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Fluctuation test data on 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB) and Benzyl Chloride (BC) using Salmonella typhimurium TA98 and TA100
Mutation Research, 100 (1982) 87-90 Elsevier Biomedical Press
87
F L U C T U A T I O N T E S T DATA ON 4 - C H L O R O M E T H Y L B I P H E N Y L (4CMB), 4-HYDROXYMETHYLBIPHENYL (4HMB) AND BENZYL CHLORIDE (BC) USING Salmonella typhimurium TA9$ AND TA100
A.W. SARGENT and A.P. REGNIER The British Petroleum Company Ltd., BP Research Centre, Chertsey Road, Sunbury-on-Thames, Middx. TW16 7LN (Great Britain)
(Received 4 July 1981) (Accepted tl August 1981)
Bacterial fluctuation tests (Green et al., 1976) were performed both with and without metabolic activation using the 'Ames' Salmonella typhimurium strains TA98 and TA100 (Ames et al., 1975) to assay the mutagenic potential of 4CMB, 4HMB and BC. 4CMB and 4HMB were tested on the same occasion. However, 4CMB was odly compared to BC in one assay. The results also show an independent test of BC.
MATERIALS AND METHODS
Bacterial strains
The strains were checked regularly for stability of the deep rough mutation rfa, and for the presence of the R factor using methods described in Ames et al. (1975). Background spontaneous, mutation was also checked and fell within the range 5-20 hist ÷ revertants for TA98 and 60-90 hist ÷ revertants for TA100. Stocks o f these strains were kept in liquid nitrogen prior to use (Green et al., 1977). Media Non-activated assay.
The method used was essentially that described by Green et al. (1976), however, tryptophan and casamino acids were replaced by L-histidine 0.25/zg/ml and D-biotin 0.4 ttg/ml. Bromo-cresol-purple (BCP) 5/~g/ml was used to indicate acid production.
Abbreviations: AAF, 2-acetylaminofluorene; DBPP, tris-(2,2-dibromopropyl) phosphate; DMSO, dimethyl sulphoxide; G6P, glucose 6-phoshate; G6PDH, glucose-6-phosphate dehydrogenase; MNNG, N-methyloN'-nitro-N-nitrosoguanidine; NADP, nicotinamide adenine dinucleotide phosphate; 2NF, 2-nitrofluorene.
0165-1218/82/0000-0000/$02.75 © Elsevier Biomedic~il Press
88
Activated assay. The media and methods were those used by Green et al. (1977) for Ca2÷-precipitated liver microsomes. However, various alterations in the activation mix were incorporated which are detailed below. Growth medium. 100 ml DM* salts; 4 ml glucose (20°70 w/v); 1.5 ml L-histidine (0.1°70); 0.8 ml D-biotin (0.01°70). Activation mix. 2 ml microsomes; 8 ml solution C*; 2 ml NADP (5 mg/ml); 2 ml G6P (8 mg/ml); 12 ml DM salts; 160 #1 G6PDH (100 units/ml). To 5.5 ml of the activation mix was added 1 ml Growth Medium containing 107 bacteria/ml and test compound (or DMSO control). The whole mixture was then dispensed in 100-~1 aliquots into each of 50 wells contained in a 'Repli' Dish (Sterilin Ltd., Teddington, Middx.). This procedure was repeated for each test concentration and the control. Maintenance medium* was then added after overnight incubation at 37 °, and the wells were scored for growth (purple to yellow colour change) after a further 72 h at 37 ° Microsomes. Aroclor 1254 induced rat-liver microsomes were obtained frozen from Litton Bionetics (Uniscience Ltd., Cambridge) and kept in liquid nitrogen until required.
TABLE 1 B A C T E R I A L F L U C T U A T I O N TEST: NUMBER OF POSITIVE WELLS P ER 50 AFTER 72 h INCUBATION AT 37 °. DATA FOR 4CMB, 4HMB ( N O N - A C T I V A T E D A N D A C T I V A T E D TA100) A N D BC ( A C T I V A T E D TA100) TAI00 Non-activated
TA I00 Activated
TA100 Activated
4CMB
4HMB
4CMB
4HMB
4CMB
BC
0.1 1.0 10.0 50.0
39 a 43 a 33 a 49 a
5 20 30 a 40 a
26 21 29 45 a
30 28 38 31
25 29 20 37
28 32 21 18
100.0 250.0
47 a 0
0 0
40 44 a
23 14
47 a 9
36 25
Concentration ~g/ml)
Controls DMSO MNNG DBPP
250 ~tg/ml 10/xg/ml 20/zg/ml
16 43 a NT
32 NT 48 a
28 NT 41 a
NT, not tested. a Significant at 1% level of probability.
