United States
Environmental Protection
Agency
Toxic Substances
Office of
Toxic Substances
Washington DC 20460
EPA-560/5-86-011
January, 1986
Dermal Absorption of
"C-Labeled
4,4' -Methylenedianiline
(4,4-MDA)
in Rats, Guinea Pigs,
and Monkeys
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DERMAL ABSORPTION OF 14C-LABELED 4,4'-METHYLENEDIANILINE (4,4'-MDA)
IN RATS, GUINEA PIGS, AND MONKEYS
by
Monaem El-hawari, Maxine Stoltz, Diane Czarnecki, and Patricia Aim
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
FINAL REPORT
January 15, 1986
EPA Prime Contract No. 68-02-3938
Work Assignment No. 21
MRI Project No. 8501-A(21)
For
U.S. Environmental Protection Agency
Office of Pesticides and Toxic Substances
Field Studies Branch, TS-798
401 M Street, SW
Washington, DC 20460
Attn: Dr. Joseph Breen, Project Officer
Ms. Janet Remmers, Work Assignment Manager
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DISCLAIMER
This document has been reviewed and approved for publication by the
Office of Toxic Substances, Office of Pesticides and Toxic Substances, U.S.
Environmental Protection Agency. The use of trade names or commerical products
does not constitute Agency endorsement or recommendation for use.
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PREFACE
This report includes the results of studies conducted to assess the
percutaneous absorption of 4, 4' -methyl enedi aniline (4,4'-MDA) and to examine
the effect of several factors on the dermal penetration process. These studies
were conducted under MRI Project No. 8501-A, Work Assignment No. 21, for EPA's
Office of Toxic Substances (EPA Prime Contract No. 68-02-3938). These studies
were performed by Monaem El-hawari (Study Director), Maxine Stoltz, Patricia
Aim, and Diane Czarnecki with assistance from Edward Williams, Jack Hoi comb,
Cristin Mansfield, and Kathy Howe. This report incorporates changes in re-
.sponse to reviewers' comments on the draft final report dated June 28, 1985.
The final report was submitted on December 30, 1985.
MIDWEST RESEARCH INSTITUTE
Monaem El-hawari
Task Leader
John E. Going
Program Manager
m
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QUALITY ASSURANCE STATEMENT
DERMAL ABSORPTION OF 14OLABELED 4,4'-METHYLENEDIANILINE
(4,4'-MDA) IN RATS, GUINEA PIGS, AND MONKEYS
This study was inspected by the Quality Assurance Unit as follows:
Phase Date
Guinea pig quarantine , 5/23/84
Rat receipt and quarantine 5/23/84
Animal quarters environmental check 5/24/84
Guinea pig, rat quarantine 6/28/84
Tissue oxidizer operation 7/26/84
Rat, dermal application 8/16/84
Monkey, environmental conditions 8/29/84
Monkey, dermal application 10/10/84
Guinea pig, i.v., dermal application 10/15/84
Guinea pig, dermal application 10/29/84
Monkey, urine, feces collection 12/3/84
Monkey, dermal, i.v. dosing 1/16/85
;Data audit 3/12/85
Final report review 6/25/85
Reports were submitted to the study director and management on
June 4, 1984, November 27, 1984, and March 12, 1985. This study was done
in compliance with the EPA Good Laboratory Practice Standards of November 28,
1983 (Federal Register 48:53922-53944). The methods described were the
methods followed and the data presented accurately represent the data
generated during the study.
The final report and all records are stored in the MRI Archives.
Manager, Quality Assurance Unit
"IV
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EXECUTIVE SUMMARY
Dermal absorption is a prevalent route of exposure for many in-
dustrial chemicals. To assist in determining workplace exposure to 4,4'-
methylenedianiline (4,4'-MDA), an aromatic amine with known toxic and car-
cinogenic properties, studies were performed in male Fischer 344 rats,
Hartley guinea pigs, and Rhesus monkeys treated topically with 14C-labeled
4,4'-MDA. Conditions of treatment including dosage, concentration, dose
regimens, and occlusion were assessed. The studies in rats and guinea pigs
were performed at two dose levels: a low dose (~ 2 mg/kg) and a high dose
(•v 20 mg/kg). The monkeys were treated with the low dose only. The disposi-
tion of 4,4'-MDA was also examined following intravenous (i.v.) administra-
tion of the low dose to the three species. Excreta from rats and guinea pigs
were collected for 96 h, then the animals were sacrificed for tissue sampling.
The studies in monkeys were extended to 168 h and only excreta were sampled.
During 96 h after continuous dermal application of the low dose to
rats, 43% of dose was recovered in urine, 10% in feces, and 2% in tissues.
Only 25% of dose was removed by washing the skin with soap and water. The
remainder (26%) was recovered by skin extraction (methanol) and solubilizing
(perchloric acid and hydrogen peroxide). In rats treated dermally with the
low dose for 24 h then the dose removed, recoveries in excreta, tissue, and
the skin application areas were lower. In rats with the application areas
nonoccluded, only 7% of the low dose was recovered in excreta and tissue at
6 h, compared to 12% in rats with occluded skin. After continuous application
of the high dose, only 4.8 and 1.3% of dose were eliminated in the urine and
feces, respectively. About 0.4% was recovered in tissue, 62% in the skin wash
and 24% from the application area. Although the percent of dose absorbed de-
creased by increasing the dose, the total amounts absorbed (0.2-0.25 mg/rat)
remained similar after both doses.
In guinea pigs, 10% of the low dose was excreted in urine and 18%
in feces during 96 h following continuous dermal application. About 1% was
recovered in tissue, 41% in the skin wash and 29% from the application area.
Guinea pigs treated dermally with low doses that were washed at 24 h had lower
amounts of 14C in excreta, tissue, and skin application areas. In guinea pigs
with nonoccluded application areas, recoveries in excreta and tissue (3% in
6 h) were similar to those with occluded skin. After the high dose, recovery
in urine, feces, and tissue averaged 3, 4, and 1%, respectively; 70% was re-
covered in the dose wash and 14% was removed from the application area. Al-
though the percent of dose absorbed decreased following application of the
high dose, the amount absorbed (in ug/kg) was doubled. In monkeys, 19 and 2%
of the low doses were eliminated in urine and feces, respectively, during a
168 hr period. Under similar conditions of exposure (dermal application of
the low dose for 24 h followed by excreta collection for 96 h) comparable
absorption was demonstrated in guinea pigs and monkeys (~ 18%) and higher
absorption in rats (^ 43%).
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In rats treated i.v., most of the dose was eliminated by 24 h; re-
covery at 96 h averaged 67% in urine and 31% in feces. Most of the i.v.
doses were eliminated by 48 h in guinea pigs and monkeys. In guinea pigs,
35% of dose was excreted in urine and 57% in feces during a 96 h period.
Monkeys excreted 84% of dose in urine and 10% in feces during a 168 h period.
In rats, 14C in blood and tissue averaged 19% of dose at 6 h which declined
to 2% by 96 h. The highest levels were demonstrated in liver which was 5-9
times higher than blood. In guinea pigs, * 3% of the dose was recovered in
blood and tissue at 96 h. The highest content was demonstrated in the spleen
(6 times higher than blood) followed by liver. The data suggest considerable
elimination through the biliary route especially for guinea pigs.
When 4,4'-MDA applied to the skin of rats, guinea pigs, and monkeys
was washed immediately with soap or acetone solutions, as low as 53% of the
applied doses was recovered. A post-exposure wash at 6 h, 24 h, and 96 h re-
moved less of the applied material. The studies showed higher and more con-
sistent recoveries when the application areas were washed with soap and water
versus acetone and water. However, both washing methods were incapable of
removing all the applied material from the skin. Significant amounts of the
applied doses remained associated with the skin available for delayed absorp-
tion. In addition, acetone facilitated absorption. Since 4,4'-MDA associated
with the skin is available for systemic circulation, the extent of dermal
absorption in rats, guinea pigs, and monkeys should be considerably higher
than calculated from the excretory and tissue distribution data only.
VI
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TABLE OF CONTENTS
Preface iii
Quality Assurance Statement iv
Executive Summary v
List of Tables viii
I. Introduction 1
II. Background 2
III. Materials and Methods 5
A. Animals 5
B. Chemicals 5
C. Dosage and Treatment. . 7
D. Experimental Design 8
E. Sample Collection 9
F. Determination of Total Radioactivity 10
IV. Results 12
A. Dermal Washing Efficiency Studies 12
B. Preliminary Studies 13
C. Rat Studies 14
D. Guinea Pig Studies 17
E. Monkey Studies 19
V. Discussion 20
VI. Conclusions and Recommendations 23
VII. Summary 25
A. Objectives and Study Design 25
B. Dermal Washing Efficiency Studies 25
C. Rat Studies 26
D. Guinea Pig Studies 27
E. Monkey Studies 28
F. Conclusions 28
VII. References 36
Tables 1-38 39
Appendix I - Study Protocol 1-1
Appendix II - Dermal Washing Efficiency Studies, Individual
Animal Data II-l
Appendix III - Rat Studies, Individual Animal Data III-l
Appendix IV - Guinea Pig Studies, Individual Animal Data IV-1
Appendix V - Monkey Studies, Individual Animal Data V-l
Appendix VI - Synthesis of 14C-Labeled 4,4'-MDA VI-1
viv
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LIST OF TABLES
Table Page
A Dermal Washing Efficiency of 4,4'-MDA in Rats, Guinea
Pigs, and Monkeys 30
B Recovery of Radioactivity in Rats Treated i.v. or
Dermally with 4,4'-MDA : 30
C Recovery of Radioactivity in Rats Treated Dermally with
4,4'-MDA under Different Conditions 31
D Recovery of Radioactivity in Guinea Pigs Treated i.v.
or Dermally with 4,4'-MDA 31
E Recovery of Radioactivity in Guinea Pigs Treated Dermally
with 4,4'-MDA under Different Conditions 32
F Recovery of Radioactivity in Monkeys Treated i.v. or
Dermally with 4,4'-MDA 32
G Percutaneous Absorption of 4,4'-MDA Based on Urinary
Excretion Data Only 33
H Percutaneous Absorption of 4,4'-MDA Based on Recoveries in
Excreta, Tissue and Skin Application Areas 34
1 Dermal Washing Efficiency of 14C-Labeled 4,4'-MDA in
Male Fischer 344 Rats, Hartley Guinea Pigs and Rhesus
Monkeys 39
2 Urinary and Fecal Excretion of Radioactivity in Male
Fischer 344 Rats Treated Dermally or Intravenously with
14C-Labeled 4,4'-MDA (0.4 or 4.0 mg/Rat):
Preliminary Study 40
3 Radioactivity in Blood, Tissue, and Excreta of Male Fischer
344 Rats at 96 h Following a Dermal or Intravenous Dose
of 14C-Labeled 4,4'-MDA (0.4 or 4.0 mg/Rat):
Preliminary Study. 41
4 Urinary and Fecal Excretion of Radioactivity in Male
Hartley Guinea Pigs Treated Dermally or Intravenously
with 14C-Labeled 4,4'-MDA (1 or 10 mg/Guinea Pig):
Preliminary Study 42
5 Radioactivity in Blood, Tissue, and Excreta of Male Hartley
Guinea Pigs at 96 h Following a Dermal or Intravenous
Dose of 14C-Labeled 4,4'-MDA (1 or 10 mg/Guinea Pig):
Preliminary Study 43
vm
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LIST OF TABLES (continued)
Table Page
6 Urinary and Fecal Excretion of Radioactivity in Male
Fischer 344 Rats Treated Intravenously with 14C-Labeled
4,4'-MDA (0.4 rag/Rat) 44
7 Radioactivity in Blood, Tissue, and Excreta at 6, 24, and
96 h Following Intravenous Treatment of Male Fischer 344
Rats with 14C-Labeled 4,4'-MDA (0.4 mg/Rat) 45
8 Tissue-to-Blood Concentration Ratios from Male Fischer 344
Rats at 6, 24, or 96 h Following Intravenous Treatment
with 14C-Labeled 4,4'-MDA (0.4 mg/Rat) 46
9 Recovery of Radioactivity in Blood, Tissue, and Excreta at
6, 24, or 96 h Following Intravenous Treatment of Male
Fischer 344 Rats with 14C-Labeled 4,4'-MDA (0.4 mg/Rat). . 46
10 Urinary and Fecal Excretion of Radioactivity in Male
Fischer 344 Rats Treated Dermally with 14C-Labeled 4,4'-MDA
(0.4 mg/Rat) 47
11 Radioactivity in Blood, Tissue, and Excreta at 6, 24, and
96 h Following Dermal Treatment of Male Fischer 344 Rats
with 14C-Labeled 4,4'-MDA (0.4 mg/Rat) 48
12 Tissue-to-Blood Concentration Ratios from Male Fischer 344
Rats at 6, 24, or 96 h Following Dermal Treatment with
14C-Labeled 4,4'-MDA (0.4 mg/Rat) 49
13 Recovery of Radioactivity in Blood, Tissue, and Excreta at
6, 24, or 96 h Following Dermal Treatment of Male
Fischer 344 Rats with 14C-Labeled 4,4'-MDA (0.4 mg/Rat). . 49
14 Urinary and Fecal Excretion of Radioactivity Following
Dermal Application of 14C-Labeled 4,4'-MDA (0.4 mg/Rat)
to Male Fischer 344 Rats: Application Area Washed at
24 h 50
15 Radioactivity in Blood, Tissue, and Excreta of Male Fischer
344 Rats Treated Dermally with 14C-Labeled 4,4'-MDA
(0.4 mg/Rat): Nonoccluded Skin and Skin Washed at 24 h. . 51
16 Tissue-to-Blood Concentration Ratios from Male Fischer 344
Rats Following Dermal Treatment with 14C-Labeled 4,4'-MDA
(0.4 mg/Rat): Nonoccluded Skin and Skin Washed at 24 h. . 52
ix.
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LIST OF TABLES (continued)
Table Page
17 Recovery of Radioactivity in Blood, Tissue, and Excreta
Following Dermal Treatment of Male Fischer 344 Rats
with 14C-Labeled 4,4'-MDA (0.4 mg/Rat): Nonoccluded Skin
and Skin Washed at 24 h 52
18 Urinary and Fecal Excretion of Radioactivity in Male
Fischer 344 Rats Treated Dermally with 14C-Labeled 4,4'-MDA
(4.0 mg/Rat): Continuous Application for 96 h versus Skin
Washing at 24 h 53
19 Radioactivity in Blood, Tissue, and Excreta of Male Fischer
344 Rats 96 h Following Dermal Treatment with 14C-Labeled
4,4'-MDA (4.0 mg/Rat): Continuous Application for 96 h
versus Washing at 24 h 54
20 Tissue-to-Blood Concentration Ratios from Male Fischer 344
Rats at 96 h Following Dermal Treatment with 14C-Labeled
4,4'-MDA (4.0 mg/Rat): Continuous Application for 96 h
versus Skin Washing at 24 h 55
21 Recovery of Radioactivity in Blood, Tissue, and Excreta at
96 h Following Dermal Treatment of Male Fischer 344 Rats
with 14C-Labeled 4,4'-MDA (4.0 mg/Rat): Continuous
Application for 96 h versus Skin Washing at 24 h 55
22 Urinary and Fecal Excretion of Radioactivity in Male
Hartley Guinea Pigs Treated Intravenously with 14C-Labeled
4,4'-MDA (1.0 mg/Guinea Pig) 56
23 Radioactivity in Blood, Tissue, and Excreta at 96 h
Following Intravenous Treatment of Male Hartley
Guinea Pigs with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig). 57
24 Tissue-to-Blood Concentration Ratios from Male Hartley
Guinea Pigs at 96 h Following Intravenous Treatment with
14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig) 58
25 Recovery of Radioactivity in Blood, Tissue, and Excreta at
96 h Following Intravenous Treatment of Male Hartley
Guinea Pigs with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig). 58
26 Urinary and Fecal Excretion of Radioactivity in Male
Hartley Guinea Pigs Treated Dermally with 14C-Labeled
4,4'-MDA (1.0 mg/Guinea Pig) 59
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LIST OF TABLES (continued)
Table Page
27 Radioactivity in Blood, Tissue, and Excreta at 6, 24, and
96 h Following Dermal Treatment of Male Hartley Pigs
with 14C-Labeled 4,4'-MDA (1.0 nig/Guinea Pig) 60
28 Tissue-to-Blood Concentration Ratios from Male Hartley
Guinea Pigs at 6, 24, or 96 h Following Dermal Treatment
with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig) 61
29 Recovery of Radioactivity in Blood, Tissue, and Excreta at
6, 24, or 96 h Following Dermal Treatment of Male Hartley
Guinea Pigs with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig). 61
30 Urinary and Fecal Excretion of Radioactivity in Male
Hartley Guinea Pigs Treated Dermally with 14C-Labeled
4,4'-MDA (1.0 mg/Guinea Pig): Application Area Washed
at 24 h 62
31 Radioactivity in Blood, Tissue, and Excreta Following
Dermal Treatment of Male Hartley Guinea Pigs with
14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig): Nonoccluded
Skin and Skin Washed at 24 h 63
32 Tissue-to-Blood Concentration Ratios from Male Hartley
Guinea Pigs Following Dermal Treatment with 14C-Labeled
4,4'-MDA (1.0 mg/Guinea Pig): Nonoccluded Skin and Skin
Washed at 24 h 64
33 Summary of Radioactivity in Blood, Tissue, and Excreta
Following Dermal Treatment of Male Hartley Guinea Pigs
with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig): Nonoccluded
Skin and Skin Washed at 24 h 64
34 Urinary and Fecal Excretion of Radioactivity in Male
Hartley Guinea Pigs Treated Dermally with 14C-Labeled
4,4'-MDA (10.0 mg/Guinea Pig,): Continuous Application
for 96 h versus Washing at 24 h 65
35 Radioactivity in Blood, Tissue, and Excreta at 96 h
Following Treatment of Male Hartley Guinea Pigs with
14C-Labeled 4,4'-MDA (10.0 mg/Guinea Pig): Continuous
Application for 96 h versus Washing at 24 h 66
36 Tissue-to-Blood Concentration Ratios from Male Hartley
Guinea Pigs at 96 h Following Dermal Treatment with
14C-Labeled 4,4'-MDA (10.0 mg/Guinea Pig): Continuous
Application for 96 h versus Washing at 24 h 67
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LIST OF TABLES (continued)
Table Page
37 Recovery of Radioactivity in Blood, Tissue, and Excreta at
96 h Following Dermal Treatment of Male Hartley Guinea
Pigs with 14C-Labeled 4,4'-MDA (10.0 mg/Guinea Pig):
Continuous Application for 96 h versus Washing at 24 h . . 67
38 Urinary and Fecal Excretion of Radioactivity in Male Rhesus
Monkeys Treated Dermally or Intravenously with 14C-Labeled
4,4'-MDA (10.0 mg/Monkey) , 68
xn
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I. INTRODUCTION
Under the Toxic Substances Control Act (TSCA), a manufacturer or
processor must submit to the Environmental Protection Agency a premanufactur-
ing notice (PMN) for every new chemical prior to commercial production. The
Agency has 90 days to review the PMN and to take any regulatory actions deemed
necessary. As part of this review, the potential workplace exposure and dose
associated with the manufacturing and processing of the new chemical must be
estimated. The estimate of dose is particularly relevant for risk assessments.
Dose, however, is quite difficult to estimate, especially for dermal exposure,
which may be the prevalent exposure route in many manufacturing and processing
operations. Currently, very little information is available that would allow
the prediction or estimation of dermal penetration rates.
The overall objective of this task was to develop dermal penetra-
tion data for 4,4'-methylenedianiline (4,4'-MDA, Figure I.), an aromatic amine
with known toxic and carcinogenic properties. The specific aims were to se-
lect and compare techniques and animal models for measuring dermal penetration
rates of this compound. The studies were performed iji vivo with three animal
models: the rat, guinea pig, and monkey. Several experimental conditions
were considered and assessed in the study protocol including skin type (rat,
guinea pig, and monkey), dosage (low, high), exposure regimen (limited, con-
tinuous), occlusion (exposed, covered), and carrier. In addition, initial
studies were performed to assess the dermal washing efficiency following ap-
plication to rats, guinea pigs, and monkeys.
Figure I. 4,4'-Methylenedianiline (4,4'-MDA)
(* The position of the 14C label)
Limited experiments were performed to assess the elimination of
4,4'-MDA following intravenous (i.v.) administration to utilize the data
generated in determining the absolute absorption of 4,4'-MDA following dermal
application. The utility of this methodology was assessed by comparing the
data generated to those resulting from excretory data in urine and feces of
animals exposed by dermal application.
The species selected were the rat, a species for which historical
data on the toxicity and carcinogenicity of aromatic amines are available and
which is used extensively in dermal absorption studies, the guinea pig, and
the monkey, species which have skin characteristics that resemble human skin.
The studies in rats and guinea pigs were performed at two dose levels, "low"
and "high." The monkey studies were carried out with the low dose only.
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II. BACKGROUND
The skin is a complex and heterogeneous membrane which provides, in
general, a significant resistance to chemical ingress. Nevertheless, the skin
is a primary body contact with the environment and the route by which many
chemicals enter the body.25'32'33 Some chemicals applied to the skin have
proved to be toxic. Thus, percutaneous absorption is an important route of
entry for potentially hazardous and toxic material. The ability to predict
dermal penetration would, therefore, be of considerable benefit in the ques-
tion of cutaneous chemical exposure for which direct testing in humans is not
feasible.
The potential for large quantities of toxic chemicals entering the
systemic circulation via the dermal route is quite high. Many factors deter-
mine the toxic potential of a topically applied chemical.42 The most obvious
is that the chemical be inherently toxic. The systemic availability is then
determined by the rate of dermal absorption, concentration of applied dose,
surface area covered, and length of exposure. Other factors such as occlusion,
hydration, and anatomical site exposed will also affect the absorption.24'42
Percutaneous absorption is a process by which a chemical moves se-
quentially from the surface of the skin through the stratum corneum, epidermis,
papillary dermis, and finally into the bloodstream. Resistance to movement
through this barrier is not equally distributed among all layers, but several
studies have indicated that the stratum corneum is the rate limiting portion.18
The rate of flux of chemicals from the vehicle into the epidermis and subse-
quently into the systemic circulation follows a process which can be divided
into a lag phase, a rising phase, and a falling phase.18 The total amounts
of chemical absorbed during this process can be calculated from excretion or
blood levels data. Although it is theoretically possible for the applied dose
to be completely (100%) absorbed, existing data on percutaneous absorption
suggest that this is not achieved.11 1S It is now clear that the skin is a
barrier of variable permeability with some compounds being well absorbed.
This contrasts with the older notion that skin is a relatively impervious
barrier.32'33
A definitive structure-activity relationship for percutaneous ab-
sorption of many xenobiotics has not yet been established. Although attempts
were made to relate chemical structure and chemical partition coefficients to
skin penetration, no clear principles are yet available to predict the j_n vivo
absorption in man.5'18 The rate of absorption of topically applied chemicals
can vary greatly. The major variables that account for differences in absorp-
tion appear to be the chemical concentration, its partition coefficient between
the stratum corneum and the vehicle, and its diffusion coefficient into the
stratum corneum. Measurements of the rate and extent of absorption of topic-
ally applied chemicals in man are difficult since most of these chemicals are
potentially toxic. In addition, these studies generally require the applica-
tion of radioactive tracers.18 When these studies are conducted, the radio-
active chemical, dissolved in a suitable vehicle, is applied to the skin and
urine is collected and analyzed for radioactivity. When only urinary excre-
tion is used for measuring percutaneous absorption, correction is made for
excretion of the tracer by other than the urinary route. This is done in a
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separate series of experiments in which the percentage of an intravenously
(i.v.) administered dose of the same compound excreted in urine is determined.42
Although animal skin is unlikely to have permeability properties
identical to that of human skin, animals are used for dermal toxicity and ab-
sorption evaluations since many of these studies cannot be performed in humans.
Penetration of toxic compounds can also be measured satisfactorily with excised
skin in diffusion cells.8'9'16'17 No one animal will simulate the penetration
in humans of all compounds, although the rhesus monkey and miniature pig yielded
the best correlation with human penetration.28'35'38"41'44 Rabbit skin is
generally recognized as the most permeable of the commonly used lab animals.38
Mouse and rat skin also showed high permeability.42 Although data on guinea
pig skin are limited,1 this species is used for dermal toxicity studies and
is considered a sensitive indicator of potential dermal toxicity. _0ther animal
models examined include the hairless dog and the hairless mouse.29 31
Several studies have shown that values for even the most permeable
skin, such as rabbit or mouse, are often well within an order of magnitude of
values for human skin. Thus, it appears that, depending on the compound of
interest and the vehicle used, values that are not too dissimilar from those
with human skin might be obtained with the skin of a number of animal species.
In studies by Bronaugh et al.,8 several test compounds were ranked in the same
order of permeability with all four types of animal skin employed (pig, rat,
mouse, and hairless mouse). The animal model of choice, however, may depend
on the compound of interest. They suggested that an initial comparison using
human skin may be made jri vitro before an animal model is selected for routine
testing. It should be noted that only a limited amount of human and animal
permeability data is available and that generalizations cannot be made.
Laboratory rodents are more convenient than the monkey or the pig
for dermal toxicity and absorption studies because of ease in handling and
lower cost. The skin of the rat has been considered to be more permeable
than human, pig, or monkey skin. However, in several studies with caffeine,
butter yellow, and N-acetylcysteine, rat skin appeared to be at least as good
a model for human skin as pig skin.3'4 When rat skin has been found to be
more permeable than human skin, the differences have not been large. It was
found that the stratum corneum of female rats is as thick as that of human
skin. The stratum corneum of male rat back skin is almost twice as thick as
that of the female rat and represents a better barrier against absorption.8
Recently, the hairless mouse has had increasing use because of the reported
similarity of its skin to human skin in the absorption of antiinflammatory
agents and aliphatic alcohols.42
Percutaneous skin absorption can be measured in living animals or
humans (in vivo) or HI vitro by using excised skin in diffusion cells.7'16'23'27
The studies with excised skin seem feasible since passage through the skin is
a passive diffusion process and the primary barrier, the stratum corneum, is
composed of nonliving tissue. The selection of whether to measure skin pene-
tration i_n vivo or by i_n vitro techniques depends at least on the utility of
the data obtained. Ir\ vivo studies measure the absorption rate indirectly,
e.g., by determining the excretion of a compound from the body, or the blood,
or tissue levels of the compound. The resulting data include physiological
3 •
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effects on an absorbed compound such as metabolism, distribution, and
excretion.20'21
In vitro absorption methods have been used recently in percutaneous
absorption studies using excised human skin. In initial studies performed by
Franz,16 the permeability of 12 organic compounds were evaluated and compared
to those obtained J_n vivo by Feldman and Maibach.12 15 Although care was taken
to ensure that both conditions of exposure were similar, some differences in
dosage and anatomical site of skin were noted. The iji vitro methods distin-
guished compounds of low or high permeability, but quantitatively the iji vivo
and iji vitro data did not agree. Although these differences suggest that j_n
vitro .methods would not always be a reliable or accurate predictor of percu-
taneous absorption in living humans, more recent studies8'9'17 suggest that
HI vitro methods can provide useful preliminary information that can be later
expanded using in vivo experiments. In addition, jm vitro methods are useful
for studies that separate the percutaneous absorption from other pharmaco-
kinetic factors associated with the uptake of compounds by the topical route.
Large numbers of experiments can be performed simultaneously and sampling is
performed directly under the surface of the skin. Only i_n vitro methods can
be used to obtain permeability data on highly toxic compounds with human skin.
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III. MATERIALS AND METHODS
A. Animals
Adult male Fischer 344 rats, Hartley guinea pigs, and rhesus monkeys
were used in the studies. The rats and guinea pigs were purchased from Charles
River Breeding Laboratories, North Wilmington, Massachusetts. The monkeys
were obtained from Hazleton Research Animals, Alice, Texas. Upon arrival,
the rats and guinea pigs were identifed by metal eartags. The identification
numbers (tattooing performed by supplier) on the chest of the monkeys were
confirmed and recorded. The animals were housed in environmentally controlled
rooms with 10 to 15 air changes per hour. Temperature and humidity were main-
tained at 72 ± 2°F and 50 ± 10%, respectively, except for brief excursions
outside these ranges. The rooms were kept on a 12-h light/dark cycle per day.
The rats and guinea pigs were housed in quarantine for at least 7 days prior
to use. The monkeys were acclimated for 1 month in quarantine, during which
time they were subjected to tuberculin tests 11 days after arrival and 1 month
later. All monkeys tested negative for tuberculosis.
During quarantine, the rats and guinea pigs were housed in polycar-
bonate cages on Ab-Sorb-Dri® (Ab-Sorb-Dri Company, Garfield, New Jersey) hard-
wood chip bedding. The rats were provided with Purina Certified Rodent Chow
(Ralston Purina Company, Richmond, Indiana). The guinea pigs were fed Purina
Certified Guinea Pig Chow, and their diets were supplemented with ascorbic
acid (0.4 g/L) in the drinking water. The monkeys were housed in stainless
steel cages and were fed Purina Certified Primate Chow, No. 5048, supplemented
with apples. Tap water was available at all times. No contaminants were known
or assumed to have been present in the food or water which could have inter-
fered with or affected the results of this study.
The attendant veterinarian examined the animals during the quaran-
tine period. All animals were determined to be in good health as evidenced
by normal growth and appearances, and absence of clinical signs. Prior to
testing, the rats and guinea pigs were selected at random using a body weight
stratification procedure. The number of rodents used, their ages and body
weights are summarized below:
Study
Washing efficiency
(dermal)
Preliminary
(dermal and i . v. )
Definitive
(dermal and i. v. )
Species
Rat
Rat
Guinea pig
Rat
Guinea pig
Rat
Guinea pig
No. of
animals
6
6
6
3
3
30
24
Age3
(days)
79b
56
66
56
45
56-72
37-46
Weight3
(9)
195-252b
169-227
436-588
187-208
357-384
149-218
323-456
flJpon initiation of dosing.
Initial study.
5 '
-------
Two monkeys were randomly assigned to each treatment group for the
first washing efficiency study. The same monkeys were used for the same treat-
ments in the second washing efficiency study. For the definitive study, one
monkey from each of the two treatment groups was chosen for treatment either
dermally or intravenously. One of these two monkeys were then chosen for the
second definitive study. This is summarized below along with ages and body
weights:
Treatment
Experiment Date
Washing 8/27/84a
efficiency
(dermal)
10/10/84
Soap and
water wash
No.
No.
No.
No.
529T
604T
529T
604T
Dermal
Definitive 11/27/84
(dermal and
i.v.)
1/16/85
No.
No.
No.
604T
619T
604T
Acetone and
water wash
No.
No.
No.
No.
554T
619T
554T
619T
Age Weight
(years) (kg)
3.2 to 3.7 4.6 to 5.9
3.4 to 3.8 4.9 to 6.4
Intravenous
No.
No.
No.
529T
554T
529T
3.5 to 3.9 4.7 to 6.4
3.8 to 4.1 4.7 to 6.4
alnitial studies.
Care and handling of animals was in accordance with the guidelines
in "Guide for the Care and Use of Laboratory Animals," 1978 Revision, prepared
by the Institute of Laboratory Animal Resources, DHEW Publication No. (NIH)
78-23, and the MRI manual for animal care.
B. Chemicals
Nonlabeled methylenedianiline (Lot No. 1707PK) was purchased from
Aldrich Chemical Company, Milwaukee, Wiconsin, and 100 g of the material was
received by Midwest Research Institute (MRI) on January 27, 1984. Ring labeled
14C-methylenedianiline (Lot No. 83-25-78) was prepared by the Radiochemical
Synthesis Section of MRI. The compound was received June 15, 1984, and
September 20, 1984, as the free base (3.2 mCi) in 100% ethanol or as the hydro-
chloride (8.18 mCi) with a specific activity of 14.8 mCi/mmol. The hydrochlo-
ride was converted to the base when required. The radiochemical purity was
found to be > 99% as determined by high pressure liquid chromatography and
thin-layer chromatography. (For details of synthesis and analysis, see
Appendix VI.)
-------
C. Dosage and Treatment
Two dose levels were used in these studies, a "low" dose, *> 2 mg/kg,
and a "high" dose, ~ 20 mg/kg. Appropriate mixtures of the 14C-labeled and
nonlabeled 4,4'-MDA were dissolved in an ethanol:water mixture and were applied
to the skin or administered intravenously (i.v.) to rats, guinea pigs, and
monkeys.
For dermal treatment, the doses were administered in a mixture of
ethanol:water (95:5) and applied at a volume of about 0.25 to 0.5 mL/kg (0.1
mL/rat, 0.25 ml/guinea pig, and 1.25 mL/monkey). For monkeys, the volume used
in the initial dermal washing efficiency study was 2.5 mL/monkey. The backs
of the rats and guinea pigs and the lateral forearm of the monkeys were lightly
shaven with electric clippers shortly before dosing. The dose was applied
with a disposable micropipette on a specific area (2 cm2 for rats, 5 cm2 for
guinea pigs, and 50 cm2 for monkeys) on the freshly shaven skin. Except when
indicated otherwise (see experimental design), the dosed areas were covered
with a wax-lined cup held in place by adhesive tape.
For intravenous treatment, a mixture of the 14C-labeled and non-
labeled 4,4'-MDA was dissolved in ethanol:water (25:75) and administered at
the low dose level in a volume of ^ 2.0 ml/kg (0.4 mL/rat and 1.0 mL/guinea
pig) or ^ 1.0 mL/kg (5.0 mL/monkey). Intravenous dosing for all three species
was performed through the saphenous vein.
Aliquots of each dosing solution were counted to determine the
amounts of radioactivity. These amounts are summarized below:
Study
Washing
efficiency
(dermal)
Preliminary
(dermal and
i.v.)
Definitive
(dermal and
i.v.)
Species
Rat
Guinea pig
Monkey
Monkey
Rat
Guinea pig
Rat
Guinea pig
Monkey
Dose
(•v- mg/kg)
2
2
2
2
2
20
2
2
20
2
2
20
2
2
20
2
2
2
Route
Dermal
Dermal
Dermal
Dermal
Dermal
Dermal
i.v.
Dermal
Dermal
i.v.
Dermal
Dermal
i.v.
Dermal
Dermal
i.v.
Dermal
i.v.
Radioactivity
(uCi/animal)
8.7-10.4
26.0
115.2
127.8
10.9
11.4
12.3
31.3
26.6
33.6
25.2
31.2
26.7-32.8
43.6
48.0
29.4
190.5-231.7
127.5-259.3
Specific
activity
(dpm/ug)
48,511-57,769
57,769
25,568
28,374
60,489
6,347
68,185
69,491
5,894
74,680
139,299
17,299
147,971-182,149
96,873
10,663
65,265
42,281-51,426
28,308-57,567
Initial study.
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D. Experimental Design
1. Washing Efficiency Studies
These studies were performed to assess the efficiency of removal of
the applied 14C-labeled material by washing with soap and water or acetone
and water. Six rats,* six guinea pigs, and four monkeys** were lightly anes-
thetized (rats and guinea pigs with an intraperitoneal dose of sodium pento-
barbitol, 45 mg/kg and 30 mg/kg, respectively, and monkeys with an intramus-
cular dose of ketamine hydrochloride, 5 mg/kg). The animals were then treated
dermally with 4,4'-MDA at the low dose level. The doses were allowed to dry
(5 min) and were then washed with soap and water or acetone and water (three
rats, three guinea pigs, and two monkeys for each treatment). Washing was
accomplished by soaking a gauze pad in either soap (Sani-Fresh® lotion cleanser,
Sani-Fresh International, Inc., San Antonio, Texas) or acetone (certified,
Fisher Scientific Company, Fair Lawn, New Jersey) and scrubbing the application
areas for ^ 30 s. Gauze pads soaked in tap water were then used to scrub the
application areas; this step was repeated five or six times. Each pad was
placed in a labeled screw-cap jar and known volumes of methanol were added
for extraction of the radioactive material.*** Aliquots (0.25 ml) of these
methanol extractions were measured and counted for determination of
radioactivity.
Following washing, the application areas were covered and the animals
were placed in individual metabolism cages (rats and guinea pigs) or metabolic
chairs (for the first 24 h in monkeys) and urine and feces were collected at
6, 12, 24, 48, 72, and 96 h for rats and guinea pigs, and 6, 12, 24, 48, 72,
96, and 168 h for monkeys. At the end of the collection period, rats and
guinea pigs were sacrificed by ether inhalation, and the covers and the appli-
cation areas (skin) were removed and individually extracted with methanol.
Later the skins were solubilized in perchloric acid:hydrogen peroxide (2:4)
and total radioactivity was determined.
2. Preliminary Studies
These studies were performed to assist in selecting the appropriate
sampling times for the definitive studies and to test and assure effective-
ness of methods to be used for treatment of rats and guinea pigs. The studies
utilized three male rats and three male guinea pigs; one of each species was
treated dermally with the low or high dose level of 4,4'-MDA, or i.v. at the
low dose level. The application areas were covered (dermally treated), and
the animals were placed in individual metabolism cages for collection of urine
and feces at 6, 12, 24, 48, 72, and 96 h following dosing. Radioactivity re-
maining on the skin of dermally treated animals was washed with soap and water
as described above and the animals were sacrificed for tissue sampling (see
below, Sample Collection).
*Experiment was repeated due to the low recoveries.
** Experiment was repeated due to the high recovery in the excreta and
low recovery in the washing solution.
*** Extraction with methanol was assumed to remove all the radioactivity from
the gauze pads.
8
-------
3. Rat Studies
These studies were performed with 21 rats treated dermally at the
low or high dose levels and 9 rats treated i.v. at the low dose level. The
dermal dose of 4,4'-MDA was applied and kept on the skin for the duration of
the study (6, 24, or 96 h) or kept on the skin for 24 h then removed. After
the application area was covered, the animals were placed in metabolism cages
for collection of urine and feces at 6, 12, 24, 48, 72, and 96 h. At the spe-
cified times, the applied doses were washed with soap and water, then the ani-
mals were sacrificed or returned to the metabolic cages for sacrifice at a
later time point. A group of three rats were treated dermally at the low dose
level, but the application areas were kept nonoccluded by fitting the animal
with wax-lined cups with the tops removed; these animals were sacrificed at
6 h.
