Document 3Qm8KQXzy141nZzYBaD1Gj6K0
SRPT T-7&
J
MIN 314l022208
PERJ%UOROO~AlVE2WLFONYLFLUORIDE
T-7 b
,OSF)
t .a
BACTERJAL REVERSE MUTATION TEST
Sponsor
3M Center 3M Corporate Toxicology Building 220-2E-02 St Paul
I"55133-3220 USA
Research Laboratory
HuntingdonLife SciencesLtd. Woolley Road Alconbury Huntingdon Cambridgeshire
PE28 4HS ENGLAND
Report issued 23 October 2002
Page 1 of 29
001570
. .
.....................
MIN 3141022208
CONTENTS
Page
COMPLIANCE WITH GOOD LABORATORYPRACTICE STANDARDS ............................. 3 QUALITY ASSURANCE STATEMENT ..................................................................................... 4 RESPONSIBLE PERSONNEL ...................................................................................................... 5 SUMMARY ................................................................................................................................... 6 INTRODUCTION .......................................................................................................................... 7 TEST SUBSTANCE ...................................................................................................................... 9 EXPERIMENTALPROCEDURE ................................................................................................ 10 ASSESSMENT OF RESULTS ...................................................................................................... 14 DEVIATIONS FROM PROTOCOL .............................................................................................. 14 MAINTENANCEOF RECORDS ................................................................................................. 15 RESULTS ....................................................................................................................................... 16 CONCLUSION .............................................................................................................................. 16 REFERENCES ............................................................................................................................... 17
TABLES
1. Results obtained with S. typhimuriumTA98: test 1 (range-finding) ..................................... 18 2. Results obtained with S. typhimurium TA98: test 2, with pre-incubation ............................. 19 3. Results obtained with S. typhimuriumTAIOO: test 1 (range-finding).................................... 20 4. Results obtained with S.typhimuriumTAI00:test 2, with pre-incubation ........................... 21 5. Results obtained with S.typhimuriumTA1535: test 1 (range-finding).................................. 22 6. Results obtained with S. typhimurium TA1535: test 2, with pre-incubation ......................... 23 7. Results obtained with S. typhimuriumTA1537: test 1(range-finding).................................. 24 8. Results obtained with S. typhimuriumTA1537: test 2, with pre-incubation ......................... 25
9. Results obtained with E. coli WP2mA/pKh4101 (CM891): test 1 (range-finding).............. 26 10. Results obtained with E. coli WP2uvrA/pKh4101(CM891):test 2, with pre-incubation ..... 27
APPENDICES
1. Historicalcontrol data ............................................................................................................. 28
2. Eye Research Centre GLP ComplianceStatement2001 ........................................................ 29
2:
MIN 3 141022208
COMPLIANCE WITH GOQD LABORATORY PRACIlCE STANDGRDS
The study described in this report was conducted in compliance with the following Good Laboratory Practice Standards, with the exceptionsstated below, and I considerthe data generated to be valid.
The UK Good Laboratory Practice Regulations 1999 (Statutory Instrument No. 3106). EC Commission Directive 1999111EC of 8 March 1999(Official Journal No. L 77/8). 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. The expiry date of the test sample was the Sponsor's responsibility.
eR/Q ............tz,....................... 9..........
Kenneth May, B.Sc.,C.Biol., lkf.Biol.,
Study Director, Huntingdon Life SciencesLtd.
.2..3..........O.....c....k....O....llb....c.. .l..r...f..mL
Date
:3:
-- .
. . _- _ _ -. . . . .
.......
.....
MIN 314/022208 QUALITY ASSURANCE STATEMENT The following inspectionsand audits have been b e d out in relation to this study:
Study Phase
Protocol Audit
Process Based Inspections
S9 Preparation Formulation and Treatment Plate Scoring
Report Audit
Date of Inspection 16 October 200 1
15 January 2002 31 October 2001 24 October 2001 4 February 2002
Date of Reporting 16 October 2001
15 January 2002 31 October 2001 24 October 2001 5 February 2002
Protocol Audit: An audit of the protocol for this study was conducted and reported to the Study Director and Company Managementas indicated above.
Process Based Inspections: At or about the time this study was in progress inspections of routine and repetitive procedures employed on this type of study were carried out. These were conducted and reported to appropriateCompany Managementas 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 Managementas indicated above.
The methods, procedures and observations were found to be accurately described and the reported results of this study to reflect the raw data.
&"".""h"'r;" 4.....f....e........*.........3.....J........................
Angela Myennings, B.Sc., MSc., Ph.D., M.R.Q.A.,
Date
Group Manager,
Department of Quality Assurance,
Huntingdon Life Sciences Ltd.
001573
:4:
RESPONSIBLE PERSONNEL
Kenneth May, B.Sc., C.Biol.,M.J.Bio1. Study Director Elizabeth Farrall, B.Sc. Scientist
MM 3 141022208
001574
:5:
MIN 3141022208 SUMMARY In this in viiro assessment of the mutagenic potential of Perfluorooctanesulfonyl fluoride (POSF), histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535,TA1537,TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain wP2uvrNpKM101 (CM89l), were exposedto the test substance. Two independentmutation tests were performed in the presence and absence of liver preparationsfrom Aroclor 1254induced rats (S9 mix). Both tests involved a pre-incubation stage in airtightvessels.
