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Toxicologic studies of alkylphenol polyoxyethylene surfactants
TOXICOLOGY
AND
Toxicologic
APPLIED
PHARMACOLOGY
Studies
14, 3l5-334(1969)
of Alkylphenol
Polyoxyethylene
Surfactants
HENRY F. SMYTH, JR. AND JOSEPH C. CALANDRA Carnegie-Mellon Unicersity, Mellon Institute, Pittsburgh, Industrial Bio-Test Laboratories, Northbrook,
Pennsyhania Illinois 60062
15213, and
ReceiL>ed July 10, I568
Toxicologic Studiesof Alkylphenol Polyoxyethylene Surfactants.SMYTH, H. F., JR.,andCALANDRA, J. C. (1969). Toxicol. Appl. Pharmucol. 14,3 15-334. In order to determinethe suitability of a large group of commercialsurfactants for applicationswhere smallamountsmay enter food, the toxicity of 22 products wasstudied. Ethylene oxide chain lengthsrangedfrom 4 to 40, and alkylphenols included di(secondary butyl), octyl, nonyl, and dodecyl. Three products wereadministeredto rats and dogsin the diet for 2 years, 13 to rats for 90 days, and 8 to dogsfor 90 days. Absorption and elimination of 2 products wasstudiedin rats. No effect wasfound asa result of 90-day feedingof: nonyl4-rats and dogs 0.04 g/kg/day; nonyl brats ~0.04, dogs 0.04; dodecyl 6-~0.04; octyl 9-rats 0.2, dogs ~0.04; nonyl 9-rats 0.01, dogs 0.040; in diet; dodecyl 9--rats 0.04; nonyl 15-rats and dogs0.04; nonyl 20-rats 1.O, dogs ~0.04; nonyl 30-rats 5.0, dogs 1.0; octyl40-rats and dogs5:; in diet; nonyl4@-rats 0.3 % in diet; di(sec-butyl)40-rats 1% in diet; dodecyl 40-rats 5.0. The effect at the next highestlevel fed wasfocal myocardial necrosisfor nonyl 20, but only trivial for the other samples;softnessof intestinal contents, low food intake, emesis,somewhatelevated liver to body weight ratio. No effect wasfound in 2 yearsfrom nonylphenol ethyleneoxide 9 on rats at 0.14 or dogs at 0.03 g/kg/day in the diet; from nonylphenol ethylene oxide 4 at 0.2 and 0.04, respectively; or from octylphenol ethylene 40 on rats at 0.7 g/kg/day. No carcinogeniceffect wasfound in any feeding test. In dogsbut not in rats there wasnonprogressivemyocardialtoxicity from oral doses,apparentlya direct pharmacodynamiceffect on the heart muscle, proportional to the content of etherswith ethylene oxide chains20 units in length. This wasmarkedin products with an average chain length of 20, but not detectedwith averagesof 12 or 25. Starting in 1961, manufacturers 1’ia of nonionic surface active agents of the classof alkylphenol ethylene oxide condensation products cooperated in the sponsorshipof an investigation of the toxicity of that family of products, relevant to the safety of their usein materials which contact food. The investigations were carried out in five toxicol1 All the unpublished reports cited have been abstracted with the request and by permission of both the authors and the commercial sponsors. la Allied Chemical Corporation, The Dow Chemical Company, General Aniline and Film Corporation, Jefferson Chemical Company, Division of American Cyanamid Corporation, Monsanto Company, Olin Mathieson Chemical Corporation, Rohm and Hass Company, and Union Carbide Corporation. 315
316
SMYTH
AND
CALANDRA
ogy laboratories,2 overseen by a committee of toxicologists,3 and supplemented by analytical studies in the laboratories of several of the manufacturers. The present paper is a report on the toxicologic study, incorporating summaries of certain parts which were originally published elsewhere. Because of the differences in methods among the five cooperating laboratories, clarity dictates presenting results of each item of the study immediately following the description of its methods, rather than reserving all results for presentation together. It was clearly impractical to perform extensive studies on each of the many commercial products in this class of surfactants. Average chain lengths ranged from 4 to 40 ethylene oxide units and the aikyl groups included secondary buty1, octyl, nonyl, and dodecyl. The combination representing the most used products was nonylphenol ethylene oxide 9. In order to bracket the entire range, products with chain lengths of 4, 9, and 40 were administered in the diets of rats and dogs for 2 years. These and five intermediate chain lengths were administered in the diets of rats and dogs for 90 days. Six products matching these in chain length but with other alkylphenols or made in other plants, were fed to rats for 90 days. Single-dose toxicity studies in rats were done on 7 products. Absorption and metabolism were studied on 2 products. The subacute feeding studies revealed an unexpected effect in dogs from a product with a chain length of 20. In order to understand this effect and to define the range ofchain lengths which can produce it, 8 additional products were fed for brief periods and physiological tests were performed. MATERIALS
STUDIED
The materials studied consist of a hydrophobic alkylphenol moiety comprising a mixture of isomers, and a hydrophilic moiety which is a chain of linearly condensed ethylene oxide units. The ethylene oxide chain length of individual molecules is distributed over a range by the thermodynamics of linear condensation (Flory, 1940), but the average chain length by which the materials are designated is the most frequent chain length of the individual molecules present. All the materials were produced in commercial-scale equipment by the usual processes of the various manufacturers. Most of the samples were marketed products at the time the study was begun. Average chain lengths had been verified by the manufacturer’s usual specification tests. Some of the samples were from selected production batches, the chain lengths of which had been found by acetyl number determination to average those desired, somewhat different from marketed products. The only sample which was prepared in a different way was a blend of 2 marketed products whose average chain lengths were 9 and 40, mixed in such proportions that acetyl number determination showed the average chain length to be 20 ethylene oxide units, while thin-plate chromatography showed that not more than 0.2% of the molecules had an actual chain length of 20. For brevity the materials are referred to in the text by symbols indicating the hydrophobic moiety and the average number of ethylene oxide units in the hydrophilic chain. Table 1 lists the materials studied, with their commercial designations. 2 Biochemical Research Laboratory of the Dow Chemical Company, Chemical Hygiene Fellowship of Mellon Institute, Industrial Biology Laboratories, Industrial Bio-Test Laboratories, and the Medical College of Virginia, 3 HUNT, W.H., ROWE, V. K.,SHAFFER, C.B., SPERLING, F.,and SMYTH, H.F.,Jr.,Chairman.
317
ALKYLPHENOLPOLYOXYETHYLENESURFACTANTS
TABLE 1 MATERIALS STUDIED
Symbol”
Hydrophobe (mixed isomers)
Commercial designation
Nony14 Nony16 Dodecyl6 Octyl9 Nonyl9d Nonyl9i Nonyl9t Dodecyl9 Dodecyl 12 Nonyll5d Nonyl 15s Nonyl 17.5 Octyl20 Nonyl20d Nonyl20t Nonyl20B Dodecyl20 Nonyl25d Nonyl25t Nonyl30d Nonyl3Os Nony135 Octyl40 Di-set-butyl40 Nonyl40 Dodecyl40
Nonylphenol Nonylphenol Dodecylphenol Octylphenol Nonylphenol Nonylphenol Nonylphenol Dodecylphenol Dodecylphenol Nonylphenol Nonylphenol Nonylphenol Octylphenol Nonylphenol Nonylphenol Nonylphenol Dodecylphenol Nonylphenol Nonylphenol Nonylphenol Nonylphenol Nonylphenol Octylphenol Di-secondarybutylphenol Nonylphenol Dodecylphenol
IgepalbCO-430 Dowfaxb 9N6 SteroxbDF Tritonb X-100 Dowfaxb 9N9 IgepaPCO-630 Tergitolb TP-9 SteroxbDJ Experimental product Dowfaxb 9NI5 SurfonicbN-l 50 Experimental product Tritonb X-205 Dowfaxb 9N20 Tergitolb NP-40 Blend of N9t and N-40 Experimentalproduct Dowfaxb 9N25 Tergitolb NP-425 Dowfax* 9N30 SurfonicbN-300 Tergitolb NP-435 Tritonb X-405 Polyglycol89-1 Dowfaxb 9N40 SteroxbDX
’ The numberindicatesthenumberof ethyleneoxideunitsin the hydrophilicchain. bTrademarknameregistered.
Calculation based on Flory’s (1940) law for linear condensations of ethylene oxide showed the expected mole fractions having a chain length of 20 to be: Nonyl9t 0.5% Nonyl20d, nonyl20t 25% Nonyl25t 2% Nony135 0.5 % Nonyl20B 0.5% By thin-layer chromatography nonyl20t was found to have 7.0% by weight of 20 unit chains, 33.8% of 18 through 22 unit chains. The blend nonyl 20B was found to have 0.13 % by weight of 20 unit chains, 0.53 % of 18 through 22 unit chains. QUANTITATIVE
TOXICITY
Single Orat Dose
The LD50 of no@ 9t is 2.6 (2.1-3.2) ml/kg when administered undiluted to 90-120 g unfasted male albino rats. The only obvious effect from the material was depression. Death occurred within 24 hours. Autopsy within a few hours revealed congested lungs
318
SMYTH
AND
CALANDRA
with petechial hemorrhages, congested livers with surfaces in apposition to the stomach blanched, pale kidneys with similar blanched areas, congested adrenals and gastrointestinal tracts. The blanching may have been a postmorten change. Larson et al. (1963) list the acute oral LD50 values for a number of ethylene oxide adducts with octylphenol, covering a wide range of chain lengths, in 120-g male rats fasted before dosing. Most of the deaths occurred within 72 hours and were preceded by depression. The values range from greater than 28 g/kg for a chain length of 40 to a minimum of 1.7 for a chain length of 9.7, rising to 7.2 at a chain length of I. Subacute Feeding to Rats
Nine materials were fed in the diet of rats for 90 days in one laboratory during one period (Table 2). Each dosage of each material was administered to 10 male and 10 female Sprague-Dawley weanling albino rats in good condition. They were housed individually in wire-bottom cages. Food consumption was recorded weekly, separately by sex, and concentrations were adjusted to allow constant dosage in terms of grams per kilogram per day. Water was freely available. The animals were weighed weekly and their condition and behavior were noted. Hematology was studied on 5 of each sex from the highest dosage levels and the controls before treatment and after 11 weeks. Urines pooled by sex were examined for reducing substances, albumin, and microscopic elements from the same groups at the same times. All rats were studied for gross pathology, livers, kidneys, and testes were weighed, and 33 tissues were examined histopathologically from 5 rats of each sex at the highest and control dose levels. In order to discriminate between poor palatability and toxic effect, 25day paired feeding studies were performed at dose levels for which weight gains were shown to be less than those of respective control groups by Bartlett tests, analyses of variance, and/or t tests. TABLE 2 ADMINISTRATION OF ALKYLPHENOLETHYLENE
OXIDE ADDUCTS IN THE DIET OF RATS
FOR 90 DAYS’
Lowest dosage showing response in either sex” Increased liver weight Material Nony14 Nonyl6 Dodecyl6 Octyl9
Dosages fed (g/kg/day) I, 0.2,0.04 1) 0.2, 0.04 1,0.2,0.04
Nonyl 15s Nonyl20 Nonyl3Os
1) 0.2,0.04 1) 0.2,0.04 1, 0.2, 0.04 5, 1, 0.2 5, 1,0.2
Dodecyl40
5, 1,0.2
Dodecyl9
Retarded growth’ 1 1
Absolute weight 1
0.04
-
0.2 0.04 0.2
Liver/body weight ratio 0.2 0.04 0.04 0.2 0.2
0.4 0.2 5 -
1 -
-
-
-
-
11Data from Industrial Bio-Test Laboratories (1963-1965). * At confidence level of 95% or higher. c Paired feeding studies showed each due to poor palatability.
-
ALKYLPHENOL
POLYOXYETHYLENE
319
SURFACTANTS
All dosage-related responses are presented in Table 2. All effects on growth rate were shown to be due to poor palatability of the diets. Livers of both sexes receiving the shorter chain materials were heavier than those of control rats, both absolutely and in relation to body weight. Since no histopathology was found in liver sections. the response was interpreted as an increase in parenchymatous tissue resulting from increased enzyme activity in metabolizing the materials fed. NonyZ9t was fed in the diet of rats for 90 days. Four dose levels and a control level were administered to 10 male and 10 female Charles River (CFE) young albino rats in good condition. They were housed in groups of 5 of one sex in wire-bottom cages. Concentrations in Purina Laboratory Chow were kept constant. Water was freely available. Food consumption was recorded over 4-week periods, for each cage. The animals were weighed weekly and behavior was noted. All rats were studied for gross pathology at sacrifice. Livers and kidneys were weighed. Sixteen tissues were studied from 12 controls and 8 on the highest dosage, three tissues from 10 rats on the next two dosages. All livers were stained with Sudan IV, and periodic and Schiff reagent for positive discrimination between intracellular lipoid and polysaccharide deposits. Numerical data were evaluated by Fisher’s t-test, Bartlett’s homogeneity of variance, analysis of variance, and Duncan’s multiple range test. Effects referable to the material fed are shown in Table 3. Groups of 15 Sherman Wistar albino rats of mixed sexes weighing 95-210 g, with a control group of 30, received diets containing 0.01 to 5.0% of the nonyZ9i for 90 days.