In the i.v. studies, the rats were treated with the low dose of
4,4'-MDA and were placed in individual metabolism cages for excreta collection
at 6, 12, 24, 48, 72, and 96 h following administration. Three animals were
sacrificed at 6, 24, or 96 h for tissue sampling.
4. Guinea Pig Studies
These studies were performed with 21 guinea pigs treated dermally
at the low or high dose level and 3 guinea pigs treated i.v. The design was
similar to the rat studies except that i.v. dosing was limited to one group
of three animals which were treated with the low dose and sacrificed at 96 h
following dosing.
5. Monkey Studies
In these studies, four male monkeys were treated dermally (2) or
i.v. (2) with the low dose of 4,4'-MDA. After dosing, the monkeys were placed
in metabolic chairs for the first 24 h. The dermal application areas were
then washed with soap and water as described above and the monkeys were trans-
ferred to individual metabolism cages for the remainder of the study. Follow-
ing dosing, urine and feces were collected at 6, 12, 24, 48, 72, 96, 120, 144,
and 168 h. One of the dermally treated and one of the i.v. treated monkeys
were redosed approximately 6 weeks later to obtain additional excretion data.
This study was performed similarly.
E. Sample Collection
Urine was collected in containers kept on dry ice. After each col-
lection, the cages were rinsed and the cage washings were measured and ana-
lyzed. At sacrifice, the rodents were anesthetized with ether and exsanguin-
ated by withdrawal of blood from the abdominal aorta. The monkeys were not
sacrificed. The following tissues and organs were removed, washed with saline,
blotted with absorbing paper, weighed, and prepared for radiochemical analysis:
-------
- Liver - Spleen - GI tract plus contents
- Kidneys - Adrenals - Skeletal muscle
- Lungs - Testes - Retroperitoneal fat
- Brain - Urinary bladder - Skin from nontreated areas
- Application area
Portions of blood were centrifuged to separate plasma and red blood
cells (RBCs). Bladder contents were removed and the bladder was washed thor-
oughly with saline. The contents and washings were combined with the final
urine samples and analyzed. Blood and tissue were kept on ice during the ne-
cropsy procedures. Sample preparation and analyses were performed immediately
after collection, or the samples were frozen on dry ice and stored frozen until
analyzed. The remaining tissue and excreta were stored frozen.
F. Determination of Total Radioactivity
1. Sample Preparation
Blood, tissue, and excreta were analyzed in duplicate whenever pos-
sible. Aliquots (0.1 to 0.25 mL) of the whole blood, RBCs and plasma were
analyzed for total radioactivity determination. Volumes of urine and cage
rinse were measured, and samples (0.5 to 1.0 mL) were counted. Feces, GI tract
(plus contents), and tissues weighing more than 0.25 g were homogenized in
4 volumes of ethanol:water, 10:90. Aliquots of the homogenates were measured
and analyzed. Adrenals, bladders, fat, and nontreated skin samples were
weighed and assayed for 14C content. The application areas of the skin and
the covers were soaked in methanol and the extracted radioactivity was ana-
lyzed. Later, the application areas were solubilized with perchloric acid:
hydrogen peroxide (2:4) and aliquots were measured and analyzed.* Blood,
tissue, and fecal samples were combusted using a Packard Tricarb Oxidizer
Model C306. Permafluor V® in combination with Carbo-Sorb® (Packard Instru-
ment Company) was used as the scintillation cocktail. Urine and cage rinse
were counted directly in Phase Combining Scintillate (PCS, Amersham).
2. Radioactivity Measurement
Vials were cooled for a minimum of 24 h before counting in a liquid
scintillation counter (Packard Tricarb Model 3255). Correction for background
was carried out automatically by the counter. Background determinations were
obtained from the average of natural counts of several tissue homogenates from
nontreated animals. The counting efficiency was determined using the automatic
external standard (AES) method. An AES versus efficiency curve was prepared
by processing a quench curve set through the counter under the conditions used
throughout the experiment. Assays not within ± 10% of the mean of the dupli-
cates were reassayed in duplicate except when the sample was no longer avail-
able or when radioactivity counts were low and nonsignificant, i.e., less than
two times the background. For these studies, background counts ranged from
25.0 to 35.6 counts/min.
Skin solubilization with perchloric acid and hydrogen peroxide was con-
ducted with conditions not likely to cause loss of 14C by oxidation of
the ring-14C-labeled 4,4'-MDA to 14C02.
10
-------
3. Data Processing and Analysis
Carbon-14 contents in blood and tissues are presented in terms of
microgram equivalents per milliliter (blood) or gram (tissues) and percentage
of the administered dose. The percent of dose excreted in urine and feces is
also tabulated. Individual calculations for each sample were performed with
an Apple II Plus computer as follows:
1. Cpm (counts per minute) for each sample was converted to dpm
(disintegrations per minute).
efficiency = dPm/samP1e
2. Dpm per g or mL was calculated.
_ dpm/sample _ _ , / .
sample weight or volume (g or mL) ~ ^ ^ or m
3. Dpm per g or ml was divided by the specific activity of the
compound (dpm/ug) to obtain the ug equivalents/g or mL.
actity
4. This was multiplied by the total weight or volume of the organ
or excreta in order to obtain the total amounts in the organ or excreta.
ug/g or mL x total weight or volume = ug/organ or excreta
5. The ug/organ or excreta was divided by the total dose adminis-
tered in order to obtain the percentage of the administered dose.
ug/organ or excreta x 100 & f . • • . , .
total dose (in ug) - = % of administered dose
The percent of administered dose recovered in blood, muscle, and
skin was calculated based on 7, 40, and 16%, respectively, of body weight.
Percent recovery in plasma and RBCs was calculated based on 60 and 40%, re-
spectively, of the total blood volume, these estimates were based on data
from the published literature.
11
-------
IV. RESULTS
A. Dermal Washing Efficiency Studies
Prior to conducting the dermal disposition studies, initial washing
efficiency experiments were performed to assess the extent of removal of the
applied 14C-labeled material by washing with soap or acetone solutions. Six
rats, six guinea pigs, and four monkeys were lightly anesthetized, then treated
dermally with low doses (~ 2 mg/kg) of 14C-labeled 4,4'-MDA. After allowing
5 to 10 min for the applied doses to dry, the application areas were washed
with soap and water or with acetone and water (three rats, three guinea pigs,
and two monkeys for each treatment). After washing, the rats and guinea pigs
were placed in individual metabolism cages for excreta collection. Monkeys
were kept in metabolism chairs for 24 h before placing in individual metabolism
cages. Urine and feces were collected at 6, 12, 24, 48, 72, and 96 h following
application. In monkeys, excreta collection was continued for 168 h.
The data generated from the dermal wash studies are summarized in
Table 1. Data from individual animals are presented in Appendix II, Table
II-l for rats, II-2 for guinea pigs, and II-3 for monkeys.* In the rat, higher
and more consistent recoveries were obtained by washing the application areas
with soap and water (91.3%) than with acetone and water (85.1%). In addition,
lower amounts were recovered in excreta when soap and water were used: 1.6%
after soap and water versus 7.7% after acetone and water. Recoveries of radio-
activity from the application areas (methanol extraction, then skin solubiliza-
tion) were lower after washing with soap and water (3.8%) than after acetone
and water (12.7%). Similar results were obtained in guinea pigs; 84.5% of
the doses were washable with soap and water while 76.0% were washable with
acetone and water. In excreta, 0.6% of the dose was recovered during 96 h
following washing with soap and water while 2.1% was recovered after washing
with acetone and water. From the application areas, 3.8% was recovered from
animals washed with soap and water versus 5.9% after washing with acetone and
water.
The data shown do not include those generated from the initial rat and
monkey studies. The rat studies were repeated to assure that the recov-
eries obtained in the initial experiments were actual (were similar to
those described above for the repeat studies). During 96 h, 1.2% of the
dose was recovered in excreta after washing with soap and water versus
6.2% after acetone and water. In the initial studies with monkeys, the
animals were placed in metabolism cages following dose application and
washing. When the monkey studies were repeated, the animals were kept
in monkey chairs for the first 24 h following dose application to mini-
mize the possibility of ingesting any radioactivity remaining on the skin
after the application area was washed. In addition, collection of excreta
was extended for 168 h to allow for complete elimination before study
termination. In this study, slightly higher amounts were recovered in
the dose wash by the use of acetone and water, 41.9%, than after washing
with soap and water, 36.4%. Similar amounts were recovered in urine and
feces, 13.9% after soap and water and 11.5% after acetone and water.
The results, however, were more consistent in animals washed with soap
and water.
12
-------
In the monkeys, only 63.0% and 53.0% were recovered in the dose wash
after soap and water or acetone and water, respectively. In addition, housing
of the monkeys in chairs for 24 h after dose application and washing to prevent
any possible ingestion of the radioactivity remaining on the application areas,
did not significantly reduce the amounts excreted in urine and feces. In 168 h
after washing with soap and water, 8.7% was eliminated in urine and feces.
After washing with acetone and water, excretion in urine and feces averaged
16.2%. The total recoveries were still low, 71.7% in monkeys washed with soap
and water and 69.2% in monkeys washed with acetone and water. It is possible
that significant amounts of the applied doses still remained to be excreted
after the 7-day collection period. However, the rate of excretion was slow;
approximately 0.5% of the dose was eliminated every 24-h period during the
last 3 days of collection. Since no monkey tissues were sampled in this study,
it was not possible to determine the location of the unexcreted material.
These studies showed that both soap and water or acetone and water
were incapable of removing all the applied material. Significant amounts of
the applied doses remained associated with the skin available for absorption.
The recoveries were low, especially for monkeys where the study design did
not allow for a determination of 14C on the application area or in tissues.
In the rat and guinea pig studies, the amounts recovered in the application
areas were found to be significant. Even when the dose was washed 24 or 96 h
after dermal application (see below), considerable amounts were recovered in
the application areas and had to be extracted with methanol followed by skin
solubilization.
B. Preliminary Studies
These studies were performed with three rats and three guinea pigs
treated dermally with the low or high doses of 14C-labeled 4,4'-MDA or i.v.
with the low dose (one animal per treatment). Following dosing, the applica-
tion areas were covered and the animals were housed in individual metabolism
cages for collection of urine and feces at 6, 12, 24, 48, 72, and 96 h. The
radioactivity remaining on the skin of dermally treated animals was washed
with soap and water (which was selected for dose removal in all preliminary
and definitive dermal experiments); then the animals were sacrificed for sam-
pling of selected tissues. As described above, the application areas were
removed, processed, and counted. These preliminary studies assisted in select-
ing the appropriate sampling times for the definitive experiments described
below and in determining the methods of treatment and sampling.
The rat data (Tables 2 and 3) showed that most (^ 95%) of the i.v.
dose was eliminated in urine and feces during a 96-h period following dosing.
After dermal application of the low dose, ~ 43% was eliminated in urine and
feces in 96 h. The remainder was recovered in the dose wash, application area,
GI tract, and tissues. Although the skin was washed thoroughly, while only
31% was recovered in the dose wash and ~ 24% of the applied dose was recovered
from the application area. After dermal application of the high dose, ~ 15%
of the dose was recovered in excreta, suggesting limited absorption and/or
elimination. About ~ 52% of the high dose was recovered in the dose wash and
32% was removed from the application area.
13
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The guinea pig data (Tables 4 and 5) showed that about 89% of the
i.v. dose was recovered in urine and feces in 96 h. After dermal application
of the low dose, only 37% of the dose was recovered in excreta following the
same period. Slightly lower amounts (~ 30%) were recovered in excreta follow-
ing the high dose, but the distribution of 14C in excreta (urine and feces)
differed considerably from the animals treated with the low dose. The recov-
eries in the dose wash and application areas were respectively, ~ 38 and ~ 16%
following the low dose, and ~ 49 and ~ 8% following the high dose.
The tissue distribution data obtained from rats and guinea pigs in
the preliminary studies are shown in Tables 3 and 5, respectively. Since the
data are consistent with those demonstrated in the definitive i.v. and dermal
studies which are described below, no discussion of these preliminary data is
included.
C. Rat Studies
These studies were performed with nine male Fischer 344 rats treated
i.v. with the low dose and 21 rats treated dermally at the low or high doses
of 14C-labeled 4,4'-MDA. The rats treated i.v. were housed in individual
metabolism cages for excreta collection at 6, 12, 24, 48, 72, and 96 h. At
6, 24, or 96 h, groups of three rats each were sacrificed for tissue sampling.*
In the dermal studies, three groups of three rats each were treated with the
low doses, the application areas were covered (see methods), and then each
rat was placed in a metabolism cage for excreta collection at 6, 12, 24, 48,
72, and 96 h. At 6, 24, or 96 h following treatment, the application areas
were washed with soap and water, then the animals were sacrificed for skin
and tissue sampling. A fourth group of three rats was treated dermally with
the low dose which remained on the skin for 24 h, then removed (washed with
soap and water), and the animals returned to the metabolism cages until sacri-
ficed at 96 h. A fifth group of three rats was treated dermally at the low
dose and the dosing area was kept uncovered to determine the effect of non-
occlusion on the dermal penetration of 14C-labeled 4,4'-MDA. Excreta were
collected for 6 h, and then the animals were sacrificed.
For the high dose, two groups of three rats each were treated dermally
with the high dose of the test compound. The application areas were covered;
then each rat was placed in a metabolism cage for excreta collection at the
time periods indicated above. At 24 h, the application areas in one group
were washed with soap and water, and the animals were returned to the metabo-
lism cages until rats of both groups were sacrificed at 96 h following dosing.
The data generated from the i.v. studies are summarized in Tables 6
through 9. Data from individual animals are shown in Tables III-l through
III-3 in Appendix III. The data obtained from the dermal studies are shown
in Tables 10 through 21. The individual animal data are presented in
Appendix III, Tables III-4 through 111-14.
The studies with rats sacrificed at 6 and 24 h were repeated due to the
low recoveries. Data obtained from the initial studies are not included
in this report.
14
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1. Radioactivity In Excreta and Tissue
In rats treated i.v., most of the administered doses appeared in
urine, the GI tract (probably through bile) and tissues 6 h after dosing. By
24 h, most of the doses were recovered in urine (67.4%) and feces (21.8%).
Additional fecal excretion (to 30.7%) occurred between 24 and 96 h (Table 6).
In rats sacrificed at 6 h following continuous dermal application
of the low dose, 11.9% of the applied radioactivity was recovered in urine,
the GI tract, and tissues. An average of 62.1 and 30.5% were present in the
dose wash (soap and water) and the application areas, respectively. For rats
sacrificed at 24 h, 27.8% of the dose was recovered in urine, feces, the GI
tracts, and tissues; 52.1 and 25.8% were recovered in the dose wash and from
the application areas, respectively. By 96 h following continuous dermal expo-
sure to the low dose, 54.7% of the applied 14C was recovered in urine, feces,
tissues, and the GI tracts, 24.7% in the dose wash, and 25.6% from the applica-
tion area.
Rats in which the application areas were kept nonoccluded showed
lower amounts of 14C in the excreta, tissues, and the GI tracts (7.0% of the
dose in 6 hr) compared to the rats with occluded skin (Table 17). The remain-
der of the applied doses were recovered in the dose wash (70.2%) or from the
application areas (27.5%).
In rats treated dermally with the low dose and the application areas
washed at 24 h before the animals were returned to the metabolism cages for
an additional 72 h, 42.9% of the doses were recovered in urine, feces, tissues,
and the GI tracts. An average of 52.1% of the applied doses were recovered
in the dose wash at 24 h. In addition, an average of 10.7% was recovered from
the application areas at 96 h, i.e., 72 h after these areas were washed
(Tables 14 to 17).
In rats treated dermally with the high dose which was kept on the
skin for 96 h, only 6.6% of the applied doses were recovered in urine, feces,
GI tracts, and tissues. An average of 62.5% of the doses were washable and
24.0% recovered from the application areas. In the rats treated with the
high dose, then the doses washed 24 h later, similar amounts (5.3%) were re-
covered in excreta and tissues during the 96-h collection period (Tables 18
through 21).
2. Tissue Distribution
Tissue distribution studies were performed in the rats treated i.v.
or dermally and sacrificed at 6, 24, or 96 h after dosing. Following an i.v.
dose, significant amounts of the radioactive doses (23.9% at 6 h) were recovered
in the GI tract. The elimination in the GI tract, probably through bile, con-
tributed to the significant fecal excretion demonstrated at 24 and 96 h (21.8
and 30.7%, respectively). Significant amounts of the radioactive doses (19.4%)
were recovered in blood and tissues at 6 h, which declined to 1.5% at 96 h.
The highest concentrations were present in the liver, which contained 9.5% of
the dose at 6 h and was five times higher than blood (see Table 8).
15
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After continuous dermal application of the low dose, 3.8 and 4.7%
were recovered in the GI tract and tissues, respectively, at 6 h. As shown
after i.v. dosing, the liver showed the highest concentration. Higher tissue
concentrations were demonstrated following the high dose of 4,4'-MDA, although
the proportion of dose recovered in tissues was lower than found after the
low dose. The distribution in tissues, including liver, was however, similar
after the low and high doses. In animals treated dermally with the doses re-
moved 24 h later, a rapid decline in tissue concentrations was demonstrated
during the 72-h recovery period, although liver-to-blood ratios remained high
(about 7).
3. Recoveries and Dose Balance
The recoveries of radioactivity in blood, tissues, and excreta fol-
lowing treatment of rats with the low dose of 14C-labeled 4,4'-MDA are shown
in Tables 9 (i.v. studies), 13, and 17 (dermal studies). Recoveries follow-
ing dermal application of the high dose are shown in Table 21. The recoveries
of the i.v. doses ranged from 98 to 99% of the administered doses. Also,
after dermal application of the low doses, quantitative recoveries (104 to
106%) were obtained. Slightly lower recovery (92 to 93%) was obtained follow-
ing dermal application of the high dose. To achieve these recoveries, the
portions of dose remaining on the application areas following washing with
soap and water were determined. Initially, the skin was extracted over a 24-h
period with methanol, but complete extraction was not achieved. The radio-
activity that remained associated with the skin following extraction was deter-
mined by skin solubilization using perchloric acid and hydrogen peroxide.
Significant amounts of radioactivity were recovered in the skin application
area following the different treatments.
The summary data presented for the dermally treated rats include
the amounts which were considered absorbed through the skin (Tables 13, 17,
and 21). These amounts were obtained by addition of percentages of the doses
recovered in the blood, tissue, GI tract, and excreta. Radioactivity asso-
ciated with the application areas was not included. However, since the 14C
recovered from these areas appears to be strongly bound to the skin and is
probably available for systemic circulation, it may be considered in calcu-
lating the total amounts absorbed. These calculations are presented below.
In rats treated dermally with the low dose, the amounts absorbed
(recovered in excreta and tissue) averaged 11.9, 27.8, and 54.5% of the ap-
plied doses in animals sacrificed at 6, 24, and 96 h, respectively. When the
radioactivity in the dermal application areas was considered in calculating
the absorbed amounts, these values increased to 42.5, 53.6, and 80.1% of the
applied doses. In rats with the dermal dose kept nonoccluded, the amounts
absorbed averaged 7.0 and 34.5% without and with the application areas, respec-
tively. In rats receiving the low dose which was removed 24 h later, the total
absorption during a 96-h period averaged 42.9 and 53.0% without and with the
application areas, respectively. The rats treated dermally with the high dose
absorbed 6.6% of the dose in the continuously treated animals and 5.3% of the
dose in animals with the dose washed at 24 h. When the application areas were
included, these amounts increased to 30.6 and 14.2%, respectively.
16
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D. Guinea Pig Studies
These studies were performed with three guinea pigs treated intra-
venously (i.v.) and 21 male guinea pigs treated dermally with 14C-labeled
4,4'-MDA. The design was similar to the rat studies except that i.v. dosing
was limited to only three animals which were treated with the low dose of the
test compound and sacrificed at 96 h following dosing. The data from the i.v.
studies are shown in Tables 22 through 25. Data from individual guinea pigs
are presented in Appendix IV, Tables IV-1 through IV-3. The data obtained
from the dermal studies are shown in Tables 26 through 37. The individual
animal data are presented in Appendix IV, Tables IV-4 through IV-14.
1. Radioactivity in Excreta and Tissue
In guinea pigs treated i.v., most of the administered doses appeared
in feces and urine by 48 h following dosing (Table 22) with the highest rate
of excretion occurring between 12 and 24 h. By 96 h, recovery in feces (prob-
ably through bile) averaged 56.5% and in urine 35.0%.
In guinea pigs sacrificed at 6 hr following continuous application
of the low dose, 3.2% of the applied radioactivity was recovered in urine,
feces, GI tract, and tissues. An average of 80.6% and 11.4% was present in
the dose wash and the application areas, respectively. For guinea pigs sac-
rificed at 24 h, 17.5% of the dose was recovered in excreta and tissues; 58.8
and 14.8% were recovered from the dose wash and the application areas, re-
spectively. By 96 h following dermal application of the low dose, 39.9% of
the dose was recovered in excreta and tissue. As noted following i.v. dosing,
fecal elimination represents the primary excretory route in guinea pigs treated
dermally with the test compound. An average of 40.9% of the dose was recov-
ered in the dose wash and 29.4% was accounted for in the application areas
(Table 29).
Guinea pigs in which the application areas were kept nonoccluded
showed similar amounts in excreta, tissues, and the GI tract (3.1%) as animals
with the occluded skin (Table 33). The remainder of the applied doses were
recovered in the dose wash (87.5%) or from the application areas (9.4%).
In guinea pigs treated dermally with the low dose and the applied
doses removed at 24 h before the animals returned to their metabolism cages
for an additional 72 h, 18.0% of the doses were recovered in excreta, tissue,
and the GI tract. An average of 61.8% was recovered in the dose wash at 24 h
and 16.6% was associated with the skin application areas at 96 h (72 h after
washing).
In guinea pigs treated dermally with the high dose and sacrificed
at 96 h following dosing, only 7.5% of the applied dose was recovered in ex-
creta, tissue, or the GI tracts. An average of 69.9% of the dose was recov-
ered in the dose wash and 13.7% was present in the application areas. Slightly
lower amounts (4.2%) were recovered in excreta and tissues of guinea pigs that
were treated with the high dose and with the doses washed 24 h later. Recover-
ies in the dose wash and application areas averaged 80.4 and 7.3%, respectively.
17
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2. Tissue Distribution
Tissue distribution studies were performed in the guinea pigs treated
i.v. and sacrificed at 96 h or dermally and sacrificed at 6, 24, or 96 h after
dosing. At 96 h after the i.v. dose, only 0.6% of the dose was recovered in
the GI tract and 2.9% was recovered from blood and tissue. Elimination in
the GI tract at earlier times was probably significant since fecal elimination
at 96 h totaled ^ 56% of the dose. In contrast to rats, the guinea pigs showed
the highest tissue contents in the spleen (six times higher than blood), which
was followed by the liver (four times higher than blood). Blood and liver
contents were higher than demonstrated for rats.
After dermal dosing of the guinea pigs, a different tissue distri-
bution pattern was noted at the low and high doses of the test compound. Most
notable was the very high concentrations in adrenals (up to 23 times higher
than blood) which were demonstrated at 6, 24, and 96 h after dosing. As in
the i.v. dosed animals, the concentrations in the liver were also high, but
the 14C levels in spleen were considerably lower. The high adrenal levels
were also demonstrated in the guinea pigs treated dermally at the high dose
with the dose removed at 24 h. In contrast, after discontinuing the exposure
to the low dose at 24 h, the levels of 14C in adrenals were significantly re-
duced (see Tables 28, 32, and 36).
3. Recoveries and Dose Balance
Recoveries of the radioactivity in blood, tissue, and excreta follow-
ing treatment of guinea pigs with the low dose of 14C-labeled 4,4'-MDA are
shown in Tables 25 (i.v. dose), 29, and 33 (dermal doses). The recoveries
following dermal treatment with the high dose are shown in Table 37. About
95% of the i.v. dose was recovered in blood, tissue, GI tract, and excreta at
96 h following dosing. After dermal application of the low dose, recoveries
ranged from 91 to 100%. Slightly lower recoveries (91 to 92%) were obtained
from guinea pigs treated dermally with the high dose. As shown in the rat
dermal studies, the recoveries for guinea pig experiments included the dermal
application areas which were extracted and solubilized to determine the radio-
activity strongly associated with the skin.
The amounts considered to be absorbed through the skin of guinea
pigs are included in the recovery tables (Tables 29, 33, and 37). These
amounts were obtained from recoveries in the blood, tissue, GI tract, and ex-
creta. As described in the rat studies, 14C in the application areas was not
included in the absorption estimates, although the radioactivity recovered
from these areas was strongly associated with the skin. In guinea pigs treated
dermally with the low dose, 3.2, 17.5, and 29.9% of the doses were absorbed
(recovered in excreta and tissue) during 6, 24, and 96 h after dosing. By
including the application areas in the absorbed estimates, these amounts in-
creased to 14.6, 32.3, and 59.3%. In guinea pigs with the dermal dose kept
nonoccluded, the amounts absorbed averaged 3.1 and 12.6% without and with the
application areas, respectively. In animals receiving the low dose which was
removed at 24 h, the total absorption during a 96-h period following applica-
tion averaged 18.0 and 34.6% without and with the application areas, respec-
tively. The guinea pigs treated dermally with the high dose absorbed 7.5% of
18
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the dose in the continuously treated animals and 4.2% in the animals with the
dose removed at 24 h. When the application areas are included, these amounts
increased to 21.2 and 11.5%, respectively.
E. Monkey Studies
In these studies six male rhesus monkeys were treated dermally
(three) or i.v. (three) with the low dose of 14C-labeled 4,4'-MDA.* The mon-
keys, which were acclimated in metabolism chairs prior to dosing, were kept
in their chairs during the first 24 h after treatment. At this time, the
dermal application areas were washed with soap and water and all monkeys were
transferred to individual metabolism cages for the remainder of the study.
Urine and feces were collected at 6, 12, 24, 48, 72, 96, 120, 144, and 168 h
following dosing. The animals dosed i.v. were treated similarly but skin
washing was not performed. In these studies, no tissues were sampled; the
monkeys were retained for future investigation following recovery periods.
Table 38 summarizes the elimination data obtained from both groups
of monkeys treated i.v. or dermally. The data from individual animals are
presented in Appendix V, Table V-l. Almost complete recovery (93 to 98%) was
obtained from the monkeys treated i.v. Excretion of 14C occurred primarily
in urine (84.3%) and to a lesser extent in feces (9.8%). Peak elimination
occurred at 6 to 12 h, although one animal showed considerable 14C elimina-
tion between 24 and 48 h. For the dermally treated monkeys, only 36 to 56%
(average 47.3%) of the applied doses was recovered in the washing solutions.
An average of 18.8% were excreted in urine and 1.4% in feces during the 7-day
collection period. Excretion peaked at 24 h for one monkey and at 48 h for
the other two monkeys. Total recoveries in skin wash and excreta averaged
only 68% (62 to 73%).
Based on the excretory data only, the amounts considered absorbed
through monkey skin averaged 20.8% of the dose. Since the application areas
were not sampled, it was not possible to determine the total amounts absorbed
or available for absorption. On the other hand, 14C associated with the appli-
cation areas and tissues can be estimated to be 32% of the dose if quantita-
tive recovery (100%) is achievable. If this estimate is considered, the
amounts absorbed or available for absorption increase to 52.8% of the applied
dose.
Since only four monkeys were used in the present studies, two experiments
were performed. The first included four monkeys treated dermally or i.v.
(two each), and the second included two of the same monkeys treated
dermally or i.v. (one each). A recovery period of 50 days after dosing
was allowed between the two experiments.
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V. DISCUSSION
The possibility of percutaneous penetration of environmental toxi-
cants increases the importance of dermal exposure risk assessment and preven-
tion. The penetration process is more complex than previously thought, and
some early generalizations regarding this process are being reassessed.
Little research has been published on the relevance of experimental models to
man.2'11"15 In addition, percutaneous absorption of chemicals that come in
contact with the skin depends on many parameters, including the concentration
of applied dose, the vehicle, and the surface area of application.24'42'43'46
Variations in absorption also occur due to the anatomical site of application,
occlusion, skin condition, and multiple applications. Therefore, the study
design of a skin absorption study in any species must take into consideration
these and other variables.
With the extensive use of 4,4'-MDA and other aromatic amines, dermal
exposure to these compounds seems obvious. Information on the dermal disposi-
tion of 4,4'-MDA is not available. Dermal penetration data on other related
aromatic amines are also limited.5'10'19'26'34 The present studies were,
therefore, undertaken to assess the disposition characteristics of 4,4'-MDA
following its dermal and intravenous administration to three animal models.
Our objective in this study was to determine the dermal absorption, systemic
elimination, and dermal washing efficiency of 4,4'-MDA. The animal models
selected were the rat, a species for which historical data on the toxicity
and carcinogenicity of aromatic amines are available and which is used exten-
sively in dermal absorption studies, and the guinea pig and monkey, species
which have been shown to have some relevance to humans as models for percu-
taneous absorption. In these studies, our goals were to establish experimen-
tal protocols and data by which the ultimate objective may be reached.
Percutaneous absorption iji vivo is usually determined by measuring
radioactivity in excreta following topical application of a radioactive dose.
The amount of radioactivity retained in the body or excreted by some route
not assayed (e.g., feces, expired air, sweat) is corrected by determining the
amount of radioactivity excreted in urine,37 following parenteral administra-
tion. Blood (or plasma) radioactivity can also be measured and the percent
absorption determined by the ratio of the areas under the blood concentration
versus time curves following topical and i.v. administration. This method
has generally given results similar to those obtained from urinary excretion
data.42 Determination of percutaneous absorption by those methods does not
take into account the differences in the route and rates of disposition aris-
ing from differences in routes of administration.
Another approach that can be used in determining ui vivo percutane-
ous absorption is to measure the loss of the applied radioactive material from
the surface as it penetrates the skin.42 The difference between the applica-
tion and the residual dose is assumed to be the amount of the chemical absorbed.
Interpretation of these data is sometimes difficult since total recovery from
the skin is never assured. In addition, the skin may act as a reservoir for
unabsorbed material.22 While this method is not generally utilized to generate
20
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dermal penetration data, it obviously offers direct measurements that can re-
duce underestimates for dermal permeability obtained from indirect measure-
ments of excretion or blood levels. In addition, if the dermal application
area is washed thoroughly, the nonrecovered radioactivity probably represents
material that either is absorbed or strongly associated with the skin, and is
available for later absorption. In fact, when the dermal absorption was mea-
sured by sectioning the skin for autoradiographic measurements, the amounts
deposited in the skin were considered as absorbed material.10
In the present studies, both urinary and fecal elimination were
assessed. In addition, in the rat and guinea pig studies, blood, tissues,
and skin application areas were assayed. The dermal absorption of 4,4'-MDA
was quite extensive, ranging from approximately 21% of the applied doses in
the monkey to about 30 and 54% in the guinea pig and rat, respectively. Addi-
tionally, when the topical dose in guinea pigs was increased, the dermal ab-
sorption of 4,4'-MDA also showed some increases. Therefore, the potential
for large quantities of 4,4'-MDA entering the systemic circulation via the
dermal route is quite high. These, however, are the lowest estimates for
percutaneous absorption of 4,4'-MDA since only amounts excreted or recovered
in tissue were considered. When the amounts of radioactivity associated with
the skin that could not be removed by washing are included and considered as
absorbed or available for systemic absorption, the estimate for percutaneous
absorption of 4,4'-MDA is considerably higher.
The data for 4,4'-MDA are similar qualitatively to those developed
for other aromatic amines in rats. The low absorption (percent of dose) demon-
strated for 4,4'-methylene-bis-ortho-chloro-aniline (MBOCA) could be related
to the higher dosage used in the MBOCA studies.19 On the other hand, the
higher absorption demonstrated for benzidine could be due to the lower dosage
and larger application areas used in the studies.34 Our data extend the find-
ings in rats to other species and examine the effects of dosage, concentration,
occlusion and washing procedures which were not assessed in detail for MBOCA
or benzidine in the published studies.
A comparison between the disposition of 4,4'-MDA administered i.v.
to rats, guinea pigs,, and monkeys showed that in 96 h, rats eliminated 67% of
the dose in urine, and 31% in the feces (ratio of about 2:1). In guinea pigs,
the reverse occurred, 35% in urine and 57% in feces. In monkeys, most of the
radioactivity (83%) was eliminated in urine with only limited amounts (9%)
recovered in feces. Elimination data obtained following dermal application
also varied between species. Urinary and fecal elimination in rats averaged
33 and 9%, respectively (about 4:1 ratio). In guinea pigs, the 14C distribu-
tion in urine and feces (7 and 11%) was similar to that after i.v. dosing.
Also, in monkeys, the distribution of radioactivity in urine and feces (20 and
2%, respectively) was similar to that after i.v. administration.
While the effects of increased dose levels (and concentration) on
the absorption, distribution, metabolism, and elimination of xenobiotics have
been extensively studied following oral and parenteral administration, only
limited studies have been reported following dermal application.45 The effect
of increased dose levels on dermal penetration was examined in the present
studies. While both doses utilized (2 and 20 mg/kg) were relatively low, a
21
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significant difference in their disposition behavior was demonstrated. In
rats, 54 and 7% of the low and high doses, respectively, were absorbed during
a 96-h period. If 14C recovered from the application areas is considered,
the absorption estimates increase to 80% following the low doses and 31% fol-
lowing the high dose. Similar data were demonstrated for the guinea pig; 30
and 7% were absorbed following the low and high doses, respectively. When
radioactivity associated with the application areas is included, these values
increase to 59 and 21%, respectively. Although smaller percentages of the
high doses were absorbed, the absolute amounts (in ug/animal) were similar
(rats) or slightly higher (guinea pigs) than after the low doses. Since in
the published literature many of the dermal penetration experiments were
conducted with high doses of the test compounds, which are sometimes higher
than encountered under human exposure conditions, the data from these studies
may significantly underestimate the amounts absorbed. In some studies the
doses utilized (on a mg/kg basis) were not specified and, therefore, it is
difficult to interpret the findings of these studies. Dose-dependent absorp-
tion and/or elimination may not, however, apply to the disposition of all
chemicals.
Immediate washing upon skin contamination is recommended for most
industrial chemicals since it is assumed that washing will remove the chemical
from the exposed area. In the present study, extensive washing procedures
immediately after 4,4'-MDA application failed to remove all the applied doses.
A significant portion of these doses remained available for delayed absorption.
A post-exposure wash at 6 h, 24 h, or 96 h also failed to remove all the 4,4'-
MDA that was not absorbed. Presumably the remaining 14C had been strongly
associated with or was "irreversibly" bound to the skin. These findings and
those recently reported for polychlorinated biphenyls36 indicate that postcon-
tamination washing cannot be assumed to remove all chemical from the skin.
In addition, dermal washing may not only fail to remove skin contaminated
chemical, but it can also promote percutaneous absorption of the chemical.42
This was demonstrated in our study by the increased absorption of 4,4'-MDA fol-
lowing washing with acetone and water.
In the present studies, some differences were noted in the skin
penetration of 4,4'-MDA in the three animal models examined. Depending on
how dermal absorption is estimated, these differences can be significant. In
all cases, however, the rat showed higher absorption than the guinea pig and
monkey, which showed comparable absorption. The relation of these data to
human absorption of 4,4'-MDA is not known and cannot be accurately assessed.
Data related to the dermal absorption of aromatic amines in animal species
are limited, and in humans are almost absent. At present, the animal models
of choice for assessing dermal penetration of aromatic amines are not known.
The animal model of choice may vary depending on the compound of interest. A
comparison to human skin should be made using i_n vitro methods which compare
the HI vitro permeability of skin from humans and animals and relate the find-
ings to the data obtained iji vivo from these animal models.
22
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VI. CONCLUSIONS AND RECOMMENDATIONS
1. Under the conditions of the present study, dermal absorption of
14Olabeled 4,4'-MDA was extensive in the three animal models used: the rats,
the guinea pigs, and the monkeys. Under similar conditions of exposure, com-
parable absorption was demonstrated in the guinea pig and the monkey, and
higher absorpton in the the rat.
2. The studies in rats and guinea pigs, which were performed at two
dose levels (concentrations), showed only limited or no differences in the abso-
lute amounts absorbed (in pg/animal) by increasing the dose 10 times. Although
the percent of absorbed doses was significantly reduced by increasing the ap-
plied dose, the rate of absorption (in ug/h) remained the same in rats and
was doubled in guinea pigs.
3. The present studies clearly indicate that washing of the skin
following 4,4'-MDA application did not remove all of the applied material.
Some remained associated with the skin and available for later absorption and
systemic circulation. The available data indicate that 4,4'-MDA or its metab-
olites are released from the skin and recovered in excreta. Neither washing
with soap or acetone solutions which are generally used in industrial settings
were capable of removing all the applied material. In addition, washing with
acetone facilitated 4,4'-MDA absorption in the three species examined. It is
possible that other related organic solvents behave in a way similar to acetone.
4. Distribution of radioactivity in excretory products differed in
the three species examined. In monkeys, most of the dose was excreted in urine.
In rats, two-thirds of the dose was eliminated in urine, while in guinea pigs
two-thirds of the radioactivity was excreted in feces. The proportions of
14C eliminated in urine and feces after dermal application were similar to
i.v. dosing in monkeys and guinea pigs. Rats, however, showed a higher pro-
portion of the dose in urine. Similarly, while tissue concentrations in rats
were similar after i.v. and dermal dosing, with the highest levels shown in
the liver, significant differences were noted in guinea pigs. After i.v.
dosing the highest tissue levels in guinea pigs were demonstrated in the
spleen while after dermal application, the highest levels were present in the
adrenals.
5. Some j_n vivo methods used for calculating dermal absorption
should be used with caution. Calculating dermal absorption by relating the
urinary excretion following dermal and parentral dosing assumes that the com-
pound or metabolites will be eliminated in a similar way after both routes of
administration. While this appears to be the case for some compounds or in
some species, e.g., 4,4'-MDA in guinea pigs, this is not always accurate.
Relating blood levels only after dermal and i.v. dosing can also generate
significantly higher absorption values at early times. It is possible that
data obtained by calculating area under blood (plasma) concentration values
would offer a more accurate procedure, but this'was not examined in the pres-
ent study.
