 22. Suzuki K, DePaul L: Cellular degeneration in developing central nervous system of rats produced by hypocholesteremic drug AY 9944. Lab Invest 25:546-555, 1971. 23. Rawlins FA, Uzman BY: Effect of AY 9944, a cholesterol biosynthesis inhibitor on peripheral nerve myelination. Lab Invest 23:184\x=req-\ 189, 1970. 24. Green MC, Sidman RL, Pinetta OH: Carbriform degeneration (cri): A new recessive neurological mutation in the mouse. Science 176:800\x=req-\ 803, 1972. 25. Buchanan DS, Davis RL: Spongy degenerthe nervous system. Neurology 15:207\x=req-\ ation of 222, 1965. 26. Murphy TE, Constanzi JJ: Pseudotumor cerebri associated with pernicious edema. Ann Intern Med 70:777, 1969. 27. Pattison UH, Jebbett JN: Histopathological similarities between scrapie and cuprizone toxicity in mice. Nature 230:115-117, 1971. 28. Curley A, Kimbrough RD, Hawk RE, et al: Dermal absorption of hexachlorophene in infants. Lancet 2:296-297, 1971.
Gowdy, MD, Andrew G. Ulsamer, PhD
\s=b\ Vacuolization of the white matter of the brain is produced by a number of disease entities and chemicals, including hexachlorophene. Brains of 135 stillborn infants and infants dying in the neonatal period were examined for vacuole formation in the white matter to determine if any hexachlorophene-like lesions could be found. A nonsignificant excess of vacuoles was found in infants bathed in hexachlorophene at birth compared with infants not bathed in it. Analysis of 11 brains for hexachlorophene showed that detectable levels were present in five, all of which showed vacuolization. None was detected In the remaining six, three of which also showed vacuolization. In two of these there was no hexachlorophene exposure. (Am J Dis Child 130:247-250, 1976)
Gump1 discovered In hexachlorophene septic properties the 1939
that retained its anti¬ in presence of soap, and it became a standard com¬ ponent of many topically applied drugs and cosmetics. Hexachloro¬ phene and a number of products con¬ taining it were tested extensively prior to marketing, but examination of the brains of animals involved in Received for publication Sept 30, 1974; acMarch 3, 1975. From St. Elizabeths Hospital, Washington, DC (Dr Gowdy), and the Division of Physical Sciences, Consumer Product Safety Commission
cepted
(Dr Ulsamer). The views expressed do not
necessarily agencies.
are the authors' own and reflect those of the affiliated
Reprint requests to 12408 Parkton St, Oxon Hill, MD 20022 (Dr Gowdy).
these tests was omitted.2 In experiments in China on the use of hexachlorophene for human liver fluke infection (Clonorchis sinensis), the brains of rats fed this compound were examined and reported as nor¬ mal; however, trials of the drug in man revealed severe neurotoxic reac¬ tion that precluded its systemic use.3 A neurotoxic condition is also a prom¬ inent feature of accidental hexachlo¬ rophene poisoning in humans,4 and hexachlorophene was suspected as the cause of convulsions in burn patients treated with hexachlorophene soaks.5 Kimbrough et al6 first described the central nervous system (CNS) patho¬ logical findings produced by hexa¬ chlorophene after conducting feeding studies in rats to determine its safety for use as an agricultural pesticide. Further studies in monkeys, rats,7 pigs, and tadpoles8 confirmed that hexachlorophene produced CNS le¬ sions in numerous species. By light microscopy the lesions in rats are seen as cystic spaces in the white matter of the brain and spinal cord. Electron microscopy shows that these spaces are in the wall of the myelin sheath.6 Vacuoles are most prominent in the white matter of the cerebellar folia, but are distributed throughout the white matter of the cerebrum, optic tract, pons, medulla, and cord. In rats below the age of 10 to 11 days, or above the age of 21 days, no lesions are seen when they are dosed orally with 10 mg/kg of hexachlorophene, but lesions do ap-
