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mammalian microsome assay
Mutation Research, 157 (1985) 49-52
49
Elsevier MTR 00986
Evaluation of 1,3-pentadiene for mutagenicity by the Salmonella/mammalian microsome assay M i c h a e l B. L i e w e n a n d E l m e r H . M a r t h Department of Food Science, and The Food Research Institute, University of Wisconsin-Madison, Madison, W153706 (U.S.A.)
(Received30 July 1984) (Revision received24 December1984) (Accepted 11 February 1985)
Summary 1,3-Pentadiene, a food contaminant produced by some molds when they metabolize sorbic acid, was tested for mutagenicity, using variations of the Salmonella/mammalian microsome assay. The chemical was incorporated into the test system (with and without $9 mix) by 3 methods: (a) the standard plate incorporation assay, (b) a liquid preincubation procedure and (c) exposure of test bacteria in the soft agar overlay to gaseous 1,3-pentadiene. The chemical was extremely toxic to the test bacteria with amounts as low as 2.0/~g/plate causing cell death. However, none of the nonlethal concentrations tested by any of the methods was mutagenic to Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 or TA1538.
Sorbic acid is commonly used as an antimicrobial preservative in a variety of foods because of its broad spectrum of action and extremely low level of toxicity in humans. Some strains of molds can decarboxylate sorbic acid (CH3CH=CHCH= CHCOOH) to 1 , 3 - p e n t a d i e n e ( C H 3 C H = CHCH=CH2), a volatile compound with an unpleasant hydrocarbon-like odor and taste (Marth et al., 1966). Presence of 1,3-pentadiene has been reported in sorbate-treated cheeses (Finol et al., 1982; Horwood et al., 1981; Marth et al., 1966) and in a sorbate-treated noncarbonated beverage (G6tz et al., 1978). No data exist on the carcinogenicity or mutagenicity of 1,3-pentadiene; however, 1,3-butadiene is mutagenic to several strains of bacteria in the Salmonella/mammalian microsome assay (de Meester et al., 1978) and could be metabolized by mammalian tissues to 1,2-epoxybutane-3, which is carcinogenic (Malvoisin et al., 1979). Since 1,3-
pentadiene differs from 1,3-butadiene by only a methylene group, it is possible that the former could have properties similar to those of the latter. Because 1,3-pentadiene can be found in mold-contaminated sorbate-treated foods, it is important to determine if its presence poses a health or safety hazard. In this study, the method of Ames et al. (1975) was used to test 1,3-pentadiene for its mutagenicity to Salmonella typhimurium.
Materials and methods 1,3-Pentadiene (99% pure, CAS No. 2004-70-8) was obtained from Aldrich Chemical Co. (Milwaukee, WI), and before use was checked for impurities by gas and thin-layer chromatography. Dimethyl sulfoxide (DMSO, AR grade) was obtained from Fisher Scientific Co. (Fair Lawn, N J). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
0165-1218/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
50
The method of Ames et al. (1975) was followed throughout this study with slight modifications. Because of the volatility and low boiling point (42°C) of liquid 1,3-pentadiene, 3 methods for incorporation of the chemical into the test system were used: (a) the standard plate incorporation assay; (b) a liquid preincubation method similar to that described by Yahagi et al. (1975), except that preincubation was done at 25°C for 30 min; and (c) exposure of test bacteria in a soft overlay to gaseous 1,3-pentadiene as described by Rannug et al. (1974), Bartsch et al. (1975) and Ames et al. (1975). The bacterial tester strains TA97, TA98, TA100, TA1535, TA1537 and TA1538 were obtained from B. Ames, University of California, Berkeley, CA. Upon receiving the strains and before each experiment, the specific mutagenic properties of each strain were tested as recommended by Ames et al. (1975). Cultures were stored at - 70°C before use. To determine the proper dosages for mutagenic testing, lethality curves were determined for