6. These data suggest considerable absorption of 4,4'-MDA in humans
exposed dermally to 4,4'-MDA. The rate and extent of this absorption, however,
23
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cannot be accurately assessed from the present studies. Since it is not possi-
ble to test 4,4'-MDA absorption directly in humans, the available data devel-
oped with animal models in vivo along with data using animal and human excised
skin ui vitro would provide a good estimate on the rate and extent of human
skin penetration of 4,4'-MDA.
7. Also, the available i_n vivo information on 4,4'-MDA compared
with jhn vitro data on 4,4'-MDA and other related aromatic amines would assist
in establishing a definitive structure activity relationship between these
compounds. The jm vitro studies can also assist in assessing the effect of
different conditions of application including dose, concentration, vehicle,
etc., on the penetration processes.
8. While the present studies provided skin penetration data for
4,4'-MDA in several animal species and assessed the effect of washing on re-
moval of this compound from the skin, it provided no means for qualitative
analysis of the circulating or excretory metabolic product of 4,4'-MDA.
Knowledge of such products is essential for direct assessment of workers or
consumer exposure to 4,4'-MDA. Most aromatic amines are metabolized and only
limited amounts are excreted unchanged. Qualitative data on the excretory
products in different species along with studies in exposed workers would
assist in establishing animal models most relevant to man.
9. No data are available on the disposition (absorption, ditribu-
tion, and elimination) or metabolism of 4,4'-MDA administered orally (by
gavage or feed). Such information is essential to the interpretation of data
on the toxicity and carcinogenicity of this compound which was demonstrated
following oral administration to rodents.
24
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VII. SUMMARY
A. Objective and Study Design
Studies were performed with the overall objective of developing
dermal penetration data for 4,4-methylenedianiline (4,4'-MDA), an aromatic
amine with known toxic and carcinogenic properties. Techniques for measuring
dermal absorption rates were selected and compared. The studies were per-
formed HI vivo using 14Olabeled 4,4'-MDA in three animal models: the Fischer
rat, Hartley guinea pig, and Rhesus monkey. Several conditions of exposure
including dosage (low and high), exposure regimens (limited or continuous),
and occlusion status (exposed or covered) were addressed. In addition, studies
were conducted to determine the dermal washing efficiency of 4,4'-MDA follow-
ing its application in these species. Limited experiments were also performed
to assess the disposition of the test compound following intravenous (i.v.)
administration. The dermal absorption studies in rats and guinea pigs were
performed at two dose levels, "low" and "high," while the i.v. studies were
performed at the low dose. The monkey studies were carried out with the low
dose only. All application areas were kept occluded except in selected stud-
ies. The low dose used was ~ 2 mg/kg (0.4 mg/rat, 1.0 mg/guinea pig, and
10.0 mg/monkey). The high dose was ~ 20 mg/kg (4.0 mg/rat and 10.0 mg/guinea
pig). The test compound concentration was 4 mg/mL for the low dose and 40 mg/
ml for the high dose. The application areas were 2 cm2 for rats (~ 200 g each)
5 cm2 for guinea pigs (~ 500 g each) and 50 cm2 for monkeys (~ 5000 g each).
Ethanol (95%) was used for the dermal studies while ethanol:water (25:75) was
utilized as the vehicle for the i.v. studies.
B. Dermal Washing Efficiency Studies
These experiments were performed to assess the extent of removal of
dermally applied 14C-labeled 4,4'-MDA by washing with soap and water or with
acetone and water. Six male rats, six male guinea pigs, and four male monkeys
were treated dermally with low doses of the test compound, then the application
areas were washed 5 to 10 min later with either solution. After washing, the
animals were housed in individual metabolism cages for excreta (urine and
feces) collection during a 4-day (rats and guinea pigs) or 7-day period
(monkeys). At termination the skin application areas (from rats and guinea
pigs enly) were removed, extracted with methanol, then solubilized. Radio-
activity was measured in the washing solutions, in excreta, and in the appli-
cation areas.
The studies in the three species showed that higher and more con-
sistent recoveries were obtained by washing the application areas with soap
and water versus acetone and water (Table A). Recoveries (percent of applied
dose) in the dose wash were 91.3% versus 85.1% (of applied dose) in rats,
84.5% versus 76.0% in guinea pigs, and 63.0% versus 53.0% in monkeys. In
addition, lower amounts were recovered in excreta after washing with soap and
water than with acetone and water: 1.6 versus 7.7% in rats, 0.6 versus 2.1%
in guinea pigs, and 8.7 versus 16.2% in monkeys. The studies showed that
both washing methods were incapable of removing all the applied material from
the skin; significant amounts of the applied doses remained on the skin avail-
able for absorption. In addition, the results suggested that acetone facili-
tated absorption during the washing process. In rats and guinea pigs, the
25
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skin application areas contained higher amounts of radioactivity after washing
with acetone and water than after washing with soap and water (17.7 versus
3.8% in rats and 5.9 versus 3.8% in guinea pigs). The total recoveries were
lower for monkeys since the application areas were not sampled. Removal of
the applied doses from monkey skin appeared more difficult than from rat or
guinea pig skin as evidenced by the lower recoveries in the washing solutions
and the higher recoveries in excreta.
C. Rat Studies
These studies were performed with male rats treated dermally at the
low or high doses of 14C-labeled 4,4'-MDA or i.v. at the low dose. After
dermal application, the rats were placed in metabolism cages for excreta col-
lection until sacrificed at 6, 24, or 96 h for tissue sampling. At sacrifice,
the application areas were washed with soap and water. Two additional groups
were treated dermally with the low or high doses and the applied doses were
washed 24 h later, and then excreta collection continued until the animals
were sacrificed at 96 h. Another group was treated dermally with the low
dose and the application area was kept nonoccluded until sacrifice 6 h later.
In rats treated dermally with the low doses which were kept on the
skin for the duration of the study, 11.9, 27.8, and 54.5% of the applied doses
were recovered in excreta and tissue at 6, 24, and 96 h after dosing; 62.1,
52.1, and 24.7% were washed from the application areas and 30.6, 25.8, and
25.6% were recovered from the skin following methanol extraction and skin
solubilization (Table B). In rats treated dermally with the low dose for 24 h
then the dose removed, recoveries in tissue and excreta at 96 h averaged 42.9%.
In the dose wash, 52.1% was recovered at 24 h and 10.7% remained associated
with the skin 72 h after washing (Table C). In rats with the application
areas nonoccluded, 7.0% of the low dose was recovered in the excreta and tis-
sue at 6 h, 70.2% was recovered in the dose wash, and 27.5% was associated
with the skin application area.
In rats treated dermally with the high doses which were kept on the
skin for 96 h, only 7.5% of the applied doses were recovered in tissue and
excreta during this period. Slightly lower amounts (5.3%) were recovered
during the same period when the dose was washed 24 h after application. In
the first group (continuous application), 62.5% of the doses were removed with
soap and water at 96 h and 24.0% recovered from the application area. In the
second group (limited application), 77.9% was removed at 24 h and 8.9% recov-
ered from the application area at 96 h (Tables B and C).
In the i.v. studies, most of the administered doses were recovered
in excreta (urine and feces) by 24 h after dosing. Total recoveries in ex-
creta and tissue averaged 98.3, 98.1, and 99.3% at 6, 24, and 96 h after dos-
ing (Table B). During 96 h following i.v. dosing, 67.0 and 30.7% of doses
were eliminated in urine and feces, respectively. During the same time peri-
od, rats treated dermally with the low dose for 96 h excreted 43.0 and 10.0%
of doses in urine and feces, respectively. Following the high doses, excre-
tion in urine and feces during a 96-h period averaged 4.8 and 1.3%, respec-
tively.
26
-------
Tissue distribution studies following i.v. dosing showed that sig-
nificant amounts of the radioactivity (*> 24% at 6 h) were recovered in the GI
tract, probably through the bile. Blood and tissue contained ~ 19% of the
doses at 6 h, but their 14C contents declined to ~ 2% of the doses at 96 h.
The highest 14C concentrations were demonstrated in the liver, which was five
to nine times higher than blood. After continuous dermal application of the
low dose, ^ 4% was recovered in the GI tract and similar amounts were present
in blood and tissues. Also, the liver showed the highest concentration (four
to nine times higher than blood). Distribution in tissue was similar after
the high dose, but the proportion of doses (percent of doses) recovered in
tissues was lower than after the low dose.
D. Guinea Pig Studies
These studies were performed with male guinea pigs treated dermally
with the low or high doses of 14C-labeled 4,4'-MDA or i.v. at the low dose.
The study design was similar to the rat studies except that i.v. dosing was
limited to one group of guinea pigs sacrificed at 96 h. After dosing, the
animals were placed in metabolism cages for excreta collection until sacri-
ficed at 6, 24, or 96 h for tissue sampling. At sacrifice, the application
areas were washed with soap and water. As in the rat studies, two additional
groups were treated dermally with the low or high doses and the application
areas were washed 24 h later; then the animals were sacrificed at 96 h. In
addition, a group of guinea pigs were treated dermally with the low dose and
the application areas were kept nonoccluded until the animals were sacrificed
6 h later.
In guinea pigs treated dermally with the low doses which were kept
on the skin for the duration of the study, 3.2, 17.5, and 29.9% of the doses
were recovered in excreta and tissue at 6, 24, and 96 h after dosing. At
these times, 80.6, 58.8, and 40.9% were recovered in the dose wash and 11.4,
14.8, and 29.4% remained associated with the application area (Table D). In
guinea pigs treated dermally with the low dose and dose removed at 24 h, re-
coveries in tissue and excreta at 96 h averaged 18.0%; 61.8% was present in
the dose wash at 24 h and 16.6% was recovered from the skin at 96 h. In ani-
mals with the application areas nonoccluded, 3.1% of the dose was recovered
in the excreta and tissue, 87.5% was recovered in the dose wash, and 9.4% was
associated with the skin application area.
After dermal application of the high dose which was kept on the skin
for 96 h, only 7.5% of the applied doses were recovered in excreta and tissue
during this period. Slightly lower amounts (4.2%) were recovered during the
same period when the doses were washed 24 h after application. In the first
group (continuous application), 69.7% of the dose was washable with soap and
water at 96 h and 13.7% recovered from the application area. In the second
group (limited application), 80.4% of the dose was washed at 24 h and 7.3%
was recovered from the application area at 96 h (Table E).
Following i.v. dosing of guinea pigs with 14C-labeled 4,4'-MDA, most
of the dose was recovered in urine and feces by 48 h after dosing. Recoveries
in excreta and tissue averaged 95.0% at 96 h after 4,4'-MDA administration
(Table D). During 96 h following i.v. dosing, 35.0 and 56.5% of doses were
eliminated in urine and feces, respectively. Guinea pigs treated dermally
27
-------
with the low dose for 96 h excreted 10.5 and 17.6% of the doses in urine and
feces, respectively, during the same period. Excretion in urine and feces
during a 96 h period averaged 2.8 and 3.6% after dermal application of the
high dose.
Tissue distribution studies showed that only limited amounts were
recovered in the GI tract (0.6%), blood, and tissue (2.9%) at 96 h after i.v.
dosing. With the high fecal elimination after i.v. dosing, excretion into
the GI tract at earlier times appeared significant. The highest 14C contents
were demonstrated in the spleen (six times higher than blood) followed by the
liver. Blood and liver contents were higher in guinea pigs than;in rats.
After dermal dosing, a different tissue distribution pattern was noted. Very
high concentrations were noted in the adrenals (up to 23 times higher than
blood) at all sacrifice times following dermal application of the low or high
doses. The concentrations in liver were also higher than blood, but 14C levels
in the spleen were low. When exposure to the test compound was discontinued
by washing the dose at 24 h, animals treated with the high dose maintained
high adrenal contents, while these contents were significantly reduced after
discontinuing (washing off) the low dose.
E. Monkey Studies
These studies were performed with male monkeys treated dermally or
i.v. with the low dose of 14C-labeled 4,4'-MDA. Following dosing, the monkeys
were kept in metabolism chairs for 24 h, the skin was washed, and then each
monkey was housed in a metabolism cage for excreta collection during a 168-h
period. The monkeys were not sacrificed and, therefore, the skin applications
areas were not sampled. In the dermally treated monkeys, 20.8% of the applied
doses were eliminated in the urine and feces during the 168-h period. Recovery
during a 96 h period averaged 18.0%. Only 47.3% of the dose was recovered in
the dose wash at 24 h after treatment. The total recoveries for these animals
averaged 68% of the dose, which indicated that significant portions of the
doses remained on the skin available for later absorption. In contrast, re-
coveries were almost quantitative in the monkey treated i.v.; an average of
94.1% of the dose was excreted during the 7-day collection period. Most of
the radioactivity was excreted by 48 h following dosing (see Table G). Fol-
lowing both i.v. and dermal administration, 14C excretion in monkeys occurred
primarily in urine. Urinary and fecal excretion averaged 84.4 and 9.8%, re-
spectively, after i.v. dosing, and 18.8 and 1.9%, respectively, after dermal
application.
F. Conclusions
Under the conditions of these studies, dermal penetration of 4,4'-MDA
was extensive in the three animal models examined. The absorbed radioactivity
was generally distributed throughout the body and was readily eliminated in
urine and feces. Association of 4,4'-MDA and/or its metabolites to the skin
was also significant, resulting in low washing efficiency with soap or acetone
solutions. After washing, considerable amounts of the applied radioactivity
remained associated with the skin available for later absorption. When only
the amounts of radioactivity excreted in urine and feces or recovered in blood
and tissue were considered in determining dermal absorption, the rats demon-
strated the highest absorption followed by the guinea pigs and the monkeys.
28
-------
When the amounts of radioactivity associated with the skin were considered in
calculating 4,4'-MDA dermal absorption, comparable absorption was demonstrated
for the rats, guinea pigs, and monkeys.
The distribution of radioactivity recovered in urine and feces dif-
fered in the three species, allowing only direct comparison of data obtained
by both routes of elimination. Although in monkeys the application areas were
washed at 24 h following treatment and the collection period was extended to
7 days, direct comparison between the three species was possible since the
study design incorporated groups of rats and guinea pigs in which the dose
was removed at 24 h following application. Under these comparable conditions
of treatment, 52, 62, and 47% of the applied doses were washable at 24 h in
rats, guinea pigs, and monkeys, respectively. Total amounts excreted in
urine and feces during 96-h periods were 42, 17, and 18% in these three
species. These data indicate comparable absorption in monkeys and guinea
pigs and higher absorption in rats. As expected, maximum absorption (based
on percent of applied dose) occurred in animals following continuous treat-
ment with the low dose and with the application areas remained occluded.
These maximum amounts averaged 54% in rats and 30% in guinea pigs during a
96-h period. In monkeys, the maximum absorption (determined by urinary and
fecal elimination only) averaged 21% of the applied doses in 168 h. The total
amounts excreted in urine and feces and recovered in tissues provided more
accurate estimates of dermal absorption than the amounts obtained by measur-
ing urinary excretion and correcting for recovery from the parenterally ad-
ministered doses. Assessment of absorption based on recovery of 14C in blood
and/or tissue only did not provide accurate measurements of the amounts ab-
sorbed.
While increasing the 4,4'-MDA applied doses (and concentration) re-
sulted in lower percentages of the doses that penetrated the skin, the abso-
lute amounts absorbed (in (jg/animal) remained the same (in rats) or increased
(in guinea pigs) by increasing the dose. In rats, increasing the applied dose
10 times resulted in a decrease of percent of dose absorbed from 54.5 to 6.6%,
but the average absorption rate over a 96 h period was similar (2.3 ug/h and
2.8 |jg/h after the low and high doses, respectively). In guinea pigs, absorp-
tion decreased from 29.9% after the low dose to 7.9% after the high dose, but
the absorption rate increased from 3.1 to 8.0 ug/h (Table H). Occulsion in-
creased 4,4'-MDA dermal absorption in rats (11.9 versus 7.0%) but had no ef-
fect on its dermal penetration in guinea pigs (3.2 versus 3.4%). However, the
studies performed to assess occlusion were limited to 6-h sampling periods and,
therefore, definitive conclusions cannot be reached. When 4,4'-MDA applied to
the skin was washed immediately with soap and water or acetone and water, as
low as 53% was recovered in the washing solutions. A post-exposure wash at
6 h, 24 h, and 96 h removed less of the applied material. Post-contamination
washing cannot be assumed to remove all 4,4'-MDA from the skin.
29
-------
Table A. Dermal Washing Efficiency of 4.4'-MDA in
Rats, Guinea Pigs, and Monkeys '
Percent of dose
Soap and water wash
Rats G. Pigs Monkeys
Acetone and water wash
Rats G. Pigs Monkeys
Urinec
Fecesc
Dose wash
Application
area
Recovery
1.21
0.35
91.33
3.80
96.68
0.33
0.31
84.52
3.83
88.99
7.25
1.49
62.98
ND
71.71
5.89
1.80
85.09
12.71
105.49
1.36
0.76
76.04
5.91
84.07
14.47
1.76
53.00
ND
69.23
a
,10.0 mg/monkey). The doses were washed 5-10 min after application.
Mean of three for rats and guinea pigs and 2 for monkeys.
^Excreta collected for 96 h in rats and guinea pigs and 168 h in monkeys.
Application areas of skin were removed, processed, and counted. Monkey
skin was not sampled.
Table B. Recovery of Radioactivity in Rats Treated i.v
or Dermally with 4,4'-MDA
6 h
i.v.
24 h
0.4
96 h
Percent
mg/rat
6 h
of .dose
Dermal
24 h 96 h
4 mg/rat
Dermal
96 h
Blood 2.80 0.44 0.22
Tissue 16.60 4.33 1.31
GI tract 23.94 4.14 0.11
Urine 54.65 67.35 66.96
Feces 0.28 21.79 30.66
Total absorbed 98.27 98.06 99.25
Dose wash -
Application -
area
0.77
4.
3.
2.
70
.80
55
0.04
11.85
62.08
30.59
0.29
2.15
2.98
20.03
2.31
27.76
52.08
25.84
0.11
0.88
0.48
43.04
9.96
54.47
24.67
25.55
0.03
0.22
0.18
4.
1.
6.
82
34
58
62.45
24.00
Recovery
98.27 98.06 99.25 104.51 105.68 104.68
93.02
.Mean of three rats per group.
Continuous application for the period indicated.
.Recovered in blood, tissue, GI tract, and excreta.
Removed by washing with soap and water.
Removed by methanol extraction and skin solubilization.
30
-------
Table C. Recovery of Radioactivity in Rats Treated Dermally
with 4,4'-MDA under Different Conditions3
Blood
Tissue
GI tract
Urine
Feces .
Total absorbed
Dose wash ,
Application area
6 h/
nonoccluded
0.53
3.26
1.24
1.95
0.03
7.00
70.21
27.53
Percent of dose
0.4 mg/rat
24 h wash/
96 h sacrifice
0.07
0.38
0.08
33.23
9.18
42.93
52.12
10.67
4.0 mg/rat
24 h wash/
96 h sacrifice
0.01
0.13
0.07
4.00
1.06
5.27
77.92
8.93
Recovery
104.74
105.72
92.11
.Mean of 3 rats per group.
.Recovered in blood, tissue, and excreta.
^Removed by washing with soap and water.
Removed by methanol extraction and skin solubilization.
Table D. Recovery of Radioactivity in Guinea Pigs Treated
i.v. or Dermally with 4,4'-MDA
96 h/
i.v.
Percent of
1.0 mg/G. pig
6 h/ . 24 h/.
dermal dermal
dose
96 h/
dermal
10 mg/G. pig.
96 h/dermal°
Blood 0.55 0.12
Tissue 2.39 1.11
GI tract 0.61 1.51
Urine 34.98 0.35
Feces 56.45 0.10
Total absorbed0 94.98 3.19
Dose wash - 80.55
Application - 11.43
area
0.14
.02
.80
.81
,70
17.46
58.78
14.77
1.
2.
7.
5.
0.14
1.08
0.59
10.47
17.62
29.89
40.85
29.41
0.04
0.43
0.64
2.75
3.62
7.48
69.70
13.72
Recovery
94.98
95.16
91.01
100.14
90.89
.Mean of three guinea pigs per group.
Continuous application of the period indicated.
.Recoved in blood, tissue, and excreta.
Removed by washing with soap and water.
Removed by methanol extraction and skin solubilization.
31
-------
Table E. Recovery of Radioactivity in Guinea Pigs Treated Dermally
with 4,4'-MDA under Different Conditions
Blood
Tissue
GI tract
Urine
Feces .
Total absorbed
Dose wash ,
Application area
1.0
6 h/
nonoccluded
0.12
1.02
1.27
0.60
0.12
3.14
87.46
9.37
Percent of dose
mg/G . pig
24 h wash/
96 h sacrifice
0.08
0.33
0.12
6.56
10.89
17.98
61.78
16.58
10 mg/G. pig
24 h wash/
96 h sacrifice
0.02
0.13
0.28
1.54
2.18
4.15
80.44
7.29
Recovery 99.97 96.34 91.88
.Mean of three guinea pigs per group.
Recovered in blood, tissue, and excreta.
•Removed by washing with soap and water.
Removed by methanol extraction and skin solubilization.
Table F. Recovery of Radioactivity in Monkeys Treated i.v.
or Dermally with 4,4'-MDA
Percent of dose
10.0 mg/monkey
168 h/i.v.168 h/dermal1
Urine
Feces
Total absorbed
Dose wash
84.34
9.75
94.09
18.83
1.93
20.76
47.27
Recovery 94.09 68.03
.Mean of three monkeys, per group.
The dose was washed at 24 h after application.
.Recovered in excreta. Tissues and application areas of skin were not sampled.
Removed by washing with soap and water.
32
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Table G. Percutaneous Absorption of 4,4'-MDA
Based on Urinary Excretion Data Only
Cumulative
% of dose in urine
% Absorption
Dermal x 100
Absorption ratec
Time (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
120
144
168
I.V.
36.24
55.10
63.84
65.61
66.51
66.96
36.24
55.10
63.84
65.61
66.51
66.96
6.43
21.76
30.65
33.96
34.64
34.98
6.43
21.76
30.65
33.96
34.64
34.98
24.85
45.95
63.69
79.35
82.48
83.27
83.64
83.90
84.34
Dermal
Rats,
1.51
9.77
23.45
36.11
40.69
43.04
Rats ,
c 0.15
0.52
1.19
2.58
3.77
4.82
Guinea Pigs,
0.22
1.52
4.32
7.64
9.13
10.47
Guinea Pigs,
c 0.04
0.30
0.54
0.94
1.63
2.75
Monkey
0.19
1.01
3.84
11.20
15.12
16.75
17.66
18.30
18.83
I.V.
Low Dose (0.4 mq/Rat)
4.2
17.7
36.7
55.0
61.2
64.3 (54.5)D
High Dose (4 mg/Rat)
0.4
0.9
1.9
3.9
5.7 .
7.2 (6.6)D
Low Dose (1.0 g/Guinea
3.4
7.0
14.1
22.5
26.4 .
29.9 (29.9)D
High Dose (10 mg/Guinea
0.6
1.4
1.8
2.8
4.7 .
7.9C (7.5)D
Low Dose (10 mg/Monkey)
0.8
2.2
6.0
14.1
18.3
20.1 (18.1)
21.1
21.8
22.3 (20.8)
% dose/h
0.70
1.48
1.53
1.14
0.85
0.67
0.07
0.08
0.08
0.08
0.08
0.08
Pig)
0.57
0.58
0.59
0.47
0.37
0.31
Pig)
0.10
0.12
0.08
0.06
0.06
0.05
0.13
0.18
0.25
0.29
. 0.25
b 0.21
0.18
. 0.15
n
D 0.13
|jg/h
2.8
5.9
6.1
4.6
3.4
2.7
2.8
3.2
3.2
3.2
3.2
3.2
5.7
5.8
5.9
4.7
3.7
3.1
10.0
12.0
8.0
6.0
6.0
5.0
13
18
25
29
25
21
18
15
13
Average rate calculated from cumulative absorption based on the urinary
.excretion data.
Recoveries in urine, feces, and tissue (where applicable).
I.V. treatment with the low dose only.
33
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Table H. Percutaneous Absorption of 4,4'-MDA Based on Recoveries
in Excreta, Tissue, and Skin Application Areas
Group
% of dose in
Excreta
and Tissue
Absorption rate
% dose/hr (jg/h
Skin
Application
Area
Total
6 h
24 h
96 h
24/96 h
6 h nonoccluded
96 h
24/96 h
6 h
24 h
96 h
24/96 h
6 h nonoccluded
96 h
24/96 h
Rats, Low Dose (0.4 mg/Rat)
11.85 1.98 7.92 30.59
27.76 1.16 4.64 25.84
54.47 0.57 2.28 25.55
42.93 0.45 1.80 10.67
7.00 1.17 4.68 27.53
Rats, High Dose (4 mg/Rat)
6.58 0.07 2.80 24.00
5.27 0.05 2.00 8.93
Guinea Pigs, Low Dose (1.0 mg/Guinea Pig)
3.19 0.53 5.3 11.43
17.46 0.73 7.3 14.77
29.89 0.31 3.1 29.41
17.98 0.19 1.9 16.58
3.14 0.52 5.2 9.37
Guinea Pigs, High Dose (10 mg/Guinea Pig)
7.48 0.08 8.0 13.72
4.15 0.04 4.0 7.29
42.44
53.60
80.02
53.59
34.53
30.58
14.20
14.62
32.23
59.30
34.56
12.51
21.20
11.44
24/96 h
24/168
h
18
20
Monkeys ,
•04h
.76b
Low Dose
0.18
0.12
(10
mg/Monkey)
18.0
12.0
(32.
00)C
52.76
Remaining after skin washing with soap and water.
.Excreta only.
'Skin application area and tissue as estimated from differences in recovery.
34
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VIII. REFERENCES
1. Andersen KE, Maibach HI, Anjo MD. 1980. The guinea pig: an animal
model for human skin absorption of hydrocortisone, testosterone and
benzoic acid? Br J Dermatol 102:447-453.
2. Anjo DM, Feldman RJ, Maibach HI. 1980. Methods for predicting percu-
taneous penetration in man. In: Percutaneous penetration of steroids.
Mauvais-Jarvais P, ed. New York: Academic Press, pp. 31-51.
3. Bartek MJ, LaBudde JA. 1975. Percutaneous absorption HI vitro. In:
Animal models in dermatology. Maibach HI, ed. , New York: Churchill
Livingstone, pp. 103-120.
4. Bartek MJ, LaBudde JA, Maibach HI. 1972. Skin permeability i_n vivo:
comparison in rat, rabbit, pig and man. J Invest Dermatol 58:114-123.
5. Braselton WE, Williams DE, McCormack KE. 1982. Pharmacokinetics of 4,4'-
methylene(2-chloroaniline). Michigan Toxic Substance Control Commission,
Lansing, MI.
6. Bronaugh RL, Congdon ER. 1984. Percutaneous absorption of hair dyes:
correlation with partition coefficients. J Invest Dermatol 83:124-127.
7. Bronaugh RL, Maibach HI. 1983. Jji vivo percutaneous absorption. In:
Dermato toxicology, 2nd ed. Marzulli FN, Maibach HI, eds. Washington,
DC: Hemisphere, pp. 117-129.
8. Bronaugh RL, Stewart RF, Congdon ER. 1982b. Methods for _ui vitro percu-
taneous absorption studies. III. Differences in permeability of rat
skin related to sex and body site. Toxicol Appl Pharmacol (in press).
9. Bronaugh RL, Congdon ER, Scheuplein RJ. 1981. The effect of cosmetic
vehicles on the penetration of N-nitrosodiethanolamine through excised
human skin. J Invest Dermatol 76:94-96.
10. Chin B, Tobes MC, Han SS. 1983. Absorption of 4,4'-methylenebis[2-
chloroaniline] by human skin. Env.Res 32:167-178.
11. Feldman RJ, Maibach HI. 1965. Penetration of 14C-hydrocortisone through
normal skin. Arch Dermatol 91:661-666.
12. Feldman RJ, Maibach HI. 1974. Percutaneous penetration of some pesti-
cides and herbicides in man. Toxicol Appl Pharmacol 28:126-132.
13. Feldman RJ, Maibach HI. 1967. Regional variation in percutaneous pene-
tration of 14C-corth'col in man. J Invest Dermatol 48:181-183.
14. Feldman RJ, Maibach HI. 1970. Absorption of some organic compounds
through the skin in man. J Invest Dermatol. 54:399-404.
35
-------
15. Feldman RJ, Maibach HI. 1969. Percutaneous penetration of steroids in
man. J Invest Dermatol 52:89-94.
16. Franz TJ. 1978. The finite dose technique as a valid jji vitro model
for the study of percutaneous absorption. Curr Probl Dermatol 7:58-68.
17. Franz TJ. 1975. Percutaneous absorption. On relevance of J_n vitro
data. J Invest Dermatol 64:190-195.
18. Franz, TJ. 1983. Kinetics of cutaneous drug penetration. Int J
Dermatol 22:499-505.
19. Groth DH, Weigel WW, Tolos WP, Brewer DE, Cheever KL, Burg JR. 1984.
4,4'-Methylene-bis-ortho-chloro-aniline MBOCA): absorption and execre-
tion after skin application and gavage. Environ Res 34:38-54.
20. Guy RH, Hadgraft J, Maibach HI. 1982. A pharmacokinetic model for
percutaneous absorption. Int J Pharmaceut 11:119-129.
21. Hadgraft J. 1980. Theoretical aspects of metabolism in the epidermis.
Int J Pharm 4:229-239.
22. Hadgraft J. 1979. The epidermal reservoir; a theoretical approach.
Int J Pharm 2:265-274.
23. Hawkins GS Jr, Reifenrath WG. 1984. Development of an jhn vitro model
for determining the fate of chemicals applied to skin. Fund and Appl
Toxicol 4:S133-S144.
24. Idson B. 1983. Vehicle effects in percutaneous absorption. Drug Metab
Rev 14(2):207-222.
25. Idson B. 1975. Percutaneous absorption. J Pharm Sci 64:901-924.
26. Marzulli FN, Anjo DM, Maibach HI. 1981. In vivo skin penetration stud-
ies of 2,4-toluenediamine, 2,4-diaminoanisole, 2-nitro-p-phenylenediamine,
£-dioxane, and n-nitrosodiethanolamine in cosmetics. Fed Cosmet Toxicol
19:741-747.
27. Marzulli FN, Brown DWG, Maibach HI. 1969. Techniques for studying skin
penetration. Toxicol Appl Pharmacol 3(Suppl):76-83.
28. Marzulli FN, Maibach HI. 1975. Relevance of animal models: the hexa-
chlorophene story. In: Animal models in dermatology. Maibach HI, ed.
Edinburgh: Churchill Livingstone, pp. 156^167.
29. Reifenrath WG, Chellquist EM, Shipwash EA, Jederberg WW. 1984. Evalua-
tion of animal models for predicting skin penetration in man. Fund Appl
Toxicol 4:S224-S230.
36
-------
30. Reifenrath WG, Chellquist EM, Shipwash EA, Jederberg WW, Krueger GG.
1984. Percutaneous penetration in the hairless dog, weanling pig and
grafted athymic nude mouse: evaluation of models for predicting skin
penetration in man. Br J Dermatol III(Suppl 27):123-135.
31. Reinfenrath WG, Hill JA, Robinson PB, McVey DL, Akers WA, Anjo DM,
Maibach, HI. 1980. Percutaneous absorption of carbon 14 labeled insect
repellants in the hairless dog. J Environ Pathol Toxicol 4:249-256.
32. Scheuplein RJ, Blank IH. 1971. Permeability of the skin. Physiol Rev
51:702-747.
33. Scheuplein RJ. 1977. Permeability of the skin. In: Handbook of
physiology. American Physiological Society, pp. 299-322.
34. Shah PV, Guthrie FE. 1983. Dermal absorption of benzidine derivatives
in rats. Bull Environ Contam Toxicol 31:73-78.
35. Stoughton RB. 1975. Animal models for HI vitro percutaneous absorption.
In: Animal models in dermatology. Maibach HI, ed. Edinburgh: Churchill
Livingstone, pp. 121-132.
36. Wester RC, Bucks DAW, Maibach HI, Anderson J. 1983. Polychlorinated
biphenyls (PCBs): dermal absorption, systemic elimination, and dermal
wash efficiency. J Toxicol Environ Health 12:511:519.
37. Wester RC, Maibach HI. 1983. Cutaneous pharmacokinetics: 10 steps to
percutaneous absorption. Drug Metabol Rev 14:169-205.
38. Wester RC, Noonan PK. 1980. Relevance of animal models for percu-
taneous absorption. Int J Pharmacol 7:99-110.
39. Wester RC, Noonan PK, Maibach HI. 1980. Variations in percutaneous ab-
sorption of testosterone in the rhesus monkey due to anatomic site of
application and frequency of application. Arch Dermatol Res 267:229-235.
40. Wester RC, Maibach HI. 1975. Percutaneous absorption in the rhesus
monkey compared to man. Toxicol Appl Pharmacol 32:394-398.
41. Wester RC, Maibach HI. 1984. Advances in percutaneous absorption. In:
Cutaneous toxicity. Drill V, Lazar P, eds. New York: Raven Press.
42. Wester RC, Maibach HI. 1982. JTI vivo percutaneous absorption. In:
Dermatotoxicology, 2nd ed. Marzulli FN, Maibach HI, eds. Washington,
DC: Hemisphere, pp. 131-146.
43. Wester RC, Maibach HI. 1977. Percutaneous absorption in man and animal:
a perspective. In: Cutaneous toxicity. Drill V, Lazar P, eds. New York:
Academic Press, pp. 111-126.
37
-------
44. Wester RC, Mai bach HI. 1975. Rhesus monkey as an animal model for per-
cutaneous absorption. In: Animal models in dermatology. Mai bach HI,
ed. Edinburgh: Churchill-Livingston, pp. 133-137.
45. Wester RC, Maibach HI. 1976. Relationship of topical dose and percu-
taneous absorption of rhesus monkey and man. J Invest Dermatol 67:518-
520.
46. Yankell SL. 1969. The effects of various vehicles on absorption rates
in skin. In: Advances in biology of skin, Vol. 12. Montagna W,
van Scott EJ, Stoughton RB, eds. New York: Appleton-Century-Crofts,
p. 511.
38
-------
Table 1. Dermal Washing Efficiency of 14C-Labeled 4,4'-MDA in
Male Fischer 344 Rats, Hartley Guinea Pigs, and Rhesus Monkeys
Percent of dose
Time after Soap and Water Wash
Excretum dosing (h) Rats
Urine
Feces
Urine
Feces
Dose wash
Application
Recovery
6
12
24
48
72
96
120
144
168
6
12
24
48
72
96
120
144
168
6
12
24
48
72
96
120
144
168
6
12
24
48
72
96
120
144
168
area
0.23 ± 0.09
0.41 ± 0.11
0.24 ± 0.04
0.15 ± 0.01.
0.08 ± 0.02
0.09 ± 0.03
0.01 ± 0.00
0.05 ± 0.03
0.06 ± 0.01
0.14 ± 0.02
0.05 ± 0.00
0.04 ± 0.01
0.23 ± 0.09
0.64 ± 0.20
0.89 ± 0.23
1.03 ± 0.24
1.11 ± 0.25
1.21 ± 0.28
0.01 ± 0.00
0.06 ± 0.03
0.12 ± 0.04
0.26 i 0.05
0.31 ± 0.05
0.35 ± 0.05
91.33 ± 2.95
3.80 ± 0.16
96.68 ± 2.90
Acetone and water wash
Guinea pigs Monkeysc Rats
0.04 ± 0.02
0.06 ± 0.02
0.10 ± 0.03
0.07 ± 0.01
0.04 ± 0.01
0.02 ± 0.01
0.00 ± 0.00
0.01 ± 0.00
0.13 ± 0.05
0.10 ± 0.03
0.04 ± 0.01
0.03 ± 0.00
0.04 ± 0.02
0.10 ± 0.03
0.20 ± 0.06
0.28 ± 0.07
0.31 ± 0.07
0.33 ± 0.08
0.00 ± 0.00
0.01 ± 0.01
0.14 ± 0.05
0.23 ± 0.08
0.28 ± 0.08
0.31 ± 0.08
84.52 ± 3.27
3.83 ± 0.57
89.00 ± 2.94
0.28
0.42
1.39
1.65
1.49
0.69
0.53
0.40
0.41
0.00
0.01
0.02
0.26
0.36
0.28
0.22
0.16
0.19
Cumulative
0.28
0.70
2.08
3.74
5.23
5.91
6.44
6.84
7.25
0.00
0.01
0.03
0.28
0.64
0.92
1.14
1.30
1.49
62.98d
NDd
71.71
0.65 ± 0.07
2.38 ± 0.91
1.38 ± 0.53
0.75 ± 0.11
0.37 ± 0.11
0.36 ± 0.09
0.01 ± 0.00
0.09 ± 0.04
0.32 ± 0.14
0.57 ± 0.06
0.37 ± 0.15
0.45 ± 0.19
percent of dose
0.65 ± 0.07
3.03 ± 0.88
4.41 ± 1.41
5.15 ± 1.51
5.52 ± 1.61
5.89 ± 1.68
0.01 ± 0.00
0.10 ± 0.04
0.42 ± 0.15
0.99 ± 0.20
1.36 ± 0.35
1.80 ± 0.52
85.09 ± 6.17
12.71 ± 1.14
105.49 ± 4.27
Guinea pigs
0.14 ± 0.07
0.22 ± 0.15
0.34 ± 0.11
0.37 ± 0.05
0.19 ± 0.06
0.11 ± 0.02
0.00C
0.08 ± 0.07
0.21 ± 0.04
0.19 ± 0.09
0.19 ± 0.04
0.10 ± 0.01
0.14 ± 0.07
0.36 ± 0.22
0.69 ± 0.31
1.06 ± 0.35
1.26 ± 0.39
1.36 ± 0.41
0.00C
0.08 ± 0.07
0.29 ± 0.10
0.48 ± 0.16
0.66 ± 0.19
0.76 1 0.20
76.04 ± 4.13
5.91 ± 0.65
84.07 ± 3.06
Monkeys0
0.33
0.69
1.73
5.36
2.81
1.54
0.81
0.76
0.46
. 0.00
0.00
0..06
0.43
0.46
0.30
0.22
0.12
0.16
0.33
1.02
2.75
8.11
10.91
12.45
13.26
14.01
14.47
0.00
0.00
0.07
0.49
0.95
1.25
1.47
1.59
1.76
53.00
NDd
69.23
a .
