'r 

CONFIDENTIAL 

LEUCO SULPHUR BLACK 1: 

REVERSE MUTATION ASSAY •AMES TEST" 

USING SALMONEllA TYPHIMURIUM 

PROJECT NUMBER 47/1657 

Date Started: 24 January 1991 

Experimental Procedures: 

STUDY SPONSOR: 

Sandoz Chemicals Ltd. 

CH-4002 BASLE 

SWITZERLAND 

PAGE 1 OF 19 PAGES 

CONTAINS NO CBI 

Date 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 ./7 
r 

-2-

PROJECT NUMBER: 47/1657 

SAFEPHARM LABORATORIES LIMITED 

QUALITY ASSURANCE UNIT REPORT 

The routine inspection of short term toxicity studies at Safepharm Laboratories 

is carried out as a continuous process designed to ensure that where possible 

all critical phases of a particular study type are inspected at least once per 

month. Dates of inspection for this study type are given below: 

STUDY TYPE DATECSl OF INSPECTION AND 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 of the procedures followed and accurately records 

the original raw laboratory data generated in this study. 

Date of report audit: 

J.R. Pateman M.I. Biol., C. Biol. 

QUALITY ASSURANCE MANAGER 

DATE: 

JA¥: -- ... \~.: , 

~~80\'! .J :i, ~ _;;j. 

21/02/91 

........•...... ~~····· 

................... )-.~{.Qh/. ~.1. ......... . 
r 

-3-

PROJECT NUMBER: 47/1647 

GLP COMPLIANCE STATEMENT 

I, the undersigned, hereby declare that the objectives laid down in the protocol were achieved and as nothing occurred to adversely affect the quality or 

integrity of the 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 Quality Assurance Unit. 

Good Laboratory Practice, The United Kingdom Compliance Programme, Department 

of Health 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-

C 0 N T E N T S 

SUMMARY 

INTRODUCTION 

TEST MATERIAL 

METHODS 

1. Tester Strains 

2. Preparation of Test and Control Materials 

3. Microsomal Enzyme Fraction 

4. 59 Mix and Top Agar 

5. Test Procedure 

a) Preliminary Toxicity Study 

b) Mutation Study 

6. Interpretation of Results 

RESULTS 

1. Preliminary Toxicity Study 

2. Mutation Study 

CONCLUSION 

ARCHIVES 

REFERENCES 

KEY TO TABLE OF TEST RESULTS 

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 and Spontaneous Reversion Test Results 14 

2 Experiment 1 - Without Metabolic Activation 15 

3 Experiment 1 - With Metabolic Activation 16 

4 Experiment 2 - Without Metabolic Activation 17 

5 Experiment 2 - With Metabolic Activation 18 

APPENDIX 

I GLP Certificate of Compliance 19 
'r 

-5-

PROJECT NUMBER: 47/1657 

LEUCO SULPHUR BLACK 1: 

REVERSE MUTATION ASSAY •AMES TEST• 

USING SALMONELLA TYPHIMURIUM 

SUMMARY 

1. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 

were treated with Leuco Sulphur Black 1 by the Ames plate incorporation 

method at five dose levels, in triplicate, both with and without the 

addition of a rat liver homogenate metabolising system at 10% in standard 

co-factors. The dose range was determined in a preliminary toxicity assay 

and was 8 to 5000 ugjplate in the first experiment. The experiment was 

repeated on a separate day using fresh cultures of the bacterial strains 

and fresh chemical solutions. In this case the dose range of Leuco 

Sulphur Black 1 was 312.5 to 5000 ug/plate. The method used conforms with 

the OECD Guidelines for the Testing of Chemicals, 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 1 caused no reduction in the growth of the bacterial 

lawn at any of the dose levels in any of the strains of Salmonella used. 

Leuco Sulphur Black 1 was, therefore, tested up to the maximum recommended 

dose level of 5000 ugjplate. A precipitate of Leuco Sulphur Black 1 was 

seen at and above 625 ug/plate, however, this did not interfere with 

scoring of revertant colonies. 

5. No significant increase in the numbers of revertant 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 1 was 

found to be non-mutagenic under the conditions of this test. 
r 

-6-

PROJECT NUMBER: 47/1657 

LEUCO SULPHUR BLACK 1: 

REVERSE MUTATION ASSAY •AMES TEST• 

USING SALMONELLA TYPHIMURIUM 

INTRODUCTION 

This study was conducted according to Safepharm Standard Protocol OECD 26A and 

was designed to assess the mutagenic potential of the 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 of Salmonella 

typhimurium to various concentrations of the test material. The method used 

conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 

471 and also with Method 814 in Commission Directive 84/449/EEC. A copy of the 

Certificate of Compliance with GLP, issued by the U.K. Department of Health and 

Social Security, is included in Appendix I. 

