EPA-560/2-76-006
INVESTIGATION OF SELECTED
POTENTIAL ENVIRONMENTAL CONTAMINANTS:
MERCAPTOBENZOTHIAZOLES
June 1976
FINAL REPORT
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
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EPA-560/2-76-006 TR 76-502
INVESTIGATION OF SELECTED POTENTIAL
ENVIRONMENTAL CONTAMINANTS:
MERCAPTOBENZOTHIAZOLES
Joseph Santodonato
Leslie N. Davis
Philip H. Howard
Jitendra Saxena
June 1976
Final Report
Contract No. 68-01-3128
Project No. L1255-06
Project Officer
Frank J. Letkiewicz
Prepared for:
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
Document is available to the public through
the National Technical Information Service,
Springfield, Virginia 22151
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W.O.T. ICE
This report has been reviewed by the Office of Toxic Substances, EPA, and
approved for publication. Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or recom-
mendation for use.
ii
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TABLE OF CONTENTS
Executive Summary
I. Physical and Chemical Data
A. Structure and Properties
1. Chemical Structure
2. Physical Properties of the Pure Material
3. Properties of the Commercial Materials
4. Principal Contaminants of Commercial Products
B. Chemistry
1. Reactions Involved in Uses
2. Hydrolysis, Oxidation and Photochemistry
II. Environmental Exposure Factors
A. Production, Consumption
1. Quantity Produced, Exported and Imported
2. Producers and Production Sites
3. Production Methods and Processes
4. Market Price
5. Market Trends
B. Uses
1. Major Uses
2. Minor Uses
3. Discontinued Uses ,
4. Projected or Proposed Uses
5. Possible Alternatives to Use
C. Environmental Contamination Potential
1. General
2. From Production
3. From Transport and Storage
4. From Use
5. From Disposal
6. Inadvertent Production via Industrial Processes
7. Inadvertent Production in the Environment
D. Current Handling Practices and Control Technology
1. Special Handling in Use
2. Methods of Transport and Storage
X
1
1
1
3
7
9
12
12
16
18
18
18
21
21
26
28
29
29
33
35
35
35
37
37
38
38
39
41
41
42
43
43
43
111
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Table of Contents
(continued)
III.
Page
E.
3.
4.
5.
Disposal Methods
Accident Procedures
Current Controls and Control Technology Development
Monitoring and Analysis
1.
2.
Analytical Methods
Current Monitoring
Environmental Health Effects
A.
B.
Environmental Effects
1.
2.
3.
Persistence
a. Biological Degradation , Organisms and Products
b. Chemical Degradation in the Environment
Environmental Transport
Bioaccumulation and Biomagnif ication
Biological Effects
1.
2.
Toxicity and Clinical Studies in Man
a. Epidemiologic and Controlled Human Studies
b. Occupational Studies
c. Non-Occupational Exposures and Poisoning Incidents
Effects on Non-Human Mammals
a. Absorption Studies
b. Metabolism Studies
c. Pharmacology
d. Acute Toxicity
e. Subacute Toxicity
f . Sensitization
g. Teratogenicity
h. Miitagenicity
i. Carcinogenicity
j. Other Chronic Effects Studies
k. Behavioral Effects
1. Possible Synergisms
A3
44
44
45
45
48
51
51
51
51
5-2
52
53
55
55
56
70
71
74
75
76
79
86
102
109
110
110
111
113
113
113
iv
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Table of Contents
(continued)
Page
3. Effects on Other Vertebrates 114
4. Effects on Invertebrates 114
5. Effects on Plants 114
a. Fungi 114
b. Higher Plants 118
6. Effects on Microorganisms 122
7. _In Vitro and Biochemical Studies 126
IV. Regulations and Standards 128
A. Current Regulation 128
B. Consensus and Similar Standards 129
V. Summary and Conclusions 130
REFERENCES 135
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LIST OF TABLES
Number Page
1 Commercially Important Mercaptobenzothiazole Compounds 4
2 Physical Properties of 2-Mercaptobenzothiazoles and Selected 5
Derivatives
3 Commercial Specifications for 2-Mercaptobenzothiazole and Selected 8
Derivatives
4 Commercial Specifications for Sodium MET Solution, 50% Aqueous 9
5 Production and Sales of 2-Mercaptobenzothiazole Compounds 19
6 Manufacturers of Mercaptobenzothiazole 22
7 Use of MET and Derivatives 29
8 SBR/cis-Polybutadiene Passenger Tire Tread Formulation, 32
9 Sources of Safety Data for MET Compounds 44
10 Analytical Techniques for MET Compounds . 46
11 MET Compounds Identified in Finished Water 49
12 Patch Test Reactions to Accelerators and Antioxidants 58
13 Summary of Patch Test Data 59
14 Patch Tests with Rubber Accelerators 62
15 Percent of Patients Reacting to Contact Allergens 64
16 Degree of Reaction to Contact Allergens 65
17 Test Substances and Their Occurrences 66
18 Patch Test Reactions in Twelve Patients 67
19 Industrial Contactants 69
20 Positive Skin Reactions in 16 Patients with Rubber Allergy 72
21 Median Paralyzing Doses of Substituted Benzazoles on Intravenous 80
Administration to White Mice
vi
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List of Tables
(continued)
Number Page
22 Mouse Brain Catecholamine Levels 1, 2, and 4 h After 83
2-Mercaptobenzothiazole (MBT), 300 mg/kg, i.p.
23 Effect of 2-Mercaptobenzothiazole (MBT) on the Repletion of Rat 84
Myocardial Noradrenaline from Exogenous Dopamine After Its
Depletion with Metaraminol
24 Effects of 2-Phenylbenzpthiazoles and Related Compounds on Benzo- 87
pyrene Hydroxylase Activity of Rat Liver and Lung
25 LD Q Values for MBT and HMBT in Mice 89
26 Acute Animal Toxicity of Benzothiazole Derivatives 91
27 Primary Irritation Evaluation of Mildew Inhibitor Vancide 51Z 99
28 Acute Eye Irritation of Benzothiazole Derivatives in the Rabbit 101
29 Acute Dermal Toxicity of Benzothiazole Derivatives in the Rabbit 103
30 Microscopic Pathologic Findings in Mouse Tissues After Dosing 104
with MBT and HMBT for One Week
31 Effect of MBT and HMBT on Hexobarbital Narcosis in Mice 105
32 Summary of Results of 31 Daily Doses of MBTS Pellets in the 106
Diet of Male Albino Rats
33 Maximal Tolerated Doses of MBT and Derivatives in Mice 107
34 Allergenicity of MBT by the Guinea-Pig Maximization Test 110
35 Mutagenic Activity of Several Rubber Additives by Feeding to 110
Fruit Flies \
36 Administration of MBT and Several Derivatives to Mice 112
37 Summary of the Acute Toxicity of Vancide Formulations to Several 115 .
Species of Fish
38 Assessment of Fungicidal Activity 116
39 Action of Benzothiazole Derivatives on Pathogenic Strains of 117
Dermatophytes and Yeast-like Fungi
vii
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List of Tables
(continued)
Number Page
40 Repression of Cucumber Root Elongation by Azoles 118
41 Induction of Adventitious Roots in Bushbeans by Root Application 121
of 1 x 10"2M Benzothiazole
42 Effect of MET and Several Derivatives on Staphylococcus aureus 124
and Escherichia coli
43 Antimycobacterial Effects of 2-Mercaptobenzothiazole 125
44 Antibacterial Effect of MET and 2,2'-Dithiobisbenzothiazole 126
45 Cell Culture. Evaluation of MET and HMBT 127
viii
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LIST OF FIGURES
Number Page
1 UV Absorbance Curves For Aqueous Solution of 10 ppm MET 6
2 UV Absorbance Spectrum of 10 mg/1 7
3 Organic Rubber Accelerators Production and Sales 20
4 Manufacture of. Mercaptobenzothiazole 24
5 Price History of 2-Mercaptobenzothiazole Derivatives 27
6 Distribution in Age of Onset of Rubber Sensitivity 60
7 Pathway for the Metabolic Transformation of 2-Mercaptobenzothiazole 77
in the Rat, Rabbit, and Dog
8 Structural Formula of a Benzazole 80
9 Effect of 2-Mercaptobenzothiazole, 300 mg/kg I.p., Upon Spontaneous 85
Motor Activity in Mice
10 Effect of Benzothiazble Upon the Elongation of the Primary Root of 119
Cucumber Seedlings
11 Effect of Benzothiazole Upon Dry Weight of Barley Roots 119
ix
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Executive Summary
Mercaptobenzothiazole (MET) compounds are produced in over 100 million
pounds annually in the United States. They are mostly consumed as rubber
accelerators in vulcanization processes, although almost 6 million pounds per
year of the sodium salt of MET are used as a corrosion inhibitor in water-based
cooling systems (e.g., automotive cooling systems). The available information
suggests that sizable quantities of MET are being released to the environment
from discarded coolants and rubber products, as well as from particles worn
from tires. No information on the persistence of MET compounds and no experi-
mental data on bioaccumulation are available. However, physical properties of
MET compounds would suggest a low bioaccumulation potential. Some derivatives
have been detected in water but rarely have concentrations been reported. The
principal actions of MET derivatives in human and animal systems include:
(1) production of allergic contact dermatitis, (2) action on the central nervous
system, and (3) inhibition of certain metalloenzymes which contain copper.
The minimum levels to produce these physiologic actions have not been published.
Furthermore, the potential for chronic exposure at sublethal doses to produce
irreversible damage has not been investigated. :
Several of the compounds have been screened for carcinogenic potential
by long-term feeding to mice with negative results. However, mutagenesis
assays in fruit flies suggest that several MET compounds may be mutagenic
(because of the deficiency of determining mutagenesis with fruit flies, these
compounds should be tested with the Ames or similar tests). Although MET
compounds have a high potential for entering the environment, the exact amount
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of exposure to biological organisms and the risk involved from such exposure
is difficult to estimate, due to a lack of information on environmental fate
and toxicological effects at possible environmental concentrations. More detailed
monitoring surveys and studies of the environmental fate of MET would seem desirable.
xi
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I. Physical and Chemical Data
A. Structure and Properties
1. Chemical Structure
The thiazole ring is a five-member unsaturated aromatic
system containing nitrogen and sulfur:
When the two adjacent carbons in a thiazole ring are also adjacent carbons of
a benzene ring, the compound is called benzothiazole:
7 1,
The S is defined as atom #1 in the benzothiazole ring. Substituent carrying
atoms are numbered clockwise around the rings, as shown above (Allen, 1966).
Although benzothiazole derivatives with substituents on the benzene ring are
known, they are not of general commercial significance. Some of these deriv-
atives occur in nature, for example, luciferin,
COOH
the enzymatic oxidation of which produces the characteristic luminescence of
the firefly (Roberts and Caserio, 1965). The most important commercial deriv-
atives are compounds in which the thiazole ring hydrogen has been replaced with
a mercapto group or other thio derivatives.
-------
2-Mercaptobenzothiazole (MET) is a bicyclic polyhetero aromatic
compound which exists in thioketo and thioenol canonical forms (Malik and
Rahmani, 1975).
-S-H
ff
thioketo-MET
-N
thioenol-MBT
Crystal structure studies show the molecule is planar and in the thioketo form
in the solid state (Chesick and Donohue, 1971). Infrared spectral studies
confirm the presence of the -N-C=S group in the solid compound, but metal
complexes show evidence of coordination through the mercapto sulfur exclu-
sively, indicating conversion of thioketo-MBT to thioenol-MBT on reaction
with metals (Khan and Malik, 1972). The absence of an N-H bond as well as
a metal-N bond has been demonstrated by IR studies on compounds of MET with
Gu(II), Ni(II), Co(II), Cd(II), Zn(II), Pb(II), Ag(I), and T1(I) (Khullar and
Agarwala, 1975), confirming the thioenol structure of metal-MBT compounds.
The products of reactions of MET involving the replacement
of the mercapto hydrogen have the general structure:
S-R
When R contains sulfur as the bonding atom, the compound is a disulfide such
as 2,2'-dithiobisbenzothiazole (MBTS):
-------
When an S-N bond is present, the compound is a sulfenamide, such as N,N-
diisopropyl-2-benzothiazolesulfenamide:
CH(CH3)2
S N
»k_
CH(CH3)2
The mercapto proton of MET can be readily replaced by metal ions, yielding
salts, typical of which is sodium mercaptobenzothiazole (NaMBT):
This review emphasizes MET and those derivatives of MET that
appear to have commercial or environmental significance. Table 1 contains
a list of the names of the commercial mercaptobenzothiazole compounds, their
chemical structures, and acronyms which have been used throughout this report
to refer to these materials. All of the compounds are produced in commercial
quantities, according to the available chemical marketing literature (SRI, 1975;
last-two years - 1972, 1973 of the U.S. International Trade Commission [USITC,
1959-73]).
2. Physical Properties of the Pure Material
2-Mercaptobenzothiazole and its derivatives are remarkably
similar in their physical properties, with the exception of subtle color
differences and melting points, as is inducated in Table 2. A relatively wide
softening-melting range is common, probably indicative of purity variations
in the commercial products. The compounds are all powders, light in color,
with characteristic odors, generally soluble in polar solvents but insoluble
3
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Table 1. Commercially Important Mercaptobenzothiazole Compounds
Chemical Name Structure Acronym
Benzothiazole
2-Mercaptobenzothiazole
2-Mercaptobenzothiazole,
sodium salt
2-Mercaptobenzothiazole,
zinc salt
KBT
2-Mercaptobenzothiazole,
zinc chloride
2-Mcrcaptobenzothlazole,
copper salt
2-Mercaptobenzothiazole,
monoethanolamine salt
•S H3N -CH2CH?OH
2,2'-Dithiobisbenzothiazole
Cyanomethylthiobenzothiazole
l,3-Bis(2-benzothiazolyl-
me reap tome thy1)urea
•S-CH2CN
/r-scH2NH c NHCHZS-SV
2-Befizothlazyl-N,N-dicthyl~
thiocarbanyl sulfide
N-te_rt-BuLyl-2-benzo-
thiazolesulfenamlde
,1—S-N-C(CH3)
N-Oxydlethylene-2-benzo
thiazolesulfenamide
N-Cyclohexyl-2-benzo-
thlazolesulfenamide
N,N-Diisoprbpyl-2-benzo-
thiazolesulfenamide
4-Morpholinyl-2-benzothiazyl
dlsulfide
N-(2,6-Dlmethylmorpholino)-
2-benzothiazolesulfenamide
y /™3
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Table 2. Physical Properties of 2-Mercaptobenzothiazoles and Selected Derivatives
2-Mercaptobenzothiazole
Molecular Physical
Weight State _
167 powder
2,2'-Dithiobisbenzothiazole 333
N-tert-Butyl-2-benzothia-
zolesulfenamide
N-Cyclohexyl-2-benzothia-r
zolesulfenamide
238
N-Oxydiethylene-2-benzothia- 252
zolesulfenamide
264
N,N-Diisopropyl-2-benzothia- 266
zolesulfenamide
Specific.
cravity Melting Nonaqueous
Colo£_ _ (^2.r -0.03 Range °C Water Polar Nonpolar
light yellow- distinct,
tan
powder cream to
light yellow
powder light tan
flakes
flakes greenish-tan
flakes greenish-
yellow
Sodium Mercaptobenzothiazole 189 solid
Zinc Mercaptobenzothiazole 398 powder cream
Sources
distinct, 1.50 177-178
characteristic
slight 1:54 160-180
characteristic 1.29 104-112
1.37 75- 90
1.29 94-108
characteristic 1.21 53- 60
1.72 >300
0.25g/100gb S
I SS
I S
I S
I SS
I S
S
I 1
SS . 1,2,3,4
SS 1,2,4
S 1,3
S 1,2,3
SS 1,4
S 1
1,2,3
SS 1,2,4
The information in this table was obtained from product bulletins supplied by:
1 American Cyanamid Company, Bound Brook, New Jersey (1970)
2 R.T. Vanderbilt Company, Norwalk, Connecticut (1974)
3 Uniroyal Chemical, Naugatuck, Connecticut (1972)
4 B.F. Goodrich Chemical Company, Cleveland, Ohio C1975)
Davis, 1930
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in .water (except for the sodium salt). All are stable materials, without
unusual storage or handling hazards.
Figure 1 shows the ultraviolet absorption spectrum of MET in
acidic, neutral, and basic aqueous solution. Figure 2 shows the UV spectrum
of MET in chloroform. The X shifts can be attributed to the different per-
max r
turbations resulting from the dissociated and undissociated forms in acidic,
basic, or neutral conditions. The characteristic peak at 329 nm in chloroform
is utilized in recently reported assay techniques for MET (Jones and Woodcock, 1975)
2-0
1-5
U
c
o
f 10
o
Wl
£t
O-5
2OO 225
25O 275 3OO 325
Wavelength (nm)
35O
Figure 1. UV Absorbance Curves For Aqueous Solution of
10 ppm MET. a. pH 1.6, b. pH 6.8, c. pH 11.7
(Jones and Woodcock, 1973)
(Reprinted with permission from the Department
of Energy, Mines & Resources)
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1-5
t 10
OS
25O 275
3OO 325 35O
Wovclcnglh, nm
4OO 45O
Figure 2. UV Absorbance Spectrum of 10 mg/1. MET
in Chloroform Using 1-cm Cells
(Jones and Woodcock, 1975)
(Reprinted with permission from the
American Chemical Society)
3. Properties of the Commercial Materials
MET commercial materials have the same physical properties
listed in Table 2. In addition, the specifications listed in Table 3 apply to
commercial materials as supplied by the manufacturers.
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Table 3. Commercial Specifications for 2-Mercaptobenzothiazole and Selected Derivatives
Fi neness
iWet Method)
oo
2-Mercaptobenzothiazole
2,2'-Dithiobisbenzothiazole
N-tert-Butyl-2-benzothia-
zolesulfenamide
N-Oxydiethylene-2-benzothia- 0.2
zolesulfenamide
Zinc 2-Mercaptobenzothia- 1.0
zole
Moisture ,
% max.
0.5
1.0
1.0
0.5
0.7
1.0
0.5
0.2
0.5
0.5
1.0
1.0
2.0
Ash, Assay, Dedusting
% max.' % min. Agent, % max.
0
0
0
1
1
0
0
. 0
0
0
.i 92 2.5
.5
.5
.0 91 2.5
.0
.7
.5
.5
.5
.15
15% Zn
15-18% Zn .
15-18% Zn
(LOO mesh)
% min.
99.0
99.8
99.8
99.9
99.9
99.9
100
100
100
99.9
99.9
99.8.
Pet, Ether Manufacturer or
Extract, % Distributor
Amer. Cyanamid
1-3 Vanderbilt
Goodri ch
Amer. Cyanamid
1-3 Vanderbilt
Goodrich
Amer. Cyanamid
Vanderbilt
Goodrich
Amer. Cyanamid
Vanderbilt
Goodrich
Commercial Free MET, Free MBTS ,
Name % max. % max.
MET
Cap tax
MBT
MBTS 2
Altax
MBTS 5
TBBS 1
NOBS 3
Amax ^3
OBTS
ZMBT
Zetax
ZMBT
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Sodium 2-mercaptobenzothiazole is supplied as a 50% aqueous
solution, unlike other MET chemicals which are insoluble in water and are
supplied as solids. Specifications for the NaMBT solutions of three manu-
facturers are listed in Table 4.
Table 4. Commercial Specifications for:-Sodium HBl Solution, 50% Aqueous
Manufacturer
American Cyanamid
Uniroyal
R.T. Vanderbllt
Color (Garner)
max.
10
10
Clarity
clear
clear,
amber
Specific
Gravity
1.25-1.29
(15.5-C)
1.27
(15-C)1
1.255
(25°C)
Assay
%
50.0 ± 0.5
50.0 .
50
NaOH Cl", I
% max.
. 0.1-0.5 0.1
0.3 nil
0.5
so,'2- *
max.
0.5
0.15
4. Principal Contaminants of Commercial Products
The major starting material from which MET. and its derivatives
are manufactured is aniline (Kouris and Northcott, 1963). Aniline heated
with equimolar amounts of sulfur and carbon disulfide at temperatures of 200-
275°C (Allen, 1966) yields MET and possibly other products whose contamination
of the major product is dependent on the temperature range of the reaction
medium. Besides by-products, unreacted starting materials, if any, may con-
taminate the desired product.
Ivanova and Shebuev (1957) have analyzed the main and side
reactions which lead to the formation of MET. The first reaction is observed
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when a mixture of aniline, CS_, and S is heated to 170°C. Aniline reacts with
CS2 to form thiocarbanilide:
+ H2S
This reaction is reversible as long as H^S is present and, in fact, tends to
shift to the left because thiocarbanilide is unstable above 160°C. Above 170°C,
the removal of thiocarbanilide is further favored as aniline reacts irreversibly
with sulfur to form 2,2'-diaminodiphenyl disulfide, which in the presence of
H9S forms 2-aminothiophenol:
H2S
^fo
Both the disulfide and 2-aminothiophenol react readily with CS9 to form MET.
At temperatures above 220°C, MET reacts with aniline to form anilinobenzothiazole:
NH
^s^
S\ ? / /^\ \
H2S
\
Above 260°C MET is unstable and decomposes to form benzothiazole. Above 200°C,
aniline and CS react to form, in addition to thiocarbanilide, phenylisothiocyanate,
which is very unstable in the presence of H S, and reacts to form MET:
I=C=S
CS2
10
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Since most of the side reactions lead to by-products which
are unstable under the conditions in which they are formed and decompose to form
the original starting materials or MBT, it is not surprising that the by-
products are notably absent from the final product when the reaction temperatures
are carefully controlled. If the reaction medium were allowed to rise above
260 C, benzothiazole would be an expected contaminant of the desired product.
If the reaction is quenched by rapidly lowering the temperature, the by-
products shown in the side reactions can be identified (Ivanova and Shebuev,
1957).
MBT and its derivatives are considered to have excellent storage
and handling stability; contamination due to decomposition during storage is
not likely (American Cyanamid Co., 1970).
There remains then the possibility of contamination due to
impurities in the starting materials as well as the presence of unreacted
starting materials. American Cyanamid Co. (1970) is the only manufacturer
of these materials which publishes minimum purity specifications (for MBT and
MBTS only, 92% and 91% minimum assay, respectively [other manufacturers have
indicated that the assay is usually between 95-97% purity]). The minimum
purity specifications indicate that if approximately 5% of the contents is
assumed to be ash, moisture, and dedusting agent, approximately 3% of the
product contents remain unaccounted for. This 3% may consist of unreacted
starting materials and by-products. MBT may be a major contaminant of the
derivatives (up to 5%, see Table 3). American Cyanamid Co. (1970) also speci-
fies the possible presence of small quantities of Cu (< 10 ppm) and Mn (< 10 ppm)
in the compounds listed in Table 3, as well as Fe (< 0.1%) in all except ZMBT.
It is probable that the products of other manufacturers have comparable purity
and contamination levels since the other published specifications are essentially
identical.
11
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B. Chemistry
1. Reactions Involved in Uses
2-Mercaptobenzothiazole finds major uses in three distinct and
quite different areas. It is used in the rubber industry as a vulcanization
accelerator (Nathan, 1965) and antioxidant; in metal processing and applications
(especially in the automobile industry) as an anticorrosion agent (Turk, 1969) ;
and as a fungicide and bacteriostatic agent (Turner, 1966). In addition, there
are a number of minor uses for MBT and its derivatives and related compounds,
as a chemical intermediate and in analytical applications as a metal chelating
agent (Shimidzu and Uno, 1973). Its chemistry as an intermediate has been of
theoretical and practical interest because many natural products and drugs
contain the thiazole ring (e.g., penicillin, sulfathiazole, sulfonamides,
thiamine) (Allen, 1966).
Vulcanization is a cross linking process in which sulfur bridges
are formed between the unsaturated portions of polymers which make up an un-
vulcanized rubber (Winspear, 1958). In order for vulcanization to proceed
readily, elemental sulfur, which exists at ambient temperatures in the ring
form, S0» must be converted into a biradical chain. This can be accomplished
o
by heating rubber and sulfur at high temperatures for long periods of time,
but such treatment often leads to unpredictable and undesirable properties
in the vulcanized rubber. It is the function of an accelerator such as MBT
to reduce both the temperature and time requirements of vulcanization, thus
aiding in the production of a product with uniform and predictable properties.
12
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The ideal accelerator allows vulcanization to occur reproducibly and rapidly
at a given temperature, while restraining vulcanization during preparative
operations (i.e.,. milling, compounding, etc.) (Shaver, 1968).
MET and its derivatives all function in similar,fashion as
accelerators. The main difference between these compounds is the temperature
at which vulcanization begins, and the delay time before vulcanization at a
given temperature. The mechanism of vulcanization involves the decomposition
and recombination of these compounds at high temperatures (Dogadkin et_ al.,
1961). In the presence of elemental sulfur, S_, polysulfides are also formed,
an example of which for MET accelerator systems is (Coran, 1965):
These polysulfides split and recombine to form atomic sulfur and other free
radicals (Winspear, 1958):
R-S-S-R *• 2R-S' >• R-S-R + -S-
The free radicals attack Sfi rings forming sulfur chains (Winspear, 1958):
R-S- + S >• R-S7-S-
The chains eventually fragment into atomic sulfur which reacts immediately
with the rubber polymer, forming the crosslinks characteristic of vulcanized
rubber (Winspear, 1958):
13
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2(-C-OC-) + 2 -S-
I
-c-c-c-
I
s-
-c-c-c-
-c-c-c-
-c-c-c-
A complex mixture of MET compounds and MET by-products results
from the vulcanization step. However, no quantitative study of MET products
formed during vulcanization has been undertaken. MET will thermally decompose
to benzothiazole at temperatures greater than 260°C (Ivanova and Shebuev, 1957),
and this compound has been detected in volatiles from simulated vulcanization
processes (Rappaport, 1975) and in rubber processing plant effluents (Webb et al.,
1975). Rappaport (1975) also detected J>butylisothiocyanate which he suggested
was a breakdown product of ^-butyl-2-benzothiazolesulfenamide.
