209
Mutation Research, 257 (1991) 209-227 © 1991 Elsevier Science Publishers B.V. 0165-1110/91/$03.50 ADONIS 0165111091000596
MUTREV 00071
Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 39 chemicals tested for carcinogenicity by the U.S. National Toxicology Program Raymond W. Tennant a and John Ashby b a National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.) and b ICI Central Toxicology Laboratory, Alderley Park, Cheshire (Great Britain)
(Accepted 9 October 1990)
Keywords: Rodent carcinogenicity bioassay; DNA reactivity; Classification according to chemical structure/mutagenicity
Summary This paper is an extension of compilations published previously in this journal. (Ashby and Tennant, 1988; Ashby et al., 1989). A s u m m a r y of the rodent carcinogenicity bioassay data on a further 39 chemicals tested by the U.S. National Toxicology Program (NTP) is presented. An evaluation of each chemical for structural alerts to DNA-reactivity is also provided, together with a s u m m a r y of its mutagenicity to Salmonella. Chemicals with an aliphatic nitro group ( - C - N O / ) have been added to the composite structure of DNA-reactive sub-groups. The 39 chemicals were numbered and evaluated as an extension of the earlier analysis of 264 N T P chemicals. The activity patterns and conclusions derived from the earlier studies are followed by these 39 chemicals, albeit a detailed analysis of the total database of 301 chemicals is reserved for the succeeding paper.
In two earlier papers we have summarized the rodent carcinogenicity data reported by the U.S. National Toxicology Program (NTP) for 264 chemicals (Ashby and Tennant, 1988; Ashby et al., 1989). The present paper describes the test data for a further 39 chemicals, and this brings the total number reviewed to 301 (2 chemicals have been tested on two separate occasions). The criteria for selection and presentation of data are essentially as described in the two earlier publications. All of the studies considered here were peer re-
Correspondence: Dr. J. Ashby, ICI Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ (Great Britain).
viewed in 1989-1990 and are due to be pubfished during 1990 or early 1991. A detailed analysis of the data for the present 39 chemicals was avoided because a review of the complete database of 301 chemicals is presented in the accompanying paper of this special issue.
Methods Criteria for selecting studies for incorporation in this review were as described earlier (Ashby et al., 1989). In the case of the two derivatives of benzidine ([268] and [269]) only rat studies were available. However, because these were potent and multi-site genotoxic carcinogens they were entered
210
Table I SUMMARY OF CARCINOGENICITY, MUTAGENICITY AND CHEMICAL DATA FOR 39 COMPOUNDS TESTED FOR CARCINOGENICITY IN F344 RATS AND B6C3F1 MICE BY THE U.S. NATIONAL TOXICOLOGY PROGRAM. Criteria for selection of the data displayed below are described in the text. The carcinogenicity bioassay tumor incidence fig u res shown reflect the conclusions of the Peer Review Panel. Not all categories of tumor are displayed for each chemical. For example, if a positive response is recorded both for liver nodules and liver carcinomas, the latter are generally the ones shown. Likewise, only the tumor data that led to the classification of an agent as carcinogenic are displayed, even though equivocal evidence of carcinogenicity may have been recorded for another sex or species of test animal. In most cases the numerical tumor incidences displayed in the Table were those selected for discussion by the Peer Review Panel and the bioassay report authors. This means that tumor incidences
CHEMICALS ASSOCIATED CARCINOGENIC STATUS
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Chemical Name
Salmonella assay response (Zeiger)l-
Level of effect across Route~ species, (F, G, I, sexes and o~M (~M tissues(A-E){} SK, orW)
NTP Technical Report Conclusions o~R
?R
NE
SE
CE
CE
A
G
+
SE
EE
EE
CE
A
/
+
+
CE
EE
SE
NE
A
G
+
+
CE
CE
NT
NT
A
W
[265] 271-89-6 370(1989)
Benzofuran
[266] 74-96-4 363(1989)
CH3CH2Br
Bromoethane [267] 20265-96-7 351 (1989)
p-Chloroaniline .HCI
NH 2
C[
[268] 20325-40-0 372(1990)
H2N " K ~ ~ ~ CH30
NH2 OCH 3
3,3'-Dimethoxybenzidine .2HCI
211
adjusted for mortality are generally employed. In some cases the incidence of carcinomas in a tissue is shown for male animals and of carcinomas plus adenomas for female animals (for example) - again, this is consistent with the decision of the authors of the bioassay report. This Table therefore only provides information on the level of carcinogenicity for each agent. In instances where the carcinogenicity of a specific chemical is of interest, it is important that reference to the original bioassay report be made. For a description of the symbols used in this Table, see the legend at its end. The assistance provided by John Reece in the construction of this table is gratefully acknowledged.
WITH CARCINOGENICITY CARCINOGENICITY DATA Maximum dose
Duration of study (weeks)
Rats
Mice
Rats/Mice
120
240
103
0.04
0.04
Tumor site •
0
2
8
8 24 4 20
104/103
103
MG SK ZG PG OC I/C L* MT
B CG U
71 77 27 40
2
46
45
4 4
18 19
10 30
C L M H
0
L M H 0 2 6 76 27 29 31 53
C
L
M
H
22 43 40 42 8 8 2 20
C L M H
84
62 77 28 23
C L M H 0 12 2 2 0 0 8 2 17 49 36 43
c
SP AG L* CS
9 C ] L }H
o"
DILI
L* S* LU
B
0.033
9
DILI
K
LU U
30
o"
CILI
L**
AG
18
Tumor data identified in Summary of NTP Technical Report Rats (% TBA)~ Mice (% TBA)
0 0 27 2 0 2 3 0
29 22 28 18 2 9 2 5
37 33 45 13 11 9 9 4
37 50 49 18 13 13 10 2
C L M H 2 4 1933 0 9 4 3 2 27 28 27 3 4 8 8 0 2 1 5 0 2 0 5
12616575 0 9 3 3
SEE TEXT
8
11 56
2.]2
Table1
(Continued) CARCINOGENIC STATUS
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Salmonella assay response (Zeiger)~
Chemical Name ÷
[269] 612-82-8
÷
Level of Route~ NTP Technical i effect across Report Conclusions species, (F, G, I, sexes and W) I oTR ~R oTM ~M~tissues(A-E)§ SK, or CE
CE
NT
NT
A
W
SE
NE
CE
SE
A
G
CE
CE
CE
CE
A
G
CE
NE
P
P
A
G G
390(1990)
H2N ~
N
H
2
OH 3
CH3
3,3'-Dimethylbenzidine .2HCI
[270] 98-01-1 382(1990)
~ C H O Furfural +
[271] 556-52-5 374(1990)
÷
CHJt,.,%CH _ CH2OH
Glycido] [272] 67-72-1 361 (1989) 68(1978)
CI
CI
I
[
C I - - C - - C - - CI
Hexachloroethane
I
I
CI
CI
213
CARCINOGENICITY DATA Maximum dose
Rats
Mice
0.015
Duration of study (weeks) Rats/Mice
Tumor data identified in Summary of NTP Technical Report
64 SK ZG PG L* OC I/C LU MT CG MG
60
175
Rats (% TBA)~
Tumor site~
Mice (% TBA)
9
c¢
cl'l"
clLI"
C 0 2 3 0 0 0 2 0
C L M H 0 7 12 20 0 13 43 70
L M H 4 23 45 7 43 60 9 8 15 0 47 55 0 5 8 0 8 25 0 11 10 0 4 7
0 0 0 2
0 7 2 2
0 0
31 56 54 2 4 10
9 7 1222 8 7 4 7
BD L*
0
0
L
M
H
50
103
TV
6
68
78
I
6
16
14
128
68
74
B
0
10
12
0
8
8
2
6
14
S*
2
4
12
0
8
22
SK
0
10
8
ZG
2
6
12
2
8
12
OC
CG HS HG
160 1179
103 78
L
M
H
2
6
10 16
32 44 35 64
MG
TG
C
4
L**
75
cl'l"
SEE TEXT
C
103
9
o1'1"
10
18
0
2
6
26
28
40
2
4
20
0
0
8
17
29
50
4
12
30
0
0
4
40
24
9
26
U
0
6
6
SB
0
6
18
10
40
31
L*
48
62
70
LU
26
22
42
15
30
63
K L*
2
4
14
214 Table 1
(Continued)
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Salmonella assay response (Zeiger)l-
Chemical Name [273] 123-31-9 366(1989)
-
-
-
CARCINOGENIC STATUS Level of NTP Technical effect across Report Conclusions species, o"R
~R d'M
Route~
(F, G, I,
sexes and SK, or W) ~M'tissues(A-E)§
SE
SE
NE
SE
A
G
+
SE
NE
NE
SE
A
G
--
NT
NT
CE
CE
A
F
--
EE
SE
SE
NE
A
G
-
OH
OH Hydroquinone [274]
+
5634-39 9
340(1989) CH 2 -- C H - - CH21 I
I
OH
OH
Iodinated Glycerol [275] 87-86-5 349(1989)
OH
CI Pentachlorophenol [276] 50-33-9 367(1990)
~
Q~
N/N~c//O
I
[
C -II O
C -I H
CH 2 -- CH 2 -- CH 2 -- CH3
Phenylbutazone [277] 509-14-8 386 (1990)
4-
4-
CE
CE
CE
CE
A
/
+
+
CE
CE
CE
CE
A
SK
NO2
I I
O2N - - C - - N O 2 NO2 Tetranitromethane [278] 106-87-6 362(1989)
0 ~
S0%
-CH - -
CH 2
4-Vinyl-l-cyclohexene Diepoxide
215
CARCINOGENICITY DATA Maximum dose
Rats 50
250
0.02
Mice 100
250
0.06
Duration of study (weeks) Rats/Mice 103
103
Tumor site ~
Tumor data identified in Summary of NTP Technical Report Rats (% TBA) ° Mice (% TBA)
c¢
K HS L*
0
HS TG PTG HG
28 0
7
300
clLIH
58 10
27
0.0005
0.0002
103
C L M H 17 40 44 69 3 8 44 92
30 mg/ animal
10 mg/ animal
105/103
SEE TEXT
SK 0 LU
0
2
0
5
29
24
21 12
33 20
52 28
48 2
K L*
LU
?
