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The absorption, tissue distribution, and excretion of dodecyldimethylamine oxide (DDAO) in selected animal species and the absorption and excretion of DDAO in man
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
39,377-389
(1977)
The Absorption, Tissue Distribution, and Excretion of Dodecyldimethylamine Oxide (DDAO) in Selected Animal Species and the Absorption and Excretion of DDAO in Man1 D. P. Rice The Procter & Gamble Company, Sharon Woods Technical Center, Cincinnati, Ohio 45241 Received May 26,1976; accepted September 9,1976
The Absorption, Tissue Distribution, and Excretion of DodecyldimethylamineOxide (DDAO) in SelectedAnimal Speciesandthe Absorption and Excretion of DDAO in Man. RICE, D. P. (1977). Tuxicol. Appl. Pharmacol. 39, 377-389.The absorption tissuedistribution, and excretion pattern of [methyl-14C]DDA0 and [I-dodecyZJ4C]DDA0 administered orally or cutaneously to rats, mice, and rabbits were investigated. The excretion pattern of radioactivity from [I-dodec~&‘~ClDDAO administered orally and cutaneouslyto man wasalsoinvestigated.An oral doseof DDAO is rapidly and extensivelyabsorbedand excretedby rats and man. Peak tissuelevels of radioactivity resulting from oral administration of [methyl-14C]DDA0 to rats occur within 1 hr after dosing. Cutaneously administeredDDAO is absorbedby man, rats, rabbits, and mice.In man, the rate of DDAO absorption through the skin is at least one order of magnitudelessthan that observedin rats, mice,and rabbits. Dodecyldimethylamine oxide (DDAO) is a semipolar surfactant used primarily in light-duty liquid detergents. In view of the potential ingestion or dermal exposure to DDAO through product use, it wasappropriate to establishwhether dermally or orally administered DDAO is absorbed, how any DDAO that is abosrbed is distributed among the body’s tissues,and how it is excreted. To these ends, the following human and animal experiments were performed. [mefh,vl-14C]- or [l-dodecyl-14C]DDA0 was applied to the skin of rats, mice, and rabbits, and the absorption of DDAO was characterized through assay of the radioactivity appearing in tissues and excrement after 72 hr of exposure. [mefhyl-‘“CIDDAO and [l-dodec~l-14C]DDA0 were administered orally to male and female rats and the disposition of radioactivity was determined. [l-dodecyZ-‘4C]DDA0 was administered orally or dermally to human subjects, and the extent of absorption was determined through the appearance of radioactivity in excretion products during the next 144 hr. METHODS Materials
[metl~~&‘~C]DDA0 and [l-dodecyl-‘4C]DDA0 were prepared by oxidation of the appropriate tertiary amine with hydrogen peroxide (Kolp ef al., 1963). [me?&Z-14C]1 Presented, in part, at the 14th Annual Meeting of the Society of Toxicology, Williamsburg, March 1975. 377 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction Printed in Great Britain
in any form
reserved.
Virginia,
1SSN 0041-008X
378
D. P. RICE
Dodecyldimethylamine (DDA) was prepared by reaction of I-bromododecane with [methyZ-14C]dimethylamine. [I-dodec.rl-14C]DDA0 was similarly prepared from l-[l-‘4C]bromododecane and dimethylamine. The chemical and radiochemical purity of these substances was shown to be greater than 98.5 % by thin layer and high pressure liquid chromatographic procedures (T. Turan, private communication). Animal Dosing and Handling Procedures
Rats were given oral doses of DDAO in several experiments; the compositions and quantity of the doses are shown in the footnotes to Tables 1-5. The amount of each dose was determined by weighing. The doses were administered intragastrically with a flexible rubber tube fitted to a glass syringe. Rats, rabbits, and mice were prepared for dermal dosing by clipping an area on the back free of hair with a small animal clipper. The clipped area was inspected for breaks in the skin, and the application site was delineated by marking with a felt-tipped pen. The dermal dose was administered from a syringe fitted with a blunt needle with lateral openings. Dermally dosed rats and mice were restrained in small animal restrainers, and rabbits were fitted with collars (Newman, 1963) to restrict oral ingestion of the applied dose. Intraperitoneal doses of [methyZ-‘4C]DDA0 were administered to rats by injection in the lower left quadrant of the abdomen. Four rats received intraperitoneally doses of 22 mg of DDAO/kg (sp act, 1.3 mCi/g) and their excreta and tissues were analyzed for radioactivity. Immediately after dosing, the animals were placed in individual, stainless steel metabolism cages for collection of expired carbon dioxide, urine, and feces. Except as otherwise noted, all experiments lasted 72 hr. Biliary excretion of radioactivity from orally ingested [meth$-14C]DDA0 was investigated in rats. The rats were prepared by surgical insertion of a biliary fistula under pentobarbital anesthesia. A dose of 100 mg of [methyZ-‘4C]DDAO/kg was administered by stomach intubation 1 day after the surgical procedure, and the bile and other excreta were collected for 72 hr. The bile-duct-cannulated rats were kept in small animal restrainers during the predose and excreta collection periods. Because of the design of the restrainers, feces frequently accumulated within them, causing cross-contamination of urine, feces, and carcass residues. Human Metabolism Procedures Generalprocedures. The subjects employed were volunteers who remained at the test facility throughout the course of the study. Each subject was given a complete physical examination before and after the study. The physical examination included clinical chemistry, ophthalmoscopic eye examination, EKG, hematology, and blood pressure. Recommended procedures for informed consent and experimental review were followed. Before dosing, subjects fasted for 12 hr. Zero time blood samples were taken immediately before dosing. Procedures for oral dosing of human subjects. Each subject drank 200 ml of an aqueous solution that contained 50 mg of [ I-dodecyl-r4C]DDA0 (100 &i of 14C). The
[i40]DD~0 AB~OR~JTIONBY bi.4~ AND ANIMALS plastic dosing cup was immediately subject drank the rinsings.
