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Carcinogenicity testing of selected food additives by parenteral administration to infant swiss mice
TOXICOLOGYANDAPPLIED
Carcinogenicity Parenteral
PHARMACOLOGYl6,321-334(1970)
Testing of Selected Food Additives-by Administration to Infant Swiss Mice1
S. S. EPSTEIN, K. Fu.m,J.
ANDREA, AND N. MANTEL
Laboratories of Environmental Pathology and Carcinogenesis, The Children’s Cancer Research Foundation, Inc., and Department of Pathology, Harvard Medical School, Boston, Massachusetts; Biometry Branch, National Cancer Institute, Bethesda, Maryland Received December 26, 1968
Carcinogenicity Testing of Selected Food Additives by Parenteral Administration to Infant SwissMice. EPSTEIN, S. S., FUJII, K., ANDREA, J., and MANTEL, N. (1970). Toxicol. Appl. Pharmacol. 16, 321-334. The toxicity and carcinogenicity of 6 food additives, safrole; alginic acid; polyoxyethylene-(20)-sorbitanmonostearate(Tweena 60); 6-ethoxy-2,2,4trimethyl-1,Zdihydroquinoline (Santoquin@); 1,3,5-trimethyl-2,4,6-tris(3,5-di-tert-butyl-4-hydroxybenzyl)benzene (Ionox@ 330); 2,4-bis-(4-hydroxy-3,5-di-tert-butylphenoxy)-6-(n-octylthio)-1,3,5-triazine (RA-858) weretestedby 4 consecutivesubcutaneousinjectionsin infant Swissalbino mice,aged1,7,14, and 21 days.Dosesof safrole,alginic acid, Tween60, or Santoquin in excessof 1 mg on day 1 of life were acutely toxic; Ionox and RA-858 were nontoxic at dosesof 10mg. Surviving mice weresacrificedat 1 year. After a total dosageof 6.6 mg safrole,a low incidenceof multiple pulmonary adenomas,6 %, and pulmonary adenocarcinomas,10%, and a high incidenceof hepatomas,58x, developedin 31 male mice alive at weaning,in contrastwith a zero incidenceof multiple pulmonary adenomas and pulmonary adenocarcinomas,and a 5-6 ‘A incidenceof hepatomasin uninjectedand solvent-injectedcontrols, based,respectively,on 36 and 78 malesalive at weaning.In groupstestedwith alginicacidand RA-858, tumor incidencesfell within control ranges;carcinogenicitydata for Santoquinand lonox wereequivocal.Theseresultsconfirm the practical utility of neonates for carcinogenicitytesting in that remote tumorsdeveloprapidly following restricted parenteral administration of relatively small quantites of test materials. The utility of neonates has been adequately demonstrated for testing a wide variety of carcinogens (Klein, 1959a, b; Roe et al., 1961; Fiore-Donati et al., 1961; Pietra et al., 1961; Toth and Shubik, 1963,1967; Nishizuka et al., 1964, 1965; O’Gara et al., 1965; Boiato et al., 1966; Gorrod eta/., 1968; Kelley et al., 1968; Della Porta, 1968)and, more restrictedly, for testing crude organic extracts of atmospheric pollutants (Epstein et al., 1966), therapeutic drugs (Epstein et al., 1967a),herbicides (Epstein and Mantel, 1968), and combinations of piperonyl butoxide, an insecticide synergist, with simple fluorocarbons (Epstein et al., 1967b). Tumors, remote or local, generally develop relatively rapidly after parenteral administration of minute quantities of carcinogens to neonatal rodents. Despite thesepractical considerations, there is no literature on carcinogenicity testing of food additives in neonates. I Supported by NIH Research Grants Nos. C-6516 andFR-0.5526. 321
322
EPSTEIN
El‘ AL.
We therefore tested the carcinogenicity-and incidentally the acute toxicity-of 6 food additives, selected to represent wide structural and functional types, by parenteral administration to neonatal mice. These were 3,4-methylenedioxyallylbenzene (safrole)z; alginic acids; polyoxyethylene-(20)~sorbitan monostearate (Tween @ 60)4; 6-ethoxy2,2,4-trimethyl,2-dihydroquinoline (Santoquin @) 5; 1,3,5-trimethyl-2,4,6-tris(3,5ditert-butyl-4-hydroxybenzyl)benzene (Ionox @ 330)6; 2,4-bis(4-hydroxy-3,5-di-tertbutylphenoxy)-6-(n-octylthio)-1,3,5triazine (RA-858)‘. Safrole is the principal constituent of sassafras oil, whose use as a natural flavoring agent, particularly in soft drinks, was prohibited after it proved to be hepatocarcinogenic in adult rats (Homburger et al., 1961, 1962; Long et al., 1963; Hagen et al., 1965). Moreover, safrole is a potent inhibitor of hepatic microsomal enzyme function (Jaffe et al., 1968). Alginic acid is a colloidal uranic acid polymer from brown seaweed used to stabilize creams and jellies (British Food Standards Committee Report, 1956). Tween 60, used as a dispersing agent in various food and as an antibloom agent in chocolate, is a weak promoting agent (Set&la, 1956; Set% et al., 1956) and a weak carcinogen (Shubik et al., 1959) after topical application to mouse skin; it also produces local sarcomas after subcutaneous injection in rats (Lusky and Nelson, 1957). While Tween 60 is not carcinogenic on feeding (Oser and Oser, 1957), the related emulsifier polyoxyethylene(8)stearate, Myrj @45,4 produced bladder tumors in rats after feeding at high levels (Fitzhugh et al., 1959; Hueper and Payne, 1963). Santoquin is an antioxidant used for stabilizing carotenoids in alfalfa poultry feeds (Livingston et al., 1955); although there are no published data on the carcinogenicity testing of Santoquin, a polymer of the closely related compound 1,2-dihydro-2,2,4-trimethylquinoline, Flectol H,4 which is used as a rubber antioxidant, produced cholangiofibrotic nodules (Panner and Tacker, 1961), pulmonary adenomas, and lymphomas after chronic feeding in rats (Hodge et al., 1966). Ionox 330 is an antioxidant used in polymers for food contact application (Code of Federal Regulations, 1967). RA-858 is an antioxidant, still at the investigative level. No published data on carcinogenicity testing are available for alginic acid, Santoquin, Ionox 330, and RA-858. MATERIALS
AND
METHODS
Materials. Stock solutions of safrole at 1.1, 11, 110, and 1100 mg/ml; Tween 60 at 11,110, and 1100 mg/ml; Santoquin at 10,50, and 100 mg/ml; and stock suspensions of alginic acid, Ionox 330, and RA-858 at 10 and 100 mg/ml were stored in sealed ampules at 4°C and used within 1 week of preparation; tricaprylin was the solvent for all drugs except Tween 60, which was dissolved in 0.9 y0 saline. Methods. By described techniques (Epstein et al., 1966, 1967a, 1968), random-bred infant Swiss albino mice [ICR/Ha]s were injected subcutaneously in the nape of the neck with solutions or suspensions of drugs or with solvent alone in volumes of 0.1, 0.1, 0.2, and 0.2 ml on days, 1,7,14, and 21, respectively, after birth. For practical considerations, all mice in each litter were treated alike. Numbers of mice injected, and the z K & K Laboratories, Inc., Plainview, New York. 3 Alginic Industries, Ltd., London, England. 4 Atlas Powder Company, Wilmington, Delaware. J Monsanto Chemical Company, St. Louis, Missouri. 6 Ethyl Corporation, Baton Rouge, Louisiana. 7 Geigy Chemical Corporation, Ardsley, New York. s Charles River Breeding Laboratories, Wilmington, Massachusetts.
CARCINOGENICITY
OF FOOD
ADDITIVES
FOR
NEONATES
323
corresponding number of litters for each test group are indicated in Table 1. The solvent and uninjected control groups were the largest as these also served for other drugs concurrently tested. Although 2 litters were initially randomly allocated to each group, the uneven number of litters finally assigned reflected an attempt to concentrate testing at the highest possible subtoxic drug concentration. This stepwise dose selection was accomplished over a few days, during which successive mice were littering, so that all tests may be considered as simultaneous. Mice were inspected daily and weighed weekly for the first month of life and at approximately monthly intervals thereafter. Following weaning at 1 month, groups of 5 or fewer mice from each litter and sex were housed in hanging metal cages with wire grid floors and given Purina laboratory chow and water ad libitum. Mice were allowed to survive, with the exception of those killed when moribund, until experiments were terminated between 49 and 53 weeks. With occasional and defined exceptions due to cannibalism, autolysis. or accidental loss, all mice were autopsied and tissues from any lesion or tumor, and usually also from thymus, heart, lungs, liver, spleen, adrenals, kidneys, lymph nodes, and sternal marrow, were fixed in Tellyesniczky’s solution, sectioned at 5 p and stained with hematoxylin and eosin. RESULTS
Acute toxicity, as indicated by mortality, in all groups was largely limited to the first few days of life. Mortality prior to weaning waslow (~20 %) and comparable in controls and in neonatesreceiving 0.11 or 1.1 mg of safrole, 1 mg of alginic acid, 1.1mg of Tween 60, 1 mg of Santoquin, 1 or 10 mg of Ionox, 1 or 10 mg of RA-858, on day 1 of life; higher mortality (74-100 %) was observed, however, in the remaining groups (Table 1). Relative to controls, there wasnoevidence ofweight lossat any time in drug-treated mice. The total number of histologically examined tumors is listed in Table 2 for mice in specified age ranges in control and various test groups. Hepatomas.A high incidence of hepatomasoccurred in malesreceiving a total safrole dosageof 0.66 mg, 50 % basedon 12 mice alive at weaning,9 and 6.6 mg, 58% basedon 31 mice alive at weaninglo; this contrasts with an incidence of 5% in 114 solvent injected and uninjected male controls. For mice receiving 60 mg of Ionox, the incidence of hepatomas, 14% based on 29 malesalive at weaning,I1 is not significantly different from control values. Statistical significance remains essentially unchanged12when litter variation (Epstein et al., 1967a)is taken into account. Litters in a treatment group were relatively uniform in their hepatoma rates; every one of 7 safrole-treated litters with 3 or more male survivors at weaning showed at least 2 mice with hepatomas; of 20 control litters with sufficient male survivors, only 6 showed any mice with hepatomasand in no casemore than 1per litter; of the 6 litters dosed with 60 mg Ionox, 1 showed2 mice with hepatomas, 2 showed 1 such mouse, and 3 showed no hepatomas. No hepatomas occurred in males in other treatment groups or in any female mice; with 4 exceptions in safrole-treated groups, no hepatomas were manifest before sacrifice at termination ot p Continuity
corrected x2, 1 d.jI, = 18.8, p e 0.001.
