FATTY ACIDS, DETERGENTS AND OTHER SURFACTANTS  '
    CARCINOGENICITY AND STRUCTURE-ACTIVITY
RELATIONSHIPS.  OTHER BIOLOGICAL PROPERTIES.
  METABOLISM.  ENVIRONMENTAL SIGNIFICANCE.
            David Y. Lai, Ph. D.
            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;

5.2.2.9    -      Fatty Acids, Detergents and Other Surfactants

  5.2.2.9.1       Introduction

  5.2.2.9.2       Physical and Chemical Properties.  Biological
                  Effects

    5.2.2.9.2.1   Physical and Chemical Properties

    5.2.2.9.2.2   Biological Effects Other Than Carcinogenic

  5.2.2.9.3       Carcinogenicity and Its Structure.  Activity
                  Relationships

    5.2.2.9.3.1   Fatty Acids

    5.2.2.9.3.2   Detergents and  Other Surfactants

  5.2.2.9.4.       Metabolism and  Mechanism of Action

  5.2.2.9.5       Environmental Significance

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 5.2.2.9  Fatty Acids, Detergents and Other Surfactants









     5.2.2.9.1  Introduction.





     A number of epidemiologic and laboratory studies have associated




 increased incidence of gastric carcinomas with the consumption of heated fats




 (1-3; see also Section 5.1.1.3.2 in Vol. IIA).  However, subsequent studies




 (4, 5) failed to provide any convincing evidence for a causal relationship.




 The role of dietary fat in the induction of cancer has been critically




 reviewed by Arffmann (6) and Cooper (7), and was the subject of a workshop in




 1981 sponsored by the U.S. National Cancer Institute (8).





     Already during the early studies on skin carcinogenesis it was estab-




 lished that certain lipophilic-hydrophilic substances, used as vehicles for




 carcinogen administration, can considerably modify the effect of carcinogens




 (9).  For instance, many polar-nonpolar compounds, in the group of non-ionic




 detergents ^e_.£.,  Tweens and Spans) were shown to be potent promoters of skin




 tumorigenesis (9,  10).  In connection with these findings, some long-chain




 fatty acids, which are present in the molecules of certain Tweens and Spans,




 were themselves found to have carcinogenic, cocarcinogenic, and/or tumori-




 genesis-promoting  properties (11-15).  These studies generated concern about




 the possible carcinogenicity of certain lipids.  Because of the increasing




growth of the synthetic detergent industry and the widespread application of




many of its products (see Section 5.2.2.9.5), the possible carcinogenicity of




lipophilic-hydrophilic (surface active) agents has become a focus of




concern.  The toxicological properties and carcinogenic activities of this




interesting group  of compounds have been extensively reviewed (16-20).

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      5.2.2.9.2  Physical and Chemical Properties..   Biological Effects.





      5.2.2.9.2.1  PHYSICAL AND CHEMICAL PROPERTIES.


      Fatty acids are saturated or unsaturated straight-chain monocarboxylic


acids, usually with an even number of carbons.  Short-chain fatty acids are


miscible with water; fatty acids with 10 or more carbon atoms are, on the


other hand, virtually insoluble in water, but readily soluble in nonpolar sol-


vents.  The boiling points and melting points of fatty acids increase with the


chain length.  Esterification of the carboxyl group and reaction of the double


bond(s) (if any) are the most important chemical reactions of fatty acids.


The common fatty acids (around 18 carbon atoms) exist mostly in the form of


esters as fats, oils, or waxes in animals and plants.  Oxidation of the double


bond(s) in unsaturated fatty acids yields unstable hydroperoxides which


further break down to keto and hydroxy-keto acids.


      In order to understand the biological activities of detergents and sur-


face  active agents ("surfactants") in general, their physico-chemical proper-


ties must be considered.  As shown in Tables I and III, all these agents


contain characteristic water-insoluble (hydrophobic, lipophilic, or non-polar)


groups such as alkyl-, alkylaryl-, or other more complex hydrocarbon moieties


and water-soluble (hydrophilic, lipophobic, or polar) moieties such as

                                      +
-(CH2-CH20)n-, -OH,  -SOj"", -COO , or -NR^ in their molecules.  For a detailed


discussion of the molecular mechanism of action of surface active agents, see


Sections 3.3.2.1 and 3.3.2.2 in Volume I.


     In water/oil or water/air systems, surfactants tend to form oriented


monolayers at interfaces; the hydrophilic portion extends into the aqueous


phase whereas the lipophilic part of the molecule is directed toward the lipid

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      TABLE 7  Structural Formulas of Surfactants Tested for Carcinogenic Activity
   Hh
                                            (CnHzn+i)^^


                                           Alky I benzene sulfonotes
            Tweens and Spans
              I R-fatty acids
              JT«ens-x,y,z"5to 100
              (Spons-x,y,z'0
Cholesterol
?"3    CH,


       <>V
                                  Deoxycholic acid
                                           CH,
                                                           CI©
                                                                  Triethano famine
                                                                         CH,
                                                                         CHj
                 Benzethonium chloride
                                                       Cetyldimettiylbenzylammonium chloride
                                                            (R-CeHs; X-CI)
                                                       Cetyltrimethylammonium bromide
                                                            (R'H;X'6r)

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Table II.  Acute Toxicity of Some Fatty Acids,
       Detergents and Other Surfactants
Compound Species and Route
A.






B.
(1)



(2)









(3)












Fatty Acids
Laurie acid

Palmitic acid
Stearic acid
Oleic acid ,
sodium salt

Rat, oral
Mouse, i.v.
Mouse, i.v.
Mouse, i.v.
Mouse, i.v.

LD5Q (mg/kg)

12,000
131
57
23
152

Reference

(21)
(22)
(22)
(22)
(21)

Detergents and Other Surfactants
Anionics
Alkylbenzene sulfonates

Deoxycholic acid
Cationics
Triethanolamine

Benzethonium chloride


Cetyldimethylbenzyl-
ammonium chloride
Cetyltrimethylammonium
bromide
Nonionics
Tween 20



Tween 40
Tween 60

Tween 80
Span 20
Span 40
Span 60
Span 80

Rat, oral
Mouse, oral
Mouse, i.p.

