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CONFIDENTIAL

LEUCO SULPHURBLACK 1: REVERSEMUTATION ASSAY •AMES TEST" USING SALMONEllATYPHIMURIUM PROJECT NUMBER 47/1657



### Date Started: 24 January 1991 Experimental Procedures:

STUDY SPONSOR: Sandoz Chemicals Ltd. CH-4002 BASLE SWITZERLAND

PAGE 1OF 19 PAGES 

### CONTAINSNOCBIDate Completed: 14 February 1991

AUTHOR: P.C. Jenkinson B.Sc., Ph.D.

ISSUED BY: Safepharm Laboratories Limited 

### P.O. Box No. 45 DERBY DEI 2BT U.K. Telephone: DERBY (0332) 792896 Facsimile: (0332) 799018 Telex: 377079 SAFPHM G

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### r -2- 

### PROJECT NUMBER: 47/1657

SAFEPHARM LABORATORIES LIMITED QUALITYASSURANCE UNIT REPORT

The routine inspection of short term toxicity studies at Safepharm Laboratories is carried out as acontinuous process designed to ensure thatwhere possible all critical phases ofaparticular study type are inspected at least once per month. Dates of inspection for this study type are given below: STUDY TYPE DATECSl OF INSPECTIONAND REPORTING Ames Test: 10/01/91, 25/01/91, 31/01/91 This report has been audited by Safepharm Laboratories Quality Assurance Unit and is an accurate account ofthe procedures followed and accurately records the original raw laboratorydata generated in this study. Date ofreport audit:

J.R. Pateman M.I. Biol., C. Biol. QUALITY ASSURANCE MANAGER

DATE:

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### 21/02/91



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-3- PROJECT NUMBER: 47/1647

GLP COMPLIANCE STATEMENT

I, the undersigned, hereby declare that the objectives laiddown in the protocol were achieved and as nothing occurred to adversely affect the quality or integrity ofthe study, I consider the data generated to be valid. This report fully and accurately reflects the procedures used and data generated in the study, and the work was performed in compliance with the following principles of Good Laboratory Practice; except that the concentration, homogeneity and stability of test material preparations were not determined by analysis; and that this specific study may not have been subject to procedure inspection by the QualityAssurance Unit. Good Laboratory Practice, The United Kingdom Compliance Programme, Department ofHealth 1989. Organisation for Economic Co-operation and Development, ISBN 92-64-12367-9, Paris 1982. United States Environmental Protection Agency, Title 40, Code of Federal Regulations, Part 160 and Part 792, and United States Food and Drug Administration, Title 21 Code of Federal Regulations Part 58.

Head of Genetic Toxicology for Safepharm Laboratories

### r
-4- C0 NT E N T S
SUMMARY INTRODUCTION TESTMATERIAL METHODS 1. TesterStrains 2. Preparation ofTest and Control Materials 3. Microsomal Enzyme Fraction 4. 59Mix and Top Agar 5. Test Procedure a) Preliminary Toxicity Study b) Mutation Study 6. Interpretation ofResults RESULTS 1. Preliminary ToxicityStudy 2. Mutation Study CONCLUSION ARCHIVES REFERENCES KEYTO TABLEOFTESTRESULTS TABLES 

### PROJECT NUMBER: 47/1657 PAGElS} 5 6 7 7 7 7 8 8 9 9 9 10

11 11 11 12 12 13
1 Viability andSpontaneous Reversion Test Results 14 2 Experiment 1
- Without MetabolicActivation 15 3 Experiment 1
- With MetabolicActivation 16 4 Experiment 2
- Without MetabolicActivation 17 5 Experiment 2
- With MetabolicActivation 18 APPENDIX I GLP Certificate ofCompliance 19