379
rinsed twice with 200 ml of tap water, and the
Procedures for cutaneous dosing qf human subjects. An area 4 x 15 cm was marked off on the outer surface of the subjects’ forearms, which had been clipped free of hair and inspected for breaks. One-half milliliter of an aqueous solution containing 10 mg of [1-dodecyZ-14C]DDA0 (100 @i of 14C) was applied evenly over the site from a plastic syringe. After the site had dried by evaporation, it was covered with a nonocclusive, plastic shield. After 8 hr, the DDAO remaining at the application site was removed by repeatedly (10x) swabbing the skin with water-moistened gauze pads. The portion of the radioactive dose retained in the stratum corneum was assayed by repeatedly (10x) stripping a 7.2-cm2 area within the area application with cellophane tape. Normal handling and exposure of the application site were permitted after these procedures. Collection of human excreta. Urine was collected in polyethylene bottles over the intervals O-6 and 6-24 hr and subsequently over 24 hr intervals through 144 hr. During the collection period, samples were stored at 0°C; upon completion of a collection interval, the urine samples were frozen on dry ice. Fecal samples were collected individually as available in plastic bags and were stored frozen. Expired CO2 was collected in a modified Hanks (Budny and Arnold, 1973) apparatus. The air-flow rate through the collection box was 12 liters/min. Carbon dioxide was collected from the orally dosed subjects for 15 min at 0, 1,2,3,4,5,6,8, l&24,36,48, and 72 hr after dosing. Collections on dermally dosed subjects were made for 30 min at 0, 2, 4, 6, 8, 12, 24, 36, 48, and 72 hr after dosing. Total expired radioactivity was computed from these samples of expired gases. Radiochemistry
The 14C in the excretion products and tissue samples was assayed by standard radiochemical techniques as described by Budny (1972). RESULTS An oral dose of [methyl-14C]- or [l-dodecyl-‘4C]DDA0 was absorbed extensively and rapidly by rats (Tables 1 and 2, Fig. 1). In male and female rats, the fraction and distribution of administered radioactivity excreted during the first 48 hr after dosing were similar. Further, although male rats were sacrificed 72 hr after dosing (Table 3) and female rats were sacrificed 48 hr after dosing, the distribution among body tissues of radioactivity from an oral dose of [l-dodecyl-‘4C]DDA0 was similar for male and female rats. Of the various tissues analyzed, the greatest amount and the highest concentration of radioactivity were found in the liver (Tables 3 and 4). As Fig. 1 indicates, the fractions of dosed radioactivity appearing in the liver, kidney, and blood reached maxima within 1 hr after an oral dose of [methyZ-14C]DDA0. The excretion of radioactivity from orally administered DDAO was rapid. Within 24 hr after dosing, 73.9 and 76.4% of the radioactivity from orally dosed [methyl-‘4C]DDAO and [l-dodecyl-14C]DDA0, respectively, were excreted by male rats. More than two-thirds of the radioactivity excreted as expired 14C02 appeared within 12 hr after oral administration of either [methyZ-14C] or [ l-dodecyl-14C]DDA0. Notably, the
380
D. P. RICE
TABLE EXCRETION
OF 14C (MEAN
+ SE) BY MALE
1
SPRAGUE-DAWLEY OF
RATS AFTER A SINGLE
ORAL
DOSE
[‘4C]DDAO” Percentage of administered dose
[methyZ-‘4C]DDA0 Urine co2 Feces Total excreted Tissues + carcass Total recovered [ 1-do&$-14C]DDA0 Urine co2 Feces
Total excreted Tissues + carcass Total recovered
O-24 hr
24-48 hr
48-72 hr
55.3 f 4.5 10.4 + 0.6 8.1 f 0.7 73.9
8.5 i 1.8 1.2kO.2 2.8 +- 0.8 12.5
3.0 + 0.6 0.7 + 0.1 1.2 + 0.5 4.9
42.1 + 9.1 19.8 k 0.6 5.4 + 1.8 67.4
6.6 f 1.7 1.2f0.16 2.6+ 1.3 10.4
2.2 + 0.8 0.5 + 0.1 1.o + 0.5 3.8
Total
71.0+ 4.5b 13.1 + 1.1’ 12.1 + l.8d
96.2+ 3.7 6.1 i- 0.3 102.3+ 6.7 53.5+ 6.9b 23.12 0.63” 9.4 k 3.06 86.0& 3.7 3.5 + 0.83 89.5-f. 1.1
’ [methyl-“C]DDAO, 100mgof DDAO/kg, spact= 1.3mCi/g; rat weight= 204.8+ 4, four rats. [I-dodecyl-“CIDDAO, 100mgof DDAO/kg, spact = 1mCi/g; rat weight= 184.8+ 5, four rats. b Daily ’ Daily
collections
plus final
cage wash.
collections plus contents of safety traps. d Daily collections plus contents of GI tract at sacrifice.
TABLE 2 EXCRETION OF DURING THE 48-~~
14C (MEAN
+ SE) BY FEMALE SPRAGUE-DAWLEY PERIOD AFTER ADMINISTRATION OF A SINGLE ORAL OF [ 1-dodecyl-14C]DDAO”
RATS DOSE
Percentageof administeredr4C
Urine co2 Feces Total excretion Carcass+ tissues Total recovered
O-24 hr
2448 hr
Total
31.7? 7.8 20.1i- 0.9 7.3 f 1.2 59.1
9.6 + 4.7 2.0 * 0.4 3.7 * 1.0 14.8
53.6+ 3.0b 23.4 _+1.2’ 12.0+ 1.6d 89.1+ 1.4 8.0 * 0.9 96.5 &-1.O
’ Four female rats, body weight = 202 + 1.5 g; DDAO
DDAO/kg, spact= &i/mg. bDaily collections plustotal cagewash. ’ Daily collections plus contents of safety trap. * Daily collections plus wash of GI tract at sacrifice.
dose = 100 mg of
[14C]DDA0
ABSORPTION
20 -
BY MAN
AND
ANIMALS
381
LIVER
16 12 - x X = Absorbed 8 -’
‘< ,
y
x x j( x
40.
level
in individual
x x
I
n
.o -
c
x
.6
-
‘.2
-x
rat
KIDNEY
.d
x
x
X = Absorbed x x xx :< x
x .4-x
x
0.
I
level
in individual
I
I
rat
3 ,
.5 TESTES .4 X = Absorbed .3
-
level
in individual
rat
level
in individual
rat
#
.O< .I2
-
.08
-
x
WHOLE
x
X = Absorbed
-x .04-
BLOOD
x x
x x
x
X
X
x 0.
1 2
I 4
x 8 6
8
10
12
14
f 16
18
20
22
I 24
HOURS FIG. 1. Variation in the percentage of dosed radioactivity present in liver, kidney, testes, and whole blood with time elapsed after oral dosing with (merhyPC]DDAO in rats (indicated points represent individual values).
382
D. P. RICE
percentage of administrated radioactivity excreted as 14C0, by rats dosed po with [methyl-14C]DDA0 was consistently greater than that appearing in expired 14C02 from rats dosed po with [mer/qG4C]DDA0. An average of 12.1 or 9.4 % of the radioactivity administered po as [methyl-14C]- or [l-dodecyl-i4C]DDA0,
respectively,
the bile-duct-cannulated
was excreted in the feces. Although
rats resulted in extensive cross-contamination TABLE
DISTRIBUTION AFTER ORAL
OF RADIOACTIVITY ADMINISTRATION
restraint
of
of radioactivity
3 IN TISSUES OF MALE RATS OF [mefhyZ-‘4ClDDA0
72 HR AND
[ 1-dodecyl-14C]DDAO’ Percentageof dose Tissue
methylJ4Cb
Liver Intestines Stomachand esophagus Testes Kidney Lungs Spleen Heart Brain and spinalcord Pancreas Carcass
1-dodecyl-14Cb
1.25 0.33 0.098 0.12 0.09 0.048 0.031 0.023 0.022 0.02 3.95
1.1
0.4 0.4 0.07 0.08 0.04 -c -c 0.02 -c 1.9
Percentageof doseper gram Leg muscle Subcutaneousfat Bone marrow Whole blood
0.014 0.014 0.04 0.013
0.013 0.062 0.05 0.015
’ Mean,n = 4; SEof all means did not exceed 20% of corresponding mean values. bAmountof dose= 20mgof DDAO (100mgof DDAOjkg bodywt);
concentration = 20 mgof DDAO/ml in water; spact of [methyl-Y]DDAO
= 1.3 mCi/g,
c Not analyzd
[I-dodecyf-Y]DDAO
= 1 mCi/g.
separately.
in urine, feces, and carcass, the data in Table 5 indiate that the bile is a minor excretory pathway for the radioactive compound administered orally as [methyl-14C]DDA0, since bile excreted over the 48 hr period after dosing contained less than 4% of the administered dose. Intraperitoneally administered [methyl-“C]DDAO was also excreted rapidly by rats. Over the first 24 hr after dosing, 67, 8, and 6% of the radioactivity from a dose of [methyl-14C]DDA0 administered ip were excreted in the urine, expired 14C02, and
feces, respectively. The rate of 14C0, production
was initially
rapid, but this excretion
[14~]~~~~
ABSORPTION
BY MAN
AND
ANIMALS
383
rate declined to a low value within 8 hr after dosing (Fig. 2). No significant differences are observed between the distributions of radioactivity among the urine, feces, and 14C02 when results for intragastric and ip administration of [methyl-14C]DDA0 in rats are compared. This suggests that microbial metabolism by gastrointestinal flora does not play a major role in the absorption and excretion of DDAO in rats. TABLE 4 DISTRIBUTION OF RADIOACTIVITY IN THE TISSUES OF FEMALE RATS 48 HR AFTER ORAL ADMINISTRATION OF [ 1-dodecyZ-14C]DDAOa
Tissue
Percentage of dose*
Liver Intestines Stomach and esophagus Ovaries and tubules Kidney Lungs Spleen Heart Brain and spinal cord Pancreas Adrenals Capsule of eye Fluid of eye Lens of eye Carcass
1.48 0.48 0.18 0.12 0.09 0.06 0.02 0.02 0.02 0.02 0.008 0.002 0.0003 0.0003 4.82 Percentage of dose per gram
Leg muscle Subcutaneous fat Bone marrow Blood cells Blood plasma
0.013 0.13 0.07 0.01 0.019
’ Mean values, n = 4; SE of all means did not exceed 20% of corresponding mean values. *Amount of dose= 100mg of DDAO/kg; body weight =202 + 1.5 g; dose solution = 20 mg of DDAO/ml of water; sp act = mCi/g.