‘0 x’ = 44.4, p -s 0.001. 1’ x* = 1.2, p > 0.2.
12The 3 X* values of 18.8, 44.4 and I .2, then become F values of 53.0, with 1 and 23 d.J, 109.9, with 1 and 26 d.f, and 2.3, with I and 27 d.jI, respectively.
INDUCED
0.11
1.1
-
0.11
1.1
11 110
Safrole
1
10
1
10
-
-
-
7
Uninjected controls
1
-
Alginic acid
IN SWISS ALBINO
MICE
-
20
2
2.2
0.22
--
-
14
-
20
2
2.2
0.22
-
-
21
60
6
11 110
6.6
0.66
-
-
Total
Drug dosageon specifieddays (mg)
TOXICITY
Solvent controls
Treatment
ACUTE
1
79
20
22 26
71
24
90
170
(7)
(2)
(2) (2)
(7)
(2)
(9)
(16)
Initial number of mice injected (No. litters)
BY NEONATAL
TABLE
1.6
1.5
1.6 1.7
1.6
1.3
1.6
1.6
1
9.8
9.5
-
7.9
8.9
7.8
0.9
21
Average weight c&dof mice at specified days
AND PERINATAL
OF
80
20
100 100
16
13
19
14
Mortality prior to weaning (%)
INJECTION
M F M F
M F M F -
M F
M F
Sex
8 8 7 9
12 9 31 29 0 0
36 37
78 69
g q k
68 31 31 11 9 19 24 -
g
50
At 49+ weeks
Number of survivors ---At weaning
FCKID ADDITIVES
E
@
“RA”
Ionox @330
Santoquin
Twem @60
1
10
10
10
10
1
1
1
-
5
5
10
1
1
-
20
2
20
2
10
2
-
20
2
20
2
10
2
-
-
-
11 110 -
2.2
2.2
1.1
1.1
60
6
60
6
10
30
6
11 110
6.6
TABLE
(5)
(2)
17 49
(6)
(2)
(2)
(4)
(5)
(2) (1)
(4)
63
22
28
53
57
22 10
49
l-continued
1.9
1.9
1.8
1.9
1.7
1.6
1.7
1.8 1.6
1.7
8.5
10.2
5.54
6.9
-
9.8
8.0
-
8.5
M F M F
2
4
0
M F M F
M F M F
5 3
-
100
74
100 100
M F -
2
9 8 24 23
15 6 29 32
25 31 9 5 0
25 23 0 0
4 5 13 19
8 4 21 26
-
21 29 5 3
16 22
M
F M F
60
F M F M F M F
M
L F M F M F
F M F M F M
77 6) 36 37 12 9 31 28 8 8 7 9 25 23 25 31 9 5 15 6 27 32 9 8 24 23
72 69 36 35 I2 9 31 28 8 8 7 9 22 23 25 31 9 5 14 6 26 32 8 8 22 23
2130
62 69 34 34 12 9 24 27 5 8 5 8 19 23 25 30 9 4 14 6 22 30 4 8 18 23
3140
8 17 23 23 29 6 4 9 5 22 28 4 8 14 21
55 68 32 33 12 9 23 25 5 :
4148
50 68 31 31 11 9 19 24 2 8 4 7 16 22 21 29 5 3 8 4 21 26 4 5 13 19
491
-
0’ 2 2 - -
4 2 4 1 3 1 5 4 0 0 0 0 4 2 0 4 4 1 0 2 2 1
Pq0.
381-S 410
il 0 0
ii 0
x 0 0 0 2 0 0
i 0 0 0 0 0
1 0 0 I
78 69 ii 37 I2 9 31 29 8 8 7 9 25 23 25 31 9 5 15 6 29 32 9 8 24 23
1120
8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0
4148 3 1 46 0 If I 0 3 0 0 0 0 3= 2 0 4 1c Id 0 2 2 1 1 0 2 2
491
autopsied (weeks)
z 0
1 0 0 0 0 0 0
0” 0 0 0 0 0 0 0 0 0 0 0 0
0 0
31-s 10 _---._ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4148
8 0 0 0 0 0
8 0 0 0 0 0 0 0 0 0
0 1 0 0 0 0 2h 0
49+
Multiple adenoma ( b 2)
of mice with tumor at each period
tumorsa
BY NEONATAL
Pulmonary
MICE
Solitary adenoma -_-~
M
410
I-
Number
SWISS ALBINO -
- --
-
IN
-
6
60
6
30
6
6.6
60
6
6.6
0.66
-
INDUCED
Number of mice, later autopsied, alive at the beginning of each weekly period
TUMORS
0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0. 0
.,..a I8
Adeno-
; 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 -
0 0 0 0 2a 0 2i .i
-_
49. t
-I-
--
-
4 0 2 0 6 0 18 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 -
INo.
INJECTIONS
OF FOOD
-
8 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 I 0 0 0 0 0 0 0
2.1-q 30 -
: 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
3l40
Solitary
0 0 0 0 I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4148
8 0
8 0 0 0 0 0 0 0 0 0 4 0 0
4 0 2b 0 28 0 7j 0 0
491
-
0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4II-Q 118
Multiple
of mice with tumor autopsied sacrificed at each period (weeks)
1Number
PERINATAL
or sacrificed
AND
2
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -
8h.Lk
0 0 0 0 3f 0
491
or
-
I
Lymphomas”
!I-4
0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
3140
:, 0 0 0 0 0
0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 1 0 0
48
Ol-
:Ff 1 0 0 0 0 0 0 0
1 0 0 0 0 0 2k 1 0 1 0 0 1e 1 0 3
491
of mice with tumor autopsied or sacrificed at each period (weeks)
E lumber
ADDITIVES
-
Ti-
-
pn.n
0” 0 0 0 0 10 0 0 0
1p 0
0 1) 0 0 0 0 0 0 0 0 0 0 0
Misc. 1L”“lOrS NO.
(rr) The Following instances of coexistent multiple tumors occurred, each tumor being indiwdually listed: (bl one with solitary pulmonary adenoma and solitary hepatoma; (c) one with solitary pulmonary adenoma and malignant lymphoma; (d) one with rolita-y pulmonary adenoma and malignant lymphoma; (c) one with solitary pulmonary adenoma and malignant lymphoma; (f) one with solitary pulmonary adenoma and multiple hepatoma; (a) two with pulmonary adenocarcinoma and solitary hepatoma; (h) two with multiple pulmonary adenoma and multiple hepatoma; (i) one with pulmonary adenocarcinoma with multiple hepatoma; (j) one with pulmonary adenocarcinoma and solitary hepatoma; (k) one with malignant lymphoma and multiple hepatoma. (I) mammary carcinoma at 51 weeks; (nz) mammary carcinoma at 48 weeks; (n) subcutaneous carcinosarcoma at 53 weeks; (0) ovary fibrosarcoma at 50 weeks; (p) thyroid adenoma at 50 weeks. (q) For each class of lesion, the first time interval shown in which the first tumor of that class arose in at least one of the treatment groups.
“RA”
lonox@
330
60
TWX”@
Santoquine
acid
Alginic
Solvent control Uninjected control Safrole
Treatment
r0td I dose E;e> (m3)
-
TABLE
h
z
; z;. E z
CARCINOGENICITY
OF FOOD
ADDITIVES
FOR
NEONATES
327
FIGS. 14. Lesions in male Swiss mice injected in infancy with a total dose of 6.6 mg of safrole. Figs. 2-4, from mice sacrificed at 1 year. All sections stained with hematoxylin and eosin. FIG. 1. Multiple hepatomas.