each of the 3 methods used to incorporate 1,3-pentadiene into the test system (Figs. 1 and 2). About 1000 bacteria were exposed to various concentrations of 1,3-pentadiene by the 3 methods of incorporation that we used. The chemical was tested to concentrations in the toxic range. With the standard plate assay and liquid preincubation, the bacteria were overlayed onto nutrient agar in petri dishes and then incubated at 37°C for 48 h. We used nutrient agar because all viable bacteria grow on this medium rather than just the histidine revertants. Strain TA100 was most sensitive to 1,3pentadiene and was used for lethality determinations. In the standard plate incorporation and the liquid preincubation mutagenicity assays, 1,3-pentadiene was tested at concentrations ranging from 0.1 to 5.0 /~g/plate. To facilitate working with such small volumes, a 1:10 dilution (w/v) of 1,3-pentadiene/DMSO was used. When plates were exposed to gaseous 1,3-pentadiene, a 1-ml (0.68 mg) or 5-ml (3.41 mg) frozen portion of the chemical was added to a 10-1 desiccator containing test plates. The dessicator was immediately sealed and partially evacuated to allow the solid chemical to vaporize. Air was then introduced until atmospheric pressure was reached. A magnetic stirring
bar was included in the sealed container and rotated at 60 rpm during the incubation period of 48 h at 37°C. Each concentration of 1,3-pentadiene was tested on all 6 Salmonella tester strains both with (50 ~1 microsomal fraction per plate) and without metabolic activation. The rat-liver microsomal fraction in the $9 mix was purchased from Litton Bionetics, Inc. (Kensington, MD). The Aroclor 1254induced microsomal fraction was prepared from 8-10-week-old Sprague-Dawley male rats and Litton Bionetics, Inc. reported the protein content to be 21 m g / m l and the benzo[a]pyrene hydrolase activity to be 22.7 nmoles hydroxybenzopyrene/20 m i n / m g protein. All other components of the $9 mix were purchased from Sigma. Four positive mutagenesis controls were included in each experiment. 2-Aminofluorene and aflatoxin B 1 are strong mutagens when metabolically activated by the enzymes in the $9 mix which confirms that the $9 mix is active. N-Methyl-N'nitro-N-nitrosoguanidine ( M N N G ) and 9aminoacridine are strong mutagens which do not require metabolic activation. Sterility controls of all solutions, test chemicals and $9 mix were routinely included in each experiment. Results and discussion
A significant increase in revertant colonies per plate was not observed at any concentration of 1,3-pentadiene tested by any of the methods used. Results obtained using the liquid preincubation and desiccator incubation techniques are shown in
10001 800 600 400 200
~iilllllllll 10
20
30
40
b'b
60
70-80
90 1~0
1,3-PENTADIENE [ L / 6 ]
Fig. 1. Viability of Salmonella typhimurium strain TA100 when exposed to 1,3-pentadiene dissolved in DMSO. O, liquid preincubation assay; A, standard plate incorporation. L / 6 = microliters.
51 TABLE 1 MUTAGENICITY TEST FOR 1,3-PENTADIENE USING LIQUID PREINCUBATION AND DESICCATOR EXPOSURE IN THE PRESENCE ( + ) OR ABSENCE ( - ) OF $9 MIX Dose (/t g/plate)
Controls No additions DMSO, 100/~1 Aflatoxin B1 2-Aminofluorene 9-Aminoacridine MNNG
1 10 10 2
1,3-Pentadiene b
0.1 0.5 1.0 2.5 5.0
1,3-Pentadiene ¢.d
a b c d *
1 5
Revertant colonies/plate a TA97
TA98
TA100
TA1535
TA1537
TA1538
--
+
--
+
--
+
--
+
--
+
--
+
174 169 212 185 2500 980
259 230 373 581 -
26 23 41 33 37 53
45 52 2200 2500 -
125 159 131 122 183 2500
140 139 2500 2500 -
26 18 23 19 1550
29 18
12 15
14 21
24 -
19 42 35
23
25 31 19 23 27
34 29 2200 -
175 193 211 52 18
253 278 220 142 37
26 32 25 28 7
36 41 37 21 32
116 111 103 16
87 82 113
24 18 22 18 7
19 19 18 23 3
17 26 16 * *
28 26 21 * *
26 31 14 6 *
31 34 39 22
156 189
161 136
35 29
22 34
11 15
24 14
8 9
11 15
30 22
* 135 128
* * 117 93
* 35 38
Average of at least 4 plates. Liquid preincubation. Desiccator exposure. ml/10-1 desiccator. Denotes no growth of bacterial strains.
