DRUG AND CHEMICAL TOXICOLOGY, 1(3),

259-275 (1978)

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HEALTH ASPECTS OF CHLOROFORM

-A

REVIEW

S . G. Winslow and H. B . Gerstner Toxicology Information Response Center ,InformationCenter Complex Informatiod Division Oak Ridge National Laboratory* Oak Ridge, Tennessee 37830

To die, to sleep; to sleep: perchance to dream: ay, there's the rub; Hamlet ABSTRACT First prepared in 1831, chloroform was initially employed as an "ideal" anesthetic. Chloroform was later found to cause heart and liver damage and, with the discovery of safer agents, lost importance as a medical anesthetic. Today chloroform is used primarily as a solvent in industry, in the production of antibiotics, as a cleaning agent, and as an ingredient in pharmaceutical and veterinary formulations. A recent report issued by the National Cancer Institute implicates chloroform as a carcinogen in laboratory rodents. These findings, in conjunction with enforcement of the Delaney Clause have generated renewed interest in chemicals such as chloroform which are not only used in a variety of industrial applications, but which are also available to the general public in a number of food and cosmetic products. *Work supported by the Toxicology Information Program, National Library of Medicine, National Institutes of Health, Department of Health, Education, and Welfare under NLM Interagency Agreement No. 40-274-71. Oak Ridge National Laboratory is operated by Union Carbide Corporation for the U.S. Department of Energy under Contract No. W-7405-eng-26.

259 Copyright 0 1978 by Marcel Dekker, Inc All Rights Reserved Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher

WINSLOW AND GERSTNER

260

INTRODUCTION Chloroform, a halogenated hydrocarbon, was prepared independently in 1831 by Liebig in Germany, Soubeirian in France, Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Guelph on 07/01/12 For personal use only.

and Guthrie in the United States.l

In 1847, Flourens first used

chloroform as an anesthetic for animals and during that same year Simpson performed surgery on human patients using chloroform anes-

*

thesia.

Although widely accepted initially, chloroform later was

found to cause heart and liver damage.ly2 With the discovery of safer agents, chloroform lost its importance as a medical anesthetic. Today, chloroform is primarily used in industry as a solvent for certain lacquers, for extraction and purification of penicillin and other antibiotics, in the manufacture of artificial silk and plastics, as a cleaning agent, as a constituent of floor polishes, and as an ingredient of pharmaceutical formulations. Other applications include use as a pesticide, a base for fluorocarbon refrigerants, and, in conjunction with carbon tetrachloride, as a fire extinguisher. Chloroform enjoys limited use as an anesthetic, a carminitic, and an antiemetic in veterinary pra~tice.~ According to data from the National Cancer Institute (NCI) the amount of chloroform to which people are exposed in pharmaceutical products alone is estimated to be more than 300,000 pounds per year.

Of this, about 98,000 pounds is ingested orally; about

117,000 pounds goes into inhalational drugs; and about 86,000

pounds is in preparations applied to the skin. Aside from its general toxicity, chloroform was found to cause cancer in animals, as demonstrated by sevetal studies performed throughout the past few decades. Because of these facts, chloroform has joined the ranks of numerous commercial chemicals in the human environment which require close scrutiny. The objective of this overview is to provide a brief, but inclusive, summary of the reference literature. Topics include physical and chemical properties, metabolism, toxicity, carcinogenic potential, and conclusions.

HEALTH ASPECTS OF CHLOROFORM

261

PHYSICAL AND CHEMICAL PROPERTIES Chloroform (CHC13) is a colorless, volatile liquid with a sweetish, burning taste and a characteristic odor. a molecular weight of 119.38.

Chloroform has

Practically insoluble in water yet

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readily soluble in organic solvents - such as ethers, benzene, and alcohols - chloroform is not affected by acids, but is decomposed by alkali hydroxides yielding alkali chloride and sodium formate. In sunlight, chloroform is oxidized to phosgene and other products forming highly toxic and irritating fumes; nonflammable and nonexplosive in ambient air, chloroform will burn with a green flame at high temperat~re.~Detailed data on the physical and chemical properties of chloroform are listed in Table 1.