FIG. 2. Trabecular hepatoma with lower margin of normal liver cells. ,99
328
EPSTEIN
ET AL.
experiments. The hepatomas were solitary or more frequently multiple and generally ovoid and superficial; they varied in size from the minute to that which exceeded in size the normal liver (Fig. 1). Histologically, the tumors were classical mouse hepatomas (Stewart and Snell, 1957) with a well differentiated and trabecular pattern (Fig. 2); hyaline and vacuolar cytoplasmic inclusion bodies were generally conspicuous. Mitotic figures were rare, and no extrahepatic metastases were found. In safrole-treated animals, cellular and structural atypia were generally seen in males with tumor-free livers (Fig. 3); these atypia were characterized by mid-zonal cytoplasmic vacuolization, nuclear inclusions, focal necrosis and bile duct proliferation.
FIG. 3. Advanced hepatic atypia with marked pleomorphism, cytoplasmic vacuolization, and nuclear inclusion bodies. x 182.
Pulmonary tumors. The incidence of solitary pulmonary adenomas in males and females for uninjected and solvent controls was 1 1 %, 3 %, 5 %, and 2 %, respectively. With the exception of the 30 mg Santoquin male and female groups and 6 mg Ionox female groups, where numbers of animals involved were small, the incidence of solitary adenomas in other groups fell within control ranges. Multiple pulmonary adenomas were rare, however, occurring in 2/31, l/9, and l/69, male 6.6 mg safrole, male 30 mg Santoquin groups, and female solvent controls, respectively. Of the 7 pulmonary adenocarcinomas throughout all groups, 5 occurred in 43 safrole-treated males, one in 38 safrole-treated females, one in 25 Tween-treated males, none in 220 controls, and none in any other group. The adenocarcinomas were large, all exceeding 5 mm in diameter in contrast with adenomas, which were less than 2 mm diameter. Histologically,
CARCINOGENICITY
OF FOOD
ADDITIVES
FOR
NEONATES
329
the adenomas were alveologenic and well differentiated, although occasional structural irregularities were seen. The pulmonary adenocarcinomas were less well differentiated, with atypical cells and frequent mitotic figures and were locally invasive (Fig. 4); no metastases were found.
FIG. 4. Papillary pulmonary adenocarcinoma
in a mouse sacrificed at 41 weeks. x 182.
Lymphomus. In contrast with controls, an enhanced but low incidence of lymphocytic and reticular lymphomas was observed in males and females of the higher safrole dosage group, 3/31 and 5/29, respectively; females of both alginic acid dosage groups, l/8 and l/9; males and females of the Tween 60 dosage group, l/25 and l/23, respectively; females of the lower Santoquin dosage group, 4/3 1; males and females of the higher Santoquin dosage group, Z/9 and 2/5, respectively; males and females of the lower Ionox dosage group, l/l5 and 2/6, respectively. Miscellaneous tumors. Details of these and the individual disposition of all mice surviving weaning are listed in Table 3.
DISCUSSION The utility of neonatal systems for testing of proximate carcinogens such as polycyclics, which are carcinogenic per se as they do not require metabolic activation, is generally accepted. Questions have, however, been raised about nonproximate carcinogens such as the aromatic amines, which are only carcinogenic after they have been metabolically activated, whose activation may be reduced by the immaturity of hepatic microscomal enzyme function in the neonate. Such immaturity is probably not limiting, (NMU) as o-aminoazotoluene (AAT) (Nishizuka et al., 1965), nitrosomethylurea
acid
330
29
8 8
M
F M
F
M F M F M
0.66
6.6
24
23
F
instances lymphoma: adenoma;
8
60
32 9
M
F M
F
60
6
6 29
F M
6
23 25 31 9
5 I5
M
F
M
9
25
F M
30
6
6.6
F
9 31
M F
7
69 36 37 12
F
6
78
weaning
1
0 1 0 0
2 0
0 0
0 0 1 4
4 0 0 0 I 0
0 5
1 1
0 0
I
NO.
48MC
3SSPA
40ML,
26ML 34SPA+ 40SPA
30ML
28ML,
45ML
44ML,
II 3
6 4 3
0 8
2 7
4 I 0
1
0 7
1 5 5 0
27
NO.
during
and
losses
INITIALLY
(17A).
29U
26U
* 2,35U,
33, 39N,
42
12, 15,30U+PN, (3OL),3OU 32Ux2.33, 39U.41
41.46
x 2,
24, 35U z 2,
46U
36U+PN,
24U,
45U,
z 2, 33, (38L)
40,46 43 x 2
10Ux2,25,31U,35U+PN,38~2, 43u 28, (29A) ,‘34A), (4OL), 24. 35 x 3 41:(43L)r2
23.46 l9N, 3lU 36U, 43U
17. 18U. 34U x 2, 39, 41U, 45.46.47,
x 2,43U,
x 2,29
28U, 29U, 31U 43 17, 19U x 2,2lU, 45 (42A) 35U x 2,43,46 45
10 22IJ,
24U,
(3OL) Y 2, (39L), 14, (26L), (3lA),
0.U)
25U x 2,28,29U, (39L) x 2,4OU, 47u
2iUk3,(i2A),23U,25,
4. IS. ISU.
Individual week of death and associated lesion, if any
tumor
without
Deaths
INJECTED
of experiments
MICE
course
SWISS ALBINO
or sacrificed
U, 39MPA+U, U, 42ML
38ML,
41PAC. 47MH
41 ML
t
tumor
dying
IN
week ofdeath and tumors or lesions
3SML,
24SH +U, 45MH+U,
31SPA 45SH
34SPAtu
Individual associated
with
Mice
DEVELOPING
Deaths
LESIONS
3
2 6
I
2
1 I6
8
NO.
Mice
tumor
tumors
with
at conclusion
WITH
lesions at death: SC, subcutaneous
SC, SPA x 2
SPA%2
MC.
MH, solitary
N
Nx2
N N U
ux2
(A) (A)
u, u-1
Associated lesions, if any
r;nrrary carcinoma: carcinosarcoma; SH,
24 3 5 11
2 15
SPAx2 SH,SPAz2,SHtU, SHx2 SPA,Ov SPA
I8 21 22 4
19 2 7 4 7 I2
8 3
65 26 31 6
42
NO.
I 7
Th
or lesions
weeks)
Deaths without tumor and losses
(49-53
ADDITIVES~
of experiments
FOOD
ML,SPA+ML ML
SPAx4,MLx3 SPA+ML
ML,SPAx2,
SPAtML.SPAr2,PAC
ML
PAC+SHx2,MHX2, SPA+MH SPA SHx6,MHr4,ML, MPA+MHx2,PAC+MH, PAC+SH,ML+MH SPAx3,PAC,ML
SPA,MPA.MC SPAx3,SPA+SH,SH
SH+U,SHx3,SPAx2. MLtlJ,SPA
Associated
Deaths
sacrificed
AS NEONATES
where no autopsy was possible due to autolysis (A) or accidental loss (L). Code for tumors or other MPA, multiple pulmonary adenoma; Ov. ovary fibrosarcoma; PAC. pulmonary adenocarcinoma; Th, thyroid adenoma; PN, pyelonephritis; U, obstructive uropathy.
OTHER
Sexand No. at
AND
M
60
-
Total dose (me)
TUMORS,
~Parenthescs are used to indicate multiple hepatoma; ML, malignant hepatoma; SPA, solitary pulmonary
"RA"
Ionox@
conrrols
controls
Tween@'60
Alginic
Safrole
Uninjected
Solvent
Treatment
SURVIVAL,
TABLE
3 i .P
6
22
CARCINOGENICITY
OF FOOD
ADDITIVES
FOR NEONATES
331
(Terracini et al., 1967), and acetylaminofluorene (AAF) (Epstein, unpublished data) are carcinogenic following single administration only to l-day-old mice. It must be stressed that there are no reports in the available literature of “false negatives,” based on comparisons with conventional adult systems, after repeated administration of nonproximate carcinogens during the first month of life. In view of the negative results reported for dimethylnitrosamine (Terracini et al., 1966), urethane (Trainin et al.. 1964), and AAF (Walters et al., 1967), single administration of nonproximate carcinogens on day 1 of life should not be regarded as an adequate test procedure. While there have been relatively few experiments designed specifically for simultaneous comparison of dose-response relationships for carcinogens in neonatal and adult systems (Toth, 1968), there is, however, sufficient composite and nonconcurrent data to consider this question. Single subcutaneous injection of 0.4 mg AAT (Nishizuka et al., 1965), and 0.05 mg NMU (Terracini and Stramignoni, 1967), and multiple injections totaling0.6 mg Aminobiphenyl (ABP) (Gorrod et al., 1968), and 3 mg griseofulvin (GF) (Epstein et al.. 1967a), and feeding of a total dose of 7 mg AAF (Klein, 1959b), were all carcinogenic to infant mice. In adult rodents, however, many of these compounds were carcinogenic only after prolonged feeding at high dietary levels. With GF, for instance, feeding of the adult mouse for about 9 months at the 1% dietary level was required to produce hepatomas (Hurst and Paget, 1963). On a total dosage basis, the infant is thus about 4500-fold more sensitive than the adult mouse to the carcinogenic effects of GF; on an mg/kg basis, however, the corresponding sensitivity is reduced to the order of 1100. The enhanced sensitivity of infant rodents is of practical importance, Even relatively large hazards due to weak carcinogens, for example, affecting l/ 10,000 of the population, would probably escape detection by conventional carcinogenicity tests involving test groups of even as many as 50 adult animals per given dosage; assuming rats and man have the same sensitivity to weak carcinogens, a test group of 10,000 rats would be required to induce tumors in 1 animal only. Thus, to demonstrate weak carcinogens in the environment, it is essential that the most sensitive test system available to be used and that the factor of comparability of route and schedule of drug administration te subordinated to the requirement of sensitivity. Once a hazard has been clearly established, the quantitative relevance of the experimental data to the human situation must be considered before limits can be reasonably proposed, and only at this stage does it become appropriate to weight factors such as age of test animal and route of drug administration. To use such factors in a limiting sense, and to insist on precise comparability between test systems and human exposure before the problem of hazard is established, will effectively preclude all possibility of detecting weak environmental carcinogens; such limitations would also challenge the human implications of data on experimental tobacco carcinogenesis. There would not appear to be any advantage in holding mice for more than 1 year following treatment; no instances of “false negatives” have been reported after sacrifice of mice at 1 year of age under these conditions. High incidences of hepatomas have been recorded in mice 1 year after neonatal administration of a wide variety of carcinogens, including AAF, ABP, AAT, GF, and maleic hydrazide (see above for references); multiple pulmonary adenomas and thymic lymphomas develop more rapidly still. The relatively high yield of remote tumors, such as hepatomas, pulmonary adenomas, and thymic lymphomas, following single or repeated subcutaneous injection of low
332
EPSTEIN ETAL.