T a b l e 1. S i m i l a r r e s u l t s w e r e a l s o o b t a i n e d u s i n g the standard plate assay (data not shown). The number of revertant colonies per plate did not i n c r e a s e w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f 1,3pentadiene nor was there a mutagenic response after exposure to the mixed-function oxidase enz y m e s o f t h e $9 f r a c t i o n . 1 , 3 - P e n t a d i e n e w a s ext r e m e l y t o x i c to t h e b a c t e r i a t e s t e r s t r a i n s ; 2.0 # g / p l a t e c a u s e d cell d e a t h in t h e l i q u i d p r e i n c u b a t i o n m e t h o d (Fig. 1). T h e r e l a t i v e s e n s i t i v i t y o f t h e
1000,
• %
800 600
40O 200
2
4
6
~
8
10 12
14 16
i
18 ~0 2 2
1,3-PENTADIENE IN IO-L DESICCATOR
~
tester strains was TA100 > TA37 > TA38 > TA35 > TA97 > TA98 with TA100 being most sensitive a n d T A 9 8 l e a s t s e n s i t i v e to 1 , 3 - p e n t a d i e n e . A g r e a t e r c o n c e n t r a t i o n w a s r e q u i r e d t o c a u s e cell death in the standard plate assay, than in the liquid preincubation method, possibly because in t h e p l a t e a s s a y t h e c h e m i c a l is q u i c k l y v o l a t i l i z e d by the molten top agar. The gaseous state of 1,3-pentadiene was also very toxic to the bacteria ( F i g . 2). T h e t o x i c i t y o f 1 , 3 - p e n t a d i e n e t o h u m a n s is u n k n o w n ; h o w e v e r , it is u n l i k e l y t h a t m o r e t h a n very minute quantities of the chemical would be c o n s u m e d b e c a u s e o f its v e r y u n p l e a s a n t s m e l l a n d taste. N e g a t i v e r e s u l t s w i t h t h e test s y s t e m s u s e d c o u l d m e a n e i t h e r t h a t 1 , 3 - p e n t a d i e n e is n o t m u t a g e n i e o r t h a t if it is m u t a g e n i c , t h a t p r o p e r t y w a s not detected by the procedures we used.
i
24
Acknowledgement
(ml)
Fig. 2. Viability of Salmonella typhimurium strain TA100 when exposed to gaseous 1,3-pentadiene.
Research supported by the College of Agricultural and Life Sciences, University of WisconsinMadison.
52
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian microsome mutagenicity test, Mutation Res., 31,347-364. Bartsch, H., C. Malaveille and R. Montesano (1975) Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in S. typhimurium strains, Int. J. Cancer, 15, 429-437. de Meester, C., F. Poncelet, M. Roberfroid and M. Mercier (1978) Mutagenicity of butadiene and butadiene monoxide, Biochern. Biophys. Res. Commun., 80, 298-305. Finol, M.L., E.H. Marth and R.C. Lindsay (1982) Depletion of sorbate from different media during growth of Penicillium species, J. Food Prot., 45, 398-404. G6tz, M., A. Heffter, I. Jaeger-Stephani and W. Nonnweiler (1978) Mikrobieller Abbau von Sorbins~iure in Erfrischungsgetr~tnken zu Pentadien-(1-3), Lebensmittelchem. Gericht. Chem., 32, 77-78.
Horwood, J.F., G.T. Lloyd, E.H. Ramshaw and W. Stark (1981) An off-flavour associated with the use of sorbic acid during feta cheese maturation, Aust. J. Dairy Technol., 36, 38-40. Malvoisin, E., G. Lhoest, F. Poncelet, M. Roberfroid and M. Mercier (1979) Identification and quantitation of 1,2epoxybutene-3 as the primary metabolite of 1,3-butadiene, J. Chromatogr., 178, 419-425. Marth, E.H., C.M. Capp, L. Hasenzahl, H.W. Jackson and R.V. Hussong (1966) Degradation of potassium sorbate by Penicillium species, J. Dairy Sci., 49, 1197-1205. Rannug, U., A. Johansson, C. Ramel and C.A. Wachtmeister (1974) The mutagenicity of vinyl chloride after metabolic activation, Ambio, 3, 194-197. Yahagi, T., M. Degawa, Y. Seino, T. Matsushima, M. Nagao, T. Sugimura and Y. Hashimoto (1975) Mutagenicity of carcinogenic azo dyes and their derivatives, Cancer Lett., 1, 91-96.
