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Umbra Limi: A model for the study of chromosome aberations in fishes
Mutation Research, 31 (I975) 235-24o © Elsevier Scientific Publication Company, A m s t e r d a m - - P r i n t e d in The Netherlands
235
LACK OF I N D U C T I O N OF DOMINANT L E T H A L S IN MICE BY ORALLY A D M I N I S T E R E D AF-2
E. R. SOARES AND WILLIAM S H E R I D A N National Institute of Environmental Health Sciences, Research Triangle Park, N.C. 277o9 ( U.S.A .) (Received December i8th, 1974) (Revision received March i2th, 1975)
SUMMARY
In a series of toxicity tests, male mice of three inbred strains were exposed to several doses of orally administered furylfuramide (AF-2). Subsequent to these tests the effects of AF-2, as measured by induced dominant lethals, were tested in strain DBA/2J mice. AF-2 at the doses used in this study was relatively non-toxic to the strains of mice tested. No indication of AF-2 induced dominant lethality was observed.
INTRODUCTION
Since 1965, trans-2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) has been used as a food preservative in such foods as soybean curd and some fish products throughout Japan. In recent years there has been considerable interest in the mutagenic effects of this compound. TONOMURA AND SASAKI~ found AF-2 to be capable of inducing chromatid aberrations in cultured human cells. CLIVE (1974, personal communication), showed AF-2 to be mutagenic in cultured mouse lymphoma cells. KADA2 and KONDO AND ICHIKAWA-RYo3, using E. coli demonstrated the production by AF-2 of induced reverse mutations. Similarly, TAZIMAAND ONIMARU6 found AF-2 to be mutagenic in their test system using silkworm oocytes, and ONG AND SHAHIN~ found AF-2 to be nmtagenic when tested in Neurospora. That AF-2 is capable of inducing mutations in bactecia and some lower invertebrates is fairly well established. However, to date, no mutagenicity testing using whole mammals has been repoLted. MIYAjII4 reported the results of chronic and acute toxicity tests of orally administered AF-2 in rats and mice. LD~0 values from these tests were 475 mg/kg for mice and 15oo mg/kg for rats. Also, MIYAjII4 showed that AF-2 was not teratogenic and that AF-2 had no effect on male fertility. In our study we have considered the effects in mice of several doses of orally administered AF-2 Effects were measured by the dominant lethal test. Abbreviations: AF-2, trans-2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; DMSO, dimethylsulfoxide.
230
E.R.
SOARES, W. S H E R I D A N
METHODS AND M A T E R I A L S
Several tests of the toxicity of AF-2 were run prior to the start of the dominant lethal test. Table I outlines the methodology and results of these tests. In test i and test 4a, food and water were withheld from the nfice for 24 h prior to administration of the compound. In all cases, the AF-2 was administered by gavage in o.25 cm ~ of solvent. All controls were given an identical amount of solvent. In conducting the initial toxicity tests, we encountered some difficulty in selecting an appropriate solvent for the AF-2. Of the three solvents used, distilled water, 4o% ethanol, and pure dimethylsulfoxide (DMSO), only the latter permitted ioo% solubility. Consequently, in the first two tests, the AF-2 was administered as a suspension and only in the last two tests was it completely dissolved. TABLE
1
R E S U L T S OF A F - 2
Test
Strain
T O X I C I T Y T E S T S IN T H R E E STRAINS OF MICE
Sample size
ia
C3H/He J
12
2
C3H/He J
3°
3 (a) (b) 4(a) a (b)
DBA/2J b CBA/J DBA/2J DBA/zJ
t66 16 9 8
Solvent
Deaths c according to dose i n m g / k g ( d e a t h s / t o t a l ) 0.0
Distilled water 40% ethyl alcohol DMSO DMSO DMSO DMSO
2/6
200
300
-
35 °
400
6/6
450
500
600
-
--
I/6 i/6 I/6 2/6 O/6 . . o/3o 0/30 0/30 0/30 0/30 0/4 . . . . . . . . . o/4 I/4 . . . . . o/4 -
.
0/4 o/4 5/5 i/4
75 °
.
900
.
0/4 o/4
0/4 o/4 "
a I n t h i s t e s t food a n d w a t e r w e r e r e m o v e d f r o m t h e m i c e 24 h. p r i o r t o i n j e c t i o n . b T h e s e m i c e w e r e u s e d in t h e d o m i n a n t l e t h a l t e s t . e D e a t h s w i t h i n 48 h f o l l o w i n g t r e a t m e n t . -, This dose was not tested.