*For composition of DM salts, solution C and Maintenance medium see Green et al. (1977).
89
TABLE 2 BACTERIAL FLUCTUATION TEST: NUMBER OF POSITIVE WELLS PER 50 AFTER 72 h INCUBATION AT 37 °. DATA FOR 4CMB A N D 4HMB (NON-ACTIVATED A N D ACTIVATED TA98) Concentration (/zg/ml)
0.1 1.0 10.0 50.0 100.0 250.0 Controls DMSO 2NF AAF
TA98 Non-activated
TA98 Activated
4CMB
4HMB
4CMB
4HMB
10 31 a 43 a 18 0 0
8 11 1 1 0 0
20 15 16 44 a 49 a 49 a
15 8 10 22 14 17
250 #g/ml 10 t~g/ml 20 #g/ml
10 50 a NT
19 NT 50 a
NT, not tested. aSignificant at 1% level of probability.
TABLE 3 BACTERIAL FLUCTUATION TEST: NUMBER OF POSITIVE WELLS PER 50 AFTER 72 h INCUBATION AT 37 o. DATA FOR BC (NON-ACTIVATED AND ACTIVATED TA100 AND TA98) Concentration (p.g/ml)
TA100 Nonactivated BC
TA100 Activated BC
TA98 Nonactivated BC
TA98 Activated BC
0.1 1.0 10.0 50.0 100.0 250.0
8 20 11 8 18 NT
NT 37 23 39 a 32 28
1 4 7 2 5 NT
6 9 11 6 3 11
12 29 a NT NT NT
25 NT 46 a NT NT
2 NT NT 50 a NT
9 NT NT NT 46 a
Controls DMSO MNNG DBPP 2NF AAF
250 #g/ml 10 #g/ml 20 #g/ml 10/zg/ml 20 #g/ml
NT, not tested. a Significant at 1% level of probability.
90 Protocol. 4 C M B and 4 H M B were tested in parallel (Tables 1 and 2), i.e. on the same occasion using the same activation mix and growth medium. The data for BC is shown in Tables 1 and 3. Positive controls were used in all assays to determine whether the activation mix a n d / o r the bacterial strains were functioning correctly. D M S O was used as solvent and negative control in all assays (see Tables 1, 2 and 3).
RESULTS AND DISCUSSION The overall results showed 4 C M B to be mutagenic both with and without metabolic activation using T A 1 0 0 and TA98, although the d o s e - r e s p o n s e with activation, especially with T A I 0 0 , was less m a r k e d than without activation. The data for 4 H M B was essentially negative, although a rise in reversion rate, significant at the 1 °7o level o f probability (Green and Muriel, 1976), was obtained using non-activated TA100 (Table 1). The data for BC indicated it to be non-mutagenic. However, at 50 # g / m l BC (TA100 activated) the reversion rate was significantly greater than that o f the control (1070 level o f probability) (Table 3). The toxicity o f both 4 C M B and 4 H M B to TA100 and T A 9 8 was apparent at concentrations o f 250 # g / m l , and to a lesser extent at 100 # g / m l , in the non-activated assays (Tables 1 and 2).
ACKNOWLEDGEMENTS The authors would like to t h a n k Mr. B.M. Aitken for excellent technical help, the British P e t r o l e u m G r o u p Occupational Health Centre for their support, and The British Petroleum Co. Ltd. for permission to publish this work.
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. Green, M.H.L., and W.J. Muriel (1976) Mutagen testing using Trp ÷ reversion in Escherichia coli, Mutation Res., 38, 3-32. Green, M.H.L., W.J. Muriel and B.A. Bridges (1976) Use of a simplified fluctuation test to detect low levels of mutagens, Mutation Res., 38, 33-42. Green, M.H.L., B.A. Bridges, A.M. Rogers, G. Horspool, W.J. Muriel, J.W. Bridges and J.R. Fry (1977) Mutagen screening by a simplified bacterial fluctuation test: use of microsomal preparations and whole liver cells for metabolic activation, Mutation Res., 48,287-294.