c] LI H
40
103
103
o"
C I L IH
15 16
L* AG CS
100
9
CILIH
0
66
92
0
44
66
72
0
32
C 3 0 0
L 8 4 2
M H 12 65 4 78 6 16
4 32
28
62
100 I 2 4
52
92
68
C L M H 0 28 78 84
8
48
98
C L M H 0 12 74 82 2 0 35 36 8 18 22 14
2]6 Table 1
(Continued)
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Salmonella assay response
Chemical Name
(Zeiger)t
[279] 924-42-5 352(1989)
--
CARCINOGENIC STATUS Level of Route~ NTP Technical effect across Report Conclusions species, (F, G, I, sexes and SK, or W) o~R ~ a o7M ~ M tissues(A-E)§ NE
NE
CE
CE
B
CE
CE
EE
NE
B
G
O II @ H 2 = CH -- C - - NH-- CH2OH
N-Methylolacrylamide 389-08-2 368(1989) [280]
CH 2 CH 3 I
H
3
C
Nalidixic Acid
~ II
C-
O
0
OH
NE
[281] 100-52-7 378(1990)
NE
SE
SE
C
G
CHO
0
Benzaldehyde [282]
4-
75-25-2
SE
CE
NE
NE
C
CE
CE
EE
EE
C
EE
NE
SE
EE
D
350(1989) H I
Br -- C -- Br I
Br Tribromomethane (bromoform) [283]
115-96-8
391 (1990) O=P(OCH2CH2CI)3
Tris (2-chloroethyl)phosphate 106-92-3 376(1990) [284]
0 1% C H 2,.,. C H -CH2OCH2CH = CH2
AIlyl Glycidyl Ether
4-
+
G
217
CARCINOGENICITY DATA Maximum dose
Duration of study (weeks)
Tumor site"
Rats
Mice
Rats/Mice
12
50
103
HG L** LU O
0.4
0.4
103
PG CG
Tumor data identified in Summary of NTP Technical Report Rats (% TBA)~ Mice (% TBA)
o"
?
01,]H
6
39
o"
c],]H
?
clL] 2 24 10
29 34 20
58 52 36
11 6 12 0
18 8 16 11
42 35 27 11
2
4
10
0
10
12
43 11
33
34
400
600
103
S**
200
200
103
I/C
0
0
6
0
2
16
88
350
103
K
2
10
48
0
4
10
0.001
0.001
103
N
0
0
6
218
Table 1
(Continued)
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
CARCINOGENIC STATUS Level of NTP Technical effect across Report Conclusions species,
Route ~
(F, G, I,
sexes and SK, or W) (~R o~M (~M tissues(A-E)§
(Zeiger)t
(::/'R
+
÷
EE
EE
IS
CE
D
+
--
SE
NE
NE
EE
D
EE
NE
NE
SE
D
CE
NE
NE
NE
D
SE
NE
NE
NE
D
NE
EE
NE
NE
E
Chemical Name
[285] 75-00-3
Salmonella assay response
I
346(1989)
CH 3 CH2CI
Chloroethane
[286] 121-69-7 360(1989)
H3C ~ 1 ,p CH3
N,N-Dimethylaniline [287] 54-31-9 356(1989)
COOH
u
NH2SO2- -x~ Cl Furosemide [288] 5989-27-5 347(1990)
CH 3
H2C~ C "-OH 3 d- Limonene
[289] 98-85-1 369(1989)
OH
I HC--CH 3
© c~-Methylbenzyl Alcohol
+
[290] 532-27-4 379(1990)
COCH2CI
2-Chloroacetophenone
--
G
219
CARCINOGENICITY DATA Maximum dose
Duration of study (weeks)
Tumor site~
Tumor data identified in Summary of NTP Technical Report Rats (% TBA) • Mice (% TBA)
o"
?
o"
C IL IH
C IL IH
C IL IH
Rats
Mice
Rats/Mice
1.5
1.5
102/100
30
30
103
SP
0.07
0.14
103/104
MG
600
1000
103
K
0
16
22
750
750
103
K
0
4
10
2 mg/m 3
4 mg/m 3
103
MG
U
0
0
? C I 0
L I H 86
8
10
24
38
46
220 Table 1
(Continued) CARCINOGENIC STATUS
[Code No} CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Salmonella assay response (Zeiger)t
Chemical Name
NTP Technical Report Conclusions o ~R
~R CM
Level of effect across species,
~M
sexes and tissues(A-E)§
Route' (F,G,I, SK, or W)
NE
NE
EE
NE
E
G
EE
EE
NE
NE
E
F
--
NE
NE
EE
NE
E
+
--
EE
EE
NE
NE
E
+
--
EE
EE
NE
NE
E
EE
NE
NE
NE
[291] 828-00-2 354(1989) H3C'h~OTCH3
,yo Q--C--CH 3 II O
Dimethoxane
[292] 147-24-0 355(1989) H L /OH3 ~ C--O--CH2--CH2-- N\ CH3
Diphenhydramine .HCI [293] 58-93-5 357(1989)
O_....~O
NH2SO2~
S-.~NH
H Hydrochlorothiazide [294] 78-11-5 365(1989) CH2ONO2
I O2NOCH 2 -- C -- CH2ONO 2 /
CH2ONO 2 Pentaerythritol Tetranitrate [295] 9 8 9 - 3 8 - 8 364(1989)
H3C
NC H
H3C
NHC2Hs
Rhodamine 6G. HCI [296] 7681-49-4 393(1990)
NaF
Sodium fluoride
W
221
CARCINOGENICITY DATA Maximum dose
Duration of study (weeks)
Tumor
Rats
Mice
Rats/Mice
site"
250
500
103
S*
0.0625
0.0313
103/105
2
0
8
2
12
10
106/104
L*
5.0
5.0
104/103
ZG
0.0175
0.0175
103
~
iS AG
103 OST
46
0
7
2
4
o"
oiL1,
B
0.5
0.2
o~
cl,l,
LU PTG
0.2
0.025
Tumor data identified in Summary of NTP Technical Report Rats (% TBA)° Mice (% TBA)
7
0
52
3
C
L
M
H
0
0
2
4
6
ol lH
4
6
16
15
20
42
70
9
16 6
(~
olLI,
20
222
Table 1
(Continued) CHEMICALS
ASSOCIATED
WITH NON-CARCINOGENICITY CARCINOGENIC STATUS
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Salmonella assay response (Zeiger)t
Chemical Name
NTP Technical Report Conclusions oTR NE
[297] 60-13-9 387(1990)
~Rro"M ~U t NE NE NE
Route¢
Duration of study (weeks)
(F,G,I. SK, orW)
Rats/Mice
F
103
I
CH 3
I ~
~[. ,-CH2-CH-NH2
D-L- Amphetamine Sulfate
[298] 2698-41-1
NE
NE
NE
NE
NE
NE
NE
NE
103
377(1990)
[~
/CN CH --C ~'CN CI
o-Chlorobenzalmelanonitrile
[299]
120 83-2
353(1989)
F
103
OH CI
2.4 Dichlorophenol
CI
[300] 55-31-2 380(1990)
103
NE NE (IS) (IS)
NE NE (IS) (iS)
NE
NE
NE
NE
G
103
NE
NE
NE
NE
I
104
[
CHOHCH2NHCH 3
L- Epinephrine Hydrochloride
[301] 108 30-5 373(1990)
//O OH 2 - - C CH2--C O
Succinic Anhydride
[302]
108 88-3 371(1989)
Toluene
223
CARCINOGENIC STATUS
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold) Chemical Name
Structural alert
Salmonella assay response (Zeiger)1"
[3031
NTP Technical Report Conclusions o"R
~R
NE
NE
o'~M ( ~ U NE
NE
Route ~
Duration of study (weeks)
(F,G,I, SK, or W)
Rats/Mice
/
103
375(1990)
CH - CH 2 Vinyl Toluenes (meta/para 70:30)
1 ¶
Salmonella mutation data provided by Dr. E. Zeiger of the NTP, and presented in the appropriate technical report. Questionable responses have been regarded as positive. G = gavage dosing (mg/Kg); I = exposure via inhalation (%); F = exposure via food (%); SK= skin painting; W= in drinking water
(%).