TABLE 1 Identifying Characteristics of Chloroform Chemical Abstracts Service (CAS) Registry Number: 67-66-3 Merck Index Monograph Number (Ninth Edition): 2120 National Cancer Institute (NCI) Number: 2686 CAS Preferred Names: Chloroform (Eighth Collective Index) Methane, trichloro- (Ninth Collective Index) Common NamesfSynonyms: Formyl trichloride; Methenyl trichloride; Methyl trichloride; Trichloroform; Trichloromethane Empirical Formula: CHC13 Molecular Weight: 119.38 Wiswesser Line Notation: GYGG Percentage Composition: C - 10.05%, C1 - 89.10%, H - 0.84% Physical State: Colorless liquid with pungent odor Melting Point: -63.5"C Boiling Point: +62"C Density: 1.4832 at 20°C Vapor Pressure: 160 mm at 2OoC Solubilities: Water - slightly soluble; Ethanol, ethyl ether, benzene - soluble in all proportions; Acetone - soluble Threshold Odor Concentration: 205-307 ppm

WINSLOW AND GERSTNER

262

METABOLISM Chloroform can be absorbed through the lungs, from the gastrointestinal tract, and to a lesser degree through the intact skin. Chloroform penetrates the placental barrier and has been Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Guelph on 07/01/12 For personal use only.

found in fetal liver.3 of entrance into man.

Inhalation is considered the primary route The compound is absorbed rapidly and dis-

tributed widely throughout the body. HUMAN STUDIES Exhalation of unchanged chloroform and its carbon dioxide metabolite accounted for a substantial amount of a single oral dose administered to humans.

Healthy male and female volunteers

received 500 mg of chloroform in capsule form and were monitored for chloroform elimination throughout an 8-hr period; blood and urine samples were examined also. After a lag time ranging from 40 to 120 min, 17.8% to 66.6% of the dose was exhaled as chloroform. Females eliminated less of the chloroform than males; the difference probably resulted from the affinity of chloroform for adipose tissue which is more prevalent in w ~ m e n . ~In two volunteers given 13CHC13, about 50% of the dose was exhaled as 13C02, with the maximal concentration in expired air occurring at 75 to 210 min after administration; no chlorinated intermediates were found

.

ANIMAL STUDIES Experiments by Paul and Rubinsteln9 showed that 70% of a chloroform dose administered intraduodenally t o rats was recovered unchanged through the lungs, while 4% was converted to carbon dioxide. Van Dyke, Chenoweth, and Van PoznaklO reported similar findings in rats. When injected intraperitoneally with 0.1 ml

of

%labeled

chloroform, the animals exhaled approximately

4% to 5% of the dose as carbon dioxide within a 12-hr post anes-

thetic period; urinary metabolites ranged from 0% to 2% of the

HEALTH ASPECTS OF CHLOROFORM a d m i n i s t e r e d amount.

263

With an improved method which recovered

about 95% of t h e a d m i n i s t e r e d r a d i o a c t i v i t y , Brown, Langley, Smith, and T a y l o r ' l

found t h a t Sprague-Dawley r a t s which had

r e c e i v e d a s i n g l e 60 mgJkg d o s e of 14CHC13 c o n v e r t e d a p p r o x i -

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m a t e l y 60% i n t o carbon d i o x i d e .

The d i s c r e p a n c y of f i n d i n g s be-

tween v a r i o u s i n v e s t i g a t o r s a p p a r e n t l y was caused by d i f f e r e n c e s i n t h e recovery t e c h n i q u e .

I n t h e i r comparative s t u d y , Brown,

Langley, Smith, and T a y l o r ' t h r e e s t r a i n s of mice.

a l s o i n c l u d e d s q u i r r e l monkeys and

For t h e monkeys t h e y o b t a i n e d a c o n v e r s i o n

r a t e of o n l y 20%; t h e mice, r e g a r d l e s s of s t r a i n (CBA, CF/LP, and C57BL) c o n s i s t e n t l y had t h e h i g h e s t c o n v e r s i o n rate among t h e tested species.