Concentrations of Perfluorooctanesulfonyl fluoride (POSF)up to 5000 pglplate were tested in the mutation tests. This is the standard limit concentration recommended in the regulatory guidelines that
this assay follows. No signs of toxicity were observed towards the tester strains in either mutation test. No evidence of mutagenic activity was seen at any concentration of Perfluorooctanesulfonyl fluoride
(POSF)in either mutationtest.
The concurrent positive controls demonstratedthe sensitivityof the assay and the metabolising activity of the liver preparations.
It is concluded that, under the test conditions employed, Perfluomtanesulfonyl fluoride (POSF)
showed no evidence of mutagenic activity in this bacterial system.
001575
:6:
MIN 3 14/022208
INTRODUCTION
This report describes a study designed to assess the mutagenic potential of Pedluorooctanesulfonyl
fluoride POSF) in a bacterial system. The study was conducted in compliance with the following guidelines:
OECD Guidelines for the Testing of Chemicals. (1997) Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline471.
EC Commission Directive 2000/32/EC Annex 4D-B.13/14. Mutagenicity - Reverse
mutation test in bacteria. No. L 136/57.
US EPA (1998) Health Effects Test Guidelines. OPPTS 870.5100 Bacterial reverse
mutation test. EPA 712-C-98-247.
Japan Ministry of Agriculture, Forestry and Fisheries. (1985) Notification of Director General, Agricultural Production Bureau. NohSan No. 4200.
Joint Directives of J EPA, J M H W and J MITI. (31 October 1997) Kanpoan No. 287, Eisei No. 127 and Kikyoku No. 2 (31 October 1997).
JMHW Genotoxicity Testing Guideline, PAB Notification No. 1604 (1 November
1999).
Official Notice of J MOL. (8 February 1999).
The method described was also designed to comply with ICH (1995 & 1997), and followed the recommendationsof the United Kingdom Environmental Mutagen Society (Gatehouse et all990).
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.typhimuriumis 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 independentform 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 of biotin synthesis).
A techniquebased on similar principleshas also been described by Green (1984). This system employs mutant strains of Escherichiacoli that are incapable of synthesisingthe amino acid, tryptophan, which is required for growth.
The strains used cany additional mutations that 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 (pKh4101), which introduces an error-prone repair process, resulting in increased sensitivityto some mutagens.
001576
:7:
MM 3 141022208 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 hction (S9 mix) prepared from rats previously treated with a substance (Aroclor 1254) known to induce a high level of enzyme activity. The protocol was approved by Huntingdon Life SciencesManagementon 18 July 2001, by the Sponsor
on 31 July 2001 and by the Study Director on 15 October 2001. The study was conducted at Huntingdon Life Sciences Ltd., Eye Research Centre, Eye, Suffolk, IP23 7PX, England. Experimental start date: 21 November 2001.
Experimentalcompletiondate: 31January 2002.
001577
. .
Identity: Appearance: Storage conditions: Lot number: Expiry date: Purity: Specific gravity: Date received:
MM 3 141022208 TEST SUBSTANCE
Perfluorooctanesulfonylfluoride (POSF) Clear liquid Room temperature 040227 Sponsor'sresponsibility;assumed stable for duration of study
ca 1.8 14 June 2001
001578
EXPERIMENTAL PROCEDURE
MIN 314l022208
BACTERIALSTRAINS
The following strains were used:
S.typhimurium TA1535: contains a histidine missensemutation (hFFG46) but is also deficient in a DNA repair system (WB) and has a defective lipopolysaccharide coat
on the cell wall ($I mutation). It is reverted by many agents causing base-pair substitutions,but is not sensitiveto frameshiftmutagens.
S.fyphimuriumTAIOO:
is the same as TA1535 but contains a resistance transfer factor conferring ampicillin resistance and increasing sensitivity to some mutagens (plasmid pKMI01). 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.
S.typhimurium TA98:
contains another histidine frameshift mutation (hisD3052). Again it has a defective DNA repair system and lipopolysaccharide coat but also contains the pKMlOl plasmid. It is reverted by agents causing deletion of two adjacent base-pairs (double frameshift mutations), but not by simple alkylatingagents causing base-pair substitutions.
E. coli W u v r A l p K M 101: (CM891)
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 (uvrA), and is more readily reverted by certain mutagens than its parent strain WP2. It also contains the pKM101 plasmid.
The strains of S.typhimuriumwere obtained from the National Collection of Type Cultures, London,
England.
The Strain of E. coli was obtained from the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland.
Batches of the strains were obtained fkom master stocks held in liquid nitrogen. The test batches were
aliquots of nutrient broth cultures and were stored at -8OOC. Dimethyl sulphoxide (DMSO) was added to the cultures at 8% vlv as a cryopreservative. Each batch of frozen strain was tested, where
applicable, for cell membrane permeability (rfa mutation), sensitivity to W light and the pKMlOl
plasmid, which confers resistance to ampicillin. The responses of the strains to a series of diagnostic mutagens were also assessed.