doses of a wide variety of carcinogens in neonatal mice is of particular interest. Production of remote tumors under these conditions obviously avoids the issue of “Oppenheimer” effects (Oppenheimer et al., 1956), which have been incriminated in suggestions as to the nonspecificity of some local sarcomas after repeated subcutaneous injection of certain food additives and other drugs in adult rats (Grass0 et al., 1966), and also minimizes the importance of the variable relating to route of drug administration. The data reported here on carcinogenicity testing of food additives in infant mice accords with the above discussion of the reported literature with regard to the practical utility of neonatal systems. The hepatocarcinogenicity of safrole after its restricted and intermittent parenteral administration to infant mice is of particular interest. Safrole also produced pulmonary tumors, solitary and multiple pulmonary adenomas and adenocarcinomas, and lymphomas in neonates in contrast with the adult rat, in which carcinogenic effects are restricted to the liver (Hornburger et al., 1962; Hagen et al., 1965). The production of pulmonary tumors, particularly adenocarcinomas, by safrole has not been previously described and is noteworthy, especially in view of reports (Fishbein et al., 1968) of acute pulmonary congestion and edema and of selective pulmonary uptake of safrole, and other methylenedioxyphenyl compounds, after parenteral administration to rats. It is interesting to contrast the low dosage of safrole, 0.66 mg, producing a 50% incidence of hepatomas, as well as some lymphomas, pulmonary adenomas and adenocarcinomas, after injection of infant male mice, with the high dosages, 1.O % dietary levels for 1 year, equivalent to about 55 g of safrole, necessary to produce hepatomas in adult rats (Hornburger et al., 1961; Long et al., 1963); this is approximately 83,000 times the dose found to be carcinogenic after injection of the infant mouse. If adjustment is made for body weight, this extreme factor is reduced to the order of 1100. Quite apart from the putatively critical age factor, such quantitative comparisons obviously do not reflect other factors, such as strain and route of drug administration, which might partially account for the observed differences in sensitivity of neonatal and adult rodents to carcinogens. The effects of Ionox 330 in relation to the production of an apparently enhanced incidence of solitary pulmonary adenomas, hepatomas, and lymphomas should be noted; however, with the exception of the 4 hepatomas developing in 29 males in the 60-mg dosage group, these incidences are based on small numbers of animals. For Santoquin, the increased and dose-related yield of solitary pulmonary adenomas and lymphomas in both sexesis of interest and is consistent with similar findings in adult rats after feeding of the related compound Flectol H (Hodge et al., 1966). However, an increase in solitary pulmonary adenomas is an unreliable carcinogenic index, especially when unaccompanied, as obtains here in the Ionox and Santoquin groups, by multiple adenomas in other animals under test. In view of these findings, it would appear appropriate to repeat testing of both these compounds, particularly Santoquin, with larger numbers of neonatal mice, in addition to conventional adult rat systems. REFERENCES BOIATO, L., MIRVISH, S. S., and BERENBLUM, I. (1966). The carcinogenic action and metabolism of N-hydroxyurethane in newborn mice. Intern. J. Cancer 1,265269. British Food Standards Committee Report, Emulsifying and Stabilizing Agents. Her Majesty’s
Stationery Office, London, 1956.
CARCINOGENICITY OF FOOD ADDITIVES FOR NEONATES
333
Code of Federal Regulations (1967). Title 21, Part 121.2566. PORTA, G. (1968). Use of newborn and infant animals in carcinogenicity testing.
DELLA
Food Cosmet. Toxicol. 6,243-252.
S. S., and MANTEL, N. (1968).Hepatocarcinogenicityof the herbicidemaleichydrazide following parenteraladministrationto infant mice. Intern. J. Cancer 3,325-335. EPSTEIN, S. S., JOSHI, S., ANDREA, J., MANTEL, N., SAWICKI, E., STANLEY, T., andTABOR,E. C. (1966).Carcinogenicity of organic particulate pollutants in urban air after administration of trace quantitiesto neonatalmice. Nature 212, 1305-l307. EPSTEIN, S. S., ANDREA, J., JOSHI,S., and MANTEL,N. (1967a). Hepatocarcinogenicityof griseofulvinfollowing parenteraladministrationto infant mice. Cancer Res. 27, 1900-1906. EPSTEIN, S. S.,JOSHI,S., ANDREA,J., CLAPP,P., FALK, H., and MANTEL, N. (1967b).Synergistic toxicity and carcinogenicityof “Freons” and piperonyl butoxide. Nature 214, 526528. FIORE-DONATI, L., CHIECO-BIANCHI, L., DEBENEDICTS, G., and MAIORANO,G. (1961). Leukemogenesis by urethanein newborn Swissmice. Nature 190, 278-279. FISHBEIN, L., FALK, H. L., FAWKES,J., and JORDAN,S. Metabolism of pesticidal synergists. PesticideManual of the Sixth Inter-AmeriFan Conferenceon Toxicology and Occupational Medicine. Coral Gables,Florida, August 2629, 1968. FITZHUGH, 0. G., BOURKE, A. R., NELSON, A. A., and FRAWLEY, J. P. (1959). Chronic oral toxicities of 4 stearicacid emulsifiers.Toxicol. Appl. Pharmacol. 1, 315-33 1. GRASSO, P., and GOLBERG,L. (1966). Subcutaneoussarcomaas an index of carcinogenic potency. Food Cosmet. Toxicol. 4,297-320. GORROD, J. W., CARTER, R. L., and ROE, F. J. C. (1968). Induction of hepatomasby 4-aminobiphenyl and three of its hydroxylated derivatives administeredto newborn mice. J. Natl. EPSTEIN,
Cancer
Inst.
41,403-410.
HAGEN,E. C., JENNER, P.-M., JONES, W. I., FITZHUGH, 0. G., LONG, E. L., BROUWER, Z. G., and WEBB, W. K. (1965).Toxic propertiesof compoundsrelatedto Safrole. Toxicol. Appl. Pharmacol. 7, 18-24. HODGE, H. C., MAYNARD, E. A., DOWNS, W. L., ASHTON, J. K., and SALERNO, L. L. (1966). Testson micefor evaluating carcinogenicity. Toxicol. Appl. Pharmacol. 9, 583-596. HOMBURGER, F., KELLEY, T., JR., FRIEDLER, G., and RUSSFIELD, A .B. (1961).Toxic andpossible carcinogeniceffectsof 4-allyl-I ,2-methylenedioxybenzene (Safrole) in rats on deficientdiets. Med. Exptl. 4, l-1 1. HOMBURGER, F., KELLEY, T., JR., BAKER, T. R., and RUSSFIELD, A. B. (1962). Sex effect on hepatic pathology from deficient diet and Safrole in rats. Arch. Puthol. 73, 118-125. HUEPER, W. C., and PAYNE, W. W. (1963). Polyoxyethylene(9)stearate:Carcinogenicstudies. Arch. Environ. Health. 6,484494.
E. W., and PAGET, G. E. (1963). Protoporphyrin, cirrhosis, and hepatomatain the livers of mice given griseofulvin. Brit. J. Dermatol. 75, 105-I 12. JAFFE, H., FUJII, K., SENGUPTA, M., GUERIN, M., and EPSTEIN, S.S. (1968).In vivo inhibition of mouse liver microsomal hydroxylating systemsby methylenedioxyphenyl insecticidal synergistsand related compounds.Life Sci. 7, 1051-1062. KELLY, M. G., O’GARA, R. W., YANCEY, S. T., and BOTKIN, C. (1968). Carcinogenicity of I-methyl-1-nitrosoureain newborn mice and rats. J. Natl. Cancer Inst. 41,619-626. KLEIN, M. (1959a).Developmentof hepatomas in inbred albino micefollowing treatmentwith 20-methylcholanthrene.Cancer Res. 19, 1109-l 113. KLEIN, M. (1959b).Influence of low doseof 2-acetylaminofluoreneon liver tumorigenesisin mice.Proc. Sot. Exptl. Biol. Med. 101, 637-638. LIVINGSTON, A. L., BICKOFF, E. M., and THOMPSON, C. R. (1955).Alfalfa carotenoids: Effects of added animal fats and antioxidants on stability of xanthophyll concentratesin mixed feeds.Agr. Food Chem. 3,439-441. LONG, E. L., NELSON, A. A., FITZHUGH, 0. G., and HANSEN, W. H. (1963). Liver tumorsproducedin rats by feedingSafrole. Arch. Pathol. 75, 595UjO4. LUSKY, L. M., and NELSON, A. A. (1957). Fibrosarcomasinduced by multiple subcutaneous injections of carboxymethylcellulose(CMC), polyvinylpyrrolidone (PVP), and polyoxyethylenesorbitan monostearate(Tween 60). Federation Proc. 16, 318. HURST,
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ET AL.
