PHENOLS AND PHENOLIC 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;
5.2.2.4             Ethylene Glycol, Diethylene Glycol, Dioxane and
                    Related Compounds
   5.2.2.4.1        Introduction
   5.2.2.4.2        Physicochemical Properties and Biological Effects
      5.2.2.4.2.1   Physical and Chemical Properties
      5.2.2.4.2.2   Biological Effects Other Than Carcinogenic
   5.2.2.4.3        Carcinogenicity and Structure-Activity Relationships
   5.2.2.4.4        Metabolism and Mechanism of Action
   5.2.2.4.5        Environmental Significance
References

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 5.2.2.4  Ethylene Glycol, Diethylene Glycol, Dioxane and Related Compounds.





     5.2.2.4.1  Introduction.





     Glycols are organic compounds characterized by two hydroxyl groups linked




 to separate carbon atoms in an aliphatic chain.  Simple glycols have the




 general formula, GnH2n^OH^2*  Polygiycols* GnH2n° ^OH^2' are adducts of simple



 glycols with intervening ether linkage(s) in the hydrocarbon chain.  Replace-




 ment of one or both hydroxyls with alkoxy groups yields glycol ethers.




jv-Dioxane, a cyclic ether, is generally considered to be a derivative of




 ethylene or diethylene glycol.  Many of these chemicals have been extensively




 used as solvents for numerous industrial, pharmaceutical and consumer




 products, as anti-freeze fluids, as surfactants, as fixatives in cosmetic




 products, as dessicants, and as reaction media in chemical synthesis.





     The concern over the potential carcinogenicity of glycol and related




 compounds arose in 1946 when Fitzhugh and Nelson (1) demonstrated that rats




 fed diets containing high concentrations of diethylene glycol developed




bladder tumors; in all but one case, rats that bore tumors also had bladder




 stones (calculi).  Subsequent studies by Weil J2£_al/ (2, 3) casted doubt on




 the carcinogenieity of diethylene glycol proper.  In fact, experiments




 designed to determine the effect of implanted calcium oxalate stones on




bladder carcinogenesis led the authors to conclude that diethylene glycol is




 not a primary carcinogen and that diethylene glycol-induced bladder  tumori-




geuesis is most likely the result of mechanical irritation by the bladder




 stones.  However, the carcinogenieity of diethylene glycol cannot be




 completely discredited.  In a 1968 study, a Russian investigator (4) reported




 that mice exposed to vapor containing low concentrations of diethylene glycol




developed mammary tumors.  Further studies of this class of compounds are




needed in view of their extensive use.

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     p-Dioxane was first reported  to be carcinogenic in rats by Argus et al.

 (5) in 1965.  This finding has been confirmed in subsequent studies (6) and by

 other investigators (7, 8).  In addition, mice and guinea pigs are also

 susceptible  to the hepatocarcinogenic action of this widely used industrial
                                 [ text-figure]
solvent.  Two related compounds, 2,3-dichloro-p_-dioxane and diiaethoxane, also

display carcinogneic activity.  The dioxanes appear to represent a new

oncogenic structural type and it is likely that other active agents may be

found among their derivatives and analogs.

     This section focuses on the carcinogenicity of glycols, dioxanes and

related compounds and their possible mechanism of action.  Mutagenicity and

developmental toxicity are also discussed in some detail.


               ./ .
     5»2.2.4.2  Physicocheraical Properties and Biological Effects.

     5.2.2.4.2.1  PHYSICAL AND CHEMICAL PROPERTIES.

     THe physical and chemical properties of various glycols, glycol ethers,

glycol ether acetates and _p_-dioxane have been reviewed by Rowe (9, 10) in 1963

and by Brown et al. (11) in 1980.  The physical properties of some glycols and

related compounds are summarized in Table I.  Simple glycols with low

molecular weight are odorless and colorless, freeze below 0°C, have low

volatility,  are very hygroscopic and ciiscible in all proportions with water,

and have excellent solvent properties.  Glycols undergo reactions common to

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   (
   •o     ci
2,3-Dichloro

 p-dioxane
            CH
                             .0
      0-C-CH
         il
         0



Dimethoxane
            Text-Figure

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             Table I.   Physical Properties of Glycols  and Related Compounds*
Compound
Ethylene glycol
monomethyl ether
dimethyl ether
monoethyl ether
Propylene glycol
3-Chloro-l , 2-
m.p.
(°C)
-13
-85
-71
-70
-60 '
—
b.p.
(°C)
197.6
124.2
85.1
134.7
187.2
213
Vapor Pressure
(mm Hg)
0.06 at 20°C
9.7 at 25°C
59 at 20°C
5.3 at 25°C
0.13 at 25°C
0.03 at 25°C
Specific Gravity
1.1155 (20/20°C)
0.963 (25/25°C)
0.869 (20/20°C)
0.928 (25/25°C)
1.038 (20/20°C)
1.3204 (20/4°C)
Solubility in
Water
miscible
miscible
miscible
niscible
miscible
miscibled
   propanediol

 2-Ethyl-l,3-
   hexanediol

 Diethylene
   glycol

|Triethylene
"  glycol

 j>-Dioxane

 Dimethoxane0
  -40     244.2     < 0.01 at 20°C  0.9422 (20/20°C)   4.2 gm/100 ml
   -8.0   245
                                                       at 20°C
< 0.01 at 20°C  1.1184 (20/20°C)   miscible
   -4.3   287.4     0.001 at 20°C   1.1254 (20/20°C)   miscible
   11.8   101.3     37 at 25°C

< -25      86
       (at 10 mm Hg)
                1.0356 (20/20°C)   miscible
                1.055-1.070
                 (25/25°C)
miscible
  Summarized from data compiled by C.H. Hine, J.K. Kodama, J.S. Wellington, M.K. Dunlap
  and H.H. Anderson [A.M.A. Arch. Ind. Hlth. 14, 250 (1956)]; V.K. Rowe, _In_ "Patty's
  Industrial Hygiene and Toxicology" (D.W. Fassett and D.D. Irish, eds.) 2nd edn., Vol.
  II, Interscience, New York, 1963, p. 1497; E.S. Brown, C.F. Hauser, B.C. Ream, and
  R.V. Berthold [Kirk-Othmer's Encycloped. Chem. Tech. (3rd edn.) 11, 933 (1980)]; G.H.
  Riesser [Kirk-Othmer's Encycloped. Chem. Tech. (3rd edn) _5_, 848 (1979)]; International
  Agency for Research on Cancer [IARC Monog. 15, 177 (1977)].

