Document rpMabzYjNZy5rrJx78qrdDbwe
AR226-3101 ^3 - GWWJ
Huntingdon
CONFIDENTIAL
DPT434/984093
BACTERIAL MUTATION ASSAY
DuPont Specialty Chemicals, Jackson Laboratory, Chambers Works,
Deepwater, NJ 08023,
USA
Research Laboratory
Huntingdon Life Sciences Ltd., Eye, Suffolk, IP237PX, ENGLAND
Final report issued 1 October 1998
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CONTENTS
Page
COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ............................ 3 QUALITY ASSURANCE STATEMENT .................................................................................. 4 SUMMARY ......................;......................;................................................................................ 5 INTRODUCTION .................................................................................................................... 6 TEST SUBSTANCE ................................................................................................................. 8 EXPERIMENTAL PROCEDURE............................................................................................. 9
ASSESSMENT OF RESULTS .................................................................................................. 13 MAINTENANCE OF RECORDS ............................................................................................. 13 RESULTS ................................................................................................................................. 14 CONCLUSION......................................................................................................................... 14 REFERENCES ......................................................................................................................... 15 TABLES
1. Results obtained with S. typhimurium TAPS following exposure tc^HI^HH^............ 16 2. Results obtained with X %?fo"wyr/uwTA 100 following exposure t^^^^^^^^l........... 18 3. Results obtained with S. typhimurium TA1535 following exposure to^HfHHB-......... 20
4. Results obtained with S. typhimurium TA1537 following exposure tcf----------B.......... 22
5. Results obtained with E. coli CM89I following exposure t(xB----^^^l^^^^_.......... 24
APPENDIX
; .<
1. Historical control data......................................................................................................... 26
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COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS
The study described in this report was conducted in compliance with the following Good Laboratory Practice standards, with the exception stated below, and I consider the data generated to be valid.
The United Kingdom Good Laboratory Practice Regulations 1997 (Statutory Instrument No .654). EC Council Directive 87/18/EEC of 18 December 1986 (Official Journal No L 15/29).
OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. In line with normal practice in this type of short-term study, the protocol did not require analysis of
the dose form.
Study Director, Huntingdon Life Sciences Ltd.
Date
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QUALITY ASSURANCE STATEMENT
The following have been inspected or audited in relation to this study:
Study Phases Inspected
Protocol
Process Based Inspections Audit Plate Scoring Treatment Formulation
Report
Date of Inspection
3 August 1998
8 January 1998 6 April 1998 15 April 1998 14 July 1998
2 September 1998
Date of Reporting
3 August 1998
8 January 1998 6 April 1998 15 April 1998 14 July 1998
2 September 1998
Protocol: An audit of the-protocol for this study was conducted and reported to the Study Director
and Company Management as indicated above.
Process based inspections: At or about the time this study was in progress inspections and audits of routine and repetitive procedures employed on this type of study were carried out. These were
conducted and reported to appropriate Company Management as indicated above.
Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above.
The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data.
G Goddard
Auditor,
Department of Quality Assurance,
Huntingdon Life Sciences Ltd.
...2&.'?.....5fep^^.Be&..B.^
Date
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SUMMARY
In this in vitro assessment of the mutagenic potential ofZonyl FS-62, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tiyptophan dependent mutant of Escherichia coli, strain CM891 (WPluvrA/pKMIOl), were exposed to the test substance diluted in purified water, which was also used as a negative control.
Two independent mutation tests were performed in the presence and absence of liver preparations from
Aroclor 1254-iBduced-rats (S9 mix). The first was a standard plate incorporation assay, the second
involved a pre-incubation stage.
- ;. -i :..- .-.'.
Concentrations of up to 5000 pg/plate were tested in the mutation tests. This is die standard limit
concentration recommended in the regulatory guidelines this assay follows. Other concentrations used
werei.a-series of GO half-Iogio dilutions of the highest concentration. No signs oftoxicity were observed
towards the tester strains in either mutation test.
No evidence of mutagenic activity was seen at any concentration oi^^^^UJ^Hin either mutation
test.
The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity ofthe liver-preparations.
It is concluded that, when tested in purified water,|l^|BB|^hows no evidence of mutagenic
activity in this bacterial system.