Mean ± SE of three animals per group.
^Average of two animals.
Not determined.
39
-------
Table 2. Urinary and Fecal Excretion of Radioactivity in Male Fischer 344 Rats Treated
Dermally or Intravenously with 14C~Labeled 4,4'-MDA (0.4 or 4.0 mg/Rat):
Preliminary Study
Percent of dose
Excretum
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
0.4 mg
dermal
No. 13
0.54
7.47
8.88
11.03
4.42
2.64
0.00
0.06
0.68
3.78
1/76
1.18
0.55
7.53
9.57
14.82
6.18
3.82
4.0 mg
dermal
No. 14
0.14
0.62
1.78
2.39
3.46
3.49
0.00
0.00
0.08
1.09
1.11
1.30
0.14
0.62
1.86
3.48
4.57
4.79
0.4 mg
i. v.
No. 15
15.77
30.09
13.35
2.02
0.66
0.56
0.11
0.36
14.91
16.37
0.65
0.38
15.88
30.45
28.26
18.39
1.31
0.94
Cumulative percent
0.4 mg
dermal
No. 13
0.54
8.01
16.89
27.93
32.34
34.98
0.00
0.07
0.75
4.53
6.29
7.47
0.55
8.08
17.64
32.46
38.64
42.45
4.0 mg
dermal
No. 14
0.14
0.76
2.54
4.93
8.39
11.88
0.00
0.01
0.08
1.17
2.28
3.58
0.14
0.76
2.62
6.10
10.67
15.46
of dose
0.4 mg
i. v.
No. 15
15.77
45.86
59.21
61.23
61.89
62.45
0.11
0.47
15.37
31.74
32.39
32.78
15.88
46.33
74.59
92.97
94.28
95.23
-------
Table 3. Radioactivity in Blood, Tissue, and Excreta of Male Fischer 344 Rats at 96 h Following a
Dermal or Intravenous Dose of 14C-|_abeled 4,4'-MDA (0.4 or 4.0 mg/Rat):
Preliminary Study
ug equivalents/g
Tissue/
excretum
Blood3 .
Plasma3'0
RBCsa'D
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
Fat°
GI tract
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
0.4 mg
dermal
No. 13
0.03
0.02
0.03
0.17
0.03
0.01
0.00
0.01
0.01
0.04
0.00
0.00
0.01
0.01
0.02
_
-
_
-
-
4.0 mg
dermal
No. 14
0.20
0.21
0.13
0.73
0.24
0.13
0.04
0.08
0.08
0.33
0.05
0.04
0.09
1.13
0.40
_
-
_
—
-
or mL
0.4 mg
i. v.
No. 15
0.07
0.04
0.09
0.35
0.05
0.17
0.00
0.16
0.01
0.10
0.00
0.00
0.01
0.02
0.01
_
-
_
—
-
Percent of dose
0.4 mg
dermal
No. 13
0.09
0.05
0.04
0.47
0.02
0.00
0.00
0.00
0.00
0.00
0.00
0.05
-
0.42
0.15
34.98
7.47
31.36
24.17
99.20
4.0 mg
dermal
No. 14
0.07
0.04
0.09
0.18
0.01
0.00
0.00
0.00
0.01
0.00
0.00
0.08
-
0.75
0.30
11.88
3.58
51.72
31.56
100.13
0.4 mg
i. v.
No. 15
0.26
0.10
0.13
0.97
0.04
0.05
0.00
0.03
0.00
0.00
0.00
0.07
—
0.15
0.09
62.45
32.78
-
~
96.89
3Percent of dose calculations are based on 7%, 16%, and 40% of body weight for blood, nontreated skin,
and muscle, respectively. Plasma and red blood cell calculations are based on 60% and 40% of blood
.volume, respectively.
Individual blood components and fat are not included in recovery estimates.
-------
Table 4. Urinary and Fecal Excretion of Radioactivity in Male Hartley Guinea Pigs Treated
Dermally or Intravenously with 14C-Labeled 4,4'-MDA (1 or 10 mg/Guinea Pig):
Preliminary Study
Percent of dose
Excretum
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
1 mg
dermal
No. 52
0.41
1.54
5.63
8.32
2.87
1.93
0.10
0.53
3.19
4.05
4.05
3.93
0.51
2.07
8.82
12.37
6.92
5.86
10 mg
dermal
No. 53
0.08
3.05
14.11
3.97
1.58
1.08
0.01
0.14
2.28
1.83
1.04
1.30
0.09
3.19
16.39
5.80
2.62
2.38
1 mg
i. v.
No. 54
2.80
18.43
6.23
3.56
0.74
0.37
0.64
20.91
18.13
11.06
4.02
1.90
3.44
39.34
24.35
14.62
4.75
2.27
Cumulative percent
1 mg
dermal
No. 52
0.41
1.95
7.58
15.90
18.77
20.70
0.10
0.62
3.81
7.86
11.91
15.83
0.51
2.58
11.39
23.76
30.67
36.53
10 mg
dermal
No. 53
0.08
3.13
17.24
21.21
22.79
23.87
0.01
0.14
2.42
4.26
5.30
6.60
0.09
3.27
19.66
25.47
28.09
30.47
of dose
1 mg
i . v.
No. 54
2.80
21.24
27.46
31.02
31.76
32.13
0.64
21.55
39.67
50.74
54.75
56.65
3.44
42.78
67.14
81.75
86.51
88.78
-------
CO
Table 5. Radioactivity in Blood, Tissue, and Excreta of Male Hartley Guinea Pigs at 96 h Following
Dermal or Intravenous Dose of 14C-Labeled 4,4'-MDA (1 or 10 mg/Guinea Pig):
Preliminary Study
jjg equivalents/CL
Tissue/
excretum
Blood3 .
Plasma3'0
RBCs3'0
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
Fat0
GI tract
Nontreated skin3
Urine
Feces
Dose wash
Application area
Recovery
1 mg
dermal
No. 52
0.05
0.05
0.05
0.22
0.12
0.03
0.00
0.02
0.06
0.64
0.01
0.01
0.02
0.22
0.04
_
-
_
-
-
10 mg
dermal
No. 53
0.18
0.18
0.15
0.61
0.32
0.11
0.03
0.06
0.08
1.79
0.02
0.05
0.13
0.94
0.68
'
-
_
-
-
or mL
1 mg
i. v.
No. 54
0.18
0.17
0.22
1.22
0.28
0.35
0.00
2.29
0.03
0.21
0.01
0.01
0.03
0.11
0.06
_
-
_
-
-
Percent of dose
1 mg
dermal
No. 52
0.12
0.08
0.05
0.37
0.06
0.01
0.00
0.00
0.01
0.01
0.00
0.12
-
1.10
0.24
20.70
15.83
38.39
15.61
92.57
10 mg
dermal
No. 53
0.05
0.03
0.02
0.10
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.07
-
0.50
0.39
23.87
6.60
49.37
8.31
89.27
1 mg
i . v.
No. 54
0.46
0.26
0.22
1.89
0.12
0.07
0.00
0.10
0.00
0.00
0.00
0.08
-
0.83
0.35
32.13
56.65
_
-
92.68
dPercent of dose calculations are based on 7%, 16%, and 40% of body weight for blood, nontreated skin,
and muscle, respectively. Plasma and red blood cell calculations are based on 60% and 40% of blood
.volume, respectively.
Individual blood components and fat are not included in recovery estimates.
-------
Table 6. Urinary and Fecal Excretion of Radioactivity in Male Fischer 344
Rats Treated Intravenously with 14C-Labeled 4,4'-MDA (0.4 mg/Rat)
Excretum
Urine
Feces
Total
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
24- h sacrifice
37.91 ± 4.65
20.67 ± 2.34
8.78 ± 1.04
0.20 ± 0.03
4.61 ± 2.81
16.99 ± 2.47
38.10 ± 4.68
25.27 ±2.22
25.76 ± 3.23
Cumulative percent
37.91 ± 4.65
58.57 ± 2.70
67.35 ± 1.71
0.20 ± 0.03
4.80 ± 2.84
21.79 ± 1.52
38.10 ±4.68
63.37 ± 5.53
89.14 ± 2.73
96-h sacrifice
36.24 ± 1.03
18.85 ± 1.24
8.74 ± 0.62
1.77 ± 0.10
0.90 ± 0.15
0.45 ± 0.01
3.11 ± 1.33
13.31 ± 0.47
11.23 ± 2.07
2.02 ± 0.08
0.51 ± 0.10
0.50 ± 0.17
39.35 ± 1.95
32.16 ± 1.27
19.96 ± 2.51
3.79 ± 0.14
1.41 ± 0.24
0.95 ± 0.17
of dose
36.24 ± 1.03
55.10 ± 0.59
63.84 ± 0.07
65.61 ± 0.13
66.51 ± 0.22
66.96 ± 0.22
3.11 ± 1.33
16.41 ± 0.86
27.64 ± 1.25
29.66 ± 1.20
30.17 ± 1.14
30.66 ± 1.05
39.35 ± 1.95
71.51 ± 1.41
91.47 ± 1.22
95.26 ± 1.08
96.67 ± 1.03
97.62 ± 0.96
Mean ± SE of three rats per group.
44
-------
Table 7. Radioactivity in Blood, Tissue, and Excreta at 6, 24, and 96 h
Following Intravenous Treatment of Male Fischer 344 Rats with
14C-Labeled 4,4'-MDA (0.4 mg/Rat)a
ug equivalents/g or mL
Tissue/excretum
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Skin
Blood .
Plasma0'0
RBCs°'c
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
GI tract
Skin0
Urine
Feces
Recovery
6 h
0.97 ± 0.06
1.14 ± 0.12
0.47 ± 0.04
4.86 ± 0.21
0.96 ± 0.06
1.15 ±0.13
0.14 ± 0.00
0.89 ± 0.06
0.63 ± 0.11
0.80 ± 0.12
0.05 ± 0.02
0.17 ± 0.01
0.02 ± 0.00
5.66 ± 0.28
0.48 ± 0.02
Percent
2.80 ± 0.10
1.97 ± 0.15
0.55 ± 0.04
9.47 ± 0.62
0.48 ± 0.03
0.26 ± 0.04
0.08 ± 0.00
0.09 ± 0.00
0.26 ± 0.03
0.01 ± 0.00
0.00 ± 0.00
2.77 ± 0.25
23.94 ± 1.70
3.19 ± 0.18
54.65 ± 2.92
0.28 ± 0.25
98.27 ± 4.04
24 h
0.15 ± 0.01
0.16 ± 0.01
0.13 ± 0.01
1.38 ± 0.21
0.09 ± 0.01
0.68 ± 0.24
0.01 ± 0.00
0.41 ± 0.02
0.02 ± 0.00
0.15 ± 0.03
0.00 ± 0.00
0.01 ± 0.00
0.01 ± 0.00
0.64 ± 0.03
0.04 ± 0.01
of administered
0.44 ± 0.03
0.28 ± 0.02
0.15 ± 0.01
3.54 ± 0.50
0.05 ± 0.00
0.24 ± 0.07
0.01 ± 0.00
0.06 ± 0.00
0.01 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.19 ± 0.03
4.14 ± 0.27
0.25 ± 0.04
67.35 ± 1.71
21.79 ± 1.52
98.06 ± 3.04
96 h
0.06 ± 0.00
0.05 ± 0.00
0.06 ± 0.00
0.31 ± 0.02
0.05 ± 0.01
0.07 ± 0.00
0.00 ± 0.00
0.20 ± 0.01
0.01 ± 0.00
0.10 ± 0.01
0.00 ± 0.00
0.01 ± 0.00
0.01 ± 0.00
0.02 ± 0.00
0.02 ± 0.00
dose
0.22 ± 0.01
0.10 ± 0.01
0.09 ± 0.00
0.86 ± 0.02
0.03 ± 0.00
0.02 ± 0.00
0.00 ± 0.00
0.02 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.21 ± 0.02
0.11 ± 0.02
0.16 ± 0.01
66.96 ± 0.22
30.66 ± 1.05
99.25 ± 1.00
.Mean ± SE of three rats per group.
Calculations are based on 7%, 16%, and 40% of body weight for blood,
nontreated skin, and muscle, respectively. Plasma and red blood cell
calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
45
-------
Table 8. Tissue-to-Blood Concentration Ratios from Male Fischer 344 Rats
at 6, 24, or 96 h Following Intravenous Treatment with
14C-Labeled 4,4'-MDA (0.4 mg/Rat)a
Tissue
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Skin
6 h
1.18
0.49
5.02
0.99
1.19
0.15
0.91
0.65
0.83
0.06
0.17
0.02
5.85
0.50
24 h
1.07
0.82
9.05
0.61
4.42
0.05
2.70
0.10
0.96
0.03
0.08
0.06
4.16
0.25
96 h
0.75
1.03
5.05
0.85
1.16
0.07
3.34
0.08
1.69
0.07
0.18
0.10
0.25
0.33
Derived from mean of three rats per group.
Table 9. Recovery of Radioactivity in Blood, Tissue, and Excreta at
6, 24, .or 96 h Following Intravenous Treatment of
Male Fischer 344 Rats with 14C-Labeled 4,4'-MDA (0.4 mg/Rat)a
Tissue/excretum
Blood
Tissue
GI tract
Urine
Feces
Recovery
6 h
2.80 ± .0.10
16.60 ± 1.00
23.94 ± 1.70
54.65 ± 2.92
0.28 ± 0.25
98.27 ± 4.04
Percent of dose
24 h
0.44 ± 0.03
4.33 ± 0.47
4.14 ± 0.27
67.35 ± 1.71
21.79 ± 1.52
98.06 ± 3.04
96 h
0.22 ± 0.01
1.31 ± 0.03
0.11 ± 0.02
66.96 ± 0.22
30.66 ± 1.05
99.25 ± 1.00
Mean ± SE of three rats per group.
46
-------
Table 10. Urinary and Fecal Excretion of Radioactivity in Male Fischer 344
Rats Treated Dermally with 14C-Labeled 4,4'-MDA (0.4 mg/Rat)
Excretum
Urine
Feces
Total
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96. .
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
24-h sacrifice
2.21 ± 0.40
4.81 ± 1.64
13.02 ± 1.91
0.03 ± 0.01
0.61°
1.87 ± 0.11
2.24 ± 0.40
5.21 ± 1.86
14.89 ± 1.80
Cumulative
2.21 ± 0.40
7.01 ± 2.00
20.03 ± 3.68
0.03 ± 0.01
0.44 ± 0.23
2/31 ± 0.21
2.24 ± 0.40
7.45 ± 2.22
22.34 ± 3.80
96-h sacrifice
1.51 ± 0.78
8.25 ± 1.59
13.68 ± 0.30
12.65 ± 1.57
4.58 ± 0.74
2.36 ± 0.36
0.02b
0.76 ± 0.19
2.69 ± 0.40
3.84 ± 0.55
1.63 ± 0.32
1.03 ± 0.13
1.52 ± 0.77
9.01 ± 1.70
16.37 ± 0.53
16.50 ± 2.12
6.21 ± 0.42
3.38 ± 0.49
percent of dose
1.51 ± 0.78
9.77 ± 0.82
23.45 ± 0.99
36.11 ± 2.43
40.69 ± 3.11
43.04 ± 3.20
0.02b
0.77 ± 0.19
3.46 ± 0.45
7.30 ± 0.39
8.93 ± 0.12
9.96 ± 0.25
1.52 ± 0.77
10.54 ± 0.93
26.91 ± 0.97
43.41 ± 2.71
49.62 ± 3.12
53.00 ± 3.25
.Mean ± SE of three rats per group.
Average of two rats.
47
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Table 11. Radioactivity in Blood, Tissue, and Excreta at 6, 24, and 96 h
Following Dermal Treatment of Male Fischer 344 Rats with
14C-Labeled 4,4'-MDA (0.4 mg/Rat)a
Tissue/excretum
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
Bloodbh
Plasma0'0
RBCsD'c
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle0
GI tract .
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
6 h
0.21 ± 0.04
0.25 ± 0.05
0.12 ± 0.02
0.86 ± 0.13
0.26 ± 0.03
0.13 ± 0.04
0.06 ± 0.01
0.10 ± 0.02
0.13 ± 0.02
0.26 ± 0.05
0.01 ± 0.01
0.06 ± 0.01
0.01 ± 0.00
0.86 ± 0.36
0.15 ± 0.03
0.77 ± 0.14
0.54 ± 0.10
0.17 ± 0.03
1.96 ± 0.31
0.15 ± 0.01
0.03 ± 0.01
0.03 ± 0.01
0.01 ± 0.00
0.07 ± 0.01
0.00 ± 0.00
0.00 ± 0.00
1.19 ± 0.18
3.80 ± 1.57
1.24 ± 0.20
2.55 ± 0.16
0.04 ± 0.01
62.08 ± 6.90
30.59 ± 2.20
104.51 ± 3.60
ug equivalents/g or
24 h
0.08 ± 0.02
0.09 ± 0.02
0.04 ± 0.01
0.55 ± 0.05
0.12 ± 0.02
0.04 ± 0.01
0.01 ± 0.00
0.03 ± o;oi
0.04 ± 0.01
0.10 ±.0.02
0.00°
0.02 ± 0.00
0.01 ± 0.01
0.74 ± 0.21
0.05 ± 0.01
Percent of administered
0.29 ± 0.06
0.18 ± 0.04
0.06 ±0.01
1.20 ± 0.12
0.07 ± 0.01
0.01 ± 0.00
0.01 ± 0.00
0.00 ± 0.00
0.02 ± 0.01
0.00 ±.0.00
r\
0.00°
0.43 ± 0.09
2.98 ± 0.97
0.41 ± 0.10
20.03 ± 3.68
2.31 ± 0.21
52.08 ± 6.09
25.84 ± 4.00
105.68 ± 3.30
mL
96 h
,0.03 ± 0.00
0.03 ± 0.00
0.03 ± 0.00
0.27 ± 0.01
0.06 ± 0.01
0.02 ± 0.00
0.00 ± 0.00
0.01 ± 0.00
0.01 ± 0.00
0.05 ± 0.01
0.01 ± 0.01
0.01 ± 0.00
0.00 ± 0.00
0.09 ± 0.01
0.02 ± 0.00
dose
0.11 ± 0.01
0.05 ± 0.01
0.04 ± 0.00
0.54 ± 0.03
0.03 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.10 ± 0.01
0.48 ± 0.07
0.20 ± 0.02
43.04 ± 3.20
9.96 ± 0.25
24.67 ± 2.85
25.55 ± 0.65
104.68 ± 1.47
.Mean ± SE of three rats per group.
Percent of dose calculations are based on 7%, 16%, and 40% of body weight
for blood, nontreated skin, and muscle, respectively. Plasma and red blood
cell calculations are based on 60% and 40% of blood volume, respectively.
.Individual blood components and fat are not included in recovery estimates.
Average of two rats.
48
-------
Table 12. Tissue-to-Blood Concentration Ratios from Male Fischer 344 Rats
at 6, 24, or 96 h Following Dermal Treatment with
14C-Labeled 4,4'-MDA (0.4 mg/Rat)
Tissue
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
6 h
1.17
0.56
4.00
1.21
0.60
0.28
0.47
0.60
1.20
0.07
0.27
0.07
4.00
0.71
24 h
1.06
0.54
6.88
1.49
0.50
0.16
0.39
0.51
1.19
0.04
0.26
0.16
9.23
0.63
96 h
0.83
0.87
9.10
1.97
0.50
0.10
0.43
0.23
1.70
0.40
0.17
0.07
3.03
0.80
aDerived from mean of three rats per group.
Table 13. Recovery of Radioactivity in Blood, Tissue, and Excreta at
6, 24, or 96 h Following Dermal Treatment of Male Fischer 344 Rats
with 14C-Labeled 4,4'-MDA (0.4 mg/Rat)
Ti ssue/excretum
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
6 h
0.77 ± 0.14
4.70 ± 0.74
3.80 ± 1.57
2.55 ± 0.16
0.04 ± 0.01
11.85 ± 2.43
Percent of dose
24 h
0.29 ± 0.06
2.15 ± 0.33
2.98 ± 0.97
20.03 ± 3.68
2.31 ± 0.21
. 27.76 ± 5.15
96 h
0.11 ± 0.01
0.88 ± 0.04
0.48 ± 0.07
43.04 ± 3.20
9.96 ± 0.25
54.47 ± 3.27
Dose wash 62.08 ± 6.90 52.08 ± 6.09 24.67 ± 2.85
Application area 30.59 ± 2.20 25.84 ± 4.00 25.55 ± 0.65
Recovery 104.51 ± 3.60 105.68 ± 3.30 104.68 ± 1.47
Mean ± SE of three rats per group.
49
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Table 14. Urinary and Fecal Excretion of Radioactivity Following
Dermal Application of 14C-Labeled 4,4'-MDA (0.4 mg/Rat)
to Male Fischer 344 Rats: Application Area Washed at 24 h
Excretum
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
2.35 ± 0.20
7.30 ± 0.42
15.89 ± 1.17
6.23 ± 0.59
0.74 ± 0.10
0.72 ± 0.17
0.03b
0.10 ± 0.04
4.45 ± 0.19
3.72 ± 0.87
0.60 ± 0.09
0.29 ± 0.04
2.37 ± 0.21
7.40 ± 0.43
20.33 ± 1.21
9.95 ± 1.46
1.34 ±0.18
1.01 ± 0.20
Cumulative percent of dose
2.35 ± 0.20
9.65 ± 0.23
25.54 ± 1.40
31.77 ± 1.99
32.51 ± 2.04
33.23 ± 1.95
0.03b
0.12 ± 0.05
4.57 ± 0.14
8.29 ± 0.90
8.89 ± 0.99
9.18 ± 0.98
2.37 ± 0.21
9.77 ± 0.25
30.11 ± 1.45
40.06 ± 2.89
41.40 ± 3.02
42.41 ± 2.92
?Mean ± SE of three rats per group.
Average of two rats.
50
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Table 15. Radioactivity in Blood, Tissue, and Excreta of Male Fischer 344
Rats Treated Dermally with 14C-Labeled 4,4'-MDA (0^4 mg/Rat):
Nonoccluded Skin and Skin Washed at 24 h
pg equivalents/g or ml
Tissue/excretum Nonoccluded/6-h sacrifice 24-h wash/96-h sacrifice
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
Blood0.
Plasma0'0
RBCsD)C
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle0
GI tract ,
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
0.15 ± 0.03
0.15 ± 0.02
0.11 ± 0.04
0.62 ± 0.06
0.17 ± 0.02
0.09 ± 0.03
0.04 ± 0.00
0.06 ± 0.00
0.09 ± 0.01
0.15 ± 0.02
0.01 ± 0.01
0.05 ± 0.00
0.01 ± 0.00
0.29 ± 0.04
0.09 ± 0.01
Percent
0.53 ± 0.09
0.33 ± 0.04
0.16 ± 0.06
1.42 ± 0.09
0.10 ± 0.01
0.02 ± 0.01
0.02 ± 0.00
0.01 ± 0.00
0.05 ± 0.01
0.00 ± 0.00
0.00 ± 0.00
0.94 ± 0.02
1.24 ± 0.18
0.71 ± 0.09
1.95 ± 0.31
0.03 ± 0.00
70.21 ± 7.98
27.53 ± 6.18
104.74 ± 2.64
0.02 ± 0.00
0.01 ± 0.00
0.02 ± 0.00
0.13 ± 0.02
0.03 ± 0.00
0.01 ± 0.00
0.00 ± 0.00
0.01 ± 0.00
0.00 ± 0.00
0.01 ± 0.01
0.00 ±0.00
0.00 ± 0.00
0.00 ± 0.00
0.03 ± 0.01
0.01 ± 0.00
of administered dose
0.07 ± 0.01
0.03 ± 0.00
0.03 ± 0.00
0.21 ± 0.01
0.01 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.05 ± 0.01
0.08 ± 0.02
0.10 ± 0.01
33.23 ± 1.95
9.18 ± 0.98
52.12 ± 3.10
10.67 ± 1.44
105.72 ± 1.28
.Mean ± SE of three rats per group.
Percent of dose calculations are based on 7%, 16%, and 40% of body weight
for blood, nontreated skin, and muscle, respectively. Plasma and red blood
ccell calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
51
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Table 16. Tissue-to-Blood Concentration Ratios from Male Fischer 344 Rats
Following Dermal Treatment with 14C-Labeled 4,4'-MDA (0.4 mg/Rat):
Nonoccluded Skin and Skin Washed at 24 h
Tissue Nonoccluded/6-h sacrifice 24-h wash/96-h sacrifice
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
1.04
0.78
4.22
1.18
0.62
0.24
0.39 .
0.59
1.02
0.05
0.31
0.06
1.95
0.59
0.63
1.21
7.00
1.68
0.37
0.05
0.37
0.11
0.47
0.05
0.16
0.05
1.47
0.68
aDerived from mean of three rats per group.
Table 17. Recovery of Radioactivity in Blood, Tissue, and Excreta Following
Dermal Treatment of Male Fischer 344 Rats with 14OLabeled 4,4'-MDA (0.4 mg/Rat):
Nonoccluded Skin and Skin Washed at 24 ha
Percent of dose ^_^_
Tissue/excretum Nonoccluded/6-h sacrifice24-h wash/96-h sacrifice
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
0.53 ± 0.09
3.26 ± 0.21
1.24 ± 0.18
1.95 ± 0.31
0.03 ± 0.00
7.00 ± 0.77
0.07 ± 0.01
0.38 ± 0.02
0.08 ± 0.02
33.23 ± 1.95
9.18 ± 0.98
42.93 ± 2.95
Dose wash 70.21 ± 7.98 52.12 ± 3.10
Application area 27.53 ± 6.18 10.67 ± 1.44
Recovery 104.74 ±2.64 . 105.72 ± 1.28
Mean ± SE of three rats per group.
52
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Table 18. Urinary and Fecal Excretion of Radioactivity in Male Fischer 344
Rats Treated Dermally with 14C-Labeled 4,4'-MDA (4.0 mg/Rat):
Continuous Application for 96 h versus Skin Washing at 24 h
Excretum
Urine
Feces
Total
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
Continuous
0.15 ± 0.02
0.37 ± 0.05
0.67 ± 0.05
1.39 ± 0.11
1.19 ± 0.21
1.05 ± 0.17
0.01 ± 0.00
0.06 ± 0.01
0.17 ±0.01
0.36 ± 0.05
0.33 ± 0.06
0.41 ± 0.08
0.16 ±0.02
0.43 ± 0.05
0.84 ± 0.05
1.75 + 0.15
1.52 ± 0.26
1.46 ± 0.25
Cumulative
0.15 ± 0.02
0.52 ± 0.05
1.19 ± 0.06
2.58 ± 0.05
3.77 ± 0.25
4.82 ± 0.42
0.01 ± 0.00
0.06 ± 0.01
0.24 ± 0.02
0.59 ± 0.04
0.92 ± 0.10
1.34 '± 0.14
0.16 ± 0.02
0.58 ± 0.06
1.43 ± 0.07
3.17 ± 0.08
4.69 ± 0.34
6.15 ± 0.56
24- h Wash
0.08 ± 0.01
0.26 ± 0.09
0.61 ± 0.05
1.45 ± 0.24
1.01 ± 0.36
0.60 ± 0.21
0.00 ± 0.00
0.05 ± 0.01
0.14 ± 0.03
0.37 ± 0.04
0.29 ± 0.07
0.21 + 0.07
0.08 ± 0.00
0.30 ± 0.11
0.75 ± 0.07
1.82 ± 0.26
1.30 ± 0.43
0.82 ± 0.29
percent of dose
0.08 ± 0.01
0.33 ± 0.09
0.94 ± 0.12
2.39 ± 0.17
3.40 ± 0.52
4.00 ± 0.73
0.00 ± 0.00
0.05 ± 0.02
0.19 ± 0.04
0.56 ± 0.06
0.85 ± 0.11
1.06 ± 0.19
0.08 ± 0.00
0.38 ± 0.11
1.13 ± 0.15
2.95 ± 0.20
4.25 ± 0.63
5.06 ± 0.91
Mean ± SE of three rats per group.
53
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Table 19. Radioactivity in Blood, Tissue, and Excreta of Male Fischer 344
Rats 96 h Following Dermal Treatment with 14C-Labeled 4,4'-MDA (4.0 mg/Rat):
Continuous Application for 96 h versus Washing at 24 h
Tissue/excretum
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
|jg equivalents/g
Continuous
0.08 ± 0.02
0.10 ± 0.02
0.05 ± 0.01
0.53 ± 0.08
0.19 ± 0.06
0.05 ± 0.01
0.01 ± 0.01
- 0.03 ± 0.01
0.04 ± 0.01
0.15 ± 0.04
0.00 ± 0.00
0.02 ± 0.01
0.01 ± 0.00
0.35 ± 0.11
0.07 ± 0.01
or mL
24-h wash
0.04 ± 0.01
0.04 ± 0.02
0.02 ± 0.01
0.35 ± 0.09
0.08 ± 0.03
0.02 ± 0.01
0.00 ± 0.00
0.01 ± 0.01
0.02 ± 0.01
0.05 ± 0.01
0.00 ± 0.00
0.01 ± 0.00
0.00 ± 0.00
0.13 ± 0.05
0.04 ± 0.01
Percent of administered dose
Bloodb.
PlasmaD'c
RBCsD'c
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle0
GI tract .
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
0.03 ± 0.01
0.02 ± 0.00
0.01 ± 0.00
0.11 ± 0.02
0.01 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.04 ± 0.01
0.18 ± 0.04
0.06 ± 0.01
4.82 ± 0.42
1.34 ± 0.14
62.45 ± 2.62
24.00 ± 4.38
93.02 ± 2.16
0.01 ± 0.00
0.01 ± 0.00
0.00 ± 0.00
0.07 ± 0.02
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.02 ± 0.01
0.07 ± 0.02
0.04 ± 0.00
4.00 ± 0.73
1.06 ± 0.19
77.92 ± 3.44
8.93 ± 3.69
92.11 ± 2.08
.Mean ± SE of three rats per group.
Calculations are based on 7%, 16%, and 40% of body weight for blood,
nontreated skin, and muscle, respectively. Plasma and red blood cell
calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
54
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Table 20. Tissue-to-Blood Concentration Ratios from Male Fischer 344 Rats
at 96 h Followig Dermal Treatment with 14C-Labeled 4,4'-MDA (4.0 mg/Rat):
Continuous Application for 96 h versus Skin Washing at 24 h
Tissue
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
Continuous
1.17
0.65
6.42
2.35
0.66
0.13
0.38
0.46
1.84
0.00
0.27
0.09
4.27
0.89
24- h wash
1.10
0.60
8.68
2.10
0.55
0.08
0.33
0.38
1.18
0.00
0.23
0.05
3.30
1.10
Derived from mean of three rats per group.
Table 21. Recovery of Radioactivity in Blood, Tissue, and Excreta at 96 h
Following Dermal Treatment of Male Fischer 344 Rats with 14C-Labeled 4,4'-MDA
(4.0 mg/Rat): Continuous Application for 96 h versus Skin Washing at 24 h
Tissue/excretum
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
Continuous
0.03 ± 0.01
0.22 ± 0.04
0.18 ± 0.04
4.82 ± 0.42
1.34 ±- 0.14
6.58 ± 0.60
Percent of dose
24- h wash
0.01 ± 0.00
0.13 ± 0.02
0.07 ± 0.02
4.00 ± 0.73
1.06 ± 0.19
5.27 ± 0.96
Dose wash
Application area
Recovery
62.45 ± 2.62
24.00 ± 4.38
93.02 ± 2.16
77.92 ± 3.44
8.93 ± 3.69
92.11 ± 2.08
Mean ± SE of three rats per group.
55
-------
Table 22. Urinary and Fecal Excretion of Radioactivity in Male Hartley
Guinea Pigs Treated Intravenously with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig)£
Excretum
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
6.43 ± 0.86
15.33 ± 2.12
8.89 ± 2.04
3.31 ± 0.43
0.68 ± 0.05
0.35 ± 0.09
1.78 ±0.17
4.05 ± 2.06
27.29 ± 1.57
18.00 ± 2.45
4.07 ± 0.16
1.27 ± 0.28
8.21 ± 1.02
19.38 ± 3.54
36.18 ± 2.14
21.31 ± 2.85
4.75 ± 0.18
1.61 ± 0.19
Cumulative percent of dose
6.43 ± 0.86
21.76 ± 2.16
30.65 ± 2.75
33.96 ± 2.97
34.64 ± 2.96
34.98 ± 3.02
1.78 ±0.17
5.83 ± 1.90
33.11 ± 2.94
51.11 ± 3.36
55.19 ± 3.45
56.45 ± 3.71
8.21 ± 1.02
27.58 ± 2.90
63.76 ± 0.78
85.07 ± 2.07
89.82 + 1.95
91.43 ± 2.11
Mean ± SE of three guinea pigs per group.
56
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Table 23. Radioactivity in Blood, Tissue, and Excreta at 96 h Following
Intravenous Treatment of Male Hartley Guinea Pigs with
14C-Labeled 4,4'-MDA (1.0 nig/Guinea Pig)
Tissue/excretum ug equivalents/g or ml Percent of dose
Bloodb,
Plasma°'c
RBCsD'c
Liver
Ki dneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle0
Fatc
GI tract
Skin0
Urine
Feces
0.21 ± 0.03
0.20 ± 0.03
0.20 ± 0.01
0.77 ± 0.08
0.24 ± 0.04
0.39 ± 0.13
0.00 ± 0.00
1.19 ± 0.10
0.02 ± 0.01
0.19 ± 0.02
0.01 ± 0.00
0.01 ± 0.00
0.03 ± 0.00
0.08 ± 0.02
0.05 ± 0.01
-
-
0.55 ± 0.07
0.31 ± 0.04
0.21 ± 0.00
1.68 ± 0.07
0.11 ± 0.00
0.10 ± 0.04
0.00 ± 0.00
0.06 ± 0.01
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.15 ± 0.04
-
0.61 ± 0.07
0.28 ± 0.02
34.98 ± 3.02
56.45 ± 3.71
Recovery - 94.98 ± 2.20
?Mean ± SE of three guinea pigs per group.
Percent of dose calculations are based on 7%, 16%, and 40% of body weight
for blood, nontreated skin, and muscle, respectively. Plasma and red blood
cell calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
57
-------
Table 24. Tissue-to-Blood Concentration Ratios from Male Hartley
Guinea Pigs at 96 h Following Intravenous Treatment with
14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig)
Tissue Ratio
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Skin
0.94
0.96
3.60
1.15
1.81
0.02
5.57
0.10
0.90
0.06
0.05
0.13
0.38
0.22
Derived from mean of three guinea pigs per group.
Table 25. Recovery of Radioactivity in Blood, Tissue, and Excreta at 96 h
Following Intravenous Treatment of Male Hartley Guinea Pigs with
14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig)a
Tissue/excretum Percent of dose
Blood
Tissue
GI tract
Urine
Feces
Recovery
0.55 ± 0.07
2.39 ± 0.04
0.61 ± 0.07
34.98 ± 3.02
56.45 ± 3.71
94.98 ± 2.20
Mean ± SE of three guinea pigs per group.
58
-------
Table 26. Urinary and Fecal Excretion of Radioactivity in Male Hartley
Guinea Pigs Treated Dermally with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig)
Excretum
Urine
Feces
Total
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24 .
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
24- h sacrifice
2.03 ± 1.64
3.43 ± 1.54
2.35 ± 0.54
0.05 ± 0.02
1.15 ± 0.30
4.50 ± 1.61
2.08 ± 1.66
4.58 ± 1.84
6.85 ± 2.12
Cumulative percent
2.03 ± 1.64
5.45 ± 2.10
7.81 ± 2.52
0.05 ± 0.02
1.20 ± 0.31
5.70 ± 1.89
2.08 ± 1.66
6.65 ± 2.35
13.51 ± 3.91
96-h sacrifice
0.22 ± 0.05
1.30 ± 0.27
2.80 ± 0.76
3.32 ± 0.75
1.50 ± 0.40
1.33 ± 0.44
0.03 ± 0.01
0.77 ± 0.34
3.87 ± 0.58
7.27 ± 1.24
3.57 ± 0.58
2.11 ± 0.28
0.25 ± 0.06
2.07 ± 0.10
6.67 ± 0.32
10.59 ± 1.88
5.07 ± 0.98
3.44 ± 0.67
of dose
0.22 ± 0.05
1.52 ± 0.30
4.32 ± 0.86
7.64 ± 1.51
9.13 ± 1.89
10.47 ± 2.21
0.03 ± 0.01
0.80 ± 0.34
4.67 ± 0.80
11.94 ± 0.63
15.52 ± 1.16
17.62 ± 1.40
0.25 ± 0.06
2.32 ± 0.05
8.99 ± 0.36
19.58 ± 1.96
24.65 ± 2.93
28.09 ± 3.45
Mean ± SE of three guinea pigs per group.
59
-------
Table 27. Radioactivity in Blood, Tissue, and Excreta at 6, 24, and 96 h
Following Dermal Treatment of Male Hartley Guinea Pigs with
i4r-iahQioH 4>4'-MDA (1.0 mg/Guinea Pig)
|jg equivalents/g or ml
Tissue/excretum
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
Blood5,
Plasma0.'0
RBCsD'c
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle0
GI tract .
