KTHYLENE GLYCOL, DIETHYLENE GLYCOL, DIOXANE AND RELATED COMPOUNDS
CARCINOGENICITY AND STRUCTURE-ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
Yin-tak Woo, Ph.D.
Joseph C. Arcos, D.Sc ., and
Mary F. Argus, Ph.D.
Preparation for the Chemical Hazard
Identification BrancH "Current
Awareness" Program
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Table -of Contents;
5.2.2.5 Phenols and Phenolic Compounds
5.2.2.5.1 Introduction
5.2.2.5.2 Physicochemical Properties and Biological Effects
5.2.2.5.2.1 Physical and Chemical Properties
5.2.2.5.2.2 Biological Effects Other Than Carcinogenic
5.2.2.5.3 Carcinogenicity and Structure-Activity Relationships
5.2.2.5.3.1 Overview
5.2.2.5.3.2 Simple (Monocyclic) Phenolics
5.2.2.5.3.2 Polynuclear Phenolics
5.3.2.5.3.3 Phenolics as Modifiers of Chemical Carcinogenesis
5.2.2.5.4 Metabolism and Mechanism of Action
5.2.2.5.5 Environmental Significance
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5.2.2.5 Phenols and Phenolic Compounds.
5.2.2.5.1 Introduction.
Phenols and phenolic compounds (hereafter collectively called phenolics)
represent an important class of compounds that helped to formulate some of the
basic concepts of chemical carcinogenesis. Depending on their chemical struc-
ture, phenolics may have complete carcinogenic, tumor-initiating, tumor-
promoting, or cocarcinogenic activity. Some phenolics inhibit the carcino-
genic activity of certain carcinogens, suggesting the possibility of their use
as chemoprophylactic agents. Phenolics are ubiquitously present in the
environment due to their natural occurrence as well as extensive industrial
uses. This section focuses on comercially used phenolics and some naturally
occurring simple phenolics; botanical phenolics with complex structures will
be discussed in Section 5.3.2.
Owing to their desirable physicochemical properties and biological
activities, phenolics have found numerous applications as antioxidants,
stabilizers, antibacterials, preservatives, solvents, redox reagents, pharma-
ceuticals, chemical intermediates, food flavoring agents, etc. Consumer
products containing phenolics include processed foods, cosmetics, dyes, drugs,
photographic chemicals, pesticides, gasoline, lubricants, adhesives, disinfec-
tants, soaps, paints and paint removers (see also Section 5.2.2.5.5). With an
annual production of 2.38 billion Ibs. in 1978, phenol ranked 38th in produc-
tion volume among U.S. chemicals (cited in ref. 1). The annual U.S. consump-
tion of phenolic antioxidants is approximately 1,400, 450 and 230 metric tons
for butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and propyl
gallate, respectively (2). Other phenolics with production volume exceeding
one million Ibs. in recent years include bisphenol A, 2,4-dichlorophenol,
-------
pentachlorophenol, cresols, resorcinol and hydroquinone. Eugenol, a widely
used food flavor and fragrance additive, had a production volume of 425,000
Ibs. in the United States in 1978 (cited in ref. 3). In addition to the above
consumer products, human exposure to phenolics may also occur through polluted
air or water, tobacco smoke, natural foodstuffs or indirectly via exposure to
aromatic compounds which may be hydroxylated to phenolics in the body.
As may be expected from their wide occurrence in consumer products and in
the environment, human'exposure to some phenolics may be extensive. The
potential carcinogenic risk of such exposure does not seem to have attracted
much attention until recently. The general lack of high concern may be due to
the facts that (a) phenolics (e.g., norepinephrine, tyramine, dopamine, homo-
gen tis tic acid) are normal constituents of animal and plant tissues where they
play a role in metabolic regulation (4), (b) aromatic hydroxylation is
generally assumed to-be "detoxifying" in nature, and (c) some phenolics may
inhibit carcinogenesis (see Section 5.2.2.5.3.4). However, recent mutagenic-
ity studies have indicated that some types of phenolics may be more hazardous
than generally assumed. Metabolic studies on benzene (see Appendix I for its
carcinogenicity) have implicated polyhydric phenols as the reactive interme-
diates. This section discusses the carcinogenicity and mutagenicity data on
phenolics according to the type of substituents present in the ring, the
possible chemical or biochemical mechanisms leading to potential reactive
intermediates, and the tumor-promoting, cocarcinogenic and inhibitory activi-
ties.
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5.2.2.5.2 Physicochemical Properties and Biological Effects.
5.2.2.5.2.1 PHYSICAL AND CHEMICAL PROPERTIES.
The physical and chemical properties of phenolics have been described in
several excellent reviews (5-16) and in standard textbooks on organic
chemistry (e.g., 17). The physicochemical properties of some widely used
simple phenolics are summarized in Table A. The structural formulas of the
phenolics discussed in this section are depicted in Table B and D. In
general, phenols are more polar, can form stronger hydrogen bonds, and have a
greater ability to act as solvents for polar organic molecules, than their
corresponding saturated alcohols (e.g., cyclohexanol). The hydroxyl group of
phenol (pKa=9.9) is considerably more acidic than that of cyclohexanol (pKa
approximately 18). The greater acidity of phenols is attributed to the
greater stability of phenoxide anion in which the unshared electron pair is
delocalized because of resonance:
V
[text figure 1]
The acidity of phenols is greatly enhanced by substitution with electron-
withdrawing atoms or groups (e.g., -NO^, -Cl), whereas electron-donating
substituents (e.g., -R, -OH, -OR, -NH2) tend to have an opposite effect (see
Table A). The partition coefficient (log P) -f phenols is increased by
substitution with halogen, alkyl or nitro group(s) whereas highly polar
substituents (such as a hydroxyl group) may lower the log P. Both pKa and log
P may affect pharmacokinetic properties and biological effects of phenolics.
-------
Table A. Physicochomical Properties of Phenol and its Derivatives'
p. 1 ot 2
Compound
Phenol
o-Cresol
m-Cresol
p-Cresol
2,4-Xylenol
2-Chlorophenol
2,4-Dichlorophenol
2,4, 5-Trichlorophenol
2,4, 6-Tr ichlorophenol
2.3,4, 6-Te trachlorophenol
Pentachlorophenol
2-Ni trophenoll
j i
1 '
2 , 4-Dini trophenol
m.p .
(°C)
40.9
30.9
12.0
34.8
27-28
8.7
43-44
68
68
69-70
190
44-45
114-115
b.p. Vapor Pressure Solubility0
(°C) (mm Hg) (gm/100 ml H20) pKa logpoct
182
191
203
202
210
175
210
245
246
64
(at 22 mm Hg)
309-310
214-216
sublimes
1.27 at 41°C
0.25 at 25°C '.
i
0.15 at 25°C
0.11 at 25°C !
I
1 at 52.8°C
1 at 12.1°C
1 at 53°C
1 at 72°C
1 at 76.5°C
—
0.12 at 100°C
1 at 49°c
—
6.6
2.5
2.2
1.9
—
<0.1
0.45
0.12
0.09
0.10
0.0014
0.032
0.079
9.89
—
—
9.82
10.6
8.49
7.68
7.43
7.42
5.38
(20°C) 4.96
(38°C) 7.13
4.0
1.46
1.95
1.96
1.94
—
2.15
3.06
3.72
3.62
4.10
5.01
1.77
1.51
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Table A. Physicochemical Properties of Phenol and Its Derivatives
(continued)
p. 2 of 2
Compound
m.p.
b.p. Vapor Pressure Solubility0
(°C) (mm Hg) (gm/100 ml HZO) pKa
Picric acid
Catechol
Resorcinol
Hydroquinone
122-123
105
111
173-174
300
245.5
280
l.at 195°C
0.67 (100 C) 0.8
volatile in steam soluble
53 at 190 C
285 4 at 150°C
(at 730 mm Hg)
58.8\
7.0
9.48
9.44
/
/ 10.0
2.03
0.78
ummarized from data compiled in Kirk-Othner Encyclo.Ch.ein. Tech. (3rd ed.)^5_, 864 (1979J) and \T_, 373
(1982); Intern. Agcy. Res. CancerTionog. , 15, 155 (1977); Ha tl.jTnst. Occup. Safety Health (HIOSH) Publ.
Nos. 78-133 (Cresol) and 78-155 (Hydroquinone); U.S. Environ. Protection Agcy. (EPA) Publ. Nos. 440/5-80-032
(Chlorinated phenols), 440/5-80-044 (2,4-Dimethylphenol) and 440/5^80-063 (Nitrophenols) ; "Merck Index" (9th
ed.), Merck and Co., Rahway, NJ, 1976; Y. Yasuda, K. tochikubo Y, Hdshicuka et ai., J.^Mied. Chem. 25j 315
(1982); C. Hansch and A. Leo, "Substituent Constants for Correlation^ Analysis in Chemistry and BioloRy,"
Wiley, New York, 1979.
For structural formulas, see Tables B and D.
cUnless specified, solubilities are measured at 25°C*
Logarithm of water-octanol -partition coefficient.
eSoluble in 2.3 parts water
-------
OH
OH
Phenol
OH OH OH
1,8,9-trihydrory-
onthracene form
BHT (
BHA (R=CH30)
Cl OH OH Cl
Cl Cl Cl Cl
Hexachlorophene
OH 0 OH
1,8-dihydroxy-
9-anthrone form
Anthralin
CH,Q
1' 2' 3'
HO-/ VcH2-CH=CH;>
Eugenol
765
Hydroxybenzo[a] pyrene
Table B. Structural Formulae of Some Phenolics that have been
Tested for Carcinogenicity.
-------
e
0
• M •
G
Text-Figure 1
-------
Phenols (and phenoxide anions) are very susceptible to electrophilic
substitution. As nay be expected from the resonance structures of phenoxide
«.
anion, electrophilic agents preferentially attack positions ortho or para to
the hydroxyl group. Polyhydric phenols are even more susceptible to electro-
philic aromatic substitution, especially if the hydroxyls are meta to one
another (e.g., resorcinol, phloroglucinol), in which case, their activating
influences reinforce each other.
One-electron oxidation generates from phenols highly reactive phenoxy
free radicals, the odd electron of which is delocalized. The positions of the
odd electron may be represented by the resonance structures:
[ text figure ?'
Depending on the nature and positions of substituents on the aromatic ring,
phenoxy radicals may undergo (a) dimerization or coupling reactions yielding
dihydroxybiphenyls, hydroxyphenol phenyl ethers or diphenyl peroxides (revers-
ible), or (b) disproportionation reaction yielding phenolic and ketonic
products. The stability of phenoxy radicals may be enhanced by substitution
with bulky alkyl groups. For example, the radical of 2,4,6-t-butylphenol is
reasonably stable in benzene as indicated by the color of the solution and the
chemical properties of the solute.
In addition to being more susceptible to electrophilic aromatic substitu-
tion than phenols, polyhydric phenols are also more easily oxidized. For 1,2-
and 1,4-dihydroxybenzene (e.g., catechol and hydroquinone), 1,2- and 1,4-
benzoquinone are the final oxidation products. In fact, quinones and poly-
-------
0
'0'
Text-Figure 2
-------
hydric phenols are often readily interconvertible. It is important to point
out that semiquinones are formed during the interconversions if the electrons
are transferred one at a time. Semiquinones are reactive electrophilic inter-
mediates; 'they may also undergo reversible dimerization to form peroxides.
[text figure 3]
In contrast to 1,2- and 1,4-dihydroxybenzene, 1,3-dihydroxybenzene does not
give rise to quinone upon oxidation because 1,3-quinone cannot assume an
unstrained planar structure. Oxidation of resorcinol yields complex products
probably by way of attack at the 4-position, which is activated by being ortho
to one hydroxyl and para to the other (17).