  Also known as glycerol X -monochlorohydrin; 
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alcohols;  they form esters, acetals, ethers and similar reaction products.




Ethylene glycol may be dehydrated to yield diethylene glycol, which gives rise




to p-dioxane following intramolecular cyclization.  ^-Dioxane and glycol




ethers have similar physicochemical properties as glycols but are considerably




more volatile.  Chlorinated derivatives of simple glycols are expected to be




more reactive than Che parent glycols themselves, if the chlorine atom and




hydroxyl group are on adjacent carbon atoms.  3-Chloro-l,2-propanediol




(-dioxane (18), and glycol derivatives (11) have since been




published.  Representative acute LDcn data of glycols and related compounds




are summarized in Table II.  As the data in the Table indicate, unsubstituted




glycols (especially propylene glycol) have very low acute toxicity.  Alkyl




ether derivatives of glycols (glycol ethers) are considerably more toxic than




their parent compounds.  Chlorination may have an even more pronounced effect




in enhancing the acute toxicity of glycol and related compounds (compare




propylene glycol vs 3-chloro-l,2-propanediol;  p-dioxane vs trans-2,3-dichloro-




p-dioxane).  A 1980 study by Woo et al.  (19) showed that the enhancement of

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Table II.  Acute Toxicity of Glycols and Related Compounds
Compound
Ethylene glycol
Ethyl ene glycol mono-
methyl ether
Ethyl glycol mono-
ethyl ether
Propylene glycol
3-Chloro-l ,2-propanediol
2-Ethyl-l , 3-hexanediol
Diethylene glycol
Diethylene glycol
dimethyl ether
Triethylene glycol
p-Dioxane
trans-2,3-Dichloro-
p-dioxane
Species & Route
Rat, oral
Rabbit, topical
Pat, oral
Rabbit, topical
Rat, oral
Rabbit, topical
Rat, oral
Rabbit, topical
Rat, oral
Rat, oral
Rat, oral
Rabbit, topical
Rat, oral
Rat, oral
Rabbit, topical
Rat, oral
Rat, i.p.
Rat, oral
Rat, i.p.
Rabbit, topical
LD50
7.4 ml/kg
20 ml/kg
2.46 g/kg
1.34 ml/kg
3.0 g/kg
3.5 ml/kg
34.6 ml /kg
20 ml/kg
0.15 g/kg
2.71 g/kg
28.3 ml/kg
11.9 ml /kg
5 g/kg
28.2 nil/kg
20 ml/kg
5.17 g/kg
5.3 g/kg
1.41 ml /kg
0.435 g/kg
0.44 ml/kg
Reference
(11)
(11)
(11)
(11)
(11)
(11)
(ID
(11)
(19)
(20)
(11)
(ID
(ID
(11)
(11)
(21)
(22)
(23)
(24)
(23)

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 toxicity of p-dioxane by chlorination is stereoselective and that a stereo-

 isomer of 2,3,5,6-tetrachloro-jv-dioxane is 1,000 times more toxic than its

 parent compound.

                                                             *
     Ethylene glycol, diethylene glycol and _p_-dioxane are known to have caused

 many cases of human poisoning (9, 10, 15, 18, 25) due to intentional, acci-

 dental or incidental misuses of  these solvents.  The principal toxic actions

 are:  severe depression of the central nervous system, damage of the kidney,

 the brain and the liver.  Judging from the cases in which the amount ingested

 could be approximated, the lethal dose of ethylene and diethylene glycol for

 humans is estimated to be 1.4 ml/kg (9) and 1.0 ml/kg (2), resepctively.  In

 addition to being relatively more toxic than glycols, a number of glycol

 ethers have been shown to exert reproductive and developmental toxicity toward

 animals.  In 1942, Morris et al. (26) reported that ethylene glycol monoethyl

 ether caused testicular enlargement, edema and tubular atrophy in rats.  Since

 1971, at least five glycol ethers and ether acetates (monomethyl ether, mono-

 methyl ether acetate, nonoethyl ether and monoethyl ether acetate of ethylene

 glycol and diethylene glycol dimethyl ether) have been demonstrated to cause

 testicular atrophy, male sterility or abnormal sperm head morphology in mice,

 rats, rabbits or dogs (27-29).  The U.S. National Institute for Occupational

 Safety and Health has recently selected 15 glycols and glycol ethers for

 testing for reproductive toxicity to elucidate possible structure-activity

 relationships.   Besides glycol ethers, 3-chloro-l,2-propanediol (glycerol

 o^-monochlorohydrin) also causes testicular atrophy in rats (30).


     Mutagenicity.  A number of glycols and glycol derivatives have been

 tested for mutagenicity in a variety of test organisms.  With the exception of

chlorinated derivatives, compounds of this chemical class are generally inac-

 tive in microbial and mammalian assays but may display some activity in cer-

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 tain plants.   In the  Ames  Salmonella  test,  ethylene glycol  (31,  32)  and  its




 mono- and  dimethyl  ether derivatives  (29,  33), propylene glycol  (32,  34,  35),




 diethylene glycol  (27,  32)  and _p_-dioxane  (33,  36,  37)  have  all been  consis-




 tently shown  to  be  inactive.  In  fact,  some of the compounds mentioned above




 are routinely used  as solvents in mutagenicity testing of other  chemicals.