ADMINISTRATION
Statistically significant effect Food eaten Weight gain Liver aspercentageof body weight Kidney aspercentageof body weight Kidney Focal tubular necrosis Focal cloudy swelling of proximal convoluted tubules Liver Focal hepatic-cell necrosis Cloudy swellingof central hepatic cords Intracellular lipoid Reducedpolysaccharide
TABLE
3
9t IN
THE DIET
OF NONYL
1.25
M F M F M F M F
Low Low Low Low High High
” Data from Mellon Institute (1959-1965).
90 DAYS
Dosagein grams/kilo/day
_--___Sex
OF RATS FOR
0.25
0.05
0.01
Low Low Low
Low
Low
High Low
+
I
-c
-L
4.
Slight
Control
SMYTH AND CALANDRA
320
Table 4.4 Body weight, diet eaten, and hematologic parameters were followed. After 8 weeks the 2 lightest male and female rats in each group were sacrificed, gross examination was made, and 19 tissues were examined histopathologically from rats receiving the greatest dosage. At the end of the 90-day feeding period all surviving rats were sacrificed and gross examination was made. Nine organs were weighed. Histopathologic study was made of 19 tissues from the 2 lightest males and females in each group. No histopathologic changes indicating toxic effects were seen. Rats receiving diets containing 2.5 and 5% surfactants were emaciated, had scanty body fat and poor organ development, interpreted as due to partial starvation. Red and white blood cell counts, differential white cell counts, and hemoglobin remained normal. The numerical data are shown in Table 4. The only statistically significant effect was retardation of weight gain, judged to be referable to poor palatability, at 0.64% and higher in the diet. TABLE 4 RESULTS
OF RATS
RECEIVING
DIETS
Concentration in the diet: 5.0xb
ADDED NONYLPHENOXY POLYETHYLENE OXIDE 9 FOR 90 DAYS”
WITH
2.5%
Rats in group 15 15 Mortality 11 2 Males Avg. weight gain (g) -5 55 Avg. food intake 10.0 10.3 (g/rat/day) Females Avg. weight gain (g) -20.5 21.3 Avg. food intake 9.4 9.7 Wrat/dw) Avg. body weight of males 194 at 90-day scarifice (g) Organ weights as percentage
Heart Liver Kidney
Testis Brain
0.64%
0.16% 0.04% 0.01% Control
15
15
0
0
104
201
11.1
12.4
48.7
85.7
10.1
10.5
228
of body weight 0.38 4.00 0.78 1.22 0.89
0.40 4.04 0.84 1.58 0.79
15 2
15 0
30 3
183 12.0
191 12.9
217 13.2
87.0 10.4
86.1 10.6
94.9
321
0.29 3.17 0.55 0.75 0.55
325 0.32 3.64 0.71 1.00 0.58
0.30 2.87 0.44 1.03 0.60
10.6 338 0.33 3.08 0.62 0.82 0.51
a Data from Shelanski (1960); see text footnote 4. b Survivors were sacrificed after 8 weeks.
NonyZ9d was added to the diet of groups of 10 male and 10 female 45- to 50-day-old albino rats at levels of 0.1 to 1.0%; nonyl 40 at 0.01 to 3.0%; and di-set-butyl40 at 0.03-3.0x; with control groups of equal size, for 90 days, Tables 5-7.5~6 4 SHELANSKI, M. V. (1960). Ninety-day feeding studies with general aniline and film corporation’s Igepal CO-630. Industrial Biology Laboratories, Inc. Unpublished Report, September 28. 5 DOW (1960). Results of dietary feeding of Dow polyglycol 89-l to rats-ninety-day experiment. Biochemical Research Laboratory, The Dow Chemical Company, Intramural Report, March 8. 6 Dow (1961). Results of go-day dietary feeding studies of Dowfax 9N40 and Dowfax 9N9 in rats. Biochemical Research Laboratory, The Dow Chemical Company.
ALKYLPHENOL
POLYOXYETHYLENE
TABLE
321
SURFACTANTS
5
RESULTSWITH RATS GIVEN DIETSCONTAINING NONYLPHENOXY ETHYLENE OXIDE9 FOR 90 DAYS’ Concentration in the diet:
1.0%
20 Rats in group 0 Mortality Males Avg. final body weight (g) 262' Avg. food eatenduring first 30 days 24 k/rat/day) Organ weightsas percentageof body weight 0.35 Heart 3.66' Liver 0.86' Kidney Spleen 0.21 1.05 Testis
0.3 7,; 20 1
0.1 7; 10 7b
327
Control 20 0 311
25
20
0.35 2.32'
0.81
0.33 2.84 0.78
0.20 0.95
0.96
0.21
Females Avg. final body weight (g) 168 22 Avg. food eaten during first 30 days (g/rat/day) Organ weightsaspercentageof body weight Heart 0.39 3.73" Liver 0.87 Kidney 0.27' Spleen
177
22
187 20
0.42 3.42' 0.92
0.25
0.41 3.21 0.94 0.24
180 20 0.39 3.13 0.85 0.23
’ Data from Dow; seetext footnote5. bSacrificedbecause of extremedehydrationdueto accidentallack of water. ’ Differencefrom control: p < 0.05.
Body weight, appearance, behavior, and mortality were followed; and food consumption was recorded for the first 30 days. Terminally hematocrit, white blood cell total and differential counts, and hemoglobin were determined on 5 females of the control and the two highest dietary levels of each material. At terminal sacrifice 6 organs were weighed and histopathology of 8 organs was studied. Blood urea nitrogen and alkaline phosphatase activity were measured terminally. There was no mortality referable to nonyl9d, and no effect whatever was seen from 0.1% in the diet. Liver weights were high from 0.3 and lx, kidney and spleen weights were high, and growth was reduced by 1%. Slight cloudy swelling of renal tubular epithelium. with slight petechial areas of central lobular granular degeneration and necrosis were seen in the livers. These reversible adaptations are commonly found in controls from the rat colony and were not considered significant. There was no mortality at any feeding level of nonyl40, and no effect whatever was seen from 0.3% or less in the diet. At 1% livers of males were numerically but not statistically heavy and at 3 % slight central lobular granular degeneration and necrosis, with general cloudy swelling were seen in the livers. There was no mortality referable to di-set-butyZ40, and no effect whatever was seen from 3 % in the diet of female rats or 1 % in the diet of male rats. At the 3 % level the
322
SMYTH
RESULTS
WITH
RATS
GIVEN
Concentration in the diet:
DIETS
AND
CALANDRA
TABLE
6
CONTAINING NONYLPHENOXY 90 DAYS”
3.0%
1.0%
Rats in group 20 20 Mortality 0 0 Males Avg. final body weight (g) 295 302 Avg. food eaten during 21 21 first 30 days (g/rat/day) Organ weights as percentage of body weight Heart 0.36 0.36 2.95 2.92 Liver Kidney 0.79 0.78 0.20 0.20 Spleen Testis
0.94
Females Avg. final body weight (g) 177 Avg. food eaten during
20
0.96 180 22
first 30 days (g/rat/day) Organ weights as percentage of body weight Heart 0.41 0.43 Liver Kidney
Spleen
3.01 0.88 0.22
3.15 0.98 0.24
ETHYLENE
OXIDE
0.3 %
0.1%
20 0
20 0
20 0
20 0
291 23
294 22
292 21
311 23
0.35 2.77 0.79 0.21 0.99 186 21 0.39 3.05 0.77 0.24
0.35 2.79 0.74 0.22 1.02 187 19 0.40 2.88 0.82 0.23
0.03 %
40 FOR
0.36 2.81 0.81 0.23 0.88 190 19 0.38 3.04 0.87 0.23
Control
0.33 2.84 0.78 0.20 0.96 180 20 0.39 3.13 0.85 0.23
a Data from Dow (1961); see text footnote 6.
livers of male rats showed very slight granular degeneration and central lobular necrosis. The spleens of females at the 1% and 3 % levels were light, and the lungs were heavy at the 0.1% level only. These organ weight changes were judged to be artifacts. Those in the lung were not dose related. In the absence of an effect on red blood cells no significance can be attached to reduced spleen weights. Important numerical data are shown in Tables 5-7. Larson et al. (1963) maintained 30 rats with appropriate controls on diets containing 5 % octyl40 for 90 days and found no effect upon food consumption, growth, urinary protein and reducing substances, hematocrit, hemoglobin, total and differential white cell count, organ to body weight ratios for livers, kidney, spleen, heart, and testes. Microscopic examination of 15 tissues revealed no structural alterations. Subacute Feeding to Dogs
Six materials were administered to dogs for 90 days (Table 8). Each dosage of each material was administered to 2 male and 2 female purebred Beagle dogs in good condition, by gelatin capsule 7 days a week following 300 g of dry diet mixed with water. Water was available freely. Animal behavior was observed daily, weights were recorded weekly. Before the first dose and after 80 doses, clinical tests were performed : hematologic, blood cytologic, blood urea nitrogen, serum alkaline phosphatase, urine specific gravity, total glucuronides, organic sulfate, total phenols, glucose, albumin, formed
ALKYLPHENOL
POLYOXYETHYLENE
TABLE RESULTS
WITH
Concentration
RATS
GIVEN
DIETS
in the diet:
Rats in group Mortality (disease or accident) Males Avg. final body weight (g)
323
SURFACTANTS
7
CONTAINING DI-SECONDARY-BUTYLPHENOXY OXIDE 40 FOR 90 DAYSO
3.0%
1.0%
10 2
10 3
3 13 278 Organ weights as percentage of body weight
Heart 0.34 0.36 Liver 2.87 2.80 Kidney 0.76 0.78 Spleen 0.31 0.29 Testis 1.08 1.07 Lung 0.55 0.58 Females Avg. final body weight (g) 195 192 Organ weights as percentage of body weight Heart 0.38 0.39 Liver 2.88 3.04 Kidney 0.84 0.86 Spleen 0.2gb 0.2gb Lung 0.69 0.70
0 *39,’ ,O
0.1 7;
ETHYLENE
0.03 9/o
Control
10 2
10 I
10 1
10 2
290
324
318
286
0.34 2.76 0.77 0.30 0.99 0.57 189 0.40 3.08 0.83 0.37 0.69
0.32 2.72 0.75 0.20 0.95 0.56 187 0.42 3.02 0.84 0.36 0.87”
0.34 2.82 0.73 0.29 0.94 0.55 197 0.41 2.96 0.84 0.38 0.73
0.36 2.83 0.79 0.29 1.02 0.59
200 0.40 2.89 0.83 0.37 0.66
a Data from Dow (1960); see text footnote 5. h Difference from control: p < 0.01.
elements, and sulfobromophthalein liver function test. Gross structures were scrutinized; liver, kidney, spleen, heart, brain, and testes were weighed; and histopathologic examination of 38 tissues was performed. Some variations in dosage and schedule with one material are not recorded because they did not yield useful information. Table 8 records dose-related responses in dogs to repeated doses. The effects in dogs seriously injured or dying from 5 g/kg/day of nonyl20d after 6 days or 1 g/kg/day after 27 days were weight loss, anorexia, emesis, salivation, gross and microscopic focal myocardial degeneration and necrosis, alterations in organ weights, and death. Single purebred Beagle dogs received diets containing 0.04, 0.64, and 5 % nonyl 9i for 90 days, with 3 dogs on the same diet without addition4 Body weight, diet eaten, and blood cells were studied. After 90 days the dogs were sacrificed, 8 organs were weighed, and 19 tissues were studied histopathologically No histopathologic change indicating a toxic effect was seen, but both control and treated dogs revealed perilobular infiltration and parasitic granulomas in the livers, referable to parasitic infestation. Red and white blood cell counts, differential white cell counts, and hemoglobin remained normal. Important numerical data appear in Table 9.
DOSAGE
OF ALKYLPHENOL
1
0.2
pData from IndustrialBio-TestLaboratories(1963-1965).
