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### UNITEDSTATES ENVIRONMENTAL PROTECTIONAGENCY WASHINGTON D
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### COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS

MR Report No. SEW 1/841223
To the best of my knowledge and belief the study described in this report was conducted in compliance with the following Good Laboratory Practice Standards: United States Food and Irrug Administration Title 21 Code of Federal Regulations Part 58 Federal Register 22 December 1978 and subsequent Amendments Japan Ministry of Health and Welfare Notification No. 313 Pharmaceutical Affairs Bureau 

### 31 March 1982 

## il orgmization for Economic Co-Cperation and Development

. 

### ISBN 92-64-12367-9, Paris 1982

4argaret Richold, B.Sc., M.Phil., Date study Director# iuntingdon Research Centre plc

QUALITY ASSURANCE STAT0EM
:ertain studies of short duration, such as that reported herein, are :onducted at HRC in a setting which involves frequent repetition of ;imilar or identical procedures. At or about the time of the study lescribed in this report, 'process-based' inspections were made by, the

uality Assurance Unit of the critical procedures relevant to this study :ype. For the inspection of any given procedure, at least one study was ;elected without bias. The findings of these inspections were reported )rouptly to the Study Director and to HRC management. .his report has been audited by the HRC Quality Assurance Unit and is :onsidered to be an accurate presentation of the data produced during the -ourse of the study.-

IAQ 

### :.W.G. Shillam, B.Sc., Ph.D., F.I.Biol. Date 

### ~Onalitv Assurance

### SEW/i BACTERIAL REVERSE GENE MUTATION ASSAY - THE AMES TEST

The object of this study is to assess the mutagenic potential of-the test materi-al in a bacterial system.

COMPOUND: Alkali blue AP 3600. Purity: 9% Storage conditions: Stored at room temperature. Appearance: Blue powder.

METHOD: The method used is as described in Appendix 1. The experiments described in this report were carried out between 20 November 1984 and 30 November 1984. Solvent: Dimethylsuiphoxide.
Dose levels: Dose range finding test: 5000, 500, 50, 5 pg/plate. Mutation tests: 5000, 1500, 500, 150, 50 jg/plate.

RESULTS The revertant colony counts for Alkali blue AP 3600 obtained? in the dose range finding test are shown in Table 1. Alkali blue AP 3600 was not toxic towards the tester strains. Therefore 5000 pg/plate was chosen as the top dose level in the mutation tests. The mean number of revertant colonies, together with the individual plate counts for Alkali blue AP 3600 obtained in the first mutation test with tester strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 are shown in Table 2. Compound sterility and positive control mutability checks are shown in Table 3. The mean number of revertant colonies, together with the individual plate counts for Alkali blue AP 3600 obtained in the second mutation test with tester strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 are shown in Table 4. Compound sterility and positive control mutability checks are shown in Table 5. No substantial increases in revertant colony numbers of any of the five testerptrains were observed following treatment with Alkali blue *AP 3600 at 'In dtjse level, either in the presence or absence of metabolicactivation (S-9 mix).CONCLUSIONS AND COMMENTS it is concluded that no evidence of mutagenic potential of Alkali blue AP'3600 was obtained in this bacterial test system at the dose levels used.

22

### X TABLE 1 Dose range finding test on Alkali blue AP 3600 -revertant colony counts obtained with S.taiurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100

----- ---- - - -------------------------------- -

Dose level Metabolic. Strain of S. typhianurium (rig/plate) activation ------------------- TA 1535 TA 1537 TA 1538 TA 98 TA 100

--------- -------------------------------- ---

5000 - 2P 6P 6P 15P 51P

500 - 7P 12P 5P 16P 59P

50 - 8 7 5 15 60

5 - a 12 3 14, 64 0 - 7 16 5 16 69 5000 + 3P 7P 16P 20P 64P

500 + 4P 12P 20P 17P 70P

s0o 4 9 10 is 68

5 + 11 .13 14 17 84 0 + 10 12 9 17 92 L------------ L---------------------------L------ j------------
*- Absence