Y., NAKAKUKI, K., and SAKAKURA, T. (1964). Induction of pulmonary tumors and leukaemia by a single injection of 4-nitroquinoline-l-oxide to newborn and infant mice. Gum. 55,495-508. NISHIZUKA, Y., ITO, K., and NAKAKUKI, K. (1965). Liver tumor induction by a single injection of o-aminoazotoluene to newborn mice. Cam 56, 135-142. O’GARA, R. W., KELLY, M. G., BROWN, J., and MANTEL, N. (1965). Induction of tumors in mice given a minute single dose of dibenz[a,h]anthracene or 3-methylcholanthrene as newborns. A dose-response study. J. Natl. Cancer Inst. 35, 1028-1042. OPPENHEIMER, B. S., OPPENHEIMER, E. T., and STOUT, A. P. (1956). Carcinogenic effect of metals in rodents. Cancer Res. 16,439441. OSER, B. L., and OSER, M. (1957). Nutritional studies on rats on diets containing high levels of partial ester emulsifiers. IV. Mortality and postmortem pathology: General conclusions. J. Nutr. 61, 235-252. PANNER, B. J., and TACKER, J. T. (1961). Hepatic alteration in rats fed 1,2-dihydro-2,2,4trimethylquinoline, Flectol H. Proc. Sot. Exptl. Biol. Med. 106, 1619. PIETRA, G., RAPPAPORT, H., and SHUBIK, P. (1961). The effectsof carcinogenicchemicalsin newborn mice. Cancer14, 308-3 17. ROE, R. J. C., ROWSON, K. E. K., and SALAMAN, M. H. (1961). Tumorsof many sitesinduced by injection of chemicalcarcinogensinto newborn mice.A sensitivetest for carcinogenesis: the implicationsof certain immunologicaltheories.Brit. J. Cancer15, 515-530. SET.XL& H. (1956).Co-carcinogeniceffectsof somenon-ionic lypophilic-hydrophilic (surface active) agents.Acta Pathol. Microbial. Stand. 115, l-93. SETALA, K., SET;~L;~, H., and HOLSTI, P. (1956). A new and physicochemicalwell-defined group of tumor-promoting (co-carcinogenic)agentsfor mouseskin.Science.120,1075-1078. SHUBIK, P., DELLA PORTS, G., andSPENCER, K. (1959).Studiesof the action of polyoxyethylene sorbitanmonostearate(Tween60) in skin carcinogenesis in the mouse.Acta Unio Intern. Contr. Cancer15, 232-241. STEWART, H. L., and SNELL, K. C. (1957). The histopathologyof experimentaltumors of the liver ofthe rat. A critical review of the histopathogenesis. Acta Unio. Intern. Contra. Cuncrum NISHIZUKA,
13,770-803.
B., and STRAMIGNONI, A. (1967). Malignant lymphomasand renal changesin Swissmicegiven nitrosomethylurea.EuropeanJ. Cancer3,435436. TERRACINI, B., PALESTRO, G., GIGLIARDI, R. M., and MONTESANO, R. (1966). Carcinogenicity of dimethylnitrosaminein Swissmice. Brit. J. Cancer20, 871-876. TOTH, B. (1968). A critical review of experimentsin chemicalcarcinogenesis usingnewborn animals.CancerRes.28, 727-738. TOTH, B., and SHUBIK, P. (1963). Carcinogenesisin Lewis rats injected at birth with 7,12dimethylbenz[a]anthracene.Brit. J. Cancer17, 540-545. TOTH, B., and SHUBIK, P. (1967). Carcinogenesis in AKR mice injected at birth with benzo[a]pyrene and dimethylnitrosamine.CancerRes.27,43-5 1. TRAININ, N., PRECERUTTI, A., and LAW, L. W. (1964). Trendsin carcinogenesis by urethane administrationto newbornmice of different strains.Nature 202, 305-306. WALTERS, M. A., ROE,F. J. C., MITCHLEY, B. C. V., and WALSH, A. (1967). Further testsfor carcinogenesisusing newborn mice: 2-Naphthylamine, 2-naphthylhydroxylamine, 2acetylaminofluorene,and ethylmethanesulphonate.Brit. J. Cancer21, 367-372.
TERRACINI,
Carcinogenicity Parenteral
PHARMACOLOGYl6,321-334(1970)
Testing of Selected Food Additives-by Administration to Infant Swiss Mice1
S. S. EPSTEIN, K. Fu.m,J.
ANDREA, AND N. MANTEL
Laboratories of Environmental Pathology and Carcinogenesis, The Children’s Cancer Research Foundation, Inc., and Department of Pathology, Harvard Medical School, Boston, Massachusetts; Biometry Branch, National Cancer Institute, Bethesda, Maryland Received December 26, 1968
Carcinogenicity Testing of Selected Food Additives by Parenteral Administration to Infant SwissMice. EPSTEIN, S. S., FUJII, K., ANDREA, J., and MANTEL, N. (1970). Toxicol. Appl. Pharmacol. 16, 321-334. The toxicity and carcinogenicity of 6 food additives, safrole; alginic acid; polyoxyethylene-(20)-sorbitanmonostearate(Tweena 60); 6-ethoxy-2,2,4trimethyl-1,Zdihydroquinoline (Santoquin@); 1,3,5-trimethyl-2,4,6-tris(3,5-di-tert-butyl-4-hydroxybenzyl)benzene (Ionox@ 330); 2,4-bis-(4-hydroxy-3,5-di-tert-butylphenoxy)-6-(n-octylthio)-1,3,5-triazine (RA-858) weretestedby 4 consecutivesubcutaneousinjectionsin infant Swissalbino mice,aged1,7,14, and 21 days.Dosesof safrole,alginic acid, Tween60, or Santoquin in excessof 1 mg on day 1 of life were acutely toxic; Ionox and RA-858 were nontoxic at dosesof 10mg. Surviving mice weresacrificedat 1 year. After a total dosageof 6.6 mg safrole,a low incidenceof multiple pulmonary adenomas,6 %, and pulmonary adenocarcinomas,10%, and a high incidenceof hepatomas,58x, developedin 31 male mice alive at weaning,in contrastwith a zero incidenceof multiple pulmonary adenomas and pulmonary adenocarcinomas,and a 5-6 ‘A incidenceof hepatomasin uninjectedand solvent-injectedcontrols, based,respectively,on 36 and 78 malesalive at weaning.In groupstestedwith alginicacidand RA-858, tumor incidencesfell within control ranges;carcinogenicitydata for Santoquinand lonox wereequivocal.Theseresultsconfirm the practical utility of neonates for carcinogenicitytesting in that remote tumorsdeveloprapidly following restricted parenteral administration of relatively small quantites of test materials. The utility of neonates has been adequately demonstrated for testing a wide variety of carcinogens (Klein, 1959a, b; Roe et al., 1961; Fiore-Donati et al., 1961; Pietra et al., 1961; Toth and Shubik, 1963,1967; Nishizuka et al., 1964, 1965; O’Gara et al., 1965; Boiato et al., 1966; Gorrod eta/., 1968; Kelley et al., 1968; Della Porta, 1968)and, more restrictedly, for testing crude organic extracts of atmospheric pollutants (Epstein et al., 1966), therapeutic drugs (Epstein et al., 1967a),herbicides (Epstein and Mantel, 1968), and combinations of piperonyl butoxide, an insecticide synergist, with simple fluorocarbons (Epstein et al., 1967b). Tumors, remote or local, generally develop relatively rapidly after parenteral administration of minute quantities of carcinogens to neonatal rodents. Despite thesepractical considerations, there is no literature on carcinogenicity testing of food additives in neonates. I Supported by NIH Research Grants Nos. C-6516 andFR-0.5526. 321
322
EPSTEIN
El‘ AL.