For use in tests, an aliquot of frozen culture was added to 25 ml of nutrient broth and incubated, with
shaking, at 37C for 10 hours. These cultureswere intended to provide approximately lo9cells per ml,
which were measured by spreadingaliquots (0.1 ml) of a dilution of the overnight cultures on the
surface of plates of nutrient agar and counting the resultant colonies.
001579
- _.
--
MIN 3141022208
POSITTVE CONTROLS
In the absence of S9 mix
Identity: CASNo.: Supplier: Lot number: purity: Appearance: Solvent: Concentration:
Identity: CASNo.: Supplier: Lot number: Purity: Appearance: Solvent: concentration:
Identity: CASNo.: Supplier: Lot number: Purity: Appearance: Solvent: Concentration:
Identity: CASNo.: Supplier: Lot number: Purity: Appearance: Solvent: Concentration:
In the presence of S9 mix
Identity: CASNo.: Supplier: Lot number: Purity: Appearance: Solvent: Concentration:
Sodium a i d e 26628-22-8 Sigma Chemical T7H0079 min. 99.5% White powder DMSO (Aldrich, A.C.S. spectrophotometricgrade) 0.5 clglplate for strains TA1535 and TAlOO
9-Aminoacridine 90-45-9 Sigma Chemical 1O6F-0668 1 > 97% Yellow powder DMSO (Aldrich,A.C.S. spectrophotometricgrade) 30 pglplate for strain TA1537
2-Nitrofluorene 607-57-8 Aldrich Chemical Company 80501-24227 98% Beige powder
DMSO (Aldrich, A.C.S. spectrophotometricgrade)
1 pglplate for strain TA98
2-(2-Furyl)3-(5-nitro-2-furyl) acrylamide (AF-2) 3688-53-7 Wako Pure Chemical Industries Ltd.
PAE 1151
98-102%
Red powder
DMSO (Aldrich, A.C.S. spectrophotometricgrade) 0.05 pglplate for strain wP2uvrNpKM101 (CM891)
2-Aminoanthracene 613-13-8 Aldrich Chemical Company 52234-024 96% Green powder
DMSO (AIdrich, A.C.S. spectrophotometricgrade) 2 pdplate for strain TA1535
I0pglplate for strain WP2wA/pKMI 01 (CM891)
: 11
001580
MTN 3141022208
Identity: CAS No.: Supplier: Lot number: Purity: Appearance: Solvent: Concentration:
Benzo[a]pyrene 50-32-8 Aldrich Chemical Company 07778-105 98% Yellow powder
DMSO (Aldrich,A.C.S. spectrophotometricgrade)
5 pg/plate for strains TA1537, TA98 and TAlOO
PREPARATIONOF S9 FRACTION
Species: Sex: Strain: source: Weight:
Rat
Male Sprague-Dawley derived Charles River UK Ltd. 400g
S9 fraction was prepared from a group of CLI I0 animals according to the method described by Ames, McCann and Yamasaki (1975). Mixed function oxidase systems in the rat livers were stimulated by Aroclor 1254, administered as a single intra-peritoneal injection in corn oil at a dosage of 500 mgkg body weight. On the fifth day after injection, following overnight fasting, the rats were killed by cervical dislocation and their livers asepticallyremoved.
The following steps were carried out at 04OC under aseptic conditions. The livers were placed in 0.15M KCI (3 ml KCI : 1 g liver) before being transferred to a Potter-Elvehjem homogeniser. Following preparation, the homogenate was centrifuged at 9000 g for 10 minutes. The supernatant fraction (S9fraction) was dispensed into aliquots and stored at -8OOC or below. Each batch of S9 fraction was tested for sterilityand efficacy.
Date of preparation: 17July 2001 (test I); 15January 2002 (test 2)
PREPARATIONOF S9 MIX
The S9 mix contained: S9 fraction (10% vh), MgCl, (8 mM), KCl(33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-&phosphate (5 mM), NADPH (4 mM) and NADH (4 mM). All the cofactors were filter-sterilised before use.
FORMULATION OF TEST SUBSTANCE
The solubilityof the test substancewas assessed in dimethyl sulphoxide(DMSO), ethanol, acetone and heme. It was insoluble in DMSO, ethanol and acetone, and was known to be insoluble in water. Although soluble in h e m e at 50 mglml, the volume of h e m e required to administerthe test substance at this concentration was found to be too toxic towards the test system. It was, therefore., decided to administerthe test substanceby direct addition withoutthe use of a solvent
All concentrations cited in this report are expressed in terms of the Perfluorooctanesulfonylfluoride (POSF) sample as received.
: 12:
MIN 3 14/022208
MUTATION TEST PROCEDURE
First test (range-finding)
The testsubstancewas added to cultures of the five tester strains at seven concentrations. The highest concentration of test substance tested was 5000 Clglplate (obtained by addition of 2.8 pl of the test substance). This is the standard limit concentration recommended in the regulatory guidelines this assay follows. The other concentrations were 2500 pdplate (1.4 pl), 1750 pgplate (1.0 pl), 1250 pglplate (0.7 pl), 700 pg/plate (0.4 pl), 350 ptglplate (0.2 pl) and 175 pglplate (0.1 pl). Untreated controlsand the appropriatepositivecontrolswere also included.