In running the dominant lethal test, we used five groups of thirty DBA/2J male mice (Table I). 24 h following treatment, each male was caged separately and was paired with three 8-week-old strain C57BL/6J females. After seven nights the females were removed from the males and replaced by an additional three females. This procedure was repeated for six consecutive weeks. 17 days after their initial exposure to a male, all females were sacrificed and examined for: (I) the total number of implants and (2) the number of post-implantation deaths. Differences between groups were analyzed by analysis of variance techniques, where appropriate, or by non-parametric techniques, where the data did not meet the requirements of the analysis of variance. RESULTS
Toxicity test In the first test and in test 4 a in which mice were deprived of food and water for 24 h prior to the treatment, all AF-2 treated animals died within 48 h (Table I). In the second test the following deaths were recorded; I death at 200 mg/kg of AF-2, I death at 300 mg/kg of AF-2, 2 deaths at 35o mg/kg of AF-2, no deaths at 40o mg/kg of AF-2, and I death at o.o mg/kg of AF-2. In the third toxicity test, we recorded no AF-2 related deaths; and in test 4 b, one AF-2 treated animal died,
TOXICITY OF AF-2 IN MICE
237
P o s t - m o r t e m s c o n d u c t e d on t h e A F - 2 t r e a t e d mice from e x p e r i m e n t 4a a n d 4 b showed, in all cases, t h a t the injected A F - 2 h a d p r e c i p i t a t e d (AF-2 is b r i g h t orange in color) a n d s u b s e q u e n t l y lodged in the fundus p o r t i o n of t h e stomach. S u r v i v i n g mice from test 4 b were sacrificed at 48 h a n d 72 h p o s t - t r e a t m e n t . A t 48 h p o s t - t r e a t m e n t , the a m o u n t of A F - 2 in t h e fundus a p p e a r e d s o m e w h a t r e d u c e d from t h a t o b s e r v e d in the d e a d mice described above. A t 72 h post-injection, t h e q u a n t i t y of A F - 2 in t h e fundus was c o n s i d e r a b l y r e d u c e d a n d in one case, the A F - 2 p r e c i p i t a t e h a d d i s a p p e a r ed from the stomach. I n addition, the urine of those animals sacrificed at 48 a n d 72 h was b r i g h t orange in color. Dominant lethal test In comparisons between the control a n d t r e a t e d groups, a n d between t h e t r e a t e d groups, three variables were a n a l y z e d ; (z) f e r t i l i t y (number of p r e g n a n t females per t o t a l n u m b e r of females), (2) n u m b e r of i m p l a n t s p e r p r e g n a n t female, a n d (3) the p e r c e n t a g e of p o s t i m p l a n t a t i o n d e a t h s (based on t o t a l i m p l a n t s per p r e g n a n t female). I n the latter, the p r o p o r t i o n r a t h e r t h a n t h e n u m b e r of d e a t h s was a n a l y z e d because t h e d a t a i n d i c a t e d t h a t as t h e n u m b e r of i m p l a n t s increased, the TABLE II THE RELATIONSHIP
BETWEEN
1--6, ALL G R O U P S
COMBINED &
POSTIMPLANTATION
DEATHS
AND
NUMBER
Number of Number of implantations litters per female
Number of resorptions