Polycyclic phenolic compounds are generally more reactive and have a
greater tendency to tautomerize from enolic to ketonic structure than mono-
cyclic phenolics (6). The naphthols share many reactions in common with
resorcinol. 1,4-Naphthaquinol exists in two tautomeric forms:
[ text figure 4]
which are stable under ambient temperature conditions and can be isolated.
The hydroxy derivatives of anthracene show a greater tendency to exist in the
tautomeric keto form. Studies by Segal _e_£al. (18) indicate that anthralin
(1,8,9-trihydroxyanthracene) exists predominantly in the ketonic anthrone form
(see Table B) .
-------
_
-e
semiqumone
Text-Figure 3
-e
0
0
OH
OH
Dienol form
Diketo form
Text-Figure
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5.2.2.5.2.2 BIOLOGICAL EFFECTS OTHER THAN CARCINOGENIC.
Toxic effects. The toxicity of phenolics has been extensively studied
because of decades of human exposure to these compounds. The literature is
replete with reviews and monographs on the toxicology of phenol (5, 15, 16,
19, 20), chlorophenols (5, 9-11, 14, 16, 21-23), nitrophenols (13, 24), alkyl-
phenols (12, 16, 25), polyhydric phenols (7, 16, 26), phenolic antioxidants
(19, 27) and hexachlorophene (28-30). Representative acute toxicity data of
some important phenolics are summarized in Table C. As the data in the Table
indicate, their toxicity is strongly influenced by the type and position(s) of
the ring substituents. In general, substitution with alkyl or alkoxy groups
tend to decrease the toxicity, especially with multiple substitution with
bulky groups. Chlorophenols are more toxic than phenol itself and toxicity
increases with an increase in the extent of chlorination. The mode of toxic
action changes from CNS effects (tremors, convulsion) by less chlorinated
phenols (which have higher pKa and are therefore largely in undissociated form
at body pH) to uncoupling of oxidative phosphorylation by highly chlorinated
phenols. Dinitrophenols are considerably more toxic than mononitrophenols.
The relative toxicity of the dinitrophenol isomers follows the order: 2,4- >
2,6- > 3,5- > 3,4- > 2,5- > 2,3-isomer (45). This order appears to correlate
with the residence time of the isomers in the body (45) and their potency as
uncouplers of oxidative phosphorylation (46). The importance of the position
of ring substitution on the toxicity of phenols is exemplified by dihydroxy-
benzenes. Both 1 ^-d^vdroxybenzene (catechol) and 1,4-dihydroxybenzene
(hydroquinone) are more toxic than their 1,3-isomer (resorcinol). It is note-
worthy in this connection that both catechol and hydroquinone can be oxidized
to quinones whereas resorcinol cannot.
-------
i Ol L
Table C. Acute Toxicity of Phenols and Phenolic Compounds
V
Compound
Phenol - -
o-Cresol
m-Cresol
p-Cresol
2,4-Xylenol
Thymol
Butyl a ted hydroxy toluene
Butylated hydroxyanisole
Eugenol
2-Chl orophenol
3-Chlorophenol
4-Chlorophenol
2 , 4-Dichlorophenol
2,4,5-Trichlorophenol
2,4, 6-Trichlorophenol
2,3,4, 6-Te trachlorophenol
Species & Route
Mouse, oral
Rat, oral
Rat, topical
Rabbit, topical
Mouse, oral
Rat, oral
Rabbit, topical
Mouse, oral
Rat, oral
Rabbit, topical
Mouse, oral
Rat, oral
Rabbit, topical
Mouse , oral
Rat, oral
Rat, oral
Rat, oral
Mouse, oral
Rat, oral
Mouse, oral
Rat, oral
Rat, oral
Rat, i.p.
Rat, i.p.
Rat, i.p.
Rat, oral
Rat, i.p.
Ra t , i.p.
Rat, i.p.
Mouse, oral
Mouse, i.p.
LD5Q (mg/kg)a
520; 436
530; 650
670
850; 1,400
344
1,470
890
828
2,010
2,830
344
1,460
300
809
3,200
980
1,700 (M); 1,970 (F)
2,000
2,200
3,000
2,680
670
230
335
250
580
430
355
276
131
82
Reference
(19,
(7,
(33)
(7,
(31)
(31)
(34)
(31)
(31)
(34)
(31)
(31)
(34)
(31)
(31)
(35)
(36)
(19)
(19)
(35)
(35)
(37)
(38)
(38)
(38)
(37)
(38)
(38)
(38)
(39)
(39)
31)
32)
34)
-------
p. 2 of 2
Table C. Acute Toxicity of Phenols and Phenolic Compounds
(continued)
Compound
Pen tachlorophenol
2-Ni trophenol
3-Ni trophenol
4-Ni trophenol
2, 4-Dini trophenol
Vanillin
Sodium salicylate
Catechol
Resorcinol
Hydroquinone
Bisphenol A
Hexachlorophene
Species & Route
Mouse , oral
Mouse, i.p.
Rat, oral
Rat, topical
House, oral
Rat, oral
Mouse , oral
Rat, oral
Mouse , oral
Rat, oral
Rat, oral
Rat, oral
Rat, i.p.
Mouse, oral
Rat, oral
Rabbit, topical
Rat, oral
Rabbit, oral
Mouse , oral
Rat, oral
Mouse, oral
Rat, oral
Rat, oral
LD5Q (mg/kg)a
74
32
146 (H); 175 (F)
320 (M); 330 (F)
1,300
2,830
1,410
930
470
620
30
1,580
780
260
300
800
980
3,360
400
320
2,500
4,240
66 (M); 56 (F)
Reference
(39)
(39)
(40)
(40)
(34)
(34)
(34)
(34)
(34)
(34)
(41)
(35)
(42)
(19)
(7)
(7)
(7)
(7)
(19)
(19)
(43)
(43)
(44)
aM = male; F = female
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Several investigators have attempted to establish a quantitative struc-
ture-toxicity relationship of phenols. In a 1970 report, Gubergrits and Kirso
(47) derived a modified form of Hammett-Taft equation* which correlates the
electron density in the reactive hydroxyl group regions with the toxicity of
phenols. Unfortunately, insufficient details of the study were given to allow
critical assessment. Stockdale and Selwyn (48), Tollenacre (49), and Motais
et al. (50) concur that a correlation exists between the activity of phenols
as uncouplers of oxidative phosphorylation and their electronic and hydro-
phobic bonding properties. Mizutani _e_t _al_. (51) have delineated the struc-
tural requirements of butylated hydroxytoluene (BHT) and related compounds as
pulmonary toxicants. The two essential requirements appear to be: (i) a
methyl group para to OH, and (ii) ortho-alkyl group(s) which represent steric
hindrance against interactions with the OH group. Based on these structure-
activity relationships and available metabolic data, they proposed the forma-
tion of _p_-quinone methide (see Section 5.2.2.5.4) as reactive toxic interme-
diate.
Mutagenicity. Phenolics have been known for decades to be mi to tic
spindle poisons^in plant tissues. Early studies on the mutagenicity of 15
^
phenols were reviewed by Dean (52) in 1978. Since then, over 100 phenolics
have been tested for mutagenicity in various test systems. The following
discussion focuses on studies using the Ames test and mammalian cells with
emphasis on structure-activity relationships.
An enormous amount of information on the mutagenicity of phenols (as
.detected in the Ames test) has become available in the past few years from the
*For a detailed discussion of Hanunett equation, see p. 271 of Volume I.
-------
testing of cosmetics ingredients, food additives, water pollutants, tobacco
smoke condensates, shale oil products and various environmental samples. The
major findings in these studies are summarized in Table D. Unsubstituted
phenol (Group A in Table D) is nonmutagenic according to most investigators,
although one study (57) indicates that phenol induces frame-shift mutation
(strain TA98) following metabolic activation. Alkyl- and alkoxyphenols (Group
B in Table D) are generally inactive with the possible exceptions of eugenol
and isoeugenol, which have an allyl and a propenyl sidechain, respectively.
It is possible that the mutagenicity of these two compounds is associated with
their unsaturated sidechains; indeed, the epoxide of eugenol (eugenol-21,3'-
oxide) is a direct-acting mutagen (61). Hone of the chlorophenols (Group C in
Table D) tested were found to be mutagenic; however, it is not clear whether
the testing of the more highly chlorinated phenols has been adequate because
of their limited solubility. Several nitro/aminophenols (Group D in Table D)
are mutagenic. These compounds all have at least two positions (one of which
ortho to the hydroxyl group) substituted with either nitro or ainino groups, or
one of each. Since a variety of substituted benzenediamines and nitrobenzenes
are mutagenic or carcinogenic (64, 67, 74, 75), the phenolic hydroxyl group
appears to play only a modifying role. With one exception, none of the
hydroxybenzaldehydes, hydroxyacetophenones and hydroxybenzoic acids (Group E
in Table D) were found rautagenic. Rapson ^_t _al_. (54) listed 3,6-dichloro-2-
hydroxybenzaldehyde, but not its 3,5-dichloro-isomer, to be mutagenic. Some-
what conflicting results have been obtained in studies on polyhydric phenols
(Group F in Table D). Ben-Gurion (73) found pyrogallol mutagenic in strains
TA100 and TA1537, but addition of liver S-9 mix reduced the activity. This
finding was supported by the data of Gocke j5£al. (57) that three polyhydric
phenols: resorcinol in TA1535A (+S9) and TA100 (-S9), hydroquinone in TA1535A
-------
p. 1 of 4
Table D. Mutagenicity of Phenols in Che Ames Test
Substituent at Position2
Compound
(A) UnaubacL Diced
Phenol
2
Phenol
a
3 4
a a
ii
a
6 A
a
itagenici tvk
- (53-56)
+ (57)
(B) Alkylphenols and Alkoxyphenola
o-, ja-, or £-Cresol
Xylenols
Trlme thylphenols
2-, 3-, or
4-Ethylphenol
Thymol
380*
Gualcol
4-Hydroxyanisole
BBAC
Eugenol
Eugenol-2 ' , 3 ' -oxide
Iso«ugenol
(C) Chlorophenols
2-, 3-, or
4-Chl o rophenol
01 chlorophenols
Trichlorophenols
CB.J at 2-, 3-, or 4-poaltion
CH, ac 2,3-, 2,
CBj at 2,
C?a5 at 2-,
i-c3a7
**&
oca3
a
4-, 2,5-, 2,6-, 3,4-,
3,5-, 2,4,5-, or 2,4,
3-, or 4-poaition
a a
n CH
a a
a oca-j
t-C,^ at 2-/3-poaid.on OCHn
OC3.J
OCHj
OCHj
Q at 2-, 3-
d at 2,3-, 2
a at 2,3.,4-, 2
B CBjCH-CH-
H (^CH-CHj
0
a CH"CHCH3
, or 4-poaition
,4-, 2,5-, 2,6-, 3,4-
,3,5-, 2,3,6-, 2.4,5-
a
or
a
3,5-poaltions
6-poaitlona
a
ca3
a
a
H
H
H
a
a
a
, or
, or
H
B
-E-C4H9
a
a
H
a
a
a ..