Following the addition of the above aliquots of the test substance (or 0.1 ml of positive control
solution) to airtight glass vessels, 0.5 ml S9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) was added,followed by 0.1 ml of a 10 hour bacterial culture. The mixtures were incubated at 37OC for 30 minutes with shaking before addition of 2 ml of agar containing histidine (0.5 mM) and tryptophan
(0.5 mM). The mixtures were thoroughly shaken and overlaid onto previously prepared Petri dishes
containing 25 ml minimal agar. Each Petri dish was individually labelled with a unique code
correspondingto a sheet, identifying the contents of the dish. 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 sodium phosphate buffer. All plates were incubated at 37OC for cu 72
hours. After this period the appearance of the background bacteria1 lawn was examined and revertant
coloniescounted using a Domino automatedcolony 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 might be chosen, ensuring that signs of bacterial inhibition are present at the top concentration. Ideally a minimum of three non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating dose levels should be obtained, unless otherwise justified by the Study Director.
Second test
The second test was an exact repeat of the first test, except that only five concentrations were used. 5000 pgtplate was again chosen as the top concentration.
STABILITYAND FORMULATION ANALYSIS
The stability of the test substance and the stability and homogeneity of the test substance in the test system were not detwmined as part of this study. Analysis of achieved concentration was not performed as part of this study.
: 13:
ASSESSMENT OF RESULTS
Acceptance For a test to be considered valid the mean of the solventlvehiclecontrol revertant colony numbers for each strain should lie within the 99% confidence limits of the current historical control range of the laboratory unless otherwisejustified by the Study Director. The historical range will be maintained as a rolling record over a maximum of five years. Also, the positive control compounds must cause at least a doubling of mean revertant colony numbers over the negative control.
Analysis The mean number of revertant colonies for all treatment groups will be compared with those obtained for the solvent/vehicle control groups.
Evaluation If exposure to a test substance produces an increase in revertant colony numbers of at least twice the concurrent solventlvehicle controls, with some evidence of a positive dose-relationship (increased revertant colony counts at concentrations below that at which the maximal increase is obtained), in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it will be considered to show evidence of mutagenic activity in this test system. No statistical analysis will be perfonned. If exposure to a test substance does not produce an increase in revertant colony numbers in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it will be consideredto show no evidence of mutagenic activity in this test system. No statistical analysis will be performed. If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after the additional testing outlined in the mutation test procedure, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et aZ(1989) and will usually be analysis of variance followed by Dunnett's test. Biological significance should always be considered along with statistical significance. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
DEVIATIONS FROM PROTOCOL
Although the protocol indicated that a solvent or vehicle would be employed, it was not possible to obtain a solution or suspension of the test substance that would be compatible with the test system. The test substance was, therefore, added directly to the test system. Since this procedure complies with the test guidelines that this study follows, this deviation does not impact on the integrity of the study.
001583
14 :
MIN 3 141022208
MAINTENANCE OF RECORDS
All raw data, samples and specimens (if appropriate) arising fiom the performance of this study will remain the property of the Sponsor. Types of sample and specimen which are unsuitable, by reason of instability, for long term retention and archiving may be disposed of after the periods stated in Huntingdon Life Sciences Standard OperatingProcedures. All other samples and specimens and all raw data will be retained by Huntingdon Life Sciences in its archive for a period of five years from the date on which the Study Director signs the finai report. After such time, the Sponsorwill be contacted and his advice soughton the return, disposal or further retention of the materials. If requested, Huntingdon Life Sciences will continue to retain the materials subjectto a reasonablefee being agreed with the Sponsor. Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and a copy of the final report in its archive indefmitely.
001584
MIN 3 14l022208
RESULTS
The results obtained with Peffluorooctanesulfonylfluoride (POSF) and positive control compounds are presented in Tables 1 to 10. The mean values quoted have been corrected to the nearest whole number. The absence of colonies on sterilitycheck plates confmed the absence of microbial contamination. The total colony counts on nutrient agar plates (see Tables) confinned the viability and high cell density of the cultures of the individual organisms. The mean revertant colony counts for the solvent controls were within the 99% cofidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains, confuming sensitivityof the cultures and activityof the S9 mix.
FIRST TEST (RANGE-FINDING) No substantial increases in revertant colony numbers over control counts were obtained with any of
the tester strains following exposure to Peffluorooctanesulfonylfluoride (POSF) at any concentration .in either the presence or absenceof S9 mix. No visible thinning of the background lawn of non-revertant cells was obtained following exposure
to Perfluorooctanesulfonyl fluoride (POSF). A maximum exposure concentration of 5000 pg/plate
was, therefore, selected for use in the second test.
SECOND TEST No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Perfluorooctanesulfonylfluoride (POSF) at any concentration in either the presence or absence of S9 mix. No visible thinning of the background lawn of non-revertant cells was obtained following exposure to Perfluorooctanesulfonylfluoride(POSF).
CONCLUSION
It is concluded that, under the test conditions employed, Perfluomtanesulfonyl fluoride (POSF) showed no evidenceof mutagenicactivity in thisbacterial system.