## 'r -5- PROJECT NUMBER: 47/1657 LEUCO SULPHURBLACK 1: REVERSEMUTATION ASSAY •AMES TEST• USING SALMONELLATYPHIMURIUM SUMMARY 1. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with Leuco Sulphur Black 1by the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of arat liverhomogenate metabolising system at 10% in standard co-factors. The dose range was determined in apreliminary toxicity assay and was 8 to 5000 ugjplate in the first experiment. The experiment was repeated on aseparate day using fresh cultures ofthe bacterial strains and fresh chemical solutions. In this case the dose range ofLeuco Sulphur Black 1was 312.5 to 5000 ug/plate. The method used conforms with the OECD Guidelines for the Testing ofChemicals, Protocol No. 471 and also with Method B14 in Commission Directive 84/449/EEC. 2. The solvent (sterile distilled water} control plates gave counts of revertant colonies within the normal range. 3. All positive control chemicals gave increases in revertants, both with and without the metabolising system, within expected ranges. 4. Leuco Sulphur Black 1caused no reduction in the growth ofthe bacterial lawn at any ofthe dose levels in any ofthe strains ofSalmonella used. Leuco Sulphur Black 1was, therefore, tested up to the maximum recommended dose level of5000 ugjplate. Aprecipitateof Leuco Sulphur Black 1was seen at and above 625 ug/plate, however, this did not interferewith scoring ofrevertant colonies.

5. No significant increase in the numbers ofrevertant colonies was recorded for any of the bacterial strains with any dose of Leuco Sulphur Black 1 either with or without metabolic activation. Leuco Sulphur Black 1was found to be non-mutagenic under the conditions ofthis test.r
-6- PROJECT NUMBER: 47/1657
LEUCO SULPHUR BLACK 1: REVERSE MUTATIONASSAY •AMES TEST• USING SALMONELLATYPHIMURIUM
INTRODUCTION This study was conducted according to Safepharm Standard Protocol OECD 26A and was designed to assess the mutagenic potential ofthe test material using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames and his co-workers (1, 2, 3) and Garner et al (4) in which mutagenic activity is assessed by exposing histidine auxotrophs ofSalmonella typhimurium to various concentrations ofthe testmaterial. The method used conforms with the OECD Guidelines for the Testing ofChemicals, Protocol No. 471 and also with Method 814 in Commission Directive 84/449/EEC. Acopy ofthe CertificateofCompliance with GLP, issued by the U.K. Department ofHealth and Social Security, is included in Appendix I. These mutant strains ofSalmonella are incapable of synthesising histidine and are, therefore, dependent for growth on an external source ofthis particular amino acid. When exposed to amutagenic agent these bacteriamay undergo a reverse mutation to histidine independent forms which are detected by their abilityto grow on ahistidine deficientmedium. Using various strains ofthis organism, revertants may be produced after exposure to achemical mutagen which have arisen as aresult of abase-pair substitution in the genetic material or as aframe-shift mutation in which genetic material is either added, deleted or miscoded. In order tomake the bacteriamore sensitiveto mutation by chemical and physical agents several additional traits have been introduced. These include a deletion through the excision repair gene (uvrB) which renders the organism incapable ofDNA excision repair and adeep rough mutation· (rfa) which increases the permeability ofthe cell wall. Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems, not available in the bacterial cell, the testmaterial and the bacteria are also incubated in the presence of alivermicrosomal fraction (S9) prepared from rats pre-treated with a compound known to induce an elevated level ofthese enzymes.-7- TESTMATERIAL 1. Sponsor's identification 2. Date received 3. Description 4. Container 5. Storage conditions PROJECT NUMBER: 47/1657 DIRESUL BLACK ROT (LEUCO SULPHUR BLACK 1) LIQUID 16 January 1991 blackliquid plastic bottlewith screw cap room temperature Data relating to the identity, purity and stabilityofthe test material are the responsibility ofthe sponsor. METHODS 1. Tester Strains The strains used in this assay were all mutants derived from Salmonella typhimurium LT2 and were those recommended for general screening. TA1535 TAlOO TA1537 TA1538 TA98 sensitive to agents inducing base-pair substitution sensitive to agents inducingframe-shift mutations These strainswere obtained from the British Industrial Biological Research Association on 14th August 1987 and were stored at -196"C in aStatebourne liquid N2 freezer, model SXR 34. Prior to being used, characterisation checks were carried out to determine the amino-acid requirement, presence ofrfa and Rfactors and the spontaneous reversion rate. In this assay, overnight sub-cultures ofthe master slopes were prepared in nutrient broth (Oxoid Limited) and incubated at 37"C for approximately 18 hours. These overnight cultures yielded approximately 108
- 109 bacteriaper ml. 2. Preparation ofTest and Control Materials Leuco Sulphur Black 1was accuratelyweighed, (with an allowance made for its purity, approximately 50%) and suspended in steriledistilledwater and appropriate dilutions made on the morning ofeach experiment. A concentration analysis ofthe testmaterial was not performed. Dilutions of Leuco Sulphur Black 1 were regularly mixed to maintain an even suspension for dosing. Negative and positive controls were used in parallel with the test material. Asolvent treatment group was used as the negative control and the positive control materials were as follows:, r
-8- 