e * §
Abbreviations for tumor site are as follows: AG = adrenal gland; B = brain; BD = bile duct; CG = clitoral gland; CS = circulatory system; HG = Harderian gland; HS = hematopoietic system; I/C = intestine/colon; IS = integumentary system; K = kidney; L = liver carcinomas; L* = liver carcinomas + adenomas; L** = liver adenomas; LU = lung; MG = mammary gland; MT = mesothelial tissue; N = nose; O = ovary; OC = oral cavity; OST = osteosarcoma in bone; PG = preputial gland; PTG = pituitary gland; S = stomach carcinomas; S* = stomach carcinomas + adenomas; S** = stomach adenomas (papilloma); SB = subcutaneous tissue; SK = skin; SP = spleen; TG = thyroid gland; TV = tunica vaginalis; U = uterus; ZG = Zymbal's gland. % TBA = % tumor bearing animals for each site of carcinogenesis. The letter C represents the concurrent control group, L the low test group and H the high dose test group. In some studies reference to a medium dose (M) is made. P: positive for carcinogenicity; IS: inadequate study; CE: clear evidence of carcinogenicity; NT: not tested; EE: equivocal evidence; NE: no evidence; SE: some evidence. Level of carcinogenic effect categorized A-F as follows: A = both species affected; B = single species with 2 or more tissues affected; C = single species with a single tissue affected; D = single sex of a single species with a single tissue affected; E = adequate study providing equivocal evidence of carcinogenicity.
into the group A category which implies carcinogenic activity in the mouse. Two other deviations were made from the earlier policy of considering only agents adequately tested in both rats and mice (Ashby and Tennant, 1988). First, epinephrine [300] was found to be noncarcinogenic in studies that subsequently were considered inadequate because a higher dose level could probably have been administered. Epinephrine was nonetheless retained in the database because it illustrates the unusually stringent standards applied to these bioassays. In the overall calculations presented in the subsequent paper, this agent is classed as a non-carcinogen. Second, pentachlorophenol [275] was only tested in mice, but in two different states of purity. Both samples of pentachlorophenol induced tumors in the adrenal gland and liver, and of particular interest, the circulatory system. In the discussion of those data in the technical report (TR 349) the probable
contribution of chlorinated dibenzodioxins to the carcinogenicity of this agent was discussed, and all such halogenated biphenyls are carcinogenic to both rat and mouse liver (see compounds [26], [42], [47], and [78] in Ashby et al., 1988). Thus, in order not to lose the unexpected tumors of the circulatory system from the database, pentachlorophenol [275] was included in the present compilation and assumed also to be a rat-liver carcinogen in the following paper, p-Chloroaniline [267] was tested by gavage as the hydrochloride in the present compilation, but had earlier been tested as the free amine in diet ([120] in Ashby et al., 1988). The structural alerts were based on the composite structure presented in revised form in Ashby et al. (1989). The Salmonella mutation data were from the N T P studies conducted by Zeiger and his colleagues (e.g. Zeiger et al., 1988) and as reported in the appropriate N T P technical report. The tumor site codes and the source of the numerical
224
Results
tumor data presented in Table 1 are described in the Introduction and Legend to the Table. Studies involved use of both sexes of Fischer 344 (F344) rats and B6C3F1 mice, except in the case of the two benzidine derivatives (rat only) and pentachlorophenol (mouse only). The 39 chemicals were stratified according to the level of carcinogenic activity as defined previously: (A) Trans-species carcinogens; (B) Carcinogens affecting 2 or more sites of a single species; (C) Carcinogens affecting a single tissue of both sexes of a single species; (D) Carcinogens whose activity is species/ sex/site specific; (E) Agents with equivocal evidence of carcinogenicity; (F) Agents non-carcinogenic to both test species.
A summary of the bioassay data abstracted from the N T P technical reports for the 39 test chemicals is shown in Table 1. Twenty-five carcinogens, 7 agents equivocal for carcinogenicity, and 7 non-carcinogens were defined by those studies. Much as observed in the earlier analyses, the proportion of mutagens decreased according to the level of carcinogenicity of the test agents. 44% of the carcinogens were both devoid of structural alerts and were negative in the Salmonella assay. The 7 new non-carcinogens were both nonmutagenic to Salmonella and non-alerting in chemical structure, a figure at variance with the earlier analysis where --30% of non-carcinogens were mutagenic to Salmonella (note small sample size and extended discussion of this point in the succeeding paper). In the previous analysis (Ashby et al., 1989),
(a) OCH 3
I I
O---S--O
(b) NO 2
OH 2
O~(d)
,c, (r)
__/ / O=N
(p)
,
N--CH 2 -
CH 3
'~/---~/ (h)
H
(q)
CH2OH '
I
(g) X\
//L----N (e)
~CH9
//
CHO
CH 3
/
I
H2N--< r ~__L__CH2__CH__ NH__N\ ( )
\\
NH2
"X(
#=,,
//~--N---N--(~\
O - - •- - - - ( \
CH (s)
~ ' ~
?--CH 2
~
OH 2 I
CICH2
0
(n)
I O~'C~NH 2 (o)
(i) CH---CH--CI
(j)
CH3
N(CH2CH2CI)2
I CH--Cm--C" NH (m)
x C H - - C H/
I NO 2 (u)
(t)
\
(') CH --OH 2
I
I
o//C
O
Halogenated methanes
C(X)4 x= H,F,CI,Br,l inany combination
Fig. 1. Revision of the multiple electrophile model chemical upon which structural alerts in the present papers are based. The new sub-structure is labelled (u) and represents an aliphatic nitro group, as present in tetranitromethane [277]. All other substructures ( a - b ) are as described in detail in A s h b y et al. (1989).