a
3,5-poaitions
2,4, 6-poai dons
- (53, 58)
- (53, 56)
- (53, 56)
- (53, 56)
- (53)
- (59, 60)
- (53, 54, 58)
- (53)
- (59, 60)
- (54, 58, 61)
+ (53)
+ (61)
- C5A) -
* (61)
- (54)
- (54, 62)
- (54, 58, 62)
-------
p. 2 of 4
Tabla 0. Mntaganlcity of Phenol* in th«
(continued)
T««t
Substttuant at Position2
Compound 2
(C) Chlorophenols (eont'd)
2,3,4,6-T«tr»-
chlorophanol Q
Pancachlorophanol . d
(D) Nitrophanola and Aainophanols
2— or 4-Hitroph«nol N02/B
2-Httro-3-
mathylphanol NOj
2,4-M.nitrophanol NC^ _
jr- or j£-A»inophanol a
Aoa Paial nnnH«i W
2-Amino-4-
nicro phenol NH-
2-Anino-5-
nieroph«nol • MB-
2-Mltro-4-
aainophanol . H02
Picric acid H02
4,6-Dinitro-
o-cr«»ol CH«
3
a
d
a
CH^
a
HBj/E
a
a
a
a
E
a
a
4 5
GL a
a a
H/HOZ a
a a
NO, a
B/HOM a ,- '
UK*.- a^
• HBCOGS* B
H02. &
a so2
£Ub^ ^L
B02 a
tin u
EIUA &
(E) Hydrox7b«nraldehyd*» , Hydrozyac* caphanona* , and Hydraxytoanzoic
2-, 3-, or 4-
hydroncTbanxaldahTda CHQ at 2-,
Vanillin OCH,
Syringealdehyde OCH^
3-, or
a
H
4-poaltion a
CBO a
CBO H
6
a
a
a
a
a
R
a
a
a
a
a
N02
N02
Acid
B
a
OCH^
Mutagenlcity"
- (62)
- (63)d
- (64, 65)
- (64)
- (64, 66, 67)
- (65. 67, 6S)
+ (67)
-(60)
+ (67, 68)
+ (64, 68)
+ (67)
+ (57)
- (64)
- (63)d
- (53, 54)
- (54)
- (54)
-------
p. 3 of 4
Table 0. Mucagenicity of Phenols In Che Ane» Tasc
(continued)
Subatituent at Position*
Coopound
( E) Bydroxyb enzaldehydea ,
3-Chloro-4-faydroxy-
benzaldehyde
3,5-D±chloro-2-
' hydroxybenzaldehyde
s
3,6-Dichloro-2-
hydroxyfoenzaldehyd*
2
3
4
5
Bydroxyaeetaphenonea, and Bydroxyb enzoic
a
CHO.
CHO
2-, 3-, or 4-
acetophenone COCE, at 2-,
Salicylic acid
j£-3ydroxyb enzoic
i acid
Vanillic acid
Syringic acid
Pro coca cachuic acid
Propyl gallate
(?) Polyhydric Phenols
CataohAl
Reaoreinol
Eydroquinone
Pyrogallol
COOH
H
OCS*
OCH.
OH
OH
OH
H
E
OH
B
E
a.
3-, or
B
E
H
E
B
OH
H
OH
H
OH
CHO
a
H
4-posl don
B
COOH
COOH
COOH
COOH
H C
H
H
OH
B
B
B
a
B
B
B
E
B
H
KXX^Ey
H
H
B
B
6
Acid
B
a.
Q
B
B
B
E
OCH,,
B
B
E
H
B
E
Miitagenicicr3
(cont'd)
- (54)
- (54)
+ (54)
• \ «*^ /
- (53, 54)
- (65)
- (54)
- (54)
- (54)
- (54)
- (59, 60)
- (53, 69)
- (53, 65, 66,
70, 71)
- (53, 54)
* (57)
- (54, 72")
± (53)
+ (57, 73)
Phloroglucinol
B
OH
E
OH
H
- (54)
-------
p. 4 of 4
Tabla 0. Mutagenicity of Phenols in Che Ames Test
(continued)
Compound
(F) Polyhydric Phenols
3,4-, 3,5-, 3,6-,
or 4,5-W.chloro—
catachol
3,4,5-Trichloro-
cacechol
4-Chlororesorcinol
Chlorohydroquinone
2 , 5-Dlchlorohydro—
quinone
2
(cont'd)
OE
OE
H
a
CL
SubstiCuent at Position3
3456
CL at 3,4-, 3,5-r 3,6- or 4,5-ooaitions
CL CL Q H
OH CL H H
H .OH H H
E OH CL E
V
Mutagenicity^
- (53, 58)
- (58)
- (68)
- (54)
- (54)
*?oaition L of all these- compounds is occupied by an OH group.
°Mutagenicityr "V • positive; "i* * equivocal; "-"' • negative. Numbers in parentheses are
reference numbers.
CBHT - butylated hydroxytoluene; BHA - butylated hydroxyanlaole.
Metabolic activation system (39 mix) not included in this study.
-------
(-S9) and pyrogallol in TA1537 (-S9), TA98 and TA100 (±S9), were mutagenic.
Florin ££jal_« (53) found pyrogallol to have marginal mutagenic activity. In
contrast to these studies, none of these and other polyhydric phenols were
found mutagenic by several other investigators (see Table D).
In addition to phenols, a number of hydroxynaphthalenes and hydroxy-
anthracenes have been tested for mutagenicity. Both 1-naphthol (53, 57, 65,
76) and 2-naphthol (53, 76) are negative in the Ames test. The introduction
of nitro groups may confer mutagenic activity. Thus, l-nitro-2-naphthol (64),
2,4-dinitro-l-naphthol (77) and its 7-sulfono derivative (64) all display
mutagenic activity. Brown and coworkers (rev, in ref. 78) have tested the
mutagenicity of several hydroxyanthracenes. An throne (9-hydroxyanthracene)
has been found inactive whereas several polyhydric anthracenes, such as
anthralin (1,8,9-trihydroxyanthracene), anthrarobin (3,4-dihydroxyanthracene)
and crysarobin (l,8,9-trihydroxy-3-niethylanthracene) are all mutagenic in
strains TA1537 (a frameshift mutant). Hexachlorophene, a bisphenol, is
nonmutagenic (57).
The genotoxic effects of phenolics in plants have prompted many investi-
gators to assess the chromosome-damaging potential of these compounds in
mammalian cells. Probst ^_t_al_. (76) showed that a number of phenolics (such
as 4-aminophenol, 4-nitrophenol1, 2,4-dinitrophenol, 2,4-dichlorophenol, resor-
cinol, I-/2-naphthol, and anthralin) are all inactive in the unscheduled DNA
synthesis (UDS) tests. Morimoto and Wolff (79) found that catechol and, to a
lesser degree, hydroquinone induce sister chromatid exchange (SCE) in cultured
human lymphocytes. Phenol is considerably less active and benzene is inac-
tive. An in vitro study by Bracher _e_t ^1_. (71) showed that resorcinol is
inactive in SCE in bone marrow cells of rats. Kawachi (cited in ref. 6)
tested six phenolics in several chromosome aberration tests; propyl gallate,
-------
acetaminophen and isobutyl-p-hydroxybenzoate were found active, whereas BHT,
BHA, and butyl-p- hydroxybenzoate were inactive. Stich _e_t_ ai. (80) asessed
the clastogenic activity of seventeen phenolics in Chinese hanster ovary (CHO)
cells by measuring the number of chroiaatid breaks and exchanges; the most
significant finding was that monohydroxylated phenolics (e.g., salicylic acid,
_p_-hydroxybenzoic acid) generally lack clastogenic activity, whereas most
dihydroxylated (e.g., catechol, resorcinol, protocatechuic acid) and tri-
hydroxylated phenolics (e.g., pyrogallol, phloroglucinol, gallic acid) exhibit
a relatively strong chronosome-datnaging potential. The clastogenic activity
of most of the phenolics is reduced by the addition of liver S-9 mix, but
i t i i
increased by transition metals such as Cu and Mn . Comparison of the dose-
response data suggests that the relative potency of polyhydric phenols follows
the order: catechol > pyrogallol > resorcinol > phloroglucinol. In the
micronucleus test (which detects chromosome aberrations in mouse bone marrow
cells) by Go eke ^_t _al_. (57), pyrogallol and hydroquinone were both active
whereas phenol, resorcinol, picric acid, 1-naphthol and hexachlorophene were
all inactive.
Thus, both the bacterial and mammalian data concur that monohydroxylated
phenolics are generally nonmutagenic unless they are ring-substituted with
functional groups which are known to confer mutagenic activity. On the other
hand, polyhydric phenolics (especially catechol, hydroquinone, pyrogallol)
have relatively strong mutagenic or chromosome-damaging potentials.
Teratogenicity. A number of phenolics have been tested for terato-
genicity; among these, hexachlorophene and salicylates are active in some
species. The safety of the use of hexachlorophene was first questioned since
the discovery in 1971 of its toxic effects on neonatal rat brain. Oakley and
Shepard (81), Gaines _e_t__al_. (44), and Kennedy _e_£_al_. (82, 83) demonstrated
10
-------
subsequently that hexachlorophene causes malformations (angulated ribs, cleft
palates, micro- and anophthalmia) in the offspring of rats given high
(maternally toxici) doses of the compound in the diet or by gavage. Terato-
genic effects were also observed in rats exposed to the compound via vaginal
treatment (84). In rabbits, oral administration of hexachlorophene led to a
relatively low incidence of rib malformation (82) whereas mice appeared to be
resistant to the teratogenic effects of the compound (85). Recently, retro-
spective epidemiological studies performed on employees of six Swedish
hospitals suggested that severe congenital malformations occurred nore
frequently in infants (25/460) born to mothers who used hexachlorophene soaps
during at least the first trimester of pregnancy than those (0/233) born to
similarly employed mothers who did not use hexachlorophene soaps (86). It is
important to note that hexachlorophene is synthesized from 2,4,5-trichloro-
phenol, the same chemical that in the manufacture of 2,4,5-trichlorophenoxy-
acetic acid is known to give rise to chlorinated dibenzodioxins.
Sodium salicylate has been shown to cause gross anomalies (rib anomalies)
in mice (87) and rats (88). Minor and Becker (88) compared the teratogenic
potential of sodium salicylate (active), sodium benzoate "(active) and phenol"
(inactive) and concluded that the teratogenicity is associated with carboxyl
moiety, while the hydroxyl group plays a contributory but not obligatory
role. It should be noted, however, that _p_-hydroxybenzoic acid was not terato-
genic in mice (87). Goldman and Yakovac (89) reported that the teratogenicity
of salicylate is probably unrelated to its activity as an uncoupler of oxida-
tive phosphorylation because 2,4-dinitrophenol, a well known uncoupler, is not
teratogenic in rats. Four other phenolics have been found to have no signifi-
cant teratogenic effects: the food additives, butylated hydroxytoluene (BHT)
and hydroxyanisole (BHA) in mice and rats (90), and nonkeys (91); the wood
11
-------
preservative, pentachlorophenol in rats (92); and the herbicide, dinitro-o_-
cresol in mice (93).
5.2.2.5^3 Carcinogenicity and Structure-Activity Relationships.
5.2.2.5.3.1 OVERVIEW. Over fifty phenolics have been tested for car-
cinogenicity. Many of these studies are, however, probably of insufficient
quality to provide unequivocal results. Available data suggest that most
monohydric nonocyclic phenols are either inactive or, at most, weakly carcino-
genic. Phenol itself is inactive by oral administration but induces skin
tumors in nice after repeated applications of high (irritating) doses.
Comparative studies by Boutwell and Bosch (94) showed that ring substitution
with chlorine (at the 2-position) or methyl groups yields compounds with skin
carcinogenicity comparable to that of phenol; substitution with bulky alkyl
group or electron-withdrawing substituents (-N02> -CHO, -COOH) at 2- or 4-
position abolishes the carcinogenicity of phenol.