Downloaded From: http://archpedi.jamanetwork.com/ by a University of Manitoba User on 06/05/2015
pear at this dose in the age range of 11 to 18 days,7 which is the period of
maximum myelinization of the axons of the white matter of the rat brain.9 The lesions produced by hexachloro¬ phene in the rat brain are not diag¬ nostic for this chemical. Vacuoles are observed in both animals and man in a variety of diseases and following the administration of a number of chemical compounds. These condi¬ tions are listed in Table 1. Electron microscopy shows that hexachloro¬ phene lesions in rats are due to accu¬ mulation of some material, probably water, in the wall of the myelin sheath,10 but this distinction cannot be seen by light microscopy. Ulsamer et al7 have reported a slight decrease in the phospholipid and sterol content of the brain in rats given hexachloro¬ phene by mouth. An attractive theory is that the spaces represent accumula¬ tion of some precursor or intermedi¬ ary of myelin; however, the spaces produced by triethyltin contain only water and electrolyte.11 Biochemical studies have shown that triethyltin and similar compounds inhibit glu¬ cose metabolism and uncouple oxida¬ tive phosphorylation.12 A similar ac¬ tion has been demonstrated for hexachlorophene,13 but it is not clear that this action is related to the pro¬ duction of vacuoles in the immature myelin sheath. Webster et al8 have reported that hexachlorophene is localized in the protein fraction of myelin where it could disrupt the myelin sheath either
mechanically or by interference with oxidative phosphorylation. Chemical compounds causing this lesion fall into two groups, those that are antiseptics for Gram-positive mi¬ croorganisms (hexachlorophene, tri¬ ethyltin, isoniazide, and cuprizone) and those that interfere with choles¬ terol synthesis (AY 9944 and related compounds). By light and electron mi¬ croscopy the lesions of cribriform de¬
the prophylaxis of staphylococcal in¬ fections in hospital nurseries. To eval¬ uate this possibility, material from the National Institutes of Health (NIH) perinatal study was reviewed. MATERIALS AND METHODS The scope and purpose of the NIH col¬ laborative study on cerebral palsy, mental retardation, and other neurological and sensory disorders of childhood have been detailed elsewhere.18 Some of the pregnancies in the NIH col¬ laborative study terminated in stillbirth or abortion. Other infants died after birth from various diseases. An attempt was made to secure an autopsy on every sub¬ ject who died. This included a detailed ex¬ amination of the brain with the prepara¬ tion of up to 60 tissue blocks from the cerebrum, medulla, pons, and spinal cord. Four sections were prepared from each block, one each for hematoxylin-eosin, gallocyanin, trichrome, and Luxol fast blue-
generation, triethyltin, isoniazid, cuprizone, and hexachlorophene ap¬ pear to be identical. Cuprizone and hexachlorophene are both chelating agents for copper. Copper deficiency in animals produces CNS symptoms
similar to those observed in the above experimental poisonings, and the ef¬ fects of hexachlorophene on CNS cop¬ per metabolism may merit further
study.