### PROJECTNUMBER: 47/1657 2. Preparation ofTest and Control Materials (contd) N-Ethy1-N'-nitro-N-nitrosoguanidine (ENNG) 3ug/p7atefor TA100 and5ug/ platefor TA1535 9-Aminoacridine (9AA) 80 ug/p1ate

### for TA1537 4-Nitro-O-pheny1enediamine (4NOPD) 5ug/p1ate 

### for TA1538 4-Nitroquino7ineN-oxide (4NQO) 0.2 ug/p1ate 

### forTA98 In addition the materials 2-Aminoanthracene {2AA) and and Benzo{a)pyrene {BP) which are non-mutagenic in the absence ofmetabolising enzymes were used atthe following concentrations in the S9 series ofplates. 2AA at 2ug/p1ate 

### for TA1535 BP at 5ug/p7ate 

### for a11 ofthe otherstrains 3. Microsomal Enzyme Fraction Lot No. Aro. S9/24/1/91A, prepared on 24/1/91 was obtained from the British Industrial Biological Research Association on 31/01/91. Itwas prepared from the livers ofmale Sprague-Dawley rats weighing- 200g. These had each received a single i.p. injection ofAroclor 1254 at 500 mg/kg, 5days before S9 preparation. The S9 was stored at -196"C in aStatebourne liquid N2 freezer, model SXR 34. 4. S9 Mix and TopAgar The S9 Mix was prepared at4"C as follows: 59 1.65M KC1 0.4M MgCJ2 0.1

### M Glucose-6-phosphate 0.1

### M NADP 0.2MSodiumphosphate buffer(pH 7.4} Steriledistilledwater 5.0m7 1.0m7 1.0m1 2.5m7 2.0m7 25.0m7 13.5m7 Top agar was prepared using 0.6% Difco Bacto agar and 0.5% sodium chloride. 5 ml of 1.0 mM h1st1d1ne/1.0mM biotin solutionwas added to each 100 ml of top agar.,.... ' -9- PROJECT NUMBER: 47/1657 5. Test Procedure a) PreliminaryToxicity Study In order to select appropriate dose levels for use in the main study, a preliminary test was carriedout to determine the toxicityofthe test material to the testerorganisms. 0.1 ml ofbacterial suspension (TA100) and 0.1 ml oftest solution were added to 2ml ofmolten, trace histidine supplemented media (histidine/biotin

### &top agar) and overlayed onto sterile plates ofVogel-Bonner agar (minimal agar - 30 ml/ plate). Five doses of the test compound and a solvent control (sterile distilled water) were tested in duplicate. After approximately 48 hours incubation at 37°C the plates were scored for revertant colonies and examined for athinning ofthe background lawn. b) Mutation Study EXPERIMENT I Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standards for mutagenicity tests using microorganisms. TestMaterial and Negative Controls 0.1 ml ofthe appropriately diluted testmaterial or negative control solution was placed in sets of sterile test tubes containing 2.0 ml of molten, trace histidine supplemented, top agar at 45°C. These sets comprised oftwo test tubes for each bacterial tester strain. A0.1 ml aliquot of one ofthe bacterial suspensions was also added to each of the two test tubes. Into one ofthe test tubes was placed 0.5 ml of the S9 livermicrosome mix; in the other tube 0.5 ml ofpH 7.4 buffer was added. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration oftest material. PositiveControls i) Without Activation 0.1 ml ofone ofthe positive control solutions (ENNG, 9AA, 4NQO or 4NOPD) was added to atest tube containing 2.0 ml ofmolten, trace histidine supplemented, top agar. 0.1 ml of the appropriate bacterial suspension and 0.5 ml ofpH 7.4 bufferwas also added to the test tube. This procedure was then repeated, in triplicate, for each ofthe positive controls.5. Test Procedure (contd) b) Mutation Studv (contd) Positive Controls (contd) ii) With Activation -10- PROJECT NUMBER: 47/1657