These mutant strains of Salmonella are incapable of synthesising histidine and 

are, therefore, dependent for growth on an external source of this particular 

amino acid. When exposed to a mutagenic agent these bacteria may undergo a 

reverse mutation to histidine independent forms which are detected by their 

ability to grow on a histidine deficient medium. Using various strains of this 

organism, revertants may be produced after exposure to a chemical mutagen which 

have arisen as a result of a base-pair substitution in the genetic material or 

as a frame-shift mutation in which genetic material is either added, deleted or 

miscoded. In order to make the bacteria more sensitive to 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 of DNA excision repair and a deep rough mutation· (rfa) which 

increases the permeability of the 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 test material and the bacteria are also 

incubated in the presence of a liver microsomal fraction (S9) prepared from 

rats pre-treated with a compound known to induce an elevated level of these 

enzymes. 
-7-

TEST MATERIAL 

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 

black liquid 

plastic bottle with screw cap 

room temperature 

Data relating to the identity, purity and stability of the test material are 

the responsibility of the 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 inducing frame-shift mutations 

These strains were obtained from the British Industrial Biological Research 

Association on 14th August 1987 and were stored at -196"C in a Statebourne 

liquid N2 freezer, model SXR 34. Prior to being used, characterisation 

checks were carried out to determine the amino-acid requirement, presence 

of rfa and R factors and the spontaneous reversion rate. 

In this assay, overnight sub-cultures of the 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 

bacteria per ml. 

2. Preparation of Test and Control Materials 

Leuco Sulphur Black 1 was accurately weighed, (with an allowance made for 

its purity, approximately 50%) and suspended in sterile distilled water 

and appropriate dilutions made on the morning of each experiment. A 

concentration analysis of the test material 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. A solvent treatment group was used as the negative control and 

the positive control materials were as follows: 
, r 

-8-

PROJECT NUMBER: 47/1657 

2. Preparation of Test and Control Materials (contd) 

N-Ethy1-N'-nitro-N-nitrosoguanidine (ENNG) 3 ug/p7ate for TA100 and 5 ug/ 

plate for TA1535 

9-Aminoacridine (9AA) 80 ug/p1ate for TA1537 

4-Nitro-O-pheny1enediamine (4NOPD) 5 ug/p1ate for TA1538 

4-Nitroquino7ine N-oxide (4NQO) 0.2 ug/p1ate for TA98 

In addition the materials 2-Aminoanthracene {2AA) and and Benzo{a)pyrene 

{BP) which are non-mutagenic in the absence of metabolising enzymes were 

used at the following concentrations in the S9 series of plates. 

2AA at 2 ug/p1ate for TA1535 

BP at 5 ug/p7ate for a11 of the other strains 

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. It was prepared 

from the livers of male Sprague-Dawley rats weighing- 200g. These had each 

received a single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before 

S9 preparation. 

The S9 was stored at -196"C in a Statebourne liquid N2 freezer, model SXR 34. 

4. S9 Mix and Top Agar 

The S9 Mix was prepared at 4"C as follows: 

59 

1.65 M KC1 

0.4 M MgCJ2 

0.1 M Glucose-6-phosphate 

0.1 M NADP 

0.2 M Sodium phosphate buffer (pH 7.4} 

Sterile distilled water 

5.0 m7 

1.0 m7 

1.0 m1 

2.5 m7 

2.0 m7 

25.0 m7 

13.5 m7 

Top agar was prepared using 0.6% Difco Bacto agar and 0.5% sodium chloride. 

5 ml of 1.0 mM h1st1d1ne/1.0 mM biotin solution was added to each 100 ml of 

top agar. 
,.... 

' 

-9-

PROJECT NUMBER: 47/1657 

5. Test Procedure 

a) Preliminary Toxicity Study 

In order to select appropriate dose levels for use in the main study, 

a preliminary test was carried out to determine the toxicity of the 

test material to the tester organisms. 0.1 ml of bacterial suspension 

(TA100) and 0.1 ml of test solution were added to 2 ml of molten, trace 

histidine supplemented media (histidine/biotin & top agar) and overlayed onto sterile plates of Vogel-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 a thinning of the 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. 

Test Material and Negative Controls 

0.1 ml of the appropriately diluted test material 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 of two test tubes for each bacterial tester strain. A 0.1 ml 

aliquot of one of the bacterial suspensions was also added to each of 

the two test tubes. Into one of the test tubes was placed 0.5 ml of 

the S9 liver microsome mix; in the other tube 0.5 ml of pH 7.4 buffer 

was added. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material. 

Positive Controls 

i) Without Activation 

0.1 ml of one of the positive control solutions (ENNG, 9AA, 4NQO 

or 4NOPD) was added to a test tube containing 2.0 ml of molten, 

trace histidine supplemented, top agar. 0.1 ml of the appropriate 

bacterial suspension and 0.5 ml of pH 7.4 buffer was also added to 

the test tube. This procedure was then repeated, in triplicate, 

for each of the 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 a test tube containing 

2.0 ml of molten, 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 of each 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, 

test material and control solutions. 