Rat , oral
Mouse, i.p.
Rat , oral
Rat, s.c.
Mouse , oral
Rat , oral

Rat, oral
Mouse, i.p.

Rat, oral
Rat, i.p.
Mouse, oral
Mouse, i.p.
Rat, i.v.
Rat, oral
Rat, i.v.
Rat, i.v.
Rat, oral
Rat , oral
Rat, oral
Rat, oral

2,200
4,600
130

8,680
1,450
665
119
485
234

410
106

> 30,000
3,500
> 30,000
2,400
1,580
> 20,000
1,220
1,790
> 20 (ml)
> 10,000
> 30,000
> 10 (ml)

(23)
(23)
(24)

(25)
(21)
(26)
(27)
(26)
(28)

(21)
(29)

(30)
(30)
(30)
(30)
(21)
(31)
(21)
(31)
(31)
(31)
(31)
(31)

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                          Table III.   Carcinogenicity of Fatty Acids and Derivatives in the Mouse
  Compound
         Structure
 Strain
Principal Organ Affected
       and Route
Reference
                          0
                          II
A.  Saturated  [CH0(CHJ -C-R]
    	     j   i n
Laurie acid


Isopropyl
myristate

Palmitic acid

Stearic acid
n=10, R=OH


n=12, R=OCH(CH3)2


n=14, R=OH

n=16, R=OH
2-, 9-, or 10-   n=16, R=OH (hydroxy group
 Hydroxystearic  at C-2, C-9, or C-10)
 acid

Methyl stearate  n=16, R=OCH3
12-Hydroxy-
 stearic acid
Methyl 12-
 hydroxy-
 stearate

4-Keto-
 stearic acid

Stearohydrox-
 amic acid
n=16, R=OH
(hydroxy group at
C-12)

n=16, R=OCH3
(hydroxy group at
C-12)

n=16, R=OH
(keto group at C-4)

n=16, R=NHOH
Albino          No significant effect, topical
Swiss-Webster   No significant effect, s.c.
Swiss
No significant effect, topical
Swiss-Webster   No significant effect, s.c.

BALB/c, ICR/Ha, No significant effect, s.c.
Swiss-Webster
                                 BALB/c
                No significant effect, s.c.
ICR/Ha          Local sarcoma, s.c
Swiss-Webster   No significant effect, s.c.

Swiss-Webster   Local sarcoma, s.c.
Swiss-Webster    Local  sarcoma,  s.c.
BALB/c
BALB/c
Local sarcoma  , s.c,
Local  sarcoma  ,  s.c,
(11) '
(12)

(32, 33)
                                      (12)

                                      (12, 13)


                                      (12)
                                      (13)
                                      (12)

                                      (12)
 (12)



 (12)


 (12)

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                          Table III.   Carcinogenicity of Fatty Acids and Derivatives in the Mouse
                                                        (continued)
  Compound
         Structure
 Strain
Principal Organ Affected
       and Route
Reference
                              0
                              II
B.  Unsaturated  [CH (CH ) -R-C-O-R']
    ^™^~™             J   ^11

Oleic acid       n=7, R=-CH=CH(CH2)7-, R1 =H

Methyl oleate    n=7, R=-CH=CH(CH2)7~, R'=Cl

Methyl 12-oxo-   n=5, R=-C-CH=CH-(CH ) -,
 trans-10-octa-          N
 decenoate       R'=CHo
Methyl 12-
 hydroxy-10-
 octadecanoate   R'=CH
n=5, R=-CH-CH=CH-(CHJ0-,
        I            •£ o
        OH
Swiss-Webster

ST/a

ST/a
ST/a


ST/a
Methyl 13-
 hydroxy-9,11-
 octadeca-
 dienoate
n=4, R=-CH-CH=CH-CH=CH-(CH ) -,  ST/a

R'=CH   ™
No significant effect, s.c.

Skin, lymphoid tissue, topical

Skin, lymphoid tissue, topical
No significant effect, oral


Skin, topical



Skin, topical
    (12)

    (14)

    (14, 15)



    (14)



    (14)
 Marginal activity

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 phase or the air.  This molecular orientation brings about a lowering of




 interfacial or surface tension.  Another characteristic of surfactants is the




 formation of micelles in aqueous solution when their concentration in the




 solution exceeds the "critical micelle concentration" (CMC).  Owing to these




 properties, solutions of surfactants exhibit, to various degrees, the




 following functional activities:  detergency, emulsifying, wetting, solu-




 bilizing, foaming, and dispersing, which are closely related to the balance




 between their hydrophilic and lipophilic properties.  For more details on the




 physico-chemical properties of surfactants the reader is referred to the




 following publications (34-37, 38a).





     On the basis of their ionizing properties, surfactants may be classified




 into anionic, cationic, and non-ionic types.  Alkylbenzene sulfonates are




 typical anionic surfactants because the electrically charged group of these




 molecules, the hydrophilic sulfonate moiety, is anionic.  Benzethonium




 chloride, cetyldimethylbenzylammonium chloride, and cetyltrimethylammoniuin




bromide are cationic surfactants since the charged moiety of these compounds,




 the hydrophilic quaternary ammonium ion, is cationic.  The Spans and Tweens,




which do not ionize, are non-ionic surfactants.  Chemically, the Spans are




 fatty acid esters of the cyclic sorbitol anhydride, sorbitan.  The fatty acid




moieties of Span 20, Span 40, Span 60, and Span 80 are lauric acid, palmitic




acid, stearic acid, and oleic acid, respectively.  Addition of polyoxyethylene




chains of variable length to the three vicinal tertiary hydroxyl groups of




 Spans results in the respective Tweens (Tween 20, Tween 40, Tween 60, and




Tween 80).  The Tweens commonly used in carcinogenicity studies contain a




total of about 5-100 ethyleneoxide units.  The Spans are lipophilic, due to




the fatty acid moieties.  The Tweens are hydrophilic and their hydrosolubility




increases with the number of the hydrophilic ethyleneoxide units.