Q
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
6 h
0.05 ± 0.01
0.05 ± 0.00
0.03 ± 0.00
0.25 ± 0.02
0.09 ± 0.00
0.05 ± 0.01
0.03 ± 0.01
0.04 ± 0.00
0.12 ± 0.02
1.10 ± 0.40
0.01 ± 0.00
0.02 ± 0.01
0.03 ± 0.00
0.28 ± 0.01
0.05 ±0.01
Percent
0.12 ± 0.02
0.08 ± 0.01
0.02 ± 0.00
0.40 ± 0.01
0.04 ± 0.00
0.01 ± 0.00
0.01 ± 0.00
0.00 ± 0.00
0.02 ± 0.00
0.02 ± 0.01
0.00 ± 0.00
0.31 ± 0.10
1.51 ± 0.11
0.30 ± 0.07
0.35 ± 0.10
0.10 ± 0.03
80.55 ± 1.49
11.43 ± 1.08
95.16 ± 2.56
24 h
0.06 ± 0.01
0.06 ± 0.01
0.04 ± 0.01
0.28 ±0.04
0.12 ± 0.02
0.06 ± 0.01
0.02 ± 0.00
0.03 ± 0.01
0.07 ± 0.02
0.98 ± 0.03
0.00 ± 0.00
0.01 ± 0.00
0.01 ± 0.00
0.67 ± 0.16
0.05 ± 0.02
of administered
0.14 ± 0.03
0.09 ± 0.02
0.04 ± 0.01
0.48 ± 0.07
0.04 ± 0.01
0.01 ± 0.00
0.01 ± 0.00
0.00 ± 0.00
0.01 ± 0.00
0.02 ± 0.00
0.00 ± 0.00
0.19 ± 0.04
2.80 ± 0.95
0.27 ± 0.12
7.81 ± 2.52
5.70 ± 1.89
58.78 ± 7.31
14.77 ± 1.73
91.01 ± 1.44
96 h
0.06 ± 0.01
0.06 ± 0.01
0.06 ± 0.01
0.19 ± 0.05
0.11 ± 0.03
0.04 ± 0.01
0.00 ± 0.00
0.02 ± 0.01
0.02 ± 0.00
0.51 ± 0.08
0.01 ± 0.00
0.03 ± 0.02
0.01 ± 0.00
0.10 ± 0.03
0.04 ± 0.02
dose
0.14 ± 0.02
0.08 ± 0.02
0.06 ± 0.01
0.40 ± 0.08
0.05 ± 0.01
0.01 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.01 ± 0.00
0.00 ± 0.00
0.37 ± 0.25
0.59 ± 0.14
0.23 ± 0.09
10.47 ± 2.21
17.62 ± 1.40
40.85 ± 4.30
29.41 ± 2.31
100.14 ± 2.69
.Mean ± SE of three guinea pigs per group.
Calculations are based on 7%, 16%, and 40% of body weight for blood,
nontreated skin, and muscle, respectively. Plasma and red blood cell
calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
60
-------
Table 28. Tissue-to-Blood Concentration Ratios from Male Hartley Guinea Pigs
at 6, 24, or 96 h Following Dermal Treatment with
14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig)a
Tissue 6 h 24 h 96 h
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
1.08
0.52
5.25
1.92
1.10
0.63
0.73
2.44
22.88
0.13
0.46
0.60
5.73
1.13
1.02
0.71
4.71
2.10
0.95
0.31
0.44
1.10
16.54
0.03
0.24
0.24
11.39
0.83
0.95
0.97
3.29
1.83
0.60
0.07
0.29
0.41
8.86
0.09
0.45
0.19
1.79
0.72
aDerived from mean of three guinea pigs per group.
Table 29. Recovery of Radioactivity in Blood, Tissue, and Excreta
at 6, 24, or 96 h Following Dermal Treatment of Male
Hartley Guinea Pigs with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig)
Percent of dose
Tissue/excretum 6 h 24 h 96 h
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
0.12 ± 0.02
1.11 ± 0.17
1.51 ± 0.11
0.35 ± 0.10
0.10 ±0.03
3.19 ± 0.18
0.14 ± 0.03
1.02 ± 0.24
2.80 ± 0.95
7.81 ± 2.52
5.70 ± 1.89
17.46 ± 4.81
0.14 ± 0.02
1.08 ± 0.29
0.59 ± 0.14
10.47 ± 2.21
17.62 ± 1.40
29.89 ± 3.72
Dose wash 80.55 ± 1.49 58.78 ± 7.31 40.85 ± 4.30
Application area • 11.43 ±1.08 14.77 ± 1.73 29.41 ± 2.31
Recovery 95.16 ± 2.56 91.01 ± 1.44 100.14 ± 2.69
Mean ± SE of three guinea pigs per group.
61
-------
Table 30. Urinary and Fecal Excretion of Radioactivity in Male Hartley
Guinea Pigs Treated Dermally with 14C-Labeled 4,4'-MQA (1.0 mg/Guinea Pig):
Application Area Washed at 24 h
Excretum
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
0.17 ± 0.03
1.53 ± 0.22
2.28 ± 0,15
2.12 ± 0.42
0.26 ± 0.04
0.19 ± 0.03
0.02 ± 0.01
1.14 ± 0.10
3.99 ± 0.99
4.94 ± 0.34
0.50 ± 0.01
0.30 ± 0.07
0.20 ± 0.03
2.67 ± 0.13
6.27 ± 1.06
7.06 ± 0.76
0.76 ± 0.04
0.49 ± 0.11
Cumulative percent of dose
0.17 ± 0.03
1.70 ± 0.21
3.99 ± 0.36
6.11 ± 0.52
6.37 ± 0.50
6.56 ± 0.48
0.02 ± 0.01
1.16 ± 0.11
5.15 ± 0.95
10.09 ± 0.61
10.59 ± 0.60
10.89 ± 0.59
0.20 ± 0.03
2.87 ± 0.10
9.14 ± 1.14
16.20 ± 0.61
16.96 ± 0.57
17.45 ± 0.48
Mean ± SE of three guinea pigs per group.
62
-------
Table 31. Radioactivity in Blood, Tissue, and Excreta Following Dermal
Treatment of Male Hartley Guinea Pigs with 14C-Labeled 4,4'-MDA
(1.0 mg/Guinea Pig): Nonoccluded Skin and Skin Washed at 24 h
ug equivalents/g or ml
Tissue/excretum Nonoccluded/6-h sacrifice24-h wash/96-h sacrifice
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brai n
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
0.05 ± 0.00
0.05 ± 0.00
0.03 ± 0.01
0.22 ± 0.02
0.19 ± 0.06
0.05 ± 0.00
0.03 ± 0.00
0.04 ± 0.00
0.10 ± 0.01
0.83 ± 0.13
0.01 ± 0.00
0.02 ± 0.00
0.02 ± 0.00
0.20 ± 0.05
0.04 ± 0.01
0.03 ± 0.00
0.03 ± 0.00
0.04 ± 0.00
0.11 ± 0.01
0.06 ± 0.00
0.02 ± 0.00
0.00 ± 0.00
0.01 ± 0.00
0.01 ± 0.00
0.07 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.01 ± 0.00
0.02 ± 0.00
0.01 ± 0.00
Percent of administered dose
Bloodb.
PlasmaD>c
RBCsD'c
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle0
GI tract .
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
0.12 ± 0.02
0.07 ± 0.01
0.03 ± 0.01
0.37 ± 0.07
0.09 ± 0.02
0.01 ± 0.00
0.01 ± 0.00
0.00 ± 0.00
0.01 ± 0.00
0.01 ± 0.00
0.00 ± 0.00
0.26 ± 0.04
1.27 ± 0.30
0.25 ± 0.05
0.60 ± 0.47
0.12 ± 0.06
87.46 ± 1.24
9.37 ± 0.61
99.97 ± 1.04
0.08 ± 0.00
0.04 ± 0.00
0.04 ± 0.00
0.22 ± 0.03
0.02 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.04 ± 0.01
0.12 ± 0.04
0.05 ± 0.01
6.56 ± 0.48
10.89 ± 0.59
61.78 ± 1.06
16.58 ±1.34
96.34 ± 0.61
.Mean ± SE of three guinea pigs per group.
Calculations are based on 7%, 16%, and 40% of body weight for blood, non-
treated skin, and muscle, respectively. Plasma and red blood cell calcu-
lations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
63
-------
Table 32. Tissue-to-Blood Concentration Ratios from Male Hartley Guinea
Pigs Following Dermal Treatment with 14C-Labeled 4,4'-MDA
(1.0 mg/Guinea Pig): Nonoccluded Skin and Skin Washed at 24 h
Tissue
Nonoccluded/6-h sacrifice 24-h wash/96-h sacrifice
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
1.02
0.71
4.96
4.22
1.16
0.67
0.87
2.20
18.44
0.16
0.40
0.33
4.36
0.93
0.76
1.15
3.27
1.76
0.48
0.06
0.27
0.15
2.00
0.09
0.09
0.24
0.55
0.27
Derived from mean of three guinea pigs per group.
Table 33. Summary of Radioactivity in Blood, Tissue, and Excreta
Following Dermal Treatment of Male Hartley Guinea Pigs with
14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig):
Nonoccluded Skin and Skin Washed at 24-h
Tissue/excretum
Percent of dose
Nonoccluded/6-h sacrifice
24-h wash/96-h sacrifice
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
0.12 ±0.02
1.02 ± 0.05
1.27 ±0.30
0.60 ± 0.47
0.12 ± 0.06
3.14 ± 0.56
0.08 ± 0.00
0.33 ± 0.03
0.12 ± 0.04
6.56 ± 0.48
10.89 ± 0.59
17.98 ±0.48
Dose wash
Application area
Recovery
87.46 ± 1.24
9.37 ± 0.61
99.97 ± 1.04
61.78 ± 1.06
16.58 ± 1.34
96.34 ± 0.61
Mean ± SE of three guinea pigs per group.
64
-------
Table 34. Urinary and Fecal Excretion of Radioactivity in Male Hartley
Guinea Pigs Treated Dermally with 14C-Labeled 4,4'-MDA (10.0 mg/Guinea Pig):
Continuous Application for 96 h versus Washing at 24 h
Excretum
Urine
Feces
Total
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
Continuous3
0.04
0.26
0.24
0.40
0.68
1.13
0.01
0.03
0.70
0.97
0.85
1.06
0.05
0.29
0.94
1.37
1.54
2.19
Cumulative percent of
0.04
0.30
0.54
0.94
1.63
2.75
0.01
0.04
0.74
1.70
2.56
3,62
0.05
0.34
1.28
2.65
4.18
6.37
24-h washb
0.05 ± 0.03
0.16 ±0.01
0.29 ± 0.03
0.60 ± 0.21
0.26 ± 0.09
0.19 ± 0.06
0.00 ± 0.00
0.06 ± 0.00
0.52 ± 0.05
0.78 ± 0.18
0.43 ± 0.15
0.39 ± 0.13
0.05 ± 0.03
0.21 ± 0.02
0.80 ± 0.08
1.38 ± 0.38
0.69 ± 0.21
0.58 ± 0.19
dose
0.05 ± 0.03
0.20 ± 0.02
0.49 ± 0.03
1.09 ± 0.18
1.35 ± 0.25
1.54 ± 0.30
0.00 ± 0.00
0.06 ± 0.00
0.58 ± 0.05
1.35 ± 0.13
1.78 ± 0.27
2.18 ± 0.39
0.05 ± 0.03
0.26 ± 0.01
1.07 ± 0.08
2.45 ± 0.30
3.14 ± 0.51
3.72 ± 0.69
.Average of two guinea pigs.
Mean ± SE of three guinea pigs.
65
-------
Table 35. Radioactivity in Blood, Tissue, and Excreta at 96 h Following
Treatment of Male Hartley Guinea Pigs with 14C-Labeled 4,4'-MDA
(10.0 nig/Guinea Pig): Continuous Application for 96 h versus Washing at 24 h
Tissue/excretum
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
ug equivalents/g
Continuous
0.16
0.17
0.12
0.59
0.31
0.12
0.01
0.04
0.43
2.18
0.01
0.04
0.06
0.89
0.40
or mL
24-h washb
0.09 ± 0.03
0.09 ± 0.03
0.08 ± 0.02
0.26 ± 0.09
0.15 ± 0.06
0.03 ± 0.01
0.01 ± 0.00
0.01 ± 0.00
0.03 ± 0.01
0.72 ± 0.38
0.02 ± 0.01
0.02 ± 0.01
0.01 ± 0.00
0.41 ± 0.17
0.08 ± 0.02
Percent of administered dose
Bloodc .
Plasma^'0
RBCsc'a
Liver
Kidneys
Lung
Brain
Spleen
Testes
Adrenals
Bladder
Musclec
GI tract
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
0.04
0.03
0.01
0.13
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.05
0.64
0.22
2.75
3.62
69.70
13.72
90.89
0.02 ± 0.01
0.01 ± 0.00
0.01 ± 0.00
0.05 ± 0.02
0.01 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.03 ± 0.02
0.28 ± 0.11
0.04 ± 0.01
1.54 ± 0.30
2.18 ± 0.39
80.44 ± 2.44
7.29 ± 1.36
91.88 ± 1.40
.Average of two guinea pigs.
Mean ± SE of three guinea pigs.
Calculations are based on 7%, 16%, and 40% of body weight for blood,
nontreated skin, and muscle, respectively. Plasma and red blood cell
.calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
66
-------
Table 36.' Tissue-to-Blood Concentration Ratios from Male Hartley Guinea Pigs
at 96 h Following Dermal Treatment with 14C-Labeled 4,4'-MDA
(10.0 mg/Guinea Pig): Continuous Application for 96 h versus Washing at 24 h
Tissue
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
Continuous
1.10
0.79
3.75
1.99
0.75
0.06
0.27
2.75
13.85
0.05
0.23
0.36
5.64
2.55
24-h washb
0.98
0.89
2.91
1.71
0.29
0.06
0.16
0.38
8.13
0.22
0.22
0.12
4.58
0.84
^Derived from average of two guinea pigs.
Derived from mean of three guinea pigs.
Table 37. Recovery of Radioactivity in Blood, Tissue, and Excreta at 96 h
Following Dermal Treatment of Male Hartley Guinea Pigs with
14C-Labeled 4,4'-MDA (10.0 mg/Guinea Pig):
Continuous Application for 96 h versus Washing at 24 h
Tissue/excretum
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
Dose wash
Application area
Recovery
Percent of
Continuous
0.04
0.43
0.64
2. 75
3.62
7.48
69.70
13.72
90.89
dose
24-h Washb
0.02 ± 0.01
0.13 ± 0.04
0.28 ± 0.11
1.54 ± 0.30
2.18 ± 0.39
4.15 ± 0.81
80.44 ± 2.44
7.29 ± 1.36
91.88 ± 1.40
.Average of two guinea pigs.
Mean ± SE of three guinea pigs.
67
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Table 38. Urinary and Fecal Excretion of Radioactivity in Male Rhesus Monkeys
Treated Dermally or Intravenously with 14C-Labeled 4,4'-MDA (10.0 mg/Monkey)
Excretum
Urine
Feces
Urine
Feces
Dose wash
Recovery
Time after
dosing (h)
6
12
24
48
72
96
120
144
168
6
12
24
48
72
96
120
144
168
6
12
24
48
72
96
120
144
168
6
12
24
48
72
96
120
144
168
Percent of dose
Dermal
0.19 ± 0.14
0.81 ± 0.20
2.84 ± 1.25
7.36 ± 1.36
3.92 ± 1.26
1.64 ± 0.50
0.90 ± 0.28
0.64 ± 0.20
0.53 ± 0.18
o.oob
0.00C
0.15 ± 0.12
0.39 ± 0.29
0.47 ± 0.04
0.37 ± 0.15
0.15 ± 0.04
0.28 ± 0.17
0.12 ± 0.05
Cumulative
0.19 ± 0.14
1.01 ± 0.32
3.84 ± 1.55
11.20 ± 1.42
15.12 ± 1.73
16.75 ± 2.09
17.66 ± 2.31
18.30 ± 2.50
18.83 ±.2.58
o.oolj
n
0.00°
0.15 ± 0.12
0.55 ± 0.41
1.02 ± 0.41
1.39 ± 0.27
1.54 ± 0.23
1.81 ± 0.21
1.93 ± 0.22
47.27 ± 5.90
68.03 ± 3.24
Intravenous
24.85 ± 6.70
21.10 ± 10.84
17.74 ± 1.60
15.66 ± 10.04
3.13 ± 1.76
0.78 ± 0.28
0.37 ± 0.06
0.27 ± 0.06
0.44 ± 0.20
0.07 ± 0.05
0.06C
3.93 ± 2.01
2.52 ± 0.87
2.37 ± 1.60
0.39 ± 0.22
0.18 ± 0.04
0.16 ± 0.04
0.11 ± 0.01
percent of dose
24.85 ± 6.70
45.95 ± 10.99
63.69 ± 11.45
79.35 ± 1.62
82.48 ± 1.14
83.27 ± 1.32
83.64 ± 1.38
83.90 ± 1.41
84.34 ± 1.57
0.07 ± 0.05
0.09 ± 0.07
4.02 ± 1.95
6.54 ± 2.81
8.92 ± 1.28
9.31 ± 1.09
9.49 ± 1.08
9.65 ± 1.12
9.75 ± 1.12
-
94.09 ± 0.97
.Mean ± SE of three monkeys per group.
Average of two monkeys.
One monkey.
68
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APPENDIX I
STUDY PROTOCOL
1-1
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Midwest 'Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
MRI Project No. 7901-A(21)
June 26, 1984
Study Protocol
DERMAL PENETRATION MODEL STUDIES: DERMAL ABSORPTION OF 14C-LABELED
METHYLENEDIANILINE (MDA) IN RATS, GUINEA PIGS, AND MONKEYS
Prepared for
U.S. Environmental Protection Agency
Office of Toxic Substances
Field Studies Branch, TS-798
Washington, DC 20460
1-2
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A. Objectives
The overall.objective of this study is to develop dermal penetration
data from representative chemical models that can be used in prediction of
dosage from exposure to toxic chemicals. The specific aims are to select and
compare techniques and animal models.for measuring dermal penetration rates
of a selected model chemical. The studies will be performed i_n vivo with
three animal models: the rat, guinea pig, and monkey.
The chemical selected is 4,4'-methylene dianiline (MDA, I.), a repre-
sentative aromatic amine with known toxic and carcinogenic properties.
H2N
I. 4,4'-Methylene dianiline (MDA)
•(* The position of the 14C-label)
Several experimental conditions will be considered and assessed in the study
protocol include skin type (rat, guinea pig, and monkey), dosage (low, high),
exposure regimen (limited, continuous), occlusion (exposed, covered) and carrier.
In addition, initial studies will be performed to assess the surface/dermal
washing efficiency following application to rats, guinea pigs, and monkeys.
Limited experiments will be performed to assess the elimination of
MDA following intravenous (i.v.) administration to utilize the data generated
in calculating the absolute absorption of MDA following dermal application.
The utility of this methodology will be assessed by comparing the data gen-
erated to those resulting from adding of excretory data in urine and feces of
animals exposed to the dermal application.
The species selected are the rat, a species for which historical
data on the toxicity and carcinogenicity of aromatic amines are available and
which is used extensively in dermal absorption studies, the guinea pig and
the monkey, species which have skin characteristics that resemble human skin.
The studies in rats and guinea pigs will be performed at two dose levels, "low"
and "high". The monkey studies will be carried out with the low dose only.
B. Animals
Adult male Fischer 344 rats, Hartley guinea pigs, and Rhesus monkeys
will be used in the studies. The rats 7 to 9 weeks old and weighing 125 to
175 g, and the guinea pigs, 5 to 7 weeks old and weighing 400 to 500 g, will
be purchased from Charles River Breeding Laboratories, North Wilmington,
Massachusetts. The monkeys, ~ 4 yr old and weighing ^ 5 kg, will be purchased
from Charles River Research Primates Corporation, Port Washington, New York.
Upon arrival, the rats and guinea pigs will be identified by metal ear tags
1-3
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and the monkeys will be tattooed across the chest. Prior to testing, the
animals will be selected at random for each group. Animals showing signs of
ill health will not be used.
C. Animal Care
Animal care and housing will be in accordance with DHEW Publication
No. (NIH)-78-23, 1978, "Guidelines for the Care and Use of Laboratory Animals,"
and the MRI Manual for Animal Care.
The animals will be housed in environmentally controlled rooms with
10 to 15 air changes per hour. The rooms will be maintained at a temperature
of 25 ± 2°C and humidity of 50 ± 10%, with a 12-hr light/dark cycle per day.
The rats and guinea pigs will be kept in the quarantine facility for at least
7 days prior to use. Monkeys will be acclimatized for a minimum of 3 weeks
and will be subjected to T.B. tests on arrival and 2 weeks later. The atten-
dant veterinarian will examine the animals prior to release for the studies.
During the acclimation period, the rats and guinea pigs will be
housed in polycarbonate cages on Ab-Sorb-Dri® hardwood chip bedding, and the
monkeys will be housed in stainless steel cages. All animals will be provided
with certified feed and tap water ad libitum. The guinea pigs diet will be
supplemented with appropriate amounts of ascorbic acid in the drinking water,
and the monkey diet will be supplemented with oranges. There are no known
contaminants in the food or water that would interfere with the study.
D. Test Compound
14C-labeled MDA will be synthesized by the BioOrganic Chemistry
Department of MRI. About 10 mCi of ring-14C-Tabeled MDA will be synthesized
with specific activity of > 10 mCi/mmol. Information on the identity, purity,
and stability of the 14C-labeled compound will be supplied. Nonlabeled MDA
will be purchsed from Aldrich Chemical Company. Identity and purity of the
nonlabeled compound will be checked by TLC. The nonlabeled and 14C-labeled
test compounds will be stored under specific conditions which minimize decom-
position.
E. Dosage and Treatment
Two dose levels will be used in the study, a "low" dose (2 mg/kg)
and a "high" dose (20 mg/kg). A mixture of the 14C-labeled and nonlabeled
MDA will be dissolved in ethanol:water mixture and applied to the skin or
administered intravenously (i.v.) to rats, guinea pigs, and monkeys. For the
dermal studies, both the low and high doses will contain ~ 200 uCi/kg
(•>• 30 uCi/rat, * 100 uCi/guinea pig, and ~ 1,000 uCi/monkey). For the i.v.
studies, the low dose will contain * 100 uCi/kg (~ 15 uCi/rat, •>• 50 uCi/
guinea pig, and ^ 500 uCi /monkey).
'^5s."
for dermaj^tratment, the doses will be administered in a mixture
of (ethano 1: water (-Sofrffind applied at a volume of 0.5 ml/kg (0.1 mL/rat,
0. 25" TII L/gTIinea pig, and2. 5 mL/monkey), The backs of the rats and guinea pigs,
1-4
-------
and the lateral forearm of the monkeys will be lightly shaved with an electric
clipper shortly before treatment. The dose will be applied with a disposable
micropipette on a specific area (~ 2 cm2 for rats, ~ 5 cm2 for guinea pigs,
and ~ 50 cm2 for monkeys) on the freshly shaven skin. Except when indicated
otherwise (see Study Design), the dosed areas will be occluded with an aluminum
foil patch which will be secured in place with adhesive tape.
For intravenous treatment, a mixture of the 14C-labeled and nonlabeled
MDA will be dissolved in ethanol:water (25:75) and administered at the low
dose level in a volume of 1.0 ml/kg (0.2 ml for rats, 0.5 ml/kg for guinea
pigs, and 5 ml for monkeys). Intravenous dosing for the three species will
be performed through the saphenous vein.
F. Study Design (See Table 1)
1. Preliminary Studies
These studies will be performed with 3 male rats and 3 male guinea
pigs treated dermally with 14C-MDA at the low or high dose levels, or i.v. at
the low dose level (1 animal per treatment). The animals will be housed in
individual metabolic cages for collection of urine and feces at 6, 12, 24,
48, 72, and 96 hr following dosing. Radioactivity remaining on the skin of
dermally treated animals will be washed with soap and water or acetone and
water (see below) then the animals will be sacrificed for sampling of selected
tissues. These studies will assist in selecting the appropriate sampling times
for the definitive experiments described below and in testing the method of
treatments for rats and guinea pigs.
2. Washing Efficiency Studies
Before initiation of the dermal disposition studies described below,
an initial washing efficiency experiment will be performed to assess the extent
of removal of the applied 14C-labeled material by washing with soap and water,
or organic solvents. Six rats, six guinea pigs, and four monkeys will be
lightly anesthetized with sodium pentobarbital then treated with dermal doses
of 14C-MDA at the low dose level. Immediately following application (5 to
10 min) the doses will be washed with soap and water or with acetone and water
(3 rats, 3 guinea pigs, 2 monkeys for each treatment) then the animals housed
in individual metabolic cages for excreta collection. Urine and feces will
be collected at 6, 12, 24, and 48 hr following dosing. Collection of excreta
may continue every 24 hr if significant amounts of radioactivity continue to
be eliminated. Determination of dermal washing efficiency at other times fol-
lowing application (6, 24, and 96 hr) will be assessed in the absorption
studies described below.
3. Rat Studies
These studies will be performed with 21 rats treated dermally at
the low or high dose levels and 9 rats treated i.v. at the low dose level.
The dermal dose of 14C-MDA will be applied for 6 or 24 hr then removed or kept
on the skin for the duration of the study (96 hr). After application, the
1-5
-------
animals will be placed in metabolic cages for excreta collection. Urine and
feces will be collected at 6, 12, 24, 48, 72, and 96 hr. At the specified
times, the applied doses will be washed with soap and water or acetone and
water as determined from the washing efficiency studies, then the animals sac-
rificed (6 and 24 hr) or returned to the metabolic cages. A group of 3 rats
will be treated dermally at the low dose level and placed in individual re-
strainers for 6 hr (until sacrifice). In this group, the dosing area will be
kept uncovered to determine the effect of nonocclusion on the dermal penetra-
tion of 14C-MDA.
In the i.v. studies the rats will be treated with the low dose of
14C-MDA and placed in individual metabolic cages for collection of excreta at
6, 12, 24, 48, 72, and 96 hr following administration. Three animals will be
sacrificed at 6, 24, or 96 hr for tissue sampling.
4. Guinea Pig Studies
These studies will be performed with 21 male guinea pigs treated
dermally at 2 dose levels and 3 guinea pigs treated i.v. The design will be
similar to the rat studies except that i.v. dosing will be limited to only
3 animals which will be treated with the low dose and sacrificed at 96 hr fol- '
lowing dosing.
5. Monkey Studies
In these studies, 4 male monkeys will be treated dermally (2), or
i.v. (2) with the low dose of 14C-MDA. The monkeys will be acclimated for
two 24-hr periods in metabolic charis during the week peri or to dosing.
After dermal application the monkeys will be placed in metabolic chairs for
the first 24 hr. The doses will be removed, washed with soap and water or
acetone and water and the animals will be transferred to individual metabolic
cages for the remainder of the studies. Following application, urine and
feces will be collected at 6, 12, 24, 48, 72 and 96 hr following dosing. The
monkeys treated i.v. will also be placed in the metabolic chairs for 24 hr
then in metabolic cages for the remainder of the study. Collection of excreta
will performed similarly.
The data collected following dermal application will be compared to
those obtained following i.v. administration of 14C-MDA. Selected tissues
may be sampled at the end of the study (96 hr) or the monkeys will be retained
for future investigations with 14C-MDA or other test chemicals. Crossover
studies can be considered if the data obtained from the 4 monkeys show signifi-
cant variability. However, the persistence of radioactivity in blood or tissue
components e.g., hemoglobin, may hinder the reutilization of these animals except
following extended periods after treatment.
G. Sample Collection
Urine will be collected in containers kept on dry ice. After each
collection, the cages will be rinsed and the cage washings will be measured
and analyzed. At sacrifice, all animals will be anesthetized by injection of
1-6
-------
sodium pentobarbital and exsanguinated by withdrawal of blood from appropriate
sites. The application areas will be washed with a suitable solvent and the
washings will be measured and analyzed. The following tissues and organs will
be removed, washed with saline, blotted with absorbing paper, weighed, and pre-
pared for radiochemical analyses:
- Liver - Spleen - GI Tract plus contents
- Kidneys - Adrenals - Skeletal muscle
- Lungs - Testes - Retroperitoneal fat
- Brain - Urinary bladder - Skin from nontreated areas
- Application area
Portions of blood will be centrifuged to separate plasma and red
blood cells (RBCs). Bladder contents will be removed and the bladder will be
washed thoroughly with saline. The contents and washings will be combined
with the final urine samples and analyzed. Blood and tissue will be kept on
ice during the necropsy procedures. Sample preparation and analyses will be
performed immediately after collection, or the samples will be frozen on dry
ice and stored frozen until analyzed. If requested by the project monitor,
tissue and excreta will be stored for future metabolic studies.
H. Sample Preparation and Analysis
Blood, tissue, and excreta will be analyzed in duplicate whenever
possible. Assays not within ± 10% of the mean of the duplicates will be re-
assayed except when no more sample is available or when radioactivity counts
are low and nonsignificant, i.e., less than two times the background.
Aliquots of the whole blood, RBCs and plasma will be analyzed for
total radioactivity determination. Volumes of urine and cage rinse will be
measured, and samples will be counted. Feces, GI tract (plus contents), and
tissues weighing more than 0.25 g will be homogenized in an appropriate sol-
vent (e.g., ethanol:water, 10:90). Aliquots of the homogenates will be mea-
sured and analyzed. Fat and nontreated skin samples will be weighed and
assayed for 14C content. The application areas of the skin and the covers
will be soaked in a suitable solvent (e.g., acetone:water), and the extracted
radioactivity will be analyzed. Organs and tissues (weighing less than 0.25 g)
will be weighed and assayed for 14C content. Blood, tissue, and fecal samples
will be combusted using a Packard Tricarb Oxidizer Model C306. Counting will
be performed in a Packard (Model 3255 or 4530) Tricarb liquid scintillation
counter. Correction for background and. efficiency will be performed.
I. Data Processing, Analysis, and Reporting
Appropriate methods will be used to present the 14C contents of blood,
tissues, and excreta in terms of microgram equivalents per gram of tissue or
milliliter of fluid and/or percentage of the dose administered to each animal.
Cumulative excretion in urine and feces will be calculated. The data from each
treatment group will be expressed as mean ± S.E.
1-7
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When applicable, statistical analyses between groups will be compared
using a one-way analysis of variance. The differences between groups will be
assessed either parametrically (Student's t-test) or nonparametrically (Mann-
Whitney U test). In both cases, the data will be expressed as means ± S.E.
and a p value of equal or less than 0.05 will be considered significant.
A final comprehensive report will be prepared describing, in detail,
the methods used and the data generated from the study. The data will be sum-
marized in tabular and/or graphical form.
J. Quality Assurance
Quality assurance of the data generated on the program will be mon-
itored both within the program and externally by MRI's Quality Assurance Unit
(QAU). All testing will be in accordance with the EPA Good Laboratory Prac-
tices Standards of November 29, 1983 (Federal Register, 48, 53922-53944. Con-
formity to the GLPs will be monitored by the QAU. After completion of the proj-
ect, all raw data will be archived at MRI and will be available for inspection
upon request. (See Appendix A for the Quality Assurance Program.)
K. Personnel Safety
The general safety policies of the Institute are established and
enforced by the Institute Safety Committee, the Carcinogen Safety Committee,
and the Radiation Safety Committee. It is anticipated that any nonroutine
hazards associated with the use of the test compound will be identified in a
project-specific health and safety plan. (See Appendix B for details of
Safety Plan.)
L. Study Personnel
The studies will be conducted in the Chemical and Biological Sciences
Department, Dr. James L. Spigarelli, Director. Dr. John Going, Project Manager,
will provide administrative oversight for the program. Dr. Monaem El-hawari,
Principal Toxicologist, will serve as the Study Director and will be responsible
for the technical aspects of the program. Ms. Maxine Stoltz, Associate Bio-
chemist, will supervise the animal studies and will be assisted by Mr. Steve
Unwin, Associate Biologist, and Ms. Patricia Aim, Technician. Mr. Edward
Williams will supervise the animal care activities under the supervision of
Dr. Clifford Templeman, Veterinarian. Dr. Eugene Podrebarac, Manager, Quality
Assurance will provide QA oversight for the program. Resumes of the key tech-
nical personnel are attached (see Appendix C).
M. Study Schedule (See Table 2)
Proposed Starting Date: July 9, 1984
Proposed Completion Date: October 22, 1984
Draft Final Report: November 12, 1984
1-8
-------
Table 1. 14C-MDA Study Designa'b'c
Species
(strain)
Rat
(Fischer)
Guinea pig
(Hartly)
Monkeys
(Rhesus)
Number Route
30 Dermal
Dermal
i.v.
24 Dermal
Dermal
i.v.
4 Dermal
i.v.
Dose
(mg/kg)
2 "low"
20 "high"
2 "low"
2 "low"
20 "high"
2 "low"
2 "low"
2 "low"
Application
period
6/24 hr
Continuous
24 hr
Continuous
Bolus
6/24 hr
Continuous
24 hr
Continuous
Bolus
24 hr
Bolus
Number of animals
per sacrifice '
6 hr 24 hr 96 hr
3+3 3g 39
- - 3
3
3
333
3 + 3f 3g 3g
3a
39
3
3
2g
2
A preliminary absorption study will be performed with 3 rats and 3 guinea pigs
which will be treated with 14C-MDA dermally at the low or high dose or intra-
venously at the low dose level (1 each treatment). Urine and feces will be
collected at 6, 12, 24, 48, 72, and 96 hr when the animals will be sacrificed
for tissue sampling.
A dermal washing efficiency study will be performed with 6 rats, 6 guinea pigs,
and 4 monkeys which will be treated with the low dose of 14C-MDA then the dose
washed immediately (5 to 10 min after application) with soap and water (half of
each group) or with acetone and water (the other half). After washing the animals
will be housed in individual metabolic cages for excreta collection at 6, 12,
24, and 48 hr.
Details of the monkey studies design will be determined based on the data
obtained from the rats and guinea pigs studies and on the information generated
during the dermal washing efficiency experiments.
For all animals kept beyond 6 hr, urine and feces will be collected at 6, 12,
|4, 48, 72, and 96 hr after dosing.
At the specified times, rats and guinea pigs will be sacrificed for tissue
sampling. Collection of tissues from monkeys will be determined based on data
generated from the rats and guinea pigs.
Groups of 3 rats and 3 guinea pigs will be exposed dermally to the low dose
of 14C-MDA and kept restrained with the skin application area nonoccluded un-
til sacrifice (6 hr). For all other rats and guinea pigs treated dermally,
the application area will be covered (see text) and the animals will be housed
in metabolic cages for excreta collection.
gFor these groups of animals, the dermal dose will be removed at 24 hr follow-
ing application. For the other groups, the dermal dose will be kept on the skin
for the duration of the study.
1-9
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N. Protocol Approvals
Study Director/Task
Leader (MRI)
Date
Stidy Monitor/Work
Assignment Manager (EPA)
Project Manager (MRI) Date Project Officer (EP&X Date
1-10
-------
APPENDIX II
DERMAL WASHING EFFICIENCY STUDIES
INDIVIDUAL ANIMAL DATA
List of Tables
Table Page
II-l Urinary and Fecal Excretion of Radioactivity in Male
Fischer 344 Rats Treated Dermally with 14C-Labeled
4,4'-MDA (0.4 mg/Rat): Washing Efficiency Study II-2
II-2 Urinary and Fecal Excretion of Radioactivity in Male
Hartley Guinea Pigs Treated Dermally with 14C-Labeled
4,4'-MDA (1.0 mg/Guinea Pig): Washing Efficiency
Study ' II-3
II-3 Urinary and Fecal Excretion of Radioactivity in Male Rhesus
Monkeys Treated Dermally with 14C-Labeled 4,4'-MDA
(10 mg/Monkey): Washing Efficiency Study 11-4
II-l
-------
Table II-l.
Urinary and Fecal Excretion of Radioactivity in Male Fischer 344 Rats Treated Dermally
with 14C-Labeled 4,4'-MDA (0.4 ng/Rat): Washing Efficiency Study
i
no
Percent of dose
Excretum
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
No. 16
0.104
0.220
0.163
0.118
0.050
0.031
0.004
0.020
0.045
0.109
0.053
0.039
0.108
0.240
0.208
0.227
0.103
0.070
Soap
No. 17
0.395
0.611
0.278
0.152
0.093
0.128
0.010
0.105
0.072
0.151
0.050
0.045
0.405
0.716
0.350
0.303
0.143
0.173
and water wash
No. 18
0.193
0.410
0.281
0.167
0.104
0.123
0.004
0.025
0.071
0.162
0.042
0.028
0.197
0.435
0.352
0.329
0.146
0.151
Mean ±
0.231
0.414
0.241
0.146
0.082
0.094
0.006
0.050
0.063
0.141
0.048
0.037
0.237
0.464
0.303
0.286
0.131
0.131
S.E.
± 0.086
± 0.113
± 0.039
± 0.014
± 0.016
± 0.032
± 0.002
± 0.028
± 0.009
± 0.016
± 0.003
± 0.005
+ 0.088
± 0.138
± 0.048
± 0.031
± 0.014
± 0.031
Cumulative
Urine
Feces
Total
Dose wash
Application
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
area
0.104
0.324
0.487
0.605
0.655
0.686
0.004
0.024
0.069
0.178
0.231
0.270
0.108
0.348
0.556
0.783
0.886
0.956
94.050
3.490
0.395
1.006
1.284
1.436
1.529
1.657
0.010
0.115
0.187
0.338
0.388
0.433
0.405
1.121
1.471
1.774
1.917
2.090
94.500
3.948
0.193
0.603
0.884
1.051
1.155
1.278
0.004
0.029
0.100
0.262
0.304
0.332
0.197
0.632
0.984
1.313
1.459
1.610
85.425
3.961
0.231
0.644
0.885
1.031
1.113
1.207
0.006
0.056
0.119
0.259
0.308
0.345
0.237
0.700
1.004
1.290
1.421
1.552
91.325
3.800
± 0.086
± 0.198
± 0.230
± 0.240
± 0.253
± 0.283
± 0.002
± 0.030
± 0.035
± 0.046
± 0.045
± 0.048
± 0.088
+ 0.226
± 0.264
± 0.286
± 0.298
± 0.329
± 2.953
+ 0.155
No. 19
0.557
1.601
0.911
0.731
0.203
0.191
0.004
0.021
0.304
0.484
0.179
0.092
0.561
1.622
1.215
1.215
0.382
0.283
percent of dose
0.557
2.158
3.069
3.800
4.003
4.194
0.004
0.025
0.329
0.813
0.992
1.084
0.565
2.183
3.398
4.613
4.995
5.278
97.425
10.703
Acetone
No. 20
0.780
1.356
0.787
0.562
0.339
0.387
0.006
0.134
0.079
0.537
0.272
0.492
0.786
1.490
0.866
1.099
0.611
0.879
0.780
2.136
2.923
3.485
3.824
4.211
0.006
0.140
0.219
0.756
1.028
1.520
0.786
2.276
3.142
4.241
4.852
5.731
78.388
14.639
and water wash
No. 21
0.604
4.184
2.444
0.944
0.566
0.508
0.007
0.128
0.570
0.688
0.660
0.750
0.611
4.312
3.014
1.632
1.226
1.258
0.604
4.788
7.232
8.176
8.742
9.250
0.007
0.135
0.705
1.393
2.053
2.803
0.611
4.923
7.937
9.569
10.795
12.053
79.463
12.801
Mean ±
0.647
2.380
1.381
0.746
0,369
0.362
0.006
0.094
0.318
0.570
0.370
0.445
0.653
2.475
1.698
1.315
0.740
0.807
0.647
3.027
4.408
5.154
5.523
5.885
0.006
0.100
0.418
0.987
1.358
1.802
0.653
3.127
4.826
6.141
6.881
7.687
85.092
12.714
S.E.