In rats CNS lesions appeared when the blood level of hexachlorophene reached 1.2/ig/ml.14 Investigation of blood levels following bathing of newborn infants with a commercial hexachlorophene preparation showed that values as high as 0.3jtig/ml can be attained after a single washing and up to 0.78µg/ml with repeated wash¬ ing.15 Thus, it is theoretically possible that lesions of the white matter of the CNS could result from the prac¬ tice of bathing newborn infants re¬ peatedly with hexachlorophene for Table 1.—Conditions
PAS-hematoxylin staining. A total of 76 specimens from liveborn, hexachlorophene-bathed subjects were available. Since only 18 specimens from nonhexachlorophene-bathed controls were available, 41 specimens from stillborn infants were reviewed for additional controls. Slides were studied by light mi¬ croscopy without knowledge of hexachloro¬ phene use, diagnosis, or cause of death. Be¬ cause of delays in processing, sections were available only until 1964. Slides from all areas were examined for lesions resem¬ bling those seen in the brains of hexachlorophene-treated rats and monkeys. The
Producing Vacuole Formation in the Brain
Source Magee et al" Katzman et al" Worden et al20
After reading of the slides pleted, clinical records of each
Triethyltin Triethyltin Pheniprazine hydrochloride
reviewed for maternal disease, type of de¬
Phenelzlne*
Indanylcarbethoxyhydrazlne* Heptylhydrazine*
These factors were then coordinated with the slide reading and hexachlorophene bathing at birth. Specimens representing 76 deaths fol¬ lowing live births were reviewed from hexachlorophene-using hospitals. Vacuoles were seen in 14 (18%). Eighteen specimens from live births were received from nonhexachlorophene-using institutions. Two (11%) were positive. These differences are
Isoniazid* Isoniazid mesylate AY 9944f AY 9944 AY 9944
Triparanolt Cribriform disease of mice of the Pernicious anemia
Spongy degeneration
nervous
system
not
Scrapie
CuprizoneJ *
Experimental
monamine oxidase inhibitors.
t Experimental cholesterol-lowering agents. Triparanol
was
marketed for
a
short time
as
MER 29.
Chelating agent used
in
was com¬ case were
livery, complications, birth weight, length of gestation, clinical course, autopsy diag¬ noses, and other pertinent information.
Tetrahydronaphthylhydrazine*
Green et al24 Buchanan and Davis25 Murphy and Constanzi26 Pattison and Jebbett27
.
study.
Compound/Condition
Smith et al2' Suzuki and DePaul22 Rawlins and Uzman23 Cremer'2
overall extent of involvement was esti¬ mated subjectively and recorded as nega¬ tive to 4 + Four plus represents vacu¬ olization of 50% or more of the white matter of the cerebellum; 3+ represents involvement of 25% to 50%; 2+ is involve¬ ment of 10% to 25%; and 1 + is involvement of less than 10%. Reliability of the diag¬ nosis varies with the percent of involve¬ ment. In the studies of Ulsamer et al,7 1 + areas of vacuolization were seen in some control animals. Sections were considered negative if there was no cerebellar involve¬ ment or if involvement was not confined to the white matter. In general, lesions in positive cases were seen in all areas of white matter in the brain and cord, but were most pronounced in the white matter of the cerebellum. The incidence of brain vacuoles in sub¬ jects washed with hexachlorophene at birth was compared to the incidence in those who were not washed in it. The use of hexachlorophene bathing in the new¬ born nurseries was determined by inquiry of the hospital involved. Two institutions used 3% hexachlorophene daily (pHisoHex) and were classified as high-level users. Most hospitals used dilute 3% daily and were considered medium-level users. Two institutions used dilute 3% hexachloro¬ phene at birth only and were classified as low-level users. The definition of dilute is somewhat vague. One formula is 15 ml of 3% hexachlorophene in 1 liter of water. In practice it is likely that the preparation was added to the bath water without ac¬ tually measuring the amount. Hexachloro¬ phene bathing of newborns was not used during the period covered by this study in two hospitals. One hospital did not use hex¬ achlorophene during the first year of the
analysis for copper.
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statistically significant.