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INTRODUCTION
f^lHUlBo This report describes a'study designed to assess the mutagenic potential
system. The study was conducted in compliance with the following guidelines:
a bacterial
<w
OECD Guidelines for Testing of Chemicals. (1997) No. 471: Genetic Toxicology:
Bacterial Reverse Mutation T^est. ..
EEC Annex to Directive 92/69/EEC. (1992) Part B : Methods for Determination of
Toxicity, B. 13. Other effects - Mutagenicity: Escherichiaeoli - Reverse Mutation Assay. O.J. No. L 383 A, 157.
EEC Annex to Directive 92/69/EEC. (1992) Part B : Methods for Determination of
Toxicity, B. 14. Other effects - Mutagenicity: Salmonella typhimurium - Reverse Mutation Assay. O.J. No. L 383 A, 160.
US EPA 40 CFR Part 799 (1997) -Toxic Substances Control Act Test Guidelines -
Sub-section 799.9510, TSCA bacterial reverse mutation test. Federal Register, Vol. 62,
No. 158.
;
..
,
The method described was also designed to comply with ICH (1996 & 1997), and followed the recommendations of the United Kingdom Environmental Mutagen Society (Gatehouse^ al 1990).
The in vitro technique described by Ames and his co-workers, (Ames, McCann and Yamasaki 1975, Maron and Ames 1983) enables the mutagenic effect of a test substance to be determined by exposing
specially selected strains of Salmonella typhimurium to the test substance. Normally S. typhimurium is
capable of synthesising the essential amino acid, histidine, but the mutant strains used in this test are incapable of this function. When these strains are exposed'to a mutagen, reverse mutation to the original histidine independent form takes place in a proportion of the population. These are referred to
as revertants, and are readily detected by their ability to grow and form colonies on a histidine deficient
medium (supplemented with biotin, since these strains are also incapable ofbiotin synthesis).
A technique based on similar principles has also been described by Green (1984). This system employs mutant strains of Escherichia coli which are incapable of synthesising the amino acid tryptophan required for growth.
The strains used carry additional mutations which render them more sensitive to mutagens. The S. typhimurium strains have a defective cell coat which allows greater permeability of test substances
into the cell. All the strains are deficient in normal DNA repair processes. In addition three of them possess a plasmid (pKMIOl) which introduces an error-prone repair process, resulting in increased
sensitivity to some mutagens.
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Many substances do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell. Therefore the bacteria and test substance are incubated in both the absence and presence of a supplemented liver fraction (S9 mix) prepared from rats previously treated wnh a substance (Aroclor 1254) known to induce a high level of enzymic activity. The protocol was approved by Huntingdon Life Sciences Management on 7 July 1998, (he Sponsor on 17 July 1998 and by the Study Director on 31 July 1998. The experimental phase of the studywas conducted between 3 and 17 August 1998.
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Identity: Chemical name:
. A r..
Appearance: Storage conditions: Lot number: Expiry date: Purity: Date received:
TEST SUBSTANCE
Room temperature 2 Years from date of receipt 23 June 1998
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w
EXPERIMENTAL PROCEDURE
BACTERIAL STRAINS
The following strains were used:-
: ~S, typhimurium TA1535: contains a histidine missense mutation (hisG46) but is also deficient in a
DNA repair system (uvrB) and has a defective lipopolysaccharide coat on
....,..-,
,
the cell wall (rfa mutation). It is reverted by many agents causing base-
pair substitutions, but is not sensitive to frameshift mutagens.
S. typhinwrium TA100: ..is the same as TA1535 but contains a resistance transfer factor conferring ampicillin resistance and increasing sensitivity to some mutagens (plasmid
pKMIOl). In addition to base-pair substitutions, it is also able to detect
certain frameshift mutagens.
S. typhimurium TA1537: bears a histidine frameshift mutation (hisC3076). Like TA1535, it is defective in a DNA repair system and lipopolysaccharide coat. It is sensitive to agents causing frameshift mutations involving insertion or deletion of a single base-pair.
5'. typhimurium TA98:
contains another histidine frameshift mutation (hisD3052). Again it has a
defective DNA repair system and lipopolysaccharide coat but also contains the pKMIOl plasmid. It is reverted by agents causing deletion of
two adjacent base-pairs (double frameshift mutations), but not by simple alkylating agents causing base-pair substitutions.