In humans, the absorption and subsequent excretion of radioactivity from a single oral dose of [l-dodecyZ-14C]DDA0 (Table 6) were rapid. In both subjects, the highest concentration of radioactivity was observed in blood samples taken 1 hr after dose administration; these levels for the two subjects were 5 x 10m4and 1.2 x 10e3 % of the dose per gram of whole blood (0.25 and 0.6 pg equivalent of DDAO/g). Comparison of data in Tables 1 and 6 shows that excretion of radioactivity in urine and expired gases is very similar for rats and humans. In the two human subjects investigated, 50 and 37% of the administered radioactivity appeared in the urine within 24 hr after dose administration. Expired 14C0, collected over this same interval contained 18 and 22 %
384
D. P. RICE
of the dosed radioactivity, respectively. The recovery of only 7680 % ofthe administered radioactivity from these subjects may be the result of the aliquot sampling procedure employed in measuring expired 14C02, loss of urine or fecal samples, or retention of radioactivity in the body beyond the collection period. 9-
a76-5 44 2’
32-
A’
(Total
.x0
lx0 I 1
0
14C02
excreted
over 72 hours = 7.37%) a
2
I
4
3
I
5
1
6
7
4
a
HOURS
ia 16 .1 14
1
I l-14C-dodecyl
E
I-DDAO
(n = 2, mean)
12 10
a 1i 14
(Total over
CO
72 hours
2
excreted = 15.5%)
l 0
I 1
2
3
4
5
6
I
7
a
HOURS
FIG. 2. The cumulative percentage of radioactivity excreted as %O, vs time after dosing. 14C was administered by intraperitoneal injection of [methyZ-14C]DDA0 and [I-dodecyl-%]DDAO to rats.
When administered alone in an aqueous solution, DDAO was absorbed through the intact skin of rats (Table 7). The amount of radioactivity found in the carcasses of rats dosed cutaneously with [methyZ-‘4C]DDA0 was greater than expected relative to the amounts of radioactivity found in excreta, liver, and kidney and was therefore probably due to external contamination of the carcasses by the cutaneous dose. Based on a comparison of the fractions of the cutaneous dose in excreta, the absorption of DDAO through skin was slightly, but not significantly, enhanced when it was applied in combina.tion with the anionic surfactant ammonium dodecyltriethoxysulfate (Table 7).
[14~]~~~~
ABSORPTION
BY MAN
TABLE EXCRETION OF SPRAGUE-DAWLEY
14C (MEAN RATS
AND
385
ANIMALS
5
IL SE) BY BILE-DUCT-CANNULATED, AFTER A SINGLE ORAL
DOSE
MALE OF
[rneth~&‘~ClDDAO” Percentage
of administered
O-24 hr Urine co2 Feces Bile Total excreted Carcass Total recovered
19.1 13.6 18.9 3.2 54.8
2448
+ 5.4 f 1.6 f 8.gd _+ 0.4
7.2 3.9 2.3 0.4 13.8
14C
hr
+ 2.5 zk 1.6 k 0.2 rf: 0.03
Total 37.8 17.9 21.2 3.6 80.0 14.8 95.6
+ f f f f + +
7.lb 1.8” 8.7” 0.4 9.0 5.8’ 5.4
a Three rats received a po dose of 40 mg of DDAO/kg; sp act of DDAO = 1.3 mCi/g; rat weight = 263 + 5.7. b Daily collection plus final cage washes. c Daily collection plus contents of safety trap. ’ High fecal value is attributable to a high fecal value for one rat caused by contamination of feces with urine under conditions of animal restraint. If high value is deleted, then fecal content is 12.0 + 4.5 (n = 2).
e Carcasses were contaminated by excretion products due to restraint of rats. TABLE PERCENTAGE OF THE URINE, FECES, AND
DOSED
6
RADIOACTIVITY DOSED
CO2 OF HUMANS [ 1-dodecyl-i4C]DDA0
APPEARING IN ORALLY WITH
Percentage of radioactivity Excretion
product
Urine 24 2448 48-72 72-144 Urine total co2 24 24-48 48-72 CO* total Feces total Total excretion
(hr)
Subject 1
dosed
Subject 2
49.8 6.2 0.8 0.5 57.3
36.8 6.0 0.7 0.4 43.9
18.16 1 ND“ 19.2 2.7 79.8
21.88 1 ND 22.9 2.5 69.3
0 ND, below detectable level (0.1%).
~
4.5 0.64 0.5 5.6
f + f f
0.3 0.02 0.08 0.3
O-24 hr ~_._
4.7 0.76 0.8 6.3
L- 1.0 _+ 0.1 f 0.10 T 0.9
24-48 hr 5.1 0.89 0.4 6.4
+ + 4 +
1.0 1.07 0.12 1.3
48-72 hr
r4Ca, DDAO
7
+ f + + + + & k f + +
1.9 0.3d 0.2 2.4 1.3’ 0.06 0.0 2.5f 2. 3.2 1.6
Total
AE,S-”
2.0 0.25 0.25 2.6
f 0.4 &- 0.03 + 0.13 + 0.5
O-24 hr
Percentage
DOSED
WITH
7.2 0.5 0.84 8.5
+ f + +
2448 1.13 0.07 0.1 1.2
hr
9.4 0.68 1.47 11.6
f 0.61 f 0.06 * 0.3 ~fr0.9
48-72 hr
-site.
Total ~~___ 18.7 + 1.9 I .6 + 0.2d 2.6 -I 0.3 22.8 j, 2.3 5.1 + 0.7’ 1.08 + 0.07 0.08 _+ 0.01 64.0 * 0.3 15.1 +0.3 56.6 + 3.3 89.5 + 1.3
with
[rneth~&‘~C]DDA
of administered 14C dose, DDAO AE3S-b,C
WASH OF MALE RATS CUTANEOUSLY THE ANIONIC SURFACTANT AE3S
14.2 2.5 1.8 18.5 6.1 0.44 0.05 15.6 16.1 48.0 88.8
without
CAGE WITH
TABLE
’ Four rats; rat weight = 188 + 8 g; application site area = 18 cm2; sp act = 1.3 mCi/g. Applied dose = 10 mg of DDAOiapplication site ’ Four rats; rat weight = 202 f 6 g; application site area = 18 cm*; sp act = 1.3 mCi/g. Applied dose = 10 mg of DDAO + 50 mg of AE3S/application ’ AE,S- = commercial grade product (Jefferson Chemical), (NH,) (CH,-(CH,),, - (OCH,-CH,),,,-OSO,-); average n = I I .7, average m = 3. d Total CO, includes radioactivity in COa safety trap. e Cage wash includes thorough wash of restrainer. / The high carcass value was probably caused by external contamination by the applied dose.