0.2
-
1
-
1
PRODUCING
0.2
ADDUCTS
Nony16 1) 0.2, 0.04 -__~ -
OXIDE
8
Nony14 I, 0.2,0.04
ETHYLENE
Death Focal myocardial necrosis (grossly) Focal myocardial necrosis or degeneration (micro) Lung hyperemia (grossly) Liver: body weight ratio increase Growth depression, anorexia Over 90-day period During first 4 weeks only Emesis, usually with salivation Severe, over 90-day period Occasionally, over 90-day period Daily during first l-3 weeks only Occasionally during first l-3 weeks only
Dosages administered (g/kg/day):
LOWEST
TABLE
1 0.04 -
-
1 -
-
Dodecyl9 1, 0.2,0.04 ~__--
EFFECTSWHEN
1 0.2
-
1 0.2
-
1 0.2 -
1-
0.04
-
FOR
Nonyl20d 5, 1, 0.2,0.04 ..~__ 1 1
OF DOGS
Nonyl 1% I, 0.2,0.04
IN THE DIET
-
Fi >
k P r
-
3
B -
-
Nonyl3Os I, 0.2
90 DAYSO
ALKYLPHENOL
POLYOXYETHYLENE
TABLE RESULTS
OF PUREBRED
BEAGLE Dms POLYETHYLENE
Concentration in the diet :
325
SURFACTANTS
9
RECEIVING DIETS WITH ADDED OXIDE 9 FOR 90 DAYS
5.0%
Dogs in group and sex 1M Mortality 0 Average weight gain (pounds) 1.5 Average food intake (total pounds) 80 Average body weight at 90-day sacrifice 17.0 (pounds) Organ weight as percentage of body weight Heart 0.63 Liver 2.87 Kidney 0.45 Testis 1.41 Brain 0.82
0.64% IF 0 1.5 91
18.0
0.93 2.79 0.53 0.87
NONYLPHENOXY
0.04%
Control
1M 0
2F,lM 0
4.0 102 19.0
5.2 102 20.0
0.87 2.82 0.57 2.00 0.83
0.63 2.30 0.45 1.09 0.77
0 Data from Shelanski (1960); seetext footnote 4. Although numerical differences are evident, it was concluded that the only statistically significant effect was retardation in weight gain related to voluntary food intake at 0.64 % and higher in the diet, and that 0.04 % was without effect. Four dogs were maintained for 90 days on diets containing 5% octyl40. The only effect found was softness of the large bowel contents {Larson et al., 1963). Chronic Feeding to Rats
Nonyl4 was administered to rats in the diet over a 2-year period. Each dosage group consisted of 35 male and 35 female weanling Sprague-Dawley albino rats, with 5 male and 5 female rats in addition on the highest and control dosage groups reserved for periodic clinical test. The rats were housed individually in wire-bottom cages. The material was added to Rockland Rat Diet in concentrations adjusted from time to time to maintain the dosages constant at 1, 0.2, 0.04, and 0 g/kg/day, presented in excess of the amount eaten. Water was freely available. Food consumption was calculated weekly for 12 weeks, irregularly thereafter. The rats were weighed weekly for 13 weeks, biweekly to 1 year, then monthly. Food utilization was calculated for the first 12 weeks. Animals were observed daily for abnormal behavior. Before the first dose, and after 3, 6, 12, 18, and 24 months clinical tests were performed on the 5 of each sex on the highest dosage and the control diet reserved for this use: hemoglobin, total red and white cell count, differential white cell count, urinary reducing substances, protein, and formed elements. All rats dying or sacrificed were carefully examined for gross pathologic changes, including tumors. After 12 months 5 of each sex from the highest dosage and from the control group were sacrificed, 3 of each sex from each of the two lower-dosage groups. After 24 months all rats were sacrificed. At sacrifice livers, kidneys, and testes were
326
SMYTH
AND
CALANDRA
weighed. Histopathologic examination of 28 tissues was done on 5 rats of each sex on the highest dosage and in the control diet groups at each sacrifice. All numerical results were evaluated by appropriate statistical tests. There were no deaths and no untoward behavior attributable to the material. At 1 g/kg/day both sexes, and at 0.2 g/kg/day the females had gained’less weight than the controls after 12 months, but did not differ from the controls after 24 months. These groups ate less than did the controls, and the growth effect was attributed to poor palatability of the diets, since a similar effect had been definitely shown to be due to voluntary limitation of food intake (see paired feeding studies in the 90-day feeding of nonyZ4 presented above). The only other dose-related effect noted was slight elevation of liver weight in relation to body weight in both sexes on an intake of I g/kg/day. Microscopically the livers were normal, hence the response was not considered to be adverse. Nonyl Pt was administered in the diet of rats over a 2-year period. Each dose group consisted of 36 male and 36 female 60-g Carworth-Elias rats. Sixteen of each sex were designated at the start of the study for serial sacrifice after 6 and 12 months of feeding. The animals were housed in groups of 5 of one sex in wire-bottom cages. The material was added to ground Purina Laboratory Chow at concentrations of 0.27, 0.09, 0.03, and Ox, presented in excess of the amount eaten. Water was freely available. Food consumption was calculated by 28-day periods. The rats were weighed biweekly for the first year, then monthly. Animals were observed at each weighing for appearance and behavior. Total red blood cell counts were made on 10 male rats of the two highest dose groups and the control group before the feeding and at 90, 180, 360, and 540 days. All rats dying or sacrificed were carefully examined grossly. Livers and kidneys were weighed at sacrifice. Histopathologic examination of 11 tissues and of all neoplasms was done. All results were evaluated by appropriate statistical methods. There was no difference between control and treated rats in any observation made-food consumption, mortality, life span, extraneous infections, liver and kidney to body weight ratios, body weight gain, maximum weight attained, red blood cell count, incidence and location of neoplasms, histopathologic lesions. Larson et al. (1963) maintained 60 rats with controls on diets containing up to 1.4 % octyl40 for 2 years and found no adverse effects upon food consumption, growth, urinary protein and reducing substances, hemoglobin and blood cells, organ to body weight ratios for liver, kidney, spleen, heart, and testes, or histologic appearance of 15 tissues. Chronic Feeding of Dogs Nony14 was administered to dogs over a 2-year period. Each dosage was given to 3 Beagle hounds of each sex, housed 3 of one sex in one kennel. Approximately 300 g of dry diet was mixed with water for each dog each day. Immediately after the feeding period uneaten diet was removed and gelatin capsules were given containing sufficient nony14 to constitute dosages of 1, 0.2, and 0.04 g/kg/day, with an untreated control group. The dogs were weighed weekly and observed daily for signs of effect. Before the study started, and after 90,180,360,540, and 720 days, clinical tests were performed on all dogs: hemoglobin, hematocrit, red and white cell total counts, and white cell differential counts; urine specific gravity, pH, glucuronides, organic sulfate, total
ALKYLPHENOL
POLYOXYETHYLENE
SURFACTANTS
327
phenol’s, glucose, albumin, formed elements; and sulfobromophthalein liver function tests. After 720 days, fasting blood glucose levels were determined. All dogs were sacrificed and carefully examined grossly; weights of liver, kidneys, spleen, heart, brain, and testes were recorded; and 28 tissues were examined microscopically. At I g/kg/day food intake was several times depressed and body weight began to fall. Each time this occurred the high protein portion of the diet was increased until anorexia was corrected and body weight was regained. During the first week of feeding occasional emesis was seen at all dose levels, but thereafter it occurred infrequently and only at the highest dosage. At 1 g/kgiday there was a moderate elevation in serum alkaline phosphatase and liver to body weight ratio, but no significant gross or microscopic pathologic change. At 0.20 g/kg/day the two effects were seen to lesser degrees, and no effect was seen at 0.04 g/kg/day. Notiyl 91 was administered to dogs in the diet over a 2-year period. Each dosage group consisted of 3 male and 3 female purebred Beagles. 6-12 months old at the start of the study. They were individually housed in cages with open-mesh floors. The material was added to Friskies Dog Food Meal in concentrations of 0.27, 0.09, 0.03. and OO,/,.The amount of food eaten was recorded each day. The calculated average intakes of nonyl9t for the treated groups were 0.088,0.028, and 0.0085 g/kg/day. Water was freely available. The condition and behavior of the dogs were observed daily, and they were weighed weekly. Before the study started and after 90,180,360,540, and 700 days serum urea nitrogen, alkaline phosphatase, bromosulfalein excretion, total red and white blood cells, differential white cell count, hemoglobin, and hematocrit were determined. After 2 years the dogs were sacrificed, liver, kidneys, and heart were weighed, gross structure was observed, and histopathologic examination of 21 tissues was done. All measurements and observations were evaluated by appropriate statistical methods. The only effect attributable to the treatment was an increased liver to body weight ratio at 0.27% nonyl9t in the diet. Microscopically the livers were normal. METABOLISM
AND EXCRETION
Metabolism and excretion were studied in male rats (Knaak ef al., 1966) at a dosage of 67 mg/kg, using eight r‘C-labeled samples: nonyZ9t labeled in the ethylene oxide chain, 7-, lo-, 12-, and 15-mole adducts separated from chain-labeled nonyl9t, nonyl9t. and nonylp’heno[ labeled uniformly in the phenol and nonane groups. Excretion was substantially complete in 2 days. Total activities found after 7 days are listed in Table 10. The nonylphenol moiety of nonyZ9t was excreted in the same pattern as nonylphenol itself. The ethylene oxide chain moiety was absorbed from the digestive tract and metabolized less completely as chain length increased. Only 1 T< of the dose was found in the urine as nonyl9t per se. Ion exchange chromatographic studies indicated the principal urinary metabolites to be carboxylic acids of polyethylene glycols and glucuronic acid conjugates of nonylphenol. The distribution and excretion of a tritiated sample of ocryl40 was studied by Larson et al. (1963). In both rats and dogs approximately 1% of the activity was found in the urine within 72 hours of the dose.
328
SMYTH
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CALANDRA
TABLE
10
EXCRETION OF 14CCOMPOUNDS BY THE MALE RAT DURING 7 DAYS AFTER AN ORAL DOSE OF LABELED NONYL 9t”
Ethylene oxide labeled 7-Mole adduct (%I
Nonyl9t (%I
Nonylphenol labeled
15-Mole adduct (%I
Nonyl9t (%I
Nonyl phenol” (%I
20 0 78
19 0 76
Activity in urine Expired air Feces
40” 3.3 36”
39b 1.2 52
26’ 0.2 56’
Total found
85’
92.2
85”
Carcass
0
0
0
-
-
98
95
’ Data from Knaak el al. (1966). bIncludescagewashings. ’ Excludescagewashings. dIdenticalexcretionwasfoundafter an intraperitonealdose.
STUDIES OF CARDIOTOXICITY
Following the discovery of focal myocardial necrosis in dogs, but not in rats, as a result of 90 days’ consumption of diets containing nonyl ZOd,additional studies were performed to elucidate and evaluate the significance of the effect. Three laboratories produced severe degrees of the condition after periods briefer than 90 days, even as brief as 5 days. Accordingly most of the additional feeding studies performed were limited to 14 days. Uniformity of Eflect
In order to investigate the possibility that some impurity or unique characteristic in the commercial product nonyl20d had caused the cardiotoxicity, new samples were obtained of 4 commercial products produced in different plants, nonyl ZOd,nonyl ZOt, octyl20, and dodecyl20. Each was given by capsule daily for 14 days to 1 male and 1 female Beagle pup, in divided doses totaling 1 g/kg/day. All animals lost weight, exhibited emesis, and developed focal myocardial necrosis. Six of the 8 dogs died after 4-14 days. The severity of the cardiac lesion was inversely as the frequency of emesis and directly as the survival time. This comparison showed no important difference between the four different products of the same chain length. SpeciesResponses
Only dogs and guinea pigs consistently developed severe focal myocardial necrosis. No myocardial lesions whatever were seen in 3 cats and 3 rabbits which were intubated on 14 successive days with approximately 0.7 g/kg of nonyl20t. Lesions interpreted as early stages of necrosis were seenin guinea pigs; eosin-staining fibers, round-cell infiltration or myocardial degeneration in 9 of 19 guinea pigs receiving 1 g/kg of nonyl20d or nonyl20t on 2 or 3 successive days. Rats did not show any heart lesions
ALKYLPHENOL
POLYOXYETHYLENE
329
SURFACTANTS
after 90 days’ feeding at 5 g/kg/day of nonyl2Od, while dogs consistently revealed the lesion after 90 days at 0.04 g/kg/day. Chain Length
Nine materials were administered to 1 male and 1 female Beagle dog daily for 14 days in one capsule, or divided between 2 or 3 capsules each day. The incidence of focal myocardial
degeneration
evident upon microscopic
study of sections is shown in
Table 11 by chain length, together with its incidence in 90-day and 2-year feeding
studies referred to earlier. TABLE CARDIOTOXICITY
IN
DOGS VERSUS ADDUCTS
11
CHAIN LENGTH OF ALKYLPHENOL ADDED TO THE DIET”
ETHYLENE
OXIDE
Focal myocardial degeneration Average ethylene oxide chain length 4
Nony14
6 9
Nony16 Nonyl9t Dodecyl9 Dodecyl 12 Nonyl 15s Nonyl 1Sd
12 15
17.5 20
Nonyl20t Dodeeyl20 Nonyl25d Nonyl3Os Octyl40
20 from
Dosage Wklday) I_ ___ 1 1 1 0.088 1 1 I 0.2 1
Nonyl 17.5 Octyl20 Nonyl20d
25 30 40
’ Data
Material
Nonyl 2OB Industrial
Bio-Test
14-Day 90-Day 2-Year feeding feeding feeding ----.--~. _ _ - .~ - ~~. 0 0 0 0 0 0 0 0 0 i
1 1
i L
0.04 + _~
t
1
0
(5 $ in diet) ___-1
Laboratories
+
1 1 1
0 0 -^--.0
0
(1963-1965).
Cardiotoxicity was evident from all materials having an average chain length of 20 ethylene oxide units, except nonyl 20B. Although the chain length of this material averaged 20, actual chain lengths constituted a bimodal distribution, and not over 0.2 % of them were actually 20 units long. Cardiotoxicity was also produced by products whose average chain length was 17.5 and 15, but not from those whose chain length was either 12 or less, or 25 or more.