We therefore tested the carcinogenicity-and incidentally the acute toxicity-of 6 food additives, selected to represent wide structural and functional types, by parenteral administration to neonatal mice. These were 3,4-methylenedioxyallylbenzene (safrole)z; alginic acids; polyoxyethylene-(20)~sorbitan monostearate (Tween @ 60)4; 6-ethoxy2,2,4-trimethyl,2-dihydroquinoline (Santoquin @) 5; 1,3,5-trimethyl-2,4,6-tris(3,5ditert-butyl-4-hydroxybenzyl)benzene (Ionox @ 330)6; 2,4-bis(4-hydroxy-3,5-di-tertbutylphenoxy)-6-(n-octylthio)-1,3,5triazine (RA-858)‘. Safrole is the principal constituent of sassafras oil, whose use as a natural flavoring agent, particularly in soft drinks, was prohibited after it proved to be hepatocarcinogenic in adult rats (Homburger et al., 1961, 1962; Long et al., 1963; Hagen et al., 1965). Moreover, safrole is a potent inhibitor of hepatic microsomal enzyme function (Jaffe et al., 1968). Alginic acid is a colloidal uranic acid polymer from brown seaweed used to stabilize creams and jellies (British Food Standards Committee Report, 1956). Tween 60, used as a dispersing agent in various food and as an antibloom agent in chocolate, is a weak promoting agent (Set&la, 1956; Set% et al., 1956) and a weak carcinogen (Shubik et al., 1959) after topical application to mouse skin; it also produces local sarcomas after subcutaneous injection in rats (Lusky and Nelson, 1957). While Tween 60 is not carcinogenic on feeding (Oser and Oser, 1957), the related emulsifier polyoxyethylene(8)stearate, Myrj @45,4 produced bladder tumors in rats after feeding at high levels (Fitzhugh et al., 1959; Hueper and Payne, 1963). Santoquin is an antioxidant used for stabilizing carotenoids in alfalfa poultry feeds (Livingston et al., 1955); although there are no published data on the carcinogenicity testing of Santoquin, a polymer of the closely related compound 1,2-dihydro-2,2,4-trimethylquinoline, Flectol H,4 which is used as a rubber antioxidant, produced cholangiofibrotic nodules (Panner and Tacker, 1961), pulmonary adenomas, and lymphomas after chronic feeding in rats (Hodge et al., 1966). Ionox 330 is an antioxidant used in polymers for food contact application (Code of Federal Regulations, 1967). RA-858 is an antioxidant, still at the investigative level. No published data on carcinogenicity testing are available for alginic acid, Santoquin, Ionox 330, and RA-858. MATERIALS
AND
METHODS
Materials. Stock solutions of safrole at 1.1, 11, 110, and 1100 mg/ml; Tween 60 at 11,110, and 1100 mg/ml; Santoquin at 10,50, and 100 mg/ml; and stock suspensions of alginic acid, Ionox 330, and RA-858 at 10 and 100 mg/ml were stored in sealed ampules at 4°C and used within 1 week of preparation; tricaprylin was the solvent for all drugs except Tween 60, which was dissolved in 0.9 y0 saline. Methods. By described techniques (Epstein et al., 1966, 1967a, 1968), random-bred infant Swiss albino mice [ICR/Ha]s were injected subcutaneously in the nape of the neck with solutions or suspensions of drugs or with solvent alone in volumes of 0.1, 0.1, 0.2, and 0.2 ml on days, 1,7,14, and 21, respectively, after birth. For practical considerations, all mice in each litter were treated alike. Numbers of mice injected, and the z K & K Laboratories, Inc., Plainview, New York. 3 Alginic Industries, Ltd., London, England. 4 Atlas Powder Company, Wilmington, Delaware. J Monsanto Chemical Company, St. Louis, Missouri. 6 Ethyl Corporation, Baton Rouge, Louisiana. 7 Geigy Chemical Corporation, Ardsley, New York. s Charles River Breeding Laboratories, Wilmington, Massachusetts.
CARCINOGENICITY
OF FOOD
ADDITIVES
FOR
NEONATES
323
corresponding number of litters for each test group are indicated in Table 1. The solvent and uninjected control groups were the largest as these also served for other drugs concurrently tested. Although 2 litters were initially randomly allocated to each group, the uneven number of litters finally assigned reflected an attempt to concentrate testing at the highest possible subtoxic drug concentration. This stepwise dose selection was accomplished over a few days, during which successive mice were littering, so that all tests may be considered as simultaneous. Mice were inspected daily and weighed weekly for the first month of life and at approximately monthly intervals thereafter. Following weaning at 1 month, groups of 5 or fewer mice from each litter and sex were housed in hanging metal cages with wire grid floors and given Purina laboratory chow and water ad libitum. Mice were allowed to survive, with the exception of those killed when moribund, until experiments were terminated between 49 and 53 weeks. With occasional and defined exceptions due to cannibalism, autolysis. or accidental loss, all mice were autopsied and tissues from any lesion or tumor, and usually also from thymus, heart, lungs, liver, spleen, adrenals, kidneys, lymph nodes, and sternal marrow, were fixed in Tellyesniczky’s solution, sectioned at 5 p and stained with hematoxylin and eosin. RESULTS
Acute toxicity, as indicated by mortality, in all groups was largely limited to the first few days of life. Mortality prior to weaning waslow (~20 %) and comparable in controls and in neonatesreceiving 0.11 or 1.1 mg of safrole, 1 mg of alginic acid, 1.1mg of Tween 60, 1 mg of Santoquin, 1 or 10 mg of Ionox, 1 or 10 mg of RA-858, on day 1 of life; higher mortality (74-100 %) was observed, however, in the remaining groups (Table 1). Relative to controls, there wasnoevidence ofweight lossat any time in drug-treated mice. The total number of histologically examined tumors is listed in Table 2 for mice in specified age ranges in control and various test groups. Hepatomas.A high incidence of hepatomasoccurred in malesreceiving a total safrole dosageof 0.66 mg, 50 % basedon 12 mice alive at weaning,9 and 6.6 mg, 58% basedon 31 mice alive at weaninglo; this contrasts with an incidence of 5% in 114 solvent injected and uninjected male controls. For mice receiving 60 mg of Ionox, the incidence of hepatomas, 14% based on 29 malesalive at weaning,I1 is not significantly different from control values. Statistical significance remains essentially unchanged12when litter variation (Epstein et al., 1967a)is taken into account. Litters in a treatment group were relatively uniform in their hepatoma rates; every one of 7 safrole-treated litters with 3 or more male survivors at weaning showed at least 2 mice with hepatomas; of 20 control litters with sufficient male survivors, only 6 showed any mice with hepatomasand in no casemore than 1per litter; of the 6 litters dosed with 60 mg Ionox, 1 showed2 mice with hepatomas, 2 showed 1 such mouse, and 3 showed no hepatomas. No hepatomas occurred in males in other treatment groups or in any female mice; with 4 exceptions in safrole-treated groups, no hepatomas were manifest before sacrifice at termination ot p Continuity
corrected x2, 1 d.jI, = 18.8, p e 0.001.
‘0 x’ = 44.4, p -s 0.001. 1’ x* = 1.2, p > 0.2.
12The 3 X* values of 18.8, 44.4 and I .2, then become F values of 53.0, with 1 and 23 d.J, 109.9, with 1 and 26 d.f, and 2.3, with I and 27 d.jI, respectively.
INDUCED
0.11
1.1
-
0.11
1.1
11 110
Safrole
1
10
1
10
-
-
-
7
Uninjected controls
1
-
Alginic acid
IN SWISS ALBINO
MICE
-
20
2
2.2
0.22
--
-
14
-
20
2
2.2
0.22
-
-
21
60
6
11 110
6.6
0.66
-
-
Total
Drug dosageon specifieddays (mg)
TOXICITY
Solvent controls
Treatment
ACUTE
1
79
20
22 26
71
24
90
170
(7)
(2)
(2) (2)
(7)
(2)
(9)
(16)
Initial number of mice injected (No. litters)
BY NEONATAL
TABLE
1.6
1.5
1.6 1.7
1.6
1.3
1.6
1.6
1
9.8
9.5
-
7.9
8.9
7.8
0.9
21
Average weight c&dof mice at specified days
AND PERINATAL
OF
80
20
100 100
16
13
19
14
Mortality prior to weaning (%)
INJECTION
M F M F
M F M F -
M F
M F
Sex
8 8 7 9
12 9 31 29 0 0
36 37
78 69
g q k
68 31 31 11 9 19 24 -
g
50
At 49+ weeks
Number of survivors ---At weaning
FCKID ADDITIVES
E
@
“RA”
Ionox @330
Santoquin
Twem @60
1
10
10
10
10
1
1
1
-
5
5
10
1
1
-
20
2
20
2
10
2
-
20
2
20
2
10
2
-
-
-
11 110 -
2.2
2.2
1.1
1.1
60
6
60
6
10
30
6
11 110
6.6
TABLE
(5)
(2)
17 49
(6)
(2)
(2)
(4)
(5)
(2) (1)
(4)
63
22
28
53
57
22 10
49
l-continued
1.9
1.9
1.8
1.9
1.7
1.6
1.7
1.8 1.6
1.7
8.5
10.2
5.54
6.9
-
9.8
8.0
-
8.5
M F M F
2
4
0
M F M F
M F M F
5 3
-
100
74
100 100
M F -
2
9 8 24 23
15 6 29 32
25 31 9 5 0
25 23 0 0
4 5 13 19
8 4 21 26
-
21 29 5 3
16 22
M
F M F
60
F M F M F M F
M
L F M F M F
F M F M F M
77 6) 36 37 12 9 31 28 8 8 7 9 25 23 25 31 9 5 15 6 27 32 9 8 24 23
72 69 36 35 I2 9 31 28 8 8 7 9 22 23 25 31 9 5 14 6 26 32 8 8 22 23
2130
62 69 34 34 12 9 24 27 5 8 5 8 19 23 25 30 9 4 14 6 22 30 4 8 18 23
3140
8 17 23 23 29 6 4 9 5 22 28 4 8 14 21
55 68 32 33 12 9 23 25 5 :
4148
50 68 31 31 11 9 19 24 2 8 4 7 16 22 21 29 5 3 8 4 21 26 4 5 13 19
491
-
0’ 2 2 - -
4 2 4 1 3 1 5 4 0 0 0 0 4 2 0 4 4 1 0 2 2 1
Pq0.
