HOECHST
JAPAN
iJVlITED
Mutagen&(.i.tp / (Experimental
T&t
of
Piroctone
Olamine
period:
Bugukt 22 - 28, 198i)
Department Oiamu Nakan
-2-
route 30 rag/kg of sodium phenobarbital (PB) (dissolved in physiological saline at a rate of 10 mg/ml) on the 1st day of experiment and 60 mg/kg each of PB (likewise, at a rate of 20 mg/ml) for the subsequent 3 days. On the 3rd day of this treatment period, they received 80 mgfkg, i-p., of 5,6-benzoflavone (dissolved in co= oil a& a rate of 10 mg/ml) as well. On the next day of the last PB administration, the animals were sacrificed, and their livers were removed. The liver was washed uith 0.15 M KCl, placed in 3 volumes of 0.15 M KC1 (3 ml/g of wet liver), and homogenized. The homogenate was centrifuged at 9000 xg for 10 min, and the snpernatant served as the S-9 fraction. It was frozen in dry ice-acetone and quickly thawed when necessary. For this procedure used were all sterilized and cooled tools and solutions. S-9 Mix was adjusted milliliter and used: to consist of the following components per
S-9 fraction MgClz (0.4 M) + KC1 (1.65 M) Glucose-6-phosphate (1 H) NADPH (0.1 M) NADH (0.1 M) Sodium phosphate buffer (q.2 M) &?O Minimal glucose agar plate:
0.1 0.02 0.005 0.04 0.04 0.5 0.295
d
Agar (60 g>, glucose (80 g>, and Vogel-Banner minimal medium (400 ml> in separate bottles received 2800, 400 and 400 ml of distilled water, respectively. These 3 solutions were autoclaved, then mixed all together, and distributed into sterile Petri dishes (30 ml/dish) to make the agar plates.
The Vogel-Bonner components per
minimal medium liter of. distilled
used contained water.
the
following
MgS04-7H~0 Citric acid K, WO‘ N-b H, PO‘ NaOH The medium prepared thawed when used. was autoclaved, stored
2 g 20 100 19.2 6.6 in a frozen state, and
-3-
Top agar: The top agar was made by mixing 10 volumes'+of"-k'autoclavid solution of agar (0.6 wtX) and sodium chloride (0.5 wt%) volume of a sterile aqueous solution of L-histidine (0.5 (0.5 mM) (0.5 mM tryptophan in place of L-histidine-biotin coli strain). Test procedure: aq&&s‘ Iii_ and one mM)-biotin for the 2. *'";'*'
The test was carried out by the preincubation method [4] as follows. Sterile test tubes received 50 ~1 of the compound solution, 0.5 ml of sodium phosphate buffer or S-9 Mix, and 0.1 ml of each bacterial and the tubes were preincubated vith shaking suspension in that order, at 30°C for 30 min. To each of them added was 2 ml of the top agar, over the minimal glucose agar and each mixture was spread evenly After the agar hardened, the plates were incubated at 37'C for plate. 48 hr, and the number of revertant colonies formed was counted. The compound solution and the following positive with 2 plates per concentration for each strain, with 4 plates. control, The sterility tests of and buffer were done with 2 plates. Positive controls (AF-2) (ENNG) controls were tested and the solvent the solvent, S-9 Mir,
2-(2-Furyl)-3-(5-nitro-2-furyljacrylamide Benzo(a)pyrene (B(a)P) N-Ethyl-N'-nitro-N-nitrosoguanidine 2-Aminoanthracene (2AA) 9-Aminoacridine (9AA) 4-Nitroquinoline-l-oxide (4NQC)
Results
growth was inhibited in any strain As Tables 1 - 6 show, bacterial the PO application at 100 and/or 200 ug/plate in the presence and absence of S-9 Mix. In addition, in the case of TAlOO and TAl535, growth was inhibited even at 50 pg/plate. At 20 pg/plate or less, numbers of colonies formed were not different, regardless of the strain, from those in the solvent control plates.
by
the
-4-
Summary and Conclusion
PO was examined for its mutagenicity by the reverse mutation test with S. typhimurium TAlOO, TA98, TA1535, TA1537, TA1538 and E. coli WP2 &A-, which is known as the most available method for zetaon of mutagens of chemicals. The tests depended on the preincubation method, and were done with the PO concentrations of 1 - 200 rig/plate in both the cases with and without S-9 Mix. At the highest 2 or 3 concentrations used, the PO application inhibited bacterial growth. At lower concentrations, however, the PO plates showed no abnormal increase in the number of revertant colonies of-any strain formed, when compared with the solvent control plates. From the results, PO is considered norrmutagenic in this test system.