87
F L U C T U A T I O N T E S T DATA ON 4 - C H L O R O M E T H Y L B I P H E N Y L (4CMB), 4-HYDROXYMETHYLBIPHENYL (4HMB) AND BENZYL CHLORIDE (BC) USING Salmonella typhimurium TA9$ AND TA100
A.W. SARGENT and A.P. REGNIER The British Petroleum Company Ltd., BP Research Centre, Chertsey Road, Sunbury-on-Thames, Middx. TW16 7LN (Great Britain)
(Received 4 July 1981) (Accepted tl August 1981)
Bacterial fluctuation tests (Green et al., 1976) were performed both with and without metabolic activation using the 'Ames' Salmonella typhimurium strains TA98 and TA100 (Ames et al., 1975) to assay the mutagenic potential of 4CMB, 4HMB and BC. 4CMB and 4HMB were tested on the same occasion. However, 4CMB was odly compared to BC in one assay. The results also show an independent test of BC.
MATERIALS AND METHODS
Bacterial strains
The strains were checked regularly for stability of the deep rough mutation rfa, and for the presence of the R factor using methods described in Ames et al. (1975). Background spontaneous, mutation was also checked and fell within the range 5-20 hist ÷ revertants for TA98 and 60-90 hist ÷ revertants for TA100. Stocks o f these strains were kept in liquid nitrogen prior to use (Green et al., 1977). Media Non-activated assay.
The method used was essentially that described by Green et al. (1976), however, tryptophan and casamino acids were replaced by L-histidine 0.25/zg/ml and D-biotin 0.4 ttg/ml. Bromo-cresol-purple (BCP) 5/~g/ml was used to indicate acid production.
Abbreviations: AAF, 2-acetylaminofluorene; DBPP, tris-(2,2-dibromopropyl) phosphate; DMSO, dimethyl sulphoxide; G6P, glucose 6-phoshate; G6PDH, glucose-6-phosphate dehydrogenase; MNNG, N-methyloN'-nitro-N-nitrosoguanidine; NADP, nicotinamide adenine dinucleotide phosphate; 2NF, 2-nitrofluorene.
0165-1218/82/0000-0000/$02.75 © Elsevier Biomedic~il Press
88
Activated assay. The media and methods were those used by Green et al. (1977) for Ca2÷-precipitated liver microsomes. However, various alterations in the activation mix were incorporated which are detailed below. Growth medium. 100 ml DM* salts; 4 ml glucose (20°70 w/v); 1.5 ml L-histidine (0.1°70); 0.8 ml D-biotin (0.01°70). Activation mix. 2 ml microsomes; 8 ml solution C*; 2 ml NADP (5 mg/ml); 2 ml G6P (8 mg/ml); 12 ml DM salts; 160 #1 G6PDH (100 units/ml). To 5.5 ml of the activation mix was added 1 ml Growth Medium containing 107 bacteria/ml and test compound (or DMSO control). The whole mixture was then dispensed in 100-~1 aliquots into each of 50 wells contained in a 'Repli' Dish (Sterilin Ltd., Teddington, Middx.). This procedure was repeated for each test concentration and the control. Maintenance medium* was then added after overnight incubation at 37 °, and the wells were scored for growth (purple to yellow colour change) after a further 72 h at 37 ° Microsomes. Aroclor 1254 induced rat-liver microsomes were obtained frozen from Litton Bionetics (Uniscience Ltd., Cambridge) and kept in liquid nitrogen until required.
TABLE 1 B A C T E R I A L F L U C T U A T I O N TEST: NUMBER OF POSITIVE WELLS P ER 50 AFTER 72 h INCUBATION AT 37 °. DATA FOR 4CMB, 4HMB ( N O N - A C T I V A T E D A N D A C T I V A T E D TA100) A N D BC ( A C T I V A T E D TA100) TAI00 Non-activated
TA I00 Activated
TA100 Activated
4CMB
4HMB
4CMB
4HMB
4CMB
BC
0.1 1.0 10.0 50.0
39 a 43 a 33 a 49 a
5 20 30 a 40 a
26 21 29 45 a
30 28 38 31
25 29 20 37
28 32 21 18
100.0 250.0
47 a 0
0 0
40 44 a
23 14
47 a 9
36 25
Concentration ~g/ml)
Controls DMSO MNNG DBPP
250 ~tg/ml 10/xg/ml 20/zg/ml
16 43 a NT
32 NT 48 a
28 NT 41 a
NT, not tested. a Significant at 1% level of probability.