Zinc mercaptobenothiazole (ZMBT) is an excellent antioxidant
when used in rubber formulations. If not actually introduced during compounding,
ZMBT will form during vulcanization if MET (or a potential precursor such as
2,2'-dithiobisbenzothiazole) and zinc oxide are in the formulation (the latter
usually is). The basis of its antioxidant activity is the ability of ZMBT to
react readily with those hydroperoxides which normally form in the rubber sub-
strate during aging and which are the cause of degradation of the rubber (Brooks,
1963). In the course of the reaction of ZMBT with hydroperoxides, the salt is
oxidized to the sulfonate, while the hydroperoxides are reduced to alcohols:
S-Zn-S
R-O-O-H
S02-Zn-S02
R-O-H
14
-------
Although insoluble in acidic or neutral aqueous media, MET
is a weak acid in water and dissolves in basic solutions:
NaOH
N'
H,0
-N'
The anion complexes readily with a wide range of metal ions, in-
+3 +2 +3+2
eluding Al , Cu , Fe , and Zn , to produce insoluble complex salts which,
when formed as coatings on a metal surface, protect the surface against further
oxidation (Prajapati et^ _al. , 1972):
S-Cu-S
N
This is believed to be the chemical basis for the activity of MET as an anti-
corrosion agent and may be the basis of its toxicity when administered intra-
+2
venously. By complexing readily with Cu , MET inhibits the action of the
enzyme dopamine 3-hydroxylase which catalyzes the conversion of dopamine to
nor adrenaline, a .neural transmitter (Johnson et^ al_. , 1970) (see Section III-B-
2, p. 83 for more details).
It is thought that, in order for MET to act as a fungicide,
the molecule must penetrate the cell membranes of the fungus and act within
the cells (Bowes ^t. _al. , 1970). In order to do this, the fungitoxic molecule
must not be bound to the substrate (textile, leather, etc.) which it is pro-
tecting. At the same time, it must not have such freedom of migration that
it is easily leached out of the substrate, affording a protection time that is
15
-------
unreasonably short. Bowes and his colleagues (1970) have shown that MET can
be bound to the collagen in leather using cyanuric chloride as an intermediary.
MET reacts with the cyanuric chloride under basic conditions to form l-(2-
mercaptobenzothiazolyl)-3,5-dichlorotriazine:
OH
+ CSL
The chlorine atoms of the MET derivative can react with the amino groups in
lysine residues in leather, thus binding the compound to the leather. Over
a period of time the derivative eventually breaks down to form MET which
protects the leather from fungal attack (Bowes
-------
or basic solution in the presence of iron, in acidic media MET is reduced in
the presence of iron to benzothiazole:
+
HqO '-
SH+2H .'--;—> ( ) />-H + H2S
MET and its derivatives are quite resistant to oxidation in air,
and NaMBT in aqueous solution is not oxidized even at temperatures of 100°C
(Squires, 1958). Under fire conditions, the principal products of combustion
are carbon dioxide, sulfur dioxide, and water (Shaffer, 1971a,b,c,d). Incomplete
combustion will produce carbon monoxide, and the combustion of sulfenamides -tends
to produce nitrogen oxides as well.
The mercapto group is subject to oxidation by hydroperoxides,
yielding sulfonates (see Section I-B-l, p. 14 ), as are the sulfur bridges in
MBTS and other polysulfide derivatives.
In the presence of ozone and potassium iodide, MET dimerizes
to MBTS (Petrenko et al., 1975). This reaction is favored by lowering the
temperature to about 0°C. The dimer can then be further oxidized to the
disulfonate if sufficient ozone is available.
MET absorbs ultraviolet light strongly at about 328 nm (see
Figures 1, 2), and, therefore, will strongly absorb sunlight (> 290 nm). How-
ever, no evidence is available to indicate whether the energy.absorbed can be
efficiently translated into photochemical processes. The fact that manufacturers
have not noted any light sensitivity for mercaptobenzothiazole compounds would
suggest that these compounds are not photochemically reactive, at least under
conditions of storage, transport, or use (usually in a solid form).
17
-------
II. Environmental Exposure Factors
A. Production, Consumption
1. Quantity Produced, Exported and Imported
More than 100 million pounds of mercaptobenzothiazole compounds
valued at over $60 million are produced annually (see Table 5) and are mainly
consumed in rubber vulcanization operations. The parent compound, 2-mercapto-
benzothiazole, accounts for less than 10% of the total production. The largest
volume compound is 2,2'-dithiobisbenzothiazole (23.2 million pounds in 1973),
followed by the sodium salt of MET (11.9 million pounds in 1973). According
to industry sources (personal communication), approximately one-half of the
NaMBT produced is used in anticorrosion applications and, therefore, is not
included in the totals of rubber-processing thiazoles. The other half is used
mostly in synthesizing other MBT derivatives. Over the years, the production
quantities of N-cyclohexyl-2-benzothiazolesulfenamide and the zinc salt of
MBT have also been enumerated (see Table 5). In 1972, the production of "other
rubber-processing thiazoles" amounted to 46 million pounds. This category
consisted of nine MBT derivatives (see Table 5 for list) and one non-MBT
derivative (thiazoline-2-thiol). However, in 1973, one MBT derivative [1,3-
bis(2-benzothiazolylmercaptome,thyl)urea] and thiazoline-2-thiol were no longer
»
included in the category (USITC, 1973) and, therefore, their production volumes
are probably quite small. Thus, the 46 million pounds noted in 1972 for the
"other rubber-processing" category is mostly attributable to eight MBT deriva-
tives. Figure 3 graphically illustrates total rubber thiazole accelerator
production trends in the United States compared to total accelerator production.
In 1971, approximately 11.6 million pounds of cyclic rubber
accelerators were exported (SRI, 1972a). Since, in that year, thiazole derivatives
18
-------
Table 5. Production and Sales of 2-Mercaptobenzothiazole Compounds (U.S.I.T.C., 1959-1973. 1974p, SRI, 1972a)
Millions of Founds
»-Cyclohexyl-2-
Sodiutn 2rMercapto— benzothiazole-
benzothiazole sulfenamide
Year
1940
1941
1942
1943
1944
1945
1946
1947
1948
1949
1950
1951
1953
1954
1955
1956
1958
1959
i960
1961
1962
1963
1964
1965
1966
1967
1968
1969
1970
1971
1972
1973
1974
Pro- Pro-
duction Sales duction
" - - - -
_ — _ _ _
_ — _ _ _
— — — — —
_ _ _ _ _
— — — — —
_ _ _ _ _
— — — — —
_ _ - _ _ _
— - — — —
_ _ _ _ _
_ — — — _
_ _ _ _ _
- - - - 6
- - - - 5
_ _ _ _ 7
- - - - 7
_ — _ _ 7
- - - - 10
_ _ 7
25. 6C- - - 6
- - - - 4
— — — - 4
- - - - 4
- - - - 5
_ _ _ _ 7
9.7 - - 8
11.9 5.8
- - _ _ 4
-
_
—
—
—
—
_
—
_
—
_
—
_
.4
.7
.4
.8
.2
.1
.2
.6
.7
.9
.9
.9
.6
.8
—
.6
Sales
- - '
_ _
— —
— —
— —
- -
_ _
- -
_ _
- —
_ _
— —
_ _
5.7
5.2
5.8
6.4
5.8
5.7
6.2
4.4
3.4
3.7
3.4
4.7
4.6
5.2
6.6
3.4
2~Mercapto-
benzothiazole
Pro-
duction
11.2
14.8
16.5
17.7
15.3
11.5
14.9
18.2
15.9
11.9
18.5
15.7
13.3
6.9
7.2
6.4
7.0
7.1
8.0
_
6.3
4.7
6. 1
6.7
6.9
6.8
6.0
7.9
6. 1
Sales
6.0
7.9
8.6
9.6
2.6
1.9
2.9
3.2
2.7
3.9
5.4
3.2
2.6
4.1
4.4
4.1
4.6
5.7
5.0
_ _
— —
— _
4.8
5.2
4.2
4.7
7.6
5.3
2,2'-Dithiobis-
benzothiazole
Pro-
duction
- -
_ _
8.3
12.4
9.8
10.3
15.0
17.1
14.9
14.7
15.4
18.2
15.2
19.5
17.5
16.7
18.1
18.4
21.0
21.3
23.3
21.9
22.8
23.5
23.2
21.1
21.3
23.2
20.7
Sales
- -
_
8.8
10.8
7.5
7.9
11.5
12.7
10.1
10.6
10.4
11.0
9.1
11.6
10.1
9.8
10.0
9.7
10.3
10.8
10.9
10.6
11.6
11.6
11.0
10.5
11.7
12.9
11.5
Zinc Other rubber—
2-Mercapto- processing
benzothiazole thiazoles '
Pro- Pro-
duction Sales duction
- - — - ' 17.6
- - - - 19.5
- - - - 12.5
- - - - 11.1
- - - - 10.5
- - - - 9.0
- - - - 10.0
- - - - 11.9
- - - - 22.0
- - - - 20.4
- - - - 21.4
- - - - 19.2
- - - - 26.8
- - - - 26.0
- - - - 25.2
- - - — 24.8
- - - - 27.6
- - - - 29.8
- - - - 35.9
- - - - 30.9
- - - - 28.9
- - - - 25.4
4.4 - - 31.9
4.6 3.6 36.5
4.8 4.1 36.4
- - - - 45.1
4.0 4.1 46.2
' - - 4.5 63.5
- - - - 52.1
Sales
- -
_
12.7
10.9
17.0
7.9
10.0
11.2
11.2
10.3
21.3
17.6
16.3
14.2
14.0
14.8
16.2
18.6
21.2
27.0
29.8
29.4
37.5
32.8
33.3
39.9
41.3
42.9
45.0
Total,
all rubber-
processing
thiazoles
Pro-
duction
28.8
34.3
37.3
41.2
35.6
30.8
39.8
47.2
52.8
47.0
55.3
53.1
55.4
58.8
55.6
55.3
60.5
62.5
75.0
. 59. 4a
65.1
56.7
70.1
76.0
77.3
80.6
86.3
94.6
83.5
Sales
23.1
26.3
30.1
31.4
19.6
17.7
24.4
27.2
24.0
24.8
37.0
31.9
28.0
35.7
33.7 .
34.5
37.2
39.8
42.2
44.1
45.1
43.4
52.8
56.3
58.4
59.2
67.0
74.5
65.3
After 1964, sodium 2-mercaptobenzothiazole was not classified as a rubber-processing chemical.
Compounds in the category of other rubber processing thiazoles vary slightly over the years. In 1972, the, category included 2-benzothiazyl-N,
N-diethylthiocarbamoyl sulfide: 1,3-bis(2-benzothiazolvImereap tome thy1)urea; N-tert-butyl-2-benzothiazolesulfenamide ; N,N-diisopropy1-2-
benzothiazolesulfenamide; N-(2,6-dimethylmorpholino)-2-benzothiazolesulfenamide; 2-mercaptobenzothiazole, copper salt; 4-morpholinyl-2-benzothiazyl
disulfide; N-oxydiethylene-2-benzothiazolesulfenamide; and thiazoline-2-thiol. Only the last compound is not a MBT derivative. When production or
sales volumes are not listed under individual compounds, they are included in the other category.
SRI (1972)
-------
t-o
o
CO
O
z
ID
o
Q.
11.
O
CO
100
90
80
70
60
50
* 40
30
20
1940
PRODUCTION
TOTAL
THIAZOLES, TOTAL
1945
1950
1955
1960
1965
1970
1975
1980
1985
Figure 3. Organic Rubber Accelerators Production and Sales (SRI, 1972a)
-------
totalled 81 million pounds compared to a 93 million pound total for cyclic
accelerators, it appears that substantial quantities of MET derivatives were
probably exported. Imports of MET compounds have been relatively small. For
example, in 1973 only 22,046 and 73,413 pounds of IffiTS and MET, respectively, were
imported (U.S. Tariff Commission, 1974).
2. Producers and Production Sites
The major manufacturers of MBT compounds of any economic signif-
icance are. listed in Table ;6; They are located mostly in the eastern .part of.
the country, close to their major market, tire manufacturers. A significant .
quantity of these chemicals is manufactured by the tire producers themselves for
their own internal consumption and never reaches the open market.
3. Production Methods and Processes
2-Mercaptobenzothiazole is manufactured by reacting aniline
with a solution of sulfur in carbon disulfide (for the chemistry of this reaction,
see Section I-A-4, p. 9 ):
:S + CS2 -^
This can be accomplished in a continuous process using the system invented by
Cooper and Mensing (1951) as illustrated in Figure 4. Column A represents a
high pressure reactor into which aniline and a solution of sulfur in carbon
disulfide are simultaneously fed, both liquids having been preheated to about
240°C. The pressure in the column is kept at about 1,000 psi. Either a curved
deflection plate or an impeller (as shown at C) assures a uniform upward flow
21
-------
Table 6. Manufacturers of Mercaptobenzothiazole (SRI, 1975)
Location
Trade Name
2-Mercaptobenzothiazole
2,2'-Dithiobisbenzothiazole
10
S3
2-Mercaptobenzothiazole, sodium salt
2-Mercaptobenzothiazole, copper salt
2-Mercaptobenzothiazole, zinc salt
American Cyanamid
DuPont .
Goodrich
Goodyear
Monsanto
Pennwalt
Uniroyal
American Cyanamid
DuPont
Goodrich
Goodyear
Monsanto
Pennwalt
Uniroyal
American Cyanamid
Buckman Labs
Goodyear
Monsanto
Uniroyal
American Cyanamid
American Cyanamid
DuPont
Goodrich
Goodyear
Uniroyal
Vanderbilt
Bound Brook, New Jersey
Deepwater Point, New Jersey
Henry, Illinois
Niagara Falls, New York
Nitro, West Virginia
Wyandotte, Michigan
Geismar, Louisiana
Bound Brook, New Jersey
Deepwater Point, New Jersey
Henry, Illinois
Akron, Ohio
Niagara Falls, New York
Nitro, West Virginia
Wyandotte, Michigan
Geismar, Louisiana
Bound Brook, New Jersey
Cadet, Missouri
Memphis, Tennessee
Akron, Ohio
Nitro, West Virginia
Geismar, Louisiana
Woodbright, New Jersey
Bound Brook, New Jersey
Deepwater Point, New Jersey
Henry, Illinois
Niagara Falls, New York
Geismar, Louisiana
Bethel, Connecticut
MET
MET
Goodrite MBT
MET
Metrax
MBT
MBT-UO
MBTS
MBTS
Goodrite MBTS
MBTS
Thiofide
MBTS
MBTS
Sodium MBT solution
Sodium MBT
Goodrite ZMBT
OXAF
Zetrax
-------
Table 6. (cont'd)
ro
2-Mercaptobenzothiazole, zinc chloride
2-Mercaptobenzothiazole,
monoethanolamine salt
Cyanomethylthiobenzothiazole
l.,3-Bis(2-benzothiazolylmercaptomethyl)
urea
2-Benzothiazyl-N,N-diethylthiocarbamyl
sulfide
N-tert-Butyl-2-benzothiozolesulfenamide
N-Oxydiethylene-2-benzothiazolesulfenamide
N-Cyclohexyl-2-benzothiazolesulfenamide
N,N-Diisopropyl-2-benzothiazolesulfenamide
4-Morpholinyl-2-benzothiazyl disulf ide.'
N-(2,6-Dimethylmorpholino)-2-
benzothiazolesulfenamide
DuPont
Vanderbilt
Millmaster Oryx
Lakeway Chemicals
Monsanto
Pennwalt
Monsanto
Pennwalt
Uniroyal
American Cyanamid
Goodrich
Goodyear
Pennwalt
American Cyanamid
DuPont
Goodrich
Monsanto
Pennwalt
Uniroyal
American Cyanamid
Goodyear
Monsanto
Location Trade Name
Deepwater Point, New Jersey
Bethel, Connecticut
Jersey City, New Jersey
Muskegon, Michigan
Nitro, West Virginia
Wyandotte, Michigan
Nitro, West Virginia
Wyandotte, Michigan
Geismar, Louisiana Delac NS
Bound Brook, New Jersey Goodrite OBTS
Henry, Illinois
Akron, Ohio
Wyandotte, Michigan
Bound Brook, New Jersey Cydac
Deepwater Point, New Jersey Conac S
Henry, Illinois
Nitro, West Virginia
Wyandotte, Michigan
Geismar, Louisiana
Bound Brook, New Jersey
Akron, Ohio
Nitro, West Virginia
Goodrite CBTS
Santocure
Delac S
-------
H2S+CS2
MBT
B
Figure 4. Manufacture of Mercaptobenzothiazole
(Cooper and Mensing, 1951)
24
-------
of the reactants and products. The evolution of hydrogen sulfide gas also
maintains agitation of the reaction medium. As reactants are continually
added to Column A, the products are discharged continually into separator
Column B. The material in Column B consists of molten MBT, H S, and CS
(an excess of CS is used in Column A). The temperature of both columns is
kept at about 260°C with high pressure steam coils which can be seen in the
figure. The pressure in Column B is kept at about 350 psi, about a third that
of Column A. The lower pressure and high temperature in Column B assure that
both the H^S and CS_ will be in the gaseous state. A weight-responsive apparatus
on Column B discharges the gases to maintain the proper pressure. After leaving
Column B, the gases are cooled to 20-40°C at constant pressure and then allowed
to expand adiabatically to atmospheric pressure, reducing the temperature to
about -40°C. This liquefies only the CS9, which is thereby separated from the
H S and recirculated. Meanwhile, liquid MBT is continually tapped from Column B
at about atmospheric pressure and cooled to the solid state.
Once MBT has been manufactured, the sodium salt is easily
obtained by dissolving the MBT in an aqueous solution of NaOH. Insoluble
metal mercaptobenzothiazoles (zinc, copper, etc.) are formed by the addition
of the appropriate cation to aqueous solutions of NaMBT, whereupon the metal-
MBT compound precipitates out of the solution.
MBT is also a starting material for producing a number of
disulfides, such as MBTS, when manufactured according to the method of Kleiman
(1950). In this method, an alkali metal mercaptide is used as a catalyst for
the reaction between MBT and disulfides (e.g., methyl disulfide); the product
25
-------
consists of MBTS and the volatile mercaptan. Other symmetrical as well as
unsymmetrical disulfides can also be synthesized by this method, depending upon
the choice of disulfide(s) (and thiol) used as starting materials.
MBTS in turn is the starting material for the manufacture of
N,N-diispropyl-2-benzothiazolesulfenamide. MBTS is reacted with diisopropyl
amine and diisopropylchloroamine on a steam bath for 30 minutes, after which
the product crystallizes out when the reaction medium is cooled on an ice bath.
It is washed with cold water and dried to give a high purity product (99%)
(Hardman, 1956).
N-Oxydiethylene-2-benzothiazolesulfenamide is prepared by
reacting MET and morpholine in benzene or toluene in the presence of a chlorin-
ating agent such as sodium hypochlorite. The reaction medium is then treated
with 50% aqueous NaOH, and the product is removed from the organic layer by
evaporation (Sullivan, 1956).
N-Cyclohexyl-2-benzothiazolesulfenamide may be prepared by
oxidizing cyclohexylamine and MBT with NaOCl at A5-70°C. The product forms as
a solid which is washed with water and dried (Lunt, 1956).
More recently, a number of sulfenamides have been manufactured
by oxidation of MBT using metal phthalocyanines as catalysts with the appro-
priate amine (Campbell and Wise, 1973).
4. Market Price
Figure 5 illustrates the past and present prices of MBT and
MBT derivatives. The prices have stayed fairly constant over the years plotted,
with the exception of N-cyclohexyl-2-benzothiazolesulfenamide. The reason for
the sharp rise in price for that compound is unknown. The cheapest product is
MBT ($0.36/lb in 1973), probably because it is the raw material for all the MBT
derivatives.
26
-------
K>
s
CM
CD
1.60-
1.50-
1.40-
1.30-
1.20-
1.10-
1.00-
1 0.90-
» 0.80-
S2
| 0.70 -<
Q
0.60
0.50-2
0.40-
0.30-
0.20-
0.10-
N — Cyclohexyl—2— Benzothiazole—Sulfenamide
Total—Thiazole Rubber Processing Chemicals
2,2* — Dithiobis (Benzothiazole)
-Q
2 —Mercaptobenzothiazole (MBT)
3 IIIIIIIIIII~TI][II
1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975
Year
Figure 5. Price History of 2-Mercaptobenzothiazole Derivatives (U.S.I.T.C., 1959-1973; Chemical
Marketing Reporter, 1975)
-------
5. Market Trends
Although there is fluctuation in the consumption of mercapto-
benzothiazole compounds, the general trend, as shown in Figure 5 and Table 5,
is toward increased consumption. The demand for individual members of the
thiazole family does not necessarily follow the general trend. The use of MET
has declined since the middle 1960?s, while the demand for sulfenamides has
increased. MET has been replaced in formulations with accelerators which
approximate its activity, but are less likely to cause scorching (premature
vulcanization). As the use of radial tires becomes more widespread, accelerator
demand will shift in favor of those compounds which impart the required properties
to these tires. In general, the use of radials is expected to decrease the
demand for accelerators because of the longer wear expectancy of radial over
conventional tires (Anon., 1974).
Since the largest use of MBT compounds is in the rubber in-
dustry and the major rubber product is vehicular tires, the mercaptobenzo-
thiazole market is expected to be affected by trends in automobile sales and
use. When auto sales are up, tire sales are up. When auto sales are down, as
they were in 1974, tire demand does not necessarily decrease since new tires
are purchased by consumers to replace worn ones on older vehicles. However,
if vehicular use should decline along with auto sales, demand for tires would
certainly fall. Such a situation could arise if, for example, gasoline prices
were to increase significantly, or in the event of a scarcity and/or rationing
of fuel.
The snow tire market also has an impact on overall tire sales.
In 1974, General Motors purchased 15 million regular tires for use as original
28
-------
equipment. That same year, 17.5 million snow tires were sold, down from
19.1 million in 1972. In 1975, GM began equipping all new cars with TPC-
Spec steel-belted radial tires, which qualify as snow tires in Idaho, Texas,
Oklahoma, and New York City. GM has been promoting the TPC-Spec "for year-
round traction performance" and this is expected to be a factor in anticipated
sales of snow tires below 15 million in 1975 (Anon., 1975a).
the market for mercaptobenzothiazole accelerators is also affected
by their cost vis-a-vis the costs of competitive chemical accelerators. The
prices and availability of raw materials needed to synthesize MET and its
derivatives must therefore be weighed in any market trend speculation. For
MET, the prospects for a steady supply of raw materials at stable prices appear
good. In 1973, aniline was in oversupply and at the lowest prices in two decades
(Anon., 1973a). There is currently an overcapacity for carbon disulfide, with
prices at their highest in 23 years and demand slackening off (due to lower
rayon production) (Anon., 1975b). These factors are likely to soften the price
of this chemical.
B. Uses
1. Major Uses
Table 7 lists the major and minor uses of MET and its derivatives.
The most important use of these chemicals is as vulcanization accelerators.
Table 7. Use of MET and Derivatives
MAJOR USES COMPOUND
Vulcanization Accelerators all, except NaMBT
Corrosion Inhibitors NaMBT, MET
MINOR USES
Fungicide Formulations MET, NaMBT, others
Reagent in Transition Metal Separations MET, NaMBT
and Analyses
29
-------
Vulcanization accelerators are selected on the basis of pre-
determined processing requirements and curing speed (Walkerj 1970). A typical
rubber compound (mixture of materials prior to vulcanization) contains approxi-
mately 0.5 - 1.5% accelerating agent. MET is the most active of the thiazole
accelerators at both processing (mixing, milling, compounding) and curing
(vulcanizing) temperatures. It is, therefore, used where high activity is
required at relatively low temperatures (below 287°F), and with slower curing
synthetic rubbers. MET is used, for example, in combination with other
accelerators in curing butyl rubber and in the manufacture of soles for shoes
(Walker, 1970). The combination of MET and tellurium diethylthiocarbamate is
the fastest known accelerator for butyl rubber. It is used extensively for
butyl inner tubes in buses (Elkin, 1969).
MBTS is used in curing systems above 287°F. It has less ten-
dency to cause premature vulcanization at higher temperatures than MET. Its
activity can be modified over a wide range when used in combination with MET
and other accelerators.
ZMBT is intermediate in curing rate and tendency to premature
vulcanization. It is used in latex foam compounding (Walker, 1970).
As a group, the sulfenamide accelerators have much less ten-
dency to cause premature vulcanization (which leads to a vulcanizate with non-
uniform physical properties) than the other thiazole accelerators. Sulfena-
mides tend to inhibit crosslinking for a time, and then accelerate it (Leib
et_ _al. , 1970). The first commercial sulfenamide accelerator was N-cyclohexyl-
2-benzothiazolesulfenamide (CBS). CBS is approximately equivalent to MBTS in
curing properties with somewhat less tendency for premature vulcanization.
30
-------
N-Oxydiethylene-2-benzothiazolesulfenamide (OBS) is a delayed-action accelerator
used with products for which premature vulcanization must be avoided at all
costs, and also when high processing temperatures may be encountered prior to
curing (Walker, 1970). N,N-Diisopropyl-2-benzothiazolesulfenamide (DIBS) also
exhibits the delayed action characteristic of the sulfenamide accelerators.
Once activated, DIBS cures very rapidly (American Cyanamid Co., 1973). N-tert-
Butyl-2-benzothiazolesulfenamide (TBBS) is a powerful accelerator above 280°F.
TBBS gives good protection from premature vulcanization and has somewhat greater
cure strength than other sulfenamide accelerators (American Cyanamid Co., 1971).
The largest single product area for vulcanized rubber is tire
and tire products for automotive vehicles. In 1972, approximately 1.5 million
tons of rubber were used for tire and tire products, accounting for 63% of all
rubber production for that year (Oosterhof, 1972). Table 8 shows a passenger
tire formulation suggested by R.T. Vanderbilt Company for tire treads. It
should be kept in mind that, while this formulation gives typical ingredients
and relative quantities, it is not meant to suggest that any particular tire
currently manufactured is made from this formulation.