225 3-nitropropionic acid was classed as structurally alerting because of the - C H 2 N O 2 substructure, and it was also mutagenic to Salmonella. The basis for this structural alert was the carcinogenicity of 2-nitropropane (Fiala et al., 1987) a compound not entered into the present analysis because it had not been tested by the NTP. In the present series of chemicals, tetranitromethane [277] again contains an aliphatic nitro group, is mutagenic to Salmonella, and is a potent lung carcinogen. Consequently, the C - N O 2 substructure has been entered into the multiple electrophile megastructure (Fig. 1), even though the mechanism of its genotoxicity is not clear. Note that pentaerythrityl tetranitrate [294] is structurally alerted because of the potential to lose nitrite ion (NO~-), but this chemical was non-mutagenic to Salmonella. The structural alert for dimethoxane [291] was based on the potential for acetaldehyde formation. Only the terminal Michael-reactive carbon atom of N-methylolacrylamide [279] was alerted. Discussion
Consideration of overall trends, such as the proportion of carcinogens that are mutagenic to Salmonella, have been avoided for the present 39 chemicals for statistical reasons; such trends and correlations are evaluated for the whole database of 301 chemicals in the accompanying paper. Several interesting carcinogens are present among the current 39 chemicals. First, three chemicals, 3,3'-dimethoxybenzidine [268], 3,3'-dimethylbenzidine [269] and glycidol [271], are probably the most extensive of carcinogens yet described by the NTP. In each case, these genotoxins are active in most of the tissues associated earlier with genotoxic carcinogenesis (Ashby and Tennant, 1989). Such carcinogens are representative of the reference carcinogens known in the early 1970's and which stimulated the N T P bioassay program. Likewise, 4-vinyl-l-cyclohexene diepoxide [278] and tetranitromethane [277] are classical potent genotoxic carcinogens of immediate relevance to exposed humans. Four chemicals induced renal tumors only in the male rat, and each was non-mutagenic to Salmonella and non-alerting in chemical structure (hexachloroethane [272], hydroquinone [273], d-
limonene [288] and a-methylbenzyl alcohol [289]). These agents may induce tumors by a-2-/~-globulin retention in the kidneys (Goldsworthy et al., 1988), and studies to evaluate this could be usefully complemented by evaluation of the putative nongenotoxin phenylbutazone [276], which affects only the female rat kidney, and tris(2-chloroethyl)phosphate [283] that is a specific renal carcinogen to both male and female rats. Three chemicals were notable for the limited extent of their carcinogenicity. Allyl glycidyl ether [284] and chloroethane [285] are overt alkylating agents and potent mutagens to Salmonella, yet each is only selectively and minimally carcinogenic to rodents when tested by inhalation. The first induced a small increase in nasal tumors in male mice ( 3 / 5 0 animals affected at the high dose level) and the second, chloroethane, induced uterine tumors selectively in female mice. The suggestion that these natural electrophiles are rapidly detoxified by reaction with non-critical macromolecules may explain the low activity of the allyl compound to the nasal epithelium, but this cannot elucidate the selective carcinogenic activity of chloroethane to the uterus upon inhalation. Both compounds would normally have been expected to induce tumors of the respiratory tract in all four test groups. Occurrences such as these emphasize our current inability to predict carcinogenesis accurately. The third chemical of interest is 2-chloroacetophenone [290]. This has a chemically reactive chlorine atom which led to the structural alert. However, it was non-mutagenic to Salmonella and gave only equivocal evidence of carcinogenicity to the female rat mammary gland. Benzofuran [265] and N-methylolacrylamide [279] are interesting on several counts. First, both are carcinogenic to tissues of the mouse associated with genotoxins, but are inactive in the rat, a very rare situation, as discussed in the following paper. Second, both are non-mutagenic to Salmonella. The acrylamide derivative is structurally alerted because of its inherent Michael reactivity, a property that can lead to chromosomal damage in the absence of mutagenicity to Salmonella (see Ashby et al., 1989). Nalidixic acid [280] induced a most unusual tumor response suggestive of a non-genotoxic hormonal mechanism of carcinogenicity (male rat
226
Male Rats
% TBA 20 -
o~
/
Female Rats
% TBA
o
PG
20
A \
\
\
x
~
10-
10
D
° ~
-A
HS
x
"A . . . . . . . . . Control
CG
\ a.
•
o
Low
,x PTG High
[] MG Control
Low
High
Fig. 2. T u m o r d a t a f o r n a l i d i x i c a c i d [280]. I n a d d i t i o n to t h e n a m e d sites o f c a r c i n o g e n e s i s [clitoral g l a n d in f e m a l e r a t s ( C G ) a n d
preputial glands in male rats (PG)] several statistically significant decreases in tumor incidences were noted in the technical report (TR 368) and are also shown here. Such a profile of tumor incidences is suggestiveof a nongenotoxic mechanism of carcinogenicity, perhaps involving hormonal disturbance on protracted dosing of this Salmonella non-mutagen [PTG = pituitary gland, HS = haemopoietic system (leukaemia), and MG = mammary gland].
preputial gland, female rat clitoral gland, no activity in the mouse). Coincident with these increases in tumor incidence were marked decreases at other sites (Fig. 2). The non-carcinogenicity of both toluene [302] and the mixed vinyl toluenes [303] emphasizes the uniqueness of the parent carcinogen benzene ([6] in Ashby and Tennant, 1988). Clearly, substitution in the benzene nucleus m a y significantly affect the subtle activation of benzene to a genotoxic species. This is a particularly important observation because the majority of organic chemicals contain an aromatic benzene or benzene-type ring, and it is inappropriate to put each under suspicion of carcinogenicity based on the unique activity of benzene itself. Attention should be given to the furan moiety as a possible carcinogenic substructure. Three carcinogens described in Table 1 contain a furan ring, benzofuran [265], furfural [270] and furosemide [287]. Each was classed as non-alerting in structure and each was non-mutagenic to Salmonella. Further, the sites of carcinogenesis (Table 1) varied dramatically a m o n g these 3 chemicals suggesting that the aromatic furan ring itself was no more important to carcinogenesis than are the benzene rings in phenylbutazone, for example. At this stage a furan ring has not been entered as a structural alert in Fig. 1. The matter should be kept under review, however, because the furan
ring in aflatoxin B1 is critical to both its mutagenicity and its carcinogenicity, facts that should not be confused with the mutagenicity and carcinogenicity of agents such as nitrofurantoin ([231] in Ashby et al., 1989) where the aromatic nitro group is the critical structural feature. Finally, two aldehyde derivatives gave rise uniquely to mouse stomach tumors (benzaldehyde [281] and dimethoxane [291], the latter a labile source of acetaldehyde, and which was equivocal for carcinogenicity). The aliphatic aldehyde [291] was structurally alerted (cf. Fig. 1) whereas benzaldehyde [281] and furfural [270] were not, these being the first carcinogenic aromatic aldehydes. Even so, only benign stomach adenomas were induced by benzaldehyde and a clear precedent for carcinogenicity is not yet considered to exist. Perhaps the most important observation to make concerning these 39 bioassay results concerns the predictability of the 25 new carcinogens defined. Four carcinogens were potent and active at sites known to be subject to chemically induced cancer in humans - - the two benzidine derivatives [268] and [269], glycidol [271] and tetranitromethane [277]. Each of these was structurally alerted and was clearly mutagenic to Salmonella. At the other extreme, 11 chemicals induced increases in tumor incidences without any alert from chemical structure or the Salmonella assay ([265], [270], [272], [273], [275], [2761, [280], [281], [287],
227
[288] and [289]). The prediction of such selective carcinogens and extrapolation of such effects to humans represents an important area for future study. References Ashby, J., and R.W. Tennant (1988) Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NTP, Mutation Res., 204, 17-115. Ashby, J., R.W. Tennant, E. Zeiger and S. Stasiewicz (1989) Classification according to chemical structure, mutagenicity
to Salmonella and level of carcinogenicity of a further 42 chemicals tested for carcinogenicity by the U.S. National Toxicology Program, Mutation Res., 223, 73-103. Fiala, E.S., R. Czerniak, A. Castonguay, C.C. Conaway and A. Rivenson (1987) Assay of 1-nitropropane, 2-nitropropane and 1-azoxypropane and 2-azoxypropane for carcinogenicity by gavage in SD rats, Carcinogenesis, 8, 1947-1949. Goldsworthy, T.L., O. Lyght, V.L. Burnett and J.A. Popp (1988) Potential role of a-2g-globuhn protein droplet accumulation and cell replication in the renal carcinogenicity of rats exposed to trichloroethylene, perchloroethylene and pentachloroethane, Toxicol. Appl. Pharmacol., 96, 367-379. Zeiger, E., B. Anderson, S. Haworth, T. Lawlor and K. Mortelmans (1988) Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals, Environ. Mol. Mutagen., 11 (Suppl. 12), 1-158.