When I 4 C H C 1 3 w a s g i v e n o r a l l y a t 60 mg/kg o v e r

a five-day p e r i o d , t h e mice c o n v e r t e d 80% of t h e d o s e i n t o carbon d i o x i d e ; a b o u t 6% w a s exhaled unchanged, w h i l e t h e rest appeared i n t h e c a r c a s s , i n t h e f e c e s , and i n t h e u r i n e which c o n t a i n e d r a d i o a c t i v e u r e a , c a r b o n a t e s and b i o c a r b o n a t e s as w e l l as s e v e r a l o t h e r u n i d e n t i f i e d compounds. Butler':!

observed a n i n v i t r o r e d u c t i o n of chloroform t o

methylene c h l o r i d e by mouse l i v e r s l i c e s o v e r a 20-hr i n c u b a t i o n period.

Upon h e a t - i n a c t i v a t i o n , breakdown proceeded a t a slower

r a t e , i n d i c a t i n g t h a t t h e p r o c e s s may n o t b e enzymatic b u t may be coupled w i t h enzymatic e l e c t r o n t r a n s p o r t systems.

P a u l and

R u b i n s t e i n 9 d i d n o t f i n d e v i d e n c e of t h i s c o n v e r s i o n i n i n v i t r o s t u d i e s with rat l i v e r t i s s u e . T a y l o r , Brown, Keeble, and Langley'

conducted a s i m i l a r

s t u d y concerning s e x d i f f e r e n c e s i n chloroform metabolism and toxicity.

A s i n d i c a t e d by 1 4 C 0 2 d e t e r m i n a t i o n s , and c l o s e l y

a g r e e i n g w i t h p r e v i o u s f i n d i n g s , m i c e m e t a b o l i z e d a b o u t 80% of a n a d m i n i s t e r e d d o s e , r e g a r d l e s s of s e x .

Tissue d i s t r i b u t i o n a t

v a r i o u s t i m e s f o l l o w i n g exposure, however, r e v e a l e d s t r i k i n g s e x differences.

I n l i v e r and k i d n e y s , t h e c o n c e n t r a t i o n of radio.-

a c t i v i t y w a s much h i g h e r f o r males t h a n f o r females.

Aside from

t h e l i v e r and i n c o n t r a s t t o males, female m i c e accumulated rad i o a c t i v i t y p a r t i c u l a r l y w i t h i n t h e i n t e s t i n e and b l a d d e r .

264

WINSLOW AND GERSTNER Because of their exceptionally high conversion rate of

chloroform into carbon dioxide, mice should not be considered suitable models for the prediction of chloroform metabolism in

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other species.

TOXICITY

EYE Exposure to concentrated chloroform vapors causes a stinging sensation.

Splashing of the substance into the eyes evokes burn-

ing, pain, and redness of the conjunctival tissue.

The corneal

epithelium is sometimes injured; however, regeneration starts rapidly and leads to full recovery within one to three days.I4 Upon inhalation, chloroform causes dilation of the pupils with reduced reaction to light as well as reduced intraocular pressure.15 HEART Chloroform depresses the cardiovascular system with a resultant fall in blood pressure.16 Vascular smooth muscles are affected.

The vagus nerve is stimulated, a condition which occasionally

leads to cardiac arrest.

Chloroform sensitizes the myocardium to

catecholamines and, as a consequence, ventricular fibrillation may be provoked through simultaneous administration of these substances.’

Prolonged chloroform anesthesia results in a defi-

ciency of prothrombin and fibrinogen, leading to an impairment of the blood clotting mechanism.

Death from chloroform adminis-

tration may occur due to stoppage of the heart either as a result of reflex stimulation of the vagus or of direct toxic action of

chloroform on the heart muscle.” KIDNEY Transient irritation of the kidneys has been observed during chloroform narcosis. Acute exposure may entail damage to the

HEALTH ASPECTS OF CHLOROFORM

265

renal tubules, mainly involving epithelium of Henle’s loops. These degenerative changes are more prevalent in adult or nephropathic animals and are manifested by the accumulation of stainable lipoid material. 3 9 l 9 Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Guelph on 07/01/12 For personal use only.