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     The emulsifying properties of surfactants is determined by the balance of




 the size and strength of the hydrophilic and lipophilic groups in the mole-

                V.


 cule, the "hydrophile-lipophile balance" (HLB).  A strongly lipophilic emulsi-




 fier has a low HLB, usually < 10; a highly hydrophilic emulsifier has a HLB




 value usually >10.  The HLB values may be determined experimentally or may be




 calculated.  The HLB values of most polyol fatty acid esters can be calculated




 with the formula:
                               HLB = 20 (1 - -I)
                                             A
where "S" is the saponification number of the ester and "A" the acid number of




the acid.  For example, the HLB values for Tween 20, Tween 40, and Tween 60




are 16.7, 15.6, and 14.9, respectively (10, 35).









     5.2.2.9.2.2  BIOLOGICAL EFFECTS  OTHER  THAN  CARCINOGENIC





     Toxicity.  Despite little direct evidence for significant toxic effects




of oxidized fats or fatty acids to humans, heated fats and oxidation products




of certain fatty acids have been reported to retard growth and induce patholo-




gical lesions, anorexia, diarrhea, and death in animals (7; cited in ref.




38b).  Administration of methyl linoleate derivatives intragastrically to mice




caused atrophy of the spleen, dilatation of the intestine, histological




changes in the lymphatic system, and leukopenia (39).  Severe inflammatory




reaction with leukocyte infiltration and necrosis were observed following




subcutaneous injection of methyl-12-oxo-trans-10-octadecenoate (38b).  Among




other lipophilic acids, linoleic acid and palmitic acid were shown to be




potent inhibitors of HeLa cell replication (40).  Linoleic acid was also




reported to be highly toxic to lung (41) and cultured liver cells of rats

-------
 (42).   Oro  and  Wretlind  (22) have determined  the LDcg values  for  saturated




 fatty acids from C2 to Cig by intravenous injection into mice.  They found




 that stearic acid  (C-^g)  was the most  toxic, with LD5Q of 23 rag/kg body weight,




 which was ten times lower than that of its unsaturated analog, oleic acid.




 Toxicity decreased with  the number of carbon  atoms in .the molecule, reaching




 the lowest  point for caproic acid (C6):>  LD5Q = 1,725 mg/kg.  With further




 decrease in the number of carbon atoms,  toxicity increased; the LDcQ for




 acetic  acid (€2) was found to be 525 mg/kg.





     In general, most surfactants are relatively innocuous.   The  oral LD^Q




 values  for  anionic surfactants in various laboratory animals  range between 500




 and 5,000 mg/kg; cationic surfactants are slightly more toxic and nonionics




 are somewhat less (17) (Table II).





     Irritation of tissues, mucous membranes, and skin is among the most




 common  local effects of  surfactants.  The degree of irritation depends largely




 on their surface active  properties which in turn depend on their  physico-




 chemical characteristics.  When tested in animals and humans, many anionic and




 nonionic surfactants which are used as household detergents do not induce skin




 or eye  irritation, or sensitization, even at high concentrations  (23, 42).




 However, exposure of the skin of mice to certain anionic and  cationic surfac-




 tants (&_•£•, cetyltrimethylammonium bromide) at a concentration of 10% results




 in cellular damage and necrosis.  Following multiple applications of 1-2.5% of




 the agents, acanthosis and hyperkeratosis were observed (44).  Toxic myocar-




dial changes and damage  in the mucous membrane of the gastrointestinal tract




have also been reported in animals receiving high doses of anionic surfactants




 (20).  Alkylbenzene sulfonates with alkyl chains of 10-12 carbon atoms are more




irritating and toxic than those with shorter or longer alkyl chains; linear




and branched chain compounds have similar acute oral toxicities (20).  Upon

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 parenteral  administration, certain anionic and cationic detergents are hemo-




 lytic, due  primarily to their capability to solubilize and elute phospholipids




 from  the  cell membrane.  The concentration required for hemolysis is related




 to  the content of phospholipids in the membrane of the erythrocytes (18).





      Mutagenicity.  There is a scarcity of information on the mutagenic




 activity  of  fatty acids and detergents.  A 1980 study showed that neither




 12-hydroxystearic acid nor the oxidized fractions of deep frying fats are




 mutagenic in different Salmonella typhimurium tester strains with or without




 metabolic activation (45).  Linoleic acid, oleic acid, and methyl oleate were




 tested for mutagenicity in the U.S. National Toxicology Program.  All three




 compounds were negative in the Ames test (46).  Tween 60 and Tween 80 did not




 induce chromosomal aberrations either in vitro in Chinese hamster cells or in




 vivo  in mouse bone marrow cells (47, 48).  They were also nonmutagenic in the




 Ames  test and in the silkworm oocyte system (47).  Consistent with these




 findings, Tween 60, Span 60, alkylbenzene sulfonates, cetyltrimethylammonium




 chloride, dicetyldimethylammonium chloride, and cetyldimethylbenzylammonium




 chloride  (49), as well as benzethonium chloride and cholesterol (50) gave




 negative  results when tested for mutagenicity in strains TA100 and TA98 of S.




 typhimuriun  in the presence and absence of S-9 activating mix.  Tween 80 was




 also not mutagenic in the dominant-lethal test in mice (51) or in the sex-




 linked recessive lethal test in Drosophila melanogaster (52).  The mutagenic




 properties of anionic surfactants have recently been reviewed by Oba (19).




 Absence of mutagenicity was reported for alkylbenzene sulfonates or other




 anionics assayed in various bacterial and mammalian systems.  Tween 60 but not




 Tween 80 was found mutagenic in strains H-17 and M-45 of Bacillus subtilis and




 strain WP-2 of Escherichia coli (47).  Moreover, synergism between Tween 60




and ethyleneimine in inducing chlorophyll mutation in barley plants was

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reported (53).  Various nmtagenic effects have also been described in cluster




beans treated with triethanolamine (54).  Hoshino and Tanooka (55), however,




found triethanolamine itself non-mutagenic to Bacillus subtilis; mutagenic




effect to the bacteria was only observed after reacting with sodium nitrite




under acidic conditions or when the mixture was heated.