~-
of administered
+- SE) AMONG TISSUES, EXCRETA AND ALONE AND IN COMBINATION
Percentage
OF 14C (MEAN
Application site Total recovered
Kidney Carcass Total tissues
Total excreta Cage wash Liver
Feces
Tissue .___~ Urine co2
DISTRIBUTION
P .? s iz
[%]DDAO
ABSORPTION
BY MAN
AND
387
ANIMALS
Cutaneously administered [methyl-14C]DDA0 was also absorbed by mice and rabbits, as is shown in the comparative Table 8. Following a continuous 72 hr cutaneous exposure to [~ethylJ4C]DDA0, 35, 36, and 51 ‘A of the administered radioactivity were found in the excreta and body tissues (excluding the application site) of rats, mice, and rabbits, respectively. A small percentage of cutaneously applied [I-dodecyZ-‘4C]DDA0 was absorbed by humans, as indicated by the appearance of 0.01 and 0.23 % of the administered radioactivity in the excretion products of the two human subjects. Notably, more than 92 % of TABLE DISTRIBUTION
OF
8
(‘“C) OVER 72 HR AFTER CUTANEOUS [methyZP4C]DDA0 TO RATS, MICE, AND RABBITS”
RADIOACTIVITY
DOSING
OF
Percentage of dosed radioactivity Sample Urine Feces 14C0, Total excreted
Liver Kidney Testes
Carcass Tissue total Skin (application site) Cage wash Material balance Blood (lug/g) Dose (mg) Dose (pmol) Application site area (cm3)
b Concentration = 20 mg of DDAO/ml, c Concentration = 10 mg of DDAO/ml, * Concentration = 20 mg of DDAO/ml, e Radioactivity is equivalent to indicated
Ratb 14.2 1.75 2.52 18.5 0.44 0.05 0.04 15.55 16.08 48.0 6.1 89.0 0.43 10 42 18
Mouse’
+ 1.9 * 0.2 f 0.3 + k + f
0.06 0.06 0.01 2.50
+ 3.25 + 1.3 rk 1.6 rl: 0.08
n = 4, sp act n = 3, sp act n = 4, sp act micrograms
11.6 1.4 5.0 18.0 0.42 0.08 0.02 17.1 17.62 48.9 10.6 95.2 1.10 1 4.2 6
k 3.5 + 0.2 k 0.6 rt f f f
0.05 0.00 0.01 1.8
+ + & +
5.5 2.0 3.0 0.32
RabbV 42.1 2.22 1.4 45.7 0.44 0.1 0.001 4.61 5.1 39.4 3.9 94.4 co.03 10 42 40
+ 3.15 + 0.33 10.35 + + + jI
0.06 0.00 0.00 0.55
+ 4.1 f 1.1 + 1.6
= 1.3 mCi/g. = 1.3 mCi/g. = 1.3 mCi/g. of DDAO per gram of whole blood at 72 hr.
the applied dose could be recovered from the site by swabbing with moist gauze 8 hr after dose application. The tape strippings of the application site showed that the stratum corneum contained less than 0.2 % of the applied dose. Blood samples drawn during the 8-hr exposure and at selected times over the following 6 days contained no detectable radioactivity (less than 3.0 x 10es % of the applied dose/g of blood). If radioactivity from percutaneously absorbed [l-dodecyl-14C]DDA0 passes through the body in a manner similar to radioactivity from orally ingested [l-dodecyl-14C]DDA0, then the finding of 0.23 % of the dose in the urine of one subject indicates an absorption of no more than 0.33 % of the cutaneously applied dose by this subject.
388
D. P. RICE
The rate of DDAO penetration through the skin (Table 9) was calculated according to the formula : (Dose applied per unit skin area x percentage absorption)/time
of exposure.
The “percentage absorption” factor employed in these calculations (Table 9) is the percentage of the administered dose found in the excrement. Since we wished to include TABLE INTERSPECIES
Species Rat” MouseC Rabbitc Man
COMPARISON
OF THE PERCUTANEOUS ABSORPTION MOUSE, AND RABBIT
Dose (nmol of DDAO/cm’) 2.33 0.7 1.05 1.05 0.8
9
Length
of
exposure (hr) 72 72 24 72 8
OF
Percentage absorption” 18.5 IL-2.4 18.1 + 3.4 29.4 + 3.7 45.7 + 3.6 <0.2*
DDAO
IN MAN,
RAT,
Transcutaneous fluxb 6.0 1.7 12.8 6.7 <0.2
f 0.8 L- 0.3 + 1.6 + 0.5
a Percentage excreted over exposure, does not include body tissues or cage wash (mean f SE). b Flux units = nanomol per hour per squared centimeter = dose administered x percentage absorption per unit area per unit time (mean + SE). c For dosing conditions, see Table 8. * Based on assumption of maximum nondetectable amounts in all urine collections falling in ND range.
humans in these comparative calculations, we were unable to use percentage of radioactivity in excreta and body tissues as a percentage absorption factor. DISCUSSION
Orally administered DDAO was rapidly and extensively absorbed by ratsand humans, and the two species showed similar excretion patterns. Therefore, the rates of clearance of radioactivity from the tissues of rats may be reasonable indicators for analogous rates in humans. On this basis, the biological half-life of DDAO in humans would appear to be less than 12 hr. Limited data indicate that sex was not a significant variable in the metabolism of DDAO, since the absorption, tissue distribution, and excretion of radioactivity from orally administered [14C]DDA0 were quite similar for male and female rats. Percutaneous absorption of dermally applied DDAO occurred in rats, mice, rabbits, and humans. The rate of percutaneous penetration of DDAO in humans appeared to be significantly lower than in other species (Table 9). Computation of the rate of DDAO penetration (flux) through the skin for man, rat, mouse, and rabbit yielded values of <0.2,6.0, 1.7, and 6.7 nmol/hr/cm2, respectively. The DDAO flux through human skin appeared to be approximately one order of mangitude less than that calculated for the animals. In these experiments, the apparent rate of DDAO percutaneous absorption for
[14~]~~~~
ABSORPTION
BY MAN
AND
ANIMALS
389
humans may have been low because the total 8 hr exposure may have been too brief. In support of our observations showing lower rates of percutaeous penetration in humans, Tregear (1966) commented that flux rates for percutaneous penetration of chemicals are three to five times greater in rats and rabbits than in man. ACKNOWLEDGMENTS The author acknowledges the assistance of the following individuals: J. E. Thompson for preparation of the radioactive substances; J. D. Arnold, M.D. (Harry S. Truman Laboratory, Kansas City, Missouri)for collaboration in the human investigation : D. J. Ketterer for technical assistance in performing the animal studies; and T. L. Davis for assay of the radioactive samples. REFERENCES BUDNY, J. A. (1972). Metabolism and blood pressureeffects of disodium nitrilotriacetate
(Na2NTA) in dogs. Toxicol. Appl. Pharmacol.22,655-660. BUDNY, J. A., AND ARNOLD, J. D. (1973).Nitrilotriacetate (NTA): Human metabolismand its
importancein the total safety evaluation program. Toxicol. Appl. Pharmacol.25,48-53. NEWMANN, E. A. (1963).A new method for restrainingrabbits for percutaneousabsorption
studies.Lab. Anim. Care 13,207. KOLP, D. G., LAUGHLIN, R. G., KRAUSE, F. P., AND ZIMMERER, R. E. (1963).Interaction of
dimethyldodecylamineoxide with sodium dodecylbenzenesulfonatein dilute solution. J. Phys. Chem.67,51-55. TREGEAR, R. T. (1966).Physical Functionsof Skin, Vol. 5, pp. 12-13. Academic Press,New York.