When receiving graded amounts of chemically pure chloroform, all males of strain A mice develop renal necrosis, regardless of dose level; however, the degree of injury rises with dose. Probably as the result of sex differences in tissue distribution, female mice do not display necrotic lesions, even at the highest exposure level.20 LIVER Chloroform exposure causes impairment of hepatic function. As early as 15 min following the start of chloroform anesthesia, fatty infiltration of the liver becomes recognizable.2 1

Prolonged

exposure may lead to further liver anomalies progressing from necrosis o f cells, particularly those surrounding central veins, to yellow subacute atrophy of the entire organ.22 Assays measuring the levels of SGOT (serum glutamic-oxaloacetic transaminase) and SGPT (serum glutamic-pyruvic transaminase), as well as the retention of certain dyes, are useful tools for determining the extent of hepatic damage. 3 9 2 2 Results of an epidemiological study among pharmaceutical workers exposed to chloroform over an extended period showed enlarged livers, fatty degeneration, and, in several cases, toxic hepatitis.23 Following improvement of work place conditions, a reduction in hepatomegaly was observed. A phenomenon associated with liver damage is that of “late

chloroform poisoning.”

For one to three days following exposure,

patients remain asymptomatic; then they enter a phase characterized by prostration, nausea and profuse vomiting, excitation alternating with apathy, and unconsciousness deepening into coma that may lead to death within four to five days.

At autopsy, degen-

erative changes are evident in various organs such as brain,

266

WINSLOW AND GERSTNER

kidneys, heart, and liver.17

The chief lesion after "delayed

poisoning" found post mortem is fatty degeneration of the liver. Animal experiments show that pathological changes return to normal in survivors.21

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Several investigators have studied the effects of various diets on the induction of liver damage by chloroform. Diets high in carbohydrates and protein lead to less fat deposition in the liver, indicating a reduced hepatic susceptibility to the toxic action of chloroform under these circumstances. RESPIRATION Generally, chloroform causes respiratory depression; occasionally, acute exposure may lead to respiratory a r r e ~ t . ~ If used at all in modern medicine, the substance is administered at low concentrations (2% to 4 % ) providing sufficiently deep anesthesia without irritating the mucosa.

In instances where symp-

toms of respiratory distress occur, mainly through inadvertent administration of high concentrations, adequate ventilation often alleviates the problem. Inhalation represents the main route of industrial exposure.19

When inhalation is discontinued, the blood level falls

to 50% within 5 min; only traces remain at 30 min.21

The thresh-

old limit value (TLV) for chloroform has been set at 50 ppm or 240 m g / n ~ ~ . * ~ Comparative inhalation studies on laboratory animals yielded the following results: mice breathing 8,000 ppm died after a 3-hr exposure; rabbits died after 2 hr at 12,500 ppm; and dogs survived higher doses.2 5

HUMAN TOXICITY SYMPTOMS Signs of chloroform poisoning include a characteristic sweetish odor on the breath, cold and clammy skin, and dilated pupils.26

Ketosis due to the incomplete oxidation of fats, as

well as a rise in blood sugar, accompanies chloroform poison-

267

HEALTH ASPECTS OF CHLOROFORM ing. 3 , 2 7

I n i t i a l e x c i t a t i o n a l t e r n a t i n g w i t h a p a t h y i s followed

by p r o s t r a t i o n , u n c o n s c i o u s n e s s , a n d , i n e x t r e m e c a s e s , by d e a t h . 1 7 Nausea and p r o f u s e v o m i t i n g a r e common c o m p l a i n t s f o l l o w i n g prolonged chloroform i n h a l a t i o n .

*

Ingested chloroform a c t s as a

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s t r o n g i r r i t a n t t o t h e mucosa of t h e g a s t r o i n t e s t i n a l t r a c t . l 7 I n d i v i d u a l s u s c e p t i b i l i t y v a r i e s considerably; an o r a l dose of 180 m l h a s been s u r v i v e d , w h i l e a d o s e o f 5 m l h a s c a u s e d s e r i o u s illness.

For a n a d u l t p e r s o n , t h e mean l e t h a l d o s e i s a b o u t 30

m l chloroform.28 Dermal a p p l i c a t i o n of c h l o r o f o r m may i r r i t a t e t h e s k i n , c a u s i n g r e d d e n i n g , b l i s t e r i n g , and b u r n s . l 7

EMBRYOTOXICITY AND FETOTOXICITY I n experiments i n v o l v i n g i n h a l a t i o n exposure of p r e g n a n t Sprague-Dawley r a t s t o c h l o r o f o r m a t t h r e e l e v e l s (30 ppm, 100 ppm, and 300 pprn) t h r o u g h o u t d a y s 6 t o 1 5 o f g e s t a t i o n , c h l o r o form proved t o be b o t h e m b r y o t o x i c and f e t o t o x i c .