Average number of resorptions per litter
13 12 II Io 9 8 7 6 5 4 3 2 I Total
9 13 52 134 277 292 193 86 28 I2 6 5 i 11o8
4.5 ° 1.62 i.z8 o.93 o.89 0.79 o.76 0.74 0.87 0.92 0.50 0.55 0.o6 0.83
2 8 44 143 3 Io 367 252 116 32 13 12 9 16 1324
OF IMPLANTATIONS,
WEEKS
a Excluding 4 totally resorbed litters: IO/IO, week I controls; 7/7, week i 300 mg/kg; 4/4, week 3 450 mg/kg; 8[8, week 5 4°0 mg/kg; all weeks and treatment groups were combined since no significant differences were found among them. average n u m b e r of d e a t h s p e r female increased (Table II). T a b l e I I I shows t h e f e r t i l i t y values for each dose group for weeks 1-6 as well as the 6-week average. Differences between groups were a n a l y z e d b y chi-square tests a n d F i s h e r ' s e x a c t test for two × two tables. In week one the 450 m g / k g group showed a significant increase in f e r t i l i t y ( P < o . o 5 ) over t h e control group. All o t h e r comparisons p r o v e d to be not significant. The m e a n values for t h e t o t a l n u m b e r of i m p l a n t s p e r p r e g n a n t female are shown in Table IV. A n a l y s i s of variance techniques were used in t h e comparisons m a d e between these values. O n l y one significant difference was found b e t w e e n groups. In week 4, t h e 35 ° m g / k g group showed a significant ( P < o . o S ) decrease in t o t a l
IV
= -__ ~ 52/87 =
5°/87 45/87 48/89 51/87
2
57% 52% - o,,o ,~4 - o0 39 60°0
z
7-84 7.74 7.56 7.77 7.68
(rag~hE)
o 3 °o 35 ° 4°0 45 °
z~ 0.22 -~- 0-24 ~ o.I8 i o,29 i 0.23
2
8.I6 7.98 7.85 8.18 7.94
i ± i i ~
o.I9 o.21 o.21 0.20 0.22
3
7.78 8.14 7.85 7.42 8.1o
i ~ ± -'+
0.28 o.2o o.26 0.28 0.26
8.38 7.93 7.41 8.24 7.54
4
37/85 41/87 46/9 o 38/87 41/87
4
4~ ~ 4 i
--= ~ =
0.35 0.30 o.28 b 0.35 0-34
44% 47°° - I oO D 440o 47°0
1
13.16 11.31 8.90 1°'3° 9"23
(mg/kg)
o 3°0 35 ° 400 45 °
y ! ± ~ --
1.98 1.54 1.77 1"7° 1.42
9.77 lO.79 lO.63 11"42 8-07
2
a E x c l u d i n g 4 totally resorbed litters.
Week
Dose
~ ~ ± ~ 7L
1-58 1.65 1.98 1.67 1.,52
lO-89 11.16 11.Ol 11-67 lO.16
3
~ ~ ~ ~ 7}:
2.I9 2.41 2.34 2.53 1-76
11.37 12.1o lO.8,5 9-9 ° 11.61
4
~ ~ ~_ ~ 2-
2.22 2.25 2.60 2.85 2.28
THE MEAN PERCENT OF P O S T I M P L A N T A T I O N DEATltS PER PREGNANT FEMALE (.~ -Z~ SE) a
TABLE V
a Excluding 4 totally resorbed litters. b p < 0.05 .
Week
Dose
) S E ) a'
3 6 / 8 7 -- 4 1 % 4 3 / 8 5 -- 5 1 ° b 4 1 / 9 ° = 46o0 4o/86 47°o 41/87 ~ 47°0
3
THE MEAN NUMBER OF IMPLANTS PER PREGNANT FEMALE ( X
TABLE
ap
<0.05.
44/89 53/89 48/89 47/90
o 3°0 35 ° 4°0
450
•
(mg/kg)
= 49% = 60°./0 = 5435 ~ " D 2 o,o 6 2 / 9 0 = 69°~'a
Week
Dose
THE PERCENT OF FERTILE MATINGS ON THE TOTAL NUMBER OF PAIRED FEMALES
"FABLE III
0.24 o.33 o,22 0.23 0.37
4__ 2.20 :h 1.73 zi 2.15 ~ 1.97 ~ 3.51
22 Z ~ ~ +
~ 54% -- 4 8 ° o 4o3; = 5 i o ,o/ -- 4 5 ° o
12.4o I2.2o I1.47 lO.O8 13,84
5
7.71 7.85 8.34 8.34 7.85
,5
45/84 4°/84 35/87 44/86 39/86
5
-
--6
8.60 lO.64 9 .20 11-29 11.82
i ~ ~ Z ~
1.56 1.7o I'99 1.72 1.58
~ 0.25 --4~ 0.29 ~ 0.28 ~ 0.29 ~ 0.29
-- 4 6 ° 0 -- 53'!,, = 4 4 o ,, = 60% -- 56°(~
. . . .