 Only jn-dioxane displays weak mutagenic  activity in one study but its  activity




 in another is questionable  (36).  As  may be expectable from its  chemical




 properties, 3-chloro-l;2-propanediol  (glycerol c<-nionochlorohydrin)  is muta-




 genic, inducing  base-pair substitution  in  Salmonella  typhimurium TA1535 both




 with and without metabolic  activation.  Esterification of the compound with




 fatty acids significantly reduces its nutagenicity (38).





      In other mutagenicity  tests, ethylene  glycol, its monomethyl  ether,  and




_p_-xlioxane  are nonmutagenic  in the yeast Schizosaccharomyces pombe  and in  V79




 Chinese hamster  cells  (39).  No chromosome  aberrations were observed  in  Swiss




 C.F.L.P. mice which received ethylene glycol orally and intraperitoneally




 (40).   Preliminary  communications by  McGregor e± al_.  (29) and by National




 Toxicology Program  (27) indicate  that ethylene glycol  monomethyl ether and




.diethylene glycol dimethyl  ether  are  both inactive in  inducing unscheduled DNA




 synthesis, are devoid of clastogenic  potential in rat  bone  marrow cells,  and




 have  marginal  activity in Drosophila  sex-linked recessive lethal test.




 Although both compounds display activity in the rat dominant lethal  assay,  the




 activity is attributed to the antifertility effects rather  than  to dominant




 lethal  mutations.   Ethylene glycol monoethyl ether is  inactive in  both  the rat




dominant lethal  and the Drosophila sex-linked recessive lethal assays (27).




 In  contrast to mammalian and inicrobial  organisms,  there is  some  evidence  that




ethylene glycol  and _p_-dioxane may be  mutagenic in certain higher plants.




Bhattacharyya, 3ose and Kandu (cited  in ref. 41) reported that ethylene glycol

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 is  nutagenic  in  rice plants and reduces germination of seeds.  Alain et al.


 (42)  showed  that treatment of wheat seeds with ethylene glycol induces chromo-

                  *.
 some  aberrations in both mi to tic and meiotic cells of the resulting plants.


 Hohl  (43) demonstrated  that _p_-dioxane causes chromosomal abnormalities in the


 root  cells of Vicia faba and Allium cepa; its activity is similar  to  that of


 urethan.


      Teratogenicity.  The potential teratogenicity of ethylene glycol, di-


 ethylene glycol,  propyiene glycol, 1,3-propanediol and several butanediol


 isomers has been tested by Gebhart (44) using chick embryo.  Only  1,3-propan-


 diol  is teratogenic, inducing micromelia; interestingly, its isomer,  propyiene


 glycol, is inactive.  The lack of teratogenicity of propyiene glycol  has been


 confirmed in  the rat (45).  In contrast to glycols, two methyl ether  deriva-


 tives of ethylene glycol have been shown to be active teratogens.  Stenger et


 al. (46) reported that ethylene glycol monomethyl ether induces skeletal


 abnormalities in the rat, but not in the mouse and the rabbit.  A  1981 study


 by  Nagano ^_£jil_.  (47) showed that the compound is also teratogenic in strain


 ICR mice.  A significant increase in the incidence of skeletal malformations


 was observed in  the fetuses of mice given daily oral dose of 31.25 mg/kg on


 days  7-14 of gestation.  At the dose of 250 mg/kg, all fetuses examined had


 skeletal malformations.  Uemura (48) demonstrated that ethylene glycol


 dimethyl ether is also  teratogenic in mice inducing skeletal abnormalities as


well as surface  deformities such as external brain, palpebral potency, caudal


defect, peritoneal hernia and cleft palate.  The doses administered ranged


 from 250-490 ng/kg.   Teratology assays conducted in the U.S. National


Toxicology Program show that monone thy1 ether, monoethyl ether and monoethyl


ether acetate derivatives of  ethylene glycol increase fetal malformations in


animals exposed by inhalational or dermal route (28).  As mentioned under

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 Toxic  Effects  section,  the  U.S.  National  Institute for Occupational  Safety  and




 Health has  expressed concern over  the  potential developmental  toxicity  of




 glycol ethers  as  a  chemical class  (27).   A monobutyl ether derivative of




 propylene glycol  has been  tested in  CD rats up to a daily oral dose  of  87.5




 mg/kg  on days  6-15  of gestation;  the compound caused minor embryotoxicity and




 is not considered teratogenic  ('49).  There is no information available  on the




 teratogenicity of j>-dioxane.   A  1964 study by Franceschini (50)  showed  that




 p-dioxane,  like  thalidomide, affects the  growth of tibial buds of  cultured




 7-day-old chick embryo; whether  this activity is indicative of potential




 teratogenicity remains  to be investigated.