2,4,6-Trichlorophenol and'4-amino-2-nitrophenol are, at the time of this
writing, the only two simple phenolics that have been unequivocally shown to
be carcinogenic in rodents after oral administration. The former induces
lymphoinas or leukemias in male F344 rats and liver tumors in B6C3F, mice while
the latter is carcinogenic toward the urinary bladder of F344 rats. There is
some suggestive evidence that butylated hydroxytoluene (BHT) and eugenol may
be carcinogenic in some strains of mice.
Bacterial and mammalian data suggest that polyhydric phenols may have
relatively high mutagenic potential (see Section 5.2.2.5.2.2). Four poly-
hydric phenols (catechol, resorcinol, hydroquinone and pyrogallol) have, thus
far, been tested for carcinogenicity on the skin of mice and rabbits and found
12
-------
to be generally inactive. There is some evidence that oral administration of
hydroquinone may increase the incidence of renal tumors; it also increases the
incidence of bladder tumors after in situ pellet implantation. Two bisphenols
(bispheno! A and hexachlorophene) have been adequately tested and consistently
found to be inactive.
Among the polycyclic phenols, anthralin (1,8,9-trihydroxyanthracene) is
generally inactive as a complete skin carcinogen but may induce lymphomas in
one strain (ICR) of mice. 2-Hydroxybenzo[a]pyrene, a phenolic derivative of
benzo[a]pyrene, has recently been shown to be an exceptionally strong skin
carcinogen; thus, the generally held assumption that phenolic metabolites of
polycyclic aromatic hydrocarbons are "detoxified" metabolites has no universal
validity.
An interesting study of the potential carcinogenicity of several phenolic
and related compounds in two tumor-prone hybrid tobacco plants (Nicotinia
hybrids) has been conducted by Andersen (95). Pyrogallol, resorcinol and
3-hydroxyanthranilic (3-hydroxy-2-aminobenzoic) acid induced tumors in the
plants at the seedling stage, whereas phenol and catechol were inactive. On
/
the molar basis, pyrogallol was the most carcinogenic. Acetylation of the '
hydroxyl groups (yielding pyrogallol triacetate) or introduction of a carboxyl
group to the ring (yielding gallic acid) completely abolishes the carcino-
genicity of pyrogallol to the plants. The author suggested that, except for
3-hydroxyanthranilic acid, the structural requirement for carcinogenicity
toward the Nicotiniama hybrids is the presence of at least two hydroxyl grou;:1.
positioned meta to each other.
5.2.2.5.3.2 SIMPLE (MONOCYCLIC) PHENOLICS. Close to forty simple
phenolics have been tested for carcinogenicity. The major findings of these
13
-------
studies are summarized in Table E according to the type of substituents
present in the aromatic ring.
Phenol. Boutwell and coworkers (94, 96) reported first that mice
topically treated with phenol for long periods of time developed skin
tumors. The finding was confirmed by Salaman and Glendenning (97). The
carcinogenicity of phenol is, however, very weak; an ulcerative concentration
(20%) of phenol is needed to elicit a weak carcinogenic action (97). Boutwell
and Bosch (94) noted that during the first 6 weeks of phenol (10 or 20% solu-
tion in benzene or dioxane) treatment many of the mice bore wounds and showed
reparative hyperplasia. Most of the skin tumors were papillomas. In the
experiments of Van Duuren and Goldschmidt (98), in the 50 ICR/Ha Swiss mice
treated topically with 3 mg phenol for 368 days only one mouse developed a
skin papilloma, so that the compound was not considered carcinogenic by the
authors.
Phenol has also been tested in rodents by oral administration. Unpub-
lished Food and Drug Administration data (19) indicate that 17/36 rats
survived over 2 years on a diet containing 0.25, 0.5 or 1.0% phenol. Histo-
pathological examination of the 6 rats that survived the highest dose did not
reveal any significant change. The U.S. National Cancer Institute (1) has
completed in 1980 a bioassay testing of phenol. Groups of 50 F344 rats and 50
B6C3F, mice were given drinking water containing 2,500 or 5,000 ppm of the
compound for 103 weeks. An increased incidence of leukemia or lymphomas was
detected in male rats (low dose group only); however, it could not be . tab-
lished whether the increased incidence was acutally due to phenol treatment.
Thus, it was concluded that, under the conditions of this bioassay, phenol was
not carcinogenic in either species of rodents.
14
-------
p. 1 of 4
Table E. Carcinogenic!t7 of Phenol and its Substituted Derivatives
Compound* Species and Strain
Route
Principal
Organ
Affected
Reference
(A) Unsubstituted Phenol
Phenol
(B) Alkyl phenol 3 and
2,4-, 2,5-, 2,6-,
3,4- or 3,5-Xylanol
Butylated hydroxy—
toluene (BHT)
Thymol
^-Phenylphenol
j^-Phenylphenol
Guaiacol
4-Hydroxyanlsole
Butylated hydroxy-
anisole (BHA)
Mouse, albino
Mouse, "3"
Mouse, ICR/Ha
Mouse, B6C3F,
R»t, —
Rat, P344-
Alkoxyphenols
Mouse, albino
Mouse, A/ He
Mouse, BALB/c
Mouse, CF-1
Mouee, B6C3F,
Rat, Wistar
Rat, CD SPP
Pat, P344
Mouse, A/He
Mouse, B6C3ZI
or B6AI7| — •••
Mouse, B6C3P,
or B6AKF,
Mouse, B6C3F*
Mouse, —
Mouse, Swiss
Rabbit, Hew
Zealand
Mouse, C3H/Anf
—
Mouse, A/He
Mouse , CD-I
Rat, —
Dog, —
topical
topical
topical
oral
oral
oral
topical
i.p.
oral
oral
oral
oral
oral
oral
i.p.
, oral or
•^ s.c»
oral
S.C.
implantation
topical
\ topical
topical .or
'' s.c*
i.p.
oral
oral
oral
Skin
Skin
None
None
None
None
Skin
Noneb
Lung
Lung
None
None
None0
None
None0
None*
None*
Heaetopoietic
system
None
None
TSunm
None
«
None0
None
None
None (15-mo
study)
(94, 96)
(97)
(98)
(1)
(PDA data, cited in
ref. 19)
(1)
(94)
(99)
(100)
(Brooks et al»,.
cited In ra1T7 101)
(102)
(36)
(103)
(102)
(99)
(104, 105)
.- -^
(104, 105)
(104)
(106)
(107)
(107)
(108)
(99)
(109)
(110)
(111)
-------
p. 2 of 4
Table E. Carcinogenlcity of. Phenol and its Substituted Derivatives
(continued)
Compound* Species and Strain
(B) Alkyl phenols and
Eugenol
X
(C) Chlorophenols
2-Cblorophenol
2,4, 5-Trichlorophenol
2,4, 6-Trichlorophenol
2,3 ,4, 6-Te trachloro-
phenol
Pen tachlorophenol
(D) Nltrophenols and
j^-flydroxyace tanilide
2-Amino-4 , 5-*ylenol
4-Dime thylaolno-
3,5-xylenol
Route
Principal
Organ
Affected
Reference
Alkoxyphenols (cont'd)
Mouse, ICR/Ha,
Mouse, B6C3F1
Rat, F344
Mouse, albino
Mouse, B6C3Fi
or B6AKF1
Mouse, B6C3Fj
Mouse-, BoAKF^
Mouse, B6C3F^
or B6AKFi
Mouse, B6C3F,
Rat, F344
Mouse, B6C3?i
Mous*, B6C3F,
or B6AKF.
Moose, B6Cj7^
Rat, Sprague-
Davley
Aminophenols
Rat, —
Mouse, —
Mouse, B6C3Ft
or B6AKF,
topical
oral
oral
topical
s.c.
oral
oral
s.c.
oral
oral
s.c*
oral
s.c. .
oral
oral
implantation
oral or
s.c.
Hone
Liver
(equivocal)
None
Skin
(papillomas)
Noned
Liver, hema-
to pole tic
system*1
Noned
None*
Liver
Hema topole tic
system
Sema topole tic
system*1
Koned
Uverd
None
Pituitary
gland
Urinary
bladder
Noned
(98)
(3) .
(3)
(94)
(104)
(104, 105)
(104)
(104)
(112)
(112)
(104)
(104, 105)
(104)
(92)
(113.)
(11.)
(104, 105)
-------
p. 3 of 4
Table E» Carcinogenicity of Phenol and its Substituted Derivatives
(continued)
Compound3 Species and Strain
Route
Principal
Organ
Affected
Reference
(D) Nitrophenols and Aninophenols (cont'd)
4-Anino- 2-ni trophenol
2-sec-Butyl-4-, 6—
dini trophenol
House, B6C3F,
Rat, F344
Mbusev B6C3Fr
or B6AKF.
oral
oral
oral or
s. c«
None
Urinary
bladder
Rone4
(114)
(114)
(104, 105)
(E) Hydrobenzaldehydes and Rydroxybenzoic Acids
Vanillin (3-oethoxy-
4-hydroxy benzalde-
hyde)
3-E thoxy-4-hydroxy-
benzaldehyde
Salicylic acid
Methyl aalicylate
Bu tyl-jv-hydroxy-
benzoate
Isobucyl-^-hydroxy-
benzoate
Propyl gallate
(F) Polyhydrie Phenols
Catechol
(pyrocatachol)
Resorcinol
House, A/ He
House,. A/ He
Mouse, —
Mouse, A/ He
Mouse, B6C3FX
or IC2,
R*tf. vazioua
strains
Mouse* B6C3F^
or ICE
Mouse,. A/He
Rac, —
Mouse, —
Mouse, ICH/Ha
Rat, —
Mouse , Swiss
Mouse, ICR/Ha
Rabbit, New
i.p.
i.p.
implantation*
i.p.
oral
oral
oral.
i.p*
oral
implantation®
topical
oral
topical
topical
topical
lfeneb
Noneb
Hone
Moneb
(tone
Hone
None
tbneb
Hone
Nonef
Skin (?)*
Sons
Hone
None
None
(99)
(99)
(106)
(99)
(60)
(60)
(60)
(99)
(19)
(106)
(98)
(FDA data, cited in
ref. 19)
(107, 115)
(98)
(107)
Zealand
-------
p. 4 of 4
Table S. Carcinogenic!t7 of Phenol and its Substituted Derivatives
(continued)
Compound*
Specie* and Strain
Route
Principal
Organ
Affected
Reference
(P) Polyhydrie Phenols (contrd)
Hydroquinone . Mouse, —
Mouse, ICR/Hs
Hat, —
implan ta tion* Urinary
bladder
topical None
oral
Kidney (?)'
Pyrogallol
(G) Biaphenola
Mouse* Swiss
Mouse, ICB/Ha
Rabbit^ New
Zealand
topical None
topical None
topical None
(106)
(98)
(FDA data, cited in
ref. 19)
(107, 115)
(98)
(107)
Bisphenol A Mouse, G6C3P,
Rat, P344
HMNPfl^nl AVrtWhamrtSB* NnnsVaV'- S»BTO aimV
Mouse,. 57U
or I7II/G
Mouse, X71I/G
(neonatal)
Mouse » Z7II/G
Mouse-^
(C57xC3H)Ft
Bat,. Sprague-
Oawley
Rat, P344
Rabbit, Sew
Zealand
oral
oral
topical
oral
3.C.
lactations!
transplacen tal
oral
oral
topical
None
None
None
None
None
None
None
None
None
None
(116)
(116)
(107, 115)
(117)
(117)
(117)
(117)
(117)
(102)
(107)
•
aSeer Tables B and C for structural formulae.
bPulmonary adenoma assay (24-week study),
C0no rat developed a aaaoary adenoeareinoma.
—j
^Preliminary screening study.
*Bladder Implantation with cholesterol pellets (25-week study).
-Erroneously reported Co have a significant carcinogenic effect by an IARC study group.
S(3ne mouse developed a aquamoua carcinoma.