AND
APPLIED
PHARMACOLOGY
39,377-389
(1977)
The Absorption, Tissue Distribution, and Excretion of Dodecyldimethylamine Oxide (DDAO) in Selected Animal Species and the Absorption and Excretion of DDAO in Man1 D. P. Rice The Procter & Gamble Company, Sharon Woods Technical Center, Cincinnati, Ohio 45241 Received May 26,1976; accepted September 9,1976
The Absorption, Tissue Distribution, and Excretion of DodecyldimethylamineOxide (DDAO) in SelectedAnimal Speciesandthe Absorption and Excretion of DDAO in Man. RICE, D. P. (1977). Tuxicol. Appl. Pharmacol. 39, 377-389.The absorption tissuedistribution, and excretion pattern of [methyl-14C]DDA0 and [I-dodecyZJ4C]DDA0 administered orally or cutaneously to rats, mice, and rabbits were investigated. The excretion pattern of radioactivity from [I-dodec~&‘~ClDDAO administered orally and cutaneouslyto man wasalsoinvestigated.An oral doseof DDAO is rapidly and extensivelyabsorbedand excretedby rats and man. Peak tissuelevels of radioactivity resulting from oral administration of [methyl-14C]DDA0 to rats occur within 1 hr after dosing. Cutaneously administeredDDAO is absorbedby man, rats, rabbits, and mice.In man, the rate of DDAO absorption through the skin is at least one order of magnitudelessthan that observedin rats, mice,and rabbits. Dodecyldimethylamine oxide (DDAO) is a semipolar surfactant used primarily in light-duty liquid detergents. In view of the potential ingestion or dermal exposure to DDAO through product use, it wasappropriate to establishwhether dermally or orally administered DDAO is absorbed, how any DDAO that is abosrbed is distributed among the body’s tissues,and how it is excreted. To these ends, the following human and animal experiments were performed. [mefh,vl-14C]- or [l-dodecyl-14C]DDA0 was applied to the skin of rats, mice, and rabbits, and the absorption of DDAO was characterized through assay of the radioactivity appearing in tissues and excrement after 72 hr of exposure. [mefhyl-‘“CIDDAO and [l-dodec~l-14C]DDA0 were administered orally to male and female rats and the disposition of radioactivity was determined. [l-dodecyZ-‘4C]DDA0 was administered orally or dermally to human subjects, and the extent of absorption was determined through the appearance of radioactivity in excretion products during the next 144 hr. METHODS Materials
[metl~~&‘~C]DDA0 and [l-dodecyl-‘4C]DDA0 were prepared by oxidation of the appropriate tertiary amine with hydrogen peroxide (Kolp ef al., 1963). [me?&Z-14C]1 Presented, in part, at the 14th Annual Meeting of the Society of Toxicology, Williamsburg, March 1975. 377 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction Printed in Great Britain
in any form
reserved.
Virginia,
1SSN 0041-008X
378
D. P. RICE
Dodecyldimethylamine (DDA) was prepared by reaction of I-bromododecane with [methyZ-14C]dimethylamine. [I-dodec.rl-14C]DDA0 was similarly prepared from l-[l-‘4C]bromododecane and dimethylamine. The chemical and radiochemical purity of these substances was shown to be greater than 98.5 % by thin layer and high pressure liquid chromatographic procedures (T. Turan, private communication). Animal Dosing and Handling Procedures
Rats were given oral doses of DDAO in several experiments; the compositions and quantity of the doses are shown in the footnotes to Tables 1-5. The amount of each dose was determined by weighing. The doses were administered intragastrically with a flexible rubber tube fitted to a glass syringe. Rats, rabbits, and mice were prepared for dermal dosing by clipping an area on the back free of hair with a small animal clipper. The clipped area was inspected for breaks in the skin, and the application site was delineated by marking with a felt-tipped pen. The dermal dose was administered from a syringe fitted with a blunt needle with lateral openings. Dermally dosed rats and mice were restrained in small animal restrainers, and rabbits were fitted with collars (Newman, 1963) to restrict oral ingestion of the applied dose. Intraperitoneal doses of [methyZ-‘4C]DDA0 were administered to rats by injection in the lower left quadrant of the abdomen. Four rats received intraperitoneally doses of 22 mg of DDAO/kg (sp act, 1.3 mCi/g) and their excreta and tissues were analyzed for radioactivity. Immediately after dosing, the animals were placed in individual, stainless steel metabolism cages for collection of expired carbon dioxide, urine, and feces. Except as otherwise noted, all experiments lasted 72 hr. Biliary excretion of radioactivity from orally ingested [meth$-14C]DDA0 was investigated in rats. The rats were prepared by surgical insertion of a biliary fistula under pentobarbital anesthesia. A dose of 100 mg of [methyZ-‘4C]DDAO/kg was administered by stomach intubation 1 day after the surgical procedure, and the bile and other excreta were collected for 72 hr. The bile-duct-cannulated rats were kept in small animal restrainers during the predose and excreta collection periods. Because of the design of the restrainers, feces frequently accumulated within them, causing cross-contamination of urine, feces, and carcass residues. Human Metabolism Procedures Generalprocedures. The subjects employed were volunteers who remained at the test facility throughout the course of the study. Each subject was given a complete physical examination before and after the study. The physical examination included clinical chemistry, ophthalmoscopic eye examination, EKG, hematology, and blood pressure. Recommended procedures for informed consent and experimental review were followed. Before dosing, subjects fasted for 12 hr. Zero time blood samples were taken immediately before dosing. Procedures for oral dosing of human subjects. Each subject drank 200 ml of an aqueous solution that contained 50 mg of [ I-dodecyl-r4C]DDA0 (100 &i of 14C). The
[i40]DD~0 AB~OR~JTIONBY bi.4~ AND ANIMALS plastic dosing cup was immediately subject drank the rinsings.