330
SMYTH
AND
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Dogs receiving for 14 days 1 g/kg/day of materials whose chain lengths were 15, 17.5, or 20 almost consistently showed anorexia, weight loss, emesis, diarrhea or loose stools, lethargy, and death. Dodecyll2 produced emesis, diarrhea, and death; nonyl25d produced emesis, diarrhea, and lethargy. Similar signs were produced by nonyl20d at 1 g/kg/day in dogs fed for 90 days. Six alkylphenoxy ethylene oxide adducts were administered by capsule daily for 30 days to 1 male and 1 female Beagle dog weighing 5-8 kg, at 1 g/kg/day.7 The behavior and condition of the animals was observed daily. Serum sodium, potassium, and magnesium levels were determined on days 0, 4 or 5, and 10. Transaminase activities of blood serum were determined on the same days. At death, or at sacrifice after 30 days, histopathology of heart, liver, and kidney was studied. Table 12 summarizes the results. At a chain length of 15 ethylene oxide units 1 of 2 dogs died after 25 doses, revealing moderate myocardial degeneration. At a chain length of 20, all 4 died after 4-10 doses, showing myocardial necrosis. At a chain length of 30 none of 4 dogs died, but 3 showed slight myocardial degeneration, perivascular myocardial round-cell infiltration, or loss of clarity in myocardial striations. At a chain length of 40, no dogs died and the heart muscle appeared normal. Gross examination of the dogs which died revealed areas of necrosis in the heart muscle of the left ventricular wall and interventricular septum. The right side of the heart was dilated and the muscle appeared flabby. The lungs showed varying degrees of edema, kidneys suggested tubular degeneration, and the livers were dark. Inactivity, elevated or irregular pulse rate, vomiting, and loose stools following the dose paralleled the myocardial histopathologic changes and mortality. Dogs receiving materials with a chain length of 30 showed swelling of eyelids, face, legs, and body suggestive of a hypersensitivity reaction to release of a histamine-like compound, or to electrolyte imbalance. Swelling was reduced by subcutaneous injection of diphenylhydramine (Benadryl). There was no significant change in serum sodium, potassium, or magnesium. Em&s The degree of correlation between emesis and myocardial degeneration in the dog feeding studies suggested the possibility that cardiotoxicity resulted from electrolyte imbalance brought about from emesis, although electrolyte measurements recorded in Table 12 did not support the hypothesis. One dog of each sex was given a nonemetic dosage of nonyl 2Od, 0.20 g/kg/day divided between two gelatin capsules several hours apart. During 34 days slight emesis occurred once in 1 dog, twice in the other. Myocardial degeneration was found in both dogs. Two dogs of each sex were given a varying dosage of nonyZ2Od, divided between three capsules a day. The daily dosage started at 0.20 mg/kg/day and increased by 0.05 g/kg/day every 5-7 days to 0.55 g/kg/day, then decreased to 0.50 g/kg/day. Emesis did not occur until the dosage reached 0.55 g/kg/day. At this dosage it was consistent, 7 SADEK, S. E. (1963). A short-term toxicity study on various nonylphenol ethylene oxide adducts. Biochemical Research Laboratory, The Dow Chemical Company, Midland, Michigan, Intramural Report, November 27.
N
OF CARDIAC
EFFECT
IN
5 TO 8 KG BEAGLE
Dots
12 FROM
1 G/KG/DAY
BY CAPSULE
FOR
a Data
from
Sadek
(1963);
see text footnote
7.
Histopathology of myocardium Moderate degeneration Moderate degeneration Necrosis Necrosis Necrosis Necrosis Necrosis Scatterednecrosis Normal Perivascularinfiltration Striations not clear Slight degeneration Normal Normal Normal Normal
30 DAYS”
Changein serumat 10days or at earlier death _____ Weight Days - __-_ change to Sodium Potassium Magnesium SGOT SGPT LDH Material Dog death (mg/lOOml) (mg/lOOml) (mg/lOOml) (SF units) (SF units) (BB units) (g) _____ -__ Nonyl 15d 657 +450 25 -6.0 -0.1 +1.8 t21 -7 +10 672 -950 +46.5 0 +lO -14 +60 12.5 Nonyl20d 658 +250 5 -1.2 12 -19 +60 +1.5 +4.1 -50 4 -216 659 +16.4 -1.4 -0.1 +I32 $68 662 -250 10 -12.7 +1.1 1-3.2 +41 i-6 +90 663 -150 4 -20.1 t-173 t52 +410 +61.2 +5.2 Nonyl20t -250 667 5 +21.1 t-2.5 +0.4 $26 -7 +475 671 -800 -23.5 0 +1.9 i-18 i-10 1-170 Nonyl30d 660 +600 -1 t20 +3.9 -4.2 t2.7 t15 +I70 664 t700 -6.4 -1.6 $5.3 t17 -12 Nonyl3Os 666 +750 -t6.7 -13.5 +3.8 il 0 rloo 670 4500 i-36.9 -6 +103 -2.7 -2.2 +24 Nonyl40 i-75 665 +1200 -8.3 -1.9 +2.3 +17 i-l .-9 669 +500 m-36.7 +0.2 +2.5 i-14 t-300 Control 661 i-1400 -6 +I80 $2.6 -3.5 -5.2 t17 668 -t1300 t 305 --30.1 -6.5 +3.0 +20 7~2
SUMMARY
TABLE
332
SMYTH
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but was less frequent later at 0.50 g/kg/day. Myocardial degeneration was seen in the dog which died after 59 days and in the 3 which survived 131 days. Four dogs were given 1 g/kg/day divided among three capsules. Two received diets to which 0.5% potassium chloride had been added, 1 received daily intramuscular injections of 1500 USP units of thiamine hydrochloride, and one received no supplements. Emesis, anorexia, weight loss, and myocardial degeneration did not differ among the 4 dogs. Five Beagle dogs were given 10 g/kg/day of nonyl20d by capsule, in divided doses three times a day to reduce emesis, 7 days a week for 12 days or until earlier death. Dogs I and II received no other treatment. Dogs III and IV received a diet to which 0.5 % potassium chloride had been added. Dog Vreceived daily subcutaneous injections of 1500 USP units of thiamine hydrochloride. Dogs I and II ate little food, vomited occasionally, lost weight and died during the second and third days. Dog III ate normally, vomited occasionally, lost weight and died during the fourth day. Dog IV ate well, vomited consistently, gained weight, but died during the second day, while dog V ate poorly, vomited seldom, lost weight and was sacrificed in extremis on the twelfth day. Focal myocardial degeneration was seen in all 5 dogs, extreme in dog V, slight in dog IV, intermediate in the others. Neither potassium chloride nor thiamine prevented the myocardial injury. Eflect on the Isolated, Perfused Cat Heart8
Isolated cat hearts obtained from lightly etherized cats were perfused through the coronary arteries in the manner of Langendorff as modified by Chenoweth and Koelle (1946). The modified Ringer-Locke solution was gassed with 95 % oxygen and 5 % carbon dioxide, and a fixed volume was permitted to recirculate throughout the system. A control period of at least 30 min was allowed before any compounds were added. To the reservoir were added aliquots of the materials under study to yield a concentration of 5 or 10 mg/l of perfusate. The apparatus adjustments remained constant throughout the tests so that the time lapse between addition of the detergent and its arrival at the heart in the perfusate was essentially the same for each preparation. Contractions were recorded with a strain gauge and Offner recorder. No@ 9d, at 5 mg/l, reduced the amplitude of contraction from 12 mm to 10 mm in 12 min. An additional 5 mg/l of nonyl9d was added. Within 5.5 min the amplitude had been reduced to 7 mm and a bigeminal rhythm occurred. Ten minutes later the amplitude was less than 50% of that during the control period and the arrhythmia apparently had ceased. The preparation continued to contract for about 10 min more before recordable contractions ceased. In the case of the nonyZ2Od, 5 mg/l reduced the contraction from 11 mm to 7 mm in 12 min. An additional 5 mg/l was added, reducing the contraction to 6.5 mm in 1.5 min more. Thus, reduction to the 50 % contractile point occurred in 13.5 min, but the heart continued to decline steadily and slowly for approximately 35 min more before recordable contraction ceased. 8 KLUG, S., MCCARTY, L. P., and CHENOWETH, M. B. (1965). Effect of Dowfax 9N9, 9M40, and Tween 20 on the isolated perfused cat heart. Biochemical Research Laboratory, The Dow Chemical Company, Intramural Report, March 5.
ALKYLPHENOL
POLYOXYETHYLENE
SURFACTANTS
333
In the case of nonyl40,5 mg/l produced very little detectable effect on the contractility. Ten minutes later an additional 5 mg/l was added. It was not until 62 min had elapsed that the amplitude of contraction was reduced to 50 y0 of the control level. In the case of Tween 20, 5 mg/l produced no decrease in amplitude for 18 min. An additional aliquot of 5 mg/l was added, and 36 min later the heart suddenly ceased to beat, although the reduction in amplitude of contractile height was not very much. being only 1 mm less than the control level of 19 mm. The cardiac toxicity shown in this experiment is the direct result of an effect upon the myocardium, and is not secondary to effect on any other organ. Among the nonylphenoxy adducts tested, cardiotoxicity is greatest at an average ethylene oxide chain length of 20, less at a length of 9, and least at a length of 40. Since the hydrophobic moiety of Tween 20 is not phenolic, and its cardiac toxicity is much less than that of nonyl ZOd, which has the same ethylene oxide chain length. chain length alone is not sufficient to produce marked cardiotoxicity. Age of Dogs
The 14-day studies which showed that molecules with 20-unit ethylene oxide chains are those responsible for cardiotoxicity were performed on dogs 4-6 months old. In order to determine whether dogs of other ages might be more sensitive, nonyZ15d was administered for 14 days to 2 dogs 8-10 weeks old and to 2 dogs more than 3 years old. The daily dosage of 0.2 g/kg/day was the greatest dosage which resulted in no cardiotoxicity in dogs 4-6 months old. No cardiotoxicity resulted in the young or old dogs. Likewise dodecyl 12 administered in identical studies at 0.2 g/kg/day showed no cardiotoxicity in young or old dogs. Fourteen- Llersus Sixty-Day
Feeding
Nonyl 15d was administered by capsule three times a day in a dosage totaling 0.2 g/kg/day to 2 male and 2 female dogs over a 60-day period. This was the greatest dosage of the product which caused no cardiotoxicity during 14 days of administration. The 60-day administration likewise caused no signs of cardiotoxicity. An identical 60-day administration ofdodecyl I2 likewise caused no cardiotoxicity. DISCUSSION
There was some rationale for considering that myocardial injury might be the result of histamine release such as Krantz et al. (1948) found in dogs from some other surfaceactive compounds with ethylene oxide chains. However, the work reported here lends no support for this hypothesis. The most prominent supporting finding was bronchospasm in the guinea pig. It is more convincingly explained as a mechanical response to intubation. Furthermore, gastric ulcer, which is an expected response to histamine in the guinea pig, was sought for and not found, while Krantz et al. (1948) found no histamine release in guinea pigs from the surfactants which did release it in dogs. No other sign of histamine release was found, unless myocardial degeneration in the dog can be considered such a symptom. It is recognized that the dog heart is unusually sensitive to histamine (while the human heart is resistant). The studies of the isolated perfused cat heart by Klug et al.8 discount this possibility by establishing a strong
334
SMYTH
AND
CALANDRA
probability that the cardiac toxicity of nonyl20d is the direct result of an effect upon the myocardium, not secondary to any other organ. The findings of a specific effect on the heart from chain lengths intermediate in a long seriesis not unprecedented. Such a finding from parenteral dosesof polypropylene glycols was reported by Shideman and Moe (1949) and Shaffer et al. (1951). REFERENCES M. B., and KOELLE, E. S. (1946).Isolated heart perfusion systemadaptedto the determination of non-gaseous metabolites.J. Lab. Clin. Med. 31, 600-608. FLORY, P. J. (1940).Molecular sizedistribution in ethylene oxide polymers.J. Am. Chem. Sot. CHENOWETH,
62, 1561-1565. J. B., ELDRIDGE, J. M., and SULLIVAN, L. J. (1966).Excretion of certain polyethylene glycol ether adductsof nonyl phenol by the rat. Toxicol. Appl. Pharmacol. 9, 331-340. KRANTZ, J. C., JR., CARR, J., BIRD, J. G., and COOK, S. (1948). Sugar alcohols, XXVI. J. Pharmacol. 93, 188-195. LARSON, P. S., BORZELLECA, J. F., BOWMAN, E. R., CRAWFORD, E. M., SMITH,R. B., and HENNIGER, G. R. (1963). Toxicologic studieson a preparation of p-tertiary octylphenoxypolyethoxy ethanol (Triton X-405). Toxicol. Appl. Pharmacol. 5,782-789. SHAFFER, C. B., CARPENTER, C. P., CRITCHFIELD, F. H., NAIR, J. R., III, and FRANKE,F. R. (1951).A toxicological study of somepolypropylene glycols. Arch. Znd. Hyg. Occupational KNAAK,
Med. 3,448-453.
F. E., and MOE, G. K. (1949).Production of ventricular extrasystolesin the dog by a polypropylene glycol. Federation Proc. 8, 322.