MM 3 141022208
REFERENCES
A M E S , B.N., McCA", J. and YAMASAKI, E. (1975) Methods for detecting carcinogens and
mutagens with the Salmonellafmammalianmicrosomemutagenicity test. Mutation Res. 31,347-364.
GATEHOUSE, D.G., ROWLAND, LR, WILCOX, P., CALLANDER,RD. and FORSTER,R (1990) Bacterial mutation assays in: KIRKLAND, D.J. (Ed.). WMS Sub-committee on Guidelines for Miuagenicity Testing. Report. Part I revised Basic Murageniciq Tests: UKElUY Recommended Procedwes, pp.13-61.CambridgeUniversityPress,Cambridge.
GREEN, M.H.L. (1984)Mutagen testing using trp+ reversion in Escherichia coli in KILBEY, B.J., LEGATOR, M., NICHOLS, W.and RAMEL, C. (Eds.). Handbook of Mutagenicity Test Procedures. Secondedition, pp.161-187.Elsevier SciencePublishersBV,Amsterdam.
ICH(1 995) Genotoxicity: Guidance on SpecificAspects of Regulatory GenotoxicityTests.
ICH (1997)Genotoxicity: A StandardBattery of GenotoxicityTesting of Pharmaceuticals.
MAHON, G.A.T., GREEN, M.H.L., MIDDLETON, B., MITCHELL, I.de G., ROBINSON, W.D.and TWEATS, DJ. (1989)Analysis of data from microbial colony assays in: Kw(LAND, D.J. (Ed.). UKEUSSub-committeeon Guidelinesfor Miitagenicity Tmting.Report. Part DI. Statistical Evaluation
of Mutageniciq TestData, pp.26-65. CambridgeUniversityPress,Cambridge.
MARON, D.M. and AMES, B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mutation Res. 113, 173-215.
MJN 3 14/022208
TABLE 1 Results obtained with S.rypAimuriurn TA98:test 1 (rangefinding)
I
'late
No.
Addition
-1+ None; S9 mix sterility check
1 None; buffer sterility check 2 POSF;
sterility check 3 POSF 4 POSF
5 POSF
6 POSF 7 POSF 8 POSF 9lPOSF 10 IUntrcated 11 POSF 12 POSF 13 POSF 14 POSF
15 POSF
16 POSF 17 POSF 18 Untreated 19 Benzo[a]pyrcnc 20 2-Nitrofluorene
None; lo4 dilution of 21 overnight culture, plated
on nutrient agar
(5of3)Pg/Platt)
(SO00 pg/plate) (2500 pg/plate) (1750 pg/platc) (1250 pg/platc) (700 pg/plate) (350 pg/plate) (175 pg/plate)
(5000 puplate) (2500 pg/plate) 1750 pg/plate) 1250 &plate) (700Pg/Pl*) (350 pg/plate) (175 pdplate)
(5 P d P W (1 Pg/PlaW
-+Sp9remseknt absent +
-
+ + + + + + +
+
-
-
-
-
+
-
r - I
Revertant colony counts* and means
B
C
Mkan sd
0
0
0
0
0
0
0
0
0
46
44
56
45
41
42
48
45
50
43
51
50
48
50
48
1 61
35
52
56
55
55
I 41
36
51
41
37
37
37
38
35
43
31
41
39
46
49
34
39
43
37
42
41
44
36
21
30
30
38
566
546
542
234
205
209
0
0
0
0
0
0
- 49
6
43
2
48
3
48
4
49
1
49
13
55
1
- 43
8
- 38
2
37
2
38
6
45
5
39
5
40
3
- 34
12
33
5
551 13
- - 216 16
133
I28
129
130 3
Except plate nos. 1,2 and 21 (total colony counts) sd Standard deviation
001587
: 18:
.
.- . ..- - .... ...
TABLE 2 Results obtained with S. typhimuriumTA98: test 2
MIN 3 141022208
Plate
Addition
No.