381-S 410
il 0 0
ii 0
x 0 0 0 2 0 0
i 0 0 0 0 0
1 0 0 I
78 69 ii 37 I2 9 31 29 8 8 7 9 25 23 25 31 9 5 15 6 29 32 9 8 24 23
1120
8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0
4148 3 1 46 0 If I 0 3 0 0 0 0 3= 2 0 4 1c Id 0 2 2 1 1 0 2 2
491
autopsied (weeks)
z 0
1 0 0 0 0 0 0
0” 0 0 0 0 0 0 0 0 0 0 0 0
0 0
31-s 10 _---._ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4148
8 0 0 0 0 0
8 0 0 0 0 0 0 0 0 0
0 1 0 0 0 0 2h 0
49+
Multiple adenoma ( b 2)
of mice with tumor at each period
tumorsa
BY NEONATAL
Pulmonary
MICE
Solitary adenoma -_-~
M
410
I-
Number
SWISS ALBINO -
- --
-
IN
-
6
60
6
30
6
6.6
60
6
6.6
0.66
-
INDUCED
Number of mice, later autopsied, alive at the beginning of each weekly period
TUMORS
0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0. 0
.,..a I8
Adeno-
; 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 -
0 0 0 0 2a 0 2i .i
-_
49. t
-I-
--
-
4 0 2 0 6 0 18 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 -
INo.
INJECTIONS
OF FOOD
-
8 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 I 0 0 0 0 0 0 0
2.1-q 30 -
: 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
3l40
Solitary
0 0 0 0 I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4148
8 0
8 0 0 0 0 0 0 0 0 0 4 0 0
4 0 2b 0 28 0 7j 0 0
491
-
0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4II-Q 118
Multiple
of mice with tumor autopsied sacrificed at each period (weeks)
1Number
PERINATAL
or sacrificed
AND
2
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -
8h.Lk
0 0 0 0 3f 0
491
or
-
I
Lymphomas”
!I-4
0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
3140
:, 0 0 0 0 0
0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 1 0 0
48
Ol-
:Ff 1 0 0 0 0 0 0 0
1 0 0 0 0 0 2k 1 0 1 0 0 1e 1 0 3
491
of mice with tumor autopsied or sacrificed at each period (weeks)
E lumber
ADDITIVES
-
Ti-
-
pn.n
0” 0 0 0 0 10 0 0 0
1p 0
0 1) 0 0 0 0 0 0 0 0 0 0 0
Misc. 1L”“lOrS NO.
(rr) The Following instances of coexistent multiple tumors occurred, each tumor being indiwdually listed: (bl one with solitary pulmonary adenoma and solitary hepatoma; (c) one with solitary pulmonary adenoma and malignant lymphoma; (d) one with rolita-y pulmonary adenoma and malignant lymphoma; (c) one with solitary pulmonary adenoma and malignant lymphoma; (f) one with solitary pulmonary adenoma and multiple hepatoma; (a) two with pulmonary adenocarcinoma and solitary hepatoma; (h) two with multiple pulmonary adenoma and multiple hepatoma; (i) one with pulmonary adenocarcinoma with multiple hepatoma; (j) one with pulmonary adenocarcinoma and solitary hepatoma; (k) one with malignant lymphoma and multiple hepatoma. (I) mammary carcinoma at 51 weeks; (nz) mammary carcinoma at 48 weeks; (n) subcutaneous carcinosarcoma at 53 weeks; (0) ovary fibrosarcoma at 50 weeks; (p) thyroid adenoma at 50 weeks. (q) For each class of lesion, the first time interval shown in which the first tumor of that class arose in at least one of the treatment groups.
“RA”
lonox@
330
60
TWX”@
Santoquine
acid
Alginic
Solvent control Uninjected control Safrole
Treatment
r0td I dose E;e> (m3)
-
TABLE
h
z
; z;. E z
CARCINOGENICITY
OF FOOD
ADDITIVES
FOR
NEONATES
327
FIGS. 14. Lesions in male Swiss mice injected in infancy with a total dose of 6.6 mg of safrole. Figs. 2-4, from mice sacrificed at 1 year. All sections stained with hematoxylin and eosin. FIG. 1. Multiple hepatomas.
FIG. 2. Trabecular hepatoma with lower margin of normal liver cells. ,99
328
EPSTEIN
ET AL.
experiments. The hepatomas were solitary or more frequently multiple and generally ovoid and superficial; they varied in size from the minute to that which exceeded in size the normal liver (Fig. 1). Histologically, the tumors were classical mouse hepatomas (Stewart and Snell, 1957) with a well differentiated and trabecular pattern (Fig. 2); hyaline and vacuolar cytoplasmic inclusion bodies were generally conspicuous. Mitotic figures were rare, and no extrahepatic metastases were found. In safrole-treated animals, cellular and structural atypia were generally seen in males with tumor-free livers (Fig. 3); these atypia were characterized by mid-zonal cytoplasmic vacuolization, nuclear inclusions, focal necrosis and bile duct proliferation.
FIG. 3. Advanced hepatic atypia with marked pleomorphism, cytoplasmic vacuolization, and nuclear inclusion bodies. x 182.
Pulmonary tumors. The incidence of solitary pulmonary adenomas in males and females for uninjected and solvent controls was 1 1 %, 3 %, 5 %, and 2 %, respectively. With the exception of the 30 mg Santoquin male and female groups and 6 mg Ionox female groups, where numbers of animals involved were small, the incidence of solitary adenomas in other groups fell within control ranges. Multiple pulmonary adenomas were rare, however, occurring in 2/31, l/9, and l/69, male 6.6 mg safrole, male 30 mg Santoquin groups, and female solvent controls, respectively. Of the 7 pulmonary adenocarcinomas throughout all groups, 5 occurred in 43 safrole-treated males, one in 38 safrole-treated females, one in 25 Tween-treated males, none in 220 controls, and none in any other group. The adenocarcinomas were large, all exceeding 5 mm in diameter in contrast with adenomas, which were less than 2 mm diameter. Histologically,
CARCINOGENICITY
OF FOOD
ADDITIVES
FOR
NEONATES
329
the adenomas were alveologenic and well differentiated, although occasional structural irregularities were seen. The pulmonary adenocarcinomas were less well differentiated, with atypical cells and frequent mitotic figures and were locally invasive (Fig. 4); no metastases were found.
FIG. 4. Papillary pulmonary adenocarcinoma
in a mouse sacrificed at 41 weeks. x 182.
Lymphomus. In contrast with controls, an enhanced but low incidence of lymphocytic and reticular lymphomas was observed in males and females of the higher safrole dosage group, 3/31 and 5/29, respectively; females of both alginic acid dosage groups, l/8 and l/9; males and females of the Tween 60 dosage group, l/25 and l/23, respectively; females of the lower Santoquin dosage group, 4/3 1; males and females of the higher Santoquin dosage group, Z/9 and 2/5, respectively; males and females of the lower Ionox dosage group, l/l5 and 2/6, respectively. Miscellaneous tumors. Details of these and the individual disposition of all mice surviving weaning are listed in Table 3.
DISCUSSION The utility of neonatal systems for testing of proximate carcinogens such as polycyclics, which are carcinogenic per se as they do not require metabolic activation, is generally accepted. Questions have, however, been raised about nonproximate carcinogens such as the aromatic amines, which are only carcinogenic after they have been metabolically activated, whose activation may be reduced by the immaturity of hepatic microscomal enzyme function in the neonate. Such immaturity is probably not limiting, (NMU) as o-aminoazotoluene (AAT) (Nishizuka et al., 1965), nitrosomethylurea
acid
330
29
8 8
M
F M
F
M F M F M
0.66
6.6
24
23
F
instances lymphoma: adenoma;
8
60
32 9
M
F M
F
60
6
6 29
F M
6
23 25 31 9
5 I5
M
F
M
9
25
F M
30
6
6.6
F
9 31
M F
7
69 36 37 12
F
6
78
weaning
1
0 1 0 0
2 0
0 0
0 0 1 4
4 0 0 0 I 0
0 5
1 1
0 0
I
NO.
48MC
3SSPA
40ML,
26ML 34SPA+ 40SPA
30ML
28ML,
45ML
44ML,
II 3
6 4 3
0 8
2 7
4 I 0
1
0 7
1 5 5 0
27
NO.
during
and
losses
INITIALLY
(17A).