SHIDEMAN,
AND
Toxicologic
APPLIED
PHARMACOLOGY
Studies
14, 3l5-334(1969)
of Alkylphenol
Polyoxyethylene
Surfactants
HENRY F. SMYTH, JR. AND JOSEPH C. CALANDRA Carnegie-Mellon Unicersity, Mellon Institute, Pittsburgh, Industrial Bio-Test Laboratories, Northbrook,
Pennsyhania Illinois 60062
15213, and
ReceiL>ed July 10, I568
Toxicologic Studiesof Alkylphenol Polyoxyethylene Surfactants.SMYTH, H. F., JR.,andCALANDRA, J. C. (1969). Toxicol. Appl. Pharmucol. 14,3 15-334. In order to determinethe suitability of a large group of commercialsurfactants for applicationswhere smallamountsmay enter food, the toxicity of 22 products wasstudied. Ethylene oxide chain lengthsrangedfrom 4 to 40, and alkylphenols included di(secondary butyl), octyl, nonyl, and dodecyl. Three products wereadministeredto rats and dogsin the diet for 2 years, 13 to rats for 90 days, and 8 to dogsfor 90 days. Absorption and elimination of 2 products wasstudiedin rats. No effect wasfound asa result of 90-day feedingof: nonyl4-rats and dogs 0.04 g/kg/day; nonyl brats ~0.04, dogs 0.04; dodecyl 6-~0.04; octyl 9-rats 0.2, dogs ~0.04; nonyl 9-rats 0.01, dogs 0.040; in diet; dodecyl 9--rats 0.04; nonyl 15-rats and dogs0.04; nonyl 20-rats 1.O, dogs ~0.04; nonyl 30-rats 5.0, dogs 1.0; octyl40-rats and dogs5:; in diet; nonyl4@-rats 0.3 % in diet; di(sec-butyl)40-rats 1% in diet; dodecyl 40-rats 5.0. The effect at the next highestlevel fed wasfocal myocardial necrosisfor nonyl 20, but only trivial for the other samples;softnessof intestinal contents, low food intake, emesis,somewhatelevated liver to body weight ratio. No effect wasfound in 2 yearsfrom nonylphenol ethyleneoxide 9 on rats at 0.14 or dogs at 0.03 g/kg/day in the diet; from nonylphenol ethylene oxide 4 at 0.2 and 0.04, respectively; or from octylphenol ethylene 40 on rats at 0.7 g/kg/day. No carcinogeniceffect wasfound in any feeding test. In dogsbut not in rats there wasnonprogressivemyocardialtoxicity from oral doses,apparentlya direct pharmacodynamiceffect on the heart muscle, proportional to the content of etherswith ethylene oxide chains20 units in length. This wasmarkedin products with an average chain length of 20, but not detectedwith averagesof 12 or 25. Starting in 1961, manufacturers 1’ia of nonionic surface active agents of the classof alkylphenol ethylene oxide condensation products cooperated in the sponsorshipof an investigation of the toxicity of that family of products, relevant to the safety of their usein materials which contact food. The investigations were carried out in five toxicol1 All the unpublished reports cited have been abstracted with the request and by permission of both the authors and the commercial sponsors. la Allied Chemical Corporation, The Dow Chemical Company, General Aniline and Film Corporation, Jefferson Chemical Company, Division of American Cyanamid Corporation, Monsanto Company, Olin Mathieson Chemical Corporation, Rohm and Hass Company, and Union Carbide Corporation. 315
316
SMYTH
AND
CALANDRA
ogy laboratories,2 overseen by a committee of toxicologists,3 and supplemented by analytical studies in the laboratories of several of the manufacturers. The present paper is a report on the toxicologic study, incorporating summaries of certain parts which were originally published elsewhere. Because of the differences in methods among the five cooperating laboratories, clarity dictates presenting results of each item of the study immediately following the description of its methods, rather than reserving all results for presentation together. It was clearly impractical to perform extensive studies on each of the many commercial products in this class of surfactants. Average chain lengths ranged from 4 to 40 ethylene oxide units and the aikyl groups included secondary buty1, octyl, nonyl, and dodecyl. The combination representing the most used products was nonylphenol ethylene oxide 9. In order to bracket the entire range, products with chain lengths of 4, 9, and 40 were administered in the diets of rats and dogs for 2 years. These and five intermediate chain lengths were administered in the diets of rats and dogs for 90 days. Six products matching these in chain length but with other alkylphenols or made in other plants, were fed to rats for 90 days. Single-dose toxicity studies in rats were done on 7 products. Absorption and metabolism were studied on 2 products. The subacute feeding studies revealed an unexpected effect in dogs from a product with a chain length of 20. In order to understand this effect and to define the range ofchain lengths which can produce it, 8 additional products were fed for brief periods and physiological tests were performed. MATERIALS
STUDIED
The materials studied consist of a hydrophobic alkylphenol moiety comprising a mixture of isomers, and a hydrophilic moiety which is a chain of linearly condensed ethylene oxide units. The ethylene oxide chain length of individual molecules is distributed over a range by the thermodynamics of linear condensation (Flory, 1940), but the average chain length by which the materials are designated is the most frequent chain length of the individual molecules present. All the materials were produced in commercial-scale equipment by the usual processes of the various manufacturers. Most of the samples were marketed products at the time the study was begun. Average chain lengths had been verified by the manufacturer’s usual specification tests. Some of the samples were from selected production batches, the chain lengths of which had been found by acetyl number determination to average those desired, somewhat different from marketed products. The only sample which was prepared in a different way was a blend of 2 marketed products whose average chain lengths were 9 and 40, mixed in such proportions that acetyl number determination showed the average chain length to be 20 ethylene oxide units, while thin-plate chromatography showed that not more than 0.2% of the molecules had an actual chain length of 20. For brevity the materials are referred to in the text by symbols indicating the hydrophobic moiety and the average number of ethylene oxide units in the hydrophilic chain. Table 1 lists the materials studied, with their commercial designations. 2 Biochemical Research Laboratory of the Dow Chemical Company, Chemical Hygiene Fellowship of Mellon Institute, Industrial Biology Laboratories, Industrial Bio-Test Laboratories, and the Medical College of Virginia, 3 HUNT, W.H., ROWE, V. K.,SHAFFER, C.B., SPERLING, F.,and SMYTH, H.F.,Jr.,Chairman.
317
ALKYLPHENOLPOLYOXYETHYLENESURFACTANTS
TABLE 1 MATERIALS STUDIED
Symbol”
Hydrophobe (mixed isomers)
Commercial designation
Nony14 Nony16 Dodecyl6 Octyl9 Nonyl9d Nonyl9i Nonyl9t Dodecyl9 Dodecyl 12 Nonyll5d Nonyl 15s Nonyl 17.5 Octyl20 Nonyl20d Nonyl20t Nonyl20B Dodecyl20 Nonyl25d Nonyl25t Nonyl30d Nonyl3Os Nony135 Octyl40 Di-set-butyl40 Nonyl40 Dodecyl40
Nonylphenol Nonylphenol Dodecylphenol Octylphenol Nonylphenol Nonylphenol Nonylphenol Dodecylphenol Dodecylphenol Nonylphenol Nonylphenol Nonylphenol Octylphenol Nonylphenol Nonylphenol Nonylphenol Dodecylphenol Nonylphenol Nonylphenol Nonylphenol Nonylphenol Nonylphenol Octylphenol Di-secondarybutylphenol Nonylphenol Dodecylphenol
IgepalbCO-430 Dowfaxb 9N6 SteroxbDF Tritonb X-100 Dowfaxb 9N9 IgepaPCO-630 Tergitolb TP-9 SteroxbDJ Experimental product Dowfaxb 9NI5 SurfonicbN-l 50 Experimental product Tritonb X-205 Dowfaxb 9N20 Tergitolb NP-40 Blend of N9t and N-40 Experimentalproduct Dowfaxb 9N25 Tergitolb NP-425 Dowfax* 9N30 SurfonicbN-300 Tergitolb NP-435 Tritonb X-405 Polyglycol89-1 Dowfaxb 9N40 SteroxbDX
’ The numberindicatesthenumberof ethyleneoxideunitsin the hydrophilicchain. bTrademarknameregistered.
Calculation based on Flory’s (1940) law for linear condensations of ethylene oxide showed the expected mole fractions having a chain length of 20 to be: Nonyl9t 0.5% Nonyl20d, nonyl20t 25% Nonyl25t 2% Nony135 0.5 % Nonyl20B 0.5% By thin-layer chromatography nonyl20t was found to have 7.0% by weight of 20 unit chains, 33.8% of 18 through 22 unit chains. The blend nonyl 20B was found to have 0.13 % by weight of 20 unit chains, 0.53 % of 18 through 22 unit chains. QUANTITATIVE
TOXICITY
Single Orat Dose
The LD50 of no@ 9t is 2.6 (2.1-3.2) ml/kg when administered undiluted to 90-120 g unfasted male albino rats. The only obvious effect from the material was depression. Death occurred within 24 hours. Autopsy within a few hours revealed congested lungs
318
SMYTH
AND
CALANDRA
with petechial hemorrhages, congested livers with surfaces in apposition to the stomach blanched, pale kidneys with similar blanched areas, congested adrenals and gastrointestinal tracts. The blanching may have been a postmorten change. Larson et al. (1963) list the acute oral LD50 values for a number of ethylene oxide adducts with octylphenol, covering a wide range of chain lengths, in 120-g male rats fasted before dosing. Most of the deaths occurred within 72 hours and were preceded by depression. The values range from greater than 28 g/kg for a chain length of 40 to a minimum of 1.7 for a chain length of 9.7, rising to 7.2 at a chain length of I. Subacute Feeding to Rats
Nine materials were fed in the diet of rats for 90 days in one laboratory during one period (Table 2). Each dosage of each material was administered to 10 male and 10 female Sprague-Dawley weanling albino rats in good condition. They were housed individually in wire-bottom cages. Food consumption was recorded weekly, separately by sex, and concentrations were adjusted to allow constant dosage in terms of grams per kilogram per day. Water was freely available. The animals were weighed weekly and their condition and behavior were noted. Hematology was studied on 5 of each sex from the highest dosage levels and the controls before treatment and after 11 weeks. Urines pooled by sex were examined for reducing substances, albumin, and microscopic elements from the same groups at the same times. All rats were studied for gross pathology, livers, kidneys, and testes were weighed, and 33 tissues were examined histopathologically from 5 rats of each sex at the highest and control dose levels. In order to discriminate between poor palatability and toxic effect, 25day paired feeding studies were performed at dose levels for which weight gains were shown to be less than those of respective control groups by Bartlett tests, analyses of variance, and/or t tests. TABLE 2 ADMINISTRATION OF ALKYLPHENOLETHYLENE
OXIDE ADDUCTS IN THE DIET OF RATS
FOR 90 DAYS’
Lowest dosage showing response in either sex” Increased liver weight Material Nony14 Nonyl6 Dodecyl6 Octyl9
Dosages fed (g/kg/day) I, 0.2,0.04 1) 0.2, 0.04 1,0.2,0.04
Nonyl 15s Nonyl20 Nonyl3Os
1) 0.2,0.04 1) 0.2,0.04 1, 0.2, 0.04 5, 1, 0.2 5, 1,0.2
Dodecyl40
5, 1,0.2
Dodecyl9
Retarded growth’ 1 1
Absolute weight 1
0.04
-
0.2 0.04 0.2
Liver/body weight ratio 0.2 0.04 0.04 0.2 0.2
0.4 0.2 5 -
1 -
-
-
-
-
11Data from Industrial Bio-Test Laboratories (1963-1965). * At confidence level of 95% or higher. c Paired feeding studies showed each due to poor palatability.
-
ALKYLPHENOL
POLYOXYETHYLENE
319
SURFACTANTS
All dosage-related responses are presented in Table 2. All effects on growth rate were shown to be due to poor palatability of the diets. Livers of both sexes receiving the shorter chain materials were heavier than those of control rats, both absolutely and in relation to body weight. Since no histopathology was found in liver sections. the response was interpreted as an increase in parenchymatous tissue resulting from increased enzyme activity in metabolizing the materials fed. NonyZ9t was fed in the diet of rats for 90 days. Four dose levels and a control level were administered to 10 male and 10 female Charles River (CFE) young albino rats in good condition. They were housed in groups of 5 of one sex in wire-bottom cages. Concentrations in Purina Laboratory Chow were kept constant. Water was freely available. Food consumption was recorded over 4-week periods, for each cage. The animals were weighed weekly and behavior was noted. All rats were studied for gross pathology at sacrifice. Livers and kidneys were weighed. Sixteen tissues were studied from 12 controls and 8 on the highest dosage, three tissues from 10 rats on the next two dosages. All livers were stained with Sudan IV, and periodic and Schiff reagent for positive discrimination between intracellular lipoid and polysaccharide deposits. Numerical data were evaluated by Fisher’s t-test, Bartlett’s homogeneity of variance, analysis of variance, and Duncan’s multiple range test. Effects referable to the material fed are shown in Table 3. Groups of 15 Sherman Wistar albino rats of mixed sexes weighing 95-210 g, with a control group of 30, received diets containing 0.01 to 5.0% of the nonyZ9i for 90 days.