+ Presence P Precipitate

3T1ABLE 2 SHw/i Test 1 Alkali blue AP 3600 -revertant colony counts obtained per plate using S. ypimrim 

### strains TA 1535, TA 1537, TA1538, TA 98 and TA 100 Strain Dose level Metabolic* Mean revertant SD Individual revertant (pg/plate) activation colony counts colony counts

- *TA 1535 5000 -3 1.5 3P,SP,2P 1500 -9 2.6 7P,8P,12P

500 - 8 2.6 11P,6P,7P 150 - a 4.5 12,8,3

50 - 6 0.6 5,6,6

0 -11 4.0 10,7,15 5000 +' 7 3.5 7P,3P,1OP 1500 + 7 2.1 5P,9P,8p

500 + 7 1.5 9P,6P,7P 150 + 9 2.1 7,8.11

50 + 9 1.5 11,9,8

0 + 8 2.1 10,6,7

--------------------------------------------------------------------------- TA 1537 5000 -6 0.6 6P,6P,7P 1500 -3 3.2 7P,2P,IP

500 -8 2.3 7P,11P,7P 150 -14 3.6 11,18,13

50 -13 3.1 16,10,14

0 - 12 2.9 14,14,9 5000 + 10 3.2 14P,9P,8P 1500 + 9 2.3 8P,12P,8P

500 + 9 3.2 5P,IOP,11P 150 + 12 4.6 17,9,9

50 + 12 1.7 13,13,10

0 + 15 1.5 15,13,16 -------------------------------------------------------------------------------- TA 1538 5000 8 1.7 9P,9P,6P 1500 6 1.0 7P,5P,6P .500 9 4.0 13P,5P,8P 150 5 2.5 3,5,8

50 5 1.2 6,4,,6

0 7 2.1 5,8,9 5000 + 12 5.0 7P,17P,11P 1500 + 11 3.5 7P,13P,13P

500 + 13 7.5' 14P,5P,20P 150 + 13 6.6 19,6,14

50 + '16 1.0 17,16,15

0 + 12 1.5 13,10,12

*-Absence + Presence SD Standard deviation

P Precipitate*TABLE 2 5KW/i (continued)

Strain Dose level Metabolic. Mean revertant SD Individual revertant (big/plate) activation colony counts colony counts

------------------------------------------------------------------- TA 98 5000 - 7 3.0 10P,4P,7P 1500 - 12 2.1 14P,11P,1OP

500 - 12 1.5 14P,llP,12P 150 - 12 1.5 13,10,12

50 - 14 3.8 12,11,18

0 - 17 2.1 16,19,15 5000 + 013 2.1 14P,15P,11P 1500 + 15 3.5 12P,15P,19P

500 + 16 4.,4 21P,14P,13P 150 + 19 4.5 15,19,24

50 + 17 7.4 25,14,11

0 + 17 2.1 19,18,15 ------------- ------------ ------------ --------------- ----- --------------------- TA 100 5000 - 29 3.1 32P,28P,26P 1500 - 55 2.6 57P,52P,56P

500 - 56 2.1 57P,58P,54P 150 - 70 14.0 66,86,59

5o - 58 1.5 57,60,58

0 - 64 7.8 62,58,73 5000 + 61 5.0 56P,66F,60P 1500 + 59 7.2 63P,51P,64P

500 + 68 1.7 69P,69P,66P 150 + 72 8.2 63,79,74

50 + 69 2.3 70,66,70 L 0 + 75 11.1 87,73,65

*-Absence + Presence SD Standard deviation

P Precipitate-~~--EEEN-EMN- 1,11 III U 

### in UU I k td J L--

### I

TI3LE 3 SEW/I Test I Mutability and sterility tests with S.tpiuimstrains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 Strain Coupoiand Concentration iBmtabolico Mlean SD Individuaal

- of coupound activation revertant revertant (pg/plate) colony colony counts conts