0.1 ml of 2AA or BP solution was added to atest tube containing 2.0 ml ofmolten, trace histidine supplemented, top agar. 0.1 ml of one of the test bacterial suspensions and 0.5 ml of S9 mix were also added to the test tube. The procedure was then repeated, in triplicate, for each tester strain. The contents ofeach test tube were equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). These plates were incubated at 37"C for approximately 48 hours and the number of revertant colonies counted. EXPERIMENT 2 The complete experiment was repeated using fresh bacterial cultures, testmaterial and control solutions.
6. InterpretationofResults For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains ofbacteria in the presence and/or absence ofthe S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained then athird experiment may be used to confirm the correct response. All data are statistically analysed using the methods recommended by the UKEMS (5). To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals ofwhich should be between 2 and 5fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of5000 ug/plate. In this case the limiting factor was the maximum recommended dose.• r -11- PROJECT NUMBER: 47/1657 RESULTS 1. PreliminaryToxicity Study The dose range of Leuco Sulphur Black 1used in the preliminary toxicity study was 0, 312.5, 625, 1250, 2500 and 5000 ug/plate. Leuco Sulphur Black 1 was non-toxic in the strain ofSalmonella used (TA100). Aprecipitate of Leuco Sulphur Black 1was observed at and above 1250 ug/plate, however, this did not interferewith scoring ofrevertant colonies. 2. Mutation Study The results for the checks on**viabilit~** and spontaneous reversion rate for each tester strain are shown in Table 1. The overnight culture ofeach strain was found to be in the required range of 108
- 109 bacteria per ml and the spontaneous reversion rate for each was found to be within the expected range. Individual plate counts together with the mean number ofrevertant colonies obtained for each tester strain following incubation with the testmaterial, with and without metabolic activation, are given in Tables 2and 3 for experiment 1and Tables 4and 5for experiment 2. The results for the positive controls are given in Tables 2to 5 .



### . No toxicitywas exhibited to any ofthe strains ofSalmonella used. A precipitatewas observed with doses ofLeuco Sulphur Black 1at and above 625 ug/plate. This did not however interferewith the scoring ofrevertant colonies. No significant increases in the numbers ofrevertant colonies ofbacteria were recorded for any ofthe strains ofSalmonella used, at any dose level eitherwith orwithout metabolic activation. The positive control substances all produced marked increases in the number of revertant colonies and the activity ofthe 59 fraction was found to be satisfactory. CONCLUSION The test material, Leuco Sulphur Black 1, was found to be non-mutagenic under the conditions ofthis test.

### '

-**,~** -12- PROJECT NUMBER: 47/1657



### ARCHIVES On completion of the test, all laboratory raw data and acopy ofthe final report were transferred to Safepharm laboratories Central Archive, london Road, Shardlow, Derbyshire, where they will be retained for aperiod often years.


### REFERENCES 1. Ames, B.N., Durston, W.E., Yamasaki, E., andLee, F.D. Proc. Nat. Acad. Sci. USA (1970), 70, 2285 2. Ames, B.N., McCann, J. and Yamasaki, E., Mutation Research (1975), 31, 347. 3. McCann, J., Coi, E., Yamasaki, E., andAmes, B.N. Proc. Nat. Acad. Sci. USA (1975) 75, 5135. 4. Garner, R.C., Miller, E.C., andMiller, J.A. CancerRes. (1972), 33, 2058. 5. Kirkland, D.J., {Ed). Statistical Evaluation ofMutagenicity Test Data. UKEMSSub-committee on Guidelines for