ADMINISTRATION
Statistically significant effect Food eaten Weight gain Liver aspercentageof body weight Kidney aspercentageof body weight Kidney Focal tubular necrosis Focal cloudy swelling of proximal convoluted tubules Liver Focal hepatic-cell necrosis Cloudy swellingof central hepatic cords Intracellular lipoid Reducedpolysaccharide
TABLE
3
9t IN
THE DIET
OF NONYL
1.25
M F M F M F M F
Low Low Low Low High High
” Data from Mellon Institute (1959-1965).
90 DAYS
Dosagein grams/kilo/day
_--___Sex
OF RATS FOR
0.25
0.05
0.01
Low Low Low
Low
Low
High Low
+
I
-c
-L
4.
Slight
Control
SMYTH AND CALANDRA
320
Table 4.4 Body weight, diet eaten, and hematologic parameters were followed. After 8 weeks the 2 lightest male and female rats in each group were sacrificed, gross examination was made, and 19 tissues were examined histopathologically from rats receiving the greatest dosage. At the end of the 90-day feeding period all surviving rats were sacrificed and gross examination was made. Nine organs were weighed. Histopathologic study was made of 19 tissues from the 2 lightest males and females in each group. No histopathologic changes indicating toxic effects were seen. Rats receiving diets containing 2.5 and 5% surfactants were emaciated, had scanty body fat and poor organ development, interpreted as due to partial starvation. Red and white blood cell counts, differential white cell counts, and hemoglobin remained normal. The numerical data are shown in Table 4. The only statistically significant effect was retardation of weight gain, judged to be referable to poor palatability, at 0.64% and higher in the diet. TABLE 4 RESULTS
OF RATS
RECEIVING
DIETS
Concentration in the diet: 5.0xb
ADDED NONYLPHENOXY POLYETHYLENE OXIDE 9 FOR 90 DAYS”
WITH
2.5%
Rats in group 15 15 Mortality 11 2 Males Avg. weight gain (g) -5 55 Avg. food intake 10.0 10.3 (g/rat/day) Females Avg. weight gain (g) -20.5 21.3 Avg. food intake 9.4 9.7 Wrat/dw) Avg. body weight of males 194 at 90-day scarifice (g) Organ weights as percentage
Heart Liver Kidney
Testis Brain
0.64%
0.16% 0.04% 0.01% Control
15
15
0
0
104
201
11.1
12.4
48.7
85.7
10.1
10.5
228
of body weight 0.38 4.00 0.78 1.22 0.89
0.40 4.04 0.84 1.58 0.79
15 2
15 0
30 3
183 12.0
191 12.9
217 13.2
87.0 10.4
86.1 10.6
94.9
321
0.29 3.17 0.55 0.75 0.55
325 0.32 3.64 0.71 1.00 0.58
0.30 2.87 0.44 1.03 0.60
10.6 338 0.33 3.08 0.62 0.82 0.51
a Data from Shelanski (1960); see text footnote 4. b Survivors were sacrificed after 8 weeks.
NonyZ9d was added to the diet of groups of 10 male and 10 female 45- to 50-day-old albino rats at levels of 0.1 to 1.0%; nonyl 40 at 0.01 to 3.0%; and di-set-butyl40 at 0.03-3.0x; with control groups of equal size, for 90 days, Tables 5-7.5~6 4 SHELANSKI, M. V. (1960). Ninety-day feeding studies with general aniline and film corporation’s Igepal CO-630. Industrial Biology Laboratories, Inc. Unpublished Report, September 28. 5 DOW (1960). Results of dietary feeding of Dow polyglycol 89-l to rats-ninety-day experiment. Biochemical Research Laboratory, The Dow Chemical Company, Intramural Report, March 8. 6 Dow (1961). Results of go-day dietary feeding studies of Dowfax 9N40 and Dowfax 9N9 in rats. Biochemical Research Laboratory, The Dow Chemical Company.
ALKYLPHENOL
POLYOXYETHYLENE
TABLE
321
SURFACTANTS
5
RESULTSWITH RATS GIVEN DIETSCONTAINING NONYLPHENOXY ETHYLENE OXIDE9 FOR 90 DAYS’ Concentration in the diet:
1.0%
20 Rats in group 0 Mortality Males Avg. final body weight (g) 262' Avg. food eatenduring first 30 days 24 k/rat/day) Organ weightsas percentageof body weight 0.35 Heart 3.66' Liver 0.86' Kidney Spleen 0.21 1.05 Testis
0.3 7,; 20 1
0.1 7; 10 7b
327
Control 20 0 311
25
20
0.35 2.32'
0.81
0.33 2.84 0.78
0.20 0.95
0.96
0.21
Females Avg. final body weight (g) 168 22 Avg. food eaten during first 30 days (g/rat/day) Organ weightsaspercentageof body weight Heart 0.39 3.73" Liver 0.87 Kidney 0.27' Spleen
177
22
187 20
0.42 3.42' 0.92
0.25
0.41 3.21 0.94 0.24
180 20 0.39 3.13 0.85 0.23
’ Data from Dow; seetext footnote5. bSacrificedbecause of extremedehydrationdueto accidentallack of water. ’ Differencefrom control: p < 0.05.
Body weight, appearance, behavior, and mortality were followed; and food consumption was recorded for the first 30 days. Terminally hematocrit, white blood cell total and differential counts, and hemoglobin were determined on 5 females of the control and the two highest dietary levels of each material. At terminal sacrifice 6 organs were weighed and histopathology of 8 organs was studied. Blood urea nitrogen and alkaline phosphatase activity were measured terminally. There was no mortality referable to nonyl9d, and no effect whatever was seen from 0.1% in the diet. Liver weights were high from 0.3 and lx, kidney and spleen weights were high, and growth was reduced by 1%. Slight cloudy swelling of renal tubular epithelium. with slight petechial areas of central lobular granular degeneration and necrosis were seen in the livers. These reversible adaptations are commonly found in controls from the rat colony and were not considered significant. There was no mortality at any feeding level of nonyl40, and no effect whatever was seen from 0.3% or less in the diet. At 1% livers of males were numerically but not statistically heavy and at 3 % slight central lobular granular degeneration and necrosis, with general cloudy swelling were seen in the livers. There was no mortality referable to di-set-butyZ40, and no effect whatever was seen from 3 % in the diet of female rats or 1 % in the diet of male rats. At the 3 % level the
322
SMYTH
RESULTS
WITH
RATS
GIVEN
Concentration in the diet:
DIETS
AND
CALANDRA
TABLE
6
CONTAINING NONYLPHENOXY 90 DAYS”
3.0%
1.0%
Rats in group 20 20 Mortality 0 0 Males Avg. final body weight (g) 295 302 Avg. food eaten during 21 21 first 30 days (g/rat/day) Organ weights as percentage of body weight Heart 0.36 0.36 2.95 2.92 Liver Kidney 0.79 0.78 0.20 0.20 Spleen Testis
0.94
Females Avg. final body weight (g) 177 Avg. food eaten during
20
0.96 180 22
first 30 days (g/rat/day) Organ weights as percentage of body weight Heart 0.41 0.43 Liver Kidney
Spleen
3.01 0.88 0.22
3.15 0.98 0.24
ETHYLENE
OXIDE
0.3 %
0.1%
20 0
20 0
20 0
20 0
291 23
294 22
292 21
311 23
0.35 2.77 0.79 0.21 0.99 186 21 0.39 3.05 0.77 0.24
0.35 2.79 0.74 0.22 1.02 187 19 0.40 2.88 0.82 0.23
0.03 %
40 FOR
0.36 2.81 0.81 0.23 0.88 190 19 0.38 3.04 0.87 0.23
Control
0.33 2.84 0.78 0.20 0.96 180 20 0.39 3.13 0.85 0.23
a Data from Dow (1961); see text footnote 6.
livers of male rats showed very slight granular degeneration and central lobular necrosis. The spleens of females at the 1% and 3 % levels were light, and the lungs were heavy at the 0.1% level only. These organ weight changes were judged to be artifacts. Those in the lung were not dose related. In the absence of an effect on red blood cells no significance can be attached to reduced spleen weights. Important numerical data are shown in Tables 5-7. Larson et al. (1963) maintained 30 rats with appropriate controls on diets containing 5 % octyl40 for 90 days and found no effect upon food consumption, growth, urinary protein and reducing substances, hematocrit, hemoglobin, total and differential white cell count, organ to body weight ratios for livers, kidney, spleen, heart, and testes. Microscopic examination of 15 tissues revealed no structural alterations. Subacute Feeding to Dogs
Six materials were administered to dogs for 90 days (Table 8). Each dosage of each material was administered to 2 male and 2 female purebred Beagle dogs in good condition, by gelatin capsule 7 days a week following 300 g of dry diet mixed with water. Water was available freely. Animal behavior was observed daily, weights were recorded weekly. Before the first dose and after 80 doses, clinical tests were performed : hematologic, blood cytologic, blood urea nitrogen, serum alkaline phosphatase, urine specific gravity, total glucuronides, organic sulfate, total phenols, glucose, albumin, formed
ALKYLPHENOL
POLYOXYETHYLENE
TABLE RESULTS
WITH
Concentration
RATS
GIVEN
DIETS
in the diet:
Rats in group Mortality (disease or accident) Males Avg. final body weight (g)
323
SURFACTANTS
7
CONTAINING DI-SECONDARY-BUTYLPHENOXY OXIDE 40 FOR 90 DAYSO
3.0%
1.0%
10 2
10 3
3 13 278 Organ weights as percentage of body weight
Heart 0.34 0.36 Liver 2.87 2.80 Kidney 0.76 0.78 Spleen 0.31 0.29 Testis 1.08 1.07 Lung 0.55 0.58 Females Avg. final body weight (g) 195 192 Organ weights as percentage of body weight Heart 0.38 0.39 Liver 2.88 3.04 Kidney 0.84 0.86 Spleen 0.2gb 0.2gb Lung 0.69 0.70
0 *39,’ ,O
0.1 7;
ETHYLENE
0.03 9/o
Control
10 2
10 I
10 1
10 2
290
324
318
286
0.34 2.76 0.77 0.30 0.99 0.57 189 0.40 3.08 0.83 0.37 0.69
0.32 2.72 0.75 0.20 0.95 0.56 187 0.42 3.02 0.84 0.36 0.87”
0.34 2.82 0.73 0.29 0.94 0.55 197 0.41 2.96 0.84 0.38 0.73
0.36 2.83 0.79 0.29 1.02 0.59
200 0.40 2.89 0.83 0.37 0.66
a Data from Dow (1960); see text footnote 5. h Difference from control: p < 0.01.
elements, and sulfobromophthalein liver function test. Gross structures were scrutinized; liver, kidney, spleen, heart, brain, and testes were weighed; and histopathologic examination of 38 tissues was performed. Some variations in dosage and schedule with one material are not recorded because they did not yield useful information. Table 8 records dose-related responses in dogs to repeated doses. The effects in dogs seriously injured or dying from 5 g/kg/day of nonyl20d after 6 days or 1 g/kg/day after 27 days were weight loss, anorexia, emesis, salivation, gross and microscopic focal myocardial degeneration and necrosis, alterations in organ weights, and death. Single purebred Beagle dogs received diets containing 0.04, 0.64, and 5 % nonyl 9i for 90 days, with 3 dogs on the same diet without addition4 Body weight, diet eaten, and blood cells were studied. After 90 days the dogs were sacrificed, 8 organs were weighed, and 19 tissues were studied histopathologically No histopathologic change indicating a toxic effect was seen, but both control and treated dogs revealed perilobular infiltration and parasitic granulomas in the livers, referable to parasitic infestation. Red and white blood cell counts, differential white cell counts, and hemoglobin remained normal. Important numerical data appear in Table 9.
DOSAGE
OF ALKYLPHENOL
1
0.2
pData from IndustrialBio-TestLaboratories(1963-1965).