*For composition of DM salts, solution C and Maintenance medium see Green et al. (1977).
89
TABLE 2 BACTERIAL FLUCTUATION TEST: NUMBER OF POSITIVE WELLS PER 50 AFTER 72 h INCUBATION AT 37 °. DATA FOR 4CMB A N D 4HMB (NON-ACTIVATED A N D ACTIVATED TA98) Concentration (/zg/ml)
0.1 1.0 10.0 50.0 100.0 250.0 Controls DMSO 2NF AAF
TA98 Non-activated
TA98 Activated
4CMB
4HMB
4CMB
4HMB
10 31 a 43 a 18 0 0
8 11 1 1 0 0
20 15 16 44 a 49 a 49 a
15 8 10 22 14 17
250 #g/ml 10 t~g/ml 20 #g/ml
10 50 a NT
19 NT 50 a
NT, not tested. aSignificant at 1% level of probability.
TABLE 3 BACTERIAL FLUCTUATION TEST: NUMBER OF POSITIVE WELLS PER 50 AFTER 72 h INCUBATION AT 37 o. DATA FOR BC (NON-ACTIVATED AND ACTIVATED TA100 AND TA98) Concentration (p.g/ml)
TA100 Nonactivated BC
TA100 Activated BC
TA98 Nonactivated BC
TA98 Activated BC
0.1 1.0 10.0 50.0 100.0 250.0
8 20 11 8 18 NT
NT 37 23 39 a 32 28
1 4 7 2 5 NT
6 9 11 6 3 11
12 29 a NT NT NT
25 NT 46 a NT NT
2 NT NT 50 a NT
9 NT NT NT 46 a
Controls DMSO MNNG DBPP 2NF AAF
250 #g/ml 10 #g/ml 20 #g/ml 10/zg/ml 20 #g/ml
NT, not tested. a Significant at 1% level of probability.
90 Protocol. 4 C M B and 4 H M B were tested in parallel (Tables 1 and 2), i.e. on the same occasion using the same activation mix and growth medium. The data for BC is shown in Tables 1 and 3. Positive controls were used in all assays to determine whether the activation mix a n d / o r the bacterial strains were functioning correctly. D M S O was used as solvent and negative control in all assays (see Tables 1, 2 and 3).
RESULTS AND DISCUSSION The overall results showed 4 C M B to be mutagenic both with and without metabolic activation using T A 1 0 0 and TA98, although the d o s e - r e s p o n s e with activation, especially with T A I 0 0 , was less m a r k e d than without activation. The data for 4 H M B was essentially negative, although a rise in reversion rate, significant at the 1 °7o level o f probability (Green and Muriel, 1976), was obtained using non-activated TA100 (Table 1). The data for BC indicated it to be non-mutagenic. However, at 50 # g / m l BC (TA100 activated) the reversion rate was significantly greater than that o f the control (1070 level o f probability) (Table 3). The toxicity o f both 4 C M B and 4 H M B to TA100 and T A 9 8 was apparent at concentrations o f 250 # g / m l , and to a lesser extent at 100 # g / m l , in the non-activated assays (Tables 1 and 2).
ACKNOWLEDGEMENTS The authors would like to t h a n k Mr. B.M. Aitken for excellent technical help, the British P e t r o l e u m G r o u p Occupational Health Centre for their support, and The British Petroleum Co. Ltd. for permission to publish this work.
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. Green, M.H.L., and W.J. Muriel (1976) Mutagen testing using Trp ÷ reversion in Escherichia coli, Mutation Res., 38, 3-32. Green, M.H.L., W.J. Muriel and B.A. Bridges (1976) Use of a simplified fluctuation test to detect low levels of mutagens, Mutation Res., 38, 33-42. Green, M.H.L., B.A. Bridges, A.M. Rogers, G. Horspool, W.J. Muriel, J.W. Bridges and J.R. Fry (1977) Mutagen screening by a simplified bacterial fluctuation test: use of microsomal preparations and whole liver cells for metabolic activation, Mutation Res., 48,287-294.