Suggestive evidence of increase in Incidence of renal tumors over spontaneous incidence
-------
Alkylphenols and alkoxyphenols. This group includes a number of commer-
/
daily important antioxidants such as butylated hydroxytoluene (BHT) and
butylated hydroxyanisole (BHA). Five isomers of xylenol (dimethylphenol) were
tested for carcinogenicity by Boutwell and Bosch (94). The compounds were
applied as 10% solutions in benzene to mouse skin twice weekly for 20 weeks.
At the end of treatment, skin papillomas were noted in each group with the
incidences for the 5 isomers as follows: 3,5- (55%) _> 2,4- (50%) > 2,4- (31%)
>_ 2,5- (24%) > 2,6- (8%). Skin carcinomas appeared at 28 weeks with inci-
dences: 14% for 3,4- and for 3,5-; 12% for 2,4-; 8% for 2,5-; and 0% for the
2,6-isoiaer.
The potential carcinogenicity of butylated hydroxytoluene (BHT) was the
subject of intensive investigations because of its wide use as a food addi-
tive. Deichmann _e_t _al^ (36) were probably the first to report the lack of
carcinogenicity of BHT in the rat. Groups of 15 rats of each sex administered
0.2, 0.5, 0.8 or 1.0% BHT in the diet for 24 months showed no signs of patho-
logical changes. Ulland e£ ^1_. (103) studied the effect of BHT on carcino-
genesis induced by a number of known carcinogens in Charles River CD SPF
rats. In the control group given BHT (6,600 ppm in diet) alone, none of 10
male rats developed tumors after 37 weeks but one of 10 female rats developed
a mammary adenocarcinoma during 44 weeks of the study. The authors considered
BHT noncarcinogenic because of the high spontaneous mammary tumor incidence in
female rats of this strain. The lack of carcinogenicity of BHT in the rat has
been further confirmed in a NCI bioassay study (102). No tum^*"" occurred in
F344 rats exposed to diets containing either 3,000 or 6,000 ppm BHT for 105
weeks.
There appears to be some evidence that BHT may have some carcinogenic
activity in certain strains of mice. Clapp ££ £l/ (100) reported that BALB/c
15
-------
mice given diets containing 0.75% BHT for 16 months had significantly higher
incidence (63.6%) of lung tumors (mainly papillary adenomas) than that (24%)
in controls. This finding was supported by Brooks _e_t _al_- (cited in ref. 101)
who observed a significnat, dose-dependent increase in lung tumors in CF-1
mice fed BHT for 100 weeks. The incidences were 53.2%, 73.8% and 75% for mice
fed 1,000, 2,500 and 5,000 ppci, .respectively, compared to 46.8% for controls.
In contrast to the above studies, a 1978 NCI bioassay (102) failed to
show a significant carcinogenic effect of BHT in B6C3Fi mice maintained on
diets containing either 3,000 or 6,000 ppm for 107: or 108 weeks. Stoner et
al. (99) found BHT inactive in pulmonary adenoma assay in strain A/He mice
receiving a. total dose of as much as 6 g/kg BHT intraperitoneally in a 24-week
period.
Like BHT, butylated hydroxyanisole (BHA) has received great attention
because of its extensive use as an antioxidant food stabilizer. Wilder and
Kraybill (110) were probably the first to report a chronic toxicity study of
BHA in the rat. Groups of 15 or more newly weaned rats were maintained on
diets containing 0.05, 0.5, 1.0 or 2.0% BHA for 21-22 months. No significant
histopathological changes attributable to BHA were observed. In at least 3
strains of mice, BHA was also consistently noncarcinogenic by several
routes. In the study of Hodge et al. (108), groups of 100 strain C3H/Anf mice
(50 of each sex) were either given single subcutaneous injections (10
mg/mouse) or weekly skin applications (0.1 mg) of BHA; no evidence of tumors
was noted up to 519 days. Brown (109) showed that BHA '. d no carcinogenic
effects in two strains (CD-I and C3H) of mice; in fact, high levels (0.5%) of
BHA appeared to decrease slightly the "spontaneous" incidence of liver tumors
in C3H mice. Moreover, S toner £t_ ai_. (99) found BHA inactive in pulmonary
adenoma assay using A/He mice. A 15-month study in dogs also produced no
evidence for the carcinogenicity of BHA (111).
16
-------
Eugenol, a widely used food additive for flavor and fragrance, has been
tested in a U.S. Rational Toxicology Program carcinogenesis bioassay at two
dose levels. A preliminary report of the study (3) showed that the compound
significantly increased the incidence of hepatocellular adenomas or carcinomas
in B6C3F, mice of the low-dose group but not the high-dose group; the results
were judged to be equivocal. In F344 rats eugenol was clearly not carcino-
genic. Also, a preliminary study (Swanson, Miller and Miller, unpublished
data cited in ref. 61)'in preweaning mice failed to provide evidence for
carcinogenici ty.
Of the other phenolics in Group (B) of Table E, thymol (99), o-phenyl-
phenol (104, 105), guaiacol (106) and 4-hydroxyanisole (107) are all noncar-
cinogenic in various screening studies. Only jj-phenylphenol increased the
incidence of reticulum cell sarcomas in male B6C3F. mice after a single sub-
cutaneous administration of 1 g/kg; however, it was inactive by oral adminis-
tration (104, 105).
Chlorophenols. Several chlorinated derivatives of phenol are carcino-
genic (Group (C) in Table E). Repeated skin application of a 20% solution of
2-chlorophenol (in dioxane) produced skin papilloiaas in 46% of albino mice
with an average of 0.64 papilloma/mouse after 12 weeks. For comparison, a 20%
solution of phenol led to skin papillomas in 63% of the animals with an
average of 0.94 papilloma/mouse (94). A preliminary screening study in 1968
of four higher chlorophenols in B6C3F, and B6AKF, mice under various condi-
tions of administration (104, 105) gave the following results. 2,4,5-Tri-
chlorophenol was inactive after a single subcutaneous injection of 1 g/kg.
2,4,6-Trichlorophenol caused an increase in the incidence of hepatomas and
reticulum cell sarcomas in B6C3F, mice receiving a diet containing 260 ppm of
the compound. 2,3,4,6-Tetrachlorophenol appeared to increase the incidence of
17
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reticulun cell sarcoma in male B6C3F1 nice that received a single subcutaneous
dose of 100 mg/kg. Pentachlorophenol (Dowcide-7) was inactive by oral
v
administration; however, a single subcutaneous administration of 46.4 tag/kg
enhanced the incidence of hepatomas in male B6C3F, mice. Pentachlorophenol
was also inactive in Sprague-Dawley rats fed the technical grade compound at
daily dose levels of 1, 3, 10 and 30 mg/kg body weight for 22-24 months (92).
The carcinbgenicity of 2,4,6-trichlorophenol has been confirmed in 1979
in a NCI bioassay study (112). Significant increases in liver tumors were
found in B6C3F1 mice of both sexes fed diets containing 5,000 and 10,000 ppm
(for males) and 5,214 and 10,428 ppm (for females) of the compound, for 105
weeks. The incidences of hepatocellular carcinomas or adenomas were 4/20
(control), 32/49 (low dose), 39/47 (high dose) for males and 1/20 (control),
12/50 (low dose), 24/48 (high dose) for females. 2,4,6-Trichlorophenol (5,000
or 10,000 ppm in diets for 107 or 108 weeks) was also carcinogenic in male
F344 rats inducing lymphoraas or leukemias (control 4/20, low dose 25/50, high
dose 29/50).
Nitrophenols and aminophenols. As previously discussed in Section
5.1.2.1 (Vol. IIB, p. 6), certain derivatives of aminophenols (e.g.,
^-hydroxyacetanilide, 2-amino-4,5-xylenol) are carcinogenic (see Group (B) of
Table E). It does not appear, however, that the phenolic hydroxyl group per
se is a structural requirement for carcinogenicity. For example, whereas
_p_-ethoxyacetanilide (phenacetin) is active (see Vol. IIB, p. 6), _p_-hydroxy-
acetanilide (acetaminophen) appears to be inactive. A preliminary NCI
carcinogenesis screening study included two amino/nitrophenols (4-dimethyl-
amino-3,5-xylenol and 2-sec-butyl-4,6-dinitrophenol); both compounds did not
display any carcinogenic activity. The NCI has recently conducted a bioassay
of 4-aniino-2-nitrophenol (1,250 or 2,500 ppm in diet for 103 weeks) in F344
18
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rats and B6C3F, mice (114). The compound was inactive in nice but carcino-
genic in male (and possibly also in female) rats inducing transitional-cell
carcinomas of the"urinary bladder. It is well documented that many disubsti-
tuted aminq/nitrobenzenes are carcinogenic/inutagenic (e.g., 74, 75). 2-Amino-
4-nitrophenol and 2-anino-5-nitrophenol are currently being tested by NCI/NTP
and a Japanese team (60) is re-evaluating the possible carcinogenic potential
of acetaminophen.
Hydroxybenzaldehydes and hydroxybenzoic acids. None of the compounds in
Group (E) of Table E appears to be carcinogenic; however, the results on
vanillin, 3-ethoxy-4-hydroxybenzaldehyde, methyl salicyclate and salicyclic
acid cannot be considered conclusive because of the preliminary nature of the
studies (pulmonary adenoma assay or bladder implantation). Butyl and isobutyl
_p_-hydroxybenzoates (60) and propyl gallate (rev. in ref. 19) have been tested
in long-term studies up to dietary levels of 0.6%, 1.2% and 5%, respectively;
no evidence of carcinogenicity was found.
Polyhydric phenols. Chemical structural considerations (Section
5.2.2.5.2.1), mutagenicity data (Section 5.2.2.5.2.2) and carcinogenicity
studies in plants (Section 5.2.2.5.3.1) suggest that this group of compounds
may be more hazardous than hitherto assumed: only limited carcinogenicity
data (Group (F) of Table E) are available at the time of this writing.
Catechol was inactive in bladder implantation study (106), induced only 1 skin
carcinoma among 50 mice after repeated topical applications for 368 days (98),
and apparently did not induce any tumor in a chronic feeding study up to a
dietary level of 1% (cited in ref. 19).
Resorcinol and pyrogallol have only been tested by topical route. Both
were inactive in Swiss mice (107, 115), ICR/Ha Swiss mice (98) and New Zealand
19
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/ -t- —•
rabbits (107). Several hair-dye formulations, which contain resorcinol
(0.4%), were also noncarcinogenic in mice after weekly or biweekly topical
applications for 18 months (118).
Hydroquinone is inactive as a complete skin carcinogen in ICR/Ha Swiss
mice (98), but active as a skin tumor initiator in mice (with croton oil as
proaotor) (119). In the bladder implantation assay, hydroquinone signifi-
cantly increased the incidence of bladder tumors in mice (106). It cannot be
ascertained, however, whether the above effect was due to the complete car-
cinogenic! ty or tumor-promoting activity of hydroquinone. Chronic oral
toxicity study of hydroquinone by the U.S. Food and Drug Administration (cited
in ref. 19) indicates increase in incidence of chronic gastrointestinal
ulceration and kidney tumors over spontaneous incidence. A variety of poly-
hydric phenols (hexylresorcinol, hydroquinone, propyl gallate and resorcinol)
are being tested or have been selected for carcinogenesis bioassay in the U.S.
Naitonal Toxicology Program at the time of this writing.