The second major use of MBT compounds, specifically NaMBT,
is as a corrosion inhibitor in water based cooling systems, especially those
of automobiles. Corrosion in ethylene glycol-water cooling mixtures is due
to oxidation of the glycol to organic acids (Collins and Higgins, 1959), which
is encouraged by excessive aeration of the antifreeze solution, high temperature
sites in the cooling system waterways, or excessively high temperature operation
of the engine. It has been shown that in a cooling system made of many types
of metals and employing ethylene glycol antifreeze, corrosion activity can be
31
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Table 8. SBR/cis-Polybutadiene Passenger Tire Tread Formulation
(Walker, 1970)
Parts
SBR (styrene-butadiene rubber) 103.1
cis-Polybutadiene 25
K-Stay G (oil soluble sulfonic acid derivative in petroleum base) 5
Stearic Acid 2
Zinc Oxide 3
AgeRite Resin D (polymerized trimethylhydroquinone) 1.5
AgeRite HP (phenyl-g-naphthylamine 65%, diphenyl-j>- 0.5
phenylenediamine 35%)
Antozite 67S (£-phenylenediamine derivative) 4
Microcrystalline Wax 1
Phi.lrich 5 7
ISAF (intermediate super abrasion furnace black) 65
Sulfur 1.8
Amax (N-oxydiethylene-2-benzothiazolesulfenamide) 1.5
TOTAL 220.4
(Press Cure: 30 minutes @ 307°F)
significantly inhibited by the addition of triethanolamine phosphate and about
0.1% NaMBT (Squires, 1958). The concentration of the NaMBT decreases with service
as it tends to form MBTS (Vanderbilt, 1974). The lower limit on concentration
for effective corrosion inhibition is about 0.01%. The length of time it takes
to reach ineffective levels of NaMBT varies considerably from vehicle to vehicle
(Weibull, 1966), hence it is recommended that antifreeze solution should be
periodically recharged with a dose of concentrated NaMBT solution (Squires,
1958).
32
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MET and NaMBT are also being used to protect metals in indus-
trial applications other than automotive. Krapivkina and Rotmistrova (1969)
formulated a corrosion inhibiting lubricant containing 1% MET for use on steel
reinforced concrete. The lubricant helped provide protection not only from the
weather, but from 6% mineral acids as well.
NaMBT has been shown to restrict corrosion in alcoholic solu-
tions (other than glycols) such as are used for aircraft deicers (Nigam and
Sanyal, 1971).
MET has been recommended to protect copper and brass fittings
against attack by chlorinated acetic acid in cellulose processing (Prajapati
et_ al_. , 1972). In the case of copper and brass, MET affords protection by
inhibiting anodic reactions (Prajapati et^ a^. , 1972) j whereas the corrosion of
steel in sulfuric acid is slowed by MET mainly by the inhibition of cathodic
reactions (Singh and Banerjee, 1972).
MET can be incorporated into paint formulations intended for
electrodeposition on mild steel. The painted steel thereby acquires a very
tough finish with high resistance to chipping and flaking as well as corrosion
(Guruswamy and Jayakrishnan, 1972).
2. Minor Uses
2-Mercaptobenzothiazole forms characteristic complexes with
transition metals (Khullar and Agarwala, 1975) and rare earths (Malik and
Rahmani, 1975), and the distinguishing properties of these complexes have been
suggested as a means of chemical analysis of the metals. The chemistry of
palladium (House and Lau, 1974), platinum, rhodium, iridium (Diamantatos, 1973b) ,
and gold (Diamantatos, 1973a) with MET has been applied to the development of
33
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a solvent extraction separation scheme which offers very high resolution, yet
is straightforward enough for routine work in the metals processing industry.
MBT has also been used in metal processing as a collector in
flotation liquors. Current applications of MBT include flotation of copper
minerals, copper-activated sphalerite, and nickel ores (Jones and Woodcock,
1973).
The benzothiazole moiety is found in some commercially im-
portant thiazole and cyanine dyes (Powell, 1969; VanLare, 1965), but these
compounds are not manufactured from MBT or MBT derivatives. In fact, although
benzothiazole is used somewhat as a dye precursor, it is not used in the above
mentioned dyes.
When applied to fabrics, MBT exhibits antifungal properties
(Darby and Kempton, 1962; Turner, 1966). MBT is used in leather processing to
prevent fungal growth on leather products (Bowes _ejt _al. ,. 1970) , especially on
those meant for tropical use. MBT is also used in antifungal formulations,
such as R.T. Vanderbilt Company's Vancide 51, which contains as active in-
gredients, 27.6% sodium dimethyldithiocarbamate and 2.4% sodium 2-mercaptobenzo-
thiazole (Berg, 1974). Vancide 51 is recommended for a wide variety of anti-
fungal applications: sweet potato seed piece treatment, industrial water
cooling slimicide, and as a textile preservative (Berg, 1974). In the case
of industrial water cooling systems (water is the heat transfer medium),
NaMBT may be present, not only as a slimicide, but also as an anticorrosion
agent. Actually, less than 1% of all NaMBT produced is used in antifungal
formulations. Most of it is used in anticorrosion applications, with more
than 50% of the amount used for anticorrosion being used to protect auto-
mobile engines (data provided by industrial sources).
34
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Several halo, alkyl, and thioalkyl derivatives of benzothiazole
have shown some anthelmintic or antifungal activity (Alaimo et^ a_l. , 1974). It
is doubtful, however, that these compounds are presently used for these purposes.
However, cyanomethylthiobenzothiazole and the monoethanolamine salt of MET have
been produced in commercial quantities for fungicide applications (USITC, 1959-73),
3. Discontinued Uses
MET causes contact dermatitis in many people. The symptoms
are usually produced by intimate contact with an article of clothing made of
or containing rubber (Fisher, 1973). Because of this effect, the use of MET in
most Spandex rubber manufactured in the United States for articles of clothing
(underwear, girdles, brassieres, etc.) has been discontinued (Jordan, 1972).
There have been shifts in uses of MET. It is used much less
now than formerly as an accelerator in the rubber industry, having been re-
placed by the sulfenamide derivatives for many applications. It is necessary
to synthesize MET, however, as an intermediate for production of sulfenamide
derivatives.
4. Projected or Proposed Uses
MET compounds will probably continue to be used in novel
organic syntheses, as for example in recent studies of 3-lactam antibiotics,
where the conversion of penicillins into cephalosporins was achieved in reactions
which employed MET as an intermediate reagent (Kamiya et^ a±., 1973). It is
difficult to predict whether such uses of MET will ever be of commercial sig-
nificance.
5. Possible Alternatives to Use
Although an important commercial chemical with widespread use,
MET could probably be replaced in some applications with other materials. In
35
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the case of vulcanization acceleration, there are other types of compounds
presently in use, although mercaptobenzothiazoles account for the majority of
all accelerator chemicals. Dithiocarbamates and tetra-R-thiouram mono and
disulfides are among the "ultra" accelerators presently in use for high tem-
perature and rapid cure vulcanization applications (Walker, 1970). Alone, or
in various combinations, these compounds could probably be substituted for
mercaptobenzothiazole accelerators in many formulations, although it is possible
that other accelerators would be a greater health hazard (e.g., thioureas) than
MET. In some applications, it might be possible to change the type of rubber
rather than the type of accelerator in a given rubber. Neoprene rubber, for
example, is presently accelerated exclusively with thiourea derivatives, not
mercaptobenzothiazoles. (Neoprene rubber represented 6.7% of all rubber types
produced in 1972 [SRI, 1972b]).
MET compounds are only one group of many which are used for
the inhibition of corrosion. Substitutes for MET derivatives include chromates,
borates, phosphates, tetraethanolainine, and mixtures of these, as well as sodium
benzoate and sodium nitrite. In the case of the cooling systems of automobiles,
however, most of the available substitutes would not be as effective as form-
ulations which include MET (Weibull, 1966). The most effective organic substi-
tute for NaMBT is probably benzotriazole and its sodium salt derivatives.
Although currently more expensive than MET compounds, benzotriazoles are effec-
tive at lower concentrations for longer periods of time, and offer superior
protection of copper and copper alloys (Davis et al., 1976).
The role of MET as a fungicide is minor, not only with respect
to other uses of MET, but with respect to all other fungicides as well. There
are many possible alternatives to MET in this application, including copper,
".MJ****
mercury, organic and inorganic zinc salts, halogenated phenols, etc. (see,
for example, Turner, 1966).
36
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C. Environmental Contamination Potential
1. General
In considering the environmental contamination potential of
MET and its related compounds, it is important to bear in mind that regardless of
which of the MET compounds is employed in a particular use, alteration fre-
quently occurs resulting in the formation of one or more other members of the
MET family. When rubber is vulcanized, for example, a sulfenamide may be the
accelerator added during compounding, but due to the chemistry of vulcanization
(see Section I-B), one is generally able to detect benzothiazole, MET, MBTS,
and ZMBT (when ZnO is present in the mix) in the vulcanizate. Moreover,
mercaptobenzothiazoles present in rubber products usually are oxidized to
sulfonates, and eventually benzothiazole, as the rubber ages (Brooks, 1963).
In the case of the use of NaMBT in anticorrosion applications, the salt
gradually oxidizes to MBTS. It is possible for the NaMBT to undergo reduction
as well to benzothiazole (which is insoluble in water) in the presence of iron
if the pH of the antifreeze solution falls below 7 (Weibull, 1962).
The most obvious source of environmental contamination potential
by MET compounds, including the parent compound, benzothiazole, is in the use and
disposal of rubber tires. The disposal of antifreeze solutions with mercapto-
benzothiazole corrosion inhibitors is probably the second largest source of
environmental contamination, especially because used antifreeze is likely to
be disposed of by simply pouring it on the ground. The compounds in solution
can then be spread widely via sewers, ground water, and streams, unlike the
case of discarded tires where the chemicals take some time to leach out of the
rubber matrix.
37
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2. From Production
MET Is usually manufactured in closed continuous systems which
include recovery and recirculation of excess CS (see Figure 4, p. 24). Under
normal circumstances, the reactants and products do not have access to the
environment. The columns and associated equipment must be able to withstand
the high temperatures and pressures required without leaking H S, CS , or aniline
into the environment. Also, there must be provisions for dealing with the H S
(which is not recirculated), so that it is kept out of air and water.
Manufacturing processes other than the continuous process de-
scribed in Section II-A-3 (p. 21) may produce side products which are potential
environmental contaminants. Ivanova and Shebuev (1957), for example, have
detected benzothiazole in the waste water effluent of a MET manufacturing
facility in the Soviet Union.
3. From Transport and Storage
Solid MET compounds are transported and stored in paper bags
and cardboard drums. Generally, the greatest hazard these present is the
possibility of dust escaping into the air during filling and emptying pro-
cedures, or if accidental breakage should occur. In the event of spills, the
recommended cleanup procedure involves the eventual burial of the chemical in
landfills or disposal down a sewer (Shaffer, 1971a,b,c,d, 1972a,b,c, 1974),
either of which would result in placing the spilled compounds directly in the
environment.
NaMBT is usually sold in aqueous solution. It is transported
and stored in tanks and drums. Spray from the liquid while being poured, or a
leak in a transport pipe are possible sources of contamination with this material.
The pH of NaMBT solution is normally quite high (ca. 13, assuming 0.3% free alkali
as NaOH) (see Table 4, p. 9), and the solution is therefore a caustic material.
38
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A. From Use
The major environmental contamination potential of MET compounds
is in the use of rubber products. Akron, Ohio, represents the area of the
largest concentration of use of vulcanization accelerators in the U.S., but
rubber processors are well scattered throughout the U.S. at more than a thousand
sites, according to industry sources. Evstifeev jet al^. (1971) have detected
MET and MBTS in the waste effluent of a rubber manufacturing plant in the USSR
and in the U.S. Webb et_ al. (1973) have identified MET and benzothiazole in
the holding ponds and aerated lagoons used for treating effluents from synthetic
rubber plants. Concentrations of 0.027 ppm and 0.049 ppm benzothiazole have
been detected in discharges of a tire plant treatment pond (Niles, 1976).
MET compounds may be emitted into the air of plants which use
these compounds in rubber manufacturing. Evidence of this was shown by
Rappaport (1975), who identified the presence of benzothiazole and fr-butyliso-
thiocyanate in the volatiles of a passenger car tire formulation undergoing
vulcanization, which included N-_t-Butyl-2-benzothiazolesulfenamide as the accelera-
tor. The techniques used were mass spectrometry and gas chromatography. He
also found an unusual assortment of other compounds discharged during vulcani-
zation, including styrene, butadiene oligomers, alkyl benzenes, naphthalenes,
and some nitrogen and sulfur compounds. The latter nitrogen and sulfur com-
pounds were not attributed to the accelerator. All identified materials appeared
to be either impurities in the rubber compound or decomposition products of the
compound ingredients.
Approximately 1.2 billion pounds of rubber dust are worn from
vehicular tires in the United States each year (Pierson and Brachaczek, 1975).
If it is assumed that the accelerators are evenly distributed in this dust,
and considering that the tire compound contains about 1% accelerators on
the average, roughly 12 million pounds of vulcanization accelerator products,
39
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including benzothiazole, ZMBT (providing ZnO is in the formulation), MET,
and MBTS (the exact kinds and quantities of MET breakdown products from
vulcanization has not been determined), get into the air and soil adjacent to
highways each year. Since gaseous emission from automobile and truck rubber
tires is negligible (Dannis, 1975), airborne particulate debris from tires is
likely to be the major source of MET in the air. Particulate matter from this
source, however, constitutes only about 1-4% of all the particulate matter in
the air (Pierson and Brachaczek, 1974, 1975) and is, therefore, not a major
source of air pollution. Most of the tire dust generated at the road settles
in the surrounding area. Under appropriate conditions in moist soil, the
rubber particles are degraded within months, thus releasing MET compounds.
Under less favorable conditions, degradation may take decades (Dannis, 1975).
The fate of MET and related compounds in these particles has not been reported,
and there is no reason to believe it is currently being investigated. However,
it is known that MET products can easily leach out of rubber which is in con-
tact with water (Aktulga, 1971a). Rubber closures which had been processed
with MBTS were used to stopper 500 ml infusion bottles filled with distilled
water. When the bottles were inverted and left undisturbed for as little a
time as one month, both MET and MBTS could be identified in the water by both
spectrophotometry and thin layer chromatography. Grushevskaya (1974) reported
similar results for water shaken with rubber samples. Thin layer chromatography
of chloroform extracts of the water demonstrated the presence of MET, MBTS, and
OBS from the rubber in the water.
Consumer exposure to MET compounds can occur when one wears
clothing containing rubber. As noted previously, such contact often leads to
dermatitis in individuals sensitive to these chemicals.
Minor potential sources of environmental contamination from
uses include antifreeze solutions (see following section) (Squires, 1958),
40
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and paints and other materials containing mercaptobenzothiazoles as anti-
fungal agents. This includes certain clothing intended for use in tropical
climates.
5. From Disposal
When used tires are discarded in garbage dumps, landfills,
or waterways rather than being reprocessed, they become a source of MBT
compounds which would be expected to enter the environment readily. It has
been shown that MBT compounds easily leach from rubber into stagnant water
(Aktulga, 1971a) (see previous section).
A major source of contamination for MBT chemicals is via
disposal of waste radiator coolants and antifreeze solutions. These materials
are likely to be discarded directly into a sewage system, as compared to rubber
tire dust and waste products, from which the accelerators must first leach out
Approximately 11 million pounds of NaMBT is produced each year, about half of
which goes into antifreeze products which will be discarded within a year or
so of their being placed into service.
Excess, contaminated, or surplus MBT compounds, if disposed
of by incineration, produce SCL (and possibly also CO and nitrogen oxides), and,
therefore, mercaptobenzothiazoles disposed of by incineration are probably not
a source of MBT contamination. However, many MBT compounds are deposited in
landfills (see Section II-D-3, p. 43), and, therefore, leaching from landfills
could be a significant source of contamination.
6. Inadvertent Production via Industrial Processes
It is clear from the discussion on vulcanization chemistry
that, although a single accelerator compound, say a sulfenamide, may be employed
in a given vulcanization operation, the nature of the process is such that
. f 41
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a variety of MET products can usually be identified in the final rubber
product, including the one actually added in compounding. It is perhaps
not quite correct to say that these other chemicals (usually benzothiazole,
MET, MBTS, and ZMBT) are inadvertently produced, since their formation is
an essential part of the vulcanization process. However, since it is not
the direct intention of the manufacturer to specifically include these
materials in the product, their presence is in that sense inadvertent.
Industrial processes other than vulcanization which might
inadvertently produce MBT compounds would have to be characterized by the high
temperatures and pressures needed to produce MBT, as well as either the
presence of the materials it is made from (aniline, CS , and sulfur), or an
MBT compound which might be a precursor to other MBT compounds (i.e., a sulfen-
amide, a metal salt, polysulfide, etc.). It is not likely that dyes containing
benzothiazole rings would act as inadvertent precursors to other MBT compounds
under the conditions in which thiazole and cyanine dyes are manufactured and
used. Phenylisothiocyanate, which is a commercial product (SRI, 1975), could
form MBT if it comes in contact with hydrogen sulfide (Ivanova and Shebuev,
1957).
7. Inadvertent Production in the Environment
Except in the depths of volcanoes, the temperatures and
pressures needed to produce MBT from its raw materials are not normally en-
countered in nature. Moreover, it is unlikely that CS and aniline would be
present in a volcano, even if sulfur were. Therefore, the possibility of MBT
compounds being produced in the environment, even in the presence of all three
starting materials, seems very remote.
42
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D. Current Handling Practices and Control Technology
1. Special Handling in Use
Respirators approved by the U.S. Bureau of Mines for nuisance
dust and safety spectacles are recommended in the event of excessive dustiness
in handling the compounds listed in Table 9. Otherwise, no special handling
in use is specified by the manufacturers of these compounds, other than ordinary
measures of personal hygiene.
Solutions of NaMBT are alkaline and, therefore, are more dangerous
than the solids with respect to skin contact and breathing of any spray from them.
Splashproof goggles and protective rubber clothing are recommended for handling
such solutions, in addition to the suggestions above. Also, an eyewash foun-
tain and safety shower should be available at the handling site.
2. Methods of Transport and Storage
The solid chemicals listed in Table 9 are supplied in 50 pound
paper bags and in 200-250 pound fiber drums. No special storage or shipping
methods are used for the solids.
Solutions of NaMBT are shipped and stored in 55 gallon drums
and tank cars. The solution is treated with the same precautions as any caustic
solution. It otherwise requires no special storage conditions.
3. Disposal Methods
The recommended disposal method for all the compounds listed
in Table 9 is burial in a landfill (see references, Table 9). Incineration
is not recommended unless provision can be made to insure that S09, CO, and
nitrogen oxides from the sulfenamides will not be emitted to the atmosphere.
-------
Table 9. Sources of Safety Data for MET Compounds
Safety Reference
2-Mercaptobenzothiazole Shaffer, 1971d
2,2'-Dithiobisbenzothiazole Shaffer 1971a
N-Oxydiethylene-2-benzothiazolesulfenamide Shaffer, 1974
N-tert-Butyl-2-benzothiazolesulfenamide Shaffer 1972a
N,N-Diisopropyl-2-benzothiazolesulfenamide Shaffer, 1972b
N-Cyclohexyl-2-benzothiazolesulfenamide Shaffer, 1971b
Zinc Mercaptoberizothiazole Shaffer 1971c
Sodium Mercaptobenzothiazole, 50% aqueous solution Shaffer, 1972c
4. Accident Procedures
The solids should be swept up and placed in a waste container
and the spill area flushed with water. Rubber gloves and splashproof goggles
should be worn to deal with spills of solutions of NaMBT. The solution spill
should be covered with a disposable absorbent material, which is then swept up
and placed in a disposal container, and the contaminated area should be flushed
with water (see references, Table 9).
5. Current Controls and Control Technology Development
MET compounds are considered relatively free of hazards with
respect to fire, reactivity, and the health of humans. The available infor-
mation indicates that no unusual efforts are made with regard to the manufacturing
and handling of these compounds to restrict their entry into the atmosphere as
dust or into waste water systems.
44
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E. Monitoring and Analysis
1. Analytical Methods
Until about 15 years ago, colorimetric techniques were
recommended for assaying MET and its derivatives. These are satisfactory
for monitoring the purity of individual compounds, but fail to distinguish
between the components of a mixture. As a practical matter, it is often im-
portant to be able to distinguish MET from its derivatives, as they will often
be found together, as for example, in rubber products, where the addition of
MET and zinc oxide in the compounding stage assures the presence in the vul-
canizate of MET, ZMBT, and also MBTS.
Table 10 summarizes the available analytical methods.
Chatterjee et^ al. (1960) developed a method for the quantitative determination
of MET, ZMBT, and MBTS in the presence of each other via amperometric titration
with silver nitrate. The latter forms a silver salt with MET, but does not react
with ZMBT or MBTS. After the determination of MET, the remaining two compounds
are converted, one at a time, into MBT and titrated. This is possible because
ZMBT is easily cleaved under acidic conditions, but MBTS requires a reducing
+2
agent (Sn ) and strong acid conditions. The accuracy of this method is better
than 2%, typically about 1%, with a detection limit of 0.2% or 2,000 ppm.
Chakravarti and Sircar (1965) extended the above technique to
include sulfenamide derivatives of MBT. The sulfenamides are also reduced to
+2
MBT in strongly acidic Sn solution. They are separated from the other com-
ponents in the sample by solvent extraction prior to cleavage and titration.
Ethylene glycol antifreeze solutions commonly contain NaMBT
as an anticorrosion agent. Usually a mixture of anticorrosion agents is used.
45
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Table 10. Analytical Techniques for MET Compounds
Technique
Amperometric titration
Paper chromatography/
UV-IR spectrescopy
Amperometric titration
Polarography
Gel chromatography
Colorimetric method
Mass spectrometry
UV spectroscopy
Filtration of air
samples, UV spec-
troscopy
XAD resin extraction
combined with GC-MS
GC-MS
GC
Compounds
MET, ZMBT, MBTS
MET
thiazole sulfenamides
NaMBT
MBT, ZMBT, MBTS
MET, MBTS
thiazole sulfenamides
MBT
MBT
benzothiazole, methy1-
benzothiazole, and
2-thiomethylbenzo-
thiazole
benzothiazole
benzothiazole
Sensitivity
2000 ppm
(qualitative)
1 ppm
2000 ppm
9-12 ppm
ppb-
Source
Chatterjee £t al., 1960
Fiorenza et al., 1963
Chakravarti and
Sircar, 1965
Woodroffe and Munro,
1970
Aktulga, 1971b
Stebletsova and
Evstifeev, 1971
Hilton and Altenau,
1973
Jones and Woodcock,
1973, 1975
Krivoruchko, 1972
Burnham et al. , 1973
Rappaport, 1975
Parsons and Mitzner,
1975
A typical antifreeze solution can be expected to contain not only NaMBT, but
also benzotriazole (triazole, not thiazole), as well as the following ions:
benzoate, borate, nitrite, and phosphate (Woodroffe and Munro, 1970). A
polarographic technique has been developed for the determination of MBT in
corrosion-inhibited glycol mixtures (Woodroffe and Munro, 1970). The method
is sensitive to 1 ppm. Benzotriazole may be determined at the same time after
separation of the NaMBT on an anion exchange resin.
46
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Paper chromatography coupled with UV and IR spectrophotometry
has been used to qualitatively identify MET in rubber product mixtures (Fiorenza
j|_t £l. , 1963). Gel chromatography has been used for quantitative determination
of materials leached from rubber bottle closures (Aktulga, 1971b). The sensi-
tivity of this method, the size of the rubber closures, and the amount of water
in the bottles (500 ml capacity) were not given, but quantities of MET as low
as 38 yg were detected, presumably per rubber closure for one month contact of
closure and distilled water.
Some monitoring of industrial waste water for MET and MBTS
has been reported in the Soviet Union. One procedure consists of benzene
extraction followed by photometric detection (as a cobalt complex). The tech-
nique is said to have an accuracy of ±14% and a sensitivity of 5 mg/1
(Stebletsova and Evstifeev, 1971).
Krivoruchko (1972) reported a technique for the monitoring of
MET in air. The samples are collected on a filter by aspiration, dissolved in
ethanol, and determined spectrophotometrically at 325 nm. The error is claimed
to be within 20%.
Hilton and Altenau (1973) have reported a very rapid mass
spectrometric procedure for the identification of sulfenamide derivatives of
MET. This method uses small samples of rubber (ca. 0.5 gm) and does not require
a solvent extraction step. Low voltage spectra are produced which can accur-
ately distinguish between the various amine moieties of the sulfenamides, thus
identifying the original accelerator. The procedure sacrifices sensitivity for
speed; its sensitivity is 2,000 ppm. Because the fragment ions typical of the
47
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original sulfenamides were not found in this study of rubber samples, the
authors concluded that very little unreacted sulfenamide accelerator remains
after vulcanization. The amine and benzothiazole products of vulcanization
are retained in the rubber matrix either physically or by hydrogen bonding
until released by heating or solvent extraction.
Jones and Woodcock (1973, 1975) have developed a method for
the determination of MET in flotation liquors. The technique consists of
extraction with chloroform followed by UV spectrometric quantitation. The
sensitivity of the method is 9-12 ppm.
Burnham jet^ al^ (1973) have used an XAD-2 polystyrene macro-
reticular resin as an adsorbent to isolate and concentrate organic compounds
from water samples. They have isolated and identified compounds using gas
chromatography and mass spectrometry (GC-MS) at ppb ranges. Using this tech-
nique, they were able to identify benzothiazole, methylbenzothiazole, and
2-thiomethylbenzothiazole in Delaware River water, but the concentrations were
not reported.
Rappaport (1975) developed a GC-MS technique which he used to
measure volatiles generated by the vulcanization of tire tread stock.
Parsons and Mitzner (1975) were able to detect benzothiazole
with a gas chromatographic apparatus developed for analysis of industrial
pollutants in stack effluents.
2. Current Monitoring
MET or related compounds have been found in drinking water and
industrial waste water, and tire dust, which will contain MET vulcanization
products, has been found in the air and soil adjacent to highways.
48
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Pierson and Brachaczek (1974, 1975) have demonstrated that
1-4% of all particulate matter in air is debris from tires. Most of the tire
dust generated at the road settles on the road or adjacent areas. The particles
will degrade at varying rates, depending upon the conditions (Dannis, 1975),
and result in possible release of MBT compounds. However, no direct monitoring
of MBT compounds in tire dust has been noted.
In 1957, Ivanova and Shebuev (1957) reported the presence of
benzothiazole in the waste effluent of a MBT manufacturing plant. Benzothiazole
has been noted as a possible contaminant of drinking water (Anon., 1973b).