     5.2.2.4.3  Carcinogenicity  and  Structure-Activity Relationships.





     Nine glycols and related  compounds have been tested for carcinogenicity;




 the results of these studies are summarized in Table III.  The data  available .




 indicate that  most  glycols have little or no carcinogenic activity.  There  is




 some suggestive  evidence that  3-chloro-l,2-propanediol (
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                           Table  III.   Carcinogenic!ty  of  Clycols and Related Compounds
Compound
Ethylene glycol
(1,2-ethanediol)
Propylene glycol
( 1 , 2-propanediol )

3-Chloro-l , 2-propanediol
(glycerol -mono-
chlorohydrin)
2-Ethyl-l , 3-hexanedlol


Diethylene glycol


Triethylene glycol
p-Dioxane







2 , 3-Dichloro-p-dioxane


Dime thoxane
Species & Strain
Rat, Sprague-Dawley
Rat, F344
Mouse, Swiss
Rat, Charles River CD
Rabbit, New Zealand
Mouse, ICR/Ha
Rat, Charles River CD

Mouse, Swiss
Rat, —
Rabbit, New Zealand
Mouse, —
Rat, Osborne-Mendel
Rat, CFN
Rat, Osborne-Mendel
Mouse, Swiss-Webster
Mouse, B6C3F.
Rat, Wistar
Rat, Charles-River CD,
Sherman or Osborne-
Mendel
Rat, Wistar
Guinea pig
Mouse, ICR/Ha
Mouse, ICR/Ha
Mouse, ICR/Ha
Rat, Wistar
Route
oral
s .c .
topical
oral
topical
topical or s.c.
oral

topical
oral
topical
inhala tion
oral
oral
oral
topical
oral
oral
oral


inhala tion
oral
topical
s.c.
i .p.
oral
Principal Organs
Affected
None
None
None
None
None
None
o
None (inconclusive)

None
None
None
Mammary gland
Urinary bladder
None
None
None
Liver
Liver
Liver, nasal cavity


None
Liver
Skin (?)c
Local sarcoma
None
Liver
Reference
(51)
(52)
(53)
(54)
(55)
(13)
(30)

(53)
(Lehman, cited in ref. 9)
(55)
(A)
(1)
(2, 3)
(1)
(56)
(8)
(5)
(6-8)


(57)
(58)
(13)
(13)
(13)
(59)
a. ,,,...., .
 With concomitant formation of bladder stones (calculi).
cSkin papilloma in 2/50 mice.
 Also known as 2,6-dime thyl-m-dioxan-4-ol acetate.

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 mammary  gland) were observed  toward  the  end of  the experiment but  the distri-

 bution of  the  tumors was such  that it was impossible  to correlate  incidence

 with  treatment.   None  of  the  tumors  were associated with  the occurrence  of

 calculi.   Ethylene glycol has  also been  tested  in the rat by subcutaneous

 route at maximum  tolerated dose; no  evidence of carcinogenicity was  found

 (52).  Ethylene glycol and its monoethyl ether  derivative are being  tested for

 carcinogenicity in the U.S. National Toxicology Program at  the  time  of  this

 wri ting.


     Propylene glycol has consistently been found to be noncarcinogenic  by

 oral or  topical route in 3 species of animals (see Table III).  In the oral

 study, rats were  fed diets containing up to 50,000 ppm propylene glycol  for  2

 years with no untoward effects.  This level is  equivalent to daily consumption

 of 2.5 g/kg body  weight (54).  3-Chloro-l,2-propanediol, a  chlorinated deriva-

 tive of propylene glycol, is also noncarcinogenic when tested by topical

 application or subcutaneous injection to ICR/Ha mice  (13).  When tested  by

 oral administration at the maximum tolerated dose, the compound induced  para-

 thyroid adenomas  in 3/26 male  rats.  The increase in  tumor  incidence was not
                                                                              s
 s-tatistically significant.  However, because of possible incomplete  reporting

 of the data,  this study was considered inconclusive (30).   2-Ethyl-l,3-hexane-

 diol, an insect repellent, is also inactive by  topical or oral route (see

 Table III).


     Diethylene glycol was first reported to be carcinogenic in Osborne-Mendel

 rats by Fitzhugh and Nelson (1) in 1946.  Groups of 12 nale rats were fed

 diets containing 1, 2, or 4% diethylene  glycol  for up to 2  years.   Six rats  of

 the 2% group and five of the 4% group developed bladder tumors.  With one

exception,  the rats that bore  tumors also had bladder stones.  The signifi-

cance of  diethylene glycol as a bladder  carcinogen was doubted by  Weil et al.


                                      8  -

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 (2,  3), who  found only one bladder  tumor among 155 CFN rats  treated with




 diethylene glycol.  Instead,  they found that a number of rats,  that were never




 fed  diethylene glycol, developed bladder tumors after receiving surgical




 implantation of either calcium oxalate stones or glass beads in the bladder.




 They concluded that diethylene glycol is not a primary carcinogen  and  that  the




 induction of bladder  tumors by the  compound is probably  the result of




 mechanical irritation by  the bladder calculi.  The formation of bladder




 calculi is now believed to be involved in bladder carcinogenesis in rodents by




 a number of  chemicals (60).  A 1968 Russian study (4) reported  that diethylene




 glycol also  fails to induce any bladder tumor in mice after long-term




 intragastric administration.  However, chronic (6-7 months) inhalational




 exposure to  low concentrations of diethylene glycol (4-5 ug/1)  caused malig-




 nant mammary gland tumors in 9 of 16 mice and one lympholeukosarcoma in  the




 cervix 2.5-11 months after the end  of treatment.  No tumors were found in 20




 control mice.





     Triethylene glycol was also tested in the study of  Fitzhugh and Nelson




 (1).  No adverse effects were observed in rats fed diets containing 1, 2, or




 4% of the compound for 2 years.  These doses are equivalent to  daily intake of




 3-4 g/kg body weight.  In a study designed to test the carcinogenicity of




 7,12-dimethylbenz[a]anthracene, triethylene glycol was used as  the vehicle.




 The glycol alone induced planocellular cancer in one and papillotnas in four of




 the  80 mice  (Shemyakina, cited in ref. 16); the significance of this finding




 is questionable,  however,  due to the lack of proper control gz^^£/.





     _p_-Dioxane was first reported to be carcinogenic in  rats by Argus et al.