8.24 7.85 8.16 8.28 8.1o
6
38/83 46/86 38/86 50/83 49/87
6
= 48~o - o~ .)2 ,b - - 54°)~
~ ~ ± 22 ~_
11.o5 Ir.33 lO.31 lO.81 lO.57
o.i O o.i1 o.io o.ii o. I I
~ 0.83 7!: 0.76 Z: 0.87 7:0.82 ~ 0.80
Tota! I-6
8,oi 7,91 7.83 8-o5 7.86
Total 1-6
256/531 270/519 284/524
2 5 o / 5 1 5 - - 49°,0 2 6 8 / 5 1 8 - - 52O/o
T o t a l a:-6
>
aZ
>
ba q.,a
TOXICITY OF A F - 2 IN MICE
239
implants as compared to the controls. All other pair wise comparisons were not significant. Since the percent resorptions did not follow a normal distribution, non-parametric procedures were used in the analyses of these data (Tables I I and V). The procedures used were the Mann-Whitney U test for pair wise comparisons and JONCI
Toxicity test In contrast to the results of others, our toxicity tests reveal a relatively low level of sensitivity to AF-2 on the part of the strains of mice used in these experiments. This is indicated by the lack of significant increases in AF-2 related deaths as well as the lack of any other obvious indications of AF-2 induced toxicity. We observed no signs of intestinal distress or diarrhea which would be expected were the AF-2 effecting a bactericidal action on the intestinal flora of the treated animals. Post-mortems and general observations of treated animals indicate a possible storage of the AF-2 in the stomach and excretion of AF-2 or a metabolic by-product of AF-2 by the kidneys. These observations are cursory in nature and further studies of the metabolic fate of AF-2 in nfice are necessary. It is important to note that in the first and fourth tests, all AF-2 injected animals which starved for 24 h prior to treatment died following treatment. Additional toxicological testing is necessary to further elucidate the cause of increased sensitivity on the part of starved mice to AF-2. Dominant lethal test TONOMURA7 showed an induction by AF-2 of chromatid aberrations in cultured human cells. It is estimated that 30% of induced fetal mortality in mammals m a y be attributed to chromosomal and/or chromatid damage (HANSMANN,1974, personal communication). Our data give no indication of AF-2 induced increases in the rate of fetal death over the six-week test period. Furthermore, our data confirm the results of MIYAjII4 in that we found AF-2 to have had no significant effect on male fertility nor did treatment with AF-2 result in any observed atrophy of the testes. Generally speaking, the results reported in this study show a lack of activity of AF-2 with regard to the induction of dominant lethality in mice. This lack of activity could be explained by one of the following: ( I ) inactivation or breakdown of AF-2 at some point in the gastro-intestinal tract prior to its entering the circulatory system, or inactivation (by the liver or kidney) after entrance into the circulatory system; or (2) AF-2 simply does not induce dominant lethals. One or both of these possibilities could explain our results. The apparent precipitation of AF-2 in the stomach, the eventual disappearance of the orange precipitate from the fundus and the excretion of orange urine would seem to signify the absorption of AF-2 or a metabolic by-product of this compound from the stomach. While our data do not necessarily negate the possibility that AF-2 could induce single gene or specific locus mutations in mice, they have clearly shown that AF-2 or its metabolic
240 by-products did not produce dominant treatment.
E. R. SOARES, W. SHERIDAN lethals during the six-week period following
ACKNOWLEDGMENT T h e a u t h o r s w i s h t o e x p r e s s t h e i r a p p r e c i a t i o n t o Dr. M. D. HOGAN a n d D r . J . K. HASEMAN for t h e i r a d v i c e r e g a r d i n g t h e s t a t i s t i c a l t r e a t m e n t of t h e s e d a t a . REFERENCES I JONCKHEERE, A. R., A distribution-free K-sample test against ordered alternatives, Biometrika, 41 (1954) 133-1452 KADA, T., Escherichia coli mutagenieity of furylfuramide, Jpn. J. Genet., 48 (1973) 3Ol-3O5. 3 KONDO, S., AND H. ICHIKAWA-RYO, Testing and classification of mutagenicity of furylfuramide in Escherichia coli, Jpn. J. Genet., 48 (1973) 295-3 oo4 MIYAJII, T., Acute and chronic toxicity of furylfuramide in rats and mice, Tohoku J. Exp. Med., lO3 (1971) 331-369. .5 ONG, TONG-MAN, AND SHAHIN, M. M., Mutagenic and recombinogenie activities of the food additive furylfuramide in eukaryotes, Science, 184 (1974) lO86-1o87. 6 TAZIMA, W., AND ONIMARU, K.. Results of mutagenicity testing for some nitrofuran derivatives in a sensitive test system with silkworm oocytes (Abstract), Mutation Res., 26 (1974) 44 TM 7 TONOMORA, A., AND SASAKI, M. S., Chromosome aberrations and DNA repair synthesis in cultured h u m a n cells exposed to nitrofurans, Jpn. J. Genet., 48 (1973) 291 294.