It has been found as a major contaminant, along with a compound identified as
"methyl benzothiazole" (probably 2-methylbenzothiazole) and 2-thiomethylbenzo-
thiazole, in water from the Delaware River sampled near an industrial plant in
the southern part of Philadelphia (Burnham et al., 1973).
The Committee Report on Organic Contaminants in Water of the
American Water Works Association (Jenkins et^ al., 1974) identified the com-
pounds listed in Table 11 in finished water. These same compounds also appear
in the latest list of organic compounds identified in drinking water which is
prepared by the Water Supply Research Laboratory in Cincinnati, Ohio (U.S.
EPA, 1975). It is unknown whether these contaminants definitely resulted from
commercial MBT compounds.
Table 11. MBT Compounds Identified in Finished Water
(Adapted from Jenkins et al., 1974)
As Listed in Jenkins et al., 1974 Probable Standard Chemical Name
benzene thiazole benzothiazole
methyl benzothiazole 2-methylbenzothiazole
thiomethylbenzothiazole 2-thiomethylbenzothiazole
-------
Evstifeev et al. (1971) have found aniline, CS , H S, MBT, and
j- — Z Z
MBTS in the waste effluent of a rubber manufacturing plant. The exact nature
of the rubber manufacturing processes was not given, nor was any quantitative
information on the substances found in the effluent. However, this calls atten-
tion to the possibility that other rubber manufacturing facilities may introduce
similar chemicals into their waste effluent.
Following toxicity studies on waste water from rubber production
as a component of reservoir water, Vaisman et^ _al. (1973) recommended that MBT
and MBTS be totally absent from waste water discharged into reservoirs. Webb
et_ al_. (.1973) found MBT in the aerated lagoon of a synthetic rubber plant and
the raw waste of a paper mill. The contents of the former had a disagreeable
odor and the contents of the latter were believed to be toxic. However, no
quantitative data were available. Rappaport (1975) monitored the air of a rubber
passenger tire press room, but was unable to detect benzothiazole, a compound
he was able to detect in the air from a simulated vulcanization process. Webb
and coworkers (1973) also found "2-benzothiazble" (probably benzothiazole) in
a latex plant's holding pond and in a synthetic rubber plant's aerated lagoon.
In the former, a concentration of 0.16 mg/£ was reported. In addition, con-
centrations of 0.027 and 0.049 ppm of benzothiazole have been detected in dis-
charges from a tire plant treatment pond (Niles, 1976).
50
-------
III. Environmental Health Effects
A. Environmental Effects
1. Persistence
a. Biological Degradation, Organisms and Products
Environmental fate of mercaptobenzothiazole and its
derivatives has not been investigated. A great majority of these compounds
are used in small concentrations as additives to various materials; for example,
they are incorporated in rubber, fungicide preparations (e.g., Vancide 51),
antifreeze for motor vehicles, etc. Although the degradation of the materials
to which they are added has been the subject of many studies, the additive
itself has received no attention, presumably because of the low concentrations
involved.
The biocidal properties of mercaptobenzothiazole and its
derivatives and the nonspecificity of their action (see Owens, 1969; Horsfall,
1956) suggest that they will not be attacked by microorganisms, at least
until the concentration has been depleted below critical toxic limits.
2+ 2+
Mercaptobenzothiazole and its derivatives may combine with Ca , Zn , or other
cations and form insoluble and relatively undissociable salts, which will
probably not be easily accessible to the enzyme systems within the microorganisms,
Mercaptobenzothiazole derivatives present in vulcanized rubber may not be
accessible to microorganisms because of the shielding provided by the rubber
matrix.
Thyasen and coworkers (1945) noted that the sulfur in
various organic sulfur compounds commonly "used in vulcanization processes were
51
-------
not susceptible to oxidation by sulfur oxidizing bacteria which converted the
elemental sulfur in rubber to sulfate. The paper does not make it clear,
however, whether mercaptobenzothiazole was one of the organic sulfur compounds
investigated.
In summary, the biodegradability of MET and its derivatives
is unknown.
b. Chemical Degradation in the Environment
The available information on the chemical reactivity of
MET and MET derivatives has been reviewed in Section I-B-2 (p. 16). Very little
information is available on which estimates of chemical stability in the environ-
ment could be based. MET compounds do not readily hydrolyze or oxidize. They
are strong ultraviolet light absorbers at sunlight wavelengths, but no photo-
chemical studies are available. Thus, the chemical stability in the environ-
ment is unknown.
2. Environmental Transport
No experimental work has been reported concerning the environ-
mental transport and behavior of mercaptobenzothiazole and its derivatives.
From the chemical and physical properties of these compounds, we have attempted
to derive some theoretical information on their environmental transport and
behavior. Mercaptobenzothiazoles are, in general, nonvolatile, and thus, it
can be suggested that they will enter and/or distribute in the atmosphere to
only a small extent.
Sodium mercaptobenzothiazole, which is used as an anticorrosive
agent in antifreeze for automobiles, may be converted to relatively insoluble
52
-------
salts of Ca, Zn, etc. upon release to the environment, and thus, the rate of
transport to the atmosphere will be significantly reduced (due to increased
molecular weight, lower volatility, and decreased solubility). At low
environmental concentrations, mercaptobenzothiazoles may be soluble enough to
migrate through soil and eventually make their way to ground water. In fact,
Aktulga (1971a, b) has demonstrated that MET is soluble enough to be leached out
of rubber with distilled water.
3. Bioaccumulation and Biomagnification
Bioaccumulation of a chemical occurs when the chemical is taken
in by an organism faster than it is eliminated. Bioaccumulation potential of a
chemical in an organism can be assessed sometimes from the partition coefficient
of the chemical. Neely and coworkers (1974) have reported a linear relation-
ship between octanol^water partition coefficients and bioconcentration of
chemicals in trout muscle (ratio of the concentration of chemical between
trout muscle and the exposure water measured at equilibrium). Unfortunately,
octanol-water partition coefficients for mercaptobenzothiazoles are not available
in the literature. For benzothiazole (partition'coefficient, 2.02), the bioconcen-
tration factor is calculated to be 16. Since the substitution of the mercapto
group on benzothiazole should increase its water solubility and reduce the
lipid solubility, the bioconcentration factor for mercaptobenzothiazoles will
probably be somewhat lower. Salts of mercaptobenzothiazole (calcium, zinc
mercaptobenzothiazoles, etc.) will be expected to have even a lower solubility
in lipids and, therefore, it is anticipated that they will bioaccumulate to
even a lesser extent. Overall, it appears unlikely that mercaptobenzothia-
zoles and their salts will bioaccumulate to a significant extent in organisms.
53
-------
Biomagnification refers to a chemical's movement through the
food chain resulting in an increase in concentration at each trophic level.
In the absence of the experimental data on biomagnification potential of mer-
captobenzothiazoles, an attempt has been made to predict the biomagnification
potential from the water solubility data. Metcalf and Lu (1973) have found that
the ecological magnification in fish of the chemicals studied in their model
ecosystem, followed a straight line relationship with water solubility. Using
this relationship, the ecological magnification for mercaptobenzothiazole is
calculated to be in the range of 60-70 (for comparison, that of DDT is approxi-
mately 170,000). From this, it appears that some biomagnification of mercapto-
benzothiazole is possible in the environment, although biomagnification to
hazardous levels seems unlikely.
54
-------
B. Biological Effects
1. Toxicity and Clinical Studies in Man
The history of human experience with the adverse effects resulting
from exposure to MET and its derivatives is an account which details numerous
cases of allergic contact dermatitis as the only consequence of exposure.
Several retrospective investigations have established that MET, particularly as
a component in rubber products, is one of the most common contact allergens
known today.
Allergic contact dermatitis is a delayed hypersensitivity reaction
that results from exposure of previously sensitized individuals to an allergenic
chemical or substance. The induction or incubation phase of contact hypersensi-
tivity begins with the initial exposure to a contact allergen when it forms an
antigenic hapten-carrier complex with epidermal proteins (Eisen et^ al., 1952;
Eisen and Tabachnick, 1958; Nakagawa et^ a^., 1971). Sensitization then takes
place in the regional lymph nodes when the antigenic substance combines with
receptor molecules on the surface bf lymphocytes (Cell and Godfrey, 1974).
Sensitized lymphocytes become blast cells and subsequently enter the circulation
and bone marrow to function as memory cells in maintaining the state of contact
sensitivity (Schneider, 1974). The incubation period may last from 5 to 21 days
and may be followed by a "spontaneous" flare-up reaction at the site of exposure,
due to minute concentrations of the allergen which may be remaining in the now
sensitized skin. Re-exposure to the allergen after the incubation period will
cause clinical symptoms of dermatitis which include redness, edema, papules,
vesiculatlon, weeping of the skin, and pruritis (Fisher, 1973). Delayed contact
hypersensitivity reactions involve only the cell-mediated immune System; humoral
55
-------
antibodies do not participate in the response to contact allergens (Catalona
et aj., 1972). Primary irritant dermatitis is distinguished from allergic con-
tact dermatitis in that skin lesions are produced upon the initial application
of the irritating substance, and a state of hypersensitivity is not induced.
An indication of the importance of the MET derivatives as
allergenic components of rubber is offered by Fisher (1973) who concluded that
the five most common causes of allergic contact dermatitis in order of frequency
are as follows:
1. Rhus (poison ivy, oak or sumac)
2. \p_-Phenylehediamine
3. Nickel compounds
4. Rubber compounds
5. Ethylenediamine
The following sections will discuss the large body of evidence
which has accumulated indicating that MBT and several related compounds are a
significant cause of allergic contact dermatitis in human populations. In
addition, the results of numerous animal studies involving various substituted
benzothiazoles will be presented in subsequent sections to demonstrate the
varied and profound pharmacologic actions of this class of compounds , which result
from simple molecular modifications of the basic benzothiazole structure.
a. Epidemiologic and Controlled Human Studies
The potential for human exposure among the general popu-
lation to MBT derivatives is extremely highj based on their widespread use as
components of rubber articles. Several studies have been undertaken to clearly
define the role of benzothiazole derivatives as causative agents in cases of
contact dermatitis.
56
-------
In an early study, Bonnevie and Marcussen (1944) investigated
74 cases of eczema caused by exposure to rubber. Among these patients, 53 (72%)
reacted positively to MET when it was applied to the skin.
Routine patch-testing for sensitivity to rubber chemicals
was conducted on a group of 401 patients who were affected by dermatitis of the
hands and/or feet (Gaul, 1957). Among these, 5 females and 6 males reacted
positively to MET. ,
Blank and Miller (1952) tested rubber adhesives in shoes
as the cause of dermatitis of the feet among a group of 24 patients. In each
of these cases, the wearing of a particular pair of shoes could be correlated
to the history of dermatitis. Patch tests were performed using 10 representa-
tive antioxidants and 17 .accelerators used by the rubber industry. These
materials were tested by application of a 1% concentration in petrolatum of
each chemical to the upper arm, left on the skin for 48 hours. Reactions were
read one hour after removal of the patch, and only vesicular reactions were
recorded as positive. Their results for 21 of these patients are summarized
in Table 12. A summary of the patch test data as presented in Table 13 clearly
reveals that more than half the patients who displayed positive reactions to
MET were also sensitive to a number of other MET type compounds. These data
suggest that cross-sensitizatibn may commonly occur among compounds containing
the mercaptobenzothiazole moiety:
57
-------
Table 12. Patch Test Reactions to Accelerators and Antioxidants* (from Blank and Miller, 1952)
Group I Croup 11 Croup II f Croup IV
Case number ' ' 1- 2- 3 4 5 6 7 8 9 10. :11 12 13 14- 15- 16 17 18 19 20 21
Accelerators - '
Mercaptobenzothiazole type . ' •
... ... r '''. 2-Mfircaptob'enzotniazole "....:• ' •.... :-..."'.' -. -"- •- - - ---•-, +.+ -'.-+ - • - • + + + -
J . . '. - "'- . 2,2'.-Behzothlazyl disulfide . .: .'.:....:' - - -'-^.- - - - - : - + + + - + "-.-.'.+ + --
. .'. '" .:" Zinc benzothiazy.1-sulfide. ............. :.......'.....'.....'..•..:. 0 - -. -- 0 0 - 0 0- -0 ' -i- •(•+.- 0. 0 - 000.0
'••.'' .-' .. • . . Benzothlazyi sulfenamide -.!....'.'.. i;.......,.- .'•-.•-•--.-'-'- -'--'- -"+...+ ..+ - + - r +. . + -
3-Anilinomethyl-2(3)-benzothiozolethi6ne . . . .' '. -'_•-:-------• + + - + + - - + +'•-.+
. Mixed • . • . .
Zinc benzothiazyl. sulfide and tetramethy Ithiuram monosulf ide . . . _______--.-_ + + + -+ + + + + --
A mercaptobenzothiazole and a dithiocarbamate ____•_____- + + + -+ - - + + --
Di thiocarbamyl type • -
Tetramethylthiuram monosulf Ide ..' ...'. - - - - --'- - -•-- - - .+ - -
Tetramethylthiuram disulfide - - - - - - - - -" - -- +
Selenium dimethyl dithiocarbamate - - - - - - - - - -
N-pentamethylene ammonium pentame thylene dithiocarbamate __-_------ _-+ --
oo
Miscellaneous . -
2-Mercaptothiazoline - - - - - - - - - -
Diphenylguanidine
Diphenylguanidine phthalate - - - - - - - - - -
Butylaldehyde-aniline condensation product • -. - - - - ~ ~ - ~ ~ ~ ~-
Triethyltrimethy lenetriamine ______--.-- ___ --
Hexamethylene tetramine '.. - - - - - - - - - ' -
Antioxidants • •
Monobenzyl eiher or hydroquinone - + + - + + + 0 0-0 0 - '- - + + + + + + + +
S-di (S-naphthyl)-p-phenyleneciamine - ---------
Mono and dioctyl dipheny lamines (mixture) - ---------
- Polymerized .trinethyl dlhydrijtj'.:incline - - ----------- - - - - - - - - - - +
Adol alpha nap:-, thvlaminc ..;..-. : •____-_---- __-•- -- - _.__-_
Phenyl beta uaphthylamine • r - - - - - - - - - - -..- - - - . -
Phenyl alpha naph thylamine ---.- ------ _-- -- - - __ + _
Reaction procurt of diphenylamine and acetone --.--- ------ - - - - - - - -- -.+
A diacry lamir.o-ket one-aldeliyvl^ r^accion product and N,N'-di-
ptieny 1-p-ph^ny lenediamine - - - - - - - - - -
Acetor.e and ai-.iiiu*; condensat: *-- product - ---------
Polyalkalatcd phenol sulfide - - - - - - - - - -
*~ = no reacl ;o:'. or ni: Id o rv: •• i ~a; -*- = vesi culation ; 0 = not tested.
r These patients were tested »•rh special shoe linings (see text).
-------
Table 13. Summary of Patch Test Data* (from Blank and Miller, 1952)
Case
No.
Group I
It
2t
3
4
5
6
7
8 ......
9
10
Group II
11
12
13
Group III
14t
15t
16
17
Group IV
18
19
20
21
Group V . .
22
23
24
• '.•• •
Shoes
+
+
+
. . . . . +
0
... 0
. +
......... +
+
. . . . . ;.. . +
+
....;.... +
...... .... +
+
...;..... +
.. .... 0
... .. +
.... +
.....:.;. +
+
..... +
. . +
......... +
Mono-
benzyl
Ether
of Hydro-
quinone
+ .
+
+
+
+
+
0
0
0
0
-
-
-
+
+
+
+
0
0
0
0
_i.
_
-
Mercapto-
benzo- Dithio-
thiazole carbamyl
Type Type Others
- ' '. . -
• '• - - .
_
- • . "
_
_
'' - -
_
.
-
+ (7)
+ (7) -
+ (5). + (5)
+(!)•-
+ (6) -
+ (2)
+ (4)
+ (6)
+ (6) ' -
+ (1) - + (1)
+ (1) - + (2)
— . _
— — *
. -
*- = no reaction or mild erythema; + = vesiculatibn; 0 = not tested
Numbers in parentheses indicate the number of accelerators of the type
indicated to which the patient reacted.
t These patients were tested with special shoe linings.
59
-------
The problem of shoe dermatitis and its relationship to
allergenic chemicals was further studied by Cronin (1966). During the 13 year
period between 1953 and 1966, a total of 100 patients were seen with dermatitis
of the feet caused by allergic reactions to rubber. Of this group, 68 were
women and 32 were men, with their ages ranging from 3 to 75 years. Patch tests
were conducted with pieces of the patient's shoe which corresponded to the areas
of dermatitis and with the rubber chemicals MET and tetramethylthiuram disulfide.
Of the 100 patients tested, 45% were sensitive to MBT, 12% were sensitive to
tetramethylthiuram disulfide, and 37% were sensitive to both. Only 2 patients
in this group reacted to rubber from their shoes without also reacting to one or
both of these rubber chemicals.
A more extensive investigation of dermatitis caused by
rubber was conducted by Wilson (1969) and involved the cases of 106 patients seen
from 1955 to 1967 who developed dermatitis from specific rubber articles such
as household gloves, condoms, footwear, and girdles. These patients, 76 women
and 30 men, developed sensitivity reactions to rubber at various ages (Figure 6)
and presented a length of history which varied from one or two weeks to as long
as 30 years.
25
20
co
Z
uj
S15
Q.
U.
0
z 10
UJ
U
a
UJ
"• 5
0
—
—
-
-
—
rif
«_
—
••
—
-
^^
-
-
Tl
5 10 IS 20 26 30 3540 45 50 55 60 66 7075
AGE OF ONSET OF SENSITIVITY TO RUBBER
Figure 6. Distribution in Age of Onset of Rubber Sensitivity (from Wilson, 1969)
60
-------
Seventy patients were sensitized by specific rubber articles;
A3 to gloves, 9 to boots and shoes, 9 to condoms, 7 to girdles, brassieres and
suspenders, and one each to bathing hats and elastic bands. The remaining 36
patients developed dermatitis from more than one rubber article and it was un-
certain which was originally responsible. These patients were grouped according
to the article which produced dermatitis and were then patch tested against
several rubber accelerators. The substances tested were dipentamethylenethiuram
disulfide (PTD), tetramethylthiuram disulfide (TMT), MET, and zinc diethyldi-
thiocarbamate (ZDC). All chemicals were applied to the skin as a 1% concentra-
tion in soft paraffin and read after 48 hours. The results of these tests are
presented in Table 14. Forty-five patients reacted to a single chemical only;
26 to MET and 19 to PTD. In addition, 8 patients reacted to MET, PTD, and TMT.
All 9 patients with dermatitis from boots were reactive to MET. Sixty-eight
percent of the tests were positive to PTD, 54% to TMT, 47% to MET and 24% to
ZDC.
In other studies on dermatitis associated with specific
rubber articles, a large percentage of allergic dermatitis cases have been associ-
ated with MET. In a study of shoe dermatitis, Calnan and Sarkany (1959) pre-
sented 37 rubber sensitive patients, 19 of whom reacted to MET. Hindson (1966)
studied 30 patients that reacted to rubber condoms. Of these, 6 were found to
react to MET.
A major survey of contact allergens in the United States
has recently been conducted (Baer et^ al., 1973) which provides convincing evi-
dence that MET is indeed one of the most common and effective dermatitis-
producing substances in use today. This report listed contact allergens which
61
-------
Table 14. Patch Tests with Rubber Accelerators (from Wilson, 1969)
Gloves Boots Condoms Girdles Mixed Total
PTD
+
Total
TMT
^ Total
K>
MET
ZDC
Total
No.
34
9
43
19
23
42
13
29
42
4
22
%
79
21
45
55
30
70
15
85
No.
3
6
9
2
7
9
9
0
9
2
4
%
33
66
22
77
100
0
33
66
No.
9
0
9
4
5
9
1
8
9
1
5
%
100
0
44
55
11
89
17
83
No.
4
3
7
6
0
6
4
2
6
3
1
%
57
43
100
0
66
33,
75
25
No.
23
13
36
22
14
36
19
17
36
7
23
%
70
30
61
39
53
47
23
77
No.
73
31
104
53
49
102
46
56
102
17
55
%
68
31
52
48
45
55
24
76
Total 26 6 64 30 72
-------
elicited positive reactions in more than 3% of 200 or more patients tested
over the period from 1968 to 1970. Almost all test subjects were suffering
from some type of skin disease, and, therefore, the subjects were a selected
group rather than a representative sample of the general population. In the
case of MET and several other chemicals, two groups of patients were selected
before testing. One group was suspected of having contact dermatitis due to
shoes or rubber, and, therefore, that group was tested with the "shoe and
rubber tray." The second group displayed clinical signs and histories which
did not specifically indicate shoe or rubber allergy, and, therefore, the
group was tested with the "diagnostic tray." Table 15 presents the percent of
patients which reacted to each of the allergens tested. In terms of overall
number of reactions, MBT ranked second among the 24 most common contact allergens.
The intensity of reactions to the allergens tested ranged from weak (1+ or 2+)
to moderate (3+) and severe (4+). The distribution of intensities of skin reac-
tions elicited with the contact allergens is presented in Table 16.
Cross-Sensitivity
With the high incidence of allergic skin reactions due
to MBT exposure having been established by the studies discussed above, the
importance of cross-sensitivity reactions to other benzothiazole derivatives is
clearly a significant problem. Fregert (1969) has undertaken to identify cross-
reacting substances in MBT-sensitized patients. Cross-sensitivity patterns were
investigated in 12 patients, 8 women and 4 men, with contact sensitivity to rubber
gloves. Allergy to MBT was established by patch testing with 2% commercial grade
MBT in petrolatum. Eleven test substances, listed in Table 17, were applied in
a 2% concentration to the back of each patient for 48 hours, and the reaction
63
-------
Table 15. Percent of Patients Reacting to Contact Allergens (from Baer £t al. , 1973)
Allergens
Mercury bichloride, 0.05% aqueous solution
Mercaptobenzothiazole , 1% in petrolatum
Shoe and rubber tray
Diagnostic tray
Paraphenylenediamine, 2% in petrolatum
Diagnostic tray
Shoe and rubber tray
Potassium dichromate, 0.5% aqueous solution
Shoe and rubber tray
Diagnostic tray
Ethylenediamine, 1% in petrolatum
Nickel sulfate, 5% aqueous solution
Diagnostic tray
Shoe and rubber tray
Turpentine, 10% in olive oil
2% aqueous formaldehyde solution
Shoe and rubber tray
Diagnostic tray
Poison ivy oleoresin, in acetone
Bismarck brown
Thiram, 1% in petrolatum
Diphenylguanidine, 0.5% in petrolatum
Peruvian balsam, 25% in petrolatum
Resorcin, 5% aqueous solution
Monobenzone, 2.5% in petrolatum
Shoe and rubber tray
Diagnostic tray
Epoxy resin, 1% in petrolatum
Ethylaminobenzoate, 5% in petrolatum
Neomycin sulfate, 20% aqueous solution
Chrysoidine brown
Epoxy hardener, 1% in petrolatum
Acrylic monomer, 25% in olive oil
2-naphthyl benzoate, 1% in petrolatum
Hexachlorophene, 1% in petrolatum
Pyre thrum
No. of Patients
Tested
540
229
540
540
229
229
540
158
540
229
540
229
540
340
229
229
229 .
340
340
229
540
340
540
540
229
340
340
229
340
540
% Reacting
22.2
18.8
7.8-
13.5
7.0
13.5
9.8
13.2
13.1
8.3
12.2 .
11.8
8.7
11.2
10.5
7.9
7.9
7.9
7.9
7.0
2.6
5.6
5.2
5.2
4.8
4.4
4.4
3.5
3.2
3.1
64
-------
Table 16. Degree of Reaction to Contact Allergens (from Baer £t al., 1973)
Allergens
No. of Patients Reacting
1+ 2+ 3+ 4+
Mercury bichloride, 0.05% aqueous solution
Mercaptobenzothiazole, 1% in petrolatum
Shoe and rubber tray
Diagnostic tray
Paraphenylenediamine, 2% in petrolatum
Diagnostic tray
Shoe and rubber tray
Potassium dichromate, 0.5% aqueous solution
Shoe and rubber tray
Diagnostic tray
Ethylenediamine, 1% in petrolatum
Nickel sulfate, 5% aqueous solution
Diagnostic tray
Shoe and rubber tray
Turpentine, 10% in olive oil
2% aqueous formaldehyde solution
Shoe and rubber tray
Diagnostic tray
Poison ivy oleoresin in acetone
Bismarck brown
Thiram, 1% in petrolatum
Diphenylguanidine, 0.5% in petrolatum
Peruvian balsam, 25% in petrolatum
Resorcin, 5% aqueous solution
Monobenzone, 2.5% in petrolatum
Shoe and. rubber tray
Diagnostic tray
Epoxy resin, 1% in petrolatum
Ethylaminobenzoate, 5% in petrolatum
Neomycin sulfate 20% aqueous solution
Chrysoidine brown
Epoxy hardener, 1% in petrolatum
Acrylic monomer, 25% in olive oil
2-naphthyl benzoate, 1% in petrolatup
Hexachlorophene , 1% in petrolatum
Pyre thrum
65
10
8
12
1
8
15
1
16
3
29
16
28
9
' 19
5
12
13
15
10
8
5
9
6
2
6
9
2
4
7
32
11
20
24
3
10
18
4
13
7
21
5'
12
6
4
2
5
12
8
5
5
8
10
10
5
5
2
2
5
7
23
18
13
26
12
10
18
12
31
6
16
5
6
19
1
8
1
2
3
'1
1
6
8
8
4
4
3
4
2
2
0
4
1
11
0
3
2
4
11
3
0
1
1
4
0
3
0
0
1
0
0
0
1
4
• 0
0
1
0
0
1
65
-------
was read 24 hours later. A reaction producing infiltration and/or vesicles or
papules was scored as positive. Each of the test substances was also applied in
the same manner to non-sensitized control subjects as a test for primary irritant
action. All the controls produced negative results. Results of the patch tests
for cross-sensitivity are presented in Table 18. Four chemically related MET
derivatives produced positive reactions in all of the patients tested. No varia-
tions were observed in this pattern of sensitivity.