Mutation Research, 257 (1991) 209-227 © 1991 Elsevier Science Publishers B.V. 0165-1110/91/$03.50 ADONIS 0165111091000596
MUTREV 00071
Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 39 chemicals tested for carcinogenicity by the U.S. National Toxicology Program Raymond W. Tennant a and John Ashby b a National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.) and b ICI Central Toxicology Laboratory, Alderley Park, Cheshire (Great Britain)
(Accepted 9 October 1990)
Keywords: Rodent carcinogenicity bioassay; DNA reactivity; Classification according to chemical structure/mutagenicity
Summary This paper is an extension of compilations published previously in this journal. (Ashby and Tennant, 1988; Ashby et al., 1989). A s u m m a r y of the rodent carcinogenicity bioassay data on a further 39 chemicals tested by the U.S. National Toxicology Program (NTP) is presented. An evaluation of each chemical for structural alerts to DNA-reactivity is also provided, together with a s u m m a r y of its mutagenicity to Salmonella. Chemicals with an aliphatic nitro group ( - C - N O / ) have been added to the composite structure of DNA-reactive sub-groups. The 39 chemicals were numbered and evaluated as an extension of the earlier analysis of 264 N T P chemicals. The activity patterns and conclusions derived from the earlier studies are followed by these 39 chemicals, albeit a detailed analysis of the total database of 301 chemicals is reserved for the succeeding paper.
In two earlier papers we have summarized the rodent carcinogenicity data reported by the U.S. National Toxicology Program (NTP) for 264 chemicals (Ashby and Tennant, 1988; Ashby et al., 1989). The present paper describes the test data for a further 39 chemicals, and this brings the total number reviewed to 301 (2 chemicals have been tested on two separate occasions). The criteria for selection and presentation of data are essentially as described in the two earlier publications. All of the studies considered here were peer re-
Correspondence: Dr. J. Ashby, ICI Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ (Great Britain).
viewed in 1989-1990 and are due to be pubfished during 1990 or early 1991. A detailed analysis of the data for the present 39 chemicals was avoided because a review of the complete database of 301 chemicals is presented in the accompanying paper of this special issue.
Methods Criteria for selecting studies for incorporation in this review were as described earlier (Ashby et al., 1989). In the case of the two derivatives of benzidine ([268] and [269]) only rat studies were available. However, because these were potent and multi-site genotoxic carcinogens they were entered
210
Table I SUMMARY OF CARCINOGENICITY, MUTAGENICITY AND CHEMICAL DATA FOR 39 COMPOUNDS TESTED FOR CARCINOGENICITY IN F344 RATS AND B6C3F1 MICE BY THE U.S. NATIONAL TOXICOLOGY PROGRAM. Criteria for selection of the data displayed below are described in the text. The carcinogenicity bioassay tumor incidence fig u res shown reflect the conclusions of the Peer Review Panel. Not all categories of tumor are displayed for each chemical. For example, if a positive response is recorded both for liver nodules and liver carcinomas, the latter are generally the ones shown. Likewise, only the tumor data that led to the classification of an agent as carcinogenic are displayed, even though equivocal evidence of carcinogenicity may have been recorded for another sex or species of test animal. In most cases the numerical tumor incidences displayed in the Table were those selected for discussion by the Peer Review Panel and the bioassay report authors. This means that tumor incidences
CHEMICALS ASSOCIATED CARCINOGENIC STATUS
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Chemical Name
Salmonella assay response (Zeiger)l-
Level of effect across Route~ species, (F, G, I, sexes and o~M (~M tissues(A-E){} SK, orW)
NTP Technical Report Conclusions o~R
?R
NE
SE
CE
CE
A
G
+
SE
EE
EE
CE
A
/
+
+
CE
EE
SE
NE
A
G
+
+
CE
CE
NT
NT
A
W
[265] 271-89-6 370(1989)
Benzofuran
[266] 74-96-4 363(1989)
CH3CH2Br
Bromoethane [267] 20265-96-7 351 (1989)
p-Chloroaniline .HCI
NH 2
C[
[268] 20325-40-0 372(1990)
H2N " K ~ ~ ~ CH30
NH2 OCH 3
3,3'-Dimethoxybenzidine .2HCI
211
adjusted for mortality are generally employed. In some cases the incidence of carcinomas in a tissue is shown for male animals and of carcinomas plus adenomas for female animals (for example) - again, this is consistent with the decision of the authors of the bioassay report. This Table therefore only provides information on the level of carcinogenicity for each agent. In instances where the carcinogenicity of a specific chemical is of interest, it is important that reference to the original bioassay report be made. For a description of the symbols used in this Table, see the legend at its end. The assistance provided by John Reece in the construction of this table is gratefully acknowledged.
WITH CARCINOGENICITY CARCINOGENICITY DATA Maximum dose
Duration of study (weeks)
Rats
Mice
Rats/Mice
120
240
103
0.04
0.04
Tumor site •
0
2
8
8 24 4 20
104/103
103
MG SK ZG PG OC I/C L* MT
B CG U
71 77 27 40
2
46
45
4 4
18 19
10 30
C L M H
0
L M H 0 2 6 76 27 29 31 53
C
L
M
H
22 43 40 42 8 8 2 20
C L M H
84
62 77 28 23
C L M H 0 12 2 2 0 0 8 2 17 49 36 43
c
SP AG L* CS
9 C ] L }H
o"
DILI
L* S* LU
B
0.033
9
DILI
K
LU U
30
o"
CILI
L**
AG
18
Tumor data identified in Summary of NTP Technical Report Rats (% TBA)~ Mice (% TBA)
0 0 27 2 0 2 3 0
29 22 28 18 2 9 2 5
37 33 45 13 11 9 9 4
37 50 49 18 13 13 10 2
C L M H 2 4 1933 0 9 4 3 2 27 28 27 3 4 8 8 0 2 1 5 0 2 0 5
12616575 0 9 3 3
SEE TEXT
8
11 56
2.]2
Table1
(Continued) CARCINOGENIC STATUS
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Salmonella assay response (Zeiger)~
Chemical Name ÷
[269] 612-82-8
÷
Level of Route~ NTP Technical i effect across Report Conclusions species, (F, G, I, sexes and W) I oTR ~R oTM ~M~tissues(A-E)§ SK, or CE
CE
NT
NT
A
W
SE
NE
CE
SE
A
G
CE
CE
CE
CE
A
G
CE
NE
P
P
A
G G
390(1990)
H2N ~
N
H
2
OH 3
CH3
3,3'-Dimethylbenzidine .2HCI
[270] 98-01-1 382(1990)
~ C H O Furfural +
[271] 556-52-5 374(1990)
÷
CHJt,.,%CH _ CH2OH
Glycido] [272] 67-72-1 361 (1989) 68(1978)
CI
CI
I
[
C I - - C - - C - - CI
Hexachloroethane
I
I
CI
CI
213
CARCINOGENICITY DATA Maximum dose
Rats
Mice
0.015
Duration of study (weeks) Rats/Mice
Tumor data identified in Summary of NTP Technical Report
64 SK ZG PG L* OC I/C LU MT CG MG
60
175
Rats (% TBA)~
Tumor site~
Mice (% TBA)
9
c¢
cl'l"
clLI"
C 0 2 3 0 0 0 2 0
C L M H 0 7 12 20 0 13 43 70
L M H 4 23 45 7 43 60 9 8 15 0 47 55 0 5 8 0 8 25 0 11 10 0 4 7
0 0 0 2
0 7 2 2
0 0
31 56 54 2 4 10
9 7 1222 8 7 4 7
BD L*
0
0
L
M
H
50
103
TV
6
68
78
I
6
16
14
128
68
74
B
0
10
12
0
8
8
2
6
14
S*
2
4
12
0
8
22
SK
0
10
8
ZG
2
6
12
2
8
12
OC
CG HS HG
160 1179
103 78
L
M
H
2
6
10 16
32 44 35 64
MG
TG
C
4
L**
75
cl'l"
SEE TEXT
C
103
9
o1'1"
10
18
0
2
6
26
28
40
2
4
20
0
0
8
17
29
50
4
12
30
0
0
4
40
24
9
26
U
0
6
6
SB
0
6
18
10
40
31
L*
48
62
70
LU
26
22
42
15
30
63
K L*
2
4
14
214 Table 1
(Continued)
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Salmonella assay response (Zeiger)l-
Chemical Name [273] 123-31-9 366(1989)
-
-
-
CARCINOGENIC STATUS Level of NTP Technical effect across Report Conclusions species, o"R
~R d'M
Route~
(F, G, I,
sexes and SK, or W) ~M'tissues(A-E)§
SE
SE
NE
SE
A
G
+
SE
NE
NE
SE
A
G
--
NT
NT
CE
CE
A
F
--
EE
SE
SE
NE
A
G
-
OH
OH Hydroquinone [274]
+
5634-39 9
340(1989) CH 2 -- C H - - CH21 I
I
OH
OH
Iodinated Glycerol [275] 87-86-5 349(1989)
OH
CI Pentachlorophenol [276] 50-33-9 367(1990)
~
Q~
N/N~c//O
I
[
C -II O
C -I H
CH 2 -- CH 2 -- CH 2 -- CH3
Phenylbutazone [277] 509-14-8 386 (1990)
4-
4-
CE
CE
CE
CE
A
/
+
+
CE
CE
CE
CE
A
SK
NO2
I I
O2N - - C - - N O 2 NO2 Tetranitromethane [278] 106-87-6 362(1989)
0 ~
S0%
-CH - -
CH 2
4-Vinyl-l-cyclohexene Diepoxide
215
CARCINOGENICITY DATA Maximum dose
Rats 50
250
0.02
Mice 100
250
0.06
Duration of study (weeks) Rats/Mice 103
103
Tumor site ~
Tumor data identified in Summary of NTP Technical Report Rats (% TBA) ° Mice (% TBA)
c¢
K HS L*
0
HS TG PTG HG
28 0
7
300
clLIH
58 10
27
0.0005
0.0002
103
C L M H 17 40 44 69 3 8 44 92
30 mg/ animal
10 mg/ animal
105/103
SEE TEXT
SK 0 LU
0
2
0
5
29
24
21 12
33 20
52 28
48 2
K L*
LU
?