 (5).  Six of the 26 male Wistar rats that received drinking water  containing




1% _p_-dioxane (equivalent to daily intake of 300 rag) for up to 63 weeks




developed hepatomas.   In addition,  one rat bore a transitional  cell carcinoma

-------
in  the kidney pelvis and one rat had  leukemia.   No  tumors were  found  in nine




control rats.  A subsequent experiment by Hoch-Ligeti £_£.£!/  (6)  showed that




_p_-dioxane is also carcinogenic  toward the nasal  cavity  of  the rat.   Six out of




120 Charles River CD rats fed _p_-dioxane in drinking water at  levels of




0.75-1.8% developed squamous cell  carcinomas  in  the nasal cavity.  Spontaneous




tumors at this tissue localization occur extremely rarely in  laboratory




animals.  Four rats with nasal  carcinoma also bore hepatocellular carci-




nomas.  The hepatic and nasal carcinogenicity of _p_-dioxane has  been confirmed




by Kociba _e_t _al_. (7) and by U.S. National Cancer  Institute (8).   In  the study




of Kociba ^e£^l_. (7), liver tumors were found in  12 and nasal carcinomas in 2




out of 66 Sherman rats  that received  1% £-dioxane in drinking water.   In rats




receiving lower doses of £-dioxane (0.1% or 0.01%), no  treatment-related tumor




induction was noted.  In the National Cancer  Institute  study  (b), in  which




Osborne-Mendel rats were given .0.5 or 1.0% £-dioxane in the drinking  water,




significant increase in the incidences of squamous cell carcinoma of  the nasal




turbinates (males:  controls 0/33, low dose 12/33, high dose  16/34; females:




controls 0/34, low dose 10/35,  high dose 8/35) and hepatocellular adenomas




(females:  controls 0/31, low dose 10/33, high dose 11/32) were noted.   The




totality of-these results clearly indicate the carcinogenicity  of j3-dioxane at




high dose.  It appears that Osborne-Mendel and Charles  River  CD rats  are more




susceptible to nasal carcinogenesis while Wistar and Sherman  rats are more




susceptible to hepatocarcinogenesis by _p_-dioxane.  j>-Dioxane  has  also been




tested in rats by inhalational  route, no significant carcinogenic effects were




observed in rats exposed to vapor containing 111 ppm j>-dioxane, 7 hours/day, 5




days/week for 2 years (57).





     In addition to rats, _p_-dioxane has been shown to be hepatocarcinogenic in




the  guinea pig and the mouse.   Hoch-Ligeti and Argus (58) showed  that of the
                                      10

-------
 22 male guinea pigs  that received drinking water containing  0.5-2% _p_-dioxane,




 two had carcinoma of the gall bladder and  three had hepatomas.   No liver




 tumors were found in 10 untreated control animals after  28 months.   The




 results of a U.S. National Cancer Institute bioassay (8) show significant




 increase in the incidence of hepatocellular carcinomas in B6C3F. mice




 (males:  controls 2/49, low dose 18/50, high dose 24/47; females:  controls




 0/50, low dose 12/48, high dose 28/37) fed 0.5 or 1.0% jv-dioxane in  the




 drinking water.  When tested by topical route in Swiss-Webster mice, King  et




 al. (56) initially reported that _p_-dioxane was inactive  as a complete




 carcinogen but highly potent as a tumorigenesis promoter, displaying an




 activity comparable  to that of croton oil.  However, a subsequent report (King




_e_t jil.,. 1974, cited in ref. 18), referring to later studies  stated  that  the




 promoting activity of jv-dioxane is substantially less than that  noted  in the




 initial study.





     In contrast to p-dioxane, trans-2,3-dichloro-p-dioxane  displays some




 complete or tumor-initiating activity toward the skin.   The  details  of these




 studies have been discussed in the Haloether section (Section 5.2.1.1.2, Vol.




 Ill A).  Dimethoxane (2,6-dimethyl-m-dioxan-4-ol acetate), an m^dioxane deri-




 vative, has also been tested by Hoch-Ligeti _e_t _al_. (59).  Administered orally,




 the compound induced hepatomas in 8 of 25 Wistar rats and tumors of  kidney,




 skin, subcutaneous and lymphoid tissues, and leukemia in 5 other rats.  The




 demonstration of carcinogenic!ty of this ^-dioxane derivative,  together with




 that of _p-dioxane, indicates that the dioxanes ui*»y represent a new  structural




 type of chemical carcinogens.
                                      11

-------
     5.2.2.4.4  Metabolism and Mechanism of Action.





     The metabolism of ethylene glycol has been studied for decades (e.g., 15,




 61-64).  The metabolites include glycolaldehyde (HOl^CCHO), glycolic acid




 (HOH2CCOOH), glyoxylic acid (OHCCOOH), oxalic acid (HOOCCOOH), carbon dioxide,




 glycine, malate and possibly glyoxal  (OHCCHO).  The major  urinary metabolites




 are glycolic and oxalic acids along with the unchanged parent compound.  Renal




 and cerebral damage produced by ethylene glycol has been generally attributed




 to the deposition of oxalate crystals in these tissues.  A number of metabolic




 intermediates are known to be more  toxic than ethylene glycol in the rat;




 these are, in decreasing order of toxicity:  glyoxylate >  glycolaldehyde >




 glycolate > ethylene glycol (64).  With the exception of glyoxal, none of




 these metabolites appear to have been shown to be genotoxic.  Glyoxal, a




 1,2-dicarbonyl compound, has been shown to be mutagenic in the Ames test (65);




 however, its role as an ethylene glycol metabolite is still debatable (63).