49
Elsevier MTR 00986
Evaluation of 1,3-pentadiene for mutagenicity by the Salmonella/mammalian microsome assay M i c h a e l B. L i e w e n a n d E l m e r H . M a r t h Department of Food Science, and The Food Research Institute, University of Wisconsin-Madison, Madison, W153706 (U.S.A.)
(Received30 July 1984) (Revision received24 December1984) (Accepted 11 February 1985)
Summary 1,3-Pentadiene, a food contaminant produced by some molds when they metabolize sorbic acid, was tested for mutagenicity, using variations of the Salmonella/mammalian microsome assay. The chemical was incorporated into the test system (with and without $9 mix) by 3 methods: (a) the standard plate incorporation assay, (b) a liquid preincubation procedure and (c) exposure of test bacteria in the soft agar overlay to gaseous 1,3-pentadiene. The chemical was extremely toxic to the test bacteria with amounts as low as 2.0/~g/plate causing cell death. However, none of the nonlethal concentrations tested by any of the methods was mutagenic to Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 or TA1538.
Sorbic acid is commonly used as an antimicrobial preservative in a variety of foods because of its broad spectrum of action and extremely low level of toxicity in humans. Some strains of molds can decarboxylate sorbic acid (CH3CH=CHCH= CHCOOH) to 1 , 3 - p e n t a d i e n e ( C H 3 C H = CHCH=CH2), a volatile compound with an unpleasant hydrocarbon-like odor and taste (Marth et al., 1966). Presence of 1,3-pentadiene has been reported in sorbate-treated cheeses (Finol et al., 1982; Horwood et al., 1981; Marth et al., 1966) and in a sorbate-treated noncarbonated beverage (G6tz et al., 1978). No data exist on the carcinogenicity or mutagenicity of 1,3-pentadiene; however, 1,3-butadiene is mutagenic to several strains of bacteria in the Salmonella/mammalian microsome assay (de Meester et al., 1978) and could be metabolized by mammalian tissues to 1,2-epoxybutane-3, which is carcinogenic (Malvoisin et al., 1979). Since 1,3-
pentadiene differs from 1,3-butadiene by only a methylene group, it is possible that the former could have properties similar to those of the latter. Because 1,3-pentadiene can be found in mold-contaminated sorbate-treated foods, it is important to determine if its presence poses a health or safety hazard. In this study, the method of Ames et al. (1975) was used to test 1,3-pentadiene for its mutagenicity to Salmonella typhimurium.
Materials and methods 1,3-Pentadiene (99% pure, CAS No. 2004-70-8) was obtained from Aldrich Chemical Co. (Milwaukee, WI), and before use was checked for impurities by gas and thin-layer chromatography. Dimethyl sulfoxide (DMSO, AR grade) was obtained from Fisher Scientific Co. (Fair Lawn, N J). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
0165-1218/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
50
The method of Ames et al. (1975) was followed throughout this study with slight modifications. Because of the volatility and low boiling point (42°C) of liquid 1,3-pentadiene, 3 methods for incorporation of the chemical into the test system were used: (a) the standard plate incorporation assay; (b) a liquid preincubation method similar to that described by Yahagi et al. (1975), except that preincubation was done at 25°C for 30 min; and (c) exposure of test bacteria in a soft overlay to gaseous 1,3-pentadiene as described by Rannug et al. (1974), Bartsch et al. (1975) and Ames et al. (1975). The bacterial tester strains TA97, TA98, TA100, TA1535, TA1537 and TA1538 were obtained from B. Ames, University of California, Berkeley, CA. Upon receiving the strains and before each experiment, the specific mutagenic properties of each strain were tested as recommended by Ames et al. (1975). Cultures were stored at - 70°C before use. To determine the proper dosages for mutagenic testing, lethality curves were determined for each of the 3 methods used to incorporate 1,3-pentadiene