An examination of the results presented in Table 18
indicates the importance of both the thiol group in the 2-position and the integ-
rity of the benzothiazole structure as determinants in producing cross-sensitizing
reactions to MET.
Table .17. Test Substances and Their Occurrences (from Fregert, 1969)
1. 2-Mercaptobenzothiazole; used as rubber accelerator and retarder
as well as anti-rust agent.
Trade names: Vulcafor MBT, Merkapto, Rotex, Captax, MBT, etc.
2. Zinc-2-tnercaptobenzothlazole; used as rubber accelerator and
ahtloxldant.
Trade names: Bantex, MBT, Vulkacit ZM, Zenite special, Zetax, etc.
3. Di-benzothiazyl-disulphide; 2,2'-dithiobisbenzothiazole; used
as a rubber accelerator and retarder.
Trade names: Altax, MBTS, Vulcafor. MBTS, Vulcacit DM, etc.
4. Benzothiazole; not used in rubber.
5. 2-Methylbenzothiazole; not used in rubber.
6. 2-Aminobenzothiazole; not used in rubber.
7. 2-Benzothiazyl-N,N-diethylthiocarbamyl sulfide; used as
rubber accelerator.
Trade name: Ethylac .
8. N,N-Diethyl-2-benzothiazolesulfenamide; used as rubber
accelerator.
Trade name: Vulkazit AZ
9. 2-Mercaptobenzoxazole; not used in rubber.
10. 2-Mercaptobenzimidazole; ethylene-thiourea; used as rubber
accelerator.
Trade names: Na-22, Vulkacit NPV .
11. 2-Mercaptothiazoline; used as rubber accelerator.
Trade name: Accelerator 2-MT
66
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Table 18. Patch Test Reactions in Twelve Patients (firom Fregert, 1969)
Substance
No.
Formula
Reaction
10
11
-SH
.--**\
Q-
^S/
67
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Human Assay of Contact Allergens
A considerable amount of work has been performed in
order to improve the standard methods of detecting contact allergens. Kligman
(1966a) has pointed out that major deficiencies exist in the standard predic- ;
tive tests used to assay moderately strong contact allergens. Notably, retro-
spective testing of known allergens resulted in high insensitivity to substances
of weak and moderate sensitizing capabilities. These testing methods, therefore, '
would be expected to fail as a means of screening potential allergens of moderate
strength.
As an alternative means of identifying contact allergens
by human assay, Kligman (1966b,c) has developed specialized procedures designed
to be free of producing false negative results. This so-called "maximization
test" involves an induction phase whereby the test site is treated 24 hours with
an occlusive patch of 1.0 ml of 5% aqueous sodium lauryl sulfate (SLS). This
procedure causes a mild inflammatory reaction which promotes sensitization. To
the same site, a 48 hour occlusive patch is then applied which contains the test
material. This sequence is repeated five times, alternating 24 hour irritant
patches and 48 hour allergen patches. Following the induction period, a challenge
test is performed which consists of pre-treating the skin occlusively with 0.4 ml
of 10% SLS for one hour and followed by occlusive application of the test sub-
stance for 48 hours. This procedure is referred to as the "SLS provocative test."
When MET was tested on healthy adult subjects according to Kligman's maximization
procedure, a high rate of sensitization was achieved. Table 19 lists these
results together with those obtained by testing of various industrial chemicals.
The sensitization grade of 3 for MET indicates that a moderate skin reaction was
68
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Table 19. Industrial Contactants (from Kligman, 1966c)
Benzene
Xylene
Pyridine
Dimethylsulfoxide (DMSO)
Hexane
Chloronaphthalene
Aniline
Mercaptobenzothiazole
Nickel sulfate
Chromium trioxide
Chromium sulfate
Cobaltous sulfate
Gold chloride
Turpentine '
Butylglycidyl ether
Beryllium sulfate
2-amino-5-diethyl-
aminotoluene HC1
Potassium dichromate
Phenyl mercuric nitrate
Thioglycerol
Diethylenetriamine
Diethylfumarate
n-Butylthiomalate /c\
Technical Malathion
Glyoxal
Krameria (Extract)
Expoxy resin
Hydrazine
Induc-
tion
concen-
tration
(%)*
50
100
50
75
100
25
20
25*
10
3
25
25
2
50
10
5
25*
2
2
50
10
1.0
5.0
25*
10
25*
25
5
Challenge
concen-
tration
(%)**
20
25
10
25
25
10
10
10**
2.5
0.5
2.0
2.5
0.005
20
10
1.0
10**
0.25
0.5
5
10
0.2
1.0
10**
2.0
10**
15
0.5
Sensiti-
zation
rate
0/25
0/24
1/24
0/23
0/25
0/25
7/25
9/24
12/25
13/23
11/23
10/25
16/23
18/25
19/24
18/22
19/25
23/23
24/25
24/24
24/25
25/25
22/25
25/25
24/24
22/22
21/25
23/23
Sensiti-
zation
grade
1
1
1
1
1
. 1
2
3
3
3
3
4
4
4
4
4
4
. 5
5
5
5
5
5
5
5
5
5
5
**
Pre-treatment of skin with SLS
SLS provocative test
69
-------
produced. The reaction grading was placed on a scale from 1 to 5 with increasing
numbers corresponding to weak, mild, moderate, strong and extreme reactions, re-
spectively. Kligman (1966c) noted that 90% of the test subjects used in this
study were Negroes, who are less susceptible to inflammatory skin reactions.
The author predicted, therefore, that an even higher rate of sensitization might
be achieved among white subjects using the same test procedures.
b. Occupational Studies
There has been comparatively little evidence of occupational
disease which can be attributed solely to contact with MET or its derivatives.
Undoubtedly, much of the reason for the lack of occupational poisonings is due
to modern hygiene practices and well-equipped manufacturing facilities. Never-
theless, several surveys of health problems in the rubber industry have revealed
that occupational diseases are not uncommon, and MET derivatives may very well
play a role in contributing to adverse health effects.
In a report on allergy in the rubber industry, Wilson ,et_ .al_. -
(1959) noted that occupational dermatitis was a common occurrence, being referred
to as "rubber itch" and "rubber poisoning." At that time, contact with chemicals
was regarded as being responsible for 90% of all occupational allergies in the
rubber industry. These chemicals would include, in addition to the benzothiazole-
type accelerators, a number of antioxidants, softening oils, phenol-formaldehyde
resins and epoxy resins.
A study which demonstrated that MBT was specifically responsi-
ble for causing occupational dermatitis has been conducted by Herrmann and Schulz
(1960). Between 1953 and 1958, 8,000 patients with contact dermatitis were seen,
70
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among which 63 cases were due to rubber. Of these, 29 were occupational in
origin, and 16 occurred in the rubber industry. Patch testing was conducted
on these 16 patients with a number of industrial and rubber chemicals to determine
the specific allergen involved (Table 20). In most cases, derivatives of MET
were the most common causative agents.
In a recent European study (Oleffe et al., 1972) 300
patients suspected of having occupational contact dermatitis were exposed by
patch tests to 20 allergenic chemicals, including MET. The group consisted of
184 men (61%) and 116 women (39%). Among these patients, 4 men (2.2%) and 5
women (4.3%) displayed positive reactions to MBT. With men and women combined
the number of persons with positive reactions to MBT was 3% of the total number
of subjects.
Product safety data sheets supplied by Uniroyal Chemical
(Uniroyal Chemical, 1971a,b,c,d) provide statements of the medical history in
the manufacture of several MBT derivatives. A record of no adverse history
based on over 20 years of production and use was claimed for 2,2'-dithiobisbenzo-
thiazole and zinc MBT. No history of medical problems was encountered based
on several years of production and use of N-cyclohexyl-2-benzothiazolesulfenamidei
and N-tert-butyl-2-benzothiazolesulfenamide..
c. Non-Occupational Exposures and Poisoning Incidents
The great majority of exposures in the general population
to derivatives of MBT undoubtedly occur by contact with clothing and rubber
articles which contain these substances. Reported incidents of adverse effects
resulting from such contact have been limited exclusively to the development of
allergic contact dermatitis. Individual cases have involved dermatitis resulting
71
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Table 20. Positive Skin Reactions in 16 Patients with Rubber Allergy
(data from Herrmann and Schulz, 1960)
No. of positive
Substance . reactions
2-Mercaptobenzothiazole (MET) 10
2,2'-Dithiobisbenzothiazole (MBTS) 4
N-Cyclohexyl-2-benzothiazolesulfenamide (CBS) 6
Mixture containing:
72% MBTS
13% Diphenylguanidine 5
15% Hexamethylenetetramine
Tetramethylthiuram disulfide 4
Diphenylguanidine 6
j>-Tolylbiguanidine 5
Hexamehylene tetramine
p_-Phenylenediamine 3
jp-Nitrosodimethylaniline -
N-Phenyl-N'-cyclohexyl-p-phenylenediamine 3
Phenyl-3-naphthylamine 2
Condensation product of acetaldehyde
and a-naphthylamine 5
4,4'-Dioxydiphenyl 2
72
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from rubber accelerators in shoes and sneakers, bathing caps, condoms, gloves
and rubber in elastic linings (Jordan, 1972).
Women are considered to be particularly vulnerable to MBT-
induced allergies due to their high degree of exposure to items containing rubber.
Bauer (1972) reported that MET may cause dermatitis in women who are in contact
with it as a component in girdles, brassieres, "falsies," elastic in underpants
and hair nets, boots, shoes, and dress shields.
Porter and Sommer (1967) described the cases of 5 women who
developed allergic contact dermatitis which corresponded to certain portions of
their brassieres. Patch tests were performed on each patient using various sub-
stances including 1% MET in petrolatum. In every case the patients were sensi-
tive to MET and to spandex, a polyurethane elastomer used in making brassieres.
A case of severe contact dermatitis was presented recently
by Epstein (1973). In this incident, a 14 year old boy developed severe derma-
titis on his feet which spread within a few months' time to involve his hands,
forearms, and legs as well. Two years later, after numerous treatments had
failed to improve his condition, the boy developed contact dermatitis under the
elastic waistband of a new pair of undershorts. Subsequent patch testing revealed
a positive sensitivity to the elastic waistband, his sneakers, another pair of
shoes, and MET. The avoidance of shoes containing rubber caused a dramatic improve-
ment in his condition. Although severe reactions to rubber, and MET in particular,
are not very common, this case emphasizes the importance of proper diagnosis in
recognizing a potentially dangerous allergy to MET.
The use of rubber products in the course of dental practice
has resulted in rubber dermatitis in the form of contact stomatitis in the mouth.
73
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Everett and Hice (1974) have presented the case of a 16 year old girl with full
orthodontic appliances, including intermaxillary rubber bands, who developed an
acute redness and swelling of both checks. Prompt recovery was achieved when
the rubber bands were removed from her mouth.
The literature has clearly indicated that MBT and other
related rubber accelerator chemicals are a threat to the general public insofar
as the development of contact dermatitis is concerned. Reports have not been
encountered, however, which offer any evidence whatsoever that benzothiazole
derivatives have caused a single human fatality or have been identified as the
causative agent of a disease other than allergic contact dermatitis. This obser-
vation in itself is remarkable in light of the data presented in reports of vari-
ous animal studies (see following sections) which demonstrate that many benzo-
thiazole derivatives elicit potent pharmacological effects. It should be noted,
however, that potential environmental exposures to MBT and its derivatives are
not likely to be sufficiently high to produce dramatic symptoms of acute toxicity.
2. Effects on Non-Human Mammals
A sizable body of data exists describing the abundance of
physiologic actions that are produced by the introduction of benzothiazole and
its derivatives into different animal systems. While many of these compounds
are not commercially important and present little environmental contamination
potential per se, they are nevertheless good examples of highly active substances
which can be derived by simple chemical substitution. Therefore, biological
data will be presented and discussed where the activity of a specific substituted
benzothiazole may be indicative of the properties of an entire group for which
data is lacking.
74
-------
Furthermore, it is not unreasonable to assume that highly toxic,.
but commercially unimportant, benzothiazole derivatives may be produced from
less toxic but more abundant substances by reaction with other environmental
chemicals. In addition, biological and non-biological oxidation, reduction and
hydrolysis reactions can significantly alter the form of a substance to which
organisms are exposed. Therefore, a cautious extrapolation of the data presented
in the following sections will, in many cases, be the best means presently availa-
ble to predict the potential harm that may result from indiscriminate exposure
to these substances.
a. Absorption Studies .
Specific studies have not been encountered which measured
either the routes or rates of absorption for compounds of the benzothiazole
class. It is not possible, therefore, to predict the toxic hazard posed by these
substances when administered by various routes, and this is suggestive of the
need for further research.
It is well-known that the relationship between toxicity and
mode of absorption for any foreign compound is largely dependent upon its molecu-
lar structure, lipid solubility, degree of ionization, and vapor pressure. Un-
fortunately, data are not available to indicate the extent of ionization of MET
and its derivatives at the various pH values along the gastrointestinal tract.
Furthermore, the fact that most MET derivatives are readily organosoluble (i.e.,
lipid soluble) is not sufficient to predict their ease of absorption by oral and
dermal routes.
With respect to the benzothiazoles used in industrial
solutions, however, the route leading to the greatest absorption is most likely
to be passage of inhaled dust through the alveoli of the lungs. In contrast to
the skin and the gastrointestinal lining, the pulmonary epithelium appears to
75
-------
be highly permeable not only to lipid-soluble molecules but also to large lipid-
insoluble molecules and ions (Enna and Schanker, 1959).
The processes of distribution and storage subsequent to
absorption of a benzothiazole compound are largely unknown. In one study (Lehman,
1965) rats were fed a mixture of MET (2.7%) and dimethyldithiocarbamate (27.6%)
at 10,000 ppm in the diet for one week. A bacteriostatic assay method sensitive
to 50 ug/g of tissue was employed to detect concentrations of these compounds in
various body tissues. Storage of MET could not be demonstrated in the liver,
kidney or spleen. This study did not attempt to define, however, the extent of
storage in the fat, which is an important consideration with lipid-soluble mole-
cules such as MBT.
b. Metabolism Studies
The biotransformation of MBT in the rat has been investigated
by Colucci and Buyske (1965). Adult male rats were administered 50 mg/kg of
2-35S-MBT by intraperitoneal injection and the urine from these animals collec-
ted quantitatively for up to two weeks. An analysis of urinary metabolites re-
vealed: (1) the presence of MBT with the same specific radioactivity as the dosed
compound, (2) benzothiazole-2-mercaptoglucuronide with the same specific radio-
activity as the dosed compound, (3) benzothiazole-2-mercaptoglucuronide with a
slightly lower specific radioactivity than the parent compound, and (4) non-
radioactive benzothiazole-2-mercapturic acid. Radioactive inorganic sulfate
was also found in the urine.
The excretion of a non-radioactive metabolite of MBT
immediately suggested the existence of a mechanism whereby the radioactive mer-
capto sulfur is detached from MBT, an idea which was supported by the presence
76
-------
of radiolabelled inorganic sulfur in the urine. The authors postulated a mechan-
ism whereby glutathione or cysteine acted as a displacing agent which attached
to the benzothiazole structure, a reaction which would lead to the formation of
a mercapturic acid conjugate.
The presence in urine of berizothiazole-2-mercaptoglucuronide
with lower specific activity than the parent compound was consistent with the
authors' theory that a dilution of radioactive MET with non-radioactive MET
would occur by the following reaction series:
R_35SH _
R-S-glutathione
R-SH
R-S-glucuronide
An overall diagrammatic representation of the metabolic
fate of MET is presented in Figure 7. It should be noted that when the same in
vivo studies were performed in the rabbit and dog, the results were similar to
those found in the rat study (Colucci and Buyske, 1965).
HH,
U H | 2
,,-C-KCO(CH ),-C-COOH
/ I ' '
' I H
V-S-CH,
Benzochlazole-2 Glutathione
2-Mercaptobenzothlazole
B«nzothiazole-2' Cyateine
Inorganic
Sulfate
B*n>ochl*tol«-2 HercapLoglucuronide
Figure 7. Pathway for the Metabolic Transformation of 2-Mercaptcbenzothiazole
in the Rat, Rabbit, and Dog (data from Colucci and Buyske, 1965)
77
-------
Colucci and Buyske (1965) were able to show further that
2-benzothiazolesulfonamide was extensively metabolized in the rat, rabbit, and
dog by the same mechanism as for MET. When 2-35S-labelled 2-benzothiazole-
sulfonamide (100 mg/kg) was administered to the rat by intraperitoneal injection,
three urinary metabolites were isolated and identified: (1) MET, (2) benzothiazole-
2-mercapturic acid, and (3) benzothiazole-2-mercaptoglucuronide. Surprisingly,
none of the metabolites were radioactive; all of the radioactivity in the urine
was due to radioactive inorganic sulfate. Similar results were also obtained
in the rabbit and dog. From this data, it was apparent that the sulfonamide
group was completely cleaved from the benzothiazole moiety. As with MET:,
the thiol group of glutathione or cysteine could be the origin of the sulfur
atom at the 2-position of the benzothiazole-containing metabolites recovered in
the urine. This conclusion was supported by investigating the metabolism in the
rat of 2-35S-benzothiazole-2-glutathione. Isolation of urinary metabolites re-
vealed that the same metabolites were produced as when 2-benzothiazolesulfonamide
was given, except that all three metabolites contained radioactive sulfur at the
2-position. Furthermore, administration of benzothiazole-2-cysteine led to the
formation of benzothiazole-2-mercapturic acid, benzothiazole-2-mercaptoglucuronide,
and MET as a minor metabolite, ^n vitro studies confirmed that the rat is capable
of enzymatlcally cleaving the sulfur-carbon bond of this conjugate to form MET.
Therefore, the metabolic scheme as pictured in Figure 7 also applies to the
sulfonamide derivative of benzothiazole.
If one extrapolates these results to other benzothiazole
derivatives having a sulfur linkage at the 2-position, it is possible that many
of the commonly used rubber accelerators may also be metabolized in the manner
portrayed by Figure 7. Considering the data which have been presented, it is
78
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not likely that MBT-type compounds will be metabolically transformed into sub-
stances of potentially high toxicity. These results should not be directly ex-
trapolated, however, to benzothiazole derivatives not having a 2-position sulfur
linkage; especially since S-glucuronide formation, although it occurs with MET,
is a relatively uncommon metabolic reaction (Miettinen and Leskinen, 1970).
c. Pharmacology
Benzothiazole and its various substituted derivatives possess
distinct pharmacologic properties which are somewhat varied and highly structure-
specific. Many of these substances were synthesized as potential chemothera-
i
peutic agents for various uses such as antimicrobial drugs, anti-inflammatory
agents, and diuretics. An examination of the data obtained in screening numerous
benzothiazole derivatives for pharmacologic activity reveals a broad spectrum of
biological activity, concerned mainly with actions on the central nervous system
and effects on enzyme function.
Neurologic Effects
A detailed investigation of the pharmacologic properties
of several benzothiazoles was undertaken by Domino et al. (1952) after it was
revealed that the benzimidazoles, a structurally-related class, could produce a
reversible flaccid paralysis in various animal species. Their examination of
the benzothiazoles began with a determination of the paralyzing potency of several
benzothiazoles upon their intravenous administration to mice. Median paralyzing
doses (PD5g) and standard errors were calculated for these and several other
chemicals described by the generic term of benzazoles (Figure 8).
79
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Benzimidazole X = NH Y = CH
Benzothiazole X = S, Y = CH
Benzoxazole X = 0, Y = CH
Benzotriazole X = NH Y = N
Figure 8. Structural Formula of a Benzazole (from Domino et^ al., 1952)
A comparison is made in Table 21 of the relative paralyzing activities of
these compounds.
Table 21\ Median Paralyzing Doses of Substituted Benzazoles on Intravenous
Administration to White Mice (Domino et al., 1952)
All doses are given In mgn/kga + standard error. Values without standard
errors are approximate.
BENZIMIDAZOLES
BENZOTHIAZOLES
BENZOXAZOLES
BENZOTRIAZOLES
PD
50
PD
50
PD
50
PD
50
NHCH
86 + 12
50
50
Convulsions
Convulsions
75
68+8
74+7
20+1
25 + 2
81+2
167 + 20
5A + 2
55 + 3
230
50
80
-------
Administration of benzothiazole was reported to cause an immediate increase in
animal activity, such as running, hopping, and squeaking, which was followed in
a few seconds by progressive ataxia leading to a loss of the righting reflex.
A semi-flaccid paralysis ensued which began in the hind limbs and was reversible
at non-lethal doses. In addition, respiratory depression was produced at para-
lyzing doses, a phenomenon which was also caused by benzoxazole but not benzi-
midazole or benzotriazole. 2-Methylbenzothiazole also caused initial running
movements and respiratory depression during the paralysis period. The 2-hydroxy
and 2-phenyl derivatives of benzothiazole were administered orally, due to their
insolubility, and produced paralysis at doses of 1,000 and 1,500 mg/kg of body
weight, respectively.
The 2-amino substitution of benzothiazole produced a
compound with the greatest paralyzing potency of any substance tested. A step-
wise replacement of the amino group hydrogens by methyl groups caused a progres-
sive reduction in paralyzing activity, but these derivatives were capable of pro-
ducing respiratory depression, as well as clonic convulsions.
Overall, it appears that the benzothiazoles are capable
of causing selective actions on the central nervous system which may result in
either stimulation or depression. These effects vary considerably depending on
the species of animal, the route of administration, and the stereo-specific addi-
tion of substituents to the molecule. For example, most of the substituted
2-aminobenzothiazoles were depressants in mice and rabbits, while in dogs the
4-position derivatives were stimulants and the 7-position derivative was a con-
vulsant. Domino and coworkers (1952) noted that the pharmacologic effects of
these compounds were related to structure, such that methyl or chloro groups
81
-------
substituted in the benzene ring of the 2-aminobenzothiazole produced depression
according to the following order: 6>5>4>7. 6-Methyl-2-aminobenzothiazole was
found to be remarkably free of any stimulating effects, whereas the 7-substituted
derivatives could produce a high degree of stimulation*
Effects on Enzymes In Vivo
The ability of MET to act as a chelating agent has
been linked to the observation that MET causes an inhibition of the oxidative
conversion of dopatnine to noradrenaline. Noradrenaline (norepinephrine, levar-
tarinol) is a neurohumoral transmitter released from sympathetic postganglionic
nerves (adrenergic nerves); Norepinephrine is synthesized in adrenergic nerves
from the amino acid tyrosine by the following sequence:
TYROSINE
tyrosine hydroxylase
DOPA
(dihydroxyphenylalanine)
dopa decarboxylase
DOPAMINE
(dihydroxyphenylamine)
dopamine (J-hydroxylase
>
NORADRENALIN
Adrenergic nerves supply stimuli primarily to the smooth muscle of the heart,
blood vessels, lungs, gastrointestinal tract, urinary bladder, and other organs,
82
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In addition, adrenergic receptors are located on arterioles in skeletal muscle.
Johnson et al. (1970) recognized that the role of cupric
ions was critical to the activity of dopamine B-hydroxylase, an enzyme which cata-
lyzes the conversion of dopamine to noradrenaline. This enzyme, therefore, would
be subject to inactivation by copper chelating agents, one of which is MET.
Johnson and his coworkers (1970) administered MET at 300 mg/kg by intraperitoneal
injection to mice and measured its effect on catecholamine levels in the brain.
As indicated in Table 22, MET reduced noradrenaline to about 60% of control
levels when measured after 1 and 2 hours. Dopamine levels, on the other hand,
were elevated by 24% at 2 hours. After 4 hours, both noradrenaline and dopamine
levels had returned to normal.
Table 22. Mouse Brain Catecholamine Levels 1, 2, and 4 h After 2-Mercaptoben-
zothiazole (MET), 300 mg/kg, i.p. (from Johnson et al., 1970)
All values are expressed as yg/g wet weight whole brain tissue
and are the average of at least three determinations + s.e.
Diluent
MET
*I = P<0
= P<0
treated controls
1 h
2-h
4 h
.01
.05
Noradrenaline
0.43 + 0.03
0.25 + 0.01*
0.27 + 0.02*
0.42 + 0.00
Dopamine
0.78 + 0.06
0.86 +0.02
0.96 + 0.03**
0.81 + 0.02
83
-------
The MET treatment quickly produced signs of extreme depression in the mice,
which was accompanied by marked ptosis (drooping of the eyelids) after 2 hours.
Symptoms of overt depression subsided as catecholamine levels returned to normal.
A further study was made in rats to determine the effects
of MET on the repletion of myocardial noradrenaline from exogenous dopamine
after the depletion of noradrenaline stores by injection of metaraminol bitar-
trate. Rats were treated with metaraminol (5 mg/kg) by intraperitoneal injec-
tion to deplete noradrenaline stores in the heart, and followed 18 hours later
by intraperitoneal injection with 300 mg/kg of MET. Thirty minutes after MET
treatment, dopamine was administered at 35 mg/kg, and three hours later the rats
were sacrificed. As indicated in Table 23, exogenous dopamine restored myo-
cardial noradrenaline concentrations to 60% of the control level. MET, however,
totally blocked the conversion of endogenous and exogenous dopamine to newly
synthesized noradrenaline.
Table 23. Effect of 2-Mercaptobenzothiazole (MBT) on the Repletion of Rat
Myocardial Noradrenaline from Exogenous Dopamine After Its Depletion
with Metaraminol (from Johnson et al., 1970)
Rats were pre treated with metaraminol bitartrate, 5 mg/kg,
18 h before each received MBT, 300 mg/kg, or diluent. Dopamine
hydrochloride, 35 mg/kg, or diluent was administered 30 rain
later and all rats were killed 3 h later. All values are
expressed as pg/g and are the average of at least three
determinations + s.e.
~~~ '. Myocardial
noradrenaline
Diluent treated controls 0.88 + 0.05
Metaraminol 0.15+0.01
Metaraminol 4- dopamine 0.52 + 0.09*
Metaraminol + MBT 0.18 +0.03
Metaraminol + MBT + dopamine 0.17+0.02
*
Significantly different from each of the other values, P<0.05.
84
-------
When Johnson and his coworkers measured the effect of
MBT on spontaneous motor activity in mice (Figure 9), their results coincided
with the previously observed time-course of depletion of brain noradrenaline
stores. Treated mice displayed almost no exploratory activity from the time they
received MBT, as determined in an actophotometer cage. Spontaneous activity in-
creased in treated mice after two hours, and coincided with the time of termina-
tion of dopamine 3-hydroxylase inhibition in the brain.