 As mentioned in Section 5.2.2.4.2.2,  ethylene glycol is mutagenic in certain




 higher plants; the-.raechanism of its mutagenic action is not known.  Ethylene




 glycol has been reported to inhibit RNA synthesis in Neurospora crassa conidia




 (41), cause s trucJtur-a-1—changes of ribosomes in Escherichia coli_.(J&-6i). and	 .




 increase DNA polymerase activity possibly by denaturing DNA primer (67);




 whether these activities may contribute to genotoxic activity, if any, of




 ethylene glycol remains to be elucidated.





     Relatively little information is available on the metabolism of di-




 ethylene glycol.  In the rat, oxalate has oeen detected in the urine or blood




 after oral dosing (1, 9, 15).  Compared to ethylene glycol, diethylene glycol




is considerably less effective in producing oxalate, and most of the compound




is excreted unchanged (15).  A 1977 study by Woo £££!_• (68) showed that di-




ethylene glycol may be metabolized  to _p_-dioxane-2-one (see Fig. 1) in the






                                      12

-------
 rat.  Under similar conditions,  ethylene  glycol,  diglycolic  acid and  oxalic

 acid did not give  rise  to  this metabolite.  Wiley et_ al.  (69)  were  unable  to

.demonstrate the presence of  oxalate in the  urine  of  rabbits  and  dogs  given

 large oral doses of diethylene glycol  indicating  significant species  dif-

 ference in the metabolism.   The  mechanism of bladder carcinogenesis by di-
              i
 ethylene glycol has generally been  attributed  to  mechanical  irritation by

 oxalate bladder stones  (see  Section 5.2.2.4.3).   However,  it is  important  to

 point out that ethylene glycol,  a better  producer of oxalate,  has thus far not

 been shown to induce bladder tumors.   Furthermore,  the  demonstration  of

 mammary carcinogenic!ty of diethylene  glycol suggests that other mechanisms

 may also be involved.  In  this respect, it  is  interesting  to note that di-

 ethylene glycol and _p_-dioxane are metabolically closely related  (see  Fig.  1);

 whether they may have a common mechanism  of action needs  further investiga-

 tion.


      The metabolism and mechanism of action of jv-dioxane have been  extensively

 studied.   The major urinary  metabolite in the  rat has been independently

 identified as ^-hydroxyethoxyacetic acid (compound  III in Fig.  1)  and

£-dioxane-2-one (compound  IV in  Fig. 1) by  Braun  and Young (70)  and by Woo et

 al.  (68),  respectively.  The two compounds  are actually readily-interconvert-

 ible depending  on  the pH of  the  solution.   It  is  believed  that hydroxyacids of

 this type  have  a narked tendency to form  lac tone  except under alkaline condi-

 tions  (71).   The possible metabolic pathways proposed by Woo £_£  jil_. (68) are

 depicted  in Fig. 1.   These include: (a)  hydrolysis  of _p-dioxane (I)  to

 diethylene glycol  (II), followed by oxidation  of  one of the  hydroxyl  group to

 form A-hydroxyethoxyacetic  acid (III) and  its lactonic form,  _p_-dioxane-2-one

 (IV),  (b)  direct conversion  via  a possible  ketoperoxyl  radical intermediate

 similar  to the  reaction scheme proposed by  Lorentzen et al.  (72) for  the
                                       13

-------
    (c)/
    /
      _ JQ) _ HOH2C     CH2_OH

 0"   \             ^^
(I)       X
                                       \
                                         \
                                           \
                                       HOH2C     COOH
          (b)
             N
                                              (ni)
Fig. 1. Proposed metabolic pathways of p-dioxane.

-------
 conversion of benzo[a]pyrene to benzo[a]pyrene diones and (c) ctf-hydroxyla-




 tion, followed by oxidation of the hemiacetal or hydroxyaldehyde interme-




 diate.  Pathway (a) is supported by the observation that IV may be detected as




 an urinary' metabolite of rats given II; however, the absence of II in the




 urine of rats given £-dioxane suggests either very rapid conversion of II to




 IV or other mechanisms.  It is interesting to note that the activating




 metabolism of a number of cyclic nitrosamines has been shown to occur via ring




 o<-hydroxylation (see Section 5.2.1.2.4.1 in Vol. Ill A).  The toxicological




.implication of _p_-dioxane neltabolisn depicted above is still not understood and




 is probably dependent on the shift of equilibrium between III and IV under




 physiological conditions.  Considering III as the sole metabolite, Young et




 al. (73) proposed that j>_-dioxane is mainly detoxified by the above pathway and




_tha.C .toxic,, .effects of jj-dioxane are manifested only after the saturation of




 metabolic capacity.   Woo et ^1_. (22), however, showed that IV is more toxic




 than _p_-dioxane_.and that there is an apparent correlation between the




 metabolism and toxicity of j)-dioxane in rats pretreated with enzyme inducers




 such as polychlorinated biphenyls or 3-methylcholanthrene.  A number of lac-




 tones, mostly o< ,A -unsatiirated,  are known to be carcinogenic (see Section




 5.2.1.1.6 in Vol. Ill A).  The mechanism of carcinogenic action of _p_-dioxane




 is not known.  In vitro (74) and in vivo (37) binding studies indicate that




jv-dioxane does not bind covalently to DNA to any significant extent.




jv-Dioxane is  also ineffective in inducing unscheduled DNA synthesis in primary




 rat hepatocytes (37).   TH--», it appears that jv-dioxane may exert its carcino-




 genic action  by some epigenetic mechanisms,  the  nature of which remains to be




 elucidated.
                                       14

-------
     5.2.2.4.5  Environmental Significance.


     Glycols, glycpl ethers, dioxanes, and related compounds have numerous


commercial applications.  Exposure may occur to workers as well as. to


consumers.  Ethylene glycol is the most extensively used simple glycol.