### Mutagenicity Testing. Report
- Part III (1989)
- Cambridge UniversityPress.-13- 

### PROJECT NUMBER: 47/1657



### KEYTO TABLEOFTESTRESULTS
NOTES: 1. "Number of revertants"
- The observedvalues and average value are shown in order, beginningwith the low concentration ofthe test substance. 2. Thefollowingpostfixes are usedwhen required:- C= contaminated P=precipitate S = sparse background lawn V= very thin background lawn T= toxic X=plate unscorable
ENNG N-Ethyl-N'-nitro-N-nitrosoguanidine

9AA 9-Aminoacridine 2AA = 2-Aminoanthracene 4NOPD

### = 4-Nitro-0-phenylenediamine 4NQO 4-NitroquinolineN-oxide BP Benzo(a)pyrene-14- PROJECT NUMBER: 47/1657 

### TABLE 1

VIABILITYANDSPONTANEOUSREVERSIONTESTS

EXPERIMENT1 STRAINOF BACTERIA PER SPONTANEOUS SALMONELLA TYPHIMURIUM 1m7 

### OFOVERNIGHT REVERSION NUTRIENTBROTH COUNTS I 

### PLATE TA1535 B1.4 X

### JOB 12 17 15 TA1537 90.4 X

### lOB 15 11 

### 13 TA153B 50.6 X

### JOB 22 13 10 TA9B 25.3 X

### JOB 20 lB lB TAlOO 11.2x1o8 Bl JOl 124

EXPERIMENT2 STRAINOF BACTERIA PER SPONTANEOUS SALMONELLA TYPHIMURIUM 1m7 

### OFOVERNIGHT REVERSION NUTRIENTBROTH COUNTS I 

### PLATE TA1535 96.4 X

### loB 19 11 14 TA1537 26.1 X

### JOB 15 10 9 TA153B 99.6 X

### JOB 15 19 27 TA9B B1.1 X

### JOB 16 34 29 TAlOO B2.9 X

### JOB 111 9B 115-15- PROJECT NUMBER: 47/1657 T A

### B l E 2 TABLE OF TEST RESULTS: EXPERIMENT 1
NAME OF TEST SUBSTANCE: lEUCO SUlPHUR BLACK 1**===============================================================~========================================================** WITH (+) OR TEST SUBSTANCE WITHOUT (-) CONCENTRATION 59 MIX (ug/plate) NUMBER OF REVERTANTS (number of colonies/plate) BASE-PAIR SUBSTITUTION TYPE FRAHESHIFT TYPE

TA100 TA1535 TA1538 TA98 TA1537 =========================================================:==============--=============================================== 111 14 16 17 11 0 94 ( 104.3 ) 13 ( 13.3 ) 14 ( 14.7) 17 ( 16.3 ) 7 ( 11.3 ) 108 13 14 15 16
102 12 19 15 7 8.0 125 ( 117.0 ) 14 ( 12.3 ) 16 ( 16.7) 26 ( 22.0 ) 7 7.7 ) 124 11 15 25 9
117 13 17 19 15 40 106 ( 112.0 ) 15 ( 13.7 ) 18 ( 15.7 ) 14 ( 18.0 ) 13 ( 14.0 ) 113 13 12 21 14
109 14 12 19 10 200 106 ( 105.3 ) 12 ( 14.3 ) 17 ( 14.7 ) 14 ( 16.0 ) 9 ( 10.0 ) 101 17 15 15 11
116P 12P 12P 24P 12P 1000 120P ( 114.3 ) 10P ( 14.0 ) 11p ( 13.3 ) 19P ( 21.3 ) 9P 12.7) 107P 20P 17P 21P 17P
100P 12P 15P 13P 11P 5000 94P C101.0) 15P ( 14.7 ) 18P ( 15.3 ) 15P ( 14.7 ) 15P ( 10.7 ) 109P 17P 13P 16P 6P ========================================================================================================================

POSITIVE CONTROL

NOT NAHE
CONCENTRATION (ug/plate) ENNG ENNG
3.0 5.0 4NOPD 4NQO 9AA
5.0 0.2 80.0 REQUIRING
----------------------------------------