± 0.068
± 0.905
± 0.533
± 0.111
± 0.106
± 0.092
± 0.001
± 0.037
± 0.142
± 0.061
± 0.147
± 0.191
± 0.068
± 0.919
± 0.666
± 0.162
± 0.252
± 0.284
+ 0.068
± 0.880
± 1.413
± 1.514
± 1.610
± 1.683
± 0.001
± 0.038
± 0.147
± 0.203
± 0.348
± 0.516
± 0.068
± 0.898
± 1.557
± 1.717
± 1.958
± 2.187
± 6.174
± 1.137
Recovery
98.496 100.538 90.996
96.677 ± 2.901
113.406
98.758 104.317
105.494 ± 4.269
-------
Table II-2. Urinary and Fecal Excretion of Radioactivity in Male Hartley Guinea Pigs Treated Dermally
with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig): Washing Efficiency Study
i
CO
Percent of dose
Time after
Excretum dosing (h) No. 1
Urine
Feces
Total
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
0.013
0.026
0.040
0.063
0.028
0.005
0.001
0.000
0.039
0.055
0.034
0.024
0.014
0.026
0.079
0.118
0.062
0.029
Soap and water
No. 2
0.026
0. 102
0.124
0.079
0.034
0.029
0.005
0.013
0.183
0.151
0.055
0.036
0.031
0.115
0.307
0.230
0.089
0.065
No. 3
0.075
0.058
0.139
0.080
0.054
0.023
0.001
0.008
0.159
0.084
0.039
0.032
0.076
0.066
0.298
0.164
0.093
0.055
wash
Mean ±
0.038 ±
0.062 ±
0.101 ±
0.074 ±
0.039 ±
0.019 ±
0.002 ±
0.007 ±
0.127 ±
0.097 ±
0.043 ±
0.031 ±
0.040 ±
0.069 ±
0.228 ±
0.171 ±
0.081 ±
0.050 ±
Acetone and water
S.E.
0.019
0.022
0.031
0.006
0.008
0.007
0.001
0.002
0.045
0.028
0.006
0.004
0.018
0.026
0.075
0.033
0.010
0.011
Cumulative
Urine
Feces
Total
Dose wash
Application area
Recovery
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
0.013
0.039
0.079
0.142
0. 170
0.175
0.001
0.001
0.040
0.095
0.129
0.153
0.014
0.040
0.119
0.237
0.299
0.328
86.540
2.741
89. 609
0.026
0.128
0.252
0.331
0.365
0.394
0.005
0.018
0.201
0.352
0.407
0.443
0.031
0.146
0.453
0.683
0.772
0.837
78.120
4.665
83.622
0.075
0.133
0.272
0.352
0.406
0.429
0.001
0.009
0.168
0.252
0.291
0.323
0.076
0.142
0.440
0.604
0.697
0.752
88.910
4.083
93.745
0.038 ±
0.100 ±
0.201 ±
0.275 ±
0.314 ±
0.333 ±
0.002 ±
0.009 ±
0.136 ±
0.233 ±
0.276 ±
0.306 ±
0.040 ±
0. 109 ±
0.337 ±
0.508 ±
0.589 ±
0.639 ±
84.523 ±
3.830 ±
88.992 ±
0.019
0.031
0.061
0.067
0.073
0.079
0.001
0.005
0.049
0.075
0.081
0.084
0.018
0.035
0.109
0.137
0.147
0.157
3.274
0.570
2.938
No. 4
0.074
0.081
0.116
0.341
0.223
0.083
0.002
0.018
0.137
0.013
0.111
0.078
0.076
0.099
0.253
0.354
0.334
0.161
percent of dose
0.074
0.155
0.271
0.612
0.835
0.918
0.002
0.020
0.157
0.170
0.281
0.359
0.076
0.175
0.428
0.782
1.116
1.277
81.510
4.652
87.439
No. 5
0.281
0.508
0.500
0.461
0.275
0.144
_a
0.212
0.277
0.227
0.198
0.100
0.281
0.720
0.777
0.688
0.473
0.244
0.281
0.789
1.289
1.750
2.025
2.169
_a
0.212
0.489
0.716
0.914
1.014
0.281
1.001
1.778
2.466
2.939
3.183
67.935
6.837
77.955
No. 6
0.067
0.055
0.391
0.315
0.079
0.088
0.000
0.006
0.214
0.319
0.256
0.112
0.067
0.061
0.605
0.634
0.335
0.200
0.067
0.122
0.513
0.828
0.907
0.995
0.000
0.006
0.220
0.539
0.795
0.907
0.067
0.128
0.733
1.367
1.702
1.902
78.670
6.250
86.822
wash
Mean
0.141
0.215
0.336
0.372
0.192
0.105
0.
0.079
0.209
0.186
0.188
0.097
0.141
0.293
0.545
0.559
0.381
0.202
0.141
0.355
0.691
1.063
1.256
1.361
0.
0.079
0.289
0.475
0.663
0.760
0.141
0.435
0.980
1.538
1.919
2.121
76.038
5.913
84.072
+ S.E.
± 0.070
± 0.147
± 0.114
± 0.045
± 0.059
± 0.020
001
+ 0.067
± 0.040
+ 0.091
± 0.042
± 0.010
± 0.070
± 0.214
± 0.154
± 0.104
± 0.046
± 0.024
± 0.070
± 0.217
± 0.307
± 0.349
± 0.385
± 0.405
.001
± 0.066
± 0.102
± 0.161
± 0.194
± 0.203
± 0.070
± 0.283
± 0.409
± 0.494
± 0.537
± 0.561
i 4.134
± 0.653
± 3.064
aNo feces collected during this time period.
-------
Table II-3. Urinary and Fecal Excretion of Radioactivity in Male Rhesus Monkeys Treated Dermally with
I4C-Labeled 4,4'-MDA (10 mg/Monkey): Washing Efficiency Study
Percent of dose
Excretum
Urine
Feces
Urine
Feces
Dose wash
Recovery
Time after
dosing (h)
6
12
24
48
72
96
120
144
168
6
12
24
48
72
96
120
144
168.
6
12
24
48
72
96
120
144
168
6
12
24
48
72
96
120
144
168
No.
0.
529
506
0.549
2.
2.
1.
1.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
3.
5.
7.
8.
9.
9.
576
209
530
008
650
509
445
002
003
017
494
538
397
260
210
251
506
055
631
840
370
378
028
537
9. 982
0.
0.
0.
0.
1.
1.
1.
1.
2.
54.
66.
002
005
022
516
054
451
711
921
172
294
448
Soap and
No.
0.
0.
0.
1.
water wash
604
054
288
193
095
1.453
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
3.
3.
3.
4.
4.
0.
0.
0.
0.
0.
0.
0.
0.
0.
71.
76.
365
407
290
364
001
Oil
018
017
175
170
184
110
120
054
342
535
630
083
448
855
145
509
001
012
030
047
222
392
576
686
806
663
978
Average
0.280
0.419
1.385
1.652
1.492
0.687
0.529
0.400
0.405
0.002
0.007
0.018
0.256
0.357
0.284
0.222
0.160
0.186
Cumulative
0.280
0.699
2.083
3.735
5.227
5.913
6.442
6.841
7.246
0.002
0.009
0.026
0.282
0.638
0.922
1.144
1.304
1.490
62.979
71.713
...No.
0.
0.
0.
3.
2.
1.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
554
272
503
914
513
281
094
502
471
355
003
002
022
210
408
299
170
081
141
Acetone and
No.
0.
0.
2.
7.
3.
1.
1.
1.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
water wash
619
394
872
544
204
329
979
108
046
564
000
003
099
640
519
294
273
164
184
Average
0.333
0.688
1.729
5.359
2.805
1.537
0.805
0.759
0.460
0.002
0.003
0.061
0.425
0.464
0.297
0.222
0.123
0.163
percent of dose
0.
0.
1.
5.
7.
8.
9.
9.
9.
272
775
689
202
483
577
079
550
905
0.003
0.
0.
0.
0.
0.
1.
1.
1.
59.
70.
005
027
237
645
944
114
195
336
741
982
0.
1.
3.
11.
14.
16.
17.
18.
19.
0.
0.
0.
0.
1.
1.
1.
1.
2.
46.
67.
394
266
810
014
343
322
430
476
040
000
003
102
742
261
555
828
992
176
266
482
0.333
1.021
2.750
8.108
10.913
12.450
13.255
14.013
14.473
0.002
0.004
0.065
0.490
0.953
1.250
1.471
1.594
1.756
53.004
69. 232
-------
APPENDIX III
RAT STUDIES
INDIVIDUAL ANIMAL DATA
List of Tables
Table Page
III-l Urinary and Fecal Excretion of Radioactivity in Male
Fischer 344 Rats Treated Intravenously with 14C-
Labeled 4,4'-MDA (0.4 mg/Rat) III-3
III-2 Radioactivity in Blood and Tissue at 6, 24, and 96 h
Following Intravenous Treatment of Male Fischer 344
Rats with 14OLabeled 4,4'-MDA (0.4 mg/Rat) III-4
III-3 Recovery of Radioactivity in Blood, Tissue, and Excreta
at 6, 24, or 96 h Following Intravenous Treatment of
Male Fischer 344 Rats with 14OLabeled 4,4'-MDA
(0.4 mg/Rat) III-6
II1-4 Urinary and Fecal Excretion of Radioactivity in Male
Fischer 344 Rats Treated Dermally with 14C-Labeled
4,4'-MDA (0.4 mg/Rat) III-7
III-5 Radioactivity in Blood and Tissue at 6, 24, and 96 h
Following Dermal Treatment of Male Fischer 344 Rats
with 14C-Labeled 4,4'-MDA (0.4 mg/Rat) III-8
III-6 Recovery of Radioactivity in Blood, Tissue, and Excreta
at 6, 24, or 96 h Following Dermal Treatment of Male
Fischer 344 Rats with 14C-Labeled 4'4'-MDA
(0.4 mg/Rat) 111-10
III-7 Radioactivity in Blood, Tissue, and Excreta at 6 hr
Following Dermal Treatment of Male Fischer 344 Rats
with 14C-Labeled 4,4'-MDA (Or4 mg/Rat): Nonoccluded
Application Area III-ll
III-8 Recovery of Radioactivity in Blood, Tissue, and Excreta at
6 h Following Dermal Treatment of Male Fischer 344 Rats
with 14C-Labeled 4,4'-MDA (0.4 mg/Rat): Nonoccluded
Application Area 111-12
III-9 Urinary and Fecal Excretion of Radioactivity Following
Dermal Application of 14OLabeled 4,4'-MDA (0.4 mg/Rat)
to Male Fischer 344 Rats: Application Area Washed at
24 h 111-13
III-l
-------
List of Tables (continued)
Table Page
I11-10 Radioactivity in Blood, Tissue, and Excreta at 96 h
Following Dermal Treatment of Male Fischer 344 Rats
with 14C-Labeled 4,4'-MDA (0.4 mg/Rat): Application
Area Washed at 24 h. 111-14
III-ll Recovery of Radioactivity in Blood, Tissue, and Excreta
at 96 h Following Dermal Treatment of Male Fischer 344
Rats with 14C-Labeled 4,4'-MDA (0.4 mg/Rat): Application
Area Washed at 24 h 111-15
II1-12 Urinary and Fecal Excretion of Radioactivity in Male
Fischer 344 Rats Treated Dermally with 14C-Labeled
4,4'-MDA (4.0 mg/Rat): Continuous 96-h Application
versus Washing at 24 h 111-16
II1-13 Radioactivity in Blood and Tissue of Male Fischer 344 Rats
at 96 h Following Dermal Treatment with 14C-Labeled
4,4'-MDA (4.0 mg/Rat): Continuous Application versus
Washing at 24 h 111-17
II1-14 Recovery of Radioactivity in Blood, Tissue, and Excreta at
96 h Following Dermal Treatment of Male Fischer 344 Rats
with 14C-Labeled 4,4'-MDA (4.0 mg/Rat): Continuous
Application versus Washing at 24 h 111-19
III-2
-------
Table III-l. Urinary and Fecal Excretion of Radioactivity in Male Fischer 344 Rats Treated Intravenously
with 14C-Labeled 4,4'-MDA (0.4 nig/Rat)
i
OJ
Excretum
Urine
Feces
Total
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
. 48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
No. 58
47.128
16.678
6.689
No. 59
34.264
20.547
9.818
No. 60
32.322
24.772
9.822
Mean ± S.E.
37.905 ± 4,646
20.666 ± 2.337
8.776 ± 1.044
No.
38.
16.
9.
49
282
554
142
1.801
0.263
9.923
13.872
47.391
26.601
20.561
-
47.128
63.806
70.495
0.263
10.186
24.058
47.391
73.992
94.553
0.164
0.387
21.867
34.428
20.934
31.685
34.264
54.811
64.629
0.164
0.551
22.418
34.428
55.362
87.047
0.168
3.507
15.222
32.490
28.279
25.044
32.322
57.094
66.916
0.168
3.675
18.897
32.490
60.769
85.813
0.198 ± 0.032
4.606 ± 2.807
16.987 ± 2.471
38.103 ± 4.678
25.271 ± 2.222
25.763 ± 3.231
Cumulative
37.905 ± 4.646
58.570 ± 2.700
67.347 ± 1.707
0.198 ± 0.032
4.804 ± 2.838
21.791 ± 1.522
38.103 ± 4.678
63.374 ± 5.534
89.138 ± 2.731
1.
0.
3.
13.
9.
2.
0.
0.
42.
29.
18.
3.
1.
1.
165
459
775
073
185
175
708
833
057
627
327
976
873
292
No. 50
35.019
19.220
9.539
1.579
0.903
0.463
0.536
14.215
15.356
1.934
0.409
0.297
35.555
33.435
24.895
3.513
1.312
0.760
No. 51
35.432
20.788
7.529
1.932
0.639
0.423
5.007
12.626
9.139
1.944
0.415
0.360
40.439
33.414
16.668
3.876
1.054
0.783
Mean ±
36.244
18.854
8.737
1.771
0.902
0.448
3.106
13.305
11.227
2.018
0.511
0.497
39.350
32.159
19.963
3.788
1.413
0.945
S.E.
± 1.026
+ 1.236
± 0.615
± 0.103
± 0.152
± 0.013
± 1.333
± 0.473
± 2.065
± 0.079
± 0.099
± 0.169
± 1.954
± 1.266
± 2.512
± 0.141
± 0.242
± 0.174
percent of dose
38.
54.
63.
65.
66.
67.
3.
16.
26.
28.
28.
29.
42.
71.
90.
93.
95.
97.
282
836
978
779
944
403
775
848
033
208
916
749
057
684
Oil
987
860
152
35.019
54.239
63.778
65.357
66.260
66.723
0.536
14.751
30. 107
32.041
32.450
32.747
35.555
68. 990
93.885
97.398
98. 710
99.470
35.432
56.220
63.749
65.681
66.320
66.743
5.007
17.633
26.772
28. 716
29.131
29.491
40.439
73.853
90.521
94.397
95.451
96.234
36.244
55. 098
63.835
65.606
66.508
66.956
3.106
16.411
27.637
29.655
30. 166
30.662
39.350
71.509
91.472
95.261
96.674
97.619
± 1.026
± 0.587
± 0.072
± 0.128
± 0.219
± 0.223
± 1.333
± 0.860
± 1.253
± 1.202
± 1.144
± 1.045
± 1.954
± 1.407
± 1.215
± 1.075
± 1.025
± 0.963
-------
Table III-2. Radioactivity in Blood and Tissue at 6, 24, and 96 h Following Intravenous Treatment
of Male Fischer 344 Rats with 14C-Labeled 4,4'-MDA (0.4 mg/Rat)
(jq equivalents/o or ml
Tissue
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Skin
No. 55
1.077
1.370
0.536
4.738
1.082
1.408
0.143
1.000
0.839
0.585
0.054
0.156
0.023
5.961
0.452
No. 56
0.916
1.039
0.411
4.578
0.871
1.061
0.140
0.795
.0.527
1.005
0.022
0.157
0.027
5.920
0.491
6 h
No. 57
0.911
1.005
0.476
5.268
0.922
0.984
0.149
0.860
0.514
0.810
0.086
0.187
0.022
5.103
0.501
Mean ± S.E.
0.968 ± 0.055
1.138 ± 0.116
0.474 ± 0.036
4.861 ± 0.209
0.958 ± 0.064
1.151 ± 0.130
0.144 ± 0.003
0.885 ± 0.060
0.627 ± 0.106
0.800 ± 0.. 121
0.054 + 0.018
0.167 ± 0.010
0.024 ± 0.002
5.661 ± 0.279
0.481 ± 0.015
No. 58
0.133
0.143
0.103
1.295
0.080
1.159
0.007
0.410
0.012
0.112
0.003
0.008
•0.007
0.698
0.028
No. 59
0.172
0.186
0.136
1.784
0.097
0.458
0.008
0.375
0.018
0.134
0.001
0.013
0.011
0.590
0.037
24 h
No. 60
0.155
0.159
0.135
1.072
0.105
0.413
0.009
0.453
0.017
0.196
0.007
0.014
0.009
0.620
0.048
Mean + S.E.
0.153 ± 0.011
0.163 ± 0.013
0.125 ± 0.011
1.384 ± 0.210
0.094 ± 0.007
0.677 ± 0.242
0.008 ± 0.001
0.413 ± 0.023
0.016 ± 0.002
0.147 ± 0.025
0.004 ± 0.002
0.012 ± 0.002
0.009 ± 0.001
0.636 ± 0.032
0.038 ± 0.006
No. 49
0.064
0.051
0.065
0.304
0.037
0.068
0.004
0.205
0.005
0.088
0.005
0.010
0.006
0.013
0.019
No. 50
0.059
0.041
0.062
0.285
0.063
0.070
0.004
0.214
0.005
0.110
0.004
0.013
0.005
0.018
0.018
96 h
No. 51
0.061
0.045
0.063
0.335
0.055
0.074
0.004
0.193
0.005
0.112
0.004
0.009
0.006
0.015
0.022
Mean ± S.E.
0.061 ± 0.001
0.046 ± 0.003
0.063 ± 0.001
0.308 ±0.015
0.052 ± 0.008
0.071 ± 0.002
0.004 ± 0.000
0.204 ± 0.006
0.005 ± 0.000
0.103 ± 0.008
0.004 ± 0.000
0.011 ± 0.001
0.006 ± 0.000
0.015 ± 0.001
0.020 ± 0.001
-------
Table III-2 (continued)
t— 1
1 1
t—t
1
en
Tissue/
excretum
Blood3
Plasma*'0
RBCsa>D
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
Urine
Feces
GI tract
Skina
Percent of administered dose
No. 55
2.978
2.273
0.593
8.445
0.468
0.329
0.080
0.095
0.325
0.004
0.001
2.457
53.856
0.770
22.279
2.854
No. 56
2.645
1.800
0.474
9.381
0.430
0.237
0.074
0.092
0.213
0.010
0.000 -
2.591
60.055
0.015
27.331
3.238
6 h
No. 57
2.774
1.836
0.580
10.577
0.537
0.200
0.082
0.092
0.248
0.006
0.001
3.254
50.043
0.056
22.204
3.487
Mean ± S.E.
2.799 ± 0.097
1.970 ± 0.152
0.549 ± 0.038
9.468 ± 0.617
0.478 ± 0.031
0.255 ± 0.038
0.079 ± 0.002
0.093 ± 0.001
0.262 ± 0.033
0.007 ± 0.002
0.001 ± 0.000
2.767 ± 0.246
54.651 ± 2.918
0.280 ± 0.245
23.938 ± 1.697
3.193 ± 0.184
No. 58
0.396
0.255
0.123
3.483
0.044
0.381
0.006
0.054
0.006
0.001
0.000
0.136
70.495
24.058
4.678
0.191
No. 59
0.480
0.312
0.152
4.437
0.051
0.167
0.006
0.051
0.008
0.002
0.000
0.200
64.629
22.418
3.777
0.237
24 h
No. 60
0.455
0.281
0.159
2.706
0.058
0.156
0.006
0.060
0.008
0.002
0.000
0.227
66.916
18.897
3.975
0.319
Mean ± S.E.
0.444 ± 0.025
0.283 ± 0.016
0.145 ± 0.011
3.542 ± 0.501
0.051 ± 0.004
0.235 ± 0.073
0.006 ± 0.000
0.055 ± 0.003
0.007 ± 0.001
0.002 ± 0.000
0.000 ± 0.000
0.188 ± 0.027
67.347 ± 1.707
21.791 ± 1.522
4.143 ± 0.273
0.249 ± 0.037
No. 49
0.232
0.111
0.094
0.887
0.025
0.016
0.002
0.025
0.003
0.001
0.000
0.207
67.403
29.749
0.109
0.153
No. 50
0.208
0.088
0.088
0.860
0.039
0.015
0.002
0.025
0.003
0.001
0.001
0.254
66.723
32.747
0.140
0.146
96 h
No. 51
0.214
0.096
0.089
0.834
0.033
0.015
0.002
0.023
0.003
0.001
0.000
0.172
66.743
29.491
0.078
0.178
Mean ± S.E.
0.218 ± 0.007
0.098 ± 0.007
0.090 ± 0.002
0.860 ± 0.015
0.032 ± 0.004
0.015 ± 0.000
0.002 ± 0.000
0.024 ± 0.001
0.003 ± 0.000
0.001 ± 0.000
0.000 ± 0.000
0.211 ± 0.024
66.956 ± 0.223
30.662 ± 1.045
0.109 ± 0.018
0.159 ± 0.010
Recovery
94.941 106.312 93.561 98.271 ± 4.040 103.929 96.463 93.785 98.059 ± 3.035 98.812 101.164 97.787 99.254 ± 0.999
Calculations are based on 7%, 16%, and 40% of body weight for blood, nontreated skin, and muscle, respectively.
^calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
Plasma and red blood cell
-------
Table III-3. Recovery of Radioactivity in Blood, Tissue, and Excreta at 6, 24, or 96 h Following Intravenous Treatment
of Male Fischer 344 Rats with 14C-Labeled 4,4'-MDA (0.4 nig/Rat)
i
en
Tissue/
excretum
Blood
Tissue
GI tract
Urine
Feces
Recovery
Percent of dose
No. 55
2.978
15.058
22.279
53.856
0.770
94.941
No. 56
2.645
16.266
27.331
60.055
0.015
106.312
6 h
No. 57
2.774
18.484
22.204
50.043
0.056
93.561
Mean ± S.E.
2.799 ± 0.097
16.603 ± 1.003
23.938 ± 1.697
54.651 + 2.918
0.280 ± 0.245
98.271 ± 4.040
No. 58
0.396
4.302
4.678
70.495
24.058
103.929
No. 59
0.480
5.159
3.777
64.629
22.418
96.463
24 h
No. 60
0.455
3.542
3.975
66.916
18.897
93.785
Mean ± S.E.
0.444 ± 0.025
4.334 ± 0.467
4.143 ± 0.273
67.347 ± 1.707
21.791 ± 1.522
98.059 ± 3.035
No. 49
0.232
1.319
0.109
67.403
29.749
98.812
No. 50
0.208
1.346
0.140
66.723
32.747
101.164
96 h
No. 51
0.214
1.261
0.078
66.743
29.491
97.787
Mean ± S.E.
0.218 ± 0.007
1.308 ± 0.025
0.109 ± 0.018
66.956 ± 0.223
30.662 ± 1.045
99.254 ± 0.999
-------
Table III-4. Urinary and Fecal Excretion of Radioactivity in Male Fischer 344 Rats Treated Dermally
with 14C-Labeled 4,4'-MDA (0.4 mg/Rat)
Excretum
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
24-h sacrifice
No. 34
2.706
5.265
16.071
0.018
0.439
1.678
2.724
5.704
17.749
No. 35
2.498
7.390
13.473
0.051
0.776
1.888
2.549
8.166
15.361
No. 36
1.415
1.764
9.507
0.024
_d
2.048
1.439
1.764
11.555
Mean ±
2.206
4.806
13.017
0.031
0.
1.871
2.237
5.211
14.888
S.E.
Percent
± 0.400
± 1.640
± 1.909
± 0.010
608
± 0.107
± 0.402
+ 1.864
+ 1.804
No. 40
of dose
1.554
8.135
14.201
11.326
5.005
3.042
0.002
1.081
3.171
3.527
1.381
1.287
1.556
9.216
17.372
14.853
6.386
4.329
96-h sacrifice
No. 41
2.840
5.555
13.164
10.846
3.146
1.850
_a
0.419
3.007
3.094
2.261
0.869
2.840
5.974
16.171
13.940
5.407
2.719
No. 42
0.145
11.071
13.686
15.791
5.594
2.175
0.027
0.777
1.888
4.909
1.249
0.930
0.172
11.848
15.574
20.700
6.843
3.105
Mean ±
1.513
8.254
13.684
12.654
4.582
2.356
0.
0.759
2.689
3.843
1.630
1.029
1.523
9.013
16.372
16.498
6.212
3.384
S.E.
± 0.778
± 1.593
± 0.299
± 1.574
± 0.738
± 0.356
015
± 0.191
± 0.403
± 0.547
± 0.318
± 0.130
± 0.770
± 1.699
± 0.529
± 2.118
± 0.424
± 0.485
Cumulative percent of dose
Urine
Feces
Total
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
2.706
7.971
24.042
0.018
0.457
2.135
2.724
8.428
26.177
2.498
9.888
23.361
0.051
0.827
2.715
2.549
10.715
26.076
1.415
3.179
12.686
0.024
0.024
2.072
1.439
3.203
14.758
2.206
7.013
20.030
0.031
0.436
2.307
2.237
7.449
22.337
± 0.400
± 1.995
± 3.677
± 0.010
± 0.232
± 0.205
± 0.402
± 2.223
± 3.790
1.554
9.689
23.890
35.216
40.221
43.263
0.002
1.083
4.254
7.781
9.162
10.449
1.556
10.772
28.144
42.997
49.383
53.712
2.840
8.395
21.559
32.405
35.551
37.401
_a
0.419
3.426
6.520
8.781
9.650
2.840
8.814
24.985
38.925
44.332
47.051
0.145
11.216
24.902
40.693
46.287
48.462
0.027
0.804
2.692
7.601
8.850
9.780
0.172
12.020
27.594
48.294
55.137
58.242
1.513
9.767
23.450
36.105
40. 686
43.042
0.
0.769
3.457
7.301
8.931
9.960
1.523
10.535
26.908
43.405
49.617
53.002
± 0.778
± 0.815
± 0.990
± 2.433
± 3.108
± 3.195
015
± 0.192
± 0.451
± 0.394
± 0.117
± 0.248
± 0.770
± 0.933
± 0.974
± 2.712
± 3.121
± 3.250
No feces collected during this time period.
-------
Table III-5. Radioactivity in Blood and Tissue at 6, 24, and 96 h Following Dermal Treatment
of Male Fischer 344 Rats with 14C-Labeled 4,4'-MDA (0.4 ing/Rat)
Co
pg equivalents/q or mL
Tissue
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
No. 31
0.154
0.179
0.075
0.626
0.218
0.061
0.039
0.075
0.092
0.181
0.002
0.043
0.015
0.326
0.108
No. 32
0.289
0.343
0.158
1.067
0.320
0.196
0.085
0.132
0.163
0.358
0.005
0.074
0.013
1.546
0.193
6 h
No. 33
0.200
0.227
0.124
0.874
0.243
0.130
0.052
0.096
0.131
0.231
0.036
0.057
0.015
0.697
0.154
Mean ± S.E.
0.214 ± 0.040
0.250 ± 0.049
0.119 ± 0.024
0.856 ± 0.128
0.260 ± 0.031
0.129 ± 0.039
0.059 ± 0.014
0.101 ± 0.017
0.129 ± 0.021
0.257 ± 0.053
0.014 ± 0.011
0.058 ± 0.009
0.014 ± 0.001
0.856 ± 0.361
0.152 ± 0.025
No. 34
0.097
0.103
0.045
0.549
0.144
0.042
0.015
0.036
0.046
0.122
_c
0.022
0.004
0.955
0.061
No. 35
0.096
0.103
0.053
0.639
0.140
0.056
0.017
0.038
0.054
0.098
0.004
0.028
0.031
0.943
0.062
24 h
No. 36
0.047
0.048
0.031
0.463
0.074
0.021
0.007
0.020
0.022
0.064
0.001
0.013
0.003
0.317
0.028
Mean ± S.E.
0.080 ± 0.017
0.085 ± 0.018
0.043 ± 0.006
0.550 ± 0.051
0.119 + 0.023
0.040 ± 0.010
0.013 ± 0.003
0.031 ± 0.006
0.041 ± 0.010
0.095 ± 0.017
0.003
0.021 ± 0.004
0.013 ± 0.009
0.738 ± 0.211
0.050 ± 0.011
No. 40
0.032
0.028
0.024
0.289
0.068
0.015
0.003
0.014
0.008
0.051
0.003
0.005
0.002
0.119
0.028
No. 41
0.027
0.021
0.025
0.254
0.050
0.014
0.002
0.011
0.005
0.041
0.002
0.006
0.002
0.084
0.025
96 h
No. 42
0.031
0.026
0.029
0.276
0.058
0.016
0.003
0.014
0.007
0.060
0.032
0.004
0.002
0.071
0.020
Mean ± S.E.
0.030 ± 0.002
0.025 ± 0.002
0.026 ± 0.002
0.273 ± 0.010
0.059 ± 0.005
0.015 ± 0.001
0.003 ± 0.000
0.013 ± 0.001
0.007 ± 0.001
0.051 + 0.005
0.012 ± 0.010
0.005 ± 0.001
0.002 ± 0.000
0.091 ± 0.014
0.024 ± 0.002
-------
Table III-5 (continued)
Tissue/
excretum
Blood3 .
Plasma3'0
RBCsS'b
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
GI tract
Nontreated skin
Urine
Feces
Dose wash
Application area
b
Recovery
Percent of
No. 31
0.537
0.373
0.105
1.364
0.125
0.015
0.021
0.010
0.051
0.002
0.000
0.856
1.397
0.856
2.652
0.050
75.675
27.900
111.511
No. 32
1.007
0.717
0.220
2.398
0.173
0.051
0.049
0.017
0.089
0.004
0.000
1.473
6.741
1.533
2.748
0.028
57.275
28.913
102.499
6 h
No. 33
0.763
0.520
0.188
2.123
0.146
0.033
0.025
0.014
0.082
0.003
0.001
. 1.243
3.246
1.343
2.242
0.035
53.275
34.950
99.524
Mean ± S.E.
0.769 ± 0.136
0.537 ± 0.100
0.171 ± 0.034
1.962 ± 0.309
0.148 ± 0.014
0.033 ± 0.010
0.032 ± 0.009
0.014 ± 0.002
0.074 ± 0.012
0.003 ± 0.001
0.000 ± 0.000
1.191 ± 0.180
3.795 ± 1.567
1.244 ± 0.202
2.547 ± 0.155
0.038 ± 0.006
62.075 ± 6.897
30.588 ± 2.201
104.511 ± 3.604
No. 34
0.355
0.226
0.066
1.300
0.085
0.010
0.009
0.004
0. 027
0.002
_C
0.462
4.028
0.513
24.042
2.135
43.050
31.510
107.532
No. 35
0.345
0.221
0.076
1.331
0.082
0.014
0.009
0.005
0.033
0.002
0.000
0.564
3.860
0.509
23.361
2.715
49.525
27.896
110.251
administered dose
24 h
No. 36
0.165
0.100
0.043
0.953
0.043
0.005
0.004
0.002
0.012
0.001
0.000
0.260
1.043
0.220
12.686
2.072
63.675
18.126
99.267
Mean ± S.E.
0.288 ± 0.062
0.182 ± 0.041
0.062 ± 0.010
1.195 ± 0.121
0.070 ± 0.014
0.010 ± 0.003
0.007 ± 0.002
0.004 ± 0.001
0.024 ± 0.006
0.002 ± 0.000
0.000
0.429 ± 0.089
2.977 ± 0.968
0.414 ± 0.097
20.030 ± 3.677
2.307 ± 0.205
52.083 ± 6.090
25.844 ± 3.998
105.683 ± 3.303
No. 40
0.116
0.061
0.035
0.580
0.028
0.003
0.002
0.002
0.005
0.001
0.000
0.095
0.629
0.236
43.263
10.449
21.775
26.242
103.426
No. 41
0.097
0.045
0.036
0.486
0.022
0.003
0.001
0.001
0.003
0.001
0.000
0.113
0.409
0.205
37.401
9.650
30.375
24.244
103.011
96 h
No. 42
0.107
0.054
0.040
0.559
0.025
0.003
0.002
0.002
0.004
0.001
0.001
0.080
0.414
0.160
48.462
9.780
21.850
26.150
107.600
Mean ± S.E.
0.107 ± 0.005
0.053 ± 0.005
0.037 ± 0.002
0.542 ± 0.028
0.025 ± 0.002
0.003 ± 0.000
0.002 ± 0.000
0.002 ± 0.000
0.004 ± 0.001
0.001 ± 0.000
0.000 ± 0.000
0.096 ± 0.010
0.484 ± 0.073
0.200 ± 0.022
43.042 ± 3.195
9.960 ± 0.248
24.667 ± 2.854
25.545 ± 0.651
104.679 ± 1.465
Calculations are based on 7%, 16%, and 40% of body weight for blood, nontreated skin, and muscle, respectively.
^calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
Sample lost.
Plasma and red blood cell
-------
I
o
Table III-6. Recovery of Radioactivity in Blood, Tissue, and Excreta at 6, 24, or 96 h Following Dermal Treatment
of Male Fischer 344 Rats with 14C-Labeled 4,4'-MDA (0.4 mg/Rat)
Tissue/
excretum
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
Dose wash
Application area
Percent of dose
No. 31
0.537
3.300
1.397
2.652
0.050
7.936
75.675
27.900
No. 32
1.007
5.787
6.741
2.748
0.028
16.311
57.275
28.913
6 h
No. 33
0.763
5.013
3.246
2.242
0.035
11.299
53.275
34.950
Mean ± S.E.
0.769 ± 0.136
4.700 ± 0.735
3.795 + 1.567
2.547 ± 0.155
0.038 ± 0.006
11.849 ± 2.433
62.075 ± 6.897
30.588 ± 2.201
No. 34
0.355
2.412
4.028
24.042
2.135
32.972
43.050
31.510
No. 35
0.345
2.549
3.860
23.361
2.715
32.830
49.525
27.896
24 h
No. 36
0.165
1.500
1.043
12.686
2.072
17.466
63.675
18.126
Mean ± S.E.
0.288 ± 0.062
2.154 ± 0.329
2.977 ± 0.968
20.030 ± 3.677
2.307 ± 0.205
27.756 ± 5.145
52.083 ± 6.090
25.844 ± 3.998
No. 40
0.116
0.952
0.629
43.263
10.449
55.409
21.775
26.242
No. 41
0.097
0.835
0.409
37.401
9.650
48. 392
30.375
24.244
96 h
No. 42
0.107
0.837
0.414
48.462
9.780
59.600
21.850
26.150
Mean ± S.E.
0.107 ± 0.005
0.875 ± 0.039
0.484 ± 0.073
43.042 ± 3.195
9.960 ± 0.248
54.467 ± 3.270
24.667 ± 2.854
25.545 ± 0.651
Recovery
111.511 102.499 99.524 104.511 ± 3.604 107.532 110.251 99.267 105.683 ± 3.303 103.426 103.011 107.600 104.679 ± 1.465
-------
Table III-7. Radioactivity in Blood, Tissue, and Excreta at 6 h Following Dermal Treatment of Male Fischer 344 Rats
with t4C-Labeled 4,4'-MDA (0.4 nig/Rat): Nonoccluded Application Area
Tissue/
excretum
Blood3
Plasma3'0
RBCs3'0
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
FatD
GI tract
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
pg equivalents/g or
No.
0.
0.
0.
28
192
176
200
0.687
0.
0.
0.
0.
0.
0.
- 0.
0.
0.
0.
0.
-
-
_
-
-
185
135
037
056
104
173
019
046
008
310
096
No. 29
0.096
0.110
0.065
0.511
0.137
0.050
0.029
0.056
0.062
0.116
0.001
0.043
0.008
0.214
0.063
-
-
-
.
-
No. 30
0.153
0.173
0.078
0.661
0.201
0.089
0.038
0.058
0.095
0.161
0.005
0.048
0.012
0.334
0.100
-
-
_
.
-
mL
Percent of dose
Mean ± S.E.
0.147 ±
0.153 ±
0.114 ±
0.620 ±
0.174 ±
0.091 ±
0.035 ±
0.057 ±
0.087 ±
0.150 t
0.008 t
0.046 ±
0.009 ±
0.286 ±
0.086 ±
-
-
.
-
-
0.028
0.022
0.043
0.055
0.019
0.025
0.003
0.001
0.013
0.017
0.005
0.001
0.001
0.037
0.012
No. 28
0.672
0.369
0.280
1.509
0.098
0.031
0.018
0.007
0.054
0.002
0.001
0.910
-
1.410
0.764
2.127
0.031
61.775
30.050
99.459
No. 29
0.360
0.249
0.098
1.243
0.085
0.014
0.015
0.007
0.038
0.002
0.000
0.914
-
0.880
0.542
1.342
0.023
86.150
15.800
107.415
No. 30
0.547
0.373
0.112
1.508
0.117
0.020
0.021
0.007
0.055
0.002
0.000
0.985
-
1.419
0.821
2.369
0.027
62.700
36.750
107.348
Mean ± S.E.