Minor e f f e c t s

were s e e n a t 30 ppm, e m b r y o t o x i c and f e t o t o x i c e f f e c t s became more s t r i k i n g a t 100 ppm, and a t 300 ppm, t h e number of l i v e f e t u s e s and t h e s i z e of f e t u s e s showed a s i g n i f i c a n t d e c r e a s e while f e t a l resorption increased.

Teratogenic e f f e c t s included

a c a u d i a and s k e l e t a l a n o m a l i e s a s w e l l as s o f t t i s s u e c h a n g e s ; t h e s e e f f e c t s grew i n number and s e v e r i t y w i t h i n c r e a s i n g c h l o r o form c o n c e n t r a t i o n . 2 9

CARCINOGENICITY More t h a n 30 y e a r s a g o , e x p e r i m e n t s c o n d u c t e d by Eschenbrenner20 i m p l i c a t e d c h l o r o f o r m a s a p o t e n t i a l c a r c i n o g e n . S t r a i n A mice r e c e i v e d g r a d e d d o s e s of c h e m i c a l l y p u r e c h l o r o form by stomach t u b e .

F o l l o w i n g a d m i n i s t r a t i o n , a dose-

dependent r e l a t i o n s h i p was o b s e r v e d between l i v e r n e c r o s i s and t h e s u b s e q u e n t i n c i d e n c e o f hepatomas.

A l t h o u g h t h e number o f

a n i m a l s used i n e a c h t e s t group was s m a l l and many m i c e d i e d w i t h i n a s h o r t period following t r e a t m e n t , a high i n c i d e n c e of

WINSLOW AND GERSTNER

268

hepatomas i n s u r v i v o r s suggested t h a t chloroform h a s c a r c i n o genic properties. On March 1, 1976, t h e N a t i o n a l Cancer I n s t i t u t e p u b l i s h e d a s t u d y t h a t f u r t h e r i n d i c t e d chloroform as a p o s s i b l e cancer-

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causing agent.30

For 78 weeks, chloroform d i s s o l v e d i n c o r n

o i l w a s a d m i n i s t e r e d f i v e t i m e s weekly t o e x p e r i m e n t a l groups of 50 r a t s o r m i c e which were a l s o grouped i n t o males and females. Two d o s e l e v e l s given by gavage w e r e t e s t e d . I n Osborne-Mendel r a t s , t h e dose l e v e l s were 90 and 180 mg/kg body weight f o r males.

Females i n i t i a l l y r e c e i v e d 1 2 5

and 250 mglkg; a f t e r 22 weeks, t h e s e d o s e s were reduced t o 90 and 180 mglkg, y i e l d i n g mean l e v e l s of 100 and 200 mglkg.

The

r e s u l t s showed t h a t b o t h s u r v i v a l t i m e and weight g a i n d e c l i n e d i n a l l t r e a t e d groups.

I n m a l e r a t s , t h e i n c i d e n c e of kidney

tumors w a s s t a t i s t i c a l l y s i g n i f i c a n t (p = 0.0016);

the incidence

amounted t o 24% i n t h e upper d o s e group, t o 8% i n t h e lower d o s e group, and t o 0% i n t h e c o n t r o l s .

Thyroid tumors seemed t o occur

more f r e q u e n t l y among t r e a t e d female r a t s ; however, t h e f i n d i n g s

w e r e n o t considered t o be s i g n i f i c a n t s t a t i s t i c a l l y .

No kidney

tumors o c c u r r e d i n c o n t r o l o r t r e a t e d females. I n i t i a l l y , male B6C3F1 m i c e r e c e i v e d d o s e s of 100 and 200 mglkg, w h i l e females r e c e i v e d 200 and 400 mglkg.

A f t e r 1 8 weeks,

t h e s e d o s e s were i n c r e a s e d t o 150 and 300 mg/kg f o r males and 250 and 500 mg/kg for females.

R e g a r d l e s s of sex, t h e a n i m a l s

d i s p l a y e d a h i g h e r i n c i d e n c e o f h e p a t o c e l l u l a r carcinoma t h a n c o n t r o l s (p =

Health aspects of chloroform--a review.

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