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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 


Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 1 (21)

Study Title

Reflexblau 3G TTR

MICRONUCLEUS TEST

IN MALE AND FEMALE NMRI MICE

AFTER ORAL ADMINISTRATION

Author

Dr. W. Muller

Report completion date July 4th, 1994

Per'formina laboratory

HOE CHST AKTIENGESELLSCH-AFT

Pharma Development Corporate Toxicology

D-65926 Frankfurt am Main

Laboratory Project ID: Study No. 94.0257

DISCLAIMER

This report is the property of Clariamt. Its cott i

confidential and not far gem oral release. It is met

allowed to cite mod reveal the report or its costeet to a

third party without prior written approval of Clarimalt.
Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 2 (21)

This report contains the unpublished research findings of

Hoechst scientists. It should not be published, in whole or in

part, or referred to in any publication without authorization

from the company.
Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 3 (21)

STATEMENT OF COMPLIANCE

To the best of my knowledge and belief, this study was

conducted in compliance with Good Laboratory Practice

regulations. No unforeseen circumstances were observed

which might have affected the quality or integrity of the study.

Study Director:.r 9 ~q'

Testing facility management:

Dr. D. Maye
Hoechst

Report No.: 94.0363

Page 4 (21)

Quality Assurance (GLP)

21.06.1994

Ouality Assurance Statement

Title: Reflexblau 3G TTR

MICRONUCLEUS TEST

IN MALE AND FEMALE NMRI MICE

AFTER ORAL ADMINISTRATION

Study: 94.0257

This study was periodically inspected and properly signed records

of these inspections were submitted to testing facility management

and the study director as shown below:

Inspection Report

29.04.1994 29.04.1994

03.05.1994 03.05.1994

21.06.1994 21.06.1994

21.06.1994 21.06.1994

M. Jhe1

Quality Assurance (GLP)
Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 5 (21)

CONTENTS

PAGE

QUALITY ASSURANCE-STATEMENT.......................................................... 4

1. SUMMARY ........................................................................................ 6

2. INTRODUCTION................................................................................ 6

3. GENERAL......................................................................................... 7

4. MATERIAL AND METHODS ..................................................................... 8-12

4.1 Test compound ................................................................................ 8

4.2 Methods............................................................................................ 8

4.3 Animal species and husbandry ................................................................... 9

4.4 Test groups and dose range finding ............................................................ 10

4.5 Assay procedure ................................................................................ 11

4.6 Preparation of the bone marrow ................................................................ 11

4.7 Evaluation.................................................................................... 11

4.8 Criteria for a positive response................................................................ 12

5. RESULTS.................................................................................... 12-13

6. PRELIMINARY STUDY........................................................................ 14

7. APPENDIX ................................................................................... 15- 21

7.1 Tables ........................................................................................ 15-20

7.1.1 Summary of findings........................................................................... 15-16

7.1.2 Individual data ................................................................................. 17-20

7.2 References and guidelines ...................................................................... 21
Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 6 (21)

1. SUMMARY

Reflexblau 3G TTR was tested in the micronucleus test. The test compound was suspended in sesame oil

and dosed once orally at 2000 mg per kg body weight to male and female mice, upon the results of the

previously conducted dose range finding assay (see preliminary study). According to the test procedure the

animals were killed 12, 24 or 48 hours after administration.

Endoxan* was used as positive control substance and was administered once orally at a dose of 50 mg per

kg body weight.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased.

The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained

unaffected by the treatment with Reflexblau 3G TTR and was statistically not different from the control

values.

Endoxan" induced in both males and females a marked statistically significant increase in the number of

polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic

erythrocytes to normocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that Reflexblau 3G TTR is not mutagenic in

the micronucleus test.

2. INTRODUCTION

Micronuclei are small secondary nuclear structures resulting from either chromosomal breakage or

malfunction of the spindle apparatus of the cell which regulates the distribution of chromosomes during mitosis. The micronucleus test originally described by Schmid [11 is a suitable in vivo method for investigating

clastogenic substances and substances which affect the mitotic spindle mechanism. Micronuclel rarely

occur in normal dividing cells. The mouse has been chosen for this study since it provides a convenient in

vivo mammalian model [2]. The study described was performed according to the OECD guideline No. 474

[3].
Pharrna Development Corporate ToxicologyHo c s

Report No. 94.0363

Page 7 (21)

3. GENERAL

Study-No. : 94.0257

Artemis-No. : MM0033

Test compound Reflexblau 3G TTR

Sponsor Hoechst AG, GB: D. F+E Pigmente, Dr. Deucker

Test system : Micronucleustest

Test species : NMR1 mouse

Route of administration : oral by gavage

initiation of the study : May 2nd, 1994

Termination of the Study : May 24th, 1994

R es po ns ib iIi t Y

Head of Genetic Toxicology : Dr. W. MOLLER

Head of Toxicology : Dr. D. MAYER

Statistical evaluation : DM R. UHL

Quality assurance unit : Ap. S.J. HARSTON

Testing facilities and archives : HOECHST AKTIENGESELLSCHAFT

Pharma Development Corporate Toxicology

D-65926 Frankfurt am Main
Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 8 (21)