Bisphenols. Two bisphenols have been tested for carcinogenic!ty (Group
(G) of Table E). Bisphenol A (4,4'-isopropylidenediphenol), a widely used
intermediate in the manufacture of phenolic resins, displays no convincing
evidence of carcinogenicity in F344 rats and B6C3F, mice at dose levels of
1,000 or 2,000 ppm for rats of either sex, 1,000 or 5,000 ppm for male mice,
and 5,000 or 10,000 ppm for female mice (116). Only a marginally significant
increase in the incidence of leukemias in male rats and in the combined inci-
dence of lymphomas and leukemias in mice have been noted, which may be
construed as representing borderline activity toward the hematopoietic
system. Hexachlorophene (2,2'-methylene-bis-3,4,6-trichlorophenol), a
commonly used antibacterial agent (prior to 1972), was found noncarcinogenic
in several bioassays (102, 107, 117).
20
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5.2.2.5.3.3 POLYNUCLEAR PHENOLICS. Few polynuclear phenolics have been
tested for carcinogenicity probably because of the pervasive belief that
».
phenolic derivatives are "detoxified" metabolites of aromatic amines and
hydrocarbons. Although 2-amino-l-naphthol was, at one time, considered to be
the proximate carcinogen of 2-naphthylamine, subsequent studies did not
substantiate this view (Section '5.1.4.2.1 in Vol. IIB) and it is now generally
accepted that N-hydroxylation is the common activating mechanism for virtually
all aromatic amines.
The carcinogenic potential of anthralin (dithranol, 1,8,9-trihydroxy-
anthracene, or l,8-dihydroxy-9-anthrone) has been explored because of its use
for the treatment of psoriasis and chronic dermatoses (120, 121). Anthralin
was consistently found to be a tumor-promo tor (see Section 5.2.2.5.3.4); it
appears to have no complete carcinogenic activity or is at most marginally
carcinogenic in a few strains of mice (18, 98, 122). The only evidence for
the complete carcinogenicity of anthralin was obtained by Yasuhira (123,
124). Topical applications of anthralin (0.033 or 0.1% solution in acetone)
alone to two groups of young ICR mice led to the induction of malignant
lymphomas in 6/40 (15%) and 4/20 (20%) of the animals. No tumors were noted
in groups given urethan alone, croton oil alone or urethan + croton oil. In
two groups of ICR mice given urethan + anthralin, the incidence of lymphomas
was 21/40 (52%) and 7/20 (35%) indicating strong synergism between anthralin
and urethan or enhancement of the carcinogenic effect of anthralin by urethan.
As mentioned above, phenolic derivatives of aromatic Ivyurocarbons were
generally regarded to be "detoxified" metabolites. However, a 1977 study by
Wislocki _e£ ^1_« (125) showed that, among the 12 possible isoraeric phenols
derived from benzo[ajpyrene, 2-hydroxybenzo[a]pyrene (2-OH-BP) is a strong
carcinogen comparable in potency to that of benzo[a]pyrene. Topical applica-
21
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tion of 0.4 umoles of 2-OH-BP to the skin of C57BL/6J mice once every 2 weeks
led to induction of squamous cell carcinomas in 100% of the animals after 53
weeks of treatment. Tested under similar conditions, 11-OH-BP was a much
weaker carcinogen, inducing skin tumors in only 14% of the mice; all the other
10 isomers were inactive (125, 126). It is interesting to note that applica-
tion of 2-OH-BP or 9-OH-BP was followed by marked epidermal hyperplasia
resembling that caused by typical tumor promo tors; the other 10 isomers were
either less active or completely inactive in inducing hyperplasia (127). It
is possible that the strong carcinogenicity of 2-OH-BP is due in part to its
strong promotor-like activity and that 9-OH-BP probably lacks tumor-initiating
activity and is, therefore, not a complete carcinogen. Following a simple
assumption, the exceptional carcinogenicity of 2-OH-BP could be due to the
fact that hydroxyl groups in the 2- and 11-positions of trans-9,10-
-------
modifiers of chemical carcinogenesis will be covered in Section 6, Vol. IV; a
bird's eye view of some key studies and structure-activity relationships is
presented here.
Phenolics as promo tors. It is well documented that phenolics are tuiaori-
genesis promoters. Boutwell and Bosch (94, 128) tested the promoting activity
of over forty simple phenols using 7,12-dimethylbenz[a]anthracene as the
initiator. Phenol itself is a relatively weak promo tor. Following initia-
tion, twice weekly applications with 2.5 mg phenol elicited approximately the
same promoting action as 0.125 mg croton oil. Structure-activity studies
indicate that a free phenolic hydroxyl is required for promoting activity,
since both the methyl ether of phenol (anisole) and acyl derivative (phenyl-
acetate) are inactive. Ring substiution with halogen atom(s) or methyl
group(s) yield compounds with comparable activity except that most highly
substituted compounds (e.g., pentachloro-, 2,4,6-trichloro- and 2,3,5-tri-
methylphenol) are inactive. Replacement of a methyl group by a higher alkyl
group tends to reduce activity. Among polyhydric phenols, catechol, hydro-
quinone and pyrogallol are inactive while the data on resorcinol are equi-
vocal. Phenols substituted with electronegative substituents, such as -N02»
-CHO, -COOH, are inactive. Monohydric naphthols (1- or 2-naphthol) are only
slightly active; introduction of a second phenolic hydroxyl, as in the 1,5-,
2,6-, 1,7- or 2,7-naphthols abolishes activity. Anthralin (1,8,9-trihydroxy-
anthracene or 1,8-dihydroxy-9-anthrone) has been consistently found to be a
skin tumor promoter (see Section 5.2.2.5.3.3). Structure-activity studies by
Van Duuren and associates (rev, in ref. 129) showed that the intactness of 1-,
8-, and 9-substituents as well as the central ring was needed for promoting
activity. Esterificatibn of the 1- and 8-positions, opening of the central
•-.
ring or introduction of an additional hydroxyl group to the 10-position all
23
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resulted in the loss of activity. Among the twelve phenolic isoners of
benzo[a]pyrene, only 2— and 9-hydroxy derivatives display strong "promotor-
like activity" (inducing epidermal hyperplasia); all the other isomers are
either weak or inactive in this regard (127).
Phenolics as cocarcinogens.* Van Duuren and Goldschmidt (98) have tested
the cocarcinogenic activity of .several phenolics in an attempt to evaluate
their role in tobacco carcinogenesis. When administered together with
benzo[a]pyrene (BP), catechol and pyrogallol enhanced the carcinogenicity of
BP remarkably. In contrast, phenol, eugenol, resorcinol and hydroquinone
partially inhibited the carcinogenicity of BP. The authors suggested that
vicinal phenolic functions may be a structural requirement of simple phenolics
as cocarcinogens. Interestingly, both catechol and pyrogallol are inactive as
promoters in the typical two-stage carcinogenesis assay, suggesting that the
mechanism of cocarcinogenesis differs from the mechanism of promoter action.
The potent cocarcinogenicity of catechol is supported by a recent study of
Hecht et al. (130) showing catechol as a major component of several smoke
condensate sub fractions that display cocarcinogenic activity.
Phenolics as tumor inhibitors. The fact that a number of phenolic anti-
oxidants (e.g., BHA, BHT) inhibit the carcinogenic effects of some carcinogens
created an intense interest toward the search among the phenolics for possible
chemoprophylactic agents against chemical carcinogenesis. Wattenberg and
associates (131a, 131b) have made significant contributions in this area.
Structure-activity studies on twenty-'-'o phenolics using benzo[a]pyrene as the
*A "cocarcinogen" is defined as an agent that enhances the potency of a
carcinogen when administered simultaneously and repeatedly with that carcino-
gen. A cocarcinogen is not carcinogenic by itself. Cocarcinogens are not
necessarily tumor-promotors and vice versa.
24
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carcinogen and the forestomach as the carcinogenic!ty target tissue showed
that: (a) substitution in position para to the OH group of the unsubstituted
phenol increases"the inhibitory activity in this order: OCH-j > SCH-j > H, (b)
among BHA-related compounds, the' position of the jt-butyl group with respect to
the OH is of considerable importance; the most active inhibitor is 3-_t_-butyl-
4-hydroxyanisole, (c) three or more substitutions on the phenol ring render
the molecule inactive as an inhibitor, (d) phenols with intercyclic bond(s)
are generally inactive; The mechanism of inhibitory action of phenolics is
not clearly understood; reduction of activating pathways (132) as well as
direct (free radical trapping) or indirect (induction of "protective" enzymes
such as GSH S-transferase, NADPH-quinone reductase) enhancement of detoxifying
pathways (133-135) have been suggested as possible mechanisms.
5.2.2.5.4 Metabolism and Mechanism of Action.
Phenol readily penetrates the mammalian body through virtually any
route. After absorption, most of the phenol is either oxidized to C02 and
water (and traces of catechol and hydroquinone) or excreted as "free" or
"conjugated" (with sulfuric, glucuronic or other acids) phenol in the urine.
It is well documented that phenol is a major metabolite of benzene (see
Appendix I of this volume). Greenlee _e_t _al_. (136) proposed that the toxic
effects of benzene may be associated with the further metabolism of phenol to
hydroquinone and 1,2,4-benzenetriol (via catechol) which may then be readily
oxidized to highly reactive semiquinone and/or quinone metabolites and bind
covalently to cellular macromolecules. This view is substantiated by the
finding of Tunek £_t al_. (137) that upon micfosomal activation, phenol binds
covalently to microsomal proteins much more efficiently than benzene itself.
It is interesting to point out, however, that phenol does not seem to bind to
microsomal RNA to any significant extent.
-------
The aajor metabolic pathway of BHT (see Figure 1) involves the oxidation
of the methyl group to yield 2,6-di-£-butyl-4-hydromethylphenol (II), 3,5-di-
».
_t-butyl-4-hydroxybenzaldehyde (III) and 3,5-di-_£-butyl-4-hydroxybenzoic acid
(IV); this' pathway is generally believed to be "detoxifying" in nature
(138). Small amounts of 1,2-bis-( 3,5-di-_t-butyl-4-hydroxyphenyl) ethane (V)
and its corresponding quinone, 3> 5,3' ,5'-tetra-_£-butyls tilbene-4,4'-quinone
(VI) were also detected (see 139 and refs. therein) suggesting possible
coupling reaction of BHT free radicals. Shaw and Chen (139) identified
*
4-hydroxy-4-methyl-2,6-di-£-butylcyclo-2,5-hexadienone (VIII) and an in vitro
metabolite of BHT and proposed the formation of the corresponding 4-hydroper-
oxy compound (VII) as an intermediate. Recently, Takahashi and Hiraga (140)
found that BHT may be oxidized in vivo to a _p_-quinone methide, 2,6-di-£-butyl-
4-methylene-2,5-cyclohexadienone (IX); quinone methides of similar structures
are known to be potent alkylating agents (141). The free radical, the
4-hydroperoxy and the quinone nethide derivatives of BHT are all electrophilic
reactive intermediates capable of attacking nucleophilic sites in cellular
macromolecules. Indeed, covalent binding of BHT to lung, liver and kidney
microsomes has been demonstrated (139, 142, 143). There is some evidence that
the covalent binding may be related to its pulmonary toxicity in mice (143);
it remains to be elucidated whether the binding is related to the possible
carcinogenicity of BHT seen in some strains of mice.
The presence of the allylic side chain in eugenol suggests that is may be
metabolically activated by epoxidation. Swans on je_tjil_. (61) showed tha.t the
2',3'-oxide of eugenol is a direct-acting mutagen in the Ames test while
eugenol itself is not. Evidence that the epoxidation of allylic side chain
does indeed occur in vivo has been obtained by Solheim and Scheline (144)
using methyl ether derivatives of eugenol.
26
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HO—(\ />-CH2OH
R
HO
COOH
f
HI
321
OH
CH3
(I)
\
0
H°~V/"CH2"°H: M-:
R 'R R
i
0
R R R
—- o=( VCH=CH-/ ")=o
R
CH2
HOO" CH3
m
HO" ^CH3
T7TTT
Fig. 1. Metabolic Pathways of Butylated Hydroxytoluene (BUT).
