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fax: 202.564.8251 

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r- Urgent I- For Review I- Please Comme - Please Reply - Please Recycle -

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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON D.C., 20460 

VIA FACSIMILE: 201-437-4801 

April 19,2012 

Attention: Glenn Merritt (201-437-4000) 

OFFICE OF 

PREVENTION, PESTICIDES AND 

TOXIC SUBSTANCES 

Subject: Follow-up to fax transmission of 4/13/12 (ePMA removal ofCBI claims regarding 

identified studies) 

Dear Mr. Merritt: 

This is sent to you pursuant our conversation of several minutes ago. In Larry Robinson's fax of 

April 13th while there is a waiver of confidential business information (CBI) claims in the 

identified studies there is a an assertion of copyright protections and some additional text that 

provides in part ... "Reproduction, reference, summarization, retransmission or other use of this 

copyrighted document. .. including electronic transmission, using the internet or email .... is 

strictly prohibited .... " 

While we understand the basis for the copyright protections, we have a concern that the language 

in the fax might be interpreted to mean that ePMA is asserting that the studies submitted per the 

TSCA activity are not to be made available in an Agency e-docket (electronic docket) or be 

included in EPA websites among the collection of TSCA health and safety studies. 

We are thinking that the identified limitations noted in the fax are not intended to apply to EPA's 

normal procedures for the handling of non-CBI materials submitted by entities subject to TSCA 

regulations (including the posting of such materials on government websites) and submitted by 

such entities in order to comply with a TSCA requirement, but are rather intended to limit 

entities outside the government from the unauthorized use or appropriation of the studies. 

Can you please confirm CPMA's intent? Many thanks. 

Scott M. Sherlock, Attorney Advisor 

Environmental Assistance Division 

Office of Pollution Prevention and Toxics 


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

3
T1ABLE 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 precipitate
TABLE 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, TA 98 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

:7
TAbLE 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~tp murumTA 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-R4

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

con tfol.
*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 M KCl (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 (S mM), 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) .12
APPENDIX 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