235
LACK OF I N D U C T I O N OF DOMINANT L E T H A L S IN MICE BY ORALLY A D M I N I S T E R E D AF-2
E. R. SOARES AND WILLIAM S H E R I D A N National Institute of Environmental Health Sciences, Research Triangle Park, N.C. 277o9 ( U.S.A .) (Received December i8th, 1974) (Revision received March i2th, 1975)
SUMMARY
In a series of toxicity tests, male mice of three inbred strains were exposed to several doses of orally administered furylfuramide (AF-2). Subsequent to these tests the effects of AF-2, as measured by induced dominant lethals, were tested in strain DBA/2J mice. AF-2 at the doses used in this study was relatively non-toxic to the strains of mice tested. No indication of AF-2 induced dominant lethality was observed.
INTRODUCTION
Since 1965, trans-2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) has been used as a food preservative in such foods as soybean curd and some fish products throughout Japan. In recent years there has been considerable interest in the mutagenic effects of this compound. TONOMURA AND SASAKI~ found AF-2 to be capable of inducing chromatid aberrations in cultured human cells. CLIVE (1974, personal communication), showed AF-2 to be mutagenic in cultured mouse lymphoma cells. KADA2 and KONDO AND ICHIKAWA-RYo3, using E. coli demonstrated the production by AF-2 of induced reverse mutations. Similarly, TAZIMAAND ONIMARU6 found AF-2 to be mutagenic in their test system using silkworm oocytes, and ONG AND SHAHIN~ found AF-2 to be nmtagenic when tested in Neurospora. That AF-2 is capable of inducing mutations in bactecia and some lower invertebrates is fairly well established. However, to date, no mutagenicity testing using whole mammals has been repoLted. MIYAjII4 reported the results of chronic and acute toxicity tests of orally administered AF-2 in rats and mice. LD~0 values from these tests were 475 mg/kg for mice and 15oo mg/kg for rats. Also, MIYAjII4 showed that AF-2 was not teratogenic and that AF-2 had no effect on male fertility. In our study we have considered the effects in mice of several doses of orally administered AF-2 Effects were measured by the dominant lethal test. Abbreviations: AF-2, trans-2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; DMSO, dimethylsulfoxide.
230
E.R.
SOARES, W. S H E R I D A N
METHODS AND M A T E R I A L S
Several tests of the toxicity of AF-2 were run prior to the start of the dominant lethal test. Table I outlines the methodology and results of these tests. In test i and test 4a, food and water were withheld from the nfice for 24 h prior to administration of the compound. In all cases, the AF-2 was administered by gavage in o.25 cm ~ of solvent. All controls were given an identical amount of solvent. In conducting the initial toxicity tests, we encountered some difficulty in selecting an appropriate solvent for the AF-2. Of the three solvents used, distilled water, 4o% ethanol, and pure dimethylsulfoxide (DMSO), only the latter permitted ioo% solubility. Consequently, in the first two tests, the AF-2 was administered as a suspension and only in the last two tests was it completely dissolved. TABLE
1
R E S U L T S OF A F - 2
Test
Strain
T O X I C I T Y T E S T S IN T H R E E STRAINS OF MICE
Sample size
ia
C3H/He J
12
2
C3H/He J
3°
3 (a) (b) 4(a) a (b)
DBA/2J b CBA/J DBA/2J DBA/zJ
t66 16 9 8
Solvent
Deaths c according to dose i n m g / k g ( d e a t h s / t o t a l ) 0.0
Distilled water 40% ethyl alcohol DMSO DMSO DMSO DMSO
2/6
200
300
-
35 °
400
6/6
450
500
600
-
--
I/6 i/6 I/6 2/6 O/6 . . o/3o 0/30 0/30 0/30 0/30 0/4 . . . . . . . . . o/4 I/4 . . . . . o/4 -
.
0/4 o/4 5/5 i/4
75 °
.
900
.
0/4 o/4
0/4 o/4 "
a I n t h i s t e s t food a n d w a t e r w e r e r e m o v e d f r o m t h e m i c e 24 h. p r i o r t o i n j e c t i o n . b T h e s e m i c e w e r e u s e d in t h e d o m i n a n t l e t h a l t e s t . e D e a t h s w i t h i n 48 h f o l l o w i n g t r e a t m e n t . -, This dose was not tested.