S9 MIX NUMBER OF COLONIES/PLATE 615 625 ( 647.7) 703 340 360 ( 357.7) 373 411 387 ( 372.7 ) 320 271 272 ( 278.7 ) 293 632 670 ( 645.0 ) 633 ========================================================================================================================! - -16- PROJECT NUMBER: 47/1657 T A B L E 3
TABLE OF TEST RESULTS: EXPERIMENT I
NAME OF TEST SUBSTANCE: LEUCO SULPHUR BLACK I ======================================================================================================================== WITH (+) OR TEST SUBSTANCE WITHOUT (-) CONCENTRATION 59 MIX (ug/plate) NUMBER OF REVERTANTS (number of colonies/plate) BASE-PAIR SUBSTITUTION TYPE FRAHESHIFT TYPE

TA100 TA1535 TA1538 TA98 TA1537 ======================================================================================================================== 163 16 28 25 17 t 0 142 ( 148.0 ) 18 ( 16.7 ) 31 25.0 ) 36 ( 27.3 ) 10 ( 13.3 ) 139 16 16 21 13
154 16 12 24 11 + 8.0 147 ( 145.0 ) 21 ( 18.7 ) 25 ( 18.7 ) 31 ( 26.3 ) 9 ( 11.3 ) 134 19 19 24 14
110 10 20 20 13 + 40 110 ( 121.7 ) 20 ( 13.7 ) 17 ( 20.3 ) 20 ( 21.3 ) 8 ( 11.7 ) 145 11 24 24 14
124 16 27 32 8 + 200 113 ( 112.0 ) 13 ( 17.3 ) 21 ( 27.0) 28 ( 31.0 ) 14 ( 11.3 ) 99 23 33 33 12
104P 24P 19P 19P 13P + 1000 95P ( 107.7 ) 12P ( 17.0 ) 25P ( 19.3 ) 16P ( 18.3 ) 12P ( 12.3 ) 124P 15P 14P 20P 12P
108P 15P 14P 15P 9? + 5000 115P ( 115.0 ) 15P ( 14.3 ) 13P ( 16.3 ) 16P ( 17.7 ) 13P 11.7 ) 122P 13P 22P 22P 13P ======================================================================================================================== NAME BP 2AA BP BP BP POSITIVE CONTROL CONCENTRATION 5.0 2.0 5.0 5.0 5.0 REQUIRING (ug/plate) S9 MIX NUMBER OF 681 286 325 380 103 + COLONIES/PLATE 632 ( 654.0 ) 310 ( 291.0 ) 397 ( 353.0 ) 379 ( 385.7 ) 114 ( 103.0 ) 649 277 337 398 92 ========================================================================================================================-17- PROJECT NUMBER: 47/1657 T A

### B L E 4 TABLE OF TEST RESULTS: EXPERIMENT 2
NAME OF TEST SUBSTANCE: LEUCO SULPHUR BLACK 1 -===================================================================z=================================================== NUMBER OF REVERTANTS (number of colonies/plate) WITH (+) OR TEST SUBSTANCE BASE-PAIR SUBSTITUTION TYPE FRAMESHIFT TYPE WITHOUT (-) CONCENTRATION TA100 TA1535 TA1538 TA98 TA1537 59 MIX (ug/plate) ======================================================================================================================== 111 17 9 17 7 0 118 ( 112.3 ) 10 ( 12.0 ) 16 ( 12.0 ) 23 ( 19.3 ) 11 ( 10.3 ) 108 9 11 18 13
91 18 16 22 11 312.5 91 ( 94.0 ) 13 ( 13.7 ) 19 ( 15.3 ) 21 ( 20.3 ) 13 ( 12.3 ) 100 10 11 18 13
95P 13P 15P 14P 10P 625 112P ( 97.0 ) 12P ( 11.7 ) 16P 13.7 ) 15P ( 16.3 ) 13P ( 11.3 ) 84P 10P 10P 20P 11P
120P 11P 12P 24P 7P 1250 118P ( 116.0 ) 12P ( 12.0 ) 11P ( 12.7 ) 18P ( 21.0 ) 7P ( 7.0 ) 110P 13P 15P 21P 7P
86P 10P 11P 18P 8P 2500 119P ( 107.0 ) 11P ( 11.7 ) 12P ( 11.3 ) 21P ( 19.3 ) 8P ( 8.7 ) 116P 14P 11p 19P 10P
103P 10P 9P 14P 9P 5000 121P ( 122.7 ) 9P ( 10.0 ) 12P ( 9.3 ) 12P ( 13.0 ) 13P ( 9.3 ) 144P 11P 7P 13P 6P ======================================================================================================================== NAME ENNG ENNG 4NOPD 4NQO 9AA POSITIVE CONTROL CONCENTRATION 3.0 5.0 5.0 0.2 80.0