- ------ ------ - ----------------- -------- TA 1535 Sodium azid. S - 686 20.0 671,709,679 TA 1537 9-aininoacridine 20 - 201 1.5 200,201,203 TA 1538f 2-nitrofluorene 

### ~ '10 - 148 21.1 124,161,160 TA 98 

## }- 293 16.3 275,299,306 TA 100 sodium azide 5 - 838 19.5 847,816,852 TA 15351 + 117 13.0 104,130,118 TA 1537~ + 55 4.9 61,52,53 TA 15381 2-aminoanthracele 2 + 419 105.0 529,407,320 TA 98 + 478 53.7 442,540,453 TA 100 

## J+ 521 70.3 539,443,580

-S-9 

### mix 500 Al 0 0

-Alkali blue 0 Op AP 3600 5000
L-----L--- --- ----------- - ------ L---------- - L-- -------

e - Absence + Presence SD standard deviation

p precipitateTABLE 4 SHW/1 Teat 2 Alkali blue AP 3600 -revertant colony counts obtained per plate using S. typhimuriuu strain. TA 1535, TA 1537, TA 13, TA98 and TA 100 Strain Dose level Metabolic. Mean revertant SD Individual revertant (gg/plate) activation colony counts colony counts TA 1535 5000 -3 1.5 3P,2P,SP 1500 -6 2.11 7P,8P,4P

500 5 2.6 8P,3P,4P 150 4 2.5 2,7,4

50 8 2.6 9,5,10

0 *9 3.1 12,8,6 5000 + 5 2.3 8P,4P,4P 1500 + 6 2. ,1 5P,8P,4P

500 + 6 1.5 7P,4P,6P 150 + 7 1.5 9,7,6

50 + 9 1.0 10,8,9 S- 0 + 12 4.6 9,17,9 TA 1537 5000 -6 2.0 8P,6P,4P 1500 - 10 2.6 12P,7P,11P

500 - 8 3.8 12P,6P,5P 150 - 13 3.61 16,9,14

50 - 8 1.7 7,10,7

0 - 13 2.1 15,11,14 5000 + 9 1.5 9P,7P,IOP 1500 + 11 1.5 11P,12P,9P

500 + 10 3.2 6P,12P,11P 150 + 18 1.7 19,16,19

50 + 7 3.6 6,4,11

0 + 18s 1.5 18,16,19 TA 1538 5000 5 1.2 4P,6P,4P 1500 -4 0.6 4P,4P,5P

500o 6 0.6 6P,7P,6P 150 -8 4.0 12,7,4

50 6 2.6 5,9,4.

0 6 2.6 5,4,9, 5000 + 10 5.2 4P,13P,13P 1500 + 15 8.5 7P,24P,14P

500 + 13 2.9 1op,15p,15P 150 + q 1.7 18,18,21

50 + 16 2.1 18,14,15

0 + 14 1.5 12,14,15

*-Absence + Presence SD Standard deviation

P Precipitate

:7TAbLE 4 S .L (continued)

Strain Dose level Metabolic. Mean revertant SD Individual revertant (jig/plate) activation colony counts colony counts TA 98 5000- 12 2.6 llP,lOP,l5P 1500 -15 2.0 13P,15P,17P

500 -15 2.0 13P,15P,17P 150 -14 0.6 14,14,15

50 -13 2.5 16,13,11

0 18i 3.6 19,21,14 5000 + # 14 2.1 16P,13P,12P 1500 + 17 2.3 16P,20P,16P

500 + 19 5.0 24P,18P,14P 150 + 22 3.1 19,25,23

50 + 20 2.1 22,18,19

0 + 18 1.0 19,18,17 TA 100 5000 -65 .3.5 62P,65P,69P 1500 -66 3.8 62P,69P,68P

500 -64 1.7 62P,65P,65P 150 - 67 14.81 54,63,83

50 - 82 15.1 70,99,77

0 - 68 4.2 63,69,71 5000 + 76 5.9 83P,,74P,72P 1500 + 78 5.5 84P,73P,78P

500 + 74 9.2 72P,66P,84P 150 + 89 1.2 88,90,90

50 + 73 2.6 74,70,75

0 + 74 8.7 84,67,72

*-Absence + Presence SD Standard deviation

P Precipitate* TABLE 5 SHN/i Teot 2 Mutability and sterility tests with S. typhizurium st~rains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 strain Conpound Concentration Metabolices Mean SD Individual of coqpound activation revertant revertant