0.526
0.330
0.163
1.420
0.100
0.022
0.018
0.007
0.049
0.002
0.000
0.936
1.236
0.709
1.946
0.027
70.208
27.533
104. 741
± 0.091
± 0.041
± 0.058
± 0.089
± 0.009
± 0.005
± 0.002
± 0.000
± 0.006
± 0.000
± 0.000
± 0.024
-
t 0.178
± 0.085
± 0.310
± 0.002
± 7.975
± 6.177
± 2.641
Percent of dose calculations are based on 7%, 16%, and 40% of body weight for blood, nontreated skin, and muscle, respectively.
^Plasma and red blood cell calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
-------
Table III-8. Recovery of Radioactivity in Blood, Tissue, and Excreta at 6 h Following Dermal Treatment of
Male Fischer 344 Rats with 14C-Labeled 4,4'-MDA (0.4 mg/Rat): Nonoccluded Application Area
Tissue/
excretum
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
Dose wash
Application area
No. 28
0.672
3.394
1.410
2.127
0.031
7.634
61.775
30.050
No. 29
0.360
2.860
0.880
1.342
0.023
5.465
86.150
15.800
Percent of dose
No. 30
0.547
3.536
1.419
2.369
0.027
7.898
62.700
36.750
Mean ± S.E.
0.526 ± 0.091
3.263 ± 0.206
1.236 ± 0.178
1.946 ± 0.310
0.027 ± 0.002
6.999 ± 0.771
70.208 ± 7.975
27.533 ± 6.177
ro
Recovery
99.459
107.415
107.348
104.741 ± 2.641
-------
Table III-9. Urinary and Fecal Excretion of Radioactivity Following Dermal Application of
14C-Labeled 4,4'-MDA (0.4 nig/Rat) to Male Fischer 344 Rats: Application Area Washed at 24 h
i
Co
Excretum
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
No. 37
2.658
6.761
15.008
5.838
0.535
0.549
0.046
0.153
4.086
3.401
0.485
0.221
2.704
6.914
19.094
9.239
1.020
0.770
No. 38
1.984
8.128
18.206
7.392
0.849
0.557
0.008
0.138
4.543
5.369
0.780
0.291
1.992
8.266
22.749
12.761
1.629
0.848
No. 39
2.413
7.005
14.443
5.458
0.840
1.058
_a
0.022
4.713
2.403
0.521
0.358
2.413
7.027
19.156
7.861
1.361
1.416
Mean ±
2.352
7.298
15.886
6.229
0.741
0.721
0.
0.104
4.447
3.724
0.595
0.290
2.370
7.402
20.333
9.954
1.337
1.011
S.E.
± 0.197
± 0.421
± 1.172
± 0.592
± 0.103
± 0.168
027
± 0.041
± 0. 187
± 0.871
± 0.093
± 0.040
± 0.207
± 0.433
± 1.208
± 1.459
± 0.176
± 0.204
No. 37
2.658
9.419
24.427
30.265
30.800
31.349
0.046
0.199
4.285
7.686
8.171
8.392
2.704
9.618
28.712
37.951
38.971
39.741
Cumulative percent
No. 38
1.984
10.112
28.318
35.710
36.559
37.116
0.008
0.146
4.689
10.058
10.838
11.129
1.992
10.258
33.007
45.768
47.397
48.245
No. 39
2.413
9.418
23.861
29. 319
30.159
31.217
_a
0.022
4.735
7.138
7.659
8.017
2.413
9.440
28. 596
36.457
37.818
39.234
of dose
Mean 1
2.352
9.650
25.535
31.765
32.506
33.227
0.
0.122
4.570
8.294
8.889
9.179
2.370
9.772
30.105
40.059
41.395
42.407
S.E.
± 0.197
± 0.231
± 1.401
± 1.991
± 2.035
± 1.945
027
± 0.052
± 0.143
± 0.896
± 0.985
± 0.981
± 0.207
± 0.248
± 1.451
± 2.887
± 3.019
± 2.923
No feces collected during this time period.
-------
Table 111-10. Radioactivity in Blood, Tissue, and Excreta at 96 h Following Dermal Treatment of Male Fischer 344 Rats
with 14C-Labeled 4,4'-MDA (0.4 mg/Rat): Application Area Washed at 24 h
Tissue/
exc return
Blood* .
as*
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
Fat"
GI tract
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
pg eguivalents/g or mL
No
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
. 37
.018
.011
.021
.139
.030
.006
.001
.007
.002
.000
.001
.004
.001
.042
.011
-
-
-
-
-
No. 38
0.023
0.013
0.027
0.157
0.036
0.008
0.001
0.007
0.002
0.024
0.002
0.002
' 0.002
0.031
0.016
_
-
-
-
-
No. 39
0.017
0.013
0.020
0.102
0.031
0.007
0.001
0.007
0.003
0.003
0.001
0.002
0.001
0.010
0.011
_
-
.
-
-
Mean
0.019
0.012
0.023
0.133
0.032
0.007
0. 001
0.007
0.002
0.009
0.001
0.003
0.001
0.028
0.013
± S.E.
± 0.002
± 0.001
± 0.002
± 0.016
± 0.002
± 0.001
± 0.000
± 0.000
± 0.000
± 0.008
± 0.000
± 0.001.
± 0.000
± 0.009
± 0.002
_
-
-
-
-
No. 37
0.066
0.023
0.031
0.201
0.011
0.001
0.001
0.001
0.001
0.000
0.000
0.074
-
0.103
0.093
31.349
8.392
54.650
9.830
104.773
Percent of dose
No. 38
0.081
0.027
0.038
0.222
0.013
0.002
0.001
0.001
0.001
0.000
0.000
0.041
-
0.092
0.127
37.116
11.129
45.950
13.477
108. 253
No. 39
0.058
0.026
0.028
0.191
0.013
0.002
0.001
0.001
0.002
0.000
0.000
0.040
-
0.053
0.084
31.217
8.017
55.750
8.694
104.123
Mean ±
0.068
0.025
0.032
0.205
0.012
0.002
0.001
0.001
0.001
0.000
0.000
0.052
0.083
0.101
33.227
9.179
52.117
10.667
105.716
S.E.
± 0.007
± 0.001
± 0.003
± 0.009
± 0.001
± 0.000
± 0.000
± 0.000
± 0.000
± 0.000
± 0.000
± 0.011
-
± 0.015
± 0.013
± 1.945
± 0.981
± 3.100
± 1.443
± 1.282
a
.Plasma and red blood cell calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
-------
Table III-ll.
1
en
Recovery of Radioactivity in Blood, Tissue, and Excreta
of Male Fischer 344 Rats with 14C-Labeled 4,4'-MDA
Application Area Washed at 24 h
at 96 h Following Dermal Treatment
(0.4 mg/Rat):
Tissue/
excretum
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
Dose wash
Application area
No. 37
0.066
0.383
0.103
31.349
8.392
40.293
54.650
9.830
No. 38
0.081
0.408
0.092
37.116
11.129
48.826
45.950
13.477
Percent of dose
No. 39
0.058
0.334
0.053
31.217
8.017
39.679
55.750
8.694
Mean ± S.E.
0.068 ± 0.007
0.375 ± 0.022
0.083 ± 0.015
33.227 ± 1.945
9.179 ± 0.981
42.933 ± 2.952
52.117 ± 3.100
10.667 ± 1.443
Recovery
104.773
108.253
104.123
105.716 ± 1.282
-------
Table 111-12. Urinary and Fecal Excretion of Radioactivity in Male Fischer 344 Rats Treated Oermally
with 14C-Labeled 4,4'-MDA (4.0 mg/Rat): Continuous 96-h Application versus Washing at 24 h
Percent of dose
Excretum
Urine
Feces
Total
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Continuous
No. 46
0.142
0.278
0.691
1.533
1.542
1.229
0.006
. 0.047
0.155
0.458
0.448
0.450
0.148
0.325
0.846
1.991
1.990
1.679
0.142
0.420
1.111
2.644
4.186
5.415
0.006
0.053
0.208
0.666
1.114
1.564
0.148
0.473
1.319
3.310
5.300
6.979
No. 47
0.127
0.443
0.577
1.466
1.197
-1.213
0.003
0.064
0.181
0.294
0.287
0.535
0.130
0.507
0.758
1.760
1.484
- 1.748
0.127
0.570
1.147
2.613
3.810
5.023
0.003
0.067
0.248
0.542
0.829
- 1.364
0.130
0.637
1.395
3.155
4.639
6.387
No. 48
0.177
0.392
0.739
1.169
0.832
0.699
0.010
0.060
0.187
0.316
0.247
0.257
0.187
0.452
0.926
1.485
1.079
0.956
0.177
0.569
1.308
2.477
3.309
4.008
0.010
0.070
0.257
0.573
0.820
1.077
0.187
0.639
1.565
3.050
4.129
5.085
Mean ± S.E.
0.149 ± 0.015
0.371 ± 0.049
0.669 ± 0.048
1.389 ± 0.112
1.190 ± 0.205
1.047 ± 0.174
0.006 ± 0.002
0.057 ± 0.005
0.174 ± 0.010
0.356 ± 0.051
0.327 ± 0.061
0.414 ± 0.082
0.155 ± 0.017
0.428 ± 0.054
0.843 ± 0.049
1.745 ± 0.146
1.518 ± 0.264
1.461 ± 0.253
Cumulative
0.149 ± 0.015
0.520 ± 0.050
1.189 ± 0.061
2.578 ± 0.051
3.768 ± 0.254
4.815 ± 0.419
0.006 ± 0.002
0.063 ± 0.005
.0.238 ± 0.015
0.594 ± 0.037
0.921 ± 0.097
1.335 ± 0.141
0.155 ± 0.017
0.583 ± 0.055
1.426 ± 0.073
3.172 ± 0.076
4.689 ± 0.339
6.150 ± 0.559
No. 43
0.068
0.221
0.695
1.084
0.302
0.188
0.003
0.029
0.157
0.297
0.149
0.075
0.071
0.250
0.852
1.381
0.451
0.263
percent of dose
0.068
0.289
0.984
2.068
2.370
2.558
0.003
0.032
0.189
0.486
0.635
0.710
0.071
0.321
1.173
2.554
3.005
3.268
No. 44
0.083
0.114
0.525
1.890
1.245
0.739
0.001
0.039
0.089
0.394
0.380
0.236
0.084
0.153
0.614
2.284
1.625
0.975
0.083
0.197
0.722
2.612
3.857
4.596
0.001
0.040
0.129
0.523
0.903
1.139
0.084
0.237
0.851
3.135
4.760
5.735
24-h Wash
No. 45
0.081
0.433
0.605
1.375
1.478
0.882
0.003
0.076
0.174
0.419
0.334
0.330
0.084
0.509
0.779
1.794 .
1.812
1.212
0.081
0.514
1.119
2.494
3.972
4.854
0.003
0.079
0.253
0.672
1.006
1.336
0.084
0.593
1.372
3.166
4.978
6.190
Mean ± S.E.
0.077 ± 0.005
0.256 ± 0.094
0.608 ± 0.049
1.450 ± 0.236
1.008 ± 0.360
0.603 ± 0.212
0.002 ± 0.001
0.048 ± 0.014
0.140 ± 0.026
0.370 ± 0.037
0.288 ± 0.071
0.214 ± 0.074
0.080 ± 0.004
0.304 ± 0.106
0.748 ± 0.070
1.820 ± 0.261
1.296 ± 0.426
0.816 ± 0.285
0.077 ± 0.005
0.333 ± 0.094
0.942 ± 0.117
2.391 ± 0.165
3.400 ± 0.516
4.003 ± 0.726
0.002 ± 0.001
0.050 ± 0.015
0.190 ± 0.036
0.560 ± 0.057
0.848 ± 0.111
1.062 ± 0.185
0.080 ± 0.004
0.384 ± 0.107
1.132 ± 0.152
2.952 ± 0.199
4.248 ± 0.625
5.064 ± 0.908
-------
Table 111-13. Radioactivity in Blood and Tissue of Male Fischer 344 Rats at 96 h Following Dermal Treatment
with 14C-Labeled 4,4'-MOA (4.0 mg/Rat): Continuous Application versus Washing at 24 h
pq equivalents/g or mL
Tissue
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
No. 46
0.079
0.088
0.051
0.494
0.159
0.042
0.006
0.034
0.032
0.197
0.001
0.019
0.007
0.256
0.061
No. 47
0.112
0.134
0.070
0.686
0.316
0.069
0.022
0.042
0.061
0.173
0.000
0.037
0.011
0.572
0.090
Continuous
No. 48
0.055
0. 066
0.037
0.399
0.104
0.051
0.006
0.016
0.022
0.083
0.000
0.011
0.002
0.222
0.068
Mean +
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
082 ±
096 ±
053 ±
526 ±
193 ±
054 ±
Oil ±
031 ±
038 ±
151 ±
000 ±
022 ±
007 ±
350 ±
073 ±
S.E.
0.017
0.020
0.010
0.084
0.064
0.008
0.005
0.008
0.012 -
0.035
0.000
0.008
0.003
0.111
0.009
No. 43
0.016
0.016
0.011
0.167
0.030
0.002
0.001
0.002
0.001
0.037
0.000
0.002
0.002
0.042
0.054
No. 44
0.047
0.050
0.028
0.427
0.102
0.031
0.002
0.019
0.014
0.046
0.000
0.011
0.002
0.205
0.036
24-h Wash
No. 45
0.056
0.067
0.033
0.446
0.121
0.034
0.006
0.018
0.030
0.057
0.000
0.014
0.002
0.148
0.043
Mean
0.040
0.044
0.024
0.347
0.084
0.022
0.003
0.013
0.015
0.047
0.000
0.009
0.002
0.132
0.044
± S.E.
± 0.012
± 0.015
± 0.007
± 0.090
± 0.028
± 0.010
± 0.002
± 0.006
± 0.008
± 0.006
± 0.000
± 0.004
± 0.000
± 0.048
± 0.005
-------
Table 111-13 (continued)
i
00
Percent of administered dose
Tissue/excretum
Blood3 .
Plasmaa'u
RBCsa'D
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
GI tract
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
No. 46
0.027
0.018
0.007
0.101
0.008
0.001
0.000
0.000
0.002
0.000
0.000
0.037
0.144,
0.048
5.415
1.564
65.513
22.702
95.562
No. 47
0.039
0.028
0.010
0.137
0.014
0.001
0.001
0.000
0.004
0.000
0.000
0.071
0.266
0.071
5.023
1.364
64.590
17.146
88.727
Continuous
No. 48
0.020
0.014
0.005
0.086
0.005
0.001
0.000
0.000
0.002
0.000
0.000
0.022
0.134
0.055
4.008
1.077
57.233
32.136
94.779
Mean ± S.E.
0.029 ± 0.006
0.020 ± 0.004
0.007 ± 0.001
0.108 ± 0.015
0.009 ± 0.003
0.001 ± 0.000
0.000 ± 0.000
0.000 ± 0.000
0.003 ± 0.001
0.000 ± 0.000
0.000 ± 0.000
0.043 ± 0.014
0.181 ± 0.042
0.058 ± 0.007
4.815 ± 0.419
1.335 ± 0.141
62.445 ± 2.620
23.995 ± 4.375
93.023 ± 2.160
No. 43
0.006
0.004
0.002
0.035
0.001
0.000
0.000
0.000
0.000
0.000
0.000
0.004
0.023
0.042
2.558
0.710 .
80.807
3.808
87.994
No. 44
0.017
0.011
0.004
0.075
0.004
0.001
0.000
0.000
0.001
0.000
0.000
0.022
0.089
0.028
4.596
1.139
81.869
6.886
94.727
24- h Wash
No. 45
0.020
0.014
0.005
0.094
0.005
0.001
0.000
0.000
0.002
0.000
0.000
0.027
0.082
0.035
4.854
1.336
71.073
16.084
93.613
Mean ± S.E.
0.014 ± 0.004
0.010 ± 0.003
0.004 ± 0.001
0.068 ± 0.017
0.003 ± 0.001
0.001 ± 0.000
0.000 ± 0.000
0.000 ± 0.000
0.001 ± 0.001
0.000 ± 0.000
0.000 ± 0.000
0.018 ± 0.007
0.065 ± 0.021
0.035 ± 0.004
4.003 ± 0.726
1.062 ± 0.185
77.916 ± 3.435
8.926 ± 3.688
92.111 ± 2.084
Calculations are based on 7%, 16%, and 40% of body weight for blood, nontreated skin, and muscle, respectively.
^red blood cell calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
Plasma and
-------
Table 111-14. Recovery of Radioactivity in Blood, Tissue, and Excreta at 96 h Following Dermal Treatment of Male Fischer 344 Rats
with 14C-Labeled 4,4'-MDA (4.0 mg/Rat): Continuous Application versus Washing at 24 h
Tissue/
excretum
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
Dose wash
Appl ication area
No.
0.
0.
0.
5.
1.
7.
65.
22.
46
027
197
144
415
,564
.347
513
,702
No. 47
0.039
0.299
0.266
5.023
1.364
6.991
64.590
17.146
Continuous
No. 48
0.020
0.171
0.134
4.008
1.077
5.410
57.233
32.136
Mean i
0.029
0.222
0.181
4.815
1.335
6.583
62.445
23.995
Percent
S.E.
± 0.006
± 0.039
± 0.042
± 0.419
± 0.141
± 0.595
± 2.620
+ 4.375
of dose
No. 43
0.006
0.082
0.023
2.558
0.710
3.379
80.807
3.808
No. 44
0.017
0.131
0.089
4.596
1.139
5.972
81.869
6.886
24-h Wash
No. 45
0.020
0.164
0.082
4.854
1.336
6.456
71.073
16.084
Mean ±
0.014 ±
0.126 ±
0.065 ±
4.003 ±
1.062 ±
5.269 ±
77.916 ±
8.926 ±
S.E.
0.004
0.024
0.021
0.726
0.185
0.955
3.435
3.688
Recovery
95.562
88.727
94.779
93.023 ± 2.160
87.994 94.727
93.613
92.111 ± 2.084
-------
APPENDIX IV
GUINEA PIG STUDIES
INDIVIDUAL ANIMAL DATA
List of Tables
Table Page
IV-1 Urinary and Fecal Excretion of Radioactivity in Male
Hartley Guinea Pigs Treated Intravenously with 14C-
Labeled 4,4'-MDA (1.0 mg/Guinea Pig) . . . IV-3
IV-2 Radioactivity in Blood, Tissue, and Excreta at 96 h
Following Intravenous Treatment of Male Hartley Guinea
Pigs with 14OLabeled 4,4'-MDA (1.0 mg/Guinea Pig) IV-4
IV-3 Recovery of Radioactivity in Blood, Tissue, and Excreta at
96 h Following Intravenous Treatment of Male Hartley
Guinea Pigs with 14C-Labeled 4,4'-MDA
(1.0 mg/Guinea Pig) IV-5
IV-4 Urinary and Fecal Excretion of Radioactivity in Male
Hartley Guinea Pigs Treated Dermally with 14C-Labeled
4,4'-MDA (1.0 mg/Guinea Pig) IV-6
IV-5 Radioactivity in Blood and Tissue at 6, 24, and 96 h
Following Dermal Treatment of Male Hartley Guinea Pigs
with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig) IV-7
IV-6 Recovery of Radioactivity in Blood, Tissue, and Excreta at
6, 24, or 96 h Following Dermal Treatment of Male Hartley
Guinea Pigs with 14C-Labeled 4,4'-MDA
(1.0 mg/Guinea Pig) IV-9
IV-7 Radioactivity in Blood, Tissue, and Excreta 6 h Following
Dermal Treatment of Male Hartley Guinea Pigs with
14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig): Nonoccluded
Application Area IV-10
IV-8 Recovery of Radioactivity in Blood, Tissue, and Excreta at
6 h Following Dermal Treatment of Male Hartley Guinea
Pigs with 14OLabeled 4,4'-MDA (1.0 mg/Guinea Pig):
Nonoccluded Application Area IV-11
IV-9 Urinary and Fecal Excretion of Radioactivity in Male
Hartley Guinea Pigs Treated Dermally with 14C-Labeled
4,4'-MDA (1.0 mg/Guinea Pig): Application Area Washed
at 24 h IV-12
IV-1
-------
LIST OF TABLES (continued)
Table Page
IV-10 Radioactivity in Blood, Tissue, and Excreta at 96 h
Following Dermal Treatment of Male Hartley Guinea Pigs
with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig):
Application Area Washed at 24 h IV-13
IV-11 Recovery of Radioactivity in Blood, Tissue, and Excreta at
96 h Following Dermal Treatment of Male Hartley Guinea
Pigs with 14OLabeled 4,4'-MDA (1.0 mg/Guinea Pig):
Application Area Washed at 24 h IV-14
IV-12 Urinary and Fecal Excretion of Radioactivity in Male
Hartley Guinea Pigs Treated Dermally with 14C-|_abeled
4,4'-MDA (10.0 mg/Guinea Pig): Continuous Application
versus Washing at 24 h IV-15
IV-13 Radioactivity in Blood and Tissue at 96 h Following
Treatment of Male Hartley Guinea Pigs with 14OLabeled
4,4'-MDA (10.0 mg/Guinea Pig): Continuous Application
versus Washing at 24 h IV-16
IV-14 Recovery of Radioactivity in Blood, Tissue, and Excreta at
96 h Following Dermal Treatment of Male Hartley Guinea
Pigs with 14C-Labeled 4,4'-MDA (10.0 mg/Guinea Pig):
Continuous Application versus Washing at 24 h IV-18
IV-2
-------
Table IV-1. • Urinary and Fecal Excretion of Radioactivity in Male Hartley Guinea Pigs Treated Intravenously
with l4C-labeled 4,4'-MDA (1.0 mg/Guinea Pig)
i
CO
Excretum
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
No. 67
7.890
12.521
12.912
4.176
0.604
0.302
2.122
0.079
26.809
22.212
3.804
- 1.453
10.012
12.600
39.721
26.388
4.408
1.755
No. 68
4.909
13.974
6.258
2.925
0.675
0.213
1.589
7.006
30.216
18.073
4.349
1.635
6.498
20.980
36.474
20.998
5.024
1.848
No. 69
6.489
19.493
7.496
2.831
0.763
0.522
1.626
5.054
24.841
13.711
4.063
0.708
8.115
24.547
32.337
16.542
4.826
1.230
Mean ±
6.429
15.329
8.889
3.311
0.681
0.346
1.779
4.046
27.289
17.999
4.072
1.265
8.208
19.376
36.177
21.309
4.753
1.611
S.E.
± 0.861
± 2.124
± 2.043
± 0.434
± 0.046
± 0.092
± 0.172
± 2.062
± 1.570
± 2.454
± 0.157
± 0.284
± 1.015
± 3.541
± 2.137
± 2.847
± 0.182
± 0.192
No. 67
7.890
20.411
33.323
37.499
38.103
38.405
2.122
2.201
29.010
51.222
55.026
56. 479
10.012
22.612
62.333
88.721
93.129
94.884
Cumulative percent
No. 68
4.909
18. 883
25.141
28.066
28.741
28.954
1.589
8.595
38.811
56.884
61.233
62.868
6.498
27.478
63.952
84. 950
89.974
91.822
No. 69
6.489
25.982
33.478
36. 309
37.072
37.594
1.626
6.680
31.521
45.232
49.295
50.003
8.115
32.662
64.999
81.541
86. 367
87.597
of dose
Mean ±
6.429
21.759
30.647
33.958
34.639
34.984
1.779
5.825
33.114
51.113
55.185
56.450
8.208
27.584
63.761
85.071
89.823
91.434
S.E.
t 0.861
± 2.157
+ 2.754
± 2.966
± 2.964
± 3.024
± 0.172
± 1.895
± 2.939
± 3.364
± 3.447
± 3.714
± 1.015
± 2.902
± 0.775
± 2.074
± 1.953
± 2.112
-------
Table IV-2. Radioactivity in Blood, Tissue, and Excreta at 96 h Following Intravenous Treatment of Male Hartley Guinea Pigs
with I4C-Labe1ed 4,4'-MDA (1.0 mg/Guinea Pig)
Tissue/
excretum
Blood3 .
Plasma3'0
RBCsa'D
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
FatD
GI tract
Skin3
Urine
Feces
Recovery
|jg equivalents/g or mL
No. 67
0.274
0.266
0.216
0.931
0.320
0.203
0.005
1.071
0.030
0.178
0.018
0.015
0.030
0.111
0.056
-
-
-
No. 68
0.196
0.169
0.199
0.684
0.232
0.314
0.003
1.376
0.023
0.168
0.007
0.010
'0.029
0.062
0.043
-
-
-
No. 69
0.168
0.169
0.196
0.687
0.180
0.640
0.003
•1.111
0.012
0.228
0.012
0.006
0.021
0.067
0.041
-
-
-
Mean ± S.E.
0.213 ± 0.032
0.201 ± 0.032
0.204 ± 0.006
0.767 ± 0.082
0.244 ± 0.041
0.386 ± 0.131
0.004 ± 0.001
1.186 ± 0.096
0.022 ± 0.005
0.191 ± 0.019
0.012 ± 0.003
0.010 ± 0.003
0.027 ± 0.003
0.080 ± 0.016
0.047 ± 0.005
-
-
-
No. 67
0.676
0.394
0.213
1.603
0.116
0.045
0.003
0.050
0.003
0.003
0.001
0.212
-
0.699
0.314
38.405
56.479
98.609
Percent of dose
No. 68
0.525
0.271
0.213
1.813
0.102
0.073
0.002
0.060
0.002
0.002
0.000
0.153
-
0.482
0.263
28.954
62.868
95.299
No. 69
0.449
0.271
0.210
1.622
0.108
0.176
0.002
0.080
0.001
0.004
0.000
0.092
-
0.641
0.249
37.594
50.003
91.021
Mean ± S.E.
0.550 ± 0.067
0.312 ± 0.041
0.212 ± 0.001
1.679 ± 0.067
0.109 ± 0.004
0.098 ± 0.040
0.002 ± 0.000
0.063 ± 0.009
0.002 ± 0.001
0.003 ± 0.001
0.000 ± 0.000
0.152 ± 0.035
'
0.607 ± 0.065
0.275 ± 0.020
34.984 ± 3.024
56.450 ± 3.714
94.976 ± 2.196
a .
.red blood cell calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
-------
Table IV-3. Recovery of Radioactivity in Blood, Tissue, and Excreta at 96 h Following Intravenous
Treatment of Male Hartley Guinea Pigs with 14OLabeled 4,4'-MDA (1.0 nig/Guinea Pig)
Tissue/
excretum
Blood
Tissue
GI tract
Urine
Feces
No. 67
0.676
2.350
0.699
38.405
56.479
No. 68
0.525
2.470
0.482
28.954
62.868
Percent of dose
No. 69
0.449
2.334
0.641
37.594
50.003
Mean ± S.E.
0.550 ± 0.067
2.385 ± 0.043
0.607 ± 0.065
34.984 ±3.024
56.450 ± 3.714
Recovery
98.609
95.299
91.021
94.976 ± 2.196
-------
Table IV-4. Urinary and Fecal Excretion of Radioactivity in Male Hartley Guinea Pigs Treated Dermally
with 14C-Labeled 4,4'-MDA (1.0 rag/Guinea Pig)
Excretum
Time after
dosing (h)
24-h sacrifice
No. 76
No. 77
No. 78
Mean ± S.E.
No. 73
96-h sacrifice
No. 74
No. 75
Mean ±
S.E.
Percent of dose
Urine
Feces
Total
Urine
Feces
Total
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
0.090
1.194
1.492
0.014
0.657
2.968
0.104
1.851
4.460
-
0.090
1.284
2.776
0.014
0.671
3.639
0.104
1.955
6.415
0.701
6.381
3.352
0.044
1.704
7.721
0.745
8.085
11.073
0.701
7.082
10.434
0.044
1.748
9.469
0.745
8.830
19.903
5.286
2.701
2.218
0.093
1.097
2.808
5.379
3.798
5.026
,
5.286
7.987
10.205
0.093
1.190
3.998
5.379
9.177
14.203
2.
3.
2.
0.
1.
4.
2.
4.
6.
2.
5.
7.
0.
1.
5.
2.
6.
13.
026 ± 1.640
425 ± 1.541
354 ± 0.541
050 ± 0.023
153 ± 0.304
499 ± 1.612
076 ± 1.662
578 ± 1.841
853 ± 2.116
Cumulative
026 ± 1.640
451 ± 2.100
805 ± 2.515
050 ± 0.023
203 ± 0.311
702 ± 1.886
076 ± 1.662
654 ± 2.352
507 ± 3.909
0.311
1.761
2.417
4.364
1.503
2.151
0.031
0.127
3.700
7.490
3.601
2.326
0.342
1.888
6.117
11.854
5.104
4.477
percent of dose
0.311
2.072
4.489
8.853
10. 356
12.507
0.031
0.158
3.858
11.348
14.949
17.275
0.342
2.230
8.347
20.201
25.305
29.782
0.215
0.820
1.719
1.875
0.799
0.628
0.048
1.257
4.958
5.027
2.558
1.548
0.263
2.077
6.677
6.902
3.357
2.176
0.215
1.035
2.754
4.629
5.428
6.056
0.048
1.305
6.263
11.290
13.848
15.396
0.263
2.340
9.017
15.919
19.276
21.452
0.124
1.319
4.259
3.730
2.185
1.214
0.012
0.924
2.963
9.296
4.563
2.441
0.136
2.243
7.222
13.026
6.748
3.655
0.124
1.443
5.702
9.432
11.617
12.831
0.012
0.936
3.899
13.195
17.758
20.199
0.136
2.379
9.601
22.627
29.375
33.030
0.217
1.300
2.798
3.323
1.496
1.331
0.030
0.769
3.874
7.271
3.574
2.105
0.247
2.069
6.672
10.594
5.070
3.436
0.217
1.517
4.315
7.638
9.134
10.465
0.030
0.800
4.673
11.944
15.518
17.623
0.247
2.316
8.988
19. 582
24.652
28. 088
± 0.054
± 0.272
± 0.758
± 0.747
± 0.400
± 0.444
± 0.010
± 0.335
± 0.582
± 1.237
± 0.579
± 0.280
± 0.060
± 0.103
± 0.319
± 1.877
± 0.979
± 0.673
± 0.054
± 0.302
± 0.855
± 1.514
± 1.888
± 2.206
± 0.010
± 0.338
± 0.795
± 0.626
± 1.164
± 1.397
± 0.060
± 0.045
± 0.362
± 1.961
± 2.934
± 3.448
-------
Table IV-5. Radioactivity in Blood and Tissue at 6, 24, and 96 h Following Dermal Treatment
of Male Hartley Guinea Pigs with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig)
ug equivalents/g or ml
Tissue
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
No. 79
0.053
0.056
0.033
0.295
0.090
0.061
0.043
0.041
0.107
0.853
0.011
0.016
0.031
0.288
0.055
No. 80
0.039
0.044
0.024
0.233
0.090
0.042
0.020
0.030
0.095
0.553
0.004
0.018
0.031
0.257
0.039
6 h
No. 81
0.053
0.057
0.019
0.227
0.096
0.057
0.028
0.033
0.148
1.887
0.005
0.031
0.026
0.281
0.067
Mean ± S.E.
0.048 ± 0.005
0.052 ± 0.004
0.025 ± 0.004
0.252 ± 0.022
0.092 ± 0.002
0.053 ± 0.006
0.030 ± 0.007
0.035 ± 0.003
0.117 ± 0.016
1.098 ± 0.404
0.006 ± 0.002
0.022 ± 0.005
0.029 ± 0.002
0.275 ± 0.009
0.054 ± 0.008
No. 76
0.049
0.053
0.033
0.231
0.100
0.053
0.017
0.023
0.048
0.944
0.002
0.012
D.013
0.509
0.029
No. 77
0.083
0.084
0.058
0.356
0.171
0.078
0.022
0.037
0.097
0.954
0.003
0.020
0.016
0.999
0.092
24 h
No. 78
0.044
0.042
0.035
0.248
0.101
0.038
0.014
0.019
0.049
1.031
0. 002
0.010
0.014
0.509
0.026
Mean ± S.E.
0.059 ± 0.012
0.060 ± 0.013
0.042 ± 0.008
0.278 ± 0.039
0.124 t 0.024
0.056 ± 0.012
0.018 ± 0.002
0.026 ± 0.005
0.065 ± 0.016
0.976 ± 0.027
0.002 ± 0.000
0.014 ± 0.003
0.014 ± 0.001
0.672 ± 0.163
0.049 ± 0.022
No. 73
0.061
0.061
0.057
0.266
0.152
0.045
0.005
0.027
0.031
0.668
0.009
0.060
0.013
0.154
0.025
No. 74
0.041
0.037
0.044
0.089
0.061
0.016
0.002
0.010
0.017
0.472
0.003
0.015
0.009
0.070
0.026
96 h
No. 75
0.073
0.067
0.067
0.217
0.105
0.044
0.005
0.015
0.024
0.403
0.004
0.004
0.012
0.089
0.075
Mean ± S.E.
0.058 ± 0.009
0.055 ± 0.009
0.056 ± 0.007
0.191 ± 0.053
0.106 ± 0.026
0.035 ± 0.010
0.004 ± 0.001
0.017 ± 0.005
0.024 ± 0.004
0.514 ± 0.079
0.005 ± 0.002
0.026 ± 0.017
0.011 ± 0.001
0.104 ± 0.025
0.042 ± 0.017
-------
Table IV-5 (continued)
i
CO
Tissue/
excretum
Blood3 .
Plasma3'"
RBCsa'D
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
GI tract
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
No. 79
0.119
0.076
0.030
0.418
0.044
0.016
0.020
0.002
0.011
0.014
0.000
0.201
1.679
0.282
0.292
0.116
83.530
13.225
99.969
No. 80
0.085
0.057
0.021
0.387
0.041
0.011
0.009
0.001
0.013
0.009
0.000
0.218
1.294
0.194
0.548
0.046
78. 900
9.481
91.237
6 h
No. 81
0.148
0.097
0.021
0.380
0.046
0.013
0.013
0.002
0.025
0.040
0.000
0.499
1.549
0.428
0.216
0.133
79.215
11.572
94.279
Percent of administered dose
Mean ± S.E.
0.117 ± 0.018
0.077 ± 0.012
0.024 ± 0.003
0.395 ± 0.012
0.044 ± 0.001
0.013 ± 0.001"
0.014 ± 0.003
0.002 ± 0.000
0.016 ± 0.004
0.021 ± 0.010
0.000 ± 0.000
0.306 ± 0.097
"1.507 ± 0.113
0.301 ± 0.068
0.352 ± 0.100
0.098 ± 0.027
80.548 ± 1.494
11.426 ± 1.083
95.162 ± 2.559
No. 76
0.121
0.079
0.033
0.401
0:638
0.013
0.007
0.001
0.005
0.013
0.000
0.163
2.301
0.164
2.776
3.639
68.980
14.89-1
93.513
No. 77
0.199
0.120
0.055
0.622
0.064
0.017
0.009
0.002
0.009
0.016
0.000
0.266
4.624
0.502
10. 434
9.469
44.605
17.700
88.538
24 h
No. 78
0.104
0.060
0.033
0.409
0.031
0.008
0.006
0.001
0.005
0.016
0.000
0.129
1.461
0.141
10.205
3.998
62.740
11.721
90.975
Mean ± S.E.
0.141 ± 0.029
0.086 ± 0.018
0.040 ± 0.007
0.477 ± 0.072
0.044 ± 0.010
0.013 ± 0.003
0.007 ± 0.001
0.001 ± 0.000
0.006 ± 0.001
0.015 ± 0.001
0.000 ± 0.000
0.186 ± 0.041
2.795 ± 0.946
0.269 ± 0.117
7.805 ± 2.515
5.702 ± 1.886
58.775 ± 7.310
14.771 ± 1.727
91.009 ± 1.436
No. 73
0.154
0.093
0.058
0.528
0.060
0.010
0.002
0.001
0.003
0.012
0.000
0.864
0.870
0.142
12.507
17.275
38.805
26.077
97.310
No. 74
0.095
0.052
0.041
0.243
0.032
0.005
0.001
0.001
0.001
0.008
0.000
0.195
0.402
0.140
6.056
15.396
49.095
33.843
105.513
96 h
No. 75
0.177
0.098
0.065
0.436
0.053
0.012
0.003
0.001
0.003
0.007
0.000
0.049
0.493
0. 415
12.831
20.199
34.635
28.294
97.608
Mean ± S.E.
0.142 ± 0.024
0.081 ± 0.015
0.055 ± 0.007
0.402 ± 0.084
0.048 ± 0.008
0.009 ± 0.002
0.002 ± 0.001
0.001 ± 0.000
0.002 ± 0.001
0.009 ± 0.002
0.000 ± 0.000
0.369 ± 0.251
0.588 ± 0.143
0.232 ± 0.091
10.465 ± 2.206
17.623 ± 1.397
40.845 ± 4.297
29.405 ± 2.310
100.144 ± 2.686
a .
^calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
-------
Table IV-6. Recovery of Radioactivity in Blood, Tissue, and Excreta at 6, 24, or 96 h Following Dermal Treatment
of Male Hartley Guinea Pigs with 14C-Labeled 4,4'-MDA (1.0 nig/Guinea Pig)
i
10
Tissue/
excretum
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
Dose wash
Application area
Percent of dose
No. 79
0.119
1.008
1.679
0.292
0.116
3.214
83.530
13.225
No. 80
0.085
0.883
1.294
0.548
0.046
2.855
78.900
9.481
6 h
No. 81
0.148
1.446
1.549
0.216
0.133
3.492
79.215
- 11.572
Mean ± S.E.
0.117 ± 0.018
1.112 ± 0.171
1.507 ± 0.113
0.352 ± 0.100
0.098 ± 0.027
3.187 ± 0.184
80.548 ± 1.494
11.426 ± 1.083
No. 76
0.121
0.805
2.301
2.776
3.639
9.516
68.980
14,891
No. 77
0.199
1.507
4.624
10.434
9.469
26.233
44.605
17.700
24 h
No. 78
0.104
0.746
1.461
10.205
3.998
16.514
62.740
11.721
Mean ± S.E.