379
rinsed twice with 200 ml of tap water, and the
Procedures for cutaneous dosing qf human subjects. An area 4 x 15 cm was marked off on the outer surface of the subjects’ forearms, which had been clipped free of hair and inspected for breaks. One-half milliliter of an aqueous solution containing 10 mg of [1-dodecyZ-14C]DDA0 (100 @i of 14C) was applied evenly over the site from a plastic syringe. After the site had dried by evaporation, it was covered with a nonocclusive, plastic shield. After 8 hr, the DDAO remaining at the application site was removed by repeatedly (10x) swabbing the skin with water-moistened gauze pads. The portion of the radioactive dose retained in the stratum corneum was assayed by repeatedly (10x) stripping a 7.2-cm2 area within the area application with cellophane tape. Normal handling and exposure of the application site were permitted after these procedures. Collection of human excreta. Urine was collected in polyethylene bottles over the intervals O-6 and 6-24 hr and subsequently over 24 hr intervals through 144 hr. During the collection period, samples were stored at 0°C; upon completion of a collection interval, the urine samples were frozen on dry ice. Fecal samples were collected individually as available in plastic bags and were stored frozen. Expired CO2 was collected in a modified Hanks (Budny and Arnold, 1973) apparatus. The air-flow rate through the collection box was 12 liters/min. Carbon dioxide was collected from the orally dosed subjects for 15 min at 0, 1,2,3,4,5,6,8, l&24,36,48, and 72 hr after dosing. Collections on dermally dosed subjects were made for 30 min at 0, 2, 4, 6, 8, 12, 24, 36, 48, and 72 hr after dosing. Total expired radioactivity was computed from these samples of expired gases. Radiochemistry
The 14C in the excretion products and tissue samples was assayed by standard radiochemical techniques as described by Budny (1972). RESULTS An oral dose of [methyl-14C]- or [l-dodecyl-‘4C]DDA0 was absorbed extensively and rapidly by rats (Tables 1 and 2, Fig. 1). In male and female rats, the fraction and distribution of administered radioactivity excreted during the first 48 hr after dosing were similar. Further, although male rats were sacrificed 72 hr after dosing (Table 3) and female rats were sacrificed 48 hr after dosing, the distribution among body tissues of radioactivity from an oral dose of [l-dodecyl-‘4C]DDA0 was similar for male and female rats. Of the various tissues analyzed, the greatest amount and the highest concentration of radioactivity were found in the liver (Tables 3 and 4). As Fig. 1 indicates, the fractions of dosed radioactivity appearing in the liver, kidney, and blood reached maxima within 1 hr after an oral dose of [methyZ-14C]DDA0. The excretion of radioactivity from orally administered DDAO was rapid. Within 24 hr after dosing, 73.9 and 76.4% of the radioactivity from orally dosed [methyl-‘4C]DDAO and [l-dodecyl-14C]DDA0, respectively, were excreted by male rats. More than two-thirds of the radioactivity excreted as expired 14C02 appeared within 12 hr after oral administration of either [methyZ-14C] or [ l-dodecyl-14C]DDA0. Notably, the
380
D. P. RICE
TABLE EXCRETION
OF 14C (MEAN
+ SE) BY MALE
1
SPRAGUE-DAWLEY OF
RATS AFTER A SINGLE
ORAL
DOSE
[‘4C]DDAO” Percentage of administered dose
[methyZ-‘4C]DDA0 Urine co2 Feces Total excreted Tissues + carcass Total recovered [ 1-do&$-14C]DDA0 Urine co2 Feces
Total excreted Tissues + carcass Total recovered
O-24 hr
24-48 hr
48-72 hr
55.3 f 4.5 10.4 + 0.6 8.1 f 0.7 73.9
8.5 i 1.8 1.2kO.2 2.8 +- 0.8 12.5
3.0 + 0.6 0.7 + 0.1 1.2 + 0.5 4.9
42.1 + 9.1 19.8 k 0.6 5.4 + 1.8 67.4
6.6 f 1.7 1.2f0.16 2.6+ 1.3 10.4
2.2 + 0.8 0.5 + 0.1 1.o + 0.5 3.8
Total
71.0+ 4.5b 13.1 + 1.1’ 12.1 + l.8d
96.2+ 3.7 6.1 i- 0.3 102.3+ 6.7 53.5+ 6.9b 23.12 0.63” 9.4 k 3.06 86.0& 3.7 3.5 + 0.83 89.5-f. 1.1
’ [methyl-“C]DDAO, 100mgof DDAO/kg, spact= 1.3mCi/g; rat weight= 204.8+ 4, four rats. [I-dodecyl-“CIDDAO, 100mgof DDAO/kg, spact = 1mCi/g; rat weight= 184.8+ 5, four rats. b Daily ’ Daily
collections
plus final
cage wash.
collections plus contents of safety traps. d Daily collections plus contents of GI tract at sacrifice.
TABLE 2 EXCRETION OF DURING THE 48-~~
14C (MEAN
+ SE) BY FEMALE SPRAGUE-DAWLEY PERIOD AFTER ADMINISTRATION OF A SINGLE ORAL OF [ 1-dodecyl-14C]DDAO”
RATS DOSE
Percentageof administeredr4C
Urine co2 Feces Total excretion Carcass+ tissues Total recovered
O-24 hr
2448 hr
Total
31.7? 7.8 20.1i- 0.9 7.3 f 1.2 59.1
9.6 + 4.7 2.0 * 0.4 3.7 * 1.0 14.8
53.6+ 3.0b 23.4 _+1.2’ 12.0+ 1.6d 89.1+ 1.4 8.0 * 0.9 96.5 &-1.O
’ Four female rats, body weight = 202 + 1.5 g; DDAO
DDAO/kg, spact= &i/mg. bDaily collections plustotal cagewash. ’ Daily collections plus contents of safety trap. * Daily collections plus wash of GI tract at sacrifice.
dose = 100 mg of
[14C]DDA0
ABSORPTION
20 -
BY MAN
AND
ANIMALS
381
LIVER
16 12 - x X = Absorbed 8 -’
‘< ,
y
x x j( x
40.
level
in individual
x x
I
n
.o -
c
x
.6
-
‘.2
-x
rat
KIDNEY
.d
x
x
X = Absorbed x x xx :< x
x .4-x
x
0.
I
level
in individual
I
I
rat
3 ,
.5 TESTES .4 X = Absorbed .3
-
level
in individual
rat
level
in individual
rat
#
.O< .I2
-
.08
-
x
WHOLE
x
X = Absorbed
-x .04-
BLOOD
x x
x x
x
X
X
x 0.
1 2
I 4
x 8 6
8
10
12
14
f 16
18
20
22
I 24
HOURS FIG. 1. Variation in the percentage of dosed radioactivity present in liver, kidney, testes, and whole blood with time elapsed after oral dosing with (merhyPC]DDAO in rats (indicated points represent individual values).
382
D. P. RICE
percentage of administrated radioactivity excreted as 14C0, by rats dosed po with [methyl-14C]DDA0 was consistently greater than that appearing in expired 14C02 from rats dosed po with [mer/qG4C]DDA0. An average of 12.1 or 9.4 % of the radioactivity administered po as [methyl-14C]- or [l-dodecyl-i4C]DDA0,
respectively,
the bile-duct-cannulated
was excreted in the feces. Although
rats resulted in extensive cross-contamination TABLE
DISTRIBUTION AFTER ORAL
OF RADIOACTIVITY ADMINISTRATION
restraint
of
of radioactivity
3 IN TISSUES OF MALE RATS OF [mefhyZ-‘4ClDDA0
72 HR AND
[ 1-dodecyl-14C]DDAO’ Percentageof dose Tissue
methylJ4Cb
Liver Intestines Stomachand esophagus Testes Kidney Lungs Spleen Heart Brain and spinalcord Pancreas Carcass
1-dodecyl-14Cb
1.25 0.33 0.098 0.12 0.09 0.048 0.031 0.023 0.022 0.02 3.95
1.1
0.4 0.4 0.07 0.08 0.04 -c -c 0.02 -c 1.9
Percentageof doseper gram Leg muscle Subcutaneousfat Bone marrow Whole blood
0.014 0.014 0.04 0.013
0.013 0.062 0.05 0.015
’ Mean,n = 4; SEof all means did not exceed 20% of corresponding mean values. bAmountof dose= 20mgof DDAO (100mgof DDAOjkg bodywt);
concentration = 20 mgof DDAO/ml in water; spact of [methyl-Y]DDAO
= 1.3 mCi/g,
c Not analyzd
[I-dodecyf-Y]DDAO
= 1 mCi/g.
separately.
in urine, feces, and carcass, the data in Table 5 indiate that the bile is a minor excretory pathway for the radioactive compound administered orally as [methyl-14C]DDA0, since bile excreted over the 48 hr period after dosing contained less than 4% of the administered dose. Intraperitoneally administered [methyl-“C]DDAO was also excreted rapidly by rats. Over the first 24 hr after dosing, 67, 8, and 6% of the radioactivity from a dose of [methyl-14C]DDA0 administered ip were excreted in the urine, expired 14C02, and
feces, respectively. The rate of 14C0, production
was initially
rapid, but this excretion
[14~]~~~~
ABSORPTION
BY MAN
AND
ANIMALS
383
rate declined to a low value within 8 hr after dosing (Fig. 2). No significant differences are observed between the distributions of radioactivity among the urine, feces, and 14C02 when results for intragastric and ip administration of [methyl-14C]DDA0 in rats are compared. This suggests that microbial metabolism by gastrointestinal flora does not play a major role in the absorption and excretion of DDAO in rats. TABLE 4 DISTRIBUTION OF RADIOACTIVITY IN THE TISSUES OF FEMALE RATS 48 HR AFTER ORAL ADMINISTRATION OF [ 1-dodecyZ-14C]DDAOa
Tissue
Percentage of dose*
Liver Intestines Stomach and esophagus Ovaries and tubules Kidney Lungs Spleen Heart Brain and spinal cord Pancreas Adrenals Capsule of eye Fluid of eye Lens of eye Carcass
1.48 0.48 0.18 0.12 0.09 0.06 0.02 0.02 0.02 0.02 0.008 0.002 0.0003 0.0003 4.82 Percentage of dose per gram
Leg muscle Subcutaneous fat Bone marrow Blood cells Blood plasma
0.013 0.13 0.07 0.01 0.019
’ Mean values, n = 4; SE of all means did not exceed 20% of corresponding mean values. *Amount of dose= 100mg of DDAO/kg; body weight =202 + 1.5 g; dose solution = 20 mg of DDAO/ml of water; sp act = mCi/g.