I200|
c
e •
o
m
3" lOOOl
400
200
01231
Time (h)
Figure 9. Effect of 2-Mercaptobenzothiazole, 300 mg/kg i.p., Upon Spontaneous
Motor Activity in Mice. Each point represents (the average 30 min.
activity for six pairs of mice + s.c. O > control;
A , MBT. (from Johnson ^t al., 1970)
(Reprinted with permission from the Pharmaceutical Society of
Great Britain)
Numerous derivatives of benzothiazole were tested by
Wattenberg et^ al. (1968) for their ability to induce the increased activity of
benzopyrene hydroxylase, an enzyme which has the capacity to metabolize foreign
compounds. The liver is thought to be highly responsive to inducers and is
generally used as a standard tissue for studying induction of increased activity
in microsomal enzyme systems. Wattenburg and coworkers (1968) measured benzo-
pyrene hydroxylase activity in the liver and also in the lung after treating rats
85
-------
by oral intubation with 0.1, mmole of one of 33 benzothiazole derivatives (Table 24),
A comparison of enzyme activity in the liver and lung offers an indication of
the possibility for selective induction in specific tissues other than the
liver.
The data presented in Table 24 clearly indicate that
MET is not an inducer of increased benzopyrene hydroxylase activity in either
the liver or lung. On the other hand, benzothiazole and several substituted
derivatives produced significant inducing effects. The greatest degree of enzyme
activity resulted from treatment with a 2-phenylbenzothiazole, especially those
with certain halogen substitutions (i.e., chloro, bromo, and iodo, but not fluoro)
in the 4'-phenyl position. As pointed out by the authors, the significance of
any compound which markedly increases liver microsomal enzyme activity is that
a potentially high number of undesirable side reactions may also be induced in
the same organ. An inducer with specific activity on tissues other than liver
(e.g., lung, gastrointestinal tract), however, would be capable of increasing
detoxification activity in that organ without producing complications which
might occur from elevated liver microsomal activity.
d. Acute Toxicity
Several animal studies have been conducted on the single-
dose toxicity of numerous benzothiazole derivatives. Simple molecular modifica-
tions of benzothiazole produce large differences in toxic potency. For this
reason, data on non-commercial benzothiazole derivatives are included in this
section for the sake of relating specific structures and substitutions to their
resultant toxic effects. Consequently, comparisons can readily be made, not
86
-------
Table 24. Effects of 2-Phenylbenzothiazoles and Related Compounds on
Benzopyrene Hydroxylase Activity of Rat Liver and Lung
(from Wattenberg et al., 1968)
Benzopyrene hydroxylase activity
Liver b
Compound tested3 (units/mg wet weight )
Controls
2-Phenylbenzothiazole
2-(2'-Broniophenyl)-benzothiazole
2-(3'-Bromophenyl)-benzothlazole
2- (4 '-Bromophenyl) -benzothiazole
2- (2 '-Chlorophenyl) -benzothiazole
2-(3'-Chlorophenyl)-benzothiazole
2- (4 '-Chlorophenyl) -benzothiazole
2-(2' ,4 '-Dichlorophenyl) -benzothiazole
2- (4 '-lodophenyl) -benzothiazole
2- (4 '-Fluorophenyl) -benzothiazole •
2- (4 ' -AminophenyD-benzothlazole
2- (4 '-Hydroxyphenyl) -benzothiazole
2- (4* -Me thoxyphenyl) -benzothiazole
2- (4 ' -Carboxyphenyl ) -benzothiazole
2-(4'-Cyanophenyl)-benzothiazole
2- (4 '-Methylphenyl) -benzothiazole
2-(4'-Formylphenyl)-benzothiazole
6-Methyl-2-phenylbenzothiazole
5-Chloro-2-phenylbenzothiazole
Benzothiazole
2-Mercaptobenzothlazole
2,2'-Bibenzothiazole
2t2'-Thiobisbenzothiazole
2, 2 ' -Dithiobisbenzothiazole
2-Benzylthlobenzothiazole
2-Benzoylthlobenzothiazole
2-Phenoxybenzothlazole
2-Benzyloxybenzothiazole
2-Benzamidobenzothiazole
2-Benzylamlnobenzothlazole
2-p-Toluenesulf onamidobenzothlazole
2- (4 ' -Pyridy 1) -benzothiazole
2-Phenylbenzimidazole
13 + 4
46 + 8
54 + 7
48 + 5
104 + 3
38 + 4
32 + 5
100+8
42 + 8
98 + 4
33 + 7
54 + 3
19 + 1
27 + 4
16 + 1
27 + 1
26+1
15 + 1
31 + 5
50+7
26 + 4
14 + 1
22 + 1
31 + 0
19 + 3
34 + 1
10 + 0
18+0
33 + 11
31 + 3
19 + 1.
16 + 0
33 + 5
15 + 2
Lung fc
(units/mg wet weight )
0.88 + 0.24
3.40 + 0.61
4.42 + 0.54
2.07 + 0.31
6.46 + 0.34
3.74 + 0.85
1.70 + 0.27
5.65 + 1.53
3.98 + 0.58
4.08 + 0.71
2.38 + 0.43
3.64 + 0.98
1.04 + 0.27
2.38 + 0.22
0.85 + 0.17
4.08 + 0.41
1.57 + 0.31
0.75 + 0.24
3.06 + Q.48
3.74 + 0.48
1.36 + 0.24
0.92 + 0.17
0.85 + 0.17
0.92 + 0.17
0.78 + 0.17
1.70 + 0.27
0.78 + 0.17
1.26 + 0.41.
2.11 + 0.44
2.38 + 0.37
1.36+0.31
0.92 + 0.17
2.14 + 0.53
1.40 + 0.24
Liver
(Ratio: test/control )
3.5
4.1
3.5
8.0
2.9
2.5
7.8
3.2
7.5
2.5
4.2
1.5
2.1
1.2
2.1
2.0
1.2
2.4
3.8
2.0
1.1
1.7
2.4
1.5
2.6
0.8
1.4
2.5
2.4
1.5
1.2
2.5
1-2
Lung
(Ratio: test/control )
3.9
5.0
2.4
7.3
4.2
1.9
6.4
4.5
4.6
2-7
4.1
1.2
2.7
1-0
4.6
1.8
0.9
3.5
4.2
1.5
1.0
1.0
1.0
0.9
1.9
0.9
1.4
2.4
2.7
1.5
1.0
2.4
1.6
t 0.1 mmole of each compound in 1 ml dimethylsulfoxide was administered by stomach'tube to 48-day-old female Sprague-Dawley rats 48- hours prior to
sacrifice. Controls received vehicle only. 3-6 rats per group except for 2-phenylbenzothlazole, 15 animals and controls, 33 animals.
b Mean + S.D.
Ratio of benzopyrene hydroxylase activity of tisau* from «nln«l receiving the Indicated compound divided by the activity of tne control.
87
-------
only among important commercial compounds but also between these substances and
structurally-related derivatives. The available evidence indicates that acute
intoxication by any of the benzothiazole derivatives will be manifested pri-
marily as disruption of central nervous system function.
Oral and Parenteral Routes
A particularly detailed investigation of the toxicity
of MET was undertaken by Guess and O'Leary (1969) to help assess the potential
public health hazards of rubber-containing items. The authors included in their
study the compound 2-(2-hydroxyethylmercapto)benzothiazole (HMBT), a substance
formed from MET contained in rubber by reaction with ethylene oxide under con-
ditions used to sterilize medical grade rubber articles.
Single-dose toxicity and LDsf) determinations were made
in mice, both by the oral and intraperitoneal routes. White male mice, 18 to
22 grams in weight, were administered either MBT or HMBT suspended in cottonseed
oil for intraperitoneal injection, or in 0.5% carboxymethyl cellulose in 0.9%
saline solution for the oral dosing. The results of the LD' evaluations are
presented in Table 25.
Deaths, when they occurred, were always within 24 hours.
The signs of toxicity were more dramatic in those animals receiving higher doses
of MBT than in those receiving HMBT by intraperitoneal injection. However, a
comparison of the LD 's and maximum tolerated doses by either the oral or paren-
teral route indicated that HMBT may be more toxic than MBT. Nevertheless, for
both compounds, doses in excess of 335 mg/kg induced a marked peripheral vasodi-
lation, extensive salivation, and convulsions. Convulsions produced by MBT
88
-------
Table 25. LD Values for MET and HMBT in Mice (from Guess and O'Leary, 1969)
Route and statistic
MET
HMBT
Intraperitoneal
LD50
Approx. 95% C.L.
Probit slope
Max. tolerated dose
Oral
LD50
Approx. 95% C.L.
Probit slope
Max. tolerated dose
437 mg/kg
415-461 mg/kg
19.05 probits/log
305 mg/kg
2000 mg/kg
1798-2225 mg/kg
9.01 probits/log
931 mg/kg
417 mg/kg
390-455 mg/kg
12.38 probits/log
239 mg/kg
1017 ms/kg
885-1146 mg/kg
6.59 probits/log
357 mg/kg
Confidence limit
became progressively severe, with intermittent clonic and tonic seizures of
prolonged duration. - HMBT, on the other hand, induced convulsions of the spas-
modic "leaping" type accompanied by long periods of quiet inactivity.
The overt symptoms of intoxication by MBT and HMBT were
clearly suggestive of central nervous system action. Other benzothiazole deriva-
tives are well-known for their central nervous effects (see Section III-B-2-c, p. 79)
Additional studies were conducted to determine whether the salivation and slight
lacrimation seen at higher dose levels were due to chblinergic activity mediated
through the autonomic system. As a test, atropine sulfate (0.2 mg/kg) was given
intraperitoneally 30 minutes prior to dosing with 550 mg/kg MBT or HMBT. This
treatment did not block the salivation activity and only slightly reduced periph-
eral vasodilation. Therefore, cn'otxriefgic activity was discounted as the mode
89
-------
of toxic action, and instead the possibility of central stimulation via the
cervical sympathetic ganglion was considered. Predictably, the intraperitoneal
injection of pentobarbital (25 mg/kg) 30 minutes prior to treatment with 550 mg/kg
MET or HMBT completely blocked the convulsions and salivation. These results con-
firmed the role of the central nervous system as a major site of toxic action for
MET or HMBT. The peripheral vasodilation, however, which was induced by MET or
HMBT treatment could be markedly reduced, but the effect persisted nevertheless.
The acute oral and parenteral toxicities of several
benzothiazole derivatives used as rubber accelerators have been investigated along
with those of many non-commercial substituted benzothiazoles. Table 26 summarizes
these results. An examination of the data contained in the table reveals that
substituents of increasing size added to the 2-position of benzothiazole tend
to decrease acute toxicity. Unsubstltuted benzothiazole and several benzene
ring-substituted 2-aminobenzothiazoles appear to be the most toxic derivatives
of the class. It is not known to what extent the parameters of absorption, tis-
sue and plasma binding, and drug-gastrointestinal tract interactions may influ-
ence the toxicity of individual compounds. In general, it has been shown that
the mouse is more susceptible than are the rat and guinea pig; sufficient data
are not available to evaluate the modifying effects of factors such as sex or
strain differences.
90
-------
Table 26. Acute Animal Toxicity of Benzothiazole Derivatives
VO
Substance
2-Aminobenzothiazole
2-Amino-4-chlorobenzothiazole
2-Amino-5-chlorobenzothlazole
2-Amlno-6-chlorobenzothiazole
'
2-Amino-7-chlorobenzothiazole
2-Amino-4,6-
dimethylbenzothiazole
2-Amln.o-4-ethoxybenzothiazole
2-Amino-4-ethylbenzothiazole
2-anino-4-
hydroxybenzothlazole
KEY
CMC « carboxymethyl cellulose;
Sex Route
4 of
Species (No.) Dose (mg/kg) Administration
Mouse M (?) 200 adjusted to i.p.
pH - 7.0 with
HO + NaOB
House ? 126 i.v.
House ? 2400 oral
House ? 71 i.v.
Mouse . ? 92 i.v.
Mouse 7 398 oral
House 1 76 i.v.
Mouse ? 77 i.v.
House ? 850 oral
Mouse ? 80 i.v.
Mouse ? 77 i.v.
House ? 160 i.v.
PC » propylene glycol
Effects
Approximate LD after 7 days.
LD temporary paralysis in survivors
LD , temporary paralysis in survivors
LD temporary paralysis in survivors
LD , temporary flaccid paralysis in
survivors
LD , temporary flaccid paralysis in
survivors
LD , temporary paralysis in survivors
LD , paralysis lasting 12 to 18 hours
with death ensuing by progressive
respiratory depression
LD , temporary paralysis in survivors
LD,.n» temporary paralysis in survivors
LD^n. temporary paralysis and marked
hyperpnea in survivors
Reference
Doull et^ al. , 1962
Domino. et al. , 1952
Domino et^ al. , 1952
Domino et^ al. , 1952
Domino et al., 1952
Domino et^ al. , 1952
Domino et^ al. , 1952
Domino et. al. , 1952
Domino et^ al. , 1952
Domino et^ al. , 1952
Domino e£ al. , 1952
LD50 " calculated dose causing death In 502 of the experimental animal species
dosages calculated from milliliter equivalents of a 50% solution
-------
Table 26. (cont'd)
VO
to
Substance
2-Amino-6-hydroxybenzothlazole
6-Amino-2-mercaptobenzothiazole
2-Amino-4-methoxybenzothlazole
2-Amino-5-methoxybenzothiazole
2-Amino-6-methoxybenzothlazole
2-Amino-4-methylbenzothiazole
2-Amino-5-methylbenzothiazole
2-Amino-6-methylbenzothiazole
KEY
CMC =* carboxyniethyl cellulose;
b
LD ^ = calculated dose causing
Sex Route
& • of
Species (No.) Dose (mg/kg) Administration
Mouse ?
Mouse M (?)
Mouse ?
Mouse ?
House ?
Mouse • ?
Mouse ?
Mouse ?
House ?
Mouse ?
Mouse ?
Mouse M (?)
Mouse ?
Mouse ?
PG = propylene glycol
death in 50% of the ez
300
150-200
adjusted to
pH - 7.0 with
H,0 + NaOH
>300 in CMC
562
46
150
140
697
54
1070
74
>100 in CMC
525
84
l.v.
i.p.
l.p.
oral
l.v.
i.v.
i.v.
oral
l.v.
oral
l.v.
i.p.
oral
i.v.
Effects
LD , clonic convulsions
Approximate LD after 7 days
Approximate LD after 7 days
LD__, temporary
U>5(), temporary
LD , temporary
paralysis in survivors
paralysis in survivors
paralysis in survivors
LD,.-, running convulsions and respiratory
depression
LD5Q, temporary
LD__, temporary
LD , temporary
LD , temporary
paralysis in survivors
paralysis in survivors
paralysis in survivors
paralysis in survivors
Approximate LD after 7 days
LD -, temporary
survivors
LD50, temporary
survivors
flaccid paralysis in
flaccid paralysis in
Reference
Domino et al . ,
Doull ec_ al. ,
Doull et_ al. ,
Domino et al.
Domino et al.
Domino et al.
Domino et al.
Domino et al.
Domino et al.
Domino et al.
Domino et al.
Doull et al. ,
Domino et al.
Domino et al.
, 1952
1962
1962
, 1952
, 1952
, 1952
, 1952
, 1952
, 1952
, 1952
, 1952
1962
, 1952
, 1952
dosages calculated from milliliter equivalents of a 50% solution
-------
Table 26. (cont'd)
u>
Substance
5-Amlno-2-phenylbenzothiazole
Benzothiazole -
H-tert-Butyl-2-
benzothlazolesulfenamlde
2-Chlorobenzothlazole
5-Chloro-2-nethylbenzothiazole
N-Cyclohexyl-2-
benzothiazolesulfenamide
KEY
CMC = carboxymetliyi cellulose; PG
b
Sex Route
4 of
Species (No.) Dose (mg/kg) Administration
Rat ? 2940 oral
Mouse M (?) 100-200 in i.p.
PG + HjO
Mouse ? 95 i.v.
Mouse ? 100° i.v.
Rat M (10) 10,000 oral
Mouse M (?) 200-300 In i.p.
PG + H20
Mouse M (?) 500 in PG i.p.
Mouse M (?) >2500 in CMC i.p.
Mouse ? >4000 in oil oral
Rabbit ? 4000 oral
Mouse ? 1870 oral
Rat M (10) 10,000 oral
» propylene glycol
Effects
"50
Approximate U>50 after 7 days
LD , temporary paralysis in survivors
"so
Mo mortality; anlaals became sluggish
within 15 minutes and displayed pllo-
erection within 45 minutes; nothing
remarkable on autopsy
Approximate LD_. after 7 days
Approximate LD - after 7 days
Approximate LD after 7 days
LD
LD
5
LD,n
50
One death. In victim, liver and spleen
mottled; kidneys mottled and congested;
adrenals slightly congested; stomach
distended, filled with a hard mass;
intestines liquid-filled, yellow and
hemorrhaged . Nothing remarkable in
survivors. No mortality produced at a
dosage of 5000 mg/kg (3 rats).
Reference
Christensen and •
Luginbyhl, 1974
Doull e_t a_l . , 1962
Domino ej: al. , 1952
Christensen and
Luginbyhl, 1974
American Cyanamld, 1975
Doull et al. , 1962
Doull et, al_. , 1962
Doull et, «1. , 1962
Vorob'eva and
Mezentseva, 1962
Christensen and
Luginbyhl, 1974
Christensen and
Lubinbyhl, 1974
American Cyanaotld, 1975
'•°5Q = calculated Jose causing death in 50% ot" the experimental, animat species
dosages calculated from mllliliter equivalents of a 50% solution
-------
Table 26. (cont'd)
VO
Sex
&
Substance Species (No.) Dose (mg/kg)
2,6-Dlamlnobenzothiazole Mouse M (?) >1000 in PG
Mouse ? 384
N,N-Dicyclohexylbenzothiazole-
sulfenamlde Rat ? 3450
2-(Dimethylamlno)-benzothiazole Mouse ? 131
N, N-Diisopropyl-2-
benzothiazolesulfenamide Rat M (10) . 10,000
Rat M (3) 5000
Mouse M (?) 3892
2,i-Dimethylbenzothiazole Rat ? 957
2,2'-Dithiobisbenzothiazole Mouse M (?) >2000
Guinea
Pig ? 2250
Rat ? >5000
KEY
CMC « carboxyraethyl cellulose; PG = propylene glycol
i.p. = intraperitoneal : i.v. *» intravenous
Route
of
Administration Effects
i.p. Approximate LD,- after 7 days
i;v. LDso' runnln8 convulsions and respiratory
depression
oral Lethal dose
i.v. LDso* tenLP°rarv paralysis in survivors
oral Three deaths (1 on day 3; 2 on day 4).
In victims, livers and spleens mottled;
kidneys speckled and slightly congested;
intestines' liquid-filled and hemorrhaged.
Nothing remarkable in survivors. No
overt signs of intoxication were noted.
oral One death on day 2. In victim, results
signs of intoxication were noted.
7 LD50
oral LD5Q
i.p. Approximate U>50 after 7 days
i.p. ^SO' Patholo8lcal changes in internal
organs
oral LD50
Reference
Doull e^ al,- . 1962
Domino et al. ,; 1952
Christensen and
Luginbyhl, 1974
Domino et^ al. , 1952
American Cyanamid, 1975
American Cyanamid, 1975
Vorob'eva, 1968
Christensen and
' Luginbyhl, 1974
Doull e_t al. , 1962
Kowalski and
Bassendovska v 1965
R. T. Vanderbilt, 1975a
LDp- = calculated dose causing death in 50% of the experimental animal species
dosages calculated from milliliter equivalents of a 50% solution
-------
Table 26. (cont'd)
VO
Substance
2-Hydrazinobenzothiazole
2-(2-Hydroxyethylmercapto)
benzothiazole
MBTS Pellets (mixture
containing: 76-80Z, 2,2'-
dithiobisbenzothiazole;
6-9Z, 2-mercaptobenzothiazole;
5-7Z, 2-benzothiazolyl-2-
amlnophenyl disulfide and
2,2' -dlaminodiphenyl
disulfide combined)
2-Mercaptobenzothiazole
Sex
&
Species (No.) Dose (mg/kg) Ado
Mouse M (?) 100^200 in PC
Mouse M (?) 1017
Mouse M (?) 417
Rat M (5) 10,000
Rat M (5) 5000, 2500,
(5) (5) 1250
Rat ? 3000
Mouse ? 2306
Mouse ? 1851
Mouse M (?) 2000
Mouse ? 1800 in oil
Mouse M (?) 437
Mouse M (?) 100-200 in PG
Mouse M (?) >300 adjusted
to pH •» 7.0 with
H20 + NaOH
Guinea ? 1680
Pig
Guinea ? 300
Pig
Route
of
linistration
I.p.
oral
i.p.
oral
oral
oral
oral
oral
oral
oral
i.p.
i.p.
i.p.
oral
I.p.
Effects
Approximate LD after 7 days
LD ; deaths within 24 hours; peripheral
vasodilation, extensive salivation,
convul sions
LD n; death within 24 hours; symptoms
same as above
100Z mortality within 24 hours; only
sign of toxicity was depression; autopsy
normal
All animals survived a 7-day observation
period.
"•so
^50
"'so
LD ; deaths within 24 hours; peripheral
vasodilation, extensive salivation, con-
vulsions; intermittent c ionic and tonic
seizures of prolonged duration
"'so
LD ; deaths within 24 hours; symptoms
same as above
Approximate LD5Q after 7 days
Approximate LD,.,, after 7 days
LD^Qj pathological changes of internal
organs
LD50' Pacnol°8ical changes of internal
Reference
Doull e£ a^. , 1962
Guess and O'Leary, 1969
Guess and O'Leary, 1969
American Cyanamld, 1975
American Cyanamid, 1975
R.T. Vanderbilt, l!i75b
Vorob'eva and
Mezentseva, 1968
R.T. Vanderbilt, 1975b
Guess and O'Leary, 1969
Vorob'eva and
Mezentseva, 1962
Guess and O'Leary, 1969
Doull et_ al. , 1962
Ooull e_t aj.. , 1962
Kowalski and
Bassendowska, 1965
Kowalnski and
Bassendowska , 1965
CMC = --arbox>-inechyl cellulose; PC = propylene glycol
i .p. ° intraperi toneal; i. v. ** intravenous
LD,
50
» calculaced dose causing death in 50% of the experimental animal species
dosages calculated from milliliter equivalents of a 50? solution
-------
Table 26. (cont'd)
VO
Substance
2-Mercaptobenzothiazole,
sodium salt
Sex
&
Species (No.)
Rat ?
Rat M (5)
Dose (tog/kg)
3968
2500 .
Route
of
Administration Effects
oral LD50
oral 100Z mortality; tremors, convulsions.
Reference
R. T. Vanderbilt,
American Cyanamld
1975c
, 1975
Rat M (5)
Rat M (5)
Rat M (5)
1250
625
312.5
oral
oral
oral
and death within 3-5 minutes; severe
depression and hematurla; hemorrhage
of stomach
3 of 5 rats died with tremors, convul-
sions, and death occurring within 3-5
minutes; severe depression and
hematuria; hemorrhage of stomach In
decedents. Survivors recovered after
2 days and autopsy was normal.
2 of 5 rats died; tremors, convulsions,
and death within 3-5 minutes; severe
depression and hematurla; hemorrhage of
stomach in decedents only
American Cyanamld, 1975
American Cyanamid, 1975
1 of 5 rats died on day 2; tremors, con- American Cyanamid, 1975
vulsions; severe depression and
hematuria noted; hemorrhage of stomach
in decedent only.
2-Mercaptobenzothiazole,
zinc Bait
Mouse M (?) 200-300 in PC i.p. Approximate U>50 after 7 days Doull et^ a_l. , 1962
Rat ? 540 oral LD";n R. T. Vanderbilt, 1975d
KEY
CMC = carboxymethyl cellulose;
i.p. - intraperitoneal; i.v. =
LD Q « calculated dose causing
PC = propylene glycol
intravenous
death in 50% of the experimental animal species
dosages calculated from milliliter equivalents of a 50% solution
-------
Table 26. (cont'd)
Substance
2-Methylbenzothiazole
2-Methyl-
mercaptobenzothiazole
Sex Route
& of
Species (No.) Dose (mg/kg) Administration Effects Reference
Mouse M (?) 300-500 in PG i.p. Approximate LD after 7 days Doull et al., 1962
+ H20
Mouse ? 105 i.v. ^«;n* temporary paralysis in survivors Doull tst_ al^. , 1962
Mouse M (?) 200-300 in PG i.p. Approximate LD after 7 days Doull et_ a^. , 1962
VO
4-Morpholinyl-2-
benzothiazyl disulfide
6-Nitro-2-
mercaptobehzothiazole
N-Oxydiethylene-2-
benzothiazolesulfenamide
Rat ? >16,000
Mouse M (?) 25-100 in CMC
Mouse M (?) 100-200 in PG
Mouse ? 1980 in oil
Mouse ? 4000
•Rat M (10) 10,000
oral
i.p.
i.p.
oral
oral
oral
LD
50
Approximate LD after 7 days
Approximate LD - after 7 days
'50
50
LD50
One death. In victim, liver and spleen
mottled; kidneys congested; stomach
transparent; pylorus hemorrhaged;
intestines pink, gas and chemical-
filled. Animals became sluggish within
15 minutes, displayed pilo-erection
within 30 minutes, and were prostrate
within 35 minutes. Spleens were dark
in survivors. No mortality produced
at dosage of 5000 mg/kg (3 rats).
R. T. Vanderbilt, 1975e
Doull et al. , 1962
Doull et^
1962
Vorob'eva and
Mezentseva, 1962
R. T. Vanderbilt, 1975 f
American Cyanamld, 1975
CMC = carboxymethyl cellulose; PG a propylene glycol
i.p. = intraperitoneal; i.v. = intravenous
LD^Q'= calculated dose causing death in 50Z of the experimental animal species
dosages calculated from milliliter equivalents of a 50% solution
-------
Skin and Eye Irritation
Guess and O'Leary (1969) looked further into the con-
sequences of acute exposure to MET and HMBT by determining their primary irritant
activity and effect on wound healing. Suspensions of MET and HMBT were prepared
in cottonseed oil or carboxymethyl cellulose-saline solution at concentrations of
4%, 2%, 1% and 0.5%. Intradermal injections were then made at four sites each
into the shaven backs of four albino rabbits at a dose of 0.2 ml per site. Only
MET at a 4% concentration in oil consistently induced intradermal irritation.