0.141 ± 0.029
1.019 ± 0.244
2.795 ± 0.946
7.805 ± 2.515
5.702 ± 1.886
17.421 ± 4.847
58.775 ± 7.310
14.771 ± 1.727
No. 73
0.154
1.622
0.870
12.507
17.275
32.428
38.805
26.077
No. 74
0.095
0.626
0.402
6.056
15.396
22.575
49.095
33.843
96 h
No. 75
0.177
0.979
0.493
12.831
20.199
34.679
34.635
28. 294
Mean ± S.E.
0.142 ± 0.024
1.076 ± 0.292
0.588 ± 0.143
10.465 ± 2.206
17.623 ± 1.397
29.894 ± 3.717
40.845 ± 4.297
29.405 ± 2.310
Recovery
99.969 91.237 94.279 95.162 ± 2.559 93.513 88.538 90.975 91.009 ± 1.436 97.310 105.513 97.608 100.144 ± 2.686
-------
Table IV-7. Radioactivity in Blood, Tissue, and Excreta 6 h Following Dermal Treatment of Male Hartley Guinea Pigs
with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea-Pig): Nonoccluded Application Area
Tissue/
excretum
Blood3 .
Plasma*'0
RBCsa>D
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
FatD
GI tract
Nontreated skin3
Urine
Feces
Dose wash
Application area
Recovery
ug equivalents/g or ml
No.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
0.
0.
0.
0.
0.
.
-
.
-
-
82
052
049
043
185
198
049
038
040
112
079
Oil
018
014
110
037
No. 83
0.040
0.047
0.021
0.259
0.087
0.059
0.027
0.031
0.100
0.650
0.006
0.016
' 0.019
0.286
0.034
.
-
.
-
-
No. 84
0.044
0.043
0.031
0.226
0.284
0.047
0.025
0.046
0.085
0.760
0.004
0.021
0.013
0.192
0.056
_
-
.
-
-
Mean ± S.E.
0.045
0.046
0.032
0.223
0.190
0.052
0.030
0.039
0.099
0.830
0.007
0.018
0.015
0.196
0.042
± 0.004
± 0.002
± 0.006
± 0.021
± 0.057
± 0.004
± 0.004
± 0.004
± 0.008
± 0.129
± 0.002
± 0.001
± 0.002
± 0.051
± 0.007
-
-
_
-
-
No. 82
0.146
0.083
0.048
0.304
0.100
0.012
0.018
0.002
0.015
0.020
0.000
0.282
-
0.859
0.239
0.116
0. 088
88.680
8.448
99.329
Percent of dose
No. 83
0.086
0.061
0.018
0.497
0.049
0.015
0.013
0.002
0.013
0.011
0.000
0.190
-
1.846
0.167
0.147
0.039
84.975
10.525
98.575
No. 84
0.120
0.070
0.034
0.299
0.107
0.010
0.012
0.002
0.013
0.011
0.000
0.319
-
1.117
0.349
1.549
0.235
88. 718
9.147
102.005
Mean ± S.E.
0.117 ±
0.071 ±
0.033 ±
0.367 ±
0.085 ±
0.012 ±
0.014 ±
0.002 ±
0.013 ±
0.014 ±
0.000 ±
0.264 ±
-
1.274 ±
0.252 ±
0.604 ±
0.121 ±
87.458 ±
9.373 ±
99.970 ±
0.017
0.006
0.009
0.065
0.018
0.001
0.002
0.000
0.001
•0.003
0.000
0.038
0.300
0.053
0.473
0.059
1.241
0.610
1.041
a .
^Plasma and red blood cell calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
-------
Table IV-8. Recovery of Radioactivity in Blood, Tissue, and Excreta at 6 h Following Dermal Treatment
of Male Hartley Guinea Pigs with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig):
Nonoccluded Application Area
Tissue/
excretum
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
Dose wash
Application area
No. 82
0.146
0.992
0.859
0.116
0.088
2.201
88.680
8.448
No. 83
0.086
0.957
1.846
0.147
0.039
3.075
84.975
10.525
Percent of dose
No. 84
0.120
1.119
1.117
1.549
0.235
4.140
88.718
9.147
Mean ± S.E.
0.117 ± 0.017
1.023 ± 0.049
1.274 ± 0.300
0.604 ± 0.473
0.121 ± 0.059
3.139 ± 0.561
87.458 ± 1.241
9.373 ± 0.610
Recovery 99.329 98.575 102.005 99.970 ± 1.041
-------
Table IV-9. Urinary and Fecal Excretion of Radioactivity in Male Hartley Guinea Pigs Treated Dermally
with 14C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig): Application Area Washed at 24 h
Excretum
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
Percent of dose
No. 70
0.168
1.191
2.012
1.788
0.325
0.253
0.045
1.308
3.988
4.771
0.494
0.442
0.213
2.499
6.000
6.559
0.819
0.695
No. 71
0.129
1.943
2.527
1.620
0.205
0.143
0.018
0.971
5.707
4.439
0.478
0.200
0.147
2.914
8.234
6.059
0.683
0.343
No. 72
0.225
1.456
2.312
2.961
0.256
0.179
0.010
1.139
2.276
5.597
0.518
0.258
0.235
2.595
4.588
8.558
0.774
0.437
Mean ± S.E.
0.174 ±
1.530 ±
2.284 ±
2.123 ±
0.262 ±
0.192 ±
0.024 ±
1.139 ±
3.990 ±
4.936 ±
0.497 ±
0.300 ±
0. 198 ±
2.669 ±
6.274 ±
7.059 ±
0.759 ±
0.492 ±
0.028
0.220
0.149
0.422
0.035
0.032
0.011
0.097
0.990
0.344
0.012
0.073
0.026
0.125
1.061
0.763
0.040
0.105
No. 70
0.168
1.359
3.371
5.159
5.484
5.737
0.045
1.353
5.341
10.112
10.606
11.048
0.213
2.712
8.712
15.271
16.090
16.785
Cumulative percent
No. 71
0.129
2.072
4.599
6.219
6.424
6.567
0.018
0.989
6.696
11. 135
11.613
11.813
0.147
3.061
11.295
17.354
18. 037
18.380
No. 72
0.225
1.681
3.993
6.954
7.210
7.389
0.010
1.149
3.425
9.022
9.540
9.798
0.235
2.830
7.418
15.976
16. 750
17.187
of dose
Mean ± S.E.
0.174
1.704
3.988
6.111
6.373
6.564
0.024
1.164
5.154
10.090
10.586
10.886
0.198
2.868
9.142
16.200
16.959
17.451
± 0.028
± 0.206
± 0.355
± 0.521
± 0.499
± 0.477
± 0.011
± 0.105
± 0.949
± 0.610
± 0.599
± 0.587
± 0.026
± 0.102
± 1.140
± 0.612
± 0.572
± 0.479
-------
Table IV-10. Radioactivity in Blood, Tissue, and Excreta at 96 h Following Dermal Treatment of
Male Hartley Guinea Pigs with l4C-Labeled 4,4'-MDA (1.0 mg/Guinea Pig):
Application Area Washed at 24 h
Tissue/
excretum
Blood3
Plasma3'0
RBCsa'b
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
FatD
GI tract
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
ug equivalents/g or mL
No.
0.
0.
0.
0.
0.
0.
0.
70
032
026
034
098
057
016
002
0.007
0.
0.
- o.
0.
0.
0.
0.
_
'
_
-
-
006
072
004
002
012
026
010
No. 71
0.031
0.020
0.038
0.129
0.057
0.015
0.002
0.008
0.005
0.065
0.003
0.004
0.005
0.014
0.006
_
-
.
-
-
No. 72
0.035
0.029
0.042
0.096
0.060
0.017
0.001
0.011
0.003
0.061
0.003
0.003
0.008
0.015
0.010
_
-
_
-
-
Mean ±
0.033
0.025
0.038
0.108
0.058
0.016
0.002
0.009
0.005
0.066
0.003
0.003
0.008
0.018
0.009
S.E.
± 0.001
± 0.003
± 0.002
± 0.011
± 0.001
± 0.001
± 0.000
± 0.001
± 0.001
± 0.003
± 0.000
± 0.001
+ 0.002
± 0.004
± 0.001
_
-
.
-
-
No. 70
0.078
0.039
0.034
0.194
0.023
0.004
0.001
0.000
0.000
0.001
0.000
0.021
-
0.191
0.054
5.737
11.048
60.535
19.255
97. 142
Percent of dose
No. 71
0.078
0.030
0.038
0.272
0.025
0.003
0.001
0.001
0.001
0.001
0.000
0.051
-
0.085
0.032
6.567
11.813
60.925
15.285
95.140
No. 72
0.082
0.041
0.039
0.190
0.023
0.004
0.000
0.000
0.000
0.001
0.000
0.034
-
0.083
0.051
7.389
9.798
63.880
15.201
96.736
Mean ±
0.079 ±
0.037 ±
0.037 ±
0.219 ±
0.024 ±
0.004 ±
0.001 ±
0.000 ±
0.000 ±
0.001 ±
0.000 ±
0.035 ±
-
0.120 ±
0.046 ±
6.564 ±
10.886 ±
61.780 ±
16.580 ±
96.339 ±
S.E.
0.001
0.003
0.002
0.027
0.001
0.000
0.000
0.000
0.000
0.000
0.000
0.009
0.036
0. 007
0.477
0.587
1.056
1.338
0.611
aD .
^Plasma and red blood cell calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
-------
Table IV-11. Recovery of Radioactivity in Blood, Tissue, and Excreta at 96 h Following Dermal Treatment
of Male Hartley Guinea Pigs with 14C-Labeled 4,4'-MDA (1.0 nig/Guinea Pig):
Application Area Washed at 24 h
Tissue/
excretum
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
Dose wash
Application area
No. 70
0.078
0.298
0.191
5.737
11.048
17.352
60.535
19.255
No. 71
0.078
0.387
0.085
6.567
11.813
18.930
60.925
15.285
Percent of dose
No. 72
0.082
0.303
0.083
7.389
9.798
17.655
63.880
15.201
Mean ± S.E.
0.079 ± 0.001
0.329 ± 0.029
0.120 ± 0.036
6.564 ± 0.477
10.886 ± 0.587
17.979 ± 0.483
61.780 ± 1.056
16.580 ± 1.338
Recovery 97.142 95.140 96.736 96.339 ± 0.611
-------
Table IV-12. Urinary and Fecal Excretion of Radioactivity in Male Hartley Guinea Pigs Treated Dermally with
14C-Labeled 4,4'-MDA (10.0 mg/Guinea Pig): Continuous Application versus Washing at 24 h
i
en
Percent of dose
Excretum
Urine
Feces
Total
Urine
Feces
Total
Time after
dosing (h)
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
6
12
24
48
72
96
No. 64
0.058
0.183
0.326
0.525
0.823
0.511
0.005
0.018
0.673
1.255
1.146
1.072
o;o63
0.201
0.999
1.780
-1.969
1.583
0.058
0.241
0.567
1.092
1.915
2.426
0.005
0.023
0.696
1.951
3.097
4.169
0.063
0.264
1.263
3.043
5.012
6.595
Continuous
No. 65
0.021
0.339
0.159
0.277
0.541
1.741
0.012
0.046
0.724
0.675
0.562
1.048
0.033
0.385
0.883
0.952
1.103
2.789
0.021
0.360
0.519
0.796
1.337
3.078
0.012
0.058
0.782
1.457
2.019
3.067
0.033
0.418
1.301
2.253
3.356
6.145
Average
0.040
0.261
0.243
0.401
0.682
1.126
0.009
0.032
0.699
0.965
0.854
1.060
0.048
0.293
0.941
1.366
1.536
2.186
Cumulative
0.040
0.301
0.543
0.944
1.626
2.752
0.009
0.041
0.739
1.704
2.558
3.618
0.048
0.341
1.282
2.648
4.184
6.370
No. 61
0.023
0.178
0.354
0.318
0.087
0.066
0.003
0.064
0.601
0.470
0.269
0.162
0.026
0.242
0.955
0. 788
0.356
0.228
percent of dose
0.023
0.201
0.555
0.873
0.960
1.026
0.003
0.067
0.668
1.138
1.407
1.569
0.026
0.268
1.223
2.011
2.367
2.595
No. 62
0.021
0.156
0.265
1.011
0.352
0.266
0.004
0.056
0.435
1.081
0.720
0.612
0.025
0.212
0.700
2.092
1.072
0.878
0.021
0.177
0.442
1.453
1.805
2.071
0.004
0.060
0.495
1.576
2.296
2.908
0.025
0.237
0.937
3.029
4.101
4.979
24-h Wash
No. 63
0.099
0.132
0.245
0.481
0.337
0.228
0.002
0.051
0.511
0.778
0.306
0.406
0.101
0.183
0.756
1.259
0.643
0.634
0.099
0.231
0.476
0.957
1.294
1.522
0.002
0.053
0.564
1.342
1.648
2.054
0.101
0.284
1.040
2.299
2.942
3.576
Mean ± S.E.
0.048 ± 0.026
0.155 ± 0.013
0.288 ± 0.034
0.603 ± 0.209
0.259 ± 0.086
0.187 ± 0.061
0.003 ± 0.001
0.057 ± 0.004
0.516 ± 0.048
0.776 ± 0.176 ..
0.432 ± 0.145
0.393 ± 0.130
0.051 ± 0.025
0.212 ± 0.017
0.804 ± 0.077
1.380 ± 0.381
0.690 ± 0.208
0.580 ± 0.190
0.048 ± 0.026
0.203 ± 0.016
0.491 ± 0.033
1.094 ± 0.181
1.353 ± 0.246
1.540 ± 0.302
0.003 ± 0.001
0.060 ± 0.004
0.576 ± 0.050
1.352 ± 0.127
1.784 ± 0.265
2.177 ± 0.391
0.051 ± 0.025
0.263 ± 0.014
1.067 ± 0.084
2.446 ± 0.303
3.137 ± 0.510
3.717 ± 0.692
-------
CT>
Table IV-13. Radioactivity in Blood and Tissue at 96 h Following Treatment of Male Hartley Guinea Pigs
with 14C-Labeled 4,4'-MDA (10.0 mg/Guinea Pigs): Continuous Application versus Washing at 24 h
ug equivalents/g or ml
Tissue
Blood
Plasma
RBCs
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle
Fat
GI tract
Nontreated skin
No. 64
0.179
0.196
0.147
0.654
0.343
0.111
0.012
0.056
0.115
2.537
0.011
0.027
0.076
0.692
0.609
Continuous
No. 65
0.135
0.149
0.100
0.524
0.283
0.122
0.006
0.028
0.749
1.812
0.005
0.045
0.035
1.077
0.191
Average
0.157
0.173
0.124
0.589
0.313
0.117
0.009
0.042
0.432
2.175
0.008
0. 036
0.056
0.885
0.400
No. 61
0.049
0.043
0.051
0.076
0.054
0.014
0.001
0.006
0.019
0.156
0.042
0.008
0.010
0.072
0.044
No. 62
0.146
0.149
0.126
0.374
0.246
0.026
0.009
0.020
0.042
1.440
0.016
0. 015
0.007
0.549
0.097
24-h Wash
No. 63
0.072
0.069
0.059
0.327
0.156
0.039
0.005
0.016
0.042
0.577
0.002
0.036
0.015
0.603
0.085
Mean ± S.E.
0.089 ± 0.029
0.087 ± 0.032
0.079 ± 0.024
0.259 ± 0.093
0.152 ± 0.056
0.026 ± 0.007
0.005 ± 0.002
0.014 ± 0.004
0.034 ± 0.008
0.724 ± 0.378
0.020 ± 0.012
0.020 ± 0.008
0.011 ± 0.002
0.408 ± 0.169
0.075 ± 0.016
-------
Table IV-13 (continued)
Tissue/excretum
Blood3 .
PlasSab
RBCs3'0
Liver
Kidneys
Lungs
Brain
Spleen
Testes
Adrenals
Bladder
Muscle3
GI tract
Nontreated skin
Urine
Feces
Dose wash
Application area
Recovery
No. 64
0.043
0.029
0.014
0.132
0.013
0.002
0.001
0.000
0.001
0.005
0.000
"0.037
0.485
0.332
2.426
4.169
66.359
16.014
90.019
Continuous
No. 65
0.036
0.024
0.011
0.126
0.013
0.003
0.000
0.000
0.007
0.003
0.000
0.068
0.791
0.116
3.078
3.067
73.030
11.430
91.768
Percent of
Average
0.040
0.027
0.013
0.129
0.013
0.003
0.001
0.000
0.004
0.004
0.000
0.053
0.638
0.224
2.752
3.618
69.695
13.722
90.894
administered dose
No. 61
0.012
0.006
0.005
0.017
0.002
0.000
0.000
0.000
0.000
0.000
0.000
0.011
0.053
0.024
1.026
1.569
81.049
5.903
89.666
No. 62
0.033
0.020
0.012
0.063
0.010
0.001
0.001
0.000
0.000
0.002
0.000
0.020
0.375
0.050
2.071
2.908
75.938
10.016
91.488
24- h Wash
No. 63
0.021
0.012
0.007
0.060
0.006
0.001
0.000
0.000
0.001
0.001
0.000
0.061
0.407
0.058
1.522
2.054
84.329
5.956
94.477
Mean ± S.E.
0.022 ± 0.006
0.013 ± 0.004
0.008 ± 0.002
0.047 ± 0.015
0.006 ± 0.002
0.001 ± 0.000
0.000 ± 0.000
0.000 ± 0.000
0.000 ± 0.000
0.001 ± 0.001
0.000 ± 0.000
0.031 ± 0.015
0.278 ± 0.113
0.044 ± 0.010
1.540 ± 0.302
2.177 ± 0.391
80.439 ± 2.441
7.292 ± 1.362
91.877 ± 1.402
Calculations are based on 7%, 16%, and 40% of body weight for blood, nontreated skin, and muscle, respectively. Plasma and red blood
.cell calculations are based on 60% and 40% of blood volume, respectively.
Individual blood components and fat are not included in recovery estimates.
-------
Table IV-14. Recovery of Radioactivity in Blood, Tissue, and Excreta at 96 h Following Dermal Treatment of Male Hartley Guinea Pigs
with 14C-Labeled 4,4'-MDA (10.0 mg/Guinea Pig): Continuous Application versus Washing at 24 h
i
00
Tissue/
excretum
Blood
Tissue
GI tract
Urine
Feces
Total absorbed
Dose wash
Application area
Recovery
Percent of dose
No. 64
0.043
0.523
0.485
2.426
4.169
7.646
66.359
16.014
90.019
Continuous
No. 65
0.036
0.336
0.791
3.078
3.067
7.308
73.030
11.430
91.768
Average
0.040
0.430
0.638
2.752
3.618
7.477
69.695
13.722
90.894
No. 61
0.012
0.054
0.053
1.026
1.569
2.781
81.049
5.903
89.666
No. 62
0.033
0.147
0.375
2.071
2.908
5.534
75.938
10.016
91.488
24-h Wash
No. 63
0.021
0.188
0.407
1.522
2.054
4.192
84.329
5.956
94.477
Mean ± S.E.
0.022 ± 0.006
0.130 ± 0.040
0.278 ± 0.113
1.540 ± 0.302
2.177 ± 0.391
4.169 ± 0.795
80.439 ± 2.441
7.292 ± 1.362
91.877 ± 1.402
-------
APPENDIX V
MONKEY STUDIES
INDIVIDUAL ANIMAL DATA
List of Tables
Table Page
i
V-l Urinary and Fecal Excretion of Radioactivity in Male Rhesus
Monkeys Treated Dermally or Intravenously with 14OLabeled
4,4'-MDA (10.0 mg/Monkey) V-2
V-l
-------
Table V-l. Urinary and Fecal Excretion of Radioactivity in Male Rhesus Monkeys Treated Dermally or Intravenously
with 14C-Labeled 4,4'-MDA (10.0 nig/Monkey)
i
ro
Percent of dose
Excretum
Urine
Feces
Urine
Feces
Dose wash
Recovery
Time after
dosing (h)
6
12
24
48
72
96
120
144
168
6
12
24
48
72
96
120
144
168
6
12
24
48
72
96
120
144
168
6
12
24
48
72
96
120
144
168
No. 604
0.069
0.939
1.919
9.984
5.633
2.370
1.305
1.018
0.635
0.004
0.001
0.009
0.036
0.548
0.589
0.192
0.613
0.216
0.069
1.008
2.927
12.911
18. 544
20.914
22.219
23.237
23.872
0.004
0.005
0.014
0.050
0.598
1.187
1.379
1.992
2.208
35.886
61.966
No. 619
0.475
1.087
5.308
5.436
1.450
0.687
- 0.362
0.348
0.183
_a
_a
0.392
0.972
0.471
0.089
0.063
0.050
0.042
0.475
1.562
6.870
12.306
13.756
14.443
14.805
15.153
15.336
_a
_a
0.392
1.364
1.835
1.924
1.987
2.037
2.079
55.631
73.046
Dermal
No. 604 R
0.038
0.415
1.277
6.659
4.659
1.853
1.043
0.557
0.773
0.003
_d
0.047
0.174
0.394
0.436
0.186
0.161
0.107
0.038
0.453
1.730
8.389
13.048
14.901
15.944
16.501
17.274
0.003
0.003
0.050
0.224
0.618
1.054
1.240
1.401
1.508
50.294
69.076
Intravenous
Mean ± S.E.
0.194 ± 0.141
0.814 ± 0.204
2.835 ± 1.250
7.360 ± 1.359
3.914 ± 1.264
1.637 ± 0.498
0.903 ± 0.281
0.641 ± 0.198
0.530 ± 0.178
0.004
0.001
0.149 ± 0.122
0.394 ± 0.292
0.471 ± 0.044
0.371 ± 0.148
0.147 ± 0.042
0.275 ± 0.172
0.122 ± 0.051
Cumulative
0.194 ± 0.141
1.008 ± 0.320
3.842 ± 1.553
11.202 ± 1.417
15.116 ± 1.726
16.753 ± 2.085
17.656 ± 2.305
18.297 ± 2.500
18.827 ± 2.584
0.004
0.004
0.152 ± 0.120
0.546 ± 0.412
1.017 ± 0.409
1.388 ± 0.271
1.535 ± 0.229
1.810 ± 0.205
1.932 ± 0.215
47.270 ± 5.897
68.029 ± 3.241
No. 529
37.661
21.258
15.641
4.850
0.862
0.360
0.257
0.209
0.165
0.034
_a
6.887
3.581
0.607
0.175
0.134
0.233
0.091
percent of dose
37.661
58.919
74.560
79.410
80.272
80.632
80.889
81.098
81.263
0.034
0.034
6.921
10.502
11. 109
11.284
11.418
11.651
11.742
-
93.005
No. 554
21.849
2.242
16.699
35.730
6.594
1.305
0.418
0.206
0.322
0.174
0.057
0.093
0.792
5.569
0.838
0.149
0.092
0.107
21.849
24.091
40. 790
76.520
83.114
84.419
84.837
85.043
85.365
0.174
0.231
0.324
1.116
6.685
7.523
7.672
7.764
7.871
-
93.236
No. 529R
15.047
39.804
20.868
6.409
1.931
0.686
0.433
0.381
0.829
0.003
a
4.820
3.191
0.940
0.165
0.252
0.155
0.119
15.047
54.851
75.719
82.128
84.059
84.745
85.178
85.559
86. 388
0.003
0.003
4.823
8.014
8.954
9.119
9.371
9.526
9.645
-
96.033
Mean ±
24.852
21.101
17.736
15.663
3.129
0.784
0.369
0.265
0.439
0.070
0.
3.933
2.521
2.372
0.393
0.178
0.160
0.106
24.852
45.954
63.690
79.353
82.482
83.265
83.635
83.900
84.339
0.070
0.089
4.023
6.544
8.916
9.309
9.487
9.647
9.753
94.091
b
±
±
±
±
±
±
±
±
±
±
.E.
6.699
10.843
1.596
10.044
1.760
0.277
0.056
0.058
0. 200
0.053
057
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±-
±
±
±
±
±
±
±
-
±
2.011
0.872
1.601
0.223
0.037
0.041
0.008
6.699
10. 994
11.455
1.619
1.138
1.320
1.376
1.409
1.566
0.053
0.071
1.946
2.807
1.277
1.090
1.083
1.124
1.119
0.973
No feces were collected during this time period.
-------
APPENDIX VI
SYNTHESIS OF 14C-LABELED 4.4'-MDA
VI-1
-------
INTEROFFICE COMMUNICATION
MIDWEST RESEARCH INSTITUTE
September 20, 1984
To: Monaem El-hawari
prom: David Chien and Bob Roth j/ljl^
Subject: [II+C]MDA Synthesis on Project No. 7901-A(21)/8201-A(21)
Attached is the synthesis procedure and analytical data for [1LfC]MDA prepared
on the subject project. A total of 3 samples were delivered: 1 x 90 mg (4.9 mCi)
and 1 x 60 mg (3.28 mCi) of [14C]MDA dihydrochloride (solid form) and 1 x 42.9 mg
(3.2 mCi) of the free base dissolved in 3 mL of ethanol.
DC/BR:cs
Attachments
VI-2
MRI2
-------
4,4'-Methylenedianiline [ring-U-14C]
Experimental
Step 1 - 4,4'-Methylenedianiline [ring-U-14C]
NHo-HCl
H20
+ ECHO
2 HC1
Literature Reference: J. T. Scanlan, J. Amer. Chem. Soc., 57, 890
(1935) [General synthesis], V. Henri, U.S. 4008275 [Purification]
MRI Reference: 83-25-73
To a solution of aniline hydrochloride [ring-U-14C] (50.0 mCi,
3.70 mmole) in 2 mL of water and 7 |JL of con. HC1 was added 36% formaldehyde
solution (171 (JL, 2.03 mmole). The resulting solution was stirred at about
10°C for 10 min and then at 55-60°C for 6 hr. The reaction mixture was
cooled to room temperature; and a-methylstyrene (194.6 mg, 1.65 mmole) and
water (1 mL) were added. The resulting mixture was stirred at 94°C for
15 hr then cooled, basified with 5 mL of ammonia water and extracted with
chloroform. The organic layer was dried (Na2S04) and the solvent removed
in vacuo to yield a dark brow oil (594 mg, 49 mCi). TLC silica gel 60,
90:25:4 benzene:dioxane:acetic acid, R = 0.30. The crude product was
estimated to be about 40% pure.
Step 2 - Purification via the N,N'-Diacetyl Derivative
(CH30)20
o=c
MRI Reference: 83-25-76
The product from the previous step was dissolved in 5 mL of acetic
anhydride and the resulting mixture was stirred at room temperature for 15 hr.
The solvent was removed under vacuum to yield 750 mg of dark brown solid.
Column chromatography on silica gel 60 (1 in. x 11 in.) eluted with chloro-
form/ ethanol 10:1 provided 4 mCi, 186 mg of light brown solid product TLC
silica gel 60, 20:3 chloroform:ethanol R = 0.34. The product was estimated
to be about 99% pure.
VI-3
-------
Step 3 - 4,4'-Methylenedianiline [ring-U-14C]
HCl2EtOH
CH3COHN-(( * )V-CH2-((r * )VNHCOCH3 >• H2N-(( * )VCH2-(( * )VNH2 ' 2 HC1
MRI Reference: 81-25-78
The product from the previous step (186 mg, 14 mCi, 0.66 mmol)
was dissolved in 20 raL of ethanol. Commercial 4,4"-dianiline methane
(57.3 mg, 0.29 mmole) and cone. HC1 (1.5 ml) were added, and the resulting
mixture was stirred at 95°C for 16 hr. Solvent was removed in vacuo and
the residue was washed with chloroform, benzene and 1.5 mL of cold ethanol
to give 210 rag (11.46 mCi) of pure 4,4'-dianiline methane dihydrochloride
(ring-U-14C). A 60-mg (3.28 mCi) sample of dihydrochloride was treated with
5 mL of cone. NH4OH and the free base extracted into CH2C12. The extract
was dried (Na2S04) and the solvent evaporated to give 42.9 mg (3.2 mCi) of
the free base as a pink solid. This material was dissolved in 3 mL of ethanol
and delivered along with 1 x 60 mg (3.28 mCi) and 1 x 90 mg (4.9 mCi) samples
of the dihydrochloride salt to Monaem El-hawari on 6/8/84.
Analysis: The radiochemical purity of the hydrochloride salt was
determined to be ^ 99% by TLC-radiochromatogram scans in two solvent systems.
A more critical evaluation of purity was attempted using HPLC. Considerable
effort was put into developing a suitable normal phase system using either
a Waters [jPoracil or Whatman Partisil 5 column and various mobile phases
derived from combinations of acetonitrile, dioxane, tetrahydrofuran, methyl
t-butyl ether, methylene chloride and hexane. The best system was found to
be a Whatman Partisil 5 column in combination with a 40:60 (v/v) hexane/methyl
t-butyl ether mobile phase. The initial analysis using this sytem on 6/19/84
•indicated both a chemical and radiochemical purity > 98%. These results
are presented in the following analytical summary.
On 7/23/84 the free base stock -solution in ethanol was reanalyzed
by the same HPLC method to check stability of the compound during storage.
Due .to changes in equilibration, the retention time of MDA was lengthened
from 13 min to approximately 26 min. All MDA chromatograms including com-
mercial and recrystallized commercial MDA's now contained a new peak, re-
tention time about 23 min with a relative area of 5%. It was subsequently
demonstrated that this 23-min peak was an artifact derived from t-butyl
methyl ether induced decompositon of MDA.
When a sample of commercial MDA was dissolved in t-butyl methyl
ether and immediately injected, only the 23-min peak as observed in the
chromatogam; the MDA peak at 26 min was completely absent.
VI-4
-------
ANALYTICAL DATA SUMMARY
MRI Project No. 7901-A(21)
COMPOUND : 4,4' -Dianiline methane dihydrochloride [ring-U-14C]
LOT NO.: 83-25-78
STRUCTURE :
' HC1
FORMULA: C13H16N2Cl2
AMOUNT: 150.0 mg (8.2 mCi)
SPECIFIC ACTIVITY: 14.8 mCi/mmole (gravimetric)
ESTIMATED PURITY: > 98% chemical and radiochemical
THIN- LAYER CHROMATOGRAPHY
Silica Gel 60, Chloroform: acetone: ammonium hydroxide, 1:8:1, R = 0.90
Benzene: acetone: ammonium hydroxide, 20:11:5, Rf = 0.60
ATTACHED : TLC radiochromatogram (2)
VI-5
-------
BIOSCAN BID 100
TLC-Radiochromatogram Scan
3
19
I 1-
12
14
3580 -•
•?=iflfi ••
5 9 S
: l
-i—i—-)_
-\—I—t-
-1 1 1 h
H—I—I—h
-I—!—I—t-
-I—I—I—1-
• '3
f
Compound:
v <-y ,
-U- C J
MCJl
a
Integration List
Label : MtTHrLtHED IrtH I Lihc HCL
0 a t ? : 3 i - y y 5 - 3 4
S t a r T -• @@ : 24 •• 33 S ' OF- •• @& • 2b ; 3i
Hccum; 9Q:92:8@
Resolution-1 Normal
Total <3S-224> = 34354
BACKGROUND REMOVED
Lot No. MRI-
Silica Gel 60
Other:
Solvent System:
i 0 • / I -' I"
/W*v**M)>u
i^vT^j.
: /)oi/Ur
r>\j?
,' tfVfi^
Label: METHYLENEDIHNILINE.
Lhans: lti? thru 134
Sum= 34857
Centroid' 121.41 Chan--
34.38 MM
'•'• of To t ( 39—224> = S'3 14
VI-6
-------
BIOSCAN BID 100
TLC-Radiochromatogram Scan
,'yyy "
I
I
y y y "'
fl tf fi - •
2 4
i i i
14
*.
a ii n
_1—,—|—|—(—^—|—f.
O
O
03
'2!'
>:••}
-r
Compound:
-o-'c J
H i-^-^T)- cH 2. - <"?>- ^^ H CJZ.
Integration List
Label: METHVLENEDIfiHILIH£
Date; 3i-yy5-84
Starr : 8@ : 94 .- 37 c; T op : ^^, .
Re cum ; MM •• 8£ : yy
Resoi u 11on: Ho rma i
Total <39-224> = 33667
B fi C K G R 0 U N D R E rt 0'.,' E D
Lot No. MRI-
^ Silica Gel 60
[] Other:
Solvent System:
Lab*!' METHYLEHEDlHHlLlNc
Char>£= U"2 thru it: 4
Sum: 39495
Cen t r-o i d •• 174.19 Chans
149.65 MM
'•; of Tot C3@-£24:1 : S3 M7
VI-7
-------
ANALYTICAL DATA SUMMARY
MRI Project No. 7901-A(21)
COMPOUND : 4,4' -Dianiline methane dihydrochloride [ring-U-14C]
LOT NO.: 83-25-80
STRUCTURE :
FORMULA: C13H14N2
AMOUNT: 42.9 mg (3.2 mCi)
SPECIFIC ACTIVITY: 14.8 mCi/mmole (gravimetric)
ESTIMATED PURITY: > 98% chemical and radiochemical
HIGH PRESSURE LIQUID CHROMATOGRAPHY
Column: Whatman Partisil 5
Mobile Phase: Hexane/methyl-t-butyl ether, 40/60
Flow Rate: 1.3 mL/min
Detection: UV 254 and collection of 20-sec fractions with
scintillation counting
ATTACHED : HPLC chromatogram (2)
VI-8
-------
(•)
»t
f
irt
I
III
in
5
a
f*
o.
10
»
Z:,'
ii. a'
i a:
;,• cu u) in f^ 01
•r 10 v
U4 '].'...
o.' d co m « o
u: oj uj ^- >-i (ii
ui mm>i>
(T A| 01 4 CQ
I- |f/ !•) (v M. •-.
**• '.'•'"
^« ^. r. 01
oi
ffi
CD
09
u.'
O
J-.
I
IL
l-l
M
HPLC Analysis of 4,4'-Dianiline methane [ring-U-^C) , Lot Mo. 83-25-80
(Whatman Partisil 5, Hexane:Methyl-t-butyl ether, 40:60)
Sample RT = 13.32 min
-------
— C
KtCC'VZ
POCKfl
VIALS
VZAt
flf.II 1530 <3H;HC
37 i1 VI DPH
<=:
i—i
i
o
I I I I I I I
OF AHDlOhCTl VI TY
*Ri O.Zl
11-C ULi HAHt H
INT -. T
fK
14
1NJ £HD 1HT DPH ZOf TOTAL
7* 477996 lOa.VSl
r ;/. IHTE&HOIIOH
TOTAL DPHS P.e.COVtRf.D *
a st;s COUKT OF ?jo i
IST-
HPLC Effluent Fraction Relative Radioactivity (6, 20-sec cuts) of
4,4'-Dianiline methane [ring-U-Uc) , Lot No. 83-25-80
-------
50372-101
REPORT DOCUMENTATION
PAGE
1. REPORT NO.
" 560/5-86-011
3. Recipient's Accession No.
4. Title end Subtitle
Dermal Absorption of ^C-Labeled 4,4'-Methylenedianiline
(4,4'-MDA) in Rats, Guinea Pigs, and Monkeys
5. Report Date
December 30, 1985
7. Author(s)
. Author(s)
Monaem El-hawari, Maxine Stoltz, Diane Czarnecki, Patricia Aim
8. Performing Organization Rept. No.
8501-A(21)
9. Performing Organization Name and Address
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
10. Project/Task/Work Unit No.
Work Assignment No. 21
11. Contract(C) or Grant(G) No.
co 68-02-3938
(G)
12. Sponsoring Organization Name and Address
U.S. Environmental Protection Agency
Field Studies Branch, TS-798
401 M Street, S.W.
Washington, D.C. 20460
13. Type of Report & Period Covered
Final
14.
IS. Supplementary Notes
The EPA Work Assignment Manager is Janet Remmers
The EPA Project Officer is Joseph Breen
To help determine workplace exposure to 4, 4'-MDA," studies were performed
pgs and monkeys treated topically with a low (2 mg/kg) or high (20 mg/kg)
•is. Abstract (Limit: 200 words)
in male rats, g
dose of 14C-4,4'-MDA. Conditions of treatment (dosage, dose regimens, and occlusion) were
assessed. The disposition of 4,4'-MDA was also examined after i.v. dosing. In rats, 43 and
10% of the low dose was recovered in urine, and feces during a 96 h period; 2% remained in
tissues and skin washing removed 25% of dose. The remainder (26%) was recovered by skin ex-
traction and solubilization. The percent of dose absorbed decreased by increasing the dose,
but the total amount absorbed (~0.25 mg/rat) was similar after both doses. In g. pigs, 10
and 18% of the low dose was excreted in urine and feces; 1% was recovered in tissue, 41% in
the skin wash and 29% from the application area. The percent of dose absorbed decreased fol-
lowing the high dose, but the amounts absorbed (in mg/animal) doubled. In monkeys, 19 and 2%
of the low dose was eliminated in urine and feces during 168 h and 47% was recovered in skin
wash. Under similar conditions (application of low dose for 24 h followed by excreta collec-
tion for 96 h) absorption was comparable in g. pigs and monkeys (~18%) and higher in rats
(~43%). When applied 4,4'-MDA was washed immediately with soap/water or acetone/water
neither washing method was capable of removing all the applied material from the skin. High-
er recoveries were obtained by washing with soap/water; in addition, acetone facilitated ab-
sorption. Since the unrecovered material represents amounts associated with the skin avail-
able for later absorption, the extent of dermal absorption should be considered higher than
calculated from excretory and tissue distribution data only. __ __
17. •Document Analysis a. Descriptors
4,4'-MDA, 4,4'-methylenedianiline, dermal absorption, washing efficiency, disposition,
species differences
b. Identifiers/Open-Ended Terms
c. COSATI Field/Group
18. Availability Statement
Unlimited
19. Security Class (This Report)
Unclassified
2O. Security Class (This Page)
UnclassifjpH
21. No. of Pages
144
22. Price
(See ANSI-Z39.18)
See Instruction* on Reverse
OPTIONAL FORM 272 (4-77)
(Formerly NTIS-35)
Department of Commerce
------- |