In running the dominant lethal test, we used five groups of thirty DBA/2J male mice (Table I). 24 h following treatment, each male was caged separately and was paired with three 8-week-old strain C57BL/6J females. After seven nights the females were removed from the males and replaced by an additional three females. This procedure was repeated for six consecutive weeks. 17 days after their initial exposure to a male, all females were sacrificed and examined for: (I) the total number of implants and (2) the number of post-implantation deaths. Differences between groups were analyzed by analysis of variance techniques, where appropriate, or by non-parametric techniques, where the data did not meet the requirements of the analysis of variance. RESULTS
Toxicity test In the first test and in test 4 a in which mice were deprived of food and water for 24 h prior to the treatment, all AF-2 treated animals died within 48 h (Table I). In the second test the following deaths were recorded; I death at 200 mg/kg of AF-2, I death at 300 mg/kg of AF-2, 2 deaths at 35o mg/kg of AF-2, no deaths at 40o mg/kg of AF-2, and I death at o.o mg/kg of AF-2. In the third toxicity test, we recorded no AF-2 related deaths; and in test 4 b, one AF-2 treated animal died,
TOXICITY OF AF-2 IN MICE
237
P o s t - m o r t e m s c o n d u c t e d on t h e A F - 2 t r e a t e d mice from e x p e r i m e n t 4a a n d 4 b showed, in all cases, t h a t the injected A F - 2 h a d p r e c i p i t a t e d (AF-2 is b r i g h t orange in color) a n d s u b s e q u e n t l y lodged in the fundus p o r t i o n of t h e stomach. S u r v i v i n g mice from test 4 b were sacrificed at 48 h a n d 72 h p o s t - t r e a t m e n t . A t 48 h p o s t - t r e a t m e n t , the a m o u n t of A F - 2 in t h e fundus a p p e a r e d s o m e w h a t r e d u c e d from t h a t o b s e r v e d in the d e a d mice described above. A t 72 h post-injection, t h e q u a n t i t y of A F - 2 in t h e fundus was c o n s i d e r a b l y r e d u c e d a n d in one case, the A F - 2 p r e c i p i t a t e h a d d i s a p p e a r ed from the stomach. I n addition, the urine of those animals sacrificed at 48 a n d 72 h was b r i g h t orange in color. Dominant lethal test In comparisons between the control a n d t r e a t e d groups, a n d between t h e t r e a t e d groups, three variables were a n a l y z e d ; (z) f e r t i l i t y (number of p r e g n a n t females per t o t a l n u m b e r of females), (2) n u m b e r of i m p l a n t s p e r p r e g n a n t female, a n d (3) the p e r c e n t a g e of p o s t i m p l a n t a t i o n d e a t h s (based on t o t a l i m p l a n t s per p r e g n a n t female). I n the latter, the p r o p o r t i o n r a t h e r t h a n t h e n u m b e r of d e a t h s was a n a l y z e d because t h e d a t a i n d i c a t e d t h a t as t h e n u m b e r of i m p l a n t s increased, the TABLE II THE RELATIONSHIP
BETWEEN
1--6, ALL G R O U P S
COMBINED &
POSTIMPLANTATION
DEATHS
AND
NUMBER
Number of Number of implantations litters per female
Number of resorptions