A total of 41 specimens from nonlive births was reviewed. Of 30 from hexachlorophene-using institutions, vacuoles were seen in six (20%). No vacuoles were seen among the 11 cases from nonuser hos-
pitáis. Table 2 gives these results quantita¬ tively. In an attempt to account for the lesions in stillborn infants, an analysis was made of the use of hexachlorophene as the anti¬ septic for sterilization of the perineum prior to delivery. Hexachlorophene is read¬ ily absorbed from this area in man and crosses the placenta to produce congenital defects in animals.17 Twelve percent of the hexachlorophene group showed lesions compared to 13.7% of the control group. There is a tendency for the percentage of lesions to increase with increasing birth weight among both liveborn and stillborn, suggesting that the phenomenon is corre¬ lated with increased maturity. As men¬ tioned above, lesions do not appear in rats until myelinization of the cerebral white matter tracts begins. In humans this is around the second month of life. In infants surviving 30 days or longer all of the posi¬ tive findings are in the hexachlorophenebathed group. However, only one infant from the control group survived 30 days. Brains, fixed in an aqueous solution of formaldehyde, from which the slides were prepared were available at NIH, and as a further check, several specimens were ana¬ lyzed for hexachlorophene content. This was determined by gas-liquid chromatog¬ raphy, using a radioactive nickel electroncapture detector as described previously by Ulsamer.18 Results are shown in Table 3. The halflife of hexachlorophene in man is given variously as 8 to 24 hours,2·10 and no hexa¬ chlorophene is found in blood 15 days after exposure stops, even though in rats the brain lesions persist for at least three months after exposure. During this period, lesions decline from 4+ to 1 + or 2+ in ex¬ tent and also decrease in size.6·10 In cases 2, 4, and 5, hexachlorophene levels and slide readings agree, and all infants were hexachlorophene-bathed. Tissue levels in case 1 could be due to postnatal contact with hexachlorophene-containing soaps, lotions, and other preparations. Although this in¬ fant was delivered in a hexachloropheneusing institution, hexachlorophene could not remain in the brain for 18 months without continued exposure. While other contact with hexachlorophene, such as pre¬ natal use of soap and cosmetics containing the antiseptic, may be a factor, case 6 could be due to the use of hexachlorophene solution for predelivery washing of the mother. The use of hexachlorophene-containing talc on infants has been implicated in hexachlorophene poisoning and death.15 The positive reading in case 3 may be due to prenatal exposure to isoniazid. Case 7 could be from maternal use of hexachloro¬ phene soaps and cosmetics or to unknown
factors not related to use of the antiseptic. In case 8 hexachlorophene levels would have fallen to zero by three months after exposure. In cases 9 through 11, levels and readings agree. It should be remembered
Table 2.—CNS Lesions in
that this tissue
fixed with formalde¬
was
hyde prior to hexachlorophene level deter¬ mination, and the effects of this on the quantitation of the chemical are not known.
Hexachlorophene-Bathed and Control Subjects Slide Reading
3+
Treatment
High Medium Low Total bathed with hexachlorophene Controls Stillborn Liveborn Total
Table 3.—Tissue
Sex
34 24
13 27
76 59 41 18 135
62 51 35 16 113
14 15 11 16
Hexachlorophene
13
Levels
Compared With Slide Readings* Hexachlor¬ Hexachlor¬
Weight,
Gestation,
Slide
gm
Weeks 42 32 30 38 35 39 40 39 39 30 40
Reading
3,360 1,310 1,414 2,545 1,225 2,980 2,955
3t
3,220 10 11
M
2,875 1,280 2,810
3+ 3+ 3+ 2+ 2+ 2+ 2+ 2+
Use
Table 4.—Association of Lesions With Various Slide Anoxia L* Anoxia Sf Premature L Premature S Atelectasis L Pneumonia L Hyaline membrane disease L Congenital defect L Congenital defect S Septicemia L
Septicemia S Digitalis L L signifies liveborn. t S signifies stillborn. One not bathed in hexachlorophene. § Two not bathed in hexachlorophene. *
j
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M|
0.70 0.40 0.0 0.34 0.20 0.10 0.0 0.0 0.0 0.0 0.0
Correlation of slide readings against tissue levels gives omitted, r = 0.71 and < .025. t M signifies medium level hexachlorophene bathing. Ì Isoniazid exposure. § H signifies high-level hexachlorophene bathing. [| signifies maternal preparation only.