(pg/plate) colony colony counts counts TA 1535 Sodium azide 5 - 736 21.1 712,750,747 TA 1537 9-aminoacridine 20 - 85 9.1 75,89.92 TA 15138} 2-nitrofluorene *10 - 213 31.7 246,209,183 TA 98 

## J- 208 8.6 206,200,217 TA 100 Sodim azide 5 - 835 69.8 859,756,889 TA 

### 15351 + 125 17.4 119,145,112 TA 153 + 60 12.5 64,70,46 TA 1538) 2-aminoanthracene 2 + 438 17.8 451,418,446 TA 98 

## J+ 366 116.9 269.,334,496 TA 100 

## J+ 850 78.7 941,810,800

-S-9 mix 500Ogl 0 0

-Alkali blue 0 OP AP 3600 5000 9 - Absence

+ Presence SD Standard deviation P Precipitate

# *jAPPENDIX 1 

## SHW/1 Experimental Protocol

1. MATERIALS 1.1 Bacterial strains / The following strains will be used in the Ames test: S~yphiimurium TA 1535 his G46 rfa-A uvr BS~tpmurumTA 1537 his C3076 rfa-A uvr BS.typhimurium TA 1538 his D3052 rfa-A uvr BS-LyEphlmurim .TA 98 his D3052 rfa-A uvr B-R 4 B~yimriwn~i TA 100 his G46 rfa-A uvr B-R+ All five strains are defective in DNA.repair capacity (A uvr B-) and have a defective lipopolysaccharide barrier on the cell wall (rfa-). These two properties confer extra sensitivity to DNA damage and also greater permeability of large molecules into the cell. Strains TA 98 and TA 100 also contain a resistance transfer factor (plasmid pIQ' 101) . This factor, which confers resistance- to ampicillin, enhances the operation of an error-prone repair system. The strains are tested routinely for cell membrane permeability and where applicable for aipicillin resistance. For use in tests sub-cultures are grown in Nutrient' Broth (Oxoid) at 37*C for 18 hours. This culture provides approximately 2 x 109 organisms per ml which is assessed by cell counting. 1.2 Positive controls (a) With S-9 mix 2-Aminoanthracene at 2 pig/plate for strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. (b) Without S-9 mix 2-Nitrofluorene at 10 p±g/plate for strains TA 1538 and

.TA 98. 9-Aminoacridine at 29 pg/plate for strain TA 1537. Sodium azide at 5 p±g/plate for strains TA'1535 and TA 100. 2. PROCEDURE 2.1 Preliminary toxicity test The following procedure is carried out on each bacterial strain: Four concentrations of test substance are assessed for toxicity using the five tester strains. The highest concentration is usually 0.05 g of test substance dissolved in 1 ml of solvent. Three 10-fold serial dilutions of the top concentration are .1'aiso.tested. The chosen solvent is used as the negative

contfol.

### *5APPENDIX 1 SHW/l (continued)

0.1 ml of an overnight bacterial culture containing approximately 2 x 109 cells/mi, and 0.5 ml S-9 