E. coli CM891: (WP2uvrA/pKM101)
contains an ochre mutation. It is reverted by many agents causing A-T base-pair substitutions at the trpE locus or by G-C base-pair substitutions in transfer RNA loci elsewhere in the chromosome. It is also deficient in a DNA repair system (uwA), and is more readily reverted by certain mutagens than its parent strain WP2. It also contains the pKMIOl
plasmid.
The strains of S. typhimurium were obtained from Professor B.N. Ames, University of California,
Berkeley, California, USA.
The strain of E. coli was obtained from the National Collections of Industrial and Marine Bacteria,
Aberdeen, Scotland.
Batches of the strains were obtained from master stocks held in liquid nitrogen. The test batches were aliquots of nutrient broth cultures and were stored,at -80C. Dimethyl sulphoxide (DMSO) was added to the cultures at 8% v/v as a cryopreservative. Each batch of frozen strain was tested, where applicable, for cell membrane permeability (rfa mutation), sensitivity to UV light and the pKMIOl plasmid which confers resistance to ampicillin. The responses of the strains to a series of diagnostic
mutagens was also assessed.
For use in tests an aliquot of frozen culture was added to 25 ml of nutrient broth (Merck No. 2) and incubated, with shaking, at 37C for 10 hours. These cultures provided at least 109 cells per ml which were measured by spreading aliquots (0.1 ml) ofalO"6 dilution of the overnight cultures on the surface of plates of nutrient agar.
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POSITIVE CONTROLS
In the absence ofS9 mix
Identity: Supplier: Lot number: Purity: Appearance: Solvent: Concentration:
//-Ethyl-^'-nitro-^-nitrosoguanidinefENNG) Sigma Chemical
20F-0235 >97%
Pale yellow crystalline powder
DMSO (Aldrich, A.C.S.^pectrophotomefric grade, ^99.9% pure) 5 pg/plate for strain TA1535 3 ug/plate for strain TA100 2 ug/plate for strain CM891
Identity: Supplier: Batch number: Purity: Appearance:
Solvent: Concentration:
Identity: Supplier: Batch number: Purity: Appearance:
Solvent: Concentration:
9-Aminoacridine Sigma Chemical tfl8-Q358 99% Yellow powder
DMSO (Aldrich, A.C.S. spectrophotometric grade, S99.9% pure) 30 ug/plate for strain TA 1537
2-Nitrofluorene Aldrich Chemical Company 012867 98% Beige powder
DMSO (Aldrich, A.C.S. spectrophotometric grade, ^99.9% pure) I ug/plate for strain TA98
In the presence ofS9 mix
Identity: Supplier: Batch number: Purity: Appearance: Solvent:
Concentration:
2-Aminoanthracene Aldrich Chemical Company 52234-024 96%
Green powder
DMSO (Aldrich, A.C.S. spectrophotometric grade, ^99.9% pure) 2 ug/plate for strain TA 1535 10 ug/plate for strain CM891
Identity: Supplier: Batch number: Purity:
Appearance: Solvent-
Concentration:
Benzo[a]pyrene Aldrich Chemical Company 67778-105 98%
Yellow powder DMSO (Aldrich, A.C.S. spectrophotometric grade, >99.9% pure) 5 ug/plate for strains TA1537, TA98 and TA100
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PREPARATION OF S9 FRACTION
Species:
Sex: Strain: Source: Age: Weight:
Rat Male -Sprague-Dawley derived HarianOlacLtd 7-8 weeks <300g
S9 fraction was prepared from'a. group of ca 10 animals. Mixed function oxidase systems in file rat
livers were stimulated by Aroclor 1254, administered as a single intra-peritoneal injection in Arachis oil at a tfosage^of 500-mg/kg-bodyweight On the fifth day after injection, following an. overnight starvation, the rats were killed and their livers aseptically removed.
The following steps were carried out at 0-4C under aseptic conditions. The livers were placed in
0.15M KC1 (3 ml :KC1: 1 g liver) before being transferred to an Ultra-Tunax homogeniser. Following
preparation, me hombgenate was centriiuged at 9000 g far 10 minutes. The supernatant fraction (S9 fraction) was dispensed into aliquots and stored at -80C until required. The efficacy of each batch
of S9 fraction was tested in a bacterial mutation assay with me mutagenic precursors 7,12-
dimethylbenzanthracene and 2-aminoanthracene before use. The sterility was also checked.