This irritation, however, was mild and transitory, less than that shown by a 20%
ethanol solution. HMBT in oil or aqueous suspension did not produce obvious skin
irritation. Histologic examination of skin tissue after treatment with 4% MET
or HMBT demonstrated a mild inflammation at 24 hours with MET, but no signs of
irritation with either substance after 48 hours.
The effect of MET and HMBT on wound healing was evalua-
ted in rabbits bearing four superficial incisions on the back, 1 cm long, 2 mm
deep, and 0.5 cm apart. Oil or aqueous suspensions of MET or HMBT were applied
to each area at concentrations of 4%, 2%, 1% and 0.5%, and the sites observed
daily for evidence of erythema, scar formation, rate of healing, and other
effects. In no instance was the normal healing process inhibited by either the
MET or HMBT treatment. Erythema and edema were absent, and hair growth and total
healing time were identical for all rabbits.
Further tests on skin and eye irritation caused by a
commercial mildew inhibitor containing MET were conducted by the Department of
the Army (Rowe, 1969). The compound tested was Vancide 51Z, a commercial fungi-
cide containing as active ingredients 90% Ziram (zinc salt of dimethyl dithio-
carbamic acid) and 7.8% zinc salt of MET. Their results are summarized in
Table 27.
98
-------
Table 27. Primary Irritation Evaluation of Mildew Inhibitor Vancide 51Z (data from Rowe, 1969)
Test
Dose
Results
Skin Irritation - single 24-hour
application to intact and
abraded skin of rabbits
vo
vo
Eye Irritation - single 24-hour
application
0.5 gram commercial grade
powder applied to each of
4 rabbits
0.5 gram commercial grade
powder in acetone (total
volume 1.5 ml) applied to
each of 4 rabbits
0.1 gram commercial grade
powder to one eye of each
of 5 rabbits
0.2 ml of a 1% suspension
of commercial grade powder
in propylene glycol to one
eye of each of 5 rabbits
Vancide 51Z did not cause primary
irritation of the intact skin or of
the skin surrounding an abrasion when
applied either as .the commercial powder
or the commercial grade formulation in
acetone
Vancide 51Z caused destruction of all
or nearly all of the corneal epithelium
as evidenced by staining with Fluorescein.
Also evident were copious ocular discharge
and severe chemosis in every eye tested.
Diluted Vancide 51Z caused slight corneal
damage in all treated eyes. This
formulation caused a moderate to heavy
ocular discharge in every eye tested and
moderate chemosis in 4 of 5 eyes tested.
-------
Interpretation of these results by the author led to
a recommendation calling for no restriction of acute application of Vancide 51Z
to human skin. It was also recommended that the technical grade powder in un-
diluted form should be used with extreme caution and should be restricted to
areas other than the face. In diluted form, it was suggested that Vancide 51Z
be used with caution around the eyes and mucosa.
Eye irritation tests have also been conducted on several
rubber accelerator chemicals belonging to the benzothiazole class (American
Cyanamid, 1975). Groups of six rabbits were treated by instillation of the test
substance directly to the eye, and the reactions evaluated at 24, 48, and 72
hours. Maximum possible scores for eye irritation (excluding necrosis) were:
cornea, 80; iris, 10; conjunctivae, 20. The results, summarized in Table 28,
indicate that sodium MET is a particularly significant hazard to the eye. Corneal
opacity was evident within 4 hours of dosing of the eyes arid did not significantly
improve during the following week.
100
-------
Table 28. Acute Eye Irritation of Benzothiazole Derivatives in the Rabbit'
Cyanamid, 1975)
(data from American
u
Sodium. mercaptobenzothiazole 50 mg.
N-Oxydiethylene-2- 100 mg.
benzothiazolesulfenamide
N,N-Diisopropyl-2- 100 mg.
benzothlazolesulfenami.de
N-Cyclohexyl-2- 100 mg.
benzothiazolesulfenamide
N-tert-Butyl-2- 100 mg.
benzothiazolesulfenamide
Cornea
Iris
Conjunctivae
Cornea
Iris
Conjunctivae
Cornea
Iris
Conj unct ivae
Cornea
Iris
Conj unct ivae
Cornea
Iris
Conjunctivae
30.0
3
13
15
5
8
2
0
3
7
3
5
10
1
5
.3
.0
.0
.0
.3
.5
.0
.0
.5
.3
.3
.0
.7
.0
38
4
13
11
2
7
0
0
0
4
0
3
6
0
4
.3
.1
.0
.7
.5
.3
.0
.0
.0
.2
.0
.3
.7
.83
.0
38
4
13
10
3
5
0
0
0
0
0
1
4
0
3
.3
.1
.0
.0
.3
.3
.0
.0
.0
.83
.0
.3
.2
.83
.0
Discernible opacity or ulceration of the cornea (other
than a slight dulling of the normal luster) ; inflam-
mation of the iris (other than slight deepening of the
folds or slight circumcorneal injection); diffuse deep-
crimson red appearance of the Conjunctivae, with indi-
vidual vessels not easily discernible; obvious swelling
of the Conjunctivae, excluding cornea and iris, with
partial eversion of the lids; destruction or .irreversi-
ble tissue change in 24 hours or less.
Discernible opacity or ulceration of the cornea (other
than a slight dulling of the normal luster) ; inflam-
mation of the iris (other than slight deepening of the
folds or slight circumcorneal injection); obvious
swelling of the Conjunctivae, excluding cornea and
iris, with partial eversion of the lids.
Discernible opacity or ulceration of the cornea (other
than a slight dulling of the normal luster).
Discernible opacity or ulceration of the cornea (other
than a slight dulling of the normal luster) ; inflam-
mation of the iris other than slight deepening of the
-folds or slight circumcorneal injection.
Discernible opacity or ulceration of the cornea (other
than a slight dulling of the normal luster).
Compounds were instilled in one eye of each of six rabbits-per treatment group, and reactions evaluated at 24, 48, and 72 hours.
Maximum possible scores for irritation (excluding necrosis) were: cornea, 80; iris, 10; Conjunctivae, 20.
Dose was given as 0.1 ml of a 50Z solution.
-------
Dermal Toxicity
Tests to measure the toxic effects from dermal exposure
to benzothiazole derivatives have only been conducted on several of the compounds
used as rubber accelerators (Table 29). From the data presented, it is obvious
that sodium MET represents a significant hazard when applied to the skin. The
apparent lack of toxicity for the other compounds tested is consistent with the
low order of toxicity observed when these substances are administered orally
(Table 26). In the absence of confirming data from parenteral or inhalation studies,
it can only be suggested that the low toxic potency of the more complex benzothia-
zole accelerators may be a reflection of their poor absorption, or the fact that
they are much less corrosive than sodium MBT.
e. Subacute Toxicity
Data from subacute studies should be interpreted with special
consideration of the fact that target organs and responses of acute bioassays are
often different than those following repeated exposure to sublethal doses. The
phenomena of storage, metabolic activation, and repeated damage to organs and or-
ganelles are potential hazards of repeated exposure, and more closely resemble
the consequences to man from environmental contamination. Massive single-dose
exposures are generally of little practical use other than for estimating the po-
tential to produce mortality.
Guess and O'Leary (1969) have shown that repeated exposure of
mice to MBT at a dosage of one-fourth the LD can produce serious liver damage
which is not detectable by gross observation. Male mice were treated daily for
one week with intraperitoneal injections of MBT or HMBT [2-(2-hydroxyethylmercap-
to)benzothiazole] as either oil or aqueous suspensions. Groups were dosed at
102
-------
Table 29. Acute Dermal Toxicity of Benzothiazole Derivatives in the Rabbit3 (data from American
Cyanimid, 1975)
Substance
Sodium mercaptobenzothiazole
N-tert-Butyl-2-
benzothiazolesulf enamide
N-Cyclohexyl-2-
benzothiazolesulf enamide
N-Oxydiethylene-2-
benzothiazolesulf enamide
N;N-Diisopropyl-2-
benzothiazolesulf enamide
MBTS Pellets (mixture containing:
76-80% , 2 , 2 '-dithiobisbenzothiazole ;
6-9%, 2-mercaptobenzothiazole;
5-7 % , 2-benzothiazolyl-2-aminophenyl
disulfide and 2,2'-diaminodiphenyl
disulfide (combined)
Sex
M
M
M
M
M
M
M
M
+ (No.)
(10)
(10)
(10)
(5)
(5)
(5)
(5)
(5)
Dose (mg/kg)
2500
1250
625
10,000
10,000
10,000
10,000
10,000
Mortality
died/dosed
4/10
1/10
0/10
0/5
0/5
0/5
0/5
0/5
• Observations
Severe depression; cold extremities; appetite
Severe degree of skin injury; area burned at
formation of hard eschar at 1-2 weeks. Gross
were normal.
No signs of intoxication or skin irritation.
showed nothing remarkable.
No signs of intoxication or skin irritation.
showed nothing remarkable .
No signs of intoxication or skin irritation.
showed nothing remarkable.
No signs of Intoxication of skin irritation.
showed nothing remarkable.
loss.
24 hours with
autopsies
Gross autopsies
Gross autopsies
Gross autopsies
Gross autopsies
No signs of toxicity during a 7-day observation period.
Autopsies were normal. 3 of 5 animals lost weight.
Compounds were applied to the clipped unabraded skin.and held in contact by occlusive bandage for 24 hours.
Dosages were calculated from milliliter equivalents of a 50Z solution.
-------
one-fourth and one-eighth the IJ>50 (110 and 55 mg/kg, respectively for MET;
104 and 52 mg/kg, respectively for HMBT). At the dose levels for both compounds
administered, no signs of overt toxicity could be detected after one week of in-
jections. Weight, gain and behavior patterns did not differ from control animals
receiving the injection vehicle only. After sacrifice at the end of one week,
gross examination of vital organs revealed no significant injury for either com-
pound at all dose levels. Microscopic examination of various tissues, however,
revealed that serious damage to the liver had occurred in those mice receiving
MET at one-fourth the LD dose. Results of the pathologic examination of all
tissues are presented in Table 30. Tissues from mice receiving one-eighth the
LD _ dose were not examined microscopically.
Table 30. Microscopic Pathologic Findings in Mouse Tissues After Dosing with
MET and HMBT for One Week (from Guess and O'Leary, 1969)
Compound
MET
HMBT
Liver
Necrosis
Cellular
swelling
Lungs
Normal
Normal
Kidney
Cloudy
swelling
Normal
Heart
Normal
Normal
Thyroid
Normal
Normal
Testes
Normal
Normal
Sections of livers from MBT-dosed animals revealed extensive liver damage in
the form of necrosis and reaction to the injury. At the cellular level, there
was cloudy swelling, opacification, accumulation of various cytoplasmic granules,
hyaline change, rupture of cell walls, and profound changes in nuclei. In addi-
tion, rupture of bile canaliculi and bile stasis had occurred which caused a
marked inflammatory reaction with a severe infiltration of lymphocytes. The
104
-------
effects of HMBT on the anatomic organization of the liver were much less severe
although some damage had occurred, primarily as cellular swelling and distinct
cell walls.
In order to determine the effect of subacute treatment with
MET and HMBT on liver function, a hexobarbital narcosis study was conducted in
mice. Aqueous saline suspensions of MET and HMBT were given daily for one week
by intraperitoneal injection at one-fourth or one-eighth the LD dose. After
one week, hexobarbital sodium was given at 75 mg/kg and the resulting time of
sleeping was judged as a measure of liver function (i.e., as liver function be-
comes impaired, sleep time will increase due to the decreased capacity of the
organ to metabolize and eliminate the hexobarbital). As indicated in table 31,
both MET and HMBT at the one-fourth LD dose caused a significant increase in
sleeping time when compared to control animals. These results confirm the micro-
scopic studies which^showed that marked damage to the liver had occurred.
Table 31. Effect of MET and HMBT on Hexobarbital Narcosis in Mice (from Guess
and O'Leary, 1969)
Number Sleep time ± SU
Dose, ip of mice (min)
MBT '
55 mg/kg 9 23.1 ± 4.94fl
110 mg/kg 8 . 25.0 ± 5.44
HMBT
52 mg/kg 8 21.0 ' 8.00
104 mg/kg 10 . 22.7 ± 2.54
Control . 8 17.0 ± 6.52
a
Significantly different from control at P = 0.05
105
-------
The results of a study have been reported which involved
the subacute feeding of MBTS Pellets, a rubber accelerator product (American
Cyanamid, 1975). MBTS Pellets is a mixture of 76-80% 2,2'-dithiobisbenzothiazole,
6-9% MET, and 5-8% 2-benzothiazolyl-2-aminophenyl disulfide and 2,2'-diaminodi-
phenyl disulfide combined. This product was added to the diet of young male
albino rats at concentrations of 0.5%, 1.0%, and 2.0% for a 31 day period.
Table 32 shows that slight, but significant, reductions in mean daily food intake
and mean total weight gain resulted in all treatment groups. There did not appear
to be any dose-related relationship to these results.
Table 32. Summary of Results of 31 Daily Doses of MBTS Pellets in the Diet of
Male Albino Rats (American Cyanamid, 1975)
Concentration in diet, %
Number of animals
Mean dosage, gm/kg/day
Mean food intake, gm/rat/day
Mean weight gain, gm/rat
Deaths
Mean no. of days to death
0
10
9
18.5
161
-
—
0.5
10
0.49
*
17.5-
*
146
-
—
1.0
10
0.94
*
16.4
*
141
-
—
2.0
10
2.05
*
17.8
*
140
-
—
Denotes a value significantly lower than control value (p = .05)
All animals were sacrificed and a gross examination performed for signs of pathology,
but no remarkable effects could be found that might be due to the treatment.
106
-------
A number of rubber accelerator chemicals related to MET were
tested for carcinogenicity and subacute toxicity in mice by Innes et_ al. (1969).
Determinations were made of the maximum tolerated dose for each chemical. The
maximal levels resulting in zero mortality were determined for a single oral
dose, then for 6 daily doses, and finally for 19 daily doses (Table 33).
Table 33. Maximal Tolerated Doses of MET and Derivatives in Mice (data from
Innes et al., 1969)
Substance
Vehicle
Daily Dosage*
(mg/kg)
2-Mercaptobenzothiazole
2,2'-Dithiobisbenzothiazole
Zinc salt of
2-mercaptobenzothiazole
N-Cyclohexyl-2-
benzothiazolesulfenamide
N-Oxydiethylene-2-
benzothiazolesulfenand.de
0.5% gelatin
0.5% gelatin
0.5% gelatin
0.5% gelatin
0.5% gelatin
100
464
1000
215
464
Given by stomach tube.
Further reports from the Russian literature (read in abstract)
have described various effects due to MET exposure. Litvinchuk (1963) reported
that repeated doses of 50-200 mg/kg of MET to rats (route unknown) caused no
serious changes in respiration, blood picture, or renal functions. The treatment
107
-------
did, however, cause an increase in bile output (rats) and a 50% enhancement of
gastric juice secretion in dogs. Vorob'eva ejt al_. (1968) noted that extensive
exposure to MET dust (species unknown) affects respiration and the liver, but has
no apparent effect on the nervous system. When Mikhailov (1973) exposed animals
(species unknown) to daily oral doses of MET at 2.5 to 25 mg/kg/day, he observed
a decreased level of sulfhydryl groups in the blood serum.
Additional toxicity studies on several benzothiazole rubber
accelerators have also been abstracted from the foreign literature. Vorob'eva
and Mezentseva (1962, 1963) allowed rats to inhale dust of N,N-diisopropyl-2-
3
benzothiazolesulfenamide at a concentration of 340-400 mg/m for two hours daily
on 15 days. No adverse changes were observed. When rabbits were treated orally
with the same compound at 20 mg/kg for four months, disturbances were noted in
the protein-forming function of the liver. Additional effects included elevation
of blood serum alkaline phosphates and aldolase activity, but no behavioral changes
or local irritant effects were noted.
Three sulfenamide rubber accelerators, N,N-diethyl-2-
benzothiazolesulfenamide, N-oxydiethylene-2-benzothiazolesulfenamide, and N-
cyclohexyl-2-benzothiazolesulfenamide (CBS) were evaluated in mice and rats by
Zaeva et al. (1966). They demonstrated that rats were more sensitive to these
compounds than mice, and that toxicity was greatest for CBS (minimum lethal oral
dose = 5000 mg/kg), with N,N-diethyl-2-benzothiazolesulfenamide being the least
toxic. All compounds inhibited thyroid function and caused dystrophic alterations
in the liver and kidneys. CBS also caused a reduction of hemoglobin and erythro-
cytes in the blood. When CBS and N-oxydiethylene-2-benzothiazolesulfenamide were
administered by intratracheal injection, they were found capable of causing patho-
logical disturbances of the lungs (i.e., interstitial productive process, emphysema,
108
-------
and bronchitis). Inhalation of N,N-diethyl-2-benzothiazolesulfenamide at a con-
centration of 0.001 to 0.002 mg/1 and repeated 25 times caused slight irritation
and increased excitability of the nervous system. This compound also produced
local irritation of the skin and mucous membranes.
f. Sensitizatiori
It has previously been established that MBT is a common
allergic contact sensitizer in humans (see Section III-B-1, p. 55). Presently,
a number of animal models and screening methods are being employed for identifi-
cation of contact allergens, and one of the most sensitive is the so-called "guinea-
pig maximization test" (Magnusson and Kligman, 1969). This procedures involves
a two-step induction phase whereby intradermal injections of the test substance,
both with and without complete Freund's adjuvant, are given in the shoulder region.
One week after the injections, the test agent is applied topically by closed
patch to the same site. Challenge is made two weeks later by topical application
with an occlusive patch to the flank region for 24 hours. The challenge site is
evaluated 24 hours after removal of the patch. Allergenic potency is determined
by percentage of animals sensitized, not intensity, and each substance tested is
assigned to one of five grades ranging from 0, to weak (I), to extreme (V).
Table 34 presents the results when MBT was assayed for allergenic capability by
the "guinea-pig maximization test." By comparison, when MBT was tested by the
conventional Landsteiner-Draize Test, the sensitization rate was zero. This
result is not in accord with clinical experience as summarized in Section III-B-1,
p. 55 , and suggests that other derivatives of MBT should also be screened by
maximization testing.
109
-------
Table 34. Allergenicity of MET by the Guinea-Pig Maximization Test
(data from Magnusson and Kligman, 1969)
Induction
Intradennal
Concentration
in Adjuvant
%
1
Topical
Concentration
in Petrolatum
%
25
Challenge
Topical
Concentration
in Petrolatum
%
15
Sensitization Grade
Rate
8/20 III
(Moderate)
g. Teratogenicity
No data are available.
h. Mutagenicity
A single report, abstracted from the Russian literature,
described studies in fruit flies on the mutagenic and morphologic effects of
several rubber additives (Revazova, 1968). These results are presented in
Table 35. Although the use of Drosophila melanogaster (fruit fly) as a mutagenic
Table 35. Mutagenic Activity of Several Rubber Additives by Feeding to Fruit
Flies (from Revazova, 1968)
Substance
2-Mercaptobenzothiazole
N-Oxydiethylene-2-
benzothiazolesulfenamide
N-Cyclohexyl-2-
benzothiazolesulfenamide
N,N-Dicyclohexyl-2-
benzothiazolesulfenamide
N,N-Hexamethylene-2-
benzothiazo]esulfenamide
3-(Diethylaminomethyl)
benzothiazole-2-thione
Concentration Number
(mg/cc) of days
20-40
20-40
20-40
20-40
20-40
20-40
8-10
8-9
12-14
12-16
10-13
8-12
% Mutations
2.5t0.49
2.9*0.38
1.4±0.47
0.4±0.26
2.1i0.54
2.6±0.41
110
-------
detection system presents several distinct advantages (short generation time,
large chromosomes, small chromosome number), a number of drawbacks must also be
considered. Large differences between man and fruit flies in such areas as for-
eign compound metabolism, physiologic processes and life span will seriously hinder
the direct extrapolation of experimental results from Drosophila to man. Further-
more, the fruit fly, due to its relatively short life span, is unsuitable for
mutagenicity testing aimed at chronic exposure to environmental chemicals. Inter-?
pretation of the above data is especially difficult since it is not possible to
determine the objectivity of the results presented due to the lack of comparison
scores between treated and control groups. This factor is very important since
it is difficult to maintain constant environmental control over critical variables
such as temperature, humidity and nutrition. The desirability of mutagenic test-
ing of MET type rubber accelerators in higher test organisms is obviously very
high if we wish to confirm their mutagenic activities as indicated by the above
results. Furthermore, although a large number of mutagens also possess carcino-
genic activity, several MET derivatives have been screened as possible carcinogens
and found to be negative.
i. Carcinogenicity
The tumorigenicity of MET and several related compounds was
\
tested by daily oral administration to both f/exes of two hybrid strains of mice
starting at the age of 7 days (Innes et_ al/, 1969). The compounds were administered
at the maximal tolerated doses as outlined in Table 36. Eighteen mice of each
sex of each strain were treated by stomach tube during days 7 to 28 of age and
thereafter given the chemical mixed in the diet through the remainder of the 18
month test period. None of the chemicals tested in this study caused a
111
-------
Table 36. Administration of MET and Several Derivatives to Mice (data from
Innes et al., 1969)
Substance
2-Mercaptobenzothiazole
2,2' -Dithiobisbenzothiazole
Zinc salt of
a
Daily Dosage
(mg/kg)
100
464
1000
Vehicle &
0.5% gelatin
0.5% gelatin
0.5% gelatin
b
ppm
323
1577
3385
2-mercaptobenzothiazole
N-Cyclohexyl-2-
benzothiazolesulfenamide
N-Oxydiethylene-2-
benzothiazolesulfenamide
215
464
0.5% gelatin 692
0.5% gelatin 1492
Used during stomach-tubing period only (7-28 days of age)
Dosage in diet, given ad libitum (after 28 days of age)
significant increase in tumor incidence above control animal values.
A 'derivative of benzothiazole has been identified by Hadidian
et aJ. (1968) as having definite tumorigenic activity. This compound, 2-(4-
dimethylaminostyryl)benzothiazole, is related to several quinoline analogs which
are also known carcinogens. Administration of the compound was made by gastric
intubation at dose levels of 0.3 to 100 mg/kg/day to male and female rats, and
was performed five times per week for a total of 260 individual doses in one year.
They found that tumors developed at all dose levels and consisted mainly of testicu-
lar interstitial cell tumors in males and mammary fibroadenomas in females.
112
-------
Two benzothiazole derivatives with potent hepatocarcinogenic
activity in male rats were reported by Brown and Sanchorawala (1968). The most
active compound tested, N,N-dimethyl-p-(6-benzthiazolylazo)aniline, when fed in
the diet at 0.03% gave a tumor incidence of 5/10 in one month and 10/10 in two
months. N,N-dimethyl-p-(7-benzothiazolylazo)aniline gave a 10/10 incidence in
three months at the 0.03% dietary level.
When benzothiazole was tested for carcinogenicity in rats by
adding it to the diet, Brown (1963) reported that no activity was demonstrated
after six months of feeding.
j. Other Chronic Effects Studies
The results of a two-year feeding study using dogs and rats
exposed to MBT have been reported by Lehman (1965). The formulation employed
was a mixture of 2.4% MBT and 27.6% dimethyldithiocarbamate. Male and female
rats were fed the mixture for two years in their diet at 500, 1580, and 5000 ppm,
which represents 12, 37.9 and 120 ppm, respectively, of MBT. Male and female dogs
were treated with the same mixture and dietary concentrations, but for a period
of one year only. In both the rat and dog studies, there was no significant
effect on mortality at any of the dosages employed. In addition, no deleterious
effects were seen for body weight gain, hematologic examinations, or histopathology.
Furthermore, in the rat, no cumulative effect could be demonstrated on reproduc-
tion and lactation through the F2 generation, and no demonstrable storage was
found in the liver, kidney, or spleen when rats were fed the mixture at 10,000
ppm for one week. Based on these results, a no-effect level for MBT in the diet
was set at 120 ppm for both rats and dogs.
k. Behavioral Effects
No data are available.
1. Possible Synergisms
No data are available.
113
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3. Effects on Other Vertebrates
A single study has been encountered (Pickering and Henderson,
1966) which presents the results of acute static toxicity studies in fish for
the MET-containing fungicides Vancide 51Z and the manganese salt of Vancide 51.
Three species of fish were tested at five different concentrations, and mortalities
were recorded at 24-, 48-, and 96-hour intervals. Table 37 presents the TL... values
m
(a statistical estimate of the concentration of a material in water that kills
50 percent of the test species) obtained in a number of bioassays, with ten fish
being used for each test concentration. As the results in Table 37 indicate,
neither Vancide 51Z nor the manganese salt of Vancide 51 is significantly more
toxic at 96 hours than at 24 hours. This observation suggests that cumulative
toxicity is not a major factor in the mortality produced by these compounds.
Furthermore, a comparison of the acute toxicity of both substances when tested
in either hard or soft dilution water did not reveal any major differences. It
is not possible to determine from this study to what extent the individual com-
ponents of the test formulation contributed to the overall toxic response. Acute
toxicity studies on fish exposed to pure MET have not been found.
4. Effects on Invertebrates
One report, read in abstract, (Miyaki and Enomoto, 1957) has
stated that MET is a powerful molluscacide against Oncomelania nosophora. No
further reports on toxicity to invertebrates have been encountered.
5. Effects on Plants
a. Fungi
Many heterocyclic mercaptan compounds have been found to be
active fungicides. More specifically, MET was shown to be active against smut
114
-------
Table 37. Summary of the Acute Toxicity of Vancide Formulations to Several Species of Fish
(data from Pickering and Henderson, 1966)
Compound
Vancide 51Z
(Dispersion 50% active
Ziram-Zn salt of
Di-Methyldithiocarbamic
acid 46% ; Zinc salt of
Mercaptobenzothiazole 4%)
Manganese Salt of
Vancide 51, 85% Active
Solvent
Carrier
Water
Water
Water
Water
Water blend
Water blend
a
Water
S
S
S
H
S
H
Fish.