-------
A comparison of in vivo covalent binding of bromobenzene and its metabo-
lite, p-broniophenol, has recently been conducted by Monks £_t^£l_« (145).
p-Bromophenol binds significantly to liver proteins and to a lesser extent to
kidney proteins. However, the level of binding of _p-broniophenol is substan-
tially lower than that of bromobenzene. This led the authors to conclude that
the toxicity of bromobenzene is'unrelated to the metabolism of _p_-bromophenol.
Extensive investigation on the bio transformation of pentachlorophenol
(PCP) have been carried out (rev, in ref. 14). Human and animal studies
indicate that PCP is excreted mainly in the urine, as such or as its
glucuronide (146). The principal metabolites are tetrachloro-jv-hydroquinone
(146, 147). Tetrachloro-p-hydroquinone is also the major urinary metabolite
(35%) of 2,3,5,6-tetrachlorophenol in the rat. The other two tetrachloro-
phenol isoraers ('2,3,4,5- and 2,3,4,6-) are excreted mainly unchanged together
with small amounts of tetrachloro-£-hydroquinone (39). The toxicological
consequence of bio transformation of chlorophenols to chloroquinones is not
clearly understood. By analogy to phenol and hydroquinone, such bio transfor-
mation may represent bioactivation.
Little information is available on the metabolic activation of amino-
phenols. It is interesting to point out that aminophenols have properties
similar to polyhydric phenols; for example, _p_-aminophenol may be oxidized to
quinonimine. In analogy to the oxidation of hydroquinone to quinone, it is
[ text figure 5j
' 27
-------
NH2
-e02H®
OH
:NH
:NH
NH
-ev
0
Text-Figure 5
-------
conceivable that seiniquinone-type of reactive intermediate may also be formed
by one-electron oxidation of aminophenols with amino group para or ortho to
the hydroxyl group.
The above brief summary serves to illustrate that electrophilic reactive
intermediates (free radicals, epoxides, peroxides, semiquinone, quinone
methides) may be generated from phenolics. It remains to be elucidated
whether these intermediates represent potential proximate carcinogens. A
systematic structure-activity relationship analysis of the effect of substi-
tuents on the stability (stable enough to reach targets) and reactivity
(active enough to react with targets) of these intermediates may help to
identify compounds with high carcinogenic potential. It is important to point
out that despite demonstration of covalent binding of phenolics to proteins,
clear evidence of covalent binding to DNA or RNA is still lacking. However,
Ts'o_e_t£l_. (148) have pointed out that reactions of free radicals with DNA
may not necessarily lead to covalent carcinogen-DNA adducts, but to other
types of damage such as strand scission and cross-linking. In this respect,
it should be noted that a number of phenolics have been shown to cause chromo-
some damage through the activated oxygen species (*OH, *0o> ^9^2^ generated
during the metabolism. Besides the above genotoxic mechanisms, phenolics may
also conceivably promote or initiate carcinogenesis by epigenetic
mechanisms. Thus, the well known effects of some phenolics as protein-
denaturants or inhibitors of energy metabolism may be related to carcino-
genesio L/y high doses of phenolics. Two recent findings, induction of
endogenous tumor virus gene expression by hexachlorophene (149) and suppres-
sion of immune response by technical grade (but not pure) pentachlorophenol
(150) are suggestive of possible mechanisms of carcinogenesis.
28
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5.2.2.5.5 Environmental Significance.
Phenolics are ubiquitously present in the environment. Human exposure
may occur via inhalation, ingestion or skin absorption of the phenolics then-
selves or of aronatic compounds which may be hydroxylated in the body to yield
phenolic metabolites. The environmental occurrence of and exposure to
phenolics have been extensively reviewed (9-15, 21, 23, 151).
Phenolics in air and tobacco smoke. Pentachlorophenol (PGP) has been
«
o o
detected in the air (average 0.26-1.89 ug/ra , max. 15 ug/m ) of a wood treat-
ment plant in Idaho (152). The occurrence of PGP appears not to be limited to
occupational setting. Cautreals £££!_• (153) reported that air samples
collected in a residential city area of Antwerp, Belgium contained 5.7-7.8
o -
ng/m PGP. For comparison, air samples collected from a relatively remote
area in Bolivia, South America contained only 0.25-0.93 ng/m . In addition to
PGP, phenol and cresols have also been detected as pollutants in urban air
(154). Lao^_t_al^ (155) reported the presence of 4-nitrophenol in an urban
air sample. Nojima^£^l_. (156) detected the formation of nitrophenols and
nitrocresols after photochemical reaction of nitrogen monoxide with benzene
and toluene. It seems likely that humans may be exposed to nitrophenols in
areas where severe photochemical smog exists.
Tobacco smoke contains a variety of phenolics. Catechol, the most abun-
dant phenolic in cigarette smoke, is believed to be formed during the incom-
plete combustion of tobacco flavonoids such as rutin, quercetin and chloro-
genic acid (157). Hoffmann and Wynder (154) reported that the mainstream
smoke of a 85 mm filterless popular U.S. cigarette contained an average of 300
^ig catechol, 100 jag phenol, 72 ug cresols and 20 ug 2,4/2,5-dimethylphenol.
Schlotzhauer et al. (158) identified catechol, resorcinol, hydroquinone and
29
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their alkyl-substituted derivatives, vanillin and its derivatives, dimethoxy-
phenols, phenylphenols and naphthols in an acidic fraction of cigarette smoke
condensate. These phenolics (especially dihydroxyphenols) are believed to
contribute to the carcinogenic effects of cigarette smoke by acting as
promoters or cocarcinogens (98, 130, 154).
Phenolics in drinking water. A number of phenolics have been detected in
the finished drinking water (FDW) of several cities. Buhler _e_t _al_. (159)
reported the occurrence of PGP (0.06 ppb) and hexachlorophene (0.01 ppb) in
FDW of Corvallis, Oregon; analysis of incoming raw river water sample showed
the presence of 0.17 ppb PGP and 0.03 ppb hexachlorophene. Phenolics were
suspected to be present in FDW of Ames, Iowa because of its taste and odor;
however, an analysis by Burnhain _e^£l.« (160) did not substantiate this view.
[This study was incorrectly cited to show a positive finding of 0.2 ppm
4-nitrophenol in an EPA Water Quality Criteria Document (13)]. ^Deinzer et al.
(161) detected 2,4,6-trichlorophenol in FDW of Cincinnati, Ohio. Shackelford
and Keith (162) listed hexachlorophene as being present in two samples of
FDW. During an accidental spillage of phenol in July of 1974, a massive
contamination of well water in a rural area of southern Wisconsin occurred
(163). An outbreak of human poisoning ensued. Analyses of a™well~"iiearest the
spill site revealed peak phenol concentrations between 15 and 126 mg/1. The
forecast is that the contamination of the well water in the area will persist
for years. Numerous other phenolics have been detected in plant effluents,
pulp and paper mill effluents, agricultural run-off, etc; these phenolics
could be present in FDW if these effluents contaminated the raw water sources.
Phenolics in foodstuffs. A variety of phenolics are present in food-
stuffs due to natural occurrence, intentional use as food additive, or inad-
vertent contamination. Fruits, vegetables (164-167), and beverages such as
30
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coffee (168) and tea (167, 169) are relatively rich in phenolics of natural
origin. Kaiser (169) identified phenol, cresols and dimethylphenols in black
fermented tea; he further showed that brewed tea may promote the skin carcino-
genic activity of benzo[a]pyrene. Many phenolics are widely used as food
additives. The most notable among these are butylated hydroxy toluene (BHT),
butylated hydroxyanisole (BHA) and propyl gallate (PG). These compounds are
used as antioxidants to prevent the rancidity of foods which contain fats, by
terminating chain reactions involving free radicals responsible for the oxida-
tive degradation of unsaturated fats. The estimated annual consumption in the
United States is 1,400, 450 and 230 metric tons for BHT, BHA and PG, respec-
tively. In 1974, the Food and Agriculture Organization of United Nations (27)
temporarily adopted an ADI (acceptable daily intake) of 0-0.5 mg/kg body
weight for BHA, BHT, or BHA and BHT combined, and 0-0.2 tag/kg body weight for
PG and related gallates. The U.S. Food and Drug Administration (101) proposed
to restrict the use of BHT in 1977 due to its possible carcinognicity in some
strains of mice. Other important food additives are eugenol and vanillin,
used to enhance food flavor and fragrance. The average maximum use levels of
eugenol in beverages, ice cream, gelatins and chewing gums range from 1.4 to
500 ppm with levels as high as 2,000 ppra used in processed meat products
(170). Inadvertent contamination of foodstuffs may occur as a result of
direct contamination or degradation of pesticides. For example, Murray et al.
(171) detected PCP levels of 2.4-8.3 ng/g (average 5.3 ng/g) in oysters from
Calves ton Bay, Texas. Johnson and Manske (172) found PCP in 13/240 food
samples collected in 20 U.S. cities; the concentration ranged from 0.01 to
0.04 mg/kg. Phenolics that have been detected in food as a result of degrada-
tion of pesticide residues include PCP, 2,4,5-/2,4,6-trichlorophenol (from
hexachlorobenzene), 2,4-dichlorophenol (from 2,4-D, 2,4,5-T, Silvex or
31
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Neraacide) and mononitrophenols (from parathion, fluoridifen or nitrofen).
Smoked meat products also contain phenolics probably due to their formation
v.
during incomplete combustion of hardwood sawdust used to generate the smoke
(173).
Phenolics as drugs or in toiletry products. Phenolics which have been
widely used as drugs are salicylates and acetaminophen (as analgesics and
antipyretics), anthralin (for treatment of psoriasis and chronic dermatoses),
thymol (as anthelmintic or antiseptic) and phenol (antiseptic, analgesic for
terminally ill cancer patients), etc. Hexachlorophene was used as a surgical
scrubbing agent, in bathing newborns to prevent skin infection and in many
toiletry products until 1972 when discovery of neurotoxicity in infants led to
the restriction of its use in the United States (174). Phenolics such as
4-ainino-2-ni trophenol, resorcinol, hydroquinone, pyrogallol, 1-naphthol,
4-hydroxyanisole are present in cosmetics and toiletry products, such as hair
dyes, depigmentation agents, soap and perfumes (57, 107).
The magnitude of carcinogenic!ty risk of human exposure to phenolics is
relatively unknown. No cancer epidemiology study for specific phenolics has
thus far been conducted. There is some evidence that humans exposed to
chlorophenoxy type herbicides (which contain chlorophenols) may incur an
increased risk for malignant lymphoma (175-177); however, it is not known
whether the increased risk can acutally be attributed to chlorophenols. One
physician (178) noted an unusual coincidence of the induction of bladder
cancer in two patients who were occupationally exposed to cresol and creosote;
thus far, no similar cases has been reported. Phenolics are present in hair
dyeing chemicals, which are suspected to be potentially hazardous to exposed
women (68). As pointed out in Section 5.2.2.5.3, a variety of phenolics are
being tested for carcinogenicity at the time of this writing.