into the test system (Figs. 1 and 2). About 1000 bacteria were exposed to various concentrations of 1,3-pentadiene by the 3 methods of incorporation that we used. The chemical was tested to concentrations in the toxic range. With the standard plate assay and liquid preincubation, the bacteria were overlayed onto nutrient agar in petri dishes and then incubated at 37°C for 48 h. We used nutrient agar because all viable bacteria grow on this medium rather than just the histidine revertants. Strain TA100 was most sensitive to 1,3pentadiene and was used for lethality determinations. In the standard plate incorporation and the liquid preincubation mutagenicity assays, 1,3-pentadiene was tested at concentrations ranging from 0.1 to 5.0 /~g/plate. To facilitate working with such small volumes, a 1:10 dilution (w/v) of 1,3-pentadiene/DMSO was used. When plates were exposed to gaseous 1,3-pentadiene, a 1-ml (0.68 mg) or 5-ml (3.41 mg) frozen portion of the chemical was added to a 10-1 desiccator containing test plates. The dessicator was immediately sealed and partially evacuated to allow the solid chemical to vaporize. Air was then introduced until atmospheric pressure was reached. A magnetic stirring
bar was included in the sealed container and rotated at 60 rpm during the incubation period of 48 h at 37°C. Each concentration of 1,3-pentadiene was tested on all 6 Salmonella tester strains both with (50 ~1 microsomal fraction per plate) and without metabolic activation. The rat-liver microsomal fraction in the $9 mix was purchased from Litton Bionetics, Inc. (Kensington, MD). The Aroclor 1254induced microsomal fraction was prepared from 8-10-week-old Sprague-Dawley male rats and Litton Bionetics, Inc. reported the protein content to be 21 m g / m l and the benzo[a]pyrene hydrolase activity to be 22.7 nmoles hydroxybenzopyrene/20 m i n / m g protein. All other components of the $9 mix were purchased from Sigma. Four positive mutagenesis controls were included in each experiment. 2-Aminofluorene and aflatoxin B 1 are strong mutagens when metabolically activated by the enzymes in the $9 mix which confirms that the $9 mix is active. N-Methyl-N'nitro-N-nitrosoguanidine ( M N N G ) and 9aminoacridine are strong mutagens which do not require metabolic activation. Sterility controls of all solutions, test chemicals and $9 mix were routinely included in each experiment. Results and discussion
A significant increase in revertant colonies per plate was not observed at any concentration of 1,3-pentadiene tested by any of the methods used. Results obtained using the liquid preincubation and desiccator incubation techniques are shown in
10001 800 600 400 200
~iilllllllll 10
20
30
40
b'b
60
70-80
90 1~0
1,3-PENTADIENE [ L / 6 ]
Fig. 1. Viability of Salmonella typhimurium strain TA100 when exposed to 1,3-pentadiene dissolved in DMSO. O, liquid preincubation assay; A, standard plate incorporation. L / 6 = microliters.
51 TABLE 1 MUTAGENICITY TEST FOR 1,3-PENTADIENE USING LIQUID PREINCUBATION AND DESICCATOR EXPOSURE IN THE PRESENCE ( + ) OR ABSENCE ( - ) OF $9 MIX Dose (/t g/plate)
Controls No additions DMSO, 100/~1 Aflatoxin B1 2-Aminofluorene 9-Aminoacridine MNNG
1 10 10 2
1,3-Pentadiene b
0.1 0.5 1.0 2.5 5.0
1,3-Pentadiene ¢.d
a b c d *
1 5
Revertant colonies/plate a TA97
TA98
TA100
TA1535
TA1537
TA1538
--
+
--
+
--
+
--
+
--
+
--
+
174 169 212 185 2500 980
259 230 373 581 -
26 23 41 33 37 53
45 52 2200 2500 -
125 159 131 122 183 2500
140 139 2500 2500 -
26 18 23 19 1550
29 18
12 15
14 21
24 -
19 42 35
23
25 31 19 23 27
34 29 2200 -
175 193 211 52 18
253 278 220 142 37
26 32 25 28 7
36 41 37 21 32
116 111 103 16
87 82 113
24 18 22 18 7
19 19 18 23 3
17 26 16 * *
28 26 21 * *
26 31 14 6 *
31 34 39 22
156 189
161 136
35 29
22 34
11 15
24 14
8 9
11 15
30 22
* 135 128
* * 117 93
* 35 38
Average of at least 4 plates. Liquid preincubation. Desiccator exposure. ml/10-1 desiccator. Denotes no growth of bacterial strains.