References
1. 2.
B.N. Ames, Acad. Sci. J. McCann, Acad. Sci.
W.E. Durston, E. Yamasaki, USA, 70, 2281-2285, 1973. N.E. Spingam, J. Kobori, USA, 72, 979-983, 1975.
and F.D.
Lee:
Proc.
Nat.
and B.N.
Ames:
Proc.
Nat.
3.
T. Matsushima, M. Sawamura, K. Hara, and T. Sugimura: In: In Vitro Metabolic Activation in/Mutagenesis Testing (eds. F.J.de Serres, J.R. Fouts, J.R. Bend, and R.M. Philpot), pp. 85-88, North-Holland Biomedical Press, Amsterdam, 1976. T. Yahagi, M. Nagao, Y. Seino, T. Matsushima, Okada: Mutation Res. 48, 121-130, 1977. T. Sugimura, and M.
4.
kn
-
-5-
Table
1 .
Effect
of piroctone
woo
olamine
an
S. ltyph imuriwn
Compound concrktration
(d/p late >
No. of revertant
-s-9Mix .
95)
coldtiies
Der Dlate + s-9 M ix
Man 8e8
Solvent
control
(DGO)
1
..
(84.
69.
70.
Mean
80 (86. 86. 87. 94)
( ( ( (
83. 96. 80. 72,
91 96 70 62 80 66 44*)
> )
87 96
( ( ( ( ( ( (
82. 87, 84, 93. 84. 56. * 28:
92 74 81 95 89 62*) 337 Killing)
) ) > > )
87 81 83 94 8.7 * 59 31* Killing
2
5 10 20 50 100 200
> 75 > 67 > > 78 66 40* Killing
( -75. ( ( (Killing, 65, 35:
Killing) AF-2
(RiIlixs.
B
(a)
P
0. 01 No. of revertant DMSO: AF-2: B(a)P:
(508. 566) 537 .-. . _ (6i2. .
5 684) 648
Dimethyl sulfoxide 2-(2-Furyl)-.3-(5nitro-2-furyl)acrylamide Benzo(a)pyrene growth in'nibition was observed.
* Bacterial
Table
2
Effect of piroctone olamine S. tzjphinnkim TA98
gn.
-
Compound
concentration
(d..platc 1
T(28,
No.
of
revertant Mean
colonies
per + s-9
plate Mix Mean .
42) 56
- s-9 Mix
2s. 15. 33) 26 (62.
Solvent
control
(DMSO)
67.
54.
1 2 5 10 20 50 100 200 -
( ( ( (
28. 28, 33. 24.
28 23 24 19 21 30 193
) )
28 26
.( ( ( ( ( ( ( :Killing.
48. 54. 58. 43. 71. 61. 34.
47 46 52. 56 59 43 52 Killing)
> 48 ) > ) ) > 50 55 50 6.5 52
> 29 ) 22
(-24, ( ( 30, 1-7:
> 23 ) 30 18*
> 43 Killing
(Killing,
KiIling)RiIling AF-2 0. 1
_ / Compound
B (a) P 5
546 (312. 304) 308
colonies/plate DMSO: AF-2: B(a)?:
(524.
568’ )
-Dimethyl sulfoxide 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide Benzo(a)pyrene growth inhibition was observed.
* Bacterial
Table 3
Effect
s. typh
of piroctone olamine imruyim TAl535
on
Compound concentration
(&/plate >
T
1 2 5 10 (
No. of revertant-colonies - s-9 Mix
Mean (18. 13. 23. 18) 18 (15.
per plate
+ s-g Mix
Mean
6. 7. 12) 10
’
Solvent
control
WSO)
12. 18. 14. 18,
18 18 19.) 15 17 7*) IlingI
) >
1.5 18 17
( (10, ( ( ( ( ( (Killing.