Average number of resorptions per litter
13 12 II Io 9 8 7 6 5 4 3 2 I Total
9 13 52 134 277 292 193 86 28 I2 6 5 i 11o8
4.5 ° 1.62 i.z8 o.93 o.89 0.79 o.76 0.74 0.87 0.92 0.50 0.55 0.o6 0.83
2 8 44 143 3 Io 367 252 116 32 13 12 9 16 1324
OF IMPLANTATIONS,
WEEKS
a Excluding 4 totally resorbed litters: IO/IO, week I controls; 7/7, week i 300 mg/kg; 4/4, week 3 450 mg/kg; 8[8, week 5 4°0 mg/kg; all weeks and treatment groups were combined since no significant differences were found among them. average n u m b e r of d e a t h s p e r female increased (Table II). T a b l e I I I shows t h e f e r t i l i t y values for each dose group for weeks 1-6 as well as the 6-week average. Differences between groups were a n a l y z e d b y chi-square tests a n d F i s h e r ' s e x a c t test for two × two tables. In week one the 450 m g / k g group showed a significant increase in f e r t i l i t y ( P < o . o 5 ) over t h e control group. All o t h e r comparisons p r o v e d to be not significant. The m e a n values for t h e t o t a l n u m b e r of i m p l a n t s p e r p r e g n a n t female are shown in Table IV. A n a l y s i s of variance techniques were used in t h e comparisons m a d e between these values. O n l y one significant difference was found b e t w e e n groups. In week 4, t h e 35 ° m g / k g group showed a significant ( P < o . o S ) decrease in t o t a l
IV
= -__ ~ 52/87 =
5°/87 45/87 48/89 51/87
2
57% 52% - o,,o ,~4 - o0 39 60°0
z
7-84 7.74 7.56 7.77 7.68
(rag~hE)
o 3 °o 35 ° 4°0 45 °
z~ 0.22 -~- 0-24 ~ o.I8 i o,29 i 0.23
2
8.I6 7.98 7.85 8.18 7.94
i ± i i ~
o.I9 o.21 o.21 0.20 0.22
3
7.78 8.14 7.85 7.42 8.1o
i ~ ± -'+
0.28 o.2o o.26 0.28 0.26
8.38 7.93 7.41 8.24 7.54
4
37/85 41/87 46/9 o 38/87 41/87
4
4~ ~ 4 i
--= ~ =
0.35 0.30 o.28 b 0.35 0-34
44% 47°° - I oO D 440o 47°0
1
13.16 11.31 8.90 1°'3° 9"23
(mg/kg)
o 3°0 35 ° 400 45 °
y ! ± ~ --
1.98 1.54 1.77 1"7° 1.42
9.77 lO.79 lO.63 11"42 8-07
2
a E x c l u d i n g 4 totally resorbed litters.
Week
Dose
~ ~ ± ~ 7L
1-58 1.65 1.98 1.67 1.,52
lO-89 11.16 11.Ol 11-67 lO.16
3
~ ~ ~ ~ 7}:
2.I9 2.41 2.34 2.53 1-76
11.37 12.1o lO.8,5 9-9 ° 11.61
4
~ ~ ~_ ~ 2-
2.22 2.25 2.60 2.85 2.28
THE MEAN PERCENT OF P O S T I M P L A N T A T I O N DEATltS PER PREGNANT FEMALE (.~ -Z~ SE) a
TABLE V
a Excluding 4 totally resorbed litters. b p < 0.05 .
Week
Dose
) S E ) a'
3 6 / 8 7 -- 4 1 % 4 3 / 8 5 -- 5 1 ° b 4 1 / 9 ° = 46o0 4o/86 47°o 41/87 ~ 47°0
3
THE MEAN NUMBER OF IMPLANTS PER PREGNANT FEMALE ( X
TABLE
ap
<0.05.
44/89 53/89 48/89 47/90
o 3°0 35 ° 4°0
450
•
(mg/kg)
= 49% = 60°./0 = 5435 ~ " D 2 o,o 6 2 / 9 0 = 69°~'a
Week
Dose
THE PERCENT OF FERTILE MATINGS ON THE TOTAL NUMBER OF PAIRED FEMALES
"FABLE III
0.24 o.33 o,22 0.23 0.37
4__ 2.20 :h 1.73 zi 2.15 ~ 1.97 ~ 3.51
22 Z ~ ~ +
~ 54% -- 4 8 ° o 4o3; = 5 i o ,o/ -- 4 5 ° o
12.4o I2.2o I1.47 lO.O8 13,84
5
7.71 7.85 8.34 8.34 7.85
,5
45/84 4°/84 35/87 44/86 39/86
5
-
--6
8.60 lO.64 9 .20 11-29 11.82
i ~ ~ Z ~
1.56 1.7o I'99 1.72 1.58
~ 0.25 --4~ 0.29 ~ 0.28 ~ 0.29 ~ 0.29
-- 4 6 ° 0 -- 53'!,, = 4 4 o ,, = 60% -- 56°(~
. . . .