-14 None; S9 mix sterility chcck
1 . None; buffer sterility check
A IPOSF;
sterility check 3 POSF
4 POSF
5 POSF 6 POSF 71POSF
8 Untrcatcd
9 POSF
10 POSF
11 POSF
12 POSF
13 FQSF
14 Untreated
15 Benzo[a]pyrene 16 2-Nitrofluorene
None; 10" dilution of 17 overnight culture, plated
on nutrient agar
Revertant co
S9 mix
+ present
C
- absent
+
0
(5000 pglplate) -
(50~Pg/PW + (2500 pglplatc) + (1750 pglplate) +
0
$ 0
39
36
43
36
(1250 pglplate) +
30
I I (700 &plate)
+
51
42
46
I +
53
44
49
- I (5000 &plate)
21
39
34
- (25OoPdPlW
34
38
23
- (I750 pglplate)
37
35
35
- (1250 &plate)
29
31
29
- (700 pglplate)
32
38
23
38
39
31
(5 PglPlate) +
449
550
567
- (1 udolatel
504
467
443
- 1 1106
96
117
* Except plate nos. 1,2 and 17(totalcolony counts) sd Standard deviation
Mean sd
0
0
0
0
0
0
43
5
38
3
40
3
36
7
46 I 5
49
5
31
9
32
8
36
I
30
1
31
8
36
4
522 64
471 31
106 11
: 19:
TABLE 3 Results obtained with S. @phimnrium TA100: test 1 (range-finding)
MN 3 141022208
IF1 Addition
I+ None; S9 mix sterility check
1 - None; buffer sterility check
2 POSF; sterility check
3 POSF 4 POSF
5 POSF 6 POSF
7 POSF
8 POSF
9 POSF 10 Untreated 11 POSF 12 POSF 13 POSF 14 POSF 15 POSF
16 POSF
17 POSF 18 Unbeated 19 Benzo[a]pyrene 20 Sodium azidc
None; lo* dilution of 21 overnightculture,plated
I [onnutrient agar
S9 mix
+ present
- absent
+
(5000 &plate)
-
(SO00 pdplate) +
(2500 pg/plate) +
(1750 &plate)
+
(1250 pglplate) +
(700 @plate)
+
(350 pglplatc) +
(175 @plate)
+
+
- (5000 &late) - (2500 &late)
- (1750 pglplatc) - (1250 &plate)
- ( 7 O o P d P W - P O P ~ / P W - (175 P d P W
- (5 P d P W
+
(0.5 pglplate)
* Except plate nos. 1,2and 21 (total colony count$
sd Standard deviation
Revertmt colony counts* and means
A
B
C
Mean sd
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
125
169
137
148
130
152
153
164
154
159
141
165
154
131
114
159
145
131
143
159
128
148
140
131
119
131
122
125
132
88
125
121
139
88
118
124
138
97
136
119
118
125
122
122
109
112
110
141
573
507
568
360
453
47 1
144 23 143 12 157 6 155 12 133 20 145 14 143 16 140 9 124 6 115 24 128 9 110 19 124 23 121 4 118 8 121 17 549 37 428 60
:20 :
TABLE 4 Results obtained with S. gphimurium TA100: test 2
MIN 3 14i022208
Plate
Addition
No.
-1+ None; S9 mix sterility check
1 None; buffer sterility check 2 POSF;
sterility check
3 POSF 4 POSF 5 POSF
6 POSF 7 POSF 8 Untreated 9 POSF
10 POSF
11 POSF
12 POSF 13 POSF
14 Untreated i 5 Benzo[a]pynne 16 Sodium azide
None; lod dilution of 17 overnight culture, plated
on nutrient agar
S9 mix +present -absent
+
(5000~glPW -
(5000 pglplate) +
(Z00Pg/PW
+
(1750 &plate)
+
(1250 pglplatc) +
(700 pdplate) +
(5000 pglplate) +-
- (2500 pglplate) - (I 750 pglplate) - (1250 pglplate) - (700 pglplate)
- (5 &plate)
+
(0.5 pglplate)
Revermnt colony counts* and means ~
A
B
C
Mean sd
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
131
I45
119
148
132
148
122
154
139
147
132
166
165
125
143
131
145
131
111
118
123
126
I37
121
135
128
147
119
128
124
150
124
125
143
123
122
752
706
705
528
50 1
549
132 13 143 9 138 16 148 17 144 20 136 8 117 6 128 8 137 10 124 5 133 15 129 12 721 27 526 24
109
110
111
110
1
* Except plate nos. 1,2 and 17(total colony counts)
sd Standard deviation
:21:
MTN 3 14l022208
TABLE 5 Results obtained with S.@phimurium TA1535: test 1(range-finding)
'I&
Addition
No.
-1+ None; S9mix sterility theck
1 None;buffer sterility check 2 POSF;
sterility check 3 POSF 4 POSF 5 POSF
6 POSF
7 POSF
8 POSF 9 POSF 10 Untreated 1 1 POSF 12 POSF 13 POSF 14 POSF I5 POSF 16 POSF 17 POSF 18 Untreated 19 2-Aminoanthraccne 20 Sodium s i d e
None; lod dilution of 21 overnight culture, plated
on nutrient agar
(5OoOWpl-1
S9 mix +present
- absent
+
-
(~O~OP~/PW+
(2500pglplate) +
(1750pglplate) +
(1250pg/plate) +
(700 @plate)
+
(350pglplate) +
(1 75 pg/plate) +
+ (5000pg/plat~) -
(2500pg/plate) -
- (I750pdplate)
- (1250pg/plate)
(700 &plate)
-
- (350pg/plate) -
(175 pg/plate)
(2P g / P W
+
(0.5pg/plate) -
Except plate nos. 1.2 and 21 (total colony counts) sd Standard deviation
Revertant colony counts* and means
I I - - A
B
C
MlXll sd
- - 0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
- 14
21
16
17
4
20
26
16
21
5
29
22
12
21
9
14
26
20
20
6
22
23
19
21
2
17
23
20
20
3
14
20
19
18
3
- - 31
28
19
26
6
- - 21
I5
22
19
4
15
19
14
16
3
20
14
24
19
5
17
15
21
I8
3
19
16
24
20
4
16
I5
19
17
2
21
20
20
- 22
20
21
- - 282
324
247
- - 231
252
25 1
20
1
21
I
284 39
245 12
184
165
175
175 10
- -
001591
: 22 :
.