29U
26U
* 2,35U,
33, 39N,
42
12, 15,30U+PN, (3OL),3OU 32Ux2.33, 39U.41
41.46
x 2,
24, 35U z 2,
46U
36U+PN,
24U,
45U,
z 2, 33, (38L)
40,46 43 x 2
10Ux2,25,31U,35U+PN,38~2, 43u 28, (29A) ,‘34A), (4OL), 24. 35 x 3 41:(43L)r2
23.46 l9N, 3lU 36U, 43U
17. 18U. 34U x 2, 39, 41U, 45.46.47,
x 2,43U,
x 2,29
28U, 29U, 31U 43 17, 19U x 2,2lU, 45 (42A) 35U x 2,43,46 45
10 22IJ,
24U,
(3OL) Y 2, (39L), 14, (26L), (3lA),
0.U)
25U x 2,28,29U, (39L) x 2,4OU, 47u
2iUk3,(i2A),23U,25,
4. IS. ISU.
Individual week of death and associated lesion, if any
tumor
without
Deaths
INJECTED
of experiments
MICE
course
SWISS ALBINO
or sacrificed
U, 39MPA+U, U, 42ML
38ML,
41PAC. 47MH
41 ML
t
tumor
dying
IN
week ofdeath and tumors or lesions
3SML,
24SH +U, 45MH+U,
31SPA 45SH
34SPAtu
Individual associated
with
Mice
DEVELOPING
Deaths
LESIONS
3
2 6
I
2
1 I6
8
NO.
Mice
tumor
tumors
with
at conclusion
WITH
lesions at death: SC, subcutaneous
SC, SPA x 2
SPA%2
MC.
MH, solitary
N
Nx2
N N U
ux2
(A) (A)
u, u-1
Associated lesions, if any
r;nrrary carcinoma: carcinosarcoma; SH,
24 3 5 11
2 15
SPAx2 SH,SPAz2,SHtU, SHx2 SPA,Ov SPA
I8 21 22 4
19 2 7 4 7 I2
8 3
65 26 31 6
42
NO.
I 7
Th
or lesions
weeks)
Deaths without tumor and losses
(49-53
ADDITIVES~
of experiments
FOOD
ML,SPA+ML ML
SPAx4,MLx3 SPA+ML
ML,SPAx2,
SPAtML.SPAr2,PAC
ML
PAC+SHx2,MHX2, SPA+MH SPA SHx6,MHr4,ML, MPA+MHx2,PAC+MH, PAC+SH,ML+MH SPAx3,PAC,ML
SPA,MPA.MC SPAx3,SPA+SH,SH
SH+U,SHx3,SPAx2. MLtlJ,SPA
Associated
Deaths
sacrificed
AS NEONATES
where no autopsy was possible due to autolysis (A) or accidental loss (L). Code for tumors or other MPA, multiple pulmonary adenoma; Ov. ovary fibrosarcoma; PAC. pulmonary adenocarcinoma; Th, thyroid adenoma; PN, pyelonephritis; U, obstructive uropathy.
OTHER
Sexand No. at
AND
M
60
-
Total dose (me)
TUMORS,
~Parenthescs are used to indicate multiple hepatoma; ML, malignant hepatoma; SPA, solitary pulmonary
"RA"
Ionox@
conrrols
controls
Tween@'60
Alginic
Safrole
Uninjected
Solvent
Treatment
SURVIVAL,
TABLE
3 i .P
6
22
CARCINOGENICITY
OF FOOD
ADDITIVES
FOR NEONATES
331
(Terracini et al., 1967), and acetylaminofluorene (AAF) (Epstein, unpublished data) are carcinogenic following single administration only to l-day-old mice. It must be stressed that there are no reports in the available literature of “false negatives,” based on comparisons with conventional adult systems, after repeated administration of nonproximate carcinogens during the first month of life. In view of the negative results reported for dimethylnitrosamine (Terracini et al., 1966), urethane (Trainin et al.. 1964), and AAF (Walters et al., 1967), single administration of nonproximate carcinogens on day 1 of life should not be regarded as an adequate test procedure. While there have been relatively few experiments designed specifically for simultaneous comparison of dose-response relationships for carcinogens in neonatal and adult systems (Toth, 1968), there is, however, sufficient composite and nonconcurrent data to consider this question. Single subcutaneous injection of 0.4 mg AAT (Nishizuka et al., 1965), and 0.05 mg NMU (Terracini and Stramignoni, 1967), and multiple injections totaling0.6 mg Aminobiphenyl (ABP) (Gorrod et al., 1968), and 3 mg griseofulvin (GF) (Epstein et al.. 1967a), and feeding of a total dose of 7 mg AAF (Klein, 1959b), were all carcinogenic to infant mice. In adult rodents, however, many of these compounds were carcinogenic only after prolonged feeding at high dietary levels. With GF, for instance, feeding of the adult mouse for about 9 months at the 1% dietary level was required to produce hepatomas (Hurst and Paget, 1963). On a total dosage basis, the infant is thus about 4500-fold more sensitive than the adult mouse to the carcinogenic effects of GF; on an mg/kg basis, however, the corresponding sensitivity is reduced to the order of 1100. The enhanced sensitivity of infant rodents is of practical importance, Even relatively large hazards due to weak carcinogens, for example, affecting l/ 10,000 of the population, would probably escape detection by conventional carcinogenicity tests involving test groups of even as many as 50 adult animals per given dosage; assuming rats and man have the same sensitivity to weak carcinogens, a test group of 10,000 rats would be required to induce tumors in 1 animal only. Thus, to demonstrate weak carcinogens in the environment, it is essential that the most sensitive test system available to be used and that the factor of comparability of route and schedule of drug administration te subordinated to the requirement of sensitivity. Once a hazard has been clearly established, the quantitative relevance of the experimental data to the human situation must be considered before limits can be reasonably proposed, and only at this stage does it become appropriate to weight factors such as age of test animal and route of drug administration. To use such factors in a limiting sense, and to insist on precise comparability between test systems and human exposure before the problem of hazard is established, will effectively preclude all possibility of detecting weak environmental carcinogens; such limitations would also challenge the human implications of data on experimental tobacco carcinogenesis. There would not appear to be any advantage in holding mice for more than 1 year following treatment; no instances of “false negatives” have been reported after sacrifice of mice at 1 year of age under these conditions. High incidences of hepatomas have been recorded in mice 1 year after neonatal administration of a wide variety of carcinogens, including AAF, ABP, AAT, GF, and maleic hydrazide (see above for references); multiple pulmonary adenomas and thymic lymphomas develop more rapidly still. The relatively high yield of remote tumors, such as hepatomas, pulmonary adenomas, and thymic lymphomas, following single or repeated subcutaneous injection of low
332
EPSTEIN ETAL.
doses of a wide variety of carcinogens in neonatal mice is of particular interest. Production of remote tumors under these conditions obviously avoids the issue of “Oppenheimer” effects (Oppenheimer et al., 1956), which have been incriminated in suggestions as to the nonspecificity of some local sarcomas after repeated subcutaneous injection of certain food additives and other drugs in adult rats (Grass0 et al., 1966), and also minimizes the importance of the variable relating to route of drug administration. The data reported here on carcinogenicity testing of food additives in infant mice accords with the above discussion of the reported literature with regard to the practical utility of neonatal systems. The hepatocarcinogenicity of safrole after its restricted and intermittent parenteral administration to infant mice is of particular interest. Safrole also produced pulmonary tumors, solitary and multiple pulmonary adenomas and adenocarcinomas, and lymphomas in neonates in contrast with the adult rat, in which carcinogenic effects are restricted to the liver (Hornburger et al., 1962; Hagen et al., 1965). The production of pulmonary tumors, particularly adenocarcinomas, by safrole has not been previously described and is noteworthy, especially in view of reports (Fishbein et al., 1968) of acute pulmonary congestion and edema and of selective pulmonary uptake of safrole, and other methylenedioxyphenyl compounds, after parenteral administration to rats. It is interesting to contrast the low dosage of safrole, 0.66 mg, producing a 50% incidence of hepatomas, as well as some lymphomas, pulmonary adenomas and adenocarcinomas, after injection of infant male mice, with the high dosages, 1.O % dietary levels for 1 year, equivalent to about 55 g of safrole, necessary to produce hepatomas in adult rats (Hornburger et al., 1961; Long et al., 1963); this is approximately 83,000 times the dose found to be carcinogenic after injection of the infant mouse. If adjustment is made for body weight, this extreme factor is reduced to the order of 1100. Quite apart from the putatively critical age factor, such quantitative comparisons obviously do not reflect other factors, such as strain and route of drug administration, which might partially account for the observed differences in sensitivity of neonatal and adult rodents to carcinogens. The effects of Ionox 330 in relation to the production of an apparently enhanced incidence of solitary pulmonary adenomas, hepatomas, and lymphomas should be noted; however, with the exception of the 4 hepatomas developing in 29 males in the 60-mg dosage group, these incidences are based on small numbers of animals. For Santoquin, the increased and dose-related yield of solitary pulmonary adenomas and lymphomas in both sexesis of interest and is consistent with similar findings in adult rats after feeding of the related compound Flectol H (Hodge et al., 1966). However, an increase in solitary pulmonary adenomas is an unreliable carcinogenic index, especially when unaccompanied, as obtains here in the Ionox and Santoquin groups, by multiple adenomas in other animals under test. In view of these findings, it would appear appropriate to repeat testing of both these compounds, particularly Santoquin, with larger numbers of neonatal mice, in addition to conventional adult rat systems. REFERENCES BOIATO, L., MIRVISH, S. S., and BERENBLUM, I. (1966). The carcinogenic action and metabolism of N-hydroxyurethane in newborn mice. Intern. J. Cancer 1,265269. British Food Standards Committee Report, Emulsifying and Stabilizing Agents. Her Majesty’s
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CARCINOGENICITY OF FOOD ADDITIVES FOR NEONATES
333
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DELLA
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TERRACINI,