32
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REFERENCES TO SECTION 5.2.2.5
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33
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12. U.S. EPA: "Ambient Water Quality Criteria for 2,4-Dimethylphenol,"
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34
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45
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Notes Added After Completion of Section 5.2.2.5
Close to 40 phenolic compounds have been tested for mutagenicity using
the Ames Salmonella test in recent studies carried out for the U.S. National
Toxicology Program (1-4). Among these, only ^-aminophenol (1), jn-aminophenol
(3), and 2,4-diaminophenol (1) were reported to give positive results. The
positive findings with ^v-aminophenol and m-aminophenol are at variance with
the results of a number of previous studies (5, 6; see also Table LX) indicat-
ing the lack of mutagenicity of all three isomeric forms of aminophenol in the
Ames test. ^-Phenylphenol was listed as positive in one publication (1) but
the accompanying data provide no support for the conclusion; the compound was
listed as a nonmutagen in a more recent publication (3). Most of the 33
compounds that yielded negative data and two that gave equivocal results have
been previously shown to be nonmutagenic (see Table LX). Negative compounds
which do not appear to have been previously tested include hydroquinone mono-
methyl ether (1), ^-benzyl-jv-chlorophenol, o-sec-butylphenol, tetrabromobis-
phenol A (2), p-tert-pentylphenol (3), and propyl gallate (4). Sodium
jv-phenylphenate (the sodium salt of ^j-phenylphenol), a bladder carcinogen, is
not mutagenic in the Ames test (7). Several phenolic compounds exhibit co-
mutagenic activity. In the presence of sufficient amount of S9 mix, catechol
substantially enhances the mutagenic activity of benzo[a]pyrene and of
4-nitroquinoline-l-oxide toward Salmonella typhimurium (8). Similarly,
butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and, to a
lesser extent, propyl gallate increases the mutagenicity of aflatoxin Bj
(9). The mechanism of the enhancement is not known. A number of phenolic
compounds enhance the mutagenicity of other compounds by inducing their acti-
vating enzymes. Pretreatment of mice with 2(3)-tert-butyl-4-hydroxyanisole
results in a 5-fold increase in the ability of liver microsomes to activate
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aflatoxin Bi to a mutagen (10) whereas 2,3,4,5-tetrachlorophenol enhances the
ability of rat liver microsomes to activate benzo[a]pyrene (11).
Among phenolic compounds tested for teratogenic activity, ^v-phenylphenol
(12), 2,4-dichlorophenol (13), chlororesorcinol (14), pyrogallol (14), and
m-aminophenol (15) are all inactive in the rat. In each of the above studies,
several dose levels were used with the highest dose level being sufficiently
high to cause some degree of maternal or fetal toxicity. ^v-Aminophenol and
jr-aminophenol are teratogenic in the Syrian golden hamster whereas the data on
m-aminophenol were equivocal (16). In this study, the compounds were admini-
stered to rats intraperitoneally, intravenously or orally on day 8 of gesta-
tion at doses of 100, 150 or 200 mg/kg; the fetuses were examined on day 13 of
gestation. The most frequently observed malformations were encaphalocele and
limb, tail and eye defects. The teratogenic effects were observed at doses
which did not produce any apparent maternal toxicity. The authors suggested
the formation of a reactive quiononeimine metabolite as a proximate (or ulti-
mate) teratogenic intermediate. This view was supported by the finding that
jv-benzoquinoneimine exhibited similar teratogenic effects as j>-aminophenol in
the hamster.
The carcinogenicity of butylated hydroxytoluene (BHT) and butylated
hydroxyanisole (BHA) has been further tested in view of their widespread use
as food additives. Two groups of Japanese investigators confirmed that BHT is
not carcinogenic in Wistar rats (17) or B6C3Fj mice (18) after long-term
feeding. In agreement with previous studies using mice as the test species,
BHA has also been reported to be noncarcinogenic in B6C3Fi mice (Yokoro, cited
in re-f. 19). However, there is strong evidence to indicate that BHA is car-
cinogenic toward the forestomach of F344 rats and Syrian golden hamsters. Ito
£t^£l^. (19) fed groups of F344 rats diets containing 0.5 or 2.0% BHA for 2
-------
years. In the high dose group, virtually all the rats developed papillomas in
the forestomach; 18 of 52 male rats and 15 of 51 female rats also had squamous
cell carcinomas in the forestomach. In the low dose group, the only, signifi-
cant change was an increase in the incidence of hyperplasia of the fore-
stomach. None of the control animals exhibited similar pathological
changes. A preliminary 24-week study (20) on Syrian golden hamsters suggested
that this species may be even more susceptible to BHA-induced forestomach
carcinogenesis; 17 of 17 hamsters maintained on diets containing 1.0 or 2.0%
BHA developed forestomach papillomas within 24 weeks. In view of these new
findings, the potential health hazard of widespread use of BHA should be
urgently reevaluated. Both BHT and BHA have been shown to be either an inhi-
bitor or a promoter of carcinogenesis depending on the target organ, the type
of carcinogen (initiator) and the temporal sequence of administration. Adding
to the existing body of literature on the ambivalence of BHA and BHT (see
Section 5.2.2.5.3.4; see also review in ref. 21), Imaida ^t^ j_l^. (22) showed
that both BHA and BHT enhance 4-hydroxybutylbutylnitrosamine-induced bladder
carcinogenesis in rats. Maeura^^. (23) found BHT to be a promotor of
2-acetylaminofluorene-induced bladder carcinogenesis in rats but an inhibitor
of hepatocarcinogenesis by the same compound. Prophylactic effects against
methylazoxymethanol-induced colon carcinogenesis (24), benzo[a]pyrene-induced
pulmonary carcinogenesis (25) in mice have been demonstrated by BHA and an
inhibition of dimethylbenz[a]anthracene-induced mammary carcinogenesis by BHT
has been reported (26).
j>-Phenyl phenol (OPP) and its sodium salt (sodium _p_-phenylphenate,
OPP-Na), widely-used broad spectrum fungicides used in protection of various
edible crops, have recently been tested for carcinogenic activity. Hiraga and
Fujii (27) reported that OPP-Na was a notably active carcinogen in male F344
-------
rats inducing tumors of the urinary system within a relatively short latency
period. In a subchronic 13-week study, 9 of 10 male rats fed 2% OPP-Na in the
diet were found to have urinary bladder tumors with five of these tumors
diagnosed as transitional cell carcinomas. Female rats appeared to be sub-
stantially less susceptible; only 2 of the 10 female rats fed 4% OPP-Na
developed bladder papillomas within this time period. A subsequent 91-week
study showed that the carcinogenic effect of OPP-Na was dose-dependent; the
incidences of transitional cell carcinomas of the urinary bladder, renal
papilla or pelvis in male rats fed 0.125, 0.25, 0.5, 1.0, 2.0 or 4.0% OPP-Na
were 0, 0, 5, 35, 95 and 85%, respectively. In a preliminary communication,
Fujii and Hiraga (28; and K. Hiraga, personal communication) reported that
dietary administration of OPP to F344 rats for 2 years also induced urinary
bladder tumors. The carcinogenic potency of OPP appeared to be less than that
of OPP-Na. A carcinogenesis bioassay of OPP by the U.S. National Toxicology
Program was near completion at the time of this writing. In a 36-week study
designed to test the tumor-promoting activity of dietary administration of 2%
OPP and OPP-Na, Fukushima _et_^l_. (29) found that OPP-Na induced urinary
bladder tumors in 8 of 44 F344 rats whereas OPP had no significant effect
within this time period. When administered after tumor initiation with a
known bladder carcinogen, 4-hydroxybutylbutylnitrosaraine, OPP-Na exhibited
potent promoting activity whereas OPP had little or no promoting activity.
The molecular basis for this apparent considerable difference between the free
phenol and its sodium salt is not clear. It may be related to the easier
generation, from the sodium salt, of phenolate (phenoxide) anion, which is
stabilized by resonant limit structures involving an extended conjugated
system through the unsubstituted phenyl ring. These structures would facili-
tate the generation of setniquinone-type reactive intermediates (see below).
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Two chlorinated phenols, 2-chlorophenol and pentachlorophenol, have no
significant carcinogenic activity in Sprague-Dawley rats after prenatal
(throughout the gestation period) and postnatal (from weaning to up-to 24
months of age) exposure to 5, 50 or 500 ppm (2-chlorophenol in drinking water
or pentachlorophenol in feed) (30; and J.H. Exon, personal communication). In
contrast to the lack of complete carcinogenic activity, both 2-chlorophenol
and, to a lesser extent, pentachlorophenol exhibited promoting or cocarcino-
genic activity enhancing the carcinogenic action of the transplacental carci-
nogen, N-ethyl-N-nitrosourea. The enhancing effect of 2-chlorophenol was
evident even at the low dose of 5 ppm administered either prenatally or
postnatally.
A carcinogenesis bioassay of propyl gallate, a polyhydric phenolic com-
pound, has recently been completed by the U.S. National Toxicology Program
(4). Groups of 50 F344/N rats and 50 B6C3Fi mice of each sex were maintained
on diets containing 6,000 or 12,000 ppm propyl gallate for 103 weeks. There
were higher incidences of preputial gland tumors, islet-cell tumors of the
pancreas and pheochromocytomas of the adrenal gland in male rats in the low
dose group, as well as an astrocytoma and a glioma (two rare types of brain
tumors) in two low-dose females. None of these tumors were found in the high-
dose group, however. Thus, under the conditions of this bioassay, propyl
gallate was concluded to be not carcinogenic in F344/N rats or B6C3Fi mice of
either sex.
The in vitro metabolism of 3-J^-butyl-4-hydroxyanisole has been studied by
Rahimtula (31). 3-£-Butyl-4-hydroxyanisole is oxidized to a variety of meta-
bolites which include formaldehyde, a dimeric product, polar metabolites, as
well as a reactive intermediate(s) that binds irreversibly to proteins. The
authors proposed that 3-^-butyl-4-hydroxyanisole is oxidized predominantly via
-------
one electron oxidation to yield reactive free radical(s) which dimerizes,
undergoes further metabolism or binds to protein. The study underscores the
importance of peroxidases in the metabolism of 3-^j-butyl-4-hydroxyanisole; the
in vivo significance of these findings remains to be investigated.
The possible molecular mechanisms involved in toxicity and bladder car-
cinogenesis by ^v-phenylphenol (OPP) or its sodium salt (OPP-Na) have been
investigated by Reitz et_ jil. (32). Both compounds appear to have little or no
genotoxic activity as demonstrated by the lack of rautagenicity in the Ames
test and the lack of in vivo covalent binding of OPP or OPP-Na to rat bladder
DNA. The metabolism of OPP or OPP-Na was shown to be dose-dependent. At
doses below 50 mg/kg, conjugation to polar metabolites (glucuronide or sulfate
conjugates) was the predominant reaction. At doses above 500 mg/kg, there was
evidence of oxidative formation of 2,5-dihydroxybiphenyl. It was postulated
that the reactive intermediates (e.g., semiquinone) generated by this oxida-
tive pathway may be associated with the toxic or carcinogenic action induced
by high concentrations of OPP or OPP-Na.
A number of investigators have recently presented evidence of the altera-
tion of immune functions of animals treated chronically with phenolic com-
pounds. Immunosuppression has been implicated as a possible mechanism of
carcinogenic or tumor-promoting action of phenolic compounds; however, the
evidence for such an association does not appear to be very strong or consis-
tent. LaVie and LaVie (33) reported that £j-phenylphenol was immunosuppressive
in mice; this finding could not be confirmed by Luster j2t^ jil_. (34). Kerkvliet
,£t_.al_» (35) demonstrated that chronic treatment of mice with technical grade
pentachlorophenol suppressed the immune functions and increased the suscepti-
bility of the animals to tumor cell challenge. Mixed findings were reported
for purified pentachlorophenol which was not immunosuppressive in mice (35),
-------
reduced humoral and cell-mediated immune functions but enhanced macrophage
activity in rats (36), and suppressed humoral immunity and T-cell responses to
mitogens in chickens (37). 2-Chlorophenol, an active tumorigenesis"promoter,
was not immunosuppressive in the rat (3fe).
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t
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