...
TABLE 6 Results obtained with S. rypirimurtkm TAI535: test 2
MIN 3 141022208
Addition
No.
1+ None; S9 mix sterility check
1 - None; buffer sterility check
2 POSF;
sterility check
3 POSF 4 POSF 5 POSF 6 POSF 7 POSF
8 Untreated
9 POSF 10 POSF 11 POSF 12 POSF 13 POSF
141Untreated 15 2-Aminoanthracene 16 Sodium azide
None; lod dilution of 17 overnight culture, plated
on nutrient agar
S9 mix +present
- absent
+
(5000 pglplate) -
RI 'ertant wlonv counts* and means
A
0
0 0
(5000PglPlW +
20
(2500 pglplate) +
19
(1750 pglplate) +
12
(1250 pglplate) +
20
(700 pglplate) +
21
+
22
- (5000 pglplate)
17
- (2500 pglplate)
15
- (1750 pglplate)
14
- (1250 pglplate)
21
- (700 pglplate)
22
27
(2 PglPlatC)
+
145
(0.5 &plate)
-
253
129
Except plate nos. 1,2 and 17 (total colony counts) sd Standard deviation
: 23 :
.
TABLE 7 Resultsobtained with S. typhimurium TA1537:test 1 (range-finding)
MIN 3 141022208
'late
Addition
VO.
1+ None; S9 mix sterility check
I - None; buffer sterility check
2 WSF; sterility check
3 POSF
4 POSF 5 POSF 6 POSF 7 POSF 8 POSF 9 POSF 10 Untreated
11 POSF 12 POSF
13 POSF 14 POSF 15 POSF 16 POSF 17 POSF
~
18 Untreated 19 Benzo[a]pyrene 20 9-Aminoacridine
None; 10" dilution of 21 overnight culture, plated
on nutrient agar
S9 mix
-+present absent +
(5000 bglplat~) -
(5000 pglplate) +
(2500 vglplate) +
(1750 pglplate) +
(I250 pdplate) +
(700PdPW
+
(350 pdpiate) +
(175 pglplate) +
+
- (5000 &plate) - (2500 pg/plate)
- (I750 pg/plate)
- (1250 pglplate)
- (700 @plate) - (350 @plate) - (175 pg/plate)
(5 P d P W
+
(30 pglplate) -
Revertant colony counts* and means
A
B
C
Mean sd
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
23
22
15
26
22
16
23
27
21
21
27
19
21
22
26
26
21
24
17
24
24
22
22
26
19
15
10
15
10
13
17
17
10
17
19
10
16
16
12
13
9
15
13
13
10
16
15
13
355
394
360
489
457
453
20
4
21
5
24
3
22
4
23
3
24
3
22
4
23
2
1s
5
13
3
15
4
15
5
15
2
12
3
12
2
15
2
370 21
466 20
103
139
107
116 20
Except place nos. 1,2 and 21 (total colony counts) sd Standard deviation
- 24 .
TABLE 8 Results obtained with S. rypAimrrrium TA1537:test 2
MIN 3 141022208
'late
Addition
No.
-1+ None; S9 mix sterility check
1 None; buffer sterility check 2 POSF;
sterility check 3 POSF 4 POSF 5 POSF
6 POSF
7 POSF 8 Untreated 9 POSF 10 POSF
11 POSF 12 POSF
13 POSF 14 Untreated
(5000 &plate)
S9 mix
-+ pnscnt absent +
-
(5000 pdplate) +
(2500 pg/plate) + (1750 pglplatc) + (1250 pglplate) +
(70OPglPW +
+
- (5000 pg/plate) - (2500 pdplate) -- ( 1 7 5 0 W ~ W
(1250 pg/plate)
(700FdPlW -
None; lod dilutionof
(30 P d P W
on nutrient agar
~
Except plate nos. 1.2 and 17 (total colony counts) sd Standard deviation
Revertant colony counts'
315 I 281 I 238
269 I 276 I 317
124 ' I 129 I 124
I0
13
4
12
2
14 I 3
-I
001594
: 25 :
MIN 3 14fO22208
TABLE 9 Results obtained with El coli wpZUvrA/pKMlOl (CM891):test 1(range-finding)
'late
Addition
No.