     Teratogenicity.  Studies on chemical teratogenesis during the past few




decades have indicated that teratogenic effects may result from a number of




different mechanisms.  Freese et_al. (40) have noted a high correlation




between inhibition of mammalian cell growth and reported teratogenicity of




certain lipophilic organic acids; it is interesting to note that palmitic and




linoleic acid are inhibitors of HeLa cell replication (see above under




"Toxicity").  It remains to be tested if exposure to unusually high levels of




these fatty acids is potentially teratogenic.  Linoleic acid was positive in




an in vitro cell surface recognition assay system for potential teratogens




(56).  An oxidized linoleic acid sample (which contained about 25% linoleic




acid hydroperoxide), but not purified linoleic acid, induced an elevated




incidence of malformations in the offspring of treated rats (57).  The terato-




genic activity of other fatty acids is still unknown.





     A considerable number of studies on the teratogenic potential of deter-




gents and other surfactants (anionics in particular) have been carried out in




various animal species by oral, dermal, and subcutaneous administration.  The




results show no conclusive evidence of teratogenicity.  A Japanese group




headed by Mikami (cited in ref. 19) reported that linear alkylbenzene sulfon-




ates and other commercial detergent formulations based on anionic surfactants




caused increased incidence of malformations in rats and mice.  However, their




data were considered to be inadequate and incomplete, and other investigators




failed to confirm their results in other strains of animals.  Studies by

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Mikani's group as well as others on the teratogenic activity of anionic sur-




factants have been critically reviewed (19, 58).  Similarly, there is no




significant evidence fo the teratogenicity of cationic surfactants.  Except




for a general increase in the incidence of variations of cervical vertebral




arches in the offspring of mice treated with dicetyldimethylammonium chloride




(50 or 200 mg/kg body weight), ho significant change in the rate of malforma-




tions was observed (59).  In rats, a high dose  (35.6 mg/kg/day) of benze-




thonium chloride produced delayed ossification; however, the effect was not




regarded to be related to teratogenicity, but to the decrease of fetal growth




secondary to maternal toxicity (60).  However, when pregnant mice were admini-




stered cetyltrimethylammonium bromide at an intraperitoneal dose of 10.5 or




35.0 mg/kg (10 or 33% of the LD5Q), a higher incidence of malformed fetuses




was found (29).  At the time of this writing, the evidence is conflicting




regarding the teratogenicity of the typical non-ionic surfactants, the




Tweens.  Verrett et al. (61) found Tween 60 and Tween 80 not teratogenic in




developing chick embryo.  However, in recent studies, Kocher-Becker et al.




(62) observed that Tween 20, which was previously regarded to be an inert




vehicle in teratogenicity assays, produced malformations in mice strikingly




similar to those produced by thalidomide.  Pregnant mice were given a single




intraperitoneal injection of Tween 20 on day 9 of gestation; the doses that




caused thaiidomide-1ike malformations were 1.0, 1.7, 2.5, and 3.3 mg/kg body




weight.

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     5.2.2.9.3  Carcinogenicity and Its Structure-Activity Relationships.









     5.2.2.9.3.1  FATTY ACIDS.





     A short-term bioassay for preliminary screening of possible carcino-




genicity of a series of fatty acids was developed by Arffmann (63) [based on




injection of the fatty acids into the tail of the newt (Triton cristatus)].




Epidermal hyperplasia and downgrowths into the dermis, suggestive of carcino-




genic activity, was observed with certain derivatives of methyl oleate and




methyl linoleate (64).  The most active compound was methyl 12-oxo-trans-10-




octadecenoate, which is derived from methyl oleate and has an oxo group in the




C^-position relative to the double bond.  Moderate activity was found with




derivatives of both methyl oleate and methyl linoleate with an Q^-hydroxy




group, namely, methyl 12-hydroxy-10-octadecenoate and methyl 13-hydroxy-9,ll-




octadecadienoate.  The corresponding derivatives having a hydroxy group in the




p-position were inactive.  On the basis.of the chemical structures, it




appears that conjugation of double bond(s) with an oxygen-containing group in




the composition may be important for the carcinogenic activity of fatty acid




derivatives.





     In studies by application of methyl oleate and two oxo- and hydroxy-




derivatives of its 10-octadecenoate isomer (20% v/v in acetone) to the skin (3




times weekly for 1 year) of ST/a mice all three compounds exhibited low level




of complete carcinogenic activity, but were potent promotors of papillomas,




lymphomas, and malignant skin tumors induced by 7, 12-dimethylbenz[a]anthra-




cene (14, 64, 65).  In another experiment, the incidence and number of fore-




stomach papillomas initiated by 4-nitroquinoline-N-oxide were also increased




in ST/a mice given methyl-12-oxo-trans-10-octadecenoate (15 mg/day) in the




diet for 300 days; when administered orally alone, however, methyl-12-oxo-

-------
 trans-10-octadecenoate did not induce tumors in the mice  (15).   Data yielded




 by  these and other studies on the complete carcinogenicity of fatty acids and




 derivatives are given in Table III.





      Swern^£jil_. (12) investigated  the carcinogenic activity of 11 fatty




 acids and their derivatives by repeated subcutaneous injection to female




 BALB/c or CFW  (Swiss-Webster) mice.  Laurie acid, palmitic acid, oleic  acid,




 stearic acid, methyl stearate, and 2-, 9-, or 10-hydroxystearic  acid were




 regarded by the authors as noncarcinogenic under the conditions  of their




 experiment.  Methyl 12-hydroxystearate and 12-hydroxystearic acid induced




 sarcomas in 8 of 27 and 9 of 28 mice, respectively, and were considered to be




 carcinogenic.  Stearohydroxamic acid and 4-ketostearic acid, which elicited  3




 and 2 sarcomas in 13 and 14 mice, respectively, were regarded to be marginally




 carcinogenic toward the subcutaneous tissue of mice.  The absence of carcino-




 genic activity of stearic acid toward the subcutaneous tissue of mice was




 later confirmed by Van Duuren et al. (13); methyl stearate, however, was found




 to be weakly carcinogenic (13).  Oleic and lauric acid did not induce tumors




 when applied to the skin of mice daily, 6 times a week for 31 weeks but




 displayed significant promoting effect in skin tumor induction (11).