6. Interpretation of Results 

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 of bacteria in the presence and/or 

absence of the S9 microsomal enzymes in both experiments at sub-toxic dose 

levels. If the two experiments give conflicting results or equivocal 

results are obtained then a third 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 of which should be 

between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 ug/plate. In this 

case the limiting factor was the maximum recommended dose. 
• r 

-11-

PROJECT NUMBER: 47/1657 

RESULTS 

1. Preliminary Toxicity Study 

The dose range of Leuco Sulphur Black 1 used 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 of Salmonella used (TA100). A precipitate 

of Leuco Sulphur Black 1 was observed at and above 1250 ug/plate, however, 

this did not interfere with scoring of revertant 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 of each 

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 of revertant colonies 

obtained for each tester strain following incubation with the test material, with and without metabolic activation, are given in Tables 2 and 3 

for experiment 1 and Tables 4 and 5 for experiment 2. The results for the 

positive controls are given in Tables 2 to 5 . 

. No toxicity was exhibited to any of the strains of Salmonella used. 

A precipitate was observed with doses of Leuco Sulphur Black 1 at and above 

625 ug/plate. This did not however interfere with the scoring of revertant 

colonies. 

No significant increases in the numbers of revertant colonies of bacteria 

were recorded for any of the strains of Salmonella used, at any dose level 

either with or without metabolic activation. 

The positive control substances all produced marked increases in the number 

of revertant colonies and the activity of the 59 fraction was found to be 

satisfactory. 

CONCLUSION 

The test material, Leuco Sulphur Black 1, was found to be non-mutagenic under the 

conditions of this test. 
' - ,~ 

-12-

PROJECT NUMBER: 47/1657 

ARCHIVES 

On completion of the test, all laboratory raw data and a copy of the final 

report were transferred to Safepharm laboratories Central Archive, london Road, 

Shardlow, Derbyshire, where they will be retained for a period of ten years. 

REFERENCES 

1. Ames, B.N., Durston, W.E., Yamasaki, E., and Lee, 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., and Ames, B.N. Proc. Nat. Acad. Sci. USA 

(1975) 75, 5135. 

4. Garner, R.C., Miller, E.C., and Miller, J.A. Cancer Res. (1972), 33, 2058. 

5. Kirkland, D.J., {Ed). Statistical Evaluation of Mutagenicity Test Data. 

UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report - Part 

III (1989) - Cambridge University Press. 
-13-

PROJECT NUMBER: 47/1657 

KEY TO TABLE OF TEST RESULTS 

NOTES: 

1. "Number of revertants" - The observed values and average value are shown 

in order, beginning with the low concentration of the test substance. 

2. The following postfixes are used when 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-Nitroquinoline N-oxide 

BP Benzo(a)pyrene 
-14-

PROJECT NUMBER: 47/1657 

TABLE 1 

VIABILITY AND SPONTANEOUS REVERSION TESTS 

EXPERIMENT 1 

STRAIN OF BACTERIA PER SPONTANEOUS 

SALMONELLA TYPHIMURIUM 1 m7 OF OVERNIGHT REVERSION 

NUTRIENT BROTH 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 

EXPERIMENT 2 

STRAIN OF BACTERIA PER SPONTANEOUS 

SALMONELLA TYPHIMURIUM 1 m7 OF OVERNIGHT REVERSION 

NUTRIENT BROTH 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 ) 11 p ( 13.3 ) 19P ( 21.3 ) 9P 12.7) 

107P 20P 17P 21P 17P 

100P 12P 15P 13P 11P 

5000 94P C 101.0) 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: LEU CO 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 11 p 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 

TABLE OF TEST RESULTS: EXPERIMENT 2 

NAME OF TEST SUBSTANCE: LEUCO SULPHUR BLACK 1 ======================--•===========================s=============================•a=========••••=•======•============== 

WITH (+) OR TEST SUBSTANCE 

WITHOUT (-) CONCENTRATION 

59 MIX (ug/plate) 

NUMBER OF 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 

THE DEPARTMENT OF HEALTII AND SOCIAL SECURITY OF THE GOVERNMENT 

OF THE UNITED KINGDOM 

LABORATORY 

DATE OF INSPECTION 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

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 out at the above laboratory as part of the UK GLP Compliance Programme. 

At the time of the inspection no deviations were found of sufficient magnitude to affect 

the validity of studies performed at these facilities. 

Date 

..... l~l~~'-'.<.~tdis.;<.<:: >"\ 

M. i. van den Heuvcl 

Director 

UK GLP Compliance Monitoring Unit 

- ; 
\ /· ........ ,...,l'\ 

CONTAINS NO CBI 

IVI.LI'C:J\.II"I"Vv YvoJ.- --PH~ 

h ftJ/J IJ