c] LI H
40
103
103
o"
C I L IH
15 16
L* AG CS
100
9
CILIH
0
66
92
0
44
66
72
0
32
C 3 0 0
L 8 4 2
M H 12 65 4 78 6 16
4 32
28
62
100 I 2 4
52
92
68
C L M H 0 28 78 84
8
48
98
C L M H 0 12 74 82 2 0 35 36 8 18 22 14
2]6 Table 1
(Continued)
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Salmonella assay response
Chemical Name
(Zeiger)t
[279] 924-42-5 352(1989)
--
CARCINOGENIC STATUS Level of Route~ NTP Technical effect across Report Conclusions species, (F, G, I, sexes and SK, or W) o~R ~ a o7M ~ M tissues(A-E)§ NE
NE
CE
CE
B
CE
CE
EE
NE
B
G
O II @ H 2 = CH -- C - - NH-- CH2OH
N-Methylolacrylamide 389-08-2 368(1989) [280]
CH 2 CH 3 I
H
3
C
Nalidixic Acid
~ II
C-
O
0
OH
NE
[281] 100-52-7 378(1990)
NE
SE
SE
C
G
CHO
0
Benzaldehyde [282]
4-
75-25-2
SE
CE
NE
NE
C
CE
CE
EE
EE
C
EE
NE
SE
EE
D
350(1989) H I
Br -- C -- Br I
Br Tribromomethane (bromoform) [283]
115-96-8
391 (1990) O=P(OCH2CH2CI)3
Tris (2-chloroethyl)phosphate 106-92-3 376(1990) [284]
0 1% C H 2,.,. C H -CH2OCH2CH = CH2
AIlyl Glycidyl Ether
4-
+
G
217
CARCINOGENICITY DATA Maximum dose
Duration of study (weeks)
Tumor site"
Rats
Mice
Rats/Mice
12
50
103
HG L** LU O
0.4
0.4
103
PG CG
Tumor data identified in Summary of NTP Technical Report Rats (% TBA)~ Mice (% TBA)
o"
?
01,]H
6
39
o"
c],]H
?
clL] 2 24 10
29 34 20
58 52 36
11 6 12 0
18 8 16 11
42 35 27 11
2
4
10
0
10
12
43 11
33
34
400
600
103
S**
200
200
103
I/C
0
0
6
0
2
16
88
350
103
K
2
10
48
0
4
10
0.001
0.001
103
N
0
0
6
218
Table 1
(Continued)
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
CARCINOGENIC STATUS Level of NTP Technical effect across Report Conclusions species,
Route ~
(F, G, I,
sexes and SK, or W) (~R o~M (~M tissues(A-E)§
(Zeiger)t
(::/'R
+
÷
EE
EE
IS
CE
D
+
--
SE
NE
NE
EE
D
EE
NE
NE
SE
D
CE
NE
NE
NE
D
SE
NE
NE
NE
D
NE
EE
NE
NE
E
Chemical Name
[285] 75-00-3
Salmonella assay response
I
346(1989)
CH 3 CH2CI
Chloroethane
[286] 121-69-7 360(1989)
H3C ~ 1 ,p CH3
N,N-Dimethylaniline [287] 54-31-9 356(1989)
COOH
u
NH2SO2- -x~ Cl Furosemide [288] 5989-27-5 347(1990)
CH 3
H2C~ C "-OH 3 d- Limonene
[289] 98-85-1 369(1989)
OH
I HC--CH 3
© c~-Methylbenzyl Alcohol
+
[290] 532-27-4 379(1990)
COCH2CI
2-Chloroacetophenone
--
G
219
CARCINOGENICITY DATA Maximum dose
Duration of study (weeks)
Tumor site~
Tumor data identified in Summary of NTP Technical Report Rats (% TBA) • Mice (% TBA)
o"
?
o"
C IL IH
C IL IH
C IL IH
Rats
Mice
Rats/Mice
1.5
1.5
102/100
30
30
103
SP
0.07
0.14
103/104
MG
600
1000
103
K
0
16
22
750
750
103
K
0
4
10
2 mg/m 3
4 mg/m 3
103
MG
U
0
0
? C I 0
L I H 86
8
10
24
38
46
220 Table 1
(Continued) CARCINOGENIC STATUS
[Code No} CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Salmonella assay response (Zeiger)t
Chemical Name
NTP Technical Report Conclusions o ~R
~R CM
Level of effect across species,
~M
sexes and tissues(A-E)§
Route' (F,G,I, SK, or W)
NE
NE
EE
NE
E
G
EE
EE
NE
NE
E
F
--
NE
NE
EE
NE
E
+
--
EE
EE
NE
NE
E
+
--
EE
EE
NE
NE
E
EE
NE
NE
NE
[291] 828-00-2 354(1989) H3C'h~OTCH3
,yo Q--C--CH 3 II O
Dimethoxane
[292] 147-24-0 355(1989) H L /OH3 ~ C--O--CH2--CH2-- N\ CH3
Diphenhydramine .HCI [293] 58-93-5 357(1989)
O_....~O
NH2SO2~
S-.~NH
H Hydrochlorothiazide [294] 78-11-5 365(1989) CH2ONO2
I O2NOCH 2 -- C -- CH2ONO 2 /
CH2ONO 2 Pentaerythritol Tetranitrate [295] 9 8 9 - 3 8 - 8 364(1989)
H3C
NC H
H3C
NHC2Hs
Rhodamine 6G. HCI [296] 7681-49-4 393(1990)
NaF
Sodium fluoride
W
221
CARCINOGENICITY DATA Maximum dose
Duration of study (weeks)
Tumor
Rats
Mice
Rats/Mice
site"
250
500
103
S*
0.0625
0.0313
103/105
2
0
8
2
12
10
106/104
L*
5.0
5.0
104/103
ZG
0.0175
0.0175
103
~
iS AG
103 OST
46
0
7
2
4
o"
oiL1,
B
0.5
0.2
o~
cl,l,
LU PTG
0.2
0.025
Tumor data identified in Summary of NTP Technical Report Rats (% TBA)° Mice (% TBA)
7
0
52
3
C
L
M
H
0
0
2
4
6
ol lH
4
6
16
15
20
42
70
9
16 6
(~
olLI,
20
222
Table 1
(Continued) CHEMICALS
ASSOCIATED
WITH NON-CARCINOGENICITY CARCINOGENIC STATUS
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold)
Structural alert
Salmonella assay response (Zeiger)t
Chemical Name
NTP Technical Report Conclusions oTR NE
[297] 60-13-9 387(1990)
~Rro"M ~U t NE NE NE
Route¢
Duration of study (weeks)
(F,G,I. SK, orW)
Rats/Mice
F
103
I
CH 3
I ~
~[. ,-CH2-CH-NH2
D-L- Amphetamine Sulfate
[298] 2698-41-1
NE
NE
NE
NE
NE
NE
NE
NE
103
377(1990)
[~
/CN CH --C ~'CN CI
o-Chlorobenzalmelanonitrile
[299]
120 83-2
353(1989)
F
103
OH CI
2.4 Dichlorophenol
CI
[300] 55-31-2 380(1990)
103
NE NE (IS) (IS)
NE NE (IS) (iS)
NE
NE
NE
NE
G
103
NE
NE
NE
NE
I
104
[
CHOHCH2NHCH 3
L- Epinephrine Hydrochloride
[301] 108 30-5 373(1990)
//O OH 2 - - C CH2--C O
Succinic Anhydride
[302]
108 88-3 371(1989)
Toluene
223
CARCINOGENIC STATUS
[Code No] CAS No NTP Tech Report No (Year) Structure or trivial name (alerting substructure in bold) Chemical Name
Structural alert
Salmonella assay response (Zeiger)1"
[3031
NTP Technical Report Conclusions o"R
~R
NE
NE
o'~M ( ~ U NE
NE
Route ~
Duration of study (weeks)
(F,G,I, SK, or W)
Rats/Mice
/
103
375(1990)
CH - CH 2 Vinyl Toluenes (meta/para 70:30)
1 ¶
Salmonella mutation data provided by Dr. E. Zeiger of the NTP, and presented in the appropriate technical report. Questionable responses have been regarded as positive. G = gavage dosing (mg/Kg); I = exposure via inhalation (%); F = exposure via food (%); SK= skin painting; W= in drinking water
(%).