14,
9 6)
>
12 8 .__ -
( ( ( .
8.
9. 9.15 8, 4%
12 IO’ )
>
10 10
> )
17 17 7* Ki lling Killing
.20 50 100 200
c- 16, ( (-fci Iling. 7:
> 7) 4f)
12 8 4* Killing
Ki
’
(Ki Iling,
Killing)
Killing) 2 AA 2
Compound
ENNC
5 il.456. 1332) 1394 (139.
157)
148
DMSO: Dimethyl sulfoxide ENNG: N-Ethyl-N' -nitro-N-niktisoguanidine 2-Aminoanthracene 2A.A: * Bacterial growth inhibition was observed.
a
Table
.4
Effect of piroctone oltine Tu.537 s- QiPh imurium
on.
:.
Compound concentration
c;9/o1atc 1
T
1
”
No. of
revertant
colonies
per
plate
Mean-
Solvent
control
(DMSO)
- s-9 Mix’ - Mean ( 5.7.a.a > 7
-I- s-9.Mix..
( 1 0. 1 0. 1 2. 1 5 > 1’ 2
( ( ( ( ( (5, ‘ (
4, 7, 3. 4. 6.12)
4) 8) 5) 9)
4 8 4 7 9
(
10,
14 11 I6
> 12
> 10
L
(
( ( ( (
8.
13-e 11. 13, IO.
5 10 20 50 100’ 200
> -1s
18 1 is
14 13 14 .) > 14 12
8) 5*, I*>
7 3*
( (Killing.
IO,
> 12
(Killing,
Killing)Killing 9AA
80
Killing)Rilling B (a> P
5
Compound Positive control Concentration Lf/pl.da) No. of revertant colonies/plate. Dimethyl sulfoxide 9-Aminoacridine Benzo(a)pyrene growth inhibition
-
(236.
258)
247
(118.
85)
102
DMSO: 9AA: B(a)P:
* Bacterial
was observed.
Table
5.'Effect
of
piroctone
olamine
on
:
s- typh i.lmLrim -ml538
, Compound concentration
(H/plate
c-
-_
>
T
(5.
No. - s-9
of revertant‘ Mix Mean
16) 11
colonies
per
plate Mean .
+ s-9
(28. 27.
Mix
32. 36) 31
Solvent
control
QIMSO)
6. 16.
1 2 5 10 20 50 100 2DO (Ki
(
( ( ( ( ( ( Iling.
88
10, 18, 11. 15, 10, 3:
-15 16 16 14 17 14 8*)
> ) > ) ) >
12 13 17 13 16 12 6* Kil ling
( ( ( ( ( ( ( (
31. 32. 28, 36. 35. 35. 14: 5:
34 35 26 28 37 31 19*) 6*)
) > ) > )
33 34 27 32 36
> 33 17* 6*
Ki liing) 4NQO 0. 25
l Compound
-_
(293.
B (a)
5
P
‘ ~ 314) 304 (144. 154) 149
DMSO: 4NQ0 : B(a)P: * Bacterial
Dimethyl sulfoxide 4-Nitroquinoline-l-oxide Benzo(a)pyrene growth inhibition was observed.
Table 6 Effect
E. coZi
of pikoctone
Wl’ uvrA2
olamine
on
Compound concentration
(pF/plate)
T
.(26.
No. of revertant
s-9
21.
colonies
+
per plate
s-9
23.
Mix
Mix MBl3
31. 26) 27 ‘ . 28 28
Mean
20. 24) 23 (277.
Solvent
control
(DMSO) .
( ( ( ( ( (
18. 17, 18. 16, 24, 18.
20 21 21 21 22 I9 15
> ) > >
19 19 20 19
( (
30. 31.
26 25 24 28 20 20 17 14*)
) )
(
( ( ( ( (
21.
25. 33, 33, 30. 12:
) .23 ) ) > > 27 27 27 24 * 13
> 21 ) ) 19 19 Killing
( 23,
(Killing,
Killing) AF-2 0. 01
2 AA
5 224 (796. 830) 813
(236.
212)
DMSO: Dimethyl sulfoxide U-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide 2AA: 2-Aminoanthracene * Bacterial growth inhibition was observed.