In humans, the absorption and subsequent excretion of radioactivity from a single oral dose of [l-dodecyZ-14C]DDA0 (Table 6) were rapid. In both subjects, the highest concentration of radioactivity was observed in blood samples taken 1 hr after dose administration; these levels for the two subjects were 5 x 10m4and 1.2 x 10e3 % of the dose per gram of whole blood (0.25 and 0.6 pg equivalent of DDAO/g). Comparison of data in Tables 1 and 6 shows that excretion of radioactivity in urine and expired gases is very similar for rats and humans. In the two human subjects investigated, 50 and 37% of the administered radioactivity appeared in the urine within 24 hr after dose administration. Expired 14C0, collected over this same interval contained 18 and 22 %
384
D. P. RICE
of the dosed radioactivity, respectively. The recovery of only 7680 % ofthe administered radioactivity from these subjects may be the result of the aliquot sampling procedure employed in measuring expired 14C02, loss of urine or fecal samples, or retention of radioactivity in the body beyond the collection period. 9-
a76-5 44 2’
32-
A’
(Total
.x0
lx0 I 1
0
14C02
excreted
over 72 hours = 7.37%) a
2
I
4
3
I
5
1
6
7
4
a
HOURS
ia 16 .1 14
1
I l-14C-dodecyl
E
I-DDAO
(n = 2, mean)
12 10
a 1i 14
(Total over
CO
72 hours
2
excreted = 15.5%)
l 0
I 1
2
3
4
5
6
I
7
a
HOURS
FIG. 2. The cumulative percentage of radioactivity excreted as %O, vs time after dosing. 14C was administered by intraperitoneal injection of [methyZ-14C]DDA0 and [I-dodecyl-%]DDAO to rats.
When administered alone in an aqueous solution, DDAO was absorbed through the intact skin of rats (Table 7). The amount of radioactivity found in the carcasses of rats dosed cutaneously with [methyZ-‘4C]DDA0 was greater than expected relative to the amounts of radioactivity found in excreta, liver, and kidney and was therefore probably due to external contamination of the carcasses by the cutaneous dose. Based on a comparison of the fractions of the cutaneous dose in excreta, the absorption of DDAO through skin was slightly, but not significantly, enhanced when it was applied in combina.tion with the anionic surfactant ammonium dodecyltriethoxysulfate (Table 7).
[14~]~~~~
ABSORPTION
BY MAN
TABLE EXCRETION OF SPRAGUE-DAWLEY
14C (MEAN RATS
AND
385
ANIMALS
5
IL SE) BY BILE-DUCT-CANNULATED, AFTER A SINGLE ORAL
DOSE
MALE OF
[rneth~&‘~ClDDAO” Percentage
of administered
O-24 hr Urine co2 Feces Bile Total excreted Carcass Total recovered
19.1 13.6 18.9 3.2 54.8
2448
+ 5.4 f 1.6 f 8.gd _+ 0.4
7.2 3.9 2.3 0.4 13.8
14C
hr
+ 2.5 zk 1.6 k 0.2 rf: 0.03
Total 37.8 17.9 21.2 3.6 80.0 14.8 95.6
+ f f f f + +
7.lb 1.8” 8.7” 0.4 9.0 5.8’ 5.4
a Three rats received a po dose of 40 mg of DDAO/kg; sp act of DDAO = 1.3 mCi/g; rat weight = 263 + 5.7. b Daily collection plus final cage washes. c Daily collection plus contents of safety trap. ’ High fecal value is attributable to a high fecal value for one rat caused by contamination of feces with urine under conditions of animal restraint. If high value is deleted, then fecal content is 12.0 + 4.5 (n = 2).
e Carcasses were contaminated by excretion products due to restraint of rats. TABLE PERCENTAGE OF THE URINE, FECES, AND
DOSED
6
RADIOACTIVITY DOSED
CO2 OF HUMANS [ 1-dodecyl-i4C]DDA0
APPEARING IN ORALLY WITH
Percentage of radioactivity Excretion
product
Urine 24 2448 48-72 72-144 Urine total co2 24 24-48 48-72 CO* total Feces total Total excretion
(hr)
Subject 1
dosed
Subject 2
49.8 6.2 0.8 0.5 57.3
36.8 6.0 0.7 0.4 43.9
18.16 1 ND“ 19.2 2.7 79.8
21.88 1 ND 22.9 2.5 69.3
0 ND, below detectable level (0.1%).
~
4.5 0.64 0.5 5.6
f + f f
0.3 0.02 0.08 0.3
O-24 hr ~_._
4.7 0.76 0.8 6.3
L- 1.0 _+ 0.1 f 0.10 T 0.9
24-48 hr 5.1 0.89 0.4 6.4
+ + 4 +
1.0 1.07 0.12 1.3
48-72 hr
r4Ca, DDAO
7
+ f + + + + & k f + +
1.9 0.3d 0.2 2.4 1.3’ 0.06 0.0 2.5f 2. 3.2 1.6
Total
AE,S-”
2.0 0.25 0.25 2.6
f 0.4 &- 0.03 + 0.13 + 0.5
O-24 hr
Percentage
DOSED
WITH
7.2 0.5 0.84 8.5
+ f + +
2448 1.13 0.07 0.1 1.2
hr
9.4 0.68 1.47 11.6
f 0.61 f 0.06 * 0.3 ~fr0.9
48-72 hr
-site.
Total ~~___ 18.7 + 1.9 I .6 + 0.2d 2.6 -I 0.3 22.8 j, 2.3 5.1 + 0.7’ 1.08 + 0.07 0.08 _+ 0.01 64.0 * 0.3 15.1 +0.3 56.6 + 3.3 89.5 + 1.3
with
[rneth~&‘~C]DDA
of administered 14C dose, DDAO AE3S-b,C
WASH OF MALE RATS CUTANEOUSLY THE ANIONIC SURFACTANT AE3S
14.2 2.5 1.8 18.5 6.1 0.44 0.05 15.6 16.1 48.0 88.8
without
CAGE WITH
TABLE
’ Four rats; rat weight = 188 + 8 g; application site area = 18 cm2; sp act = 1.3 mCi/g. Applied dose = 10 mg of DDAOiapplication site ’ Four rats; rat weight = 202 f 6 g; application site area = 18 cm*; sp act = 1.3 mCi/g. Applied dose = 10 mg of DDAO + 50 mg of AE3S/application ’ AE,S- = commercial grade product (Jefferson Chemical), (NH,) (CH,-(CH,),, - (OCH,-CH,),,,-OSO,-); average n = I I .7, average m = 3. d Total CO, includes radioactivity in COa safety trap. e Cage wash includes thorough wash of restrainer. / The high carcass value was probably caused by external contamination by the applied dose.