-I+ None; S9 mix sterilitycheck
1 None; buffer sterility check 2 POSF;
sterility check 3 POSF 4 POSF 5 POSF 6 POSF 7 POSF 8 POSF 9 POSF 10 Untreated 11 POSF 12 POSF 13 POSF 14 POSF 15 POSF 16 POSF 17 POSF 18 Untreated 19 2-Aminoanthraccne 20 *-2+
None; lod dilution of 21 overnight culture, plated
on nutrient agar
S9 mix
-+ present absent +
(5000 &plate)
-
(5000~g/platc) +
(2500 pglplate) +
(1750 @plate)
+
(1250 pglplate) +
(700 pglplate) +
(350 pglplate) +
(175 &plate)
+
+
(5000 &plate)
-
- (=00Pg/Plate)
- (1750 palate)
- (I250 pglplate)
- (700 P d P W
- (350 pglplate)
- (175 &plate)
- (10 Pg/PlW +
(0.05 &plate)
Except plate nos. 1,2 and 21 (totalcolony counts) sd Standard deviation
t 2-(2-Furyl)-3-(5-nitroro-t-furyl) acrylamidc
-Revertant colc - Y counts* and met
- A
B
C
0
0
0
0
0
0
0
0
0
107
131
87
119
136
96
126
122
116
123
96
119
122
144
123
122
138
119
117
121
119
123
121
133
101
112
99
111
82
89
132
118
116
85
138
85
110
86
110
95
74
84
-- 81
111
73
88
124
123
320
423
405
559
529
53 1
0
0
0
0
- - 0
0
108 22
117 20
121 5
113 15
130 12
I26 10
- - 119 2 126 6
104 7
94
15
122 9
103 31
102 14
84
11
- 88 20 112 21
- - - - 383 55
- 540 - 17
I62
188
217
189 28
- -
:26 :
MTN 3 14/022208
TABLE 10
Results obtained with B coli WP2uvrNpKM101 (CMS91): test 2
-
Plate
Addition
No.
-1t None;S9 mix sterility check
1. None; buffer sterility check
2 POSF;
- sterility check 3 POSF
4 POSF < POSF
f POSF
5 POSF
-8 Untreated -5 POSF
1c POSF
11 POSF
12 POSF 13 POSF
-14 Unecated
-15 2-Aminoanthracene -16 AF-2+ -None; lod dilution of
17 overnight culture, plated
on nutrient agar
S9 mix
-+present absent +
(5CJOo Pg/Pbt4
-
Rcvcrtant colony counts* and means
A
B
C
Mean sd
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
(50~Pg/PW +
125
136
122
128 7
(2500 pg/plate) +
154
122
165
147 22
(1750pg/plate) +
126
159
I45
143 17
(1250 pglplate) +
132
145
172
150 20
(700 pglplate) +
+
- (5000 pg/platc) - (2500 pg/plate) -- (1750 pg/plate)
(1250 &late)
- (700 pg/plat~)
184
143
166
137
144
165
147
148
128
121
139
155
144
131
145
13 1
13 1
139
115
110
135
164 21 149 I5 141 11 138 17 140 8 134 5 120 13
153
152
140
148 7
- (10 pglplatc) +
50 1
465
583
516 60
(0.05 pg/plate)
1041 899
957
966 71
190
175
189
185 8
Except plate nos. 1,2 and 17 (total colony counts)
sd Standard deviation
t 2-(2-Fury1>3-(5-nitro-2-furyl) acrylamide
: 27 :
MM 3 14/022208
' APPENDIX1 Historical control data Presented below are the historical control data from the period 1 April 1997to 30 September2001.
Untreated controls
Strain S9 mix
TAlOO
- +
TA1535
+
wP2Wf NpKMl 01 (CM891)
+
TA98
- f
TA1537
- +
Minimum
'78 81 12 10
79
73 23 29
7
7
Maximum
149 165 33
29
167 200 48
54 23 34
Meall
109 114 18 19 121 134 38 41 12 13
No.of values
309 313 307 311 214 218 311 315 308 312
Standard deviation
16 22
3
3
17
23
4
5
3
5
Upper 99% limit
151 168 33
29
170 204 49
55
23
35
Lower 99% limit
76 78 12
10
76
69
22 28
7
6
Positive controls
Strain S9 mix
TAlOO
4-
Maximum
860 1327 1350
Mean
405 564 561
No. of values
346 441 808
Standarddeviation 120 175 189
TA1535
+
781 1130 1145 205 419 229 341 434 796 139 196 114
wP2uvrA/pKM101
- (CM891) -i.
( c ) ( e l (j) 294 244 188 2312 1533 1704 1331 679 659 112 415 549 488 183 250
TA98
TA1537
+
4-
(f) (h) (g) (h) 123 123 39 67
993 1031 1933 543 320 517 350 246 788 810 569 810 115 165 337 80
(a) ENNG 3 pg @) m"N5 (c) m"N 2 Pg (d) Sodium azide OSpg (c) AF-2 0.05 pg (f)2-Nitrofluorme1 vg (g) 9-Aminoacridine 30 pg (h) Benzo[a]pyrene 5 pg (i) 2-Aminoanthraccne 2 pg (i) 2-Aminoanthracene 10 pg
: 28
MIN 3 14/022208
APPENDIX 2 Eye Research Centre GLP ComplianceStatement2001
T H E D E P ~ O F H E A L T OBFTRE GOVERNMENT 0FTHEU"ITEDKINGDOM
GOODLABORATORYPRACI'ICE
LABORATORY
BuntingdamLife S&ncrr Eye Reaurcb Centre Eye snffolk I n 3 7PX
TESTTYPE
'. -.
DATE OF INSPECTION
29* January 2001
A general inspection for compliance with the Principles of Good Laboratory Practice was canied out at the above laboratory as part of UKGLP Compliance Programme.
At the time of the inspection no deviuions were found of sufficient magnitude to affect the validity of non-clinical studies performedat tbae facilities.
Dr.Roger G. Akxanda Head, UK GLPMonitoringAuthority
OOlS98
29 :