8.24 7.85 8.16 8.28 8.1o
6
38/83 46/86 38/86 50/83 49/87
6
= 48~o - o~ .)2 ,b - - 54°)~
~ ~ ± 22 ~_
11.o5 Ir.33 lO.31 lO.81 lO.57
o.i O o.i1 o.io o.ii o. I I
~ 0.83 7!: 0.76 Z: 0.87 7:0.82 ~ 0.80
Tota! I-6
8,oi 7,91 7.83 8-o5 7.86
Total 1-6
256/531 270/519 284/524
2 5 o / 5 1 5 - - 49°,0 2 6 8 / 5 1 8 - - 52O/o
T o t a l a:-6
>
aZ
>
ba q.,a
TOXICITY OF A F - 2 IN MICE
239
implants as compared to the controls. All other pair wise comparisons were not significant. Since the percent resorptions did not follow a normal distribution, non-parametric procedures were used in the analyses of these data (Tables I I and V). The procedures used were the Mann-Whitney U test for pair wise comparisons and JONCI
Toxicity test In contrast to the results of others, our toxicity tests reveal a relatively low level of sensitivity to AF-2 on the part of the strains of mice used in these experiments. This is indicated by the lack of significant increases in AF-2 related deaths as well as the lack of any other obvious indications of AF-2 induced toxicity. We observed no signs of intestinal distress or diarrhea which would be expected were the AF-2 effecting a bactericidal action on the intestinal flora of the treated animals. Post-mortems and general observations of treated animals indicate a possible storage of the AF-2 in the stomach and excretion of AF-2 or a metabolic by-product of AF-2 by the kidneys. These observations are cursory in nature and further studies of the metabolic fate of AF-2 in nfice are necessary. It is important to note that in the first and fourth tests, all AF-2 injected animals which starved for 24 h prior to treatment died following treatment. Additional toxicological testing is necessary to further elucidate the cause of increased sensitivity on the part of starved mice to AF-2. Dominant lethal test TONOMURA7 showed an induction by AF-2 of chromatid aberrations in cultured human cells. It is estimated that 30% of induced fetal mortality in mammals m a y be attributed to chromosomal and/or chromatid damage (HANSMANN,1974, personal communication). Our data give no indication of AF-2 induced increases in the rate of fetal death over the six-week test period. Furthermore, our data confirm the results of MIYAjII4 in that we found AF-2 to have had no significant effect on male fertility nor did treatment with AF-2 result in any observed atrophy of the testes. Generally speaking, the results reported in this study show a lack of activity of AF-2 with regard to the induction of dominant lethality in mice. This lack of activity could be explained by one of the following: ( I ) inactivation or breakdown of AF-2 at some point in the gastro-intestinal tract prior to its entering the circulatory system, or inactivation (by the liver or kidney) after entrance into the circulatory system; or (2) AF-2 simply does not induce dominant lethals. One or both of these possibilities could explain our results. The apparent precipitation of AF-2 in the stomach, the eventual disappearance of the orange precipitate from the fundus and the excretion of orange urine would seem to signify the absorption of AF-2 or a metabolic by-product of this compound from the stomach. While our data do not necessarily negate the possibility that AF-2 could induce single gene or specific locus mutations in mice, they have clearly shown that AF-2 or its metabolic
240 by-products did not produce dominant treatment.
E. R. SOARES, W. SHERIDAN lethals during the six-week period following
ACKNOWLEDGMENT T h e a u t h o r s w i s h t o e x p r e s s t h e i r a p p r e c i a t i o n t o Dr. M. D. HOGAN a n d D r . J . K. HASEMAN for t h e i r a d v i c e r e g a r d i n g t h e s t a t i s t i c a l t r e a t m e n t of t h e s e d a t a . REFERENCES I JONCKHEERE, A. R., A distribution-free K-sample test against ordered alternatives, Biometrika, 41 (1954) 133-1452 KADA, T., Escherichia coli mutagenieity of furylfuramide, Jpn. J. Genet., 48 (1973) 3Ol-3O5. 3 KONDO, S., AND H. ICHIKAWA-RYO, Testing and classification of mutagenicity of furylfuramide in Escherichia coli, Jpn. J. Genet., 48 (1973) 295-3 oo4 MIYAJII, T., Acute and chronic toxicity of furylfuramide in rats and mice, Tohoku J. Exp. Med., lO3 (1971) 331-369. .5 ONG, TONG-MAN, AND SHAHIN, M. M., Mutagenic and recombinogenie activities of the food additive furylfuramide in eukaryotes, Science, 184 (1974) lO86-1o87. 6 TAZIMA, W., AND ONIMARU, K.. Results of mutagenicity testing for some nitrofuran derivatives in a sensitive test system with silkworm oocytes (Abstract), Mutation Res., 26 (1974) 44 TM 7 TONOMORA, A., AND SASAKI, M. S., Chromosome aberrations and DNA repair synthesis in cultured h u m a n cells exposed to nitrofurans, Jpn. J. Genet., 48 (1973) 291 294.