Date of preparation: 9 June 1998
PREPARATION OF S9 MIX
S9 mix contained: S9 fraction (10% v/v), MgC^ (8 mM), KC1 (33 mM), sodium ormophosphate buffer pH7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All me cofactors were filter-sterilised
before use.
SELECTION OF SOLVENT AND FORMUIATION OFTEST''SlJBSTAIiCK;
The solubility of the test substance was assessed at 50 mg/ml in purified water, in which it dissolved
following warming to 35-40C with gentle stirring. Therefore purified water (obtained by the reverse osmosis of tap water) was used as me solvent for this study.
All concentrations cited mthis report are expressed in terms of pureMUBI^^Bh.e. correction was made for the purity ox|BR
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MUTATION TEST PROCEDURE
First test
The test substance was added to cultures of the five tester strains at seven concentrations 'separated by
ca half-logio intervals. The highest concentration of|HHB----posted was 50 mg/ml in the chosen
solvent, which provided a final concentration of 5000 ug/plate. This is the standard limit concentration recommended in the regulatory guidelines this assay follows. The negative control was the chosen solvent, purified water. The appropriate positive controls were also included. An aHquot bfO.l ml of a lOhour bacterial culture and 0.5 nu S9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed in glass tubes. An aliquot of 0.1 ml of the test solution was added, followed immediately by 2 ml of molten agar containing 0.5mM histidine/biotin/tryptophan. The mixture was
thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Each petri dish was individually labelled with a unique code corresponding to a sheet, identifying the dish^s'cbntents. Three petri dishes were used for each concentration. Plates were also prepared without
the. addition of bacteria in order to assess the sterility of the test substance, S9 mix and phosphate buSer. AIT plates were incubated at 37C for ca 72 hours. After ttus period the appearance of the
background bacterial lawn was'examined and revertant colonies counted using a Domino automated colony counter.
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects
the top concentration normally used in the second test would be the same as that used in the first. If
toxic effects were observed a lower concentration may be chosen. It should be ensured that if a lower concentration was chosen, signs of bacterial inhibition are present at the top concentration. Ideally a minimum of three non-toxic concentrations should be obtained.
Second test
As a clear negative response was obtained in the first test, a variation to me test procedure was used for the second. The variation used was the pre-incubation assay in which the tubes were incubated at 37C for 30 minutes with shaking before the addition of the agar overlay. 5000 ug/plate was again chosen as the top concentration, but. only.five concentrations were used.
'
STABILITY AND FORMULATION ANALYSIS
The stability of the test substance and the stability of the test substance in the solvent were not determined as part of this study. Analysis of achieved conceiilraliun was not performed as part of mis
study.
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ASSESSMENT OF RESULTS
For a test to be considered' valid the mean of the solvent control revertant colony numbers for each
strain should lie in die range stated in the appropriate Standard Operating Procedure. Also, the positive
control compounds, must cause at least a doubling of mean revertant colony numbers over the negative
control.
''''
"_
_'
The mean number of revertant colonies for all treatment groups were compared wHfi those obtained for the solvent control groups. The mutagenic activity of a test substance was assessed by applying the
following criteria:
a) If treaaaent with a lest substance produces an increase in revertant colony numbers of at least
twice the concurrent solvent controls/with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it is considered to show evidence of mutagenic activity7nthis test system. No statistical analysis is
performed.
b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the
concurrent solvent controls in either mutation test it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given
in paragraphs a) and b), additional testing may be performed in order to resolve the issue of the test
substance's mutagenic activity in this test system. Should an increase in revertant colony numbers
then be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
If no clear "positive" response can be obtained, the test data may be subjected to analysis tn
determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon e( al (1989) and will usually be analysis of variance followed by Dunnett's test.
MAINTENANCE OF RECORDS
All experimental data arising from the study (including documentary raw data, records and other materials; collectively defined as the "materials") will remain the property of the Sponsor.
Huntingdon Life Sciences shall retain the materials in its archive for a period of five years from the date of issue of the final report. After such time, the Sponsor will be contacted and their advice sought on
the return, disposal or further retention of the materials. If requested, Huntingdon Life Sciences will
continue to retain the materials, subject to a reasonable fee being agreed with the Sponsor.