0.2
-
1
-
1
PRODUCING
0.2
ADDUCTS
Nony16 1) 0.2, 0.04 -__~ -
OXIDE
8
Nony14 I, 0.2,0.04
ETHYLENE
Death Focal myocardial necrosis (grossly) Focal myocardial necrosis or degeneration (micro) Lung hyperemia (grossly) Liver: body weight ratio increase Growth depression, anorexia Over 90-day period During first 4 weeks only Emesis, usually with salivation Severe, over 90-day period Occasionally, over 90-day period Daily during first l-3 weeks only Occasionally during first l-3 weeks only
Dosages administered (g/kg/day):
LOWEST
TABLE
1 0.04 -
-
1 -
-
Dodecyl9 1, 0.2,0.04 ~__--
EFFECTSWHEN
1 0.2
-
1 0.2
-
1 0.2 -
1-
0.04
-
FOR
Nonyl20d 5, 1, 0.2,0.04 ..~__ 1 1
OF DOGS
Nonyl 1% I, 0.2,0.04
IN THE DIET
-
Fi >
k P r
-
3
B -
-
Nonyl3Os I, 0.2
90 DAYSO
ALKYLPHENOL
POLYOXYETHYLENE
TABLE RESULTS
OF PUREBRED
BEAGLE Dms POLYETHYLENE
Concentration in the diet :
325
SURFACTANTS
9
RECEIVING DIETS WITH ADDED OXIDE 9 FOR 90 DAYS
5.0%
Dogs in group and sex 1M Mortality 0 Average weight gain (pounds) 1.5 Average food intake (total pounds) 80 Average body weight at 90-day sacrifice 17.0 (pounds) Organ weight as percentage of body weight Heart 0.63 Liver 2.87 Kidney 0.45 Testis 1.41 Brain 0.82
0.64% IF 0 1.5 91
18.0
0.93 2.79 0.53 0.87
NONYLPHENOXY
0.04%
Control
1M 0
2F,lM 0
4.0 102 19.0
5.2 102 20.0
0.87 2.82 0.57 2.00 0.83
0.63 2.30 0.45 1.09 0.77
0 Data from Shelanski (1960); seetext footnote 4. Although numerical differences are evident, it was concluded that the only statistically significant effect was retardation in weight gain related to voluntary food intake at 0.64 % and higher in the diet, and that 0.04 % was without effect. Four dogs were maintained for 90 days on diets containing 5% octyl40. The only effect found was softness of the large bowel contents {Larson et al., 1963). Chronic Feeding to Rats
Nonyl4 was administered to rats in the diet over a 2-year period. Each dosage group consisted of 35 male and 35 female weanling Sprague-Dawley albino rats, with 5 male and 5 female rats in addition on the highest and control dosage groups reserved for periodic clinical test. The rats were housed individually in wire-bottom cages. The material was added to Rockland Rat Diet in concentrations adjusted from time to time to maintain the dosages constant at 1, 0.2, 0.04, and 0 g/kg/day, presented in excess of the amount eaten. Water was freely available. Food consumption was calculated weekly for 12 weeks, irregularly thereafter. The rats were weighed weekly for 13 weeks, biweekly to 1 year, then monthly. Food utilization was calculated for the first 12 weeks. Animals were observed daily for abnormal behavior. Before the first dose, and after 3, 6, 12, 18, and 24 months clinical tests were performed on the 5 of each sex on the highest dosage and the control diet reserved for this use: hemoglobin, total red and white cell count, differential white cell count, urinary reducing substances, protein, and formed elements. All rats dying or sacrificed were carefully examined for gross pathologic changes, including tumors. After 12 months 5 of each sex from the highest dosage and from the control group were sacrificed, 3 of each sex from each of the two lower-dosage groups. After 24 months all rats were sacrificed. At sacrifice livers, kidneys, and testes were
326
SMYTH
AND
CALANDRA
weighed. Histopathologic examination of 28 tissues was done on 5 rats of each sex on the highest dosage and in the control diet groups at each sacrifice. All numerical results were evaluated by appropriate statistical tests. There were no deaths and no untoward behavior attributable to the material. At 1 g/kg/day both sexes, and at 0.2 g/kg/day the females had gained’less weight than the controls after 12 months, but did not differ from the controls after 24 months. These groups ate less than did the controls, and the growth effect was attributed to poor palatability of the diets, since a similar effect had been definitely shown to be due to voluntary limitation of food intake (see paired feeding studies in the 90-day feeding of nonyZ4 presented above). The only other dose-related effect noted was slight elevation of liver weight in relation to body weight in both sexes on an intake of I g/kg/day. Microscopically the livers were normal, hence the response was not considered to be adverse. Nonyl Pt was administered in the diet of rats over a 2-year period. Each dose group consisted of 36 male and 36 female 60-g Carworth-Elias rats. Sixteen of each sex were designated at the start of the study for serial sacrifice after 6 and 12 months of feeding. The animals were housed in groups of 5 of one sex in wire-bottom cages. The material was added to ground Purina Laboratory Chow at concentrations of 0.27, 0.09, 0.03, and Ox, presented in excess of the amount eaten. Water was freely available. Food consumption was calculated by 28-day periods. The rats were weighed biweekly for the first year, then monthly. Animals were observed at each weighing for appearance and behavior. Total red blood cell counts were made on 10 male rats of the two highest dose groups and the control group before the feeding and at 90, 180, 360, and 540 days. All rats dying or sacrificed were carefully examined grossly. Livers and kidneys were weighed at sacrifice. Histopathologic examination of 11 tissues and of all neoplasms was done. All results were evaluated by appropriate statistical methods. There was no difference between control and treated rats in any observation made-food consumption, mortality, life span, extraneous infections, liver and kidney to body weight ratios, body weight gain, maximum weight attained, red blood cell count, incidence and location of neoplasms, histopathologic lesions. Larson et al. (1963) maintained 60 rats with controls on diets containing up to 1.4 % octyl40 for 2 years and found no adverse effects upon food consumption, growth, urinary protein and reducing substances, hemoglobin and blood cells, organ to body weight ratios for liver, kidney, spleen, heart, and testes, or histologic appearance of 15 tissues. Chronic Feeding of Dogs Nony14 was administered to dogs over a 2-year period. Each dosage was given to 3 Beagle hounds of each sex, housed 3 of one sex in one kennel. Approximately 300 g of dry diet was mixed with water for each dog each day. Immediately after the feeding period uneaten diet was removed and gelatin capsules were given containing sufficient nony14 to constitute dosages of 1, 0.2, and 0.04 g/kg/day, with an untreated control group. The dogs were weighed weekly and observed daily for signs of effect. Before the study started, and after 90,180,360,540, and 720 days, clinical tests were performed on all dogs: hemoglobin, hematocrit, red and white cell total counts, and white cell differential counts; urine specific gravity, pH, glucuronides, organic sulfate, total
ALKYLPHENOL
POLYOXYETHYLENE
SURFACTANTS
327
phenol’s, glucose, albumin, formed elements; and sulfobromophthalein liver function tests. After 720 days, fasting blood glucose levels were determined. All dogs were sacrificed and carefully examined grossly; weights of liver, kidneys, spleen, heart, brain, and testes were recorded; and 28 tissues were examined microscopically. At I g/kg/day food intake was several times depressed and body weight began to fall. Each time this occurred the high protein portion of the diet was increased until anorexia was corrected and body weight was regained. During the first week of feeding occasional emesis was seen at all dose levels, but thereafter it occurred infrequently and only at the highest dosage. At 1 g/kgiday there was a moderate elevation in serum alkaline phosphatase and liver to body weight ratio, but no significant gross or microscopic pathologic change. At 0.20 g/kg/day the two effects were seen to lesser degrees, and no effect was seen at 0.04 g/kg/day. Notiyl 91 was administered to dogs in the diet over a 2-year period. Each dosage group consisted of 3 male and 3 female purebred Beagles. 6-12 months old at the start of the study. They were individually housed in cages with open-mesh floors. The material was added to Friskies Dog Food Meal in concentrations of 0.27, 0.09, 0.03. and OO,/,.The amount of food eaten was recorded each day. The calculated average intakes of nonyl9t for the treated groups were 0.088,0.028, and 0.0085 g/kg/day. Water was freely available. The condition and behavior of the dogs were observed daily, and they were weighed weekly. Before the study started and after 90,180,360,540, and 700 days serum urea nitrogen, alkaline phosphatase, bromosulfalein excretion, total red and white blood cells, differential white cell count, hemoglobin, and hematocrit were determined. After 2 years the dogs were sacrificed, liver, kidneys, and heart were weighed, gross structure was observed, and histopathologic examination of 21 tissues was done. All measurements and observations were evaluated by appropriate statistical methods. The only effect attributable to the treatment was an increased liver to body weight ratio at 0.27% nonyl9t in the diet. Microscopically the livers were normal. METABOLISM
AND EXCRETION
Metabolism and excretion were studied in male rats (Knaak ef al., 1966) at a dosage of 67 mg/kg, using eight r‘C-labeled samples: nonyZ9t labeled in the ethylene oxide chain, 7-, lo-, 12-, and 15-mole adducts separated from chain-labeled nonyl9t, nonyl9t. and nonylp’heno[ labeled uniformly in the phenol and nonane groups. Excretion was substantially complete in 2 days. Total activities found after 7 days are listed in Table 10. The nonylphenol moiety of nonyZ9t was excreted in the same pattern as nonylphenol itself. The ethylene oxide chain moiety was absorbed from the digestive tract and metabolized less completely as chain length increased. Only 1 T< of the dose was found in the urine as nonyl9t per se. Ion exchange chromatographic studies indicated the principal urinary metabolites to be carboxylic acids of polyethylene glycols and glucuronic acid conjugates of nonylphenol. The distribution and excretion of a tritiated sample of ocryl40 was studied by Larson et al. (1963). In both rats and dogs approximately 1% of the activity was found in the urine within 72 hours of the dose.
328
SMYTH
AND
CALANDRA
TABLE
10
EXCRETION OF 14CCOMPOUNDS BY THE MALE RAT DURING 7 DAYS AFTER AN ORAL DOSE OF LABELED NONYL 9t”
Ethylene oxide labeled 7-Mole adduct (%I
Nonyl9t (%I
Nonylphenol labeled
15-Mole adduct (%I
Nonyl9t (%I
Nonyl phenol” (%I
20 0 78
19 0 76
Activity in urine Expired air Feces
40” 3.3 36”
39b 1.2 52
26’ 0.2 56’
Total found
85’
92.2
85”
Carcass
0
0
0
-
-
98
95
’ Data from Knaak el al. (1966). bIncludescagewashings. ’ Excludescagewashings. dIdenticalexcretionwasfoundafter an intraperitonealdose.
STUDIES OF CARDIOTOXICITY
Following the discovery of focal myocardial necrosis in dogs, but not in rats, as a result of 90 days’ consumption of diets containing nonyl ZOd,additional studies were performed to elucidate and evaluate the significance of the effect. Three laboratories produced severe degrees of the condition after periods briefer than 90 days, even as brief as 5 days. Accordingly most of the additional feeding studies performed were limited to 14 days. Uniformity of Eflect
In order to investigate the possibility that some impurity or unique characteristic in the commercial product nonyl20d had caused the cardiotoxicity, new samples were obtained of 4 commercial products produced in different plants, nonyl ZOd,nonyl ZOt, octyl20, and dodecyl20. Each was given by capsule daily for 14 days to 1 male and 1 female Beagle pup, in divided doses totaling 1 g/kg/day. All animals lost weight, exhibited emesis, and developed focal myocardial necrosis. Six of the 8 dogs died after 4-14 days. The severity of the cardiac lesion was inversely as the frequency of emesis and directly as the survival time. This comparison showed no important difference between the four different products of the same chain length. SpeciesResponses
Only dogs and guinea pigs consistently developed severe focal myocardial necrosis. No myocardial lesions whatever were seen in 3 cats and 3 rabbits which were intubated on 14 successive days with approximately 0.7 g/kg of nonyl20t. Lesions interpreted as early stages of necrosis were seenin guinea pigs; eosin-staining fibers, round-cell infiltration or myocardial degeneration in 9 of 19 guinea pigs receiving 1 g/kg of nonyl20d or nonyl20t on 2 or 3 successive days. Rats did not show any heart lesions
ALKYLPHENOL
POLYOXYETHYLENE
329
SURFACTANTS
after 90 days’ feeding at 5 g/kg/day of nonyl2Od, while dogs consistently revealed the lesion after 90 days at 0.04 g/kg/day. Chain Length
Nine materials were administered to 1 male and 1 female Beagle dog daily for 14 days in one capsule, or divided between 2 or 3 capsules each day. The incidence of focal myocardial
degeneration
evident upon microscopic
study of sections is shown in
Table 11 by chain length, together with its incidence in 90-day and 2-year feeding
studies referred to earlier. TABLE CARDIOTOXICITY
IN
DOGS VERSUS ADDUCTS
11
CHAIN LENGTH OF ALKYLPHENOL ADDED TO THE DIET”
ETHYLENE
OXIDE
Focal myocardial degeneration Average ethylene oxide chain length 4
Nony14
6 9
Nony16 Nonyl9t Dodecyl9 Dodecyl 12 Nonyl 15s Nonyl 1Sd
12 15
17.5 20
Nonyl20t Dodeeyl20 Nonyl25d Nonyl3Os Octyl40
20 from
Dosage Wklday) I_ ___ 1 1 1 0.088 1 1 I 0.2 1
Nonyl 17.5 Octyl20 Nonyl20d
25 30 40
’ Data
Material
Nonyl 2OB Industrial
Bio-Test
14-Day 90-Day 2-Year feeding feeding feeding ----.--~. _ _ - .~ - ~~. 0 0 0 0 0 0 0 0 0 i
1 1
i L
0.04 + _~
t
1
0
(5 $ in diet) ___-1
Laboratories
+
1 1 1
0 0 -^--.0
0
(1963-1965).
Cardiotoxicity was evident from all materials having an average chain length of 20 ethylene oxide units, except nonyl 20B. Although the chain length of this material averaged 20, actual chain lengths constituted a bimodal distribution, and not over 0.2 % of them were actually 20 units long. Cardiotoxicity was also produced by products whose average chain length was 17.5 and 15, but not from those whose chain length was either 12 or less, or 25 or more.