T a b l e 1. S i m i l a r r e s u l t s w e r e a l s o o b t a i n e d u s i n g the standard plate assay (data not shown). The number of revertant colonies per plate did not i n c r e a s e w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f 1,3pentadiene nor was there a mutagenic response after exposure to the mixed-function oxidase enz y m e s o f t h e $9 f r a c t i o n . 1 , 3 - P e n t a d i e n e w a s ext r e m e l y t o x i c to t h e b a c t e r i a t e s t e r s t r a i n s ; 2.0 # g / p l a t e c a u s e d cell d e a t h in t h e l i q u i d p r e i n c u b a t i o n m e t h o d (Fig. 1). T h e r e l a t i v e s e n s i t i v i t y o f t h e
1000,
• %
800 600
40O 200
2
4
6
~
8
10 12
14 16
i
18 ~0 2 2
1,3-PENTADIENE IN IO-L DESICCATOR
~
tester strains was TA100 > TA37 > TA38 > TA35 > TA97 > TA98 with TA100 being most sensitive a n d T A 9 8 l e a s t s e n s i t i v e to 1 , 3 - p e n t a d i e n e . A g r e a t e r c o n c e n t r a t i o n w a s r e q u i r e d t o c a u s e cell death in the standard plate assay, than in the liquid preincubation method, possibly because in t h e p l a t e a s s a y t h e c h e m i c a l is q u i c k l y v o l a t i l i z e d by the molten top agar. The gaseous state of 1,3-pentadiene was also very toxic to the bacteria ( F i g . 2). T h e t o x i c i t y o f 1 , 3 - p e n t a d i e n e t o h u m a n s is u n k n o w n ; h o w e v e r , it is u n l i k e l y t h a t m o r e t h a n very minute quantities of the chemical would be c o n s u m e d b e c a u s e o f its v e r y u n p l e a s a n t s m e l l a n d taste. N e g a t i v e r e s u l t s w i t h t h e test s y s t e m s u s e d c o u l d m e a n e i t h e r t h a t 1 , 3 - p e n t a d i e n e is n o t m u t a g e n i e o r t h a t if it is m u t a g e n i c , t h a t p r o p e r t y w a s not detected by the procedures we used.
i
24
Acknowledgement
(ml)
Fig. 2. Viability of Salmonella typhimurium strain TA100 when exposed to gaseous 1,3-pentadiene.
Research supported by the College of Agricultural and Life Sciences, University of WisconsinMadison.
52
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian microsome mutagenicity test, Mutation Res., 31,347-364. Bartsch, H., C. Malaveille and R. Montesano (1975) Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in S. typhimurium strains, Int. J. Cancer, 15, 429-437. de Meester, C., F. Poncelet, M. Roberfroid and M. Mercier (1978) Mutagenicity of butadiene and butadiene monoxide, Biochern. Biophys. Res. Commun., 80, 298-305. Finol, M.L., E.H. Marth and R.C. Lindsay (1982) Depletion of sorbate from different media during growth of Penicillium species, J. Food Prot., 45, 398-404. G6tz, M., A. Heffter, I. Jaeger-Stephani and W. Nonnweiler (1978) Mikrobieller Abbau von Sorbins~iure in Erfrischungsgetr~tnken zu Pentadien-(1-3), Lebensmittelchem. Gericht. Chem., 32, 77-78.
Horwood, J.F., G.T. Lloyd, E.H. Ramshaw and W. Stark (1981) An off-flavour associated with the use of sorbic acid during feta cheese maturation, Aust. J. Dairy Technol., 36, 38-40. Malvoisin, E., G. Lhoest, F. Poncelet, M. Roberfroid and M. Mercier (1979) Identification and quantitation of 1,2epoxybutene-3 as the primary metabolite of 1,3-butadiene, J. Chromatogr., 178, 419-425. Marth, E.H., C.M. Capp, L. Hasenzahl, H.W. Jackson and R.V. Hussong (1966) Degradation of potassium sorbate by Penicillium species, J. Dairy Sci., 49, 1197-1205. Rannug, U., A. Johansson, C. Ramel and C.A. Wachtmeister (1974) The mutagenicity of vinyl chloride after metabolic activation, Ambio, 3, 194-197. Yahagi, T., M. Degawa, Y. Seino, T. Matsushima, M. Nagao, T. Sugimura and Y. Hashimoto (1975) Mutagenicity of carcinogenic azo dyes and their derivatives, Cancer Lett., 1, 91-96.