Huntingdon Life Sciences shall also retain a copy of the final report in its archive indefinitely.
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RESULTS
witlfUHBind The resultsObtained
positive control compounds are presented in Tables 1 to 5.
The mean values quoted have been corrected to the nearest whole number.
The absence of colonies on sterility check plates confirmed the absence of microbial contamination.
The total colony counts on nutrient agar plates (see Tables) confirmed the viability and nigh"cell density
of the cultures of the individual organisms.
,
.
.
.
.
The mean. revertant colony.. counts for me solvent controls .were within, the ranges stated in me
appropriate Standard Operating Procedure or quoted by Gatehouse et al (1990). Appropriate positive control chemicals (with S9mix where required) induced substantial increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.
FIRST TEST
"
-./.. ..
.
No substantial increases in revertant colony numbers over control counts were obtained with any of the
tester strains following exposure tolllBB|Upt any concentration in either the presence or absence
ofS9mix.
HB^BBB No visible thinning of the background lawn of non-revertant cells was obtained following exposure to A top exposure concentration of 5000 pg/plate was therefore selected for use in the
second test.
SECOND TEST
No substantial increases in revertantcolony numbers over control counts were obtained with any of the
tjHUUly: tester strains following exposure
ofS9mix.
any concentration in either the presence or absence
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to
CONCLUSION
It is concluded that, when tested in purified water.pimjU|^hows no evidence of mutagenic
activity in this bacterial system.
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REFERENCES
AMES, B.N., McCANN, J. and YAMASAKI, E. (1975) Methods for detecting carcinogens and mutageas with the Salmonella/mammalian microsome mutagenicity test. Mutation Res. 31, 347.
GATEHOUSE, D.G., ROWLAND, I.R., WILCOX, P., CALLANDER, R.D. andI FORSTER, R. (1990) Bacteria} mutation assays in: KIRKLAND, D.J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part I revised. Basic Mutagenicity Tests: UKEMS Recommended
Procedures, p. 13. Cambridge University Press, Cambridge.
GREEN, M.H.L. (1984) Mutagen testing using tvp. reversion,in Escherichia coli in_.KILBEY, B.J., LEGATOR, M., NICHOLS, W. and RAMEL, C. (Eds.). Handbook of Mutagenicity Test Procedures.
Second edition, p. 161. Elsevier Science Publishers BV, Amsterdam.
ICH (1996) GeHoHSacity: .Guidance on Specific Aspects of Regulatory Genotoxicity Tests.
ICH (1997) Genotoxicity: A Standard Battery of Genotoxicity Testing ofPharmaceuticaIs.
MAHON, G.A.T., GREEN, M.H.L., MIDDLETON, B., MITCHELL, I.DE G., ROBINSON, W.D. and TWEATS, D.J...(1989) Analysis of data from microbial colony assays in: KIRKLAND, D.J. (Ed.). UKEMS Sub-committee - on Guidelines for Mutagenicity Testing. Report. Part III. Statistical
Evaluation of Mutagenicity Test Data, p.26. Cambridge University Press, Cambridge.
MARON, D.M. and AMES, B.N. (1983)
Mutation Res. 113, 173.
Revised methods for the Salmonella mutagenicity test.
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TABLE 1
Results .obtained with Salmonella typhiiwirium TA98 following exposure l
S9mix
+ present - absent.
Revertant colony counts and means Test I
A
B
C
Mean
sd
0
0
0
None; 10'6 dilution of overnight culture, plated on nutrient agar (total counts)
4-5
37
44
--42-
44
35
43
41
31
37
42
37
31
34
36
34
38
38
31
36
32
27
34
31
41
32
48
40
+
38
43
43
41
41
34
42
39
24
41
39
35
34
34
45
38
37
38
35
37
44
36
35
38
41
50
34
42
45
30
31
35
43
39
34
39
554
580
562
565
13
184
284
218
229
51
119
143
129
130
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TABLE 1 - continued
Results obtained withSalmoneUllaattyvphimurium TA98 following-exposure
Plate No.