330
SMYTH
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CALANDRA
Dogs receiving for 14 days 1 g/kg/day of materials whose chain lengths were 15, 17.5, or 20 almost consistently showed anorexia, weight loss, emesis, diarrhea or loose stools, lethargy, and death. Dodecyll2 produced emesis, diarrhea, and death; nonyl25d produced emesis, diarrhea, and lethargy. Similar signs were produced by nonyl20d at 1 g/kg/day in dogs fed for 90 days. Six alkylphenoxy ethylene oxide adducts were administered by capsule daily for 30 days to 1 male and 1 female Beagle dog weighing 5-8 kg, at 1 g/kg/day.7 The behavior and condition of the animals was observed daily. Serum sodium, potassium, and magnesium levels were determined on days 0, 4 or 5, and 10. Transaminase activities of blood serum were determined on the same days. At death, or at sacrifice after 30 days, histopathology of heart, liver, and kidney was studied. Table 12 summarizes the results. At a chain length of 15 ethylene oxide units 1 of 2 dogs died after 25 doses, revealing moderate myocardial degeneration. At a chain length of 20, all 4 died after 4-10 doses, showing myocardial necrosis. At a chain length of 30 none of 4 dogs died, but 3 showed slight myocardial degeneration, perivascular myocardial round-cell infiltration, or loss of clarity in myocardial striations. At a chain length of 40, no dogs died and the heart muscle appeared normal. Gross examination of the dogs which died revealed areas of necrosis in the heart muscle of the left ventricular wall and interventricular septum. The right side of the heart was dilated and the muscle appeared flabby. The lungs showed varying degrees of edema, kidneys suggested tubular degeneration, and the livers were dark. Inactivity, elevated or irregular pulse rate, vomiting, and loose stools following the dose paralleled the myocardial histopathologic changes and mortality. Dogs receiving materials with a chain length of 30 showed swelling of eyelids, face, legs, and body suggestive of a hypersensitivity reaction to release of a histamine-like compound, or to electrolyte imbalance. Swelling was reduced by subcutaneous injection of diphenylhydramine (Benadryl). There was no significant change in serum sodium, potassium, or magnesium. Em&s The degree of correlation between emesis and myocardial degeneration in the dog feeding studies suggested the possibility that cardiotoxicity resulted from electrolyte imbalance brought about from emesis, although electrolyte measurements recorded in Table 12 did not support the hypothesis. One dog of each sex was given a nonemetic dosage of nonyl 2Od, 0.20 g/kg/day divided between two gelatin capsules several hours apart. During 34 days slight emesis occurred once in 1 dog, twice in the other. Myocardial degeneration was found in both dogs. Two dogs of each sex were given a varying dosage of nonyZ2Od, divided between three capsules a day. The daily dosage started at 0.20 mg/kg/day and increased by 0.05 g/kg/day every 5-7 days to 0.55 g/kg/day, then decreased to 0.50 g/kg/day. Emesis did not occur until the dosage reached 0.55 g/kg/day. At this dosage it was consistent, 7 SADEK, S. E. (1963). A short-term toxicity study on various nonylphenol ethylene oxide adducts. Biochemical Research Laboratory, The Dow Chemical Company, Midland, Michigan, Intramural Report, November 27.
N
OF CARDIAC
EFFECT
IN
5 TO 8 KG BEAGLE
Dots
12 FROM
1 G/KG/DAY
BY CAPSULE
FOR
a Data
from
Sadek
(1963);
see text footnote
7.
Histopathology of myocardium Moderate degeneration Moderate degeneration Necrosis Necrosis Necrosis Necrosis Necrosis Scatterednecrosis Normal Perivascularinfiltration Striations not clear Slight degeneration Normal Normal Normal Normal
30 DAYS”
Changein serumat 10days or at earlier death _____ Weight Days - __-_ change to Sodium Potassium Magnesium SGOT SGPT LDH Material Dog death (mg/lOOml) (mg/lOOml) (mg/lOOml) (SF units) (SF units) (BB units) (g) _____ -__ Nonyl 15d 657 +450 25 -6.0 -0.1 +1.8 t21 -7 +10 672 -950 +46.5 0 +lO -14 +60 12.5 Nonyl20d 658 +250 5 -1.2 12 -19 +60 +1.5 +4.1 -50 4 -216 659 +16.4 -1.4 -0.1 +I32 $68 662 -250 10 -12.7 +1.1 1-3.2 +41 i-6 +90 663 -150 4 -20.1 t-173 t52 +410 +61.2 +5.2 Nonyl20t -250 667 5 +21.1 t-2.5 +0.4 $26 -7 +475 671 -800 -23.5 0 +1.9 i-18 i-10 1-170 Nonyl30d 660 +600 -1 t20 +3.9 -4.2 t2.7 t15 +I70 664 t700 -6.4 -1.6 $5.3 t17 -12 Nonyl3Os 666 +750 -t6.7 -13.5 +3.8 il 0 rloo 670 4500 i-36.9 -6 +103 -2.7 -2.2 +24 Nonyl40 i-75 665 +1200 -8.3 -1.9 +2.3 +17 i-l .-9 669 +500 m-36.7 +0.2 +2.5 i-14 t-300 Control 661 i-1400 -6 +I80 $2.6 -3.5 -5.2 t17 668 -t1300 t 305 --30.1 -6.5 +3.0 +20 7~2
SUMMARY
TABLE
332
SMYTH
AND
CALANDRA
but was less frequent later at 0.50 g/kg/day. Myocardial degeneration was seen in the dog which died after 59 days and in the 3 which survived 131 days. Four dogs were given 1 g/kg/day divided among three capsules. Two received diets to which 0.5% potassium chloride had been added, 1 received daily intramuscular injections of 1500 USP units of thiamine hydrochloride, and one received no supplements. Emesis, anorexia, weight loss, and myocardial degeneration did not differ among the 4 dogs. Five Beagle dogs were given 10 g/kg/day of nonyl20d by capsule, in divided doses three times a day to reduce emesis, 7 days a week for 12 days or until earlier death. Dogs I and II received no other treatment. Dogs III and IV received a diet to which 0.5 % potassium chloride had been added. Dog Vreceived daily subcutaneous injections of 1500 USP units of thiamine hydrochloride. Dogs I and II ate little food, vomited occasionally, lost weight and died during the second and third days. Dog III ate normally, vomited occasionally, lost weight and died during the fourth day. Dog IV ate well, vomited consistently, gained weight, but died during the second day, while dog V ate poorly, vomited seldom, lost weight and was sacrificed in extremis on the twelfth day. Focal myocardial degeneration was seen in all 5 dogs, extreme in dog V, slight in dog IV, intermediate in the others. Neither potassium chloride nor thiamine prevented the myocardial injury. Eflect on the Isolated, Perfused Cat Heart8
Isolated cat hearts obtained from lightly etherized cats were perfused through the coronary arteries in the manner of Langendorff as modified by Chenoweth and Koelle (1946). The modified Ringer-Locke solution was gassed with 95 % oxygen and 5 % carbon dioxide, and a fixed volume was permitted to recirculate throughout the system. A control period of at least 30 min was allowed before any compounds were added. To the reservoir were added aliquots of the materials under study to yield a concentration of 5 or 10 mg/l of perfusate. The apparatus adjustments remained constant throughout the tests so that the time lapse between addition of the detergent and its arrival at the heart in the perfusate was essentially the same for each preparation. Contractions were recorded with a strain gauge and Offner recorder. No@ 9d, at 5 mg/l, reduced the amplitude of contraction from 12 mm to 10 mm in 12 min. An additional 5 mg/l of nonyl9d was added. Within 5.5 min the amplitude had been reduced to 7 mm and a bigeminal rhythm occurred. Ten minutes later the amplitude was less than 50% of that during the control period and the arrhythmia apparently had ceased. The preparation continued to contract for about 10 min more before recordable contractions ceased. In the case of the nonyZ2Od, 5 mg/l reduced the contraction from 11 mm to 7 mm in 12 min. An additional 5 mg/l was added, reducing the contraction to 6.5 mm in 1.5 min more. Thus, reduction to the 50 % contractile point occurred in 13.5 min, but the heart continued to decline steadily and slowly for approximately 35 min more before recordable contraction ceased. 8 KLUG, S., MCCARTY, L. P., and CHENOWETH, M. B. (1965). Effect of Dowfax 9N9, 9M40, and Tween 20 on the isolated perfused cat heart. Biochemical Research Laboratory, The Dow Chemical Company, Intramural Report, March 5.
ALKYLPHENOL
POLYOXYETHYLENE
SURFACTANTS
333
In the case of nonyl40,5 mg/l produced very little detectable effect on the contractility. Ten minutes later an additional 5 mg/l was added. It was not until 62 min had elapsed that the amplitude of contraction was reduced to 50 y0 of the control level. In the case of Tween 20, 5 mg/l produced no decrease in amplitude for 18 min. An additional aliquot of 5 mg/l was added, and 36 min later the heart suddenly ceased to beat, although the reduction in amplitude of contractile height was not very much. being only 1 mm less than the control level of 19 mm. The cardiac toxicity shown in this experiment is the direct result of an effect upon the myocardium, and is not secondary to effect on any other organ. Among the nonylphenoxy adducts tested, cardiotoxicity is greatest at an average ethylene oxide chain length of 20, less at a length of 9, and least at a length of 40. Since the hydrophobic moiety of Tween 20 is not phenolic, and its cardiac toxicity is much less than that of nonyl ZOd, which has the same ethylene oxide chain length. chain length alone is not sufficient to produce marked cardiotoxicity. Age of Dogs
The 14-day studies which showed that molecules with 20-unit ethylene oxide chains are those responsible for cardiotoxicity were performed on dogs 4-6 months old. In order to determine whether dogs of other ages might be more sensitive, nonyZ15d was administered for 14 days to 2 dogs 8-10 weeks old and to 2 dogs more than 3 years old. The daily dosage of 0.2 g/kg/day was the greatest dosage which resulted in no cardiotoxicity in dogs 4-6 months old. No cardiotoxicity resulted in the young or old dogs. Likewise dodecyl 12 administered in identical studies at 0.2 g/kg/day showed no cardiotoxicity in young or old dogs. Fourteen- Llersus Sixty-Day
Feeding
Nonyl 15d was administered by capsule three times a day in a dosage totaling 0.2 g/kg/day to 2 male and 2 female dogs over a 60-day period. This was the greatest dosage of the product which caused no cardiotoxicity during 14 days of administration. The 60-day administration likewise caused no signs of cardiotoxicity. An identical 60-day administration ofdodecyl I2 likewise caused no cardiotoxicity. DISCUSSION
There was some rationale for considering that myocardial injury might be the result of histamine release such as Krantz et al. (1948) found in dogs from some other surfaceactive compounds with ethylene oxide chains. However, the work reported here lends no support for this hypothesis. The most prominent supporting finding was bronchospasm in the guinea pig. It is more convincingly explained as a mechanical response to intubation. Furthermore, gastric ulcer, which is an expected response to histamine in the guinea pig, was sought for and not found, while Krantz et al. (1948) found no histamine release in guinea pigs from the surfactants which did release it in dogs. No other sign of histamine release was found, unless myocardial degeneration in the dog can be considered such a symptom. It is recognized that the dog heart is unusually sensitive to histamine (while the human heart is resistant). The studies of the isolated perfused cat heart by Klug et al.8 discount this possibility by establishing a strong
334
SMYTH
AND
CALANDRA
probability that the cardiac toxicity of nonyl20d is the direct result of an effect upon the myocardium, not secondary to any other organ. The findings of a specific effect on the heart from chain lengths intermediate in a long seriesis not unprecedented. Such a finding from parenteral dosesof polypropylene glycols was reported by Shideman and Moe (1949) and Shaffer et al. (1951). REFERENCES M. B., and KOELLE, E. S. (1946).Isolated heart perfusion systemadaptedto the determination of non-gaseous metabolites.J. Lab. Clin. Med. 31, 600-608. FLORY, P. J. (1940).Molecular sizedistribution in ethylene oxide polymers.J. Am. Chem. Sot. CHENOWETH,
62, 1561-1565. J. B., ELDRIDGE, J. M., and SULLIVAN, L. J. (1966).Excretion of certain polyethylene glycol ether adductsof nonyl phenol by the rat. Toxicol. Appl. Pharmacol. 9, 331-340. KRANTZ, J. C., JR., CARR, J., BIRD, J. G., and COOK, S. (1948). Sugar alcohols, XXVI. J. Pharmacol. 93, 188-195. LARSON, P. S., BORZELLECA, J. F., BOWMAN, E. R., CRAWFORD, E. M., SMITH,R. B., and HENNIGER, G. R. (1963). Toxicologic studieson a preparation of p-tertiary octylphenoxypolyethoxy ethanol (Triton X-405). Toxicol. Appl. Pharmacol. 5,782-789. SHAFFER, C. B., CARPENTER, C. P., CRITCHFIELD, F. H., NAIR, J. R., III, and FRANKE,F. R. (1951).A toxicological study of somepolypropylene glycols. Arch. Znd. Hyg. Occupational KNAAK,
Med. 3,448-453.
F. E., and MOE, G. K. (1949).Production of ventricular extrasystolesin the dog by a polypropylene glycol. Federation Proc. 8, 322.
SHIDEMAN,