 Interestingly, isopropyl myristate which was reported to  cause various




 cutaneous lesions, did not significantly increase the tumor incidence in




 female Swiss mice (32, 33) or New Zealand rabbits (33) by repeated application




 to the skin for the life-span of the animals.









     5.2.2.9.3.2  DETERGENTS AND OTHER SURFACTANTS.





     Studies on the complete carcinogenicity of these compounds  are summarized




 in Table IV.  Their cocarcinogenic and tumorigenesis-promoting activities will




be extensively discussed in Section  6, Vol. IV.






                                      10

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                             Table IV.  Carcinogenicity of Detergents and Surfactants
     Compound'
   Species and Strain
 Principal Organ Affected
        and Route
Reference
A.   Non-ionic
     Tween 60
     Tween 80
     Cholesterol
B.   Anionic
     Alkylbenzene
      sulfonates
      (ABS)
Mouse, Swiss and
unspecified

Rat, Osborne-Mendel and
 Bethesda Black

Rat, Shell and Carworth
 Farms E

Mouse, C57, MRC and CBA
Mouse, albino and Swiss
Rat, albino
Rat, Wistar
Rat, Moriyama
Mouse, Swiss
Mouse, C57
Rabbit, unspecified
Skin, topical
                                                    Local sarcoma, s.c,
Local sarcoma, s.c.
Local sarcoma, s.c.
Bladder, implantation
No significant effect, oral
No significant effect, oral
No significant effect, oral
No significant effect, topical
No significant effect, s.c.
No significant effect, topical
(10, 66-68)


(69)


(70)
(71-73)
(74-76)
(77)
(78)
(79)
(80)
(80)
(80)

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                             Table IV.  Carcinogenicity of Detergents and Surfactants
                                                   (continued)
     Compound1
   Species and Strain
 Principal Organ Affected
        and Route
Reference
B.   Anionic (cont'd)

     Deoxycholic
      acid
Rat, unspecified
Mouse, C3H
Mouse, CF-1
Local sarcoma, s.c.
Local sarcoma, s.c.
No significant effect, topical
(cited in ref. 16)
(cited in ref. 16)
(81)
C.   Cationic
     Triethanol-
      amine
     Benzethonium
      chloride
Rat, albino
Mouse, ICR-JCL

Mouse, CBA x C57

Rat, Fischer 344
     Cetyldimethyl-    Rat, Osborne-Mendel
      benzylammonium   Rat, albino
      chloride

     Cetyltrimethyl-   Rat, Sprague-Dawley
     ammonium bromide
No significant effect0, topical
Lymphoid tissue, mammary gland,
lung, ovary, oral
No significant effect , topical

Local sarcoma, s.c.
                             No significant effect, oral
                             No significant effect, oral
                             No significant effect, oral
(82)
(55)

(82)

(27)
                                          (83)
                                          (28)
                                          (84)
aSee Table 1 for structure

 The duration of the study was only 26 weeks

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     Anionics.  Because of the evident structural relationship to alkyl-




benzenes, there has been concern about the possible carcinogenicity of




mixtures of alkylbenzene sulfonates averaging twelve carbon atoms (ABS), which




are among 'the strongest surfactants known and the major components of commer-




cial detergent products.  However, experiments to date failed to demonstrate




any carcinogenic effect of these compounds.  Paynter and Weir (77) conducted a




2-year  toxicity study, in which 120 albino rats of both sexes were given




dodecylbenzene sodium sulfonate at levels of 0, 200, 1,000, and 2,000 ppm in




the diet and the authors found no pathological changes in various organs




attributable to the intake of the test substance.  Similar studies were per-




formed  by Tusing££^l_. (78) in 120 male and 120 female Wistar rats.  ABS was




administered either in the diet at levels of 0, 0.1% (1,000 ppm) and 0.5%




(5,000  ppm) or in the drinking water (about 0.05%) for 104 weeks.  Gross and




microscopic examination of the tissues revealed no evidence of toxic changes




resulting from the treatment with ABS by either route.  Saffiotti et al. (80)




tested  ABS for possible carcinogenicity in the skin of mice and rabbits.




Application of ABS twice weekly to the skin of Swiss mice or of rabbits for




110 weeks, at concentrations of 5 or 10%, induced no tumors; no carcinogenic




activity was observed either by subcutaneous injection of ABS into C57 mice




(80).   Additional evidence for the absence of carcinogenic activity of ABS has




been provided by other bioassays using rats and mice (cited in ref. 19).





     In contrast to the lack of complete carcinogenic activity, there is




evidence for the tumorigenesis-promoting properties of ABS.  Takahashi  (79)




reported significantly higher incidence of gastric cancers in rats which




received concurrent treatment of 4-nitroquinoline-N-oxide and ABS by gavage




than in those given 4-nitroquinoline-N-oxide alone; no neoplasms of the




glandular stomach or other organs were found in the control group of rats
                                      11

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 administered  only ABS.  ABS also enhanced  the  incidence of  N-niethyl-N1-nitro-


 N-nitrosoguanidine-induced gastric tumors  in rats when administered  simul-


 taneously  in  the drinking water (85).



      Deoxycholic acid, a bacterial metabolite  of bile acid  (cholic acid), has


 long  been  suspected to be a carcinogen,  cocarcinogen, or promoter in the


 pathogenesis  of human colon cancer (cited  in ref. 86).  While  several investi-
                                                                             %

 gators reported its sarcomatogenic activity following subcutaneous injection


 into  rats  and mice, others found it  not  carcinogenic (rev.  in  ref. 16).


 Painting of deoxycholic acid on the  skin of mice induced no  tumors (81).