Addition (|tg)
1 None; sterility check 2 ZonylFS-62,(5000);
sterility check
TO^rifted wa^r (0:1 nd) 15iBenzo[a]pyrene (5) 16 2-Nitrofluorene (1)
None; 10r-6 dilution of 17 fflnerni^tcuaun^plated
on nutrient agar (total counts) sdlStandard deviation
S9mix
+ present -absent
Revertant colony counts and means Test 2 (with pre-incubation)
A
B
C
Mean ! sd
0
'
0
34
38
37
37
42
34
37
42
27
39
42
42
37
20
39
36
37
36
35
38
29
28
41
38
32
37
37
30
44
37
35
37
39
43
32
41
478
471
484
246
306
304
0
36
2
38
4
35
8
41
2
.32
10
36
1
34
5
36
7
35
3
37
7
37
2
39
6
478
285
34
116
112
110
- 113 1
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TABLE 2
Results obtained with Salmonella tyyhimur'wm TA100
following exposure <
Non^JQ^dHutionof
overnight culture, plated on nutrient agar (total counts)
Revertantcolony counts and means
S9mix
Testi - ;-
+ present A
B
C
Mean
sd
-absent
0
0
.0
0
W9
93
--89-
-97-
11
100
90
94
95
5
106
96
85
96
11
100
109
102
104
5
101
100
103
101
2
86
92
103
34
9
87
102
101
97
8
110
100
96
102
94
114
99
14
99
100
86
95
8
106
90
104
100
9
92
110
92
'98
10
106
108
106
107
I
109
67
82
86
21
108
94
94
99
8
118
107
96
+
412
377
426
107
II
405
25
370
386
413
390
22
135
130
130
132
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Company
TABLE 2 - continued
Results obtained withSalmonella fyghimurium TA100
following exposure 1
Plate No.
Addition ("g)
l|None;sterility check
(sterility check
8JParifiedwater (0.1 ml)
WPurifiied water .(0:1 nd) Benzo[a]pyrene (5)
16 ENNG (3) None; 10"6 dilution of
17 overnight cutttue, plated on nutrient agar (total counts)
sd|Standard deviation
S9mix
+ present -absent
Revertant colony counts and means Test 2 (with pre-incubation)
A
B
C
Mean | sd
0
0
99
94
100
7
97
101
99
99
2
99
97
102
99
3
87
106
102
98
10
85
94
112
97
14
104
115
89
103
13
109
104
89
101
10
100
99
108
102
5
95
8S
96
93
95
109
102
102
95
90
8S
91
95
103
92
-97 -
391
480
414
428
46
384
387
395
389
106
107
107
107
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TABLE3
Results obtained with Salmonella typhinwrium TA1535
following exposure 1
S9inix
+ present - absent
Revertant colony counts and means Test i
A
B
C
j Mean | sd
0
0
0
None; 10 dilution of overnight culture, plated on nutrient agar (total counts)
19
K
47-
-IT-
15
20
23
19
15
19
16
17
22
16
16
18
.14
n
14
13
21
17
16
18
21
20
21
21
16
17
23
19
12
19
27
19
20
12
15
16
19
28
16
21
15
17
15
16
19
15
9
14
19
21
34
25
19
19
15
18
17
22
19
19
129
119
132
127
100
117
129
115
15
14i
129
139
136
. e*~~??Mi.^oe<>"Hi. DOoSss Wr-s.t cCOomnt-aasin TSCA CBI
TABLE 3 - continued
Results obtained with Salmonella typhimurium TA1535
following exposure to|
Plate No.
Addition (ug)
16 ENNG (5)
None; 101-6.dilution of 17 overnight culture,' plated
. on nutrient agar (total counts) sdjStandard deviation
Revertant colony counts and means Test 2 (with pre-incubation)
S9mix
+; present A
B
C
Mean
sd
- absent:
0
0
2fr
14
44-
46-
20
21
20
20
21
14
14
16
22
22
14
19
21
14
20
18
20
23
19
21
16
21
13
17
14
13
19
15
23
23
22
23
20
22
14
19
20
20
23
21
17
22
19
125
118
112
19"
118
90
123
117
110
18
121
111
117
116
S^ed.D,r,o.c-,-.l,lnTSCACB<
CompsW aa'
TABLE 4
Results obtained with Salmonella typhimurium TA1537
following exposure toj
S9nux
+ present - absent
+
Revertant colony counts and means
Testi
A
B
C
| Mean | sd
0 |0
0
8
10-
-T
9
10
12
10
2
10
13
12
12
2
12
12
9
11
2
15
10
10
12
3
10
15
9
11
3
13
8
8
12
12
10
15
14
13
10
JL.