NOT (ug/platel REQUIRING

59 MIX NUMBER OF 616 401 366 280 280 COLONIES/PLATE 620 ( 641.7 ) 374 ( 397.0 ) 353 ( 348.3 ) 256 ( 275.7 ) 256 ( 275.7 ) 689 416 326 291 291 ======================================================================···====···========================================-18- 

### PROJECT NUMBER: 47/1657 T A B L E 5


### TABLEOF TEST RESULTS: EXPERIMENT2
NAME OF TEST SUBSTANCE: LEUCO SULPHURBLACK 1 ======================--•===========================s=============================•a=========••••=•======•============== WITH (+) OR TEST SUBSTANCE WITHOUT (-) CONCENTRATION 59 MIX (ug/plate) NUMBEROF REVERTANTS (number of colonies/plate) BASE-PAIR SUBSTITUTION TYPE FRAMESHIFT TYPE

TA100 TA1535 TA1538 TA98 TA1537
================-•••===-••cz===========================-===========================•==========•=========================

+ 0

+ 312.5

+ 625

+ 1250

+ 2500

+ 5000 92 130 ( 116.7 ) 128
100 92 ( 96.3 ) 97
103P 95P ( 95.3 ) 88P
88P 95P ( 85.3 ) 73P
88P 110P ( 103.3 ) 112P
107P 116P ( 119.0 ) 134P 11 13 ( 12.0 ) 12
13 15 ( 13.3 ) 12
17P 11P ( 12.7 ) 10P
11P 13P ( 12.7 ) 14P
9P 12P ( 10.0 ) 9P
10P 9P ( 9.7 ) 10P 21 23 ( 21.3 ) 20
27 23 ( 25.0 ) 25
19P 18P ( 19.0 ) 20P
11P c ( 12.0 ) 13P
14P 7P ( 10.7 ) 11P
17P 13P ( 14.3 ) 13P 26 23 ( 26.3 ) 30
29 30 ( 25.3 ) 17
17P 28P ( 26.3 ) 34P
23P 17P ( 19.7 ) 19P
23P 26P ( 25.0 ) 26P
17P 20P ( 19.3 ) 21P 13 14 ( 13.0 ) 12
17 11 ( 13.0 ) 11
9P 16P ( 14.0 ) 17P
15P 10P ( 10.7 ) 7P
9P 8P ( 10.0 ) 13P
11P SP ( 9.3 ) 12P ===============================········=========Em=·================================================··================== NAME BP 2AA BP BP BP POSITIVE CONTROL CONCENTRATION 5.0 2.0 5.0 5.0 5.0 REQUIRING Cug/plate)

59 MIX NUMBER OF 598 256 144 434 176 + COLONIES/PLATE 627 ( 608.0 ) 266 ( 271.3 ) 145 ( 143.0 ) 404 ( 397.0 ) 222 ( 201.7 ) 599 292 140 353 207 ===================================================================-===============================================--=-=-19- 

### PROJECT NUMBER: 47/1657 APPENDIX I

THEDEPARTMENTOFHEALTIIANDSOCIALSECURITYOFTHEGOVERNMENT OFTHEUNITEDKINGDOM

LABORATORY

DATEOFINSPECTION GOODLABORATORYPRACTICE
STATEMENTOFCOMPLIANCE
SafePharm Laboratories Ltd P.O. Box No. 45 DERBY Derbyshire DEl 2BT
8 August 1989

A general inspection for compliance with the Principles of Good Laboratory Practice was carried outat the above laboratory as part ofthe UK GLPCompliance Programme. At the time ofthe inspection no deviations were found ofsufficient magnitude to affect the validity ofstudies performed atthese facilities.

Date 

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M. i.van den Heuvcl Director UK GLP Compliance MonitoringUnit

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### CONTAINSNO

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