 Several experiments seem to support  the  view that deoxycholic  acid or its


 metabolites ^e_.j^., 12-ketolithocholic acid) may act as a cocarcinogen or


 promoter in colon carcinogenesis (87-92).  However, studies  in the classic


 mouse skin system (93) or in the dimethylhydrazine-induced  rat  colon cancer


 model (86) showed no promoting activity  of deoxycholic acid  or related agents,



      Cationics.  The carcinogenic potential of triethanolamine  has been inves-


 tigated by Hoshino and Tanooka (55).  Following feeding to  male and  female


 ICR-JCL mice  0.03 and 0.3% of triethanolamine  in the diet,  significantly


 higher incidences of tumors of the lymphoid tissues were found  in the


 females.  Moreover, malignant tumors of  various other tissues  were also


 produced in higher rates in both sexes of  the  experimentals  than in  the


 untreated controls.  Most tumors appeared after at least 60  weeks of treat-


ment.  Kostrodymova et al. (82) conducted  a 26-week study in which triethanol-


amine (13%) was administered epicutaneously to albino rats  and  CBA x C57


mice.  However, because of the short duration  of administration no conclusion


can be made regarding the carcinogenicity or inactivity of  this  compound.
                                      12

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      Benzethonium chloride, when administered subcutaneously at doses of 0.1,
 0.3,  1.0, and 3.0 mg/kg body weight twice weekly for 52 weeks to 200 F344
 rats, gave rise to  26 fibrosarcomas at the injection site.  Only one injection
 site-related tumor was observed in the 200 controls (27).  Several struc-
 turally-related cationics such as cetyltrimethylammonium chloride, dicetyldi-
 methylammonium chloride, and cetyldimethylbenzylammonium chloride were
 reported negative in the in vitro transformation of hamster embryo cell bio-
 assay (49).  Oral administration of cetyldimethylbenzylammonium chloride (28,
 83) or cetyltrimethylammonium bromide (84) to rats for 4 to 24 months produced
 no carcinogenic effects.

      Nonionics.  In a series of studies on the tumorigenesis-promoting effects
 of Span and Tween surfactants, Setala (10) was the first to observe occasional
 papillomas on the skin of mice painted with Tween 60 alone (daily, 6 times a
 week for 52 weeks) without prior initiation by hydrocarbon carcinogens.
 Subsequently, the complete carcinogenic action of Tween 60 in mouse skin was
 observed by various investigators, reporting the induction of malignant
 squamous cell carcinomas as well as benign tumors (66-68).  The compound was
 also shown to induce local fibrosarcomas of moderate malignancy in 5 of 30
 rats  receiving 1 ml (6%) weekly by subcutaneous injection for 73 weeks (69).
 Similarly, repeated subcutaneous injections of 2 ml (6%) Tween 80 twice weekly
 for 40 weeks induced local sarcomas in 11 of 17 rats (70).  Since large doses
are required to induce tumors, these compounds are regarded as only weakly
carcinogenic.  Some authors (66, 68) interpreted the effects as due to a
physical phenomenon (the so-called "solid-state carcinogenesis") rather than
to                chemical action.  No evidence of carcinogenicity was found
with other Tween and Span surfactants                   (94, 95).  This group
of nonionic hydrophilic lipophilic substances are better known in the cancer
literature for their tumorigenesis-promoting properties.
                                      13

-------
     Indeed, as early as 1954, Setala and coworkers (9) observed that Span 20

and Tween 60, when applied topically and repeatedly to the skin of mice
                 »_
enhanced the carcinogenic action of 7,12-dimethylbenz[a]anthracene.  Subse-

quent studies on a series of Tween and Span surfactants established that these

compounds represent a new type of physico-chemically defined promoting agents

for the mouse skin (10, 96, 97).  Evidence for the tumorigenesis-promoting

action of various Tweens was also provided by other investigators  (66-68,

98).  The hydrophilic nature of as well as the fatty acid moiety in these

compounds are key features for their promoting activity.  The most potent

promoting agents in the group are Tween 40, Tween 60, and Tween 80, which all

possess high HLB values (15.6, 14.9, and 15.0, respectively) and a relatively

long-chain fatty acid in their molecules.  Compounds of the Span type, which

lack the hydrophilic polyoxyethylene moieties and have, therefore, lower HLB

values (1.8-8.6) are only weak promoters.  The optimum HLB range for skin

tumor promotion is 11.0-15.6 (10).  In order to be effective in tumorigenesis

promotion, it is also essential that they are applied in relatively large

doses and with sufficient frequency (10).  The difference between  this group

of compounds and croton oil lies mainly in the amount of substance necessary

for tumorigenesis promotion (68).  More recently, the cocarcinogenic effects

of Span 20 and several Tweens in gastric carcinogenesis, by oral administra-

tion of N-methyl-N'-nitro-N-nitrosoguanidine or methylnitrosocyanimide, have

also been shown in rats (85, 99, 100).


     The carcinogenicity of cholesterol — which may be regarded structurally

as a strongly lipophilic nonionic surfactant — has been a subject of contro-

versy and has been critically discussed by several authors (6, 16, 101,

102).   Bioassays of cholesterol in many laboratories failed to induce tumors

in experimental animals (cited in ref. 6).  An International Agency for
                                      14

-------
 Research on  Cancer working group  (102)  considered the tuiaorigenicity of




 cholesterol  difficult to evaluate in a  number of feeding experiments, because.




 of the presence of other components and impurities in the diet.   Positive




 carcinogenic effects were noted in several experiments involving  subcutaneous




 injection  (je.g_.,  71-73) or implantation (74-76) of cholesterol pellets into




 mice.  However, the effects were variably interpreted (72, 102, 103) and the




 carcinogenic action of cholesterol is still unclear.  On the other hand, the




 compound has been reported to exhibit cocarcinogenic activity with dimethyl-




 nitrosamine  in the induction of bladder tumors in hamsters (104)  as well as




 with N-hydroxy-acetylaminofluorene in the induction of liver tumors in mice




 (105).  The  carcinogenic activity of cholesterol has also been dicussed in




 Vol. IIA,  Section 5.1.1.2.1.