~-
of administered
+- SE) AMONG TISSUES, EXCRETA AND ALONE AND IN COMBINATION
Percentage
OF 14C (MEAN
Application site Total recovered
Kidney Carcass Total tissues
Total excreta Cage wash Liver
Feces
Tissue .___~ Urine co2
DISTRIBUTION
P .? s iz
[%]DDAO
ABSORPTION
BY MAN
AND
387
ANIMALS
Cutaneously administered [methyl-14C]DDA0 was also absorbed by mice and rabbits, as is shown in the comparative Table 8. Following a continuous 72 hr cutaneous exposure to [~ethylJ4C]DDA0, 35, 36, and 51 ‘A of the administered radioactivity were found in the excreta and body tissues (excluding the application site) of rats, mice, and rabbits, respectively. A small percentage of cutaneously applied [I-dodecyZ-‘4C]DDA0 was absorbed by humans, as indicated by the appearance of 0.01 and 0.23 % of the administered radioactivity in the excretion products of the two human subjects. Notably, more than 92 % of TABLE DISTRIBUTION
OF
8
(‘“C) OVER 72 HR AFTER CUTANEOUS [methyZP4C]DDA0 TO RATS, MICE, AND RABBITS”
RADIOACTIVITY
DOSING
OF
Percentage of dosed radioactivity Sample Urine Feces 14C0, Total excreted
Liver Kidney Testes
Carcass Tissue total Skin (application site) Cage wash Material balance Blood (lug/g) Dose (mg) Dose (pmol) Application site area (cm3)
b Concentration = 20 mg of DDAO/ml, c Concentration = 10 mg of DDAO/ml, * Concentration = 20 mg of DDAO/ml, e Radioactivity is equivalent to indicated
Ratb 14.2 1.75 2.52 18.5 0.44 0.05 0.04 15.55 16.08 48.0 6.1 89.0 0.43 10 42 18
Mouse’
+ 1.9 * 0.2 f 0.3 + k + f
0.06 0.06 0.01 2.50
+ 3.25 + 1.3 rk 1.6 rl: 0.08
n = 4, sp act n = 3, sp act n = 4, sp act micrograms
11.6 1.4 5.0 18.0 0.42 0.08 0.02 17.1 17.62 48.9 10.6 95.2 1.10 1 4.2 6
k 3.5 + 0.2 k 0.6 rt f f f
0.05 0.00 0.01 1.8
+ + & +
5.5 2.0 3.0 0.32
RabbV 42.1 2.22 1.4 45.7 0.44 0.1 0.001 4.61 5.1 39.4 3.9 94.4 co.03 10 42 40
+ 3.15 + 0.33 10.35 + + + jI
0.06 0.00 0.00 0.55
+ 4.1 f 1.1 + 1.6
= 1.3 mCi/g. = 1.3 mCi/g. = 1.3 mCi/g. of DDAO per gram of whole blood at 72 hr.
the applied dose could be recovered from the site by swabbing with moist gauze 8 hr after dose application. The tape strippings of the application site showed that the stratum corneum contained less than 0.2 % of the applied dose. Blood samples drawn during the 8-hr exposure and at selected times over the following 6 days contained no detectable radioactivity (less than 3.0 x 10es % of the applied dose/g of blood). If radioactivity from percutaneously absorbed [l-dodecyl-14C]DDA0 passes through the body in a manner similar to radioactivity from orally ingested [l-dodecyl-14C]DDA0, then the finding of 0.23 % of the dose in the urine of one subject indicates an absorption of no more than 0.33 % of the cutaneously applied dose by this subject.
388
D. P. RICE
The rate of DDAO penetration through the skin (Table 9) was calculated according to the formula : (Dose applied per unit skin area x percentage absorption)/time
of exposure.
The “percentage absorption” factor employed in these calculations (Table 9) is the percentage of the administered dose found in the excrement. Since we wished to include TABLE INTERSPECIES
Species Rat” MouseC Rabbitc Man
COMPARISON
OF THE PERCUTANEOUS ABSORPTION MOUSE, AND RABBIT
Dose (nmol of DDAO/cm’) 2.33 0.7 1.05 1.05 0.8
9
Length
of
exposure (hr) 72 72 24 72 8
OF
Percentage absorption” 18.5 IL-2.4 18.1 + 3.4 29.4 + 3.7 45.7 + 3.6 <0.2*
DDAO
IN MAN,
RAT,
Transcutaneous fluxb 6.0 1.7 12.8 6.7 <0.2
f 0.8 L- 0.3 + 1.6 + 0.5
a Percentage excreted over exposure, does not include body tissues or cage wash (mean f SE). b Flux units = nanomol per hour per squared centimeter = dose administered x percentage absorption per unit area per unit time (mean + SE). c For dosing conditions, see Table 8. * Based on assumption of maximum nondetectable amounts in all urine collections falling in ND range.
humans in these comparative calculations, we were unable to use percentage of radioactivity in excreta and body tissues as a percentage absorption factor. DISCUSSION
Orally administered DDAO was rapidly and extensively absorbed by ratsand humans, and the two species showed similar excretion patterns. Therefore, the rates of clearance of radioactivity from the tissues of rats may be reasonable indicators for analogous rates in humans. On this basis, the biological half-life of DDAO in humans would appear to be less than 12 hr. Limited data indicate that sex was not a significant variable in the metabolism of DDAO, since the absorption, tissue distribution, and excretion of radioactivity from orally administered [14C]DDA0 were quite similar for male and female rats. Percutaneous absorption of dermally applied DDAO occurred in rats, mice, rabbits, and humans. The rate of percutaneous penetration of DDAO in humans appeared to be significantly lower than in other species (Table 9). Computation of the rate of DDAO penetration (flux) through the skin for man, rat, mouse, and rabbit yielded values of <0.2,6.0, 1.7, and 6.7 nmol/hr/cm2, respectively. The DDAO flux through human skin appeared to be approximately one order of mangitude less than that calculated for the animals. In these experiments, the apparent rate of DDAO percutaneous absorption for
[14~]~~~~
ABSORPTION
BY MAN
AND
ANIMALS
389
humans may have been low because the total 8 hr exposure may have been too brief. In support of our observations showing lower rates of percutaeous penetration in humans, Tregear (1966) commented that flux rates for percutaneous penetration of chemicals are three to five times greater in rats and rabbits than in man. ACKNOWLEDGMENTS The author acknowledges the assistance of the following individuals: J. E. Thompson for preparation of the radioactive substances; J. D. Arnold, M.D. (Harry S. Truman Laboratory, Kansas City, Missouri)for collaboration in the human investigation : D. J. Ketterer for technical assistance in performing the animal studies; and T. L. Davis for assay of the radioactive samples. REFERENCES BUDNY, J. A. (1972). Metabolism and blood pressureeffects of disodium nitrilotriacetate
(Na2NTA) in dogs. Toxicol. Appl. Pharmacol.22,655-660. BUDNY, J. A., AND ARNOLD, J. D. (1973).Nitrilotriacetate (NTA): Human metabolismand its
importancein the total safety evaluation program. Toxicol. Appl. Pharmacol.25,48-53. NEWMANN, E. A. (1963).A new method for restrainingrabbits for percutaneousabsorption
studies.Lab. Anim. Care 13,207. KOLP, D. G., LAUGHLIN, R. G., KRAUSE, F. P., AND ZIMMERER, R. E. (1963).Interaction of
dimethyldodecylamineoxide with sodium dodecylbenzenesulfonatein dilute solution. J. Phys. Chem.67,51-55. TREGEAR, R. T. (1966).Physical Functionsof Skin, Vol. 5, pp. 12-13. Academic Press,New York.