Worldwide production of this compound in 1978 exceeded 6 million metric


tons.  About 40% of this amount is used in nonvolatile antifreeze for automo-


biles, 35% in the manufacture of polyester fibers and the remaining as de-


icing agent, heat-transfer fluid, drying agent, motor oil additive, and in


many other product applications (11).  Residue of ethylene glycol may be found


in pharmaceutical products, medical devices or food samples sterilized by


ethylene oxide (75).  Propylene glycol has an annual production volume of


about 0.38 million metric tons in United States alone; it is extensively used


in the food and pharmaceutical industries as solvent, humectant (for tobacco),


preservative, emollient (for cosmetics and Pharmaceuticals), in the manufac-


ture of plasticizers for food wraps, as lubricant or antifreeze for food


machinery or refrigeration units (11).  Residue of propylene glycol has also


been detected in potato starch that had been fumigated with propylene oxide

                          /
(76).  The—use of diethyl'ene glycol appears to be mainly confined to indus-


trial applications following the "elixir of sulfanilamide" tragedy in 1937


when more than 100 deaths occurred as a result of ingestion of the solvent


(77).  The higher homolog, triethylene glycol,/is used industrially (11) as


well as in fragrances (16).  A number of alkyl ether derivatives of glycols


are also widely pi\-ent ift a variety of consumer products such as paints,


inks, soaps, degreasing agents, cosmetics, etc. (27).  The National Institute


for Occupation Safety and Health estimates that 10,000 to 2,000,000 workers in


United States are exposed occupationally to each of at least 14 glycols,


glycol ethers and glycol ether acetates (cited in ref. 27).  Epidemiologic
                                      15

-------
 studies  of  possible health effects of  such  exposure are lacking.   A 1971


 Russian  study  (78) reported  that hematologic disorders occurred more often in
                 V

 90 workers  occupationally exposed  to diethylene glycol in  the  aromatic hydro-


 carbon industry  than in  those exposed  only  to aromatic hydrocarbons.  No


 increase in tumor induction  was observed  in workers exposed  for 1-9 years;


 however,  no firm conclusion  regarding  carcinogenicity should be made from  this


 study because  of the short duration of follow-up  time.



     _p_-Dioxane is a commonly used industrial and laboratory  solvent; its


 annual produc tion"ifi United  Spates alone  exceeded  7.4 million  kg  in 1973.  It


 has been used  as a stabilizer for  trichloroethylene, as a  solvent for cellu-


 lose, resins,  oils, waxes, dyes, adhesives, cosmetics, Pharmaceuticals, rubber


 chemicals and  surface coatings (79).   In  the laboratory, it  is employed as a


 solvent  in  chemi-calTynthesis, liquid  scintillation counting fluid and in  the


 preparation of tissues for histological studies.   The threshold limit value


 (TLV) recommended by the American Conference of Governmental and  Industrial

                                          o
 Hygienist (80) in 1980 is 25 ppm (90 mg/m  air); a lower limit of 1 ppm has


been proposed by the National Institute for Occupational Safety and Health


 (18).  Human - exposure~"to £-di ox ane is  not confined to occupational settings.


 In a 10-city survey, jv-dioxane (0.01 iig/1)  has been detected in the drinking


water of one (Lawrence, MA)  of these cities (81).  Marzulli £££l_. (82) have


 demonstrated that _p_-dioxane  may penetrate skin with relative ease; it has been


 estimated that 60 ug _p_-dioxane would be expected  to be absorbed with one


applicative, of a suntan lotion containing 600 ppm _p_-dioxane.   Two epidemio-


logic studies of workers exposed to p-dioxane have been conducted.  In a


German study of 74 current,  previous and  retired employees of  a dioxane-


manufacturing plant, Thiess £t_^l_« (83) reported  that no significant health


hazards were identified.  There were two  cancer deaths (a  lanellar epithelial
                                      16

-------
 f
\l
        carcinoma of the lumbar region and a nyelofibrotic leukemia); however, the



        incidence was not significantly different from that expected from a control

                          \

        population.  As a group, the workers were potentially exposed for an average


        of 24.9 years; the estimated (under simulated conditions) concentration of


        £-dioxane in the workplace air ranged from 0.01 to 13.3 ppm.  In another study


        of 165 employees who worked in a, dioxane plant in Texas, Buffler et al. (84)



        found no significant difference between observed cancer deaths and that
                                  o

        expected in a control population.  The authors noted, however, that the mean


        duration of exposure to dioxane for this cohort is less than 5 years and that


        the exposure of only 41% of this group occurred early enough to satisfy a



        latency criterion of 10 years.






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                                              17

-------
                                                                   .
 8.  NCI:  "Bioassay of 1,4-Dioxane for Possible Carcinogenic!ty," NCI
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     J.H.:  J. Natl. Cancer Inst. 67, 75 (1981).
                                      19

-------
31.  McCann, J., Choi, E., Yamasaki, E., and Ames, B.N.:  Proc. Nat. Acad.




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32.  Pfeiffer, E.H., and Dunkelberg, H.:  Food Cosmet. Toxicol. 18, 115




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33.  Maron, D., Katzenellenbogen, J., and Ames, B.N.:  Mutat. Res. 88, 343




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34.  Stolzenberg,. S.J., and Hine, C.H.:  J. Toxicol. Environ. Health 5, 1149




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37.  Stott, W.T., Quast, J.F., and Watanabe, P.G.:  Toxicol. Appl. Phanaacol.




     60, 287 (1981).




38.  Silhanova, L., Smid, F., Cerna, M., Davidek, H., and Velisek, J.:  Mutat.




     Res. 103, 77 (1982).




39.  Abbondandolo, A., Bonatti, S., Corsi, C., Corti, G., Fiorio, R.,




     Leporini, C., Mazzaccaro, A., Nieri, R., Barale, R., and Loprieno, N.:




     Mutat. Res. 79, 141 (1980).