3+
ophene
ophene,
*
Condition
Negative Positive
39 33
Birth Case
%
1+
2+
2+
3Í 1Í 4t
r
M
None
H§ None H None M :
0.57 and
Survival 18 mo 5 days
Stillborn 102 25 5 33 3 33 29 8
< .1 ; if
hr hr
days hr mo
hr hr weeks case
3 is
Autopsy Diagnoses
Reading
%
Negative 8 10 29 10 15 15 20 11
2§ 11
Positive
29
25 25 13 41 29 43 15
The
of death and other autopsy compared with the inci¬ dence of vacuolization. This is shown in Table 4. Among live births anoxia was not related to the lesion, being recorded in 18% of both the negative and positive groups. However, among stillbirths, anoxia was present in half the subjects in whom le¬ sions were seen. An excess of cardio¬ vascular malformations was found in the positive group, occurring in 32% compared with 7% in the negative group. Pneumonia was less common in the positive group, being 9% compared with 36% in the nega¬ tive group. Atelectasis was recorded in 27% of the positive group and 15% of the nega¬ tive group. Finally, septicemia was seen in 14% of the positive group and 7% of the negative group. If anoxia and atelectasis are omitted, the figures in Table 2 become nine out of 48 positive for the bathed group and four out of 44 for the nonbathed group, which is not statistically signifi¬ cause
diagnoses
were
cant.
Prescription drug intake of the mothers during pregnancy was reviewed for isonia¬ zid and triparanol, the only two drugs in clinical use at that time implicated in vacu¬ ole production. One mother of a stillborn with 3+ vacuolization received isoniazid during her pregnancy. Aside from this, cardiac glycosides, and the small amounts of vitamin A contained in prenatal supple¬ ments, the subjects of the study were not exposed to any of the agents listed in Table 1. Most of the agents producing vacuoliza-
tion are laboratory curiosities or industrial chemicals that need not be considered in the differential diagnosis in the present cases.
COMMENT
Comparison of hexachlorophene brain tissue levels and slide readings shows one false positive or a 10% er¬ ror in interpretation. No hexachloro¬ phene was detected in the brains of infants not washed with this sub¬ stance. Correlation of slide readings and tissue levels gives a correlation coefficient of .57, which is not signifi¬ cant (P < .1). Although some lesions appear to be associated with hexa¬ chlorophene exposure, all such lesions are not due to the practice of bathing newborns in hexachlorophene, since vacuoles were found in subjects who were not bathed with this material. Prenatal use of hexachlorophene preparations by the mother and the use of lotions and other products con¬ taining this antiseptic on infants may have produced lesions in some cases. Detectable, albeit minute, amounts of hexachlorophene have been found in the cord blood of unbathed newborns.28 During the period of the perinatal study (1960 to 1970),
hexachlorophene was widely used, and both prenatal and postnatal ex-
posure to it
might well have occurred. Under the conditions of this study there were no noteworthy differences in the incidence of CNS vacuolization between hexachlorophene-bathed and control subjects. The occurrence of vacuolization and hexachlorophene levels in the same subjects is sugges¬ tive, but does not prove a causal rela¬
tionship.
It would
seem
to avoid the
preparations
prudent to continue hexachlorophene
use of on infants
and children until a definitive answer to the ques¬ tion of toxicity can be found. Joseph S. Drage gave permission to examine clinical records of the Perinatal Research Branch of the National Institute of Neurological Diseases and Strokes. Toshio Fujikura, MD, National Institute of Neurological Diseases and Strokes, gave permission to study slides and provided advice on interpretation of slides. William Cockran, MD, Robert O. Fisch, MD, William Clark, Jr, MD, Carl Zelson, MD, Marguerite Yates, MD, Raymond Vande Weile, MD, Brigette de la Burde, MD, Janet Hardy, MD, Harry M. Beine, MD, Sheldon Korones, MD, Adolph Sellman, MD, and Harmon Jordan supplied information on hexachlorophene use in the various hospitals.
Nonproprietary Names and Trademarks of Drugs Hexachlorophene—pHisoHex. Pheniprazine-Cainm.
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