     5.2.2.9.4  Metabolism and Mechanism of Action.





     In contrast to the well established ft-oxidation pathway of  normal fatty




 acids, there is a relative paucity of information regarding the metabolism of




 the carcinogenic fatty acid derivatives.  The metabolism and tissue distribu-




 tion studies on methyl 13-hydroxy-9,ll-octadecadienoate in the rat (106, 107)




 are rather unique in this regard.  On the other hand, several important




 reviews on the absorption, metabolic degradation, and excretion of various




 detergents and other surfactants are available (18, 31, 108).  There is no




 evidence for bioactivation which might  be responsible for the weak carcino-




genic and promoting effects of these hydrophilic-lipophilic compounds toward




 the skin and subcutaneous tissue of rats and mice.  Instead, there is over-




whelming support for the hypothesis that the action of these polar-nonpolar




substances is due primarily to their surfactant properties, whereby they




structurally disorganize endoplasmic lipoprotein membranes and affect cellular
                                      15

-------
 homeostasis (16).  Interference with mitochondrial oxidative phosphorylation




 by 12-hydroxystearic acid and its methyl ester is believed to be the mechanism




 of carcinogenesis by these fatty acid derivatives (109), due possibly to




 disruption of membrane systems and denaturation of functional proteins.  Many




 surfactants have been shown to alter the intestinal permeability of various




 nutrient substances (cited in ref. 84).  "Solvent action" which facilitates




 the absorption and penetration of other carcinogens through the skin or




 gastrointestinal tract is believed to play a major role in tumorigenesis




 promotion or cocarcinogenesis (10, 11, 79, 85, 99, 110).  The promoting




 activity of individual agents, on one hand, and the nature and intensity of




 histological changes in the skin (9) and stomach wall (111), on the other




 hand, have both been correlated with the hydrophile-lipophile balance (HLB




 value) of the compounds.





     Interestingly, Rohrschneider and Boutwell (112) noted a structural




 similarity between phorbol esters, the active principles of croton oil, and




 methyl esters of polyunsaturated fatty acids.  The authors speculated that




 there might be a receptor site in the cell membrane for natural cellular




 regulators which are structurally related to fatty acids — prostaglandins,




 for instance.  The effect of phorbol esters was interpreted as being mediated




by interaction with the receptor site due to structural similarity with the




 natural cellular regulator.  The weak carcinogenic and promoting activities of




 fatty acid derivatives observed may mimic the action of the phorbol esters.




Like croton oil and other promoting agents, some fatty acids, unsaturated in




 particular, and their derivatives, have actually been found to induce mitosis




and stimulate cell division when applied to the mouse skin (10, 113).




Jirgensons (114) has recently studied, by circular dichroism, the effects of




various promoting agents on the conformation of histones, which play an impor-
                                      16

-------
 Cant role in gene regulation:  the order of conformation-modifying activity of




 phorbol esters and Tween 60 (polyoxyethylene sorbitan monostearate) was found




 to parallel their promoting activity.





     Furthermore, there are observations that Tween and Span surfactants are




 inhibitors of DNA repair (115-117), and it has been suggested that the




 carcinogenic, cocarcinogenic, and tumorigenesis-promoting activities of these




 agents are related to their inhibition of DNA repair replication (115).




 However, other studies (116, 117) failed to demonstrate any specificity of




 these compounds in the inhibition of DNA synthesis.  It is possible that the




 effects are due to the general action of these substances to denature




 proteins, including DNA repair enzymes, by virtue of their surfactant




 properties.









     5.2.2.9.5  Environmental Significance.





     Fatty acids are normal components of animal fats and plant oils.




 Unsaturated fatty acids readily undergo autooxidation to yield hydroperoxides




 and various oxygen-containing secondary products including epoxides and




 hydroxy compounds.  (The carcinogenic action of peroxides and epoxides have




been discussed in Sections 5.2.1.7.3 and 5.2.1.1.5 of Vol. IIIA).  Altered




 fatty acids may also result from polymerization and other chemical reactions




during heating.





     Certain fatty acids occur as the lipophilic moiety of soaps and synthetic




surfactants (e^g_., Tweens and Spans), which are used to formulate a wide




variety of household and industrial cleansing products.  In addition, many of




the surfactants are used as food additives as well as co-solvents,




solubilizers,  dispersants, and emulsifiers in various industries (37, 118).
                                      17

-------
Because of their relatively low cost and rapid rate of biodegradation, the




anionics are the most widely used.  An estimate in 1968 indicated that the




production rate of ABS in the U.S. was then about 540 million Ibs/yr (37).




The cationics (^.J[.,  benzethonium chloride, cetyldimethylbenzylammonium




chloride) are used principally as disinfectants and germicides, rather than as




detergents.  Many of  these are used in hospitals and restaurants, as well as




in the pharmaceutical, cosmetics, and toiletries industries (119).  Moreover,




a considerable amount of triethanolamine, which may be converted to the




carcinogenic nitrosatiines is often present in cutting fluid formulations




(e.g., 55).





     The widespread applications of this group of chemicals, both in the home




and in industry, make it almost certain that there is ubiquitous exposure of




humans either through direct contact with the skin, mucous membranes, or




indirectly by ingestion through food and water.  The intake of detergents per




person in the U.S. has been estimated to be 0.3-3.0 mg/day (17).
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                                      18

-------
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-------
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                                     21

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102.   IARC:  International Agency for Research on Cancer Monograph 10, 99




       (1976).




103.   Grasso, P. and Golberg, L.:  Food Cosmet. Toxicol. _4_, 297 (1966).




104.   Kowalewski,  K. and Todd, E.F.:  Proc. Soc. Exp. Biol. Med. 136, 482




       (1971).




105.   Enomoto,  M., Nase, S., Harada, M., Miyata, K., Saito, M. and Noguchi,




       Y.:  Japan J.  Exp. Med. 44, 37 (1974).




106.   Reber, R.J.  and Draper, H.H.:  Lipids 5,  983 (1970).




107.   Glavind,  J.  and Sylven, C.:  Acta Chem. Scand. 24, 3723  (1970).
                                      24

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108.   Black, J.B. and Howes, D.:  Absorption, Metabolism, and Excretion of




       Anionic Surfactants.  In;   "Anionic Surfactants" (C. Gloxhuber, ed.),




       Chapter 2,'Marcel Dekker,  New York, 1980, p. 51.




109.   Hadler, H.I. and Mueller,  K.W.:   J. Environ. Pathol. Toxicol. 1_, 75




       (1977).