Fatheads
Bluegills
Guppies
Guppies
Fatheads
Fatheads
TLb
.41
.85
.59
.51
.91
.97
24-hour
Conf.
.31
.74
.51
.41
.73
.77 •
limits
.52
1.1
.70
.67
1.2
1.3
TLb
.35
.85
.83
.75
48-hour
Conf.
.24
.74
.66
.58
limits
.45
1.1
1.1
.95
TLb
.35
.85
.83
.71
96-hour
Conf.
.24
.74
.66
.55
limits
.45
1.1
1.1
.90
S = soft water, H - hard water
tog of formulation/liter and 95% confidence limits
-------
and bunt of wheat, carnation rust, mildew on leather and textiles, and mycoses
of humans (Owens, 1969). It was suggested that the thiol group of MET is essential
for toxicity=, since neither benzothiazole nor 2-chlorobenzothiazole is an active
fungicide.
Several commercial fungicide formulations incorporate MET
as an active ingredient, but it is rarely used alone. The effectiveness of MET
as a primary fungicide was^tested by Chatterjee et^ al., (1961). An assay system
was employed whereby the ability of MET to inhibit the germination of spores of
Aspergillus niger and Memnoniella echinata was evaluated. Calculations were made
for the percent concentrations, expressed in mg/100 ml, required for 50 and 90
percent inhibition of spore germination (Lc50 and Lc90, respectively). Spore
suspensions used for testing were prepared with a density of 50,000 spores/ml.
Their results are presented in Table 38.
Table 38. Assessment of Fungicidal Activity (data from Chatterjee ^t_ a^., 1961)
Name of
Fungicide
Test
Organism
Lc50 Lc90
(mg/100 ml) (mg/100 ml)
Vulcafor Daw
(active ingredient
MET)
Aspergillus niger
0.06035
Memnoniella echinata 0.00605
0.8439
0.05962
116
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Several additional benzothiazole derivatives have been tested
for fungistatic activity by Merkel et^ al., (1963). These chemicals were tested
on six strains of yeast-like fungi Candida albicans, and six strains of dermato-
phytes: Trichophyton gypseum, Tr. interdigitalis, Tr. plicatile, Tr. sulfureum,
Epidermophyton rubrum, and Microsporum fulvum. Responses to chemical exposure
in liquid and solid media were expressed as molar concentrations of the substance
required to inhibit growth (Table 39 ).
Table 39 . Action of Benzothiazole Derivatives on Pathogenic Strains of
Dermatophytes and Yeast-like Fungi (data from Merkel et al., 1963)
Molar. Concentrations Inhibiting Growth
Compound Tested Dermatophytes Yeast-like fungi
2-Methylbenzothiazole 10~5 10~5
2-Methyl-4-chlorobenzothiazole 10 10
-4 -8
2-Methyl-5-chlorobenzothiazole .10 10
—8 —8
2-Methyl-6-chlorobenzothiazole 10 10
2-Methyl-7-chlorobenzothiazole 10~ 10~
The zinc salt of MET was tested against strains of
Stemphylium sarcinaeforme and Curvularia lunata (Horsfall and Rich, 1951).
The respective concentrations causing germination failure in 50% of the spores
were 8.4 and 5.0 ppm.
117
-------
b. Higher Plants
A number of compounds belonging to the benzazole class (e.g.,
benzitnidazole, benzothiazole, benzotriazole) were evaluated by Klingensmith (1961)
for their effects on growth of seedlings, established plants, and plant tissue
cultures. Because the primary root of a seedling is highly responsive to chemical
modifications, Klingensmith applied benzothiazole and mercapto-S-methylbenzothiazole
to germinating cucumber seedlings in order to determine their effects on elongation
of the primary root (Table 40). In addition, dose-response curves for repression
of root elongation in cucumber seedlings were constructed after treating the plants
with benzothiazole (Figure 10). Benzothiazole was also tested on barley to deter-
mine its effect on the growth response involving the conversion of endosperm to
dry matter in the roots (Figure 11).
Table 40. Repression of Cucumber Root Elongation by Azoles. Elongation of
Primary Root. Cucumber "Early Fortune," 96 hr. at 25°C.
(data from Klingensmith, 1961)
Concentration causing
Compound 50% repression
__
Benzothiazole 5x10 M
-4
Mercapto-S-methylbenzothiazole 4x10 M
118
-------
100
j 80
0
oc
H
Z
8 60
a?
— .
z
o
I- 40
O
z
o
J
uj 20
o
(1)
a^
\
\
\
\
\
\
\
\
\
\
\
\
N
X
\
\
\
N
— \
X
X
X
N
X
s
II II 1
1X10-5 1X10-* 1X10~3 1X10~2
5X10-
5X10-
5X10~3
MOLAR CONCENTRATION
Seedlings grown in presence of benzothiazole at 25°C in darkness for 96 hours.
Figure 10. Effect of Benzothiazole Upon the Elongation of the Primary Root of
Cucumber Seedlings (data from Klingensmith, 1961)
100
o
£E 80
z
8
— 60
X
52
UJ
> 40
oc
Q
20
(2)
-
N
. \
\
\
\
\
\
\
\
\
\
— \
\
\
\
— A
\
\
\
\
II 1 1 1
1X10-5 1X10-" 1X10~3 1X10-2
5X10-5 5X10"4 5X10~3
MOLAR CONCENTRATION
Seedlings grown in aerated deep cultures, 25°C in darkness for six days.
Figure 11. Effect of Benzothiazole Upon Dry Weight of Barley Roots (data from
Klingensmith, 1961)
119
-------
When benzotriazole and benzimidazole were tested for their
effects on inhibition of the elongation of cucumber root, they were only about
one-tenth as active as benzothiazole. In repressing the growth of barley roots,
benzimidazole was only about one-tenth as active as benzothiazole and benzotria-
zole. The mechanism of action for the inhibition of root elongation was not dis-
-4
covered, but attempts to reverse the repression by adding 5x10 M adenine were
not successful. This result indicates that benzothiazole does not act as a purine
anti-metabolite, and therefore inhibition of growth probably does not involve
interference with the synthesis of nucleic acid.
Further effects were noted when established plants were
-2
exposed to benzothiazole. When bushbeans were treated with 1x10 M benzothiazole,
adventitious roots developed along the lower portion of the stem or hypocotyl.
To further explore this phenomenon, a series of plants were subjected to environ-
mental changes around the stem, both with and without the addition of benzothiazole.
-2
The treated plants received 25 ml of 1x10 M benzothiazole applied to the ver-
miculite, and each treatment was replicated six times with two plants in each con-
tainer. The results obtained (Table 41) show that benzothiazole was responsible
for the formation of adventitious roots from the stem. Increased moisture around
the stem, as in deep-planting and when the container top was enclosed with "Saran
Wrap," further enhanced the number of roots emerging.
120
-------
Table 41. Induction of Adventitious Roots in Bushbeans by Root Application of
_2
1x10 M Benzothiazole (18 day old "Black Valentine" Bean Plants,
25 ml. solution applied to the vermiculite) (from Klingensmith, 1961)
Number of plants with adventitious roots
Environment Variable
Control
Deep planting
Stem wrapped with cotton
"Saran Wrap" moist chamber
"No benzothiazole
0
2a
0
0
Plus, benzothiazole
7
10
6
11
Each plant had 1 small adventitious root.
-2 -3
When benzothiazole in solutions of 1x10 M or 3x10
applied to tomato plants, there was an induction of root primordia to within
several inches of the apex in the stem. The application of benzothiazole at
-2
1x10 M to the root medium of tomato also caused dry, dark-brown, sunken lesions
to appear on the upper portion of the stem and on the petioles. Several days
later, the basal leaves began to turn yellow, and the death of the plant usually
resulted in 10 to 14 days.
A series of experiments was also conducted on tobacco stem
segments to which had been added benzothiazole at concentrations of 5x10 M,
-5 -4
5x10 M, and 5x10 M in the culture media. Klingensmith (1961) found that in
-4
stems treated with 5x10 M benzothiazole, not only were buds produced but root
formation was also enhanced. These results were particularly interesting because
root formation is dependent upon the ratio of adenine to auxin, with a high con-
centration of auxin favoring root development. In these experiments, however, ,
exogenous auxin was not added, yet benzothiazole nevertheless was able to induce
121
-------
root growth. Furthermore, at low auxin-high adenine concentrations, bud develop-
ment is favored. In the benzothiazole-treated tobacco segments, however, sections
which produced roots also had more buds than the controls.
-4
Observations that MET at concentrations of 10 M would
completely inhibit the browning reactions in banana pulp were shown by Palmer
and Roberts (1967) to be due to an inhibition of the enzyme banana polyphenoloxidase
(PPO). They suggested that MET inhibition of PPO, a copper metalloenzyme, could
be due to interactions between MET and the enzymically bound copper. This argu-
I |
ment is strengthened by the fact that addition of Cu to the assay system could
reduce or completely overcome the inhibition by MET. These results are consistent
with observations by Wang and Mellenthin (1974) who demonstrated that friction
discoloration of d'Anjou pears could be prevented by MET treatment, and was appar-
ently due to inactivation of the PPO of the pear. In higher animals, the ability
of MET to interact with copper metalloenzymes has also been postulated (see
Section II-B-2-c, p. 79).
6. Effects on Microorganisms
A large number of benzothiazole derivatives have been synthesized
and tested for antibacterial activity, and many of these compounds are very effec-
tive bactericidal and bacteriostatic agents. Hundreds of benzothiazole deriva-
tives have been tested as possible chemotherapeutic agents with antibacterial
or antiviral effectiveness, including benzothiazole basic ethers (Cossey et al.,
1966), substituted alky! (2-benzothiazolylthio) acetates and alkyl (6-x-2-
benzothiazolylthio) formates (Mikulasek et al., 1974), and various substituted
chlorobenzothiazoles (Logemann et al., 1961).
122
-------
In a study conducted by Foltinova and Blockinger (1970), it was
demonstrated that MET, MBT thiolates together with metals, and 6-nitro-2-
mercaptobenzothiazole are all effective against G+ and G- bacterial strains.
The inhibitory concentrations of MBT and its derivatives on bacterial growth are
presented in Table 42. Table 43 summarizes the effectiveness of MBT against a
number of mycobacterial strains.
Recent studies were conducted by Aktulga (1972) in which a number
of ingredients found in pharmaceutical rubber closures were tested for inhibitory
action on microorganisms. Among these substances were MBT and 2,2'-dithiobisben-
zothiazole. Bacteria culture media was prepared in petri dishes and inoculated
with young broth cultures of several bacterial strains. Both compounds were
sterilized and placed on the agar plates at a concentration of either 2 or 5 mg
per plate. The diameters of the zones of growth inhibition produced by the two
chemicals are presented in Table 44.
123
-------
Table 42. Effect of MET and Several Derivatives on Staphylococcus aureus and
Escherichia coli (from Foltinova and Blockinger, 1970)
Staphylococcus aureus
Compound
Growth
of
Control
I
II
III IV V
Escherichia coli
Growth
of
Control I II III IV
V
MET
6-N02-2-MBT
2Cu+2-MBT
2Ag-MBT
2Hg+2-MBT
2Pb+2-MBT
2Bi+3-MBT
4- —
I =1000 yg/ml
II = 500 ug/ml
III = 250 pg/ml
IV = 100 pg/ml
V = 50
124
-------
Table 43. Antimycobacterial Effects of 2-Mercaptobenzothiazole (data from Foltinova and Blockinger, 1970)
Undefined Media
Synthetic Media
Organism
M. tuberculosis-
H3? Rv
M. tuberculosis-
H Rv IR
(isonicotyl hydrazide
resistant variant)
M. tuberculosis-
~H Rv SR
(streptomycin
resistant variant)
BCG (attenuated strain
of M. tuberculosis)
M. bov±s
M. avium
M. kansasii
M. phlei
Growth Complete Partial Growth Complete Partial
of Inhibition Inhibition of . Inhibition Inhibition
Control (ug/ml) (yg/ml) Control (pg/ml) (ug/ml)
250
500
500
100
250
500
500
>1000
100 +
250 +
250 +
50 +
100 +
250 +
250 +
1000 + + +
100
250
250
50
100
250
250
>500
50 +
100 +
100 +
25
50
100
100
500
+
+
+
+
+ + +
-------
Table 44. Antibacterial Effect of MET and 2,2'-Dithiobisbenzdthiazole
(from Aktulga, 1972)
Test Org.ni ism
Compound
MBT
2,2' -Dithlobisbenzothiazole
Amount
2 mg
5 mg
2 mg
5 mg
Staphylococcus
aureus
zone diameter
(mm)
1
2
-
~
Escherichia
coli
zone diameter
(mm)
1
2
-
"~~
Corynebacterium
diptheriae
zone diameter
(mm)
4
large
2
2
Vseudomonas
aerufiinosa
zone diameter
(mm)
_
—
_
~
As indicated by Table 44, MBT is strongly inhibitory toward
Corynebacterium diptheriae and considerably less effective against the other bac-
terial tribes tested. Antibacterial properties for 2,2'-dithiobisbenzothiazole
could be demonstrated only against Corynebacterium diptheriae in this study.
7. In Vitro and Biochemical Studies
A cell culture evaluation of the toxicity of MBT and 2-(2-
hydroxyethylmercapto) benzothiazole (HMBT) was conducted by Guess and O'Leary (1969)
using the NCTC-929, strain L mouse fibroblast cell line (L-929) and 10-day chick
embryo cells. Test plates were made by overlaying confluent cell lines with
nutrient agar and staining with a vital dye. Toxicity was evident as an all-or-none
126
-------
response when the killed cells released the vital stain to create clear zones
around the test material. Any zone of dead cells surrounding the test material
was regarded as a positive response, regardless of its size. Table 45 summarizes
the results from testing the pure compounds, and various oil solutions and satura-
ted aqueous solutions of MBT and HMBT.
Table 45. Cell Culture Evaluation of MBT and HMBT (from Guess and O'Leary, 1969)
Sample
Pure compound
sssb
SSS, 1:2 dilution
SSS, 1:4 dilution
Oil suspensions
12 mg/ml
6 mg/ml
3 mg/ml
1.5 mg/ml
Oil control
Saline control
L cells
Pos.
Neg.
Neg.
Neg.
Pos.
• Pos.
Pos.
Neg.
Neg.
Neg.
MBT3
C.E. cells
Neg.
Neg.
Neg.
Neg.
Pos.
Pos.
Neg.
Neg.
Neg.
Neg.
L cells
Pos.
Pos.
Pos.
Neg.
Pos.
Pos.
Pos.
Neg.
Neg.
Neg.
HMBT3
C.E. cells
Pos.
Pos.
Pos.
Neg.
Pos.
Neg.
Neg.
Neg.
Neg.
Neg.
Pos. = positive, toxic response; Neg. = negative, nontoxic response.
SSS = saturated saline solution. SSS for MBT = 0.5 mg/ml; SSS for HMBT = 0.8 mg/ml.
Judging by the above results, the authors felt that MBT and
HMBT should be considered to have a fairly low order of toxicity, based on the
unusally high sensitivity to toxicants of isolated cells in culture. Where cellu-
lar damage occurred, it was generally evident as cellular irritation with HMBT
treatment, and as vacuolization with MBT treatment.
127
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IV. Regulations and Standards
A. Current Regulation
Regulation and control over derivatives of MET are provided under
several different authorities. Because this group of compounds is involved in a
number of applications, product control at the federal level is varied. Effluent
control, on the other hand, is exercised under basically the same authority for
all these chemicals.
The use of MET as an agricultural fungicide is regulated under the
Federal Environmental Pesticide Control Act of 1972, which has revised the Federal
Insecticide, Fungicide, and Rodenticide Act of 1947 (7 U.S.C7 135-135k), Toler-
ances for pesticide chemical residues in or on raw agricultural commodities have
been established under the Federal Food, Drug, and Cosmetic Act (21 U.S.C. 346a).
Specific tolerances for MET have been stated in the Federal Register (22;9258,
November 28, 1957). A tolerance of 0.1 ppm was established for MET, calculated
as 2,2'-dithiobisbenzothiazole, in or on apples.
A tolerance has also been established for residues of 2-(thiocyano-
methylthio)benzothiazole under the Federal Food, Drug, and Cosmetic Act as detailed
in 40CFR 180.288. Tolerances allow for residues of 0.1 ppm in or on barley, corn,
cotton forage, cottonseed, oats, rice, safflower, sorghum, sugarbeets, and wheat.
By amendment to the Federal Food, Drug, and Cosmetic Act, the rubber
accelerator l,3-bis(2-benzothiazolylmercaptomethyl)urea is permitted as a com-
ponent of rubber articles intended for repeated use in contact with food (Federal
Register 30:3207, March 9, 1965) and as a component of food-packaging adhesives
(Federal Register 30:7386, June 4, 1965).
128
-------
In a report abstracted from the Russian literature, Vaisman et al.
(1973) have recommended that undissolved MET and MBTS be absent from waste
waters.
The transportation of hazardous materials by rail and highway is
regulated by the Hazardous Materials Regulation Board of the Department of
Transportation. In January, 1974, the Department of Transportation proposed
extensive changes in the rules governing the transport of hazardous chemicals,
especially by air. These changes, published in the Federal Register (January 24,
1974) listed a large number of chemical substances. However, no derivatives
of MET are included on this list or classified as being hazardous.
B. Concensus and Similar Standards
Limits have been established for maximum permissible exposure to
hazardous chemicals by several agencies. Threshold limit values (TLV's) for
chemicals in the workroom environment have been established by the American
Conference of Governmental Industrial Hygienists. These TLV's, which are
revised and updated each year, do not, however, include MET or any of its
derivatives.
Exposure limits for hazardous substances have also been set by the
Occupational Safety and Health Administration and the National Institute for
Occupational Safety and Health. Their list of standards which has been pub-
lished in the Federal Register (October 19, 1972) does not include any deriva-
tives of MET.
129
-------
V. Summary and Conclusions
Mercaptobenzothiazole compounds are important commercial chemicals which
are produced in considerable quantities. There are at least sixteen compounds
which have the 2-mercaptobenzothiazole moiety and are produced in commercial
quantities. The major commercial chemicals include sodium 2-mercaptobenzothia-
zole (NaMBT), which is used mostly for anticorrosion applications; mercaptobenzo-
thiazole (MET), 2,2'-dithiobisbenzothiazole (MBTS), zinc 2-mercaptobenzothiazole
(ZMBT), N-cyclohexyl-2-benzothiazolesulfenamide (CBS), and a number of other
sulfenamide derivatives, which are used almost exclusively as vulcanization
accelerators. In general, the compounds are chemically stable, water insoluble,
solids which will decompose by free radical mechanisms at vulcanization temper-
atures to form a variety of compounds, including the parent compound, benzothia-
zole. The sodium salt is somewhat unusual in that it is water soluble (the
other salts are insoluble).
Approximately 100 million pounds of MBT compounds are produced annually.
Individual production figures are available for NaMBT (11.9 million pounds -
1973), CBS (4.6 million pounds - 1974), MBT (6.1 million pounds - 1974), MBTS
(20.7 million pounds - 1974), and ZMBT (4.0 million pounds - 1972).
The sodium salt (NaMBT) is used as a corrosion inhibitor in water based
cooling systems and as a chemical intermediate for some of the other MBT deriva-
tives. Approximately 3 million pounds per year is consumed in automobile cool-
ing systems, and since antifreeze solutions are frequently changed, the release
to the environment is probably quite substantial. The quantity lost from other
stationary cooling systems (~ 3 million pounds per year) may also be quite high,
depending upon how frequently the coolant is changed.
130
-------
The largest use of MET compounds is for vulcanization accelerators. These
chemicals are added at concentrations of 0.5 - 1.5% before vulcanization, in order
to allow the process to take place in a reproducible and uniform fashion at lower
temperatures. It appears that substantial amounts of these chemicals may be re-
leased to the environment during use in rubber processing and in the use and
disposal of rubber products. MET, MBTS, and benzothiazole have all been detected
in effluents from rubber manufacturing plants in the U.S. and USSR. The quan-
tities lost are unknown (0.027 ppm and 0.049 ppm have been detected in the dis-
charge from a tire plant treatment pond), but the rubber manufacturing capacity
in the U.S. is approximately 7,500 million pounds, so the quantities could be
substantial.
Approximately 1.2 billion pounds of rubber dust are worn from automotive
tires each year in the U.S. , which amounts to 12 million pounds of MET compounds
(including MET, MBTS, and benzothiazole) being released each year, based on an
average of 1% accelerator in the rubber. The accelerators are probably leached
out of this dust by water in the environment, since it has been shown that
accelerators can be rapidly dissolved out of rubber products with distilled
water. MET compounds may also be leached out of rubber products that are dis-
carded in garbage dumps, landfills, or waterways.
Very little is known about the environmental fate of MET compounds. No
experimental data on persistence or bioaccumulation are available. Calculations
of bioaccumulation potential based upon physical properties would suggest that
the compounds probably do not concentrate in higher food chain organisms. The
monitoring data provide little clarification of the question of environmental
fate. So far, the following compounds have been found in raw or drinking water:
benzothiazole, 2-methylbenzothiazole, and 2-thiomethylbenzothiazole. However,
in most cases, the analytical procedure and concentrations have not been men-
tioned and the chemical nomenclature is confusing.
131
-------
An accumulation of direct evidence and a cautious extrapolation of experi-
mental data indicate that MET and its derivatives may influence several important
biologic parameters in all living organisms. Principally, the actions of MET
derivatives in human and animal systems may involve at least three possible effects:
(1) the production of allergic contact dermatitis, (2) action on the central
nervous system, and (3) inhibition of certain metalloenzymes which contain
copper.
The results of acute toxicity determinations, although somewhat variable
and markedly influenced by route of administration, demonstrate that single dose
exposure and the capacity to produce immediate death is not the major threat to
health posed by these compounds. Indeed, it may be concluded that the acute
toxic hazard of MBT derivatives when administered by the routes of probable
environmental exposure (e.g., oral, dermal, inhalational) is minimal. Among
the chemicals of the benzothiazole class, unsubstituted benzothiazole and
several of the 2-amino- and benzene ring-substituted derivatives are considerably
more toxic than the rubber accelerator chemicals containing a 2-position sulfur
linkage.
The production of allergic contact dermatitis by exposure to MBT in domestic
situations has occurred with relatively high frequency. However, there is little
evidence of occupational disease which can be attributed solely to contact with
MBT or its derivatives. Both animal and man have been shown to develop sensi-
tivity to MBT when applied to the skin. The dermatitic reaction elicited by MBT,
while not particularly severe, occurs with enough frequency to warrant classifying
MBT as one of the most common contact allergens in use today. Exposure of humans
to this substance is accomplished almost exclusively through its use as a component
132
-------
in rubber products. In addition, where sensitivity to MET has been achieved
in man, cross-sensitivity may also be shown to other chemicals containing the
MET moiety.
The potent action on the central nervous system of numerous derivatives
of benzothiazole, including MET, has only been shown to occur in animals. These
effects can be manifested as either a central stimulation (convulsions) or as
selective depression (flaccid paralysis, mental depression, lack of spontaneous
motor activity). The mechanism of these actions has not been investigated, nor
have studies been performed to determine the extent of transport and accumu-
lation of benzothiazole compounds in tissues of the central nervous system.
The effects of benzothiazole derivatives on central nervous system function
may well be due, in part, to their interaction with specific enzymes. By acting
as a copper chelating agent, MET was shown in mice to inhibit a metalloenzyme
which is responsible for the conversion of dopamine to noradrenaline (an important
neurohumoral transmitter substance). Symptoms of central nervous system de-
pression could be correlated with levels of noradrenalin in the brain as in-
fluenced by MET exposure. These results are clearly suggestive of the need to
investigate other benzothiazole and MET derivatives with respect to their po-
tential role in disruption of enzyme function.
Feeding studies in mice have been conducted on benzothiazole, MET, and
several MET type rubber accelerators which demonstrated that these compounds do
not appear to present a significant carcinogenic threat. However, limited data
from mutagenesis assays in the fruit fly suggest that several MET compounds may
possess mutagenic properties. Additional testing using a more reliable system
(e.g., Ames assay) will be necessary to more clearly predict possible genetic
damage or other reproductive hazards in humans.
133
-------
In summary, it appears that substantial quantities of MET derivatives
and benzothiazole are being released to the environment. Their environmental
fate is unknown, and the available monitoring data are not detailed enough to
suggest the degree of contamination. More information in these areas is needed
before a definitive assessment of the environmental hazard associated with the
commercial use of MET compounds is possible.
134
-------
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146
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TECHNICAL REPORT DATA
(Please rend Instructions on ilic reverse before completing/
1. REPORT MO.
EPA-560/2-76-006
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE ANDSUBTITLE
Investigation of Selected Potential Environmental
Contaminants: Mercaptobenzothiazoles
5. REPORT DATE
June 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHORIS)
Joseph Santodonato, Leslie N. Davis,
Philip H. Howard, Jitendra Saxena
8. PERFORMING ORGANIZATION REPORT NO
TR 76-502
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Center for Chemical Hazard Assessment
Syracuse Research Corporation
Merrill Lane, University Heights
Syracuse, NY 13210
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
EPA 68-01-3128
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report reviews the potential environmental hazard from the commercial
use of 2-mercaptobenzothiazoles (MET). Most of the mercaptobenzothiazoles are
consumed as rubber accelerators in vulcanization processes, although the sodium
salt of MET is used as a corrosion inhibitor in water-based cooling systems.
Information on physical and chemical properties, production methods and quantities,
commercial uses and factors affecting environmental contamination, as well as
information related to health and biological effects, are reviewed.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
2-mercap tobenzothiazole
2,2'-di thiobisbenzothiazole
N-cyclohexyl-2-benzbthiazole
rubber accelerators
corrosion inhibitors
b.lDENTIFIERS/OPEN ENDED TERMS
benzothiazolesulfenamide
c. COSATI Held/Group
18. DISTRIBUTION STATEMENT
Document is available to public through
the National Technical Information
Service. Springfield, Virginia
19. SECURITY CLASS (This Report)
21. NO. OF PAGtS
146
20..SECURITY CLASS (This page.)
22. PRICE
EPA Form *ZZO-1 (9-73)
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