e * §
Abbreviations for tumor site are as follows: AG = adrenal gland; B = brain; BD = bile duct; CG = clitoral gland; CS = circulatory system; HG = Harderian gland; HS = hematopoietic system; I/C = intestine/colon; IS = integumentary system; K = kidney; L = liver carcinomas; L* = liver carcinomas + adenomas; L** = liver adenomas; LU = lung; MG = mammary gland; MT = mesothelial tissue; N = nose; O = ovary; OC = oral cavity; OST = osteosarcoma in bone; PG = preputial gland; PTG = pituitary gland; S = stomach carcinomas; S* = stomach carcinomas + adenomas; S** = stomach adenomas (papilloma); SB = subcutaneous tissue; SK = skin; SP = spleen; TG = thyroid gland; TV = tunica vaginalis; U = uterus; ZG = Zymbal's gland. % TBA = % tumor bearing animals for each site of carcinogenesis. The letter C represents the concurrent control group, L the low test group and H the high dose test group. In some studies reference to a medium dose (M) is made. P: positive for carcinogenicity; IS: inadequate study; CE: clear evidence of carcinogenicity; NT: not tested; EE: equivocal evidence; NE: no evidence; SE: some evidence. Level of carcinogenic effect categorized A-F as follows: A = both species affected; B = single species with 2 or more tissues affected; C = single species with a single tissue affected; D = single sex of a single species with a single tissue affected; E = adequate study providing equivocal evidence of carcinogenicity.
into the group A category which implies carcinogenic activity in the mouse. Two other deviations were made from the earlier policy of considering only agents adequately tested in both rats and mice (Ashby and Tennant, 1988). First, epinephrine [300] was found to be noncarcinogenic in studies that subsequently were considered inadequate because a higher dose level could probably have been administered. Epinephrine was nonetheless retained in the database because it illustrates the unusually stringent standards applied to these bioassays. In the overall calculations presented in the subsequent paper, this agent is classed as a non-carcinogen. Second, pentachlorophenol [275] was only tested in mice, but in two different states of purity. Both samples of pentachlorophenol induced tumors in the adrenal gland and liver, and of particular interest, the circulatory system. In the discussion of those data in the technical report (TR 349) the probable
contribution of chlorinated dibenzodioxins to the carcinogenicity of this agent was discussed, and all such halogenated biphenyls are carcinogenic to both rat and mouse liver (see compounds [26], [42], [47], and [78] in Ashby et al., 1988). Thus, in order not to lose the unexpected tumors of the circulatory system from the database, pentachlorophenol [275] was included in the present compilation and assumed also to be a rat-liver carcinogen in the following paper, p-Chloroaniline [267] was tested by gavage as the hydrochloride in the present compilation, but had earlier been tested as the free amine in diet ([120] in Ashby et al., 1988). The structural alerts were based on the composite structure presented in revised form in Ashby et al. (1989). The Salmonella mutation data were from the N T P studies conducted by Zeiger and his colleagues (e.g. Zeiger et al., 1988) and as reported in the appropriate N T P technical report. The tumor site codes and the source of the numerical
224
Results
tumor data presented in Table 1 are described in the Introduction and Legend to the Table. Studies involved use of both sexes of Fischer 344 (F344) rats and B6C3F1 mice, except in the case of the two benzidine derivatives (rat only) and pentachlorophenol (mouse only). The 39 chemicals were stratified according to the level of carcinogenic activity as defined previously: (A) Trans-species carcinogens; (B) Carcinogens affecting 2 or more sites of a single species; (C) Carcinogens affecting a single tissue of both sexes of a single species; (D) Carcinogens whose activity is species/ sex/site specific; (E) Agents with equivocal evidence of carcinogenicity; (F) Agents non-carcinogenic to both test species.
A summary of the bioassay data abstracted from the N T P technical reports for the 39 test chemicals is shown in Table 1. Twenty-five carcinogens, 7 agents equivocal for carcinogenicity, and 7 non-carcinogens were defined by those studies. Much as observed in the earlier analyses, the proportion of mutagens decreased according to the level of carcinogenicity of the test agents. 44% of the carcinogens were both devoid of structural alerts and were negative in the Salmonella assay. The 7 new non-carcinogens were both nonmutagenic to Salmonella and non-alerting in chemical structure, a figure at variance with the earlier analysis where --30% of non-carcinogens were mutagenic to Salmonella (note small sample size and extended discussion of this point in the succeeding paper). In the previous analysis (Ashby et al., 1989),
(a) OCH 3
I I
O---S--O
(b) NO 2
OH 2
O~(d)
,c, (r)
__/ / O=N
(p)
,
N--CH 2 -
CH 3
'~/---~/ (h)
H
(q)
CH2OH '
I
(g) X\
//L----N (e)
~CH9
//
CHO
CH 3
/
I
H2N--< r ~__L__CH2__CH__ NH__N\ ( )
\\
NH2
"X(
#=,,
//~--N---N--(~\
O - - •- - - - ( \
CH (s)
~ ' ~
?--CH 2
~
OH 2 I
CICH2
0
(n)
I O~'C~NH 2 (o)
(i) CH---CH--CI
(j)
CH3
N(CH2CH2CI)2
I CH--Cm--C" NH (m)
x C H - - C H/
I NO 2 (u)
(t)
\
(') CH --OH 2
I
I
o//C
O
Halogenated methanes
C(X)4 x= H,F,CI,Br,l inany combination
Fig. 1. Revision of the multiple electrophile model chemical upon which structural alerts in the present papers are based. The new sub-structure is labelled (u) and represents an aliphatic nitro group, as present in tetranitromethane [277]. All other substructures ( a - b ) are as described in detail in A s h b y et al. (1989).