110.   Setala, K.:  Nature (London) 174, 873 (1954).




111.   Ekwall, P., Ernala, P. and Setala, K.:   Cancer Res. 11, 753 (1951).




112.   RohrSchneider, L.R. and Boutwell, R.K.:  Nature (London) 243, 212




       (1973).




113.   Raicheva, E. and Frankfurt, O.S.:  Tsitologiya 14, 1161 (1972).




114.   Jirgensons, B.:  Cancer Biochem. Biophys. ^, 7 (1980).




115.   Gaudin, D., Gregg, R.S. and Yielding, K.L.:   Biochem. Biophys. Res.




       Commun. 45, 630 (1971).




116.   Tuschl, H., Klein, W., Kocsis, F., Bernat, E. and Altmann, H.:




       Environ. Physiol. Biochem. _5, 84 (1975).




117.   Cleaver, J.E. and Painter, R.B.:  Cancer Res. 35, 1773 (1975).




118.   Stakowski, W.B., Huang, S.K. and Liss,  R.L.:  Polyoxyethylene Esters of




       Fatty Acids.  In;  "Nonionic Surfactants" (M.J. Schick, ed.), Chapter




       5,  Marcel Dekker, New York, 1967.  p. 142.




119.   Cutler, R.A. and Drobeck,  H.P.:   Toxicology  of Cationic Surfactants.




       In;  "Cationic Surfactants" (E.  Jungermann,  ed.), Chapter 15, Marcel




       Dekker, New York, 1970. p. 527.
                                      25

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Notes Added After Completion of Section 5.2.2.9





     The promotion of experimental carcinogenesis by dietary unsaturated fatty




acids has been repeatedly demonstrated in rodents.  Sakaguchi e±_al_,' (1)




reported that rats fed a diet containig 5% linoleic acid or 4.7% stearic acid




had significantly higher incidence of colon tumors induced by azoxymethane.




Lipid analysis showed that unsaturated fat diet markedly altered the phos-




phatide fatty acyl composition of colon mucosa and increased the level of




arachidonic acid in the neutral lipid of colon tumors (1).  The altered lipid




composition of the mucosa can lead to changes in the fluidity and permeability




of cell membranes.  In line with these findings, recent research has shown




that dietary unsaturated fatty acids, as many tumorigenesis-promoting chemi-




cals, inhibit "metabolic cooperation" between Chinese V79 hamster cells (2,




3).  Interestingly, no such activity was observed with trans-oleic (elaidic)




acid and with saturated fatty acids, suggesting that the cis-double bond




orientation may be essential for the inhibition of intercellular communication




by fatty acids (3).  The excess of arachidonic acid, the precursor of prosta-




glandins, in colon tumors has been suggested to play a role in the promotion




of carcinogenesis by the unsaturated fat diet (1).  Ip et al. (4) have demon-




strated that at least part of the promoting effects of polyunsaturated fat on




7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis in rats is




mediated through increased synthesis of prostaglandins, since the addition of




indomethacin (an inhibitor of prostaglandin synthesis) to the diet completely




abolished the stimulatory effect of linoleic acid on tumorigenesis.  On the




other hand, arachidonic acid and unsaturated fatty acids (but not saturated




fatty acids) have been found to activate protein kinase C in human (5) and




mouse (6) tissues to similar extent as the tumorigenesis-promotor phorbol




esters.  McPhail et al. (5) proposed that the release of arachidonic acid

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could play the role of a second messenger in the regulation of cellular activ-




ities by the stimulation of protein kinase C.  It is interesting that the




potency of unsaturated Cjg fatty acids in activating protein kinase C paral-




lels the number of cis-double bonds, in that f -linolenic acid > linoleic




acid > oleic acid, containing three, two and one double bonds, respectively.





     Triethanolamine has been selected for carcinogenesis bioassay by the U.S.




National Toxicology Program; the study on a closely related compound




diethanolamine, has been completed and the histopathology of animal tissues




are being examined at the time of this writing (7).  The toxicological proper-




ties 'of a large number of surfactants used in cosmetic formulations has been




assessed by the Cosmetic Ingredient Review expert panel (8-11).









References for Section 5.2.2.9 Update









  1.  Sakaguchi, M., Hiramatsu, Y., Takada, H., Yamamura, M.,  Hioki, K.,




      Saito, K., and Yamamoto, M.:  Cancer Res. 44, 1472 (1984).




  2.  Aylsworth, C.F., Jone, C., Trosko, J.E.,  and Welsch, C.W.:  Proc. Am.




      Assoc. Cancer Res. 24, 109 (1983).




  3.  Aylsworth, C.F., Jone, C., Trosko, J.E.,  and Welsch, C.W.:  Proc. Am.




      Assoc. Cancer Res. 25, 152 (1984).




  4.  Ip, M.M., Carter, C.A., Milholland, R.J., and Shea, W.K.:  Proc. Am.




      Assoc. Cancer Res. 24, 97 (1983).




  5.  McPhail, L.C., Clayton, C.C., and Snyderman, R.:  Science 224, 622




      (1984).




  6..  Leach, K.L., and Blumberg, P.M.:  Proc. Am. Assoc. Cancer Res. 25, 147




      (1984).

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 7.  NTP:   "National Toxicology Program.  Fiscal Year 1984 Annual Plan" (NTP-




     84-023).   National Toxicology Program,  Research Triangle Park,  North




     Carolina,  1984.




 8.  Elder, R.L.  (ed.):  Second Report on the Cosmetic Ingredient Review




     Expert Panel.   In;  J.  Am.  College Toxicol. _H2), 1-177 (1982).




 9.  Elder, R.L.  (ed.):  Third Report on the Cosmetic Ingredient Review




     Expert Panel.   In;  J.  Am.  College Toxicol. JX4), 1-192 (1982).




10.  Elder, R.L.  (ed.):  Fourth Report on the Cosmetic Ingredient Review




     Expert Panel.   In;  J.  Am.  College Toxicol.  2(5), 1-178 (1983).




11.  Elder, R.L.  (ed.):  Fifth Report on the Cosmetic Ingredient Review




     Expert Panel.   In:  J.  Am.  College Toxicol.  2(7), 1-235 (1983).

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"Current Awareness'
      Program
   Vol.   IV.
                               January 1983

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