### mix (see Section 3) or 0.5 ml 0.1 M sodium phosphate buffer (pH 7.4) are placed,,. in glass bijou bottles. 0.1 ml of the test solution is added followed by 2 mal histidine deficient agar. The mixture is thoroughly shaken and overlaid onto previously prepared plates containing 15 ml minimal agar. Single petri dishes are used for each dose level. They are incubated at 37*C for 72 hours. After this period the plates are examined for the appearance of a complete bact~erial lawn. Revertant colonies are counted using a Biotrart Automatic Colony Counter. Any toxic effects of the test substance are detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. 2.2 Ames test procedure (a) Without metabolic activation The following procedure is carried out on each tester strain. 0.1 ml aliquots of bacterial suspension and 0.5 ml of sterile 0.1 M sodium phosphate buffer (pH 7.4) are added to each of one set of sterile bijou bottles. 0.1 ml of the test compound is added to cultures at five concentrations separated by half-log 10 intervals. The negative control is the chosen solvent. The appropriate positive control is also included. 3 bottles are used at each dose level. 2.0 ml of histidine deficient agar is added to each of the bottles, thoroughly mixed and then overlaid onto previously prepared plates containing 15 ml of minimal agar. Plates are incubated for 72 hours at 37*C. Colonies areB counted using a Biotran Automatic Colony Counter, and the mean number of revertant colonies per treatment group assessed. Mb With metabolic activation Methodology is as described in 2.2 (a) except that 0.5 ml of liver homogenate S-9 mix (see Section 3) is added to bijou bottles in place of sterile buffer. 2.3 Second mutation test The procedure outlined in Section 2.2 is repeated at a later date; though the concentrations of test substance used in the second test 'may be altered, if the results of the first test indicate this may be expedient.

4F-APPERIDIX 1 H/ (continued)

3.- PREPARATION OF LIVER HOMOGENATE S-9 FRACTION species: Rat strain: CD (Sprague-Dawley-derived). Source: Charles River UK Limited, Manston Road, Margate, Kent, England. Age range: 7-8 weeks on arrival. Weight range: 180-200 g on arrival. Diet: Spratt's Laboratory Diet No. 1. Number used: 8 3a. stimulation of rat liver enzymes Mixed-function oxidase systems in the rat liver are stimulated following a single i/p injection of Aroclor 1254 (diluted in Arachis oil to 200 mg/ml) at a dosage of 500 mg/kg. On the fifth day of induction, following an overnight starvation, the rats are killed and their livers aseptically removed. 3b. Preparation of liver homogenate "S-9' 1. All steps are at-0-4*C using sterile solutions and glassware. The livers are placed in beakers containing 0.15 M potassium chloride. After weighing, livers are transferred to a beaker containing 0.15 MKCl (the volume of KCl in ml is equivalent to 3 times the weight of liver in gram), minced with a sterile scalpel and homogenized in an MSE top-drive homogenizer. This homogenate is centrifuged for 10 minutes at 9000 x 'g' and the supernatant divided into 15 ml aliquots. These are frozen on dry ice and stored at -S80C, and tested with the carcinogen 7, 12-dimethylbenzanthracene before use. i. Preparation of "S-9 mix" M S-9 mix contains: S-9 fraction (10% v/v), Mg Cl2 (SmM), KCl (33 if4), sodium phosphate buffer pH 7.4 (100 nti, glucose-6- phosphate (5 mM), NADP (4 nM*). All the cofactors are filtersterilized before use. 4' ASSESSMENT OF RESULTS The mean number of revertant colonies for all treatment groups is coupared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group. A compound is deemed to provide evidence of mutagenic potential if (1) a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments, and (2) the increase in the number of revertant colonies is at least twice the concurrent solvent control value.

(5) .12APPENDIX 1 SHWI' (continued)

5. MAINTENANCE OF RECORDS All data are kept in a loose-leaved laboratory notebook which is held in the Department of Mutagenesis and later transferred, together with a mastercopy of the final report, to the Archive Department, Huntingdon Research Centre plc, Huntingdon, Camba, U.K. 6. REFERENCES 1. Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D., Proc. Nat. Acad. Sci. USA (1973) 70, 2281. 2. Ames, B.N., McOann, J. and Yamasaki, E., Mutation Res. (1975) 31, 347. 3. McCann, J., Choi, 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. de Serres, F.J. and Shelby, M.D., Mutation Res. (1979) 61, 159. 6. OECD Guideline for testing of chemicals no. 471:- Genetic Toxicology :Salmonella typhimurium. Reverse Mutation Assay.

(5) 13