40.  Conan, L, Foucault, B., Siou, G., Chaigneau, M., and Le Moan, G.:  Ann.-




     Falsif. Expert. Chim. 72, 141 (1979).




41.  Chaudhuri, R.K.:  Experientia 34, 735 (1978).




42.  Alam,  S., Khan, M.R., and Banu, K.:  Bengladesh J. Agric. Sci. _8_, 63




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43.  Hohl,  K.:  Experientia 3, 109 (1947).




44.  Gebhart,  D.O.E.:  Teratology 1, 153 (1968).
                                      20

-------
45.  Mackler, B., Grace, R., Tippit, D.F., Leraire, R.J., Shepard, T.H., and




     Kelley, V.C.:  Teratology 12, 291 (1975).




46.  Stenger, E.G., Aeppli, L., Mueller, D., Peheim, E., and Thomann, P.:




     Arzneim.-Forsch. 21, 880 (1971).




47.  Nagano, K., Nakayama, E., Oobayashi, H., Yamada, T., Adachi, H.,




     Nishizawa, T., Ozawa, H., Nakaichi, M., Okuda, H., Minami, K., and




     Yamazaki, K.:  Toxicology 20, 335 (1981).




48.  Uemura, K.:  Nippon Sanka Fujinka Gakkai Zasshl 32, 113 (1980).




49.  Unger, T.M., Kliethermes, J., Van Goethem, D., and Short, R.D.:




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51.  Blood, F.R.:  Food Cosmet. Toxicol. 3, 229 (1965).




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53.  Stenback, F., and Shubik, P.:  Toxicol. Appl. Pharmacol. 30, 7 (1974).




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     p.  267.

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     20, 255 (1978).  .
                                      23

-------
Notes Added After Completion of Section 5.2.2.4





     The concern over glycol ethers as teratogens and reproductive toxins has




continued to attract extensive investigations..  Hardin (1) has recently




reviewed the reproductive toxicity data of 13 glycol ethers and related com-




pounds.  The glycol ethers,  which have been thus far shown to exhibit clear




reproductive toxicity (testicular atrophy and/or teratogenicity), are mostly




methyl or ethyl ether derivatives of ethylene glycol.  Monobutyl or monophenyl




derivative of ethylene glycol failed to cause significant reproductive toxic-




ity suggesting that bulky substituents tend to mitigate the toxicity.  Both




monomethyl and dimethyl derivatives of ethylene glycol produce similar spectra




of embryotoxic and teratogenic effects suggesting in vivo metabolism to a




common active form, possibly 2-methoxyacetic acid.  The demonstration by




Foster et al. (2) of similarity between the nature and severity of testicular




toxicity obtained with 2-methoxy- and 2-ethoxyacetic acids and their corre-




sponding glycol ethers lends support to the suggestion that 2-alkoxyacetic




acids are the active metabolites of glycol ethers.  Further support has been




provided by studies showing lack of reproductive toxicity of propylene glycol




monomethyl ether in rats (3) and the inability of rats to biotransform this




glycol ether to its acetic acid derivative (4).  Unlike ethylene glycol mono-




methyl ether, propylene glycol monomethyl ether is mainly metabolized via




0-demethylation to propylene glycol, which has a low degree of toxicity.  The




final results of a study on genetic effects of monomethyl and dimethyl deriva-




tives of ethylene glycol (2-methoxyethanol and bis-2-methyoxyethyl ether) in




rats by McGregor et_ jiJL (5) have been published.  Both comopunds were negative




in the Ames test, unscheduled DNA synthesis (UDS) and chromosome aberrations




assays but showed some (albeit weak) mutagenic activity in dominant lethal




test and strong antifertility effects.  The possible molecular mechanism of

-------
teratogenesis by glycol ethers and other teratogens has been studied by Welsch




(6).  Like a number of other potent teratogens,  several glycol ethers have




been shown, by "metabolic cooperation" experiments, to inhibit intercellular




communication.  Cell-to-cell transfer, through gap junction, of regulatory




growth factors is believed to be involved in the control of cell differentia-




tion and proliferation.  A number of "epigenetic" carcinogens and potent




tumorigenesis promoters have also been shown to inhibit intercellular communi-




cation (7); it remains to be tested whether glycol ethers may have similar




properties.









References for Section 5.2.2.4 Update









  1.  Hardin, B.D.:  Toxicology 27, 91 (1983).




  2.  Foster, P.M.D., Creasy, D.M., Foster, J.R., Thomas, L.V., Cook, M.W.,




      and Gangolli, S.D.:  Toxicol. Appl. Pharmacol. 69, 385 (1983).




  3.  Doe, J.E., Samuels, D.M., Tinston, D.J., and de Silva Wickramaratne,




      G.A.:  Toxicol. Appl. Pharmacol. 69, 43 (1983).




  4.  Miller, R.R., Herman, E.A., Langvardt, P.W., McKenna, M.J., and Schwetz,




      B.A.:  Toxicologist 3, 82 (1983).




  5.  McGregor, D.B., Willins, M.J., McDonald, P., Holmstrom, M., McDonald,




      D., and Niemeier, R.W.:  Toxicol. Appl. Pharmacol. 70, 303 (1983).




  6.  Welsch, F.:  Mechanisms of Teratogenesis.  ln_ "CUT Activities," Vol. 3,




      No. 7, Chemical Industry Institute of Toxicology, Research Triangle




      Park, North Carolina, 1983.




  7.  Boreiko, C.J.:  Intercellular Communication and Tumor Promotion.  In




      "CUT Activities," Vol. 4, No.  3, Chemical Industry Institute of




      Toxicology, Research Triangle Park, North Carolina, 1984.

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