225 3-nitropropionic acid was classed as structurally alerting because of the - C H 2 N O 2 substructure, and it was also mutagenic to Salmonella. The basis for this structural alert was the carcinogenicity of 2-nitropropane (Fiala et al., 1987) a compound not entered into the present analysis because it had not been tested by the NTP. In the present series of chemicals, tetranitromethane [277] again contains an aliphatic nitro group, is mutagenic to Salmonella, and is a potent lung carcinogen. Consequently, the C - N O 2 substructure has been entered into the multiple electrophile megastructure (Fig. 1), even though the mechanism of its genotoxicity is not clear. Note that pentaerythrityl tetranitrate [294] is structurally alerted because of the potential to lose nitrite ion (NO~-), but this chemical was non-mutagenic to Salmonella. The structural alert for dimethoxane [291] was based on the potential for acetaldehyde formation. Only the terminal Michael-reactive carbon atom of N-methylolacrylamide [279] was alerted. Discussion
Consideration of overall trends, such as the proportion of carcinogens that are mutagenic to Salmonella, have been avoided for the present 39 chemicals for statistical reasons; such trends and correlations are evaluated for the whole database of 301 chemicals in the accompanying paper. Several interesting carcinogens are present among the current 39 chemicals. First, three chemicals, 3,3'-dimethoxybenzidine [268], 3,3'-dimethylbenzidine [269] and glycidol [271], are probably the most extensive of carcinogens yet described by the NTP. In each case, these genotoxins are active in most of the tissues associated earlier with genotoxic carcinogenesis (Ashby and Tennant, 1989). Such carcinogens are representative of the reference carcinogens known in the early 1970's and which stimulated the N T P bioassay program. Likewise, 4-vinyl-l-cyclohexene diepoxide [278] and tetranitromethane [277] are classical potent genotoxic carcinogens of immediate relevance to exposed humans. Four chemicals induced renal tumors only in the male rat, and each was non-mutagenic to Salmonella and non-alerting in chemical structure (hexachloroethane [272], hydroquinone [273], d-
limonene [288] and a-methylbenzyl alcohol [289]). These agents may induce tumors by a-2-/~-globulin retention in the kidneys (Goldsworthy et al., 1988), and studies to evaluate this could be usefully complemented by evaluation of the putative nongenotoxin phenylbutazone [276], which affects only the female rat kidney, and tris(2-chloroethyl)phosphate [283] that is a specific renal carcinogen to both male and female rats. Three chemicals were notable for the limited extent of their carcinogenicity. Allyl glycidyl ether [284] and chloroethane [285] are overt alkylating agents and potent mutagens to Salmonella, yet each is only selectively and minimally carcinogenic to rodents when tested by inhalation. The first induced a small increase in nasal tumors in male mice ( 3 / 5 0 animals affected at the high dose level) and the second, chloroethane, induced uterine tumors selectively in female mice. The suggestion that these natural electrophiles are rapidly detoxified by reaction with non-critical macromolecules may explain the low activity of the allyl compound to the nasal epithelium, but this cannot elucidate the selective carcinogenic activity of chloroethane to the uterus upon inhalation. Both compounds would normally have been expected to induce tumors of the respiratory tract in all four test groups. Occurrences such as these emphasize our current inability to predict carcinogenesis accurately. The third chemical of interest is 2-chloroacetophenone [290]. This has a chemically reactive chlorine atom which led to the structural alert. However, it was non-mutagenic to Salmonella and gave only equivocal evidence of carcinogenicity to the female rat mammary gland. Benzofuran [265] and N-methylolacrylamide [279] are interesting on several counts. First, both are carcinogenic to tissues of the mouse associated with genotoxins, but are inactive in the rat, a very rare situation, as discussed in the following paper. Second, both are non-mutagenic to Salmonella. The acrylamide derivative is structurally alerted because of its inherent Michael reactivity, a property that can lead to chromosomal damage in the absence of mutagenicity to Salmonella (see Ashby et al., 1989). Nalidixic acid [280] induced a most unusual tumor response suggestive of a non-genotoxic hormonal mechanism of carcinogenicity (male rat
226
Male Rats
% TBA 20 -
o~
/
Female Rats
% TBA
o
PG
20
A \
\
\
x
~
10-
10
D
° ~
-A
HS
x
"A . . . . . . . . . Control
CG
\ a.
•
o
Low
,x PTG High
[] MG Control
Low
High
Fig. 2. T u m o r d a t a f o r n a l i d i x i c a c i d [280]. I n a d d i t i o n to t h e n a m e d sites o f c a r c i n o g e n e s i s [clitoral g l a n d in f e m a l e r a t s ( C G ) a n d
preputial glands in male rats (PG)] several statistically significant decreases in tumor incidences were noted in the technical report (TR 368) and are also shown here. Such a profile of tumor incidences is suggestiveof a nongenotoxic mechanism of carcinogenicity, perhaps involving hormonal disturbance on protracted dosing of this Salmonella non-mutagen [PTG = pituitary gland, HS = haemopoietic system (leukaemia), and MG = mammary gland].
preputial gland, female rat clitoral gland, no activity in the mouse). Coincident with these increases in tumor incidence were marked decreases at other sites (Fig. 2). The non-carcinogenicity of both toluene [302] and the mixed vinyl toluenes [303] emphasizes the uniqueness of the parent carcinogen benzene ([6] in Ashby and Tennant, 1988). Clearly, substitution in the benzene nucleus m a y significantly affect the subtle activation of benzene to a genotoxic species. This is a particularly important observation because the majority of organic chemicals contain an aromatic benzene or benzene-type ring, and it is inappropriate to put each under suspicion of carcinogenicity based on the unique activity of benzene itself. Attention should be given to the furan moiety as a possible carcinogenic substructure. Three carcinogens described in Table 1 contain a furan ring, benzofuran [265], furfural [270] and furosemide [287]. Each was classed as non-alerting in structure and each was non-mutagenic to Salmonella. Further, the sites of carcinogenesis (Table 1) varied dramatically a m o n g these 3 chemicals suggesting that the aromatic furan ring itself was no more important to carcinogenesis than are the benzene rings in phenylbutazone, for example. At this stage a furan ring has not been entered as a structural alert in Fig. 1. The matter should be kept under review, however, because the furan
ring in aflatoxin B1 is critical to both its mutagenicity and its carcinogenicity, facts that should not be confused with the mutagenicity and carcinogenicity of agents such as nitrofurantoin ([231] in Ashby et al., 1989) where the aromatic nitro group is the critical structural feature. Finally, two aldehyde derivatives gave rise uniquely to mouse stomach tumors (benzaldehyde [281] and dimethoxane [291], the latter a labile source of acetaldehyde, and which was equivocal for carcinogenicity). The aliphatic aldehyde [291] was structurally alerted (cf. Fig. 1) whereas benzaldehyde [281] and furfural [270] were not, these being the first carcinogenic aromatic aldehydes. Even so, only benign stomach adenomas were induced by benzaldehyde and a clear precedent for carcinogenicity is not yet considered to exist. Perhaps the most important observation to make concerning these 39 bioassay results concerns the predictability of the 25 new carcinogens defined. Four carcinogens were potent and active at sites known to be subject to chemically induced cancer in humans - - the two benzidine derivatives [268] and [269], glycidol [271] and tetranitromethane [277]. Each of these was structurally alerted and was clearly mutagenic to Salmonella. At the other extreme, 11 chemicals induced increases in tumor incidences without any alert from chemical structure or the Salmonella assay ([265], [270], [272], [273], [275], [2761, [280], [281], [287],
227
[288] and [289]). The prediction of such selective carcinogens and extrapolation of such effects to humans represents an important area for future study. References Ashby, J., and R.W. Tennant (1988) Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NTP, Mutation Res., 204, 17-115. Ashby, J., R.W. Tennant, E. Zeiger and S. Stasiewicz (1989) Classification according to chemical structure, mutagenicity
to Salmonella and level of carcinogenicity of a further 42 chemicals tested for carcinogenicity by the U.S. National Toxicology Program, Mutation Res., 223, 73-103. Fiala, E.S., R. Czerniak, A. Castonguay, C.C. Conaway and A. Rivenson (1987) Assay of 1-nitropropane, 2-nitropropane and 1-azoxypropane and 2-azoxypropane for carcinogenicity by gavage in SD rats, Carcinogenesis, 8, 1947-1949. Goldsworthy, T.L., O. Lyght, V.L. Burnett and J.A. Popp (1988) Potential role of a-2g-globuhn protein droplet accumulation and cell replication in the renal carcinogenicity of rats exposed to trichloroethylene, perchloroethylene and pentachloroethane, Toxicol. Appl. Pharmacol., 96, 367-379. Zeiger, E., B. Anderson, S. Haworth, T. Lawlor and K. Mortelmans (1988) Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals, Environ. Mol. Mutagen., 11 (Suppl. 12), 1-158.