11
-L
14
i
16
10
16
14
3
12
12
9
11
2
12
13
13
13
1
10
12
10
11
1
10
12
6
9
3
13
9
12
10
14
13
11 _2_
12
2
206
237
202
215
19
245
189
202
212
29
115
121
119
118
Company Sanded. Doss rsof contain TSCA CBS
TABLE 4 - continued
Results obtained wMiSalmonella typhimurium TA1537 following exposure tc
Plate No.
Addition (Ug)
t4l?unfied:waterXO.T nd) 15|Benzo[a]pyrene(5) 16 9-Aniinoacridine (30)
None; 10n-6.dilution of
17 pveradght oiritare; plated on nutrient agar (total counts)
sd Standard deviation
S9nux
+ present - absent
+
Revertant colony counts and means Test 2 (with pre-incubation)
A
B
C
Mean
sd
0
9
12
10
10
16
9
6
10
12
9
8
15
12
14
12
12
12
8
7
8
12
8
8
14
8
12
13
12
10
7
12
10
-10
174
176
203
143
141
136
0
10 12
9 11 13
10
12
10
9
184
16
140
93
115
-108
105
11
C,n'" S.ni..zd. Do^ -. con..,n TSCA CB1
TABLES
Results obtained with Escherichia coll CM891 following exposure to
S9mix
+ present - absent
Revertant colony counts and means
Testi
A
B
C
Mean
sd
0
0
None;10dilution of
overnight culture, plated on nutrient agar (total counts)
-96
100
99
1--99
111
94
119
108
90
108
107
99
106
96
97
100
102
103
115
101
95
+
96
107
110
102
100
109
119
108
86
108
109
100
99
87
?2
81
112
109
104
93
88
110
88
96
107
95
112
435
474
415
768
758
740
123
110
117
-98-
-2-
102
9
106
15
105
5
100
6
102
2
104
10
104
7
104
5
104
17
106
5
93
6
101
17
95
8
98
II
105
9
441
30
755
14
117
Company ganl.^.O"'^"0""""""061
TABLE 5 - continued
Results obtained with Escherichia coli CM891
following exposure toj
Plate No.
Addition (ug)
14 Purified water <0;I nri) 15j2-Aminoanthracene (10)
16. ENNG (2) None; 10n-6 dilution of
17 overnight culture, plated on nutrient agar (total counts)
sd|Standard deviation
S9inix
+ present - absent
Revertant colony counts and means Test 2 (with pre-incubation)
A
B
C
Mean
sd
0
0
+
87
96
-96
-93-
100
93
103
99
94
96
101
97
115
125
114
118
+
102
114
115
110
102
100
109
104
102
100
78
107
118
128
93
13
118
11
101
90
107
99
9
97
104
101
101
4
115
102
114
110
7
112
m
93
112
19
443
402
465
437
32
842
886
900
876
30
123
124
111
119
Company Ssawa"n^<^- d.Oo^^"^08'
APPENDIX 1
Historical control data Presented below are the historical control data from the period 1 April 1997 to 30 June 1998.
Purified water solvent controls
Strain .
S9nux
TA 100 i- j
.
-
+
tAlJ)35
,
-
+
,^-. CM8;91
-
+
TA9 8
-
+
TA15;?7
-
+
Minimum Maximum Mean
No. of values
78
81
122 123
99:5 100.4
99 '" 99
12 33 18.7 95
10 29 18.8 95
79 164 112.0
31
73 211 121.3
31
26. . 27-
45
47
36.1 98
38.1 ?8.
7 21 12.1
--96
7
22 12.2
-.96.
Positive controls
Strain
S9mix
-'
Minimum Maximum Mean No. of values
TA 100
+
-^
190 1477 377.3 274
240 1231
510.8 273
TA1 535
-
+
37 1243 193.8 268
80 533 233.3 267
CMiS91
-
+
294 2312 1296.7
89
231 2097 723.1
89
17 .98
-
+
TA1537
-
.
-
117 649 243.7 271
(80 ug) (30 ug)
200
562
169
823 4532
384
488.8 1695.4
270
266
251.5 6
+
94 396 210.2 271
Company Sanitized. Does not contain TSCACBS