4. MATERIAL AND METHODS

4.1. Test compound

Name Refiexblau 3G TTR

Code DRAC 595

CAS No. 6417-46-5

Batch No. Pt. 41/93

Molecular formula C,HN3SO3

Molecular weight 638

Certificate of analysis dated March 31st, 1994

F+E Pigmente 2, Dr. A. Acs

Stability and homogeneity in vehicle stable for 5 hours, Pharma

Development Corporate Toxicology,

Dr. H. - J. Pletsch, dated April 27th, 1994

Storage stability stable until April 1996, guaranteed by the sponsor dated

April 5th, 1994

Storage conditions dark at approximately 200C

Appearance dark violet crystals

On the day of the experiment the test substance was suspended in sesame oil at an appropriate concentration. The preparation for application was kept homogenous by a magnetic stirrer until the completion of

dosing.

4.2 Methods

There are standard operation procedures CSOPs) applied in the "Genetic Toxicology Laboratory".

These instructions are in the hands of the scientists and technicians performing the experiments.
Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 9 (21)

4.3 Animal species and husbandry

Species : HsdlWin: NMVRI mouse

Origin Harlan - Winkelmann GmbH, 33178 Borchen

Initial age at start of study : 7 weeks

Number of animals 70 (35 males /35 females)

Body weight at start of study males : x = 37.63 g (32 - 44 g)

females: x = 29.37 g (26 - 32 g)

Acclimatization : at least 7 days

Diet : rat/mice diet Altromin 1324 (Altromin-GmbH,

Lage/Lippe), ad libitum

Water : tap water in plastic bottles, ad libitum

Housing : in air-conditioned rooms in Macrolon cages Type 3 (five

animals per cage) on soft-wood granulate

Room temperature : 22 ± 3 *C

Relative humidity : 50 +20 %

Lighting time : 12 hours daily

Animal identification : fur-marking with KMnO, and cage numbering

Randomisation : randomisation schedule 94.0244an1 94.0245
- I "U lL I

Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 10 (21)

4.4 Test aroups

Oral administration of 2000 mg Refiexblau 3G TTR per kg body weight did not cause any toxic effects in

male and female mice. This dose level was selected as the limit dose for the main study.

Group Dose Concentration () Volume Number of Animal Killing-time

(mg/kg bwt.) (Wlv) (mi/kg bwt.) animals and sex No. (hours p. a.)

1 0 0 20 5Smales I - 5 24

5 females 6 - 10

2 2000 10 20 5 males 11 - 15 24

5 females 16 - 20

4* 50 0,5 10 5 males 21 - 25 24

5 females 26 - 30

1 0 0 20 5 males 61 - 65 48

5 females 66 - 70

2 2000 10 20 5 males 31 -35 48

5 females 36 - 40

1 0 0 20 5 males 81 - 85 12

5 females 86 - 90

2 2000 10 20 5 males 41 - 45 12

5 females 46 - 50

*=Positive control: Endoxans (Batch No. 082527)

hours p.a. =hours after administration
Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 11 (21)

4.5 Assay procedure

The test substance was administered orally to the test animals in a dose of 2000 mg per kg body weight.

Sesame oil was administered in the same way to the negative control groups. Simultaneously to negative

controls and dose groups Endoxan* was used as positive control substance and was administered once

orally at a dose at 50 mg per kg body weight.

4.6 Pretparation of the bone marrow

Extraction of the bone marrow

In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 12, 24 or 48

hours after application. For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube.

Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were

opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was

then centrifuged for 5 minutes at approx. 1200 rpm and almost all the supemnatant discarded. One drop of

the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal

number and air-dried for about 12 hours.

Staining procedure

- 5 minutes in methanol

- 5 minutes in May-Gr~nwalds solution

- brief rinsing twice in distilled water - 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)

- rinsing in distilled water - drying

- coating with Entellan

4.7 Evaluation

1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was

recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were

also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic

erythrocytes was determined. All bone marrow smears for evaluation were coded to ensure that the group

which they belong to remained unknown to the investigator. The number of polychromatic erythrocytes with

micronuclei occurrng in the 1000 polychromatic erythrocytes counted, and the number of normocytes with

micronuclei occurring in the 1000 normocytes counted, were evaluated statistically.

A Wilcoxon-Test (one-sided) [6,7] was evaluated to check the validity of the study. This is done by comparing the number of polychromatic erythrocytes with micronuclei in the positive control with the negative

control.

The presupposition to make any of the following comparisons is a difference between the positive control

and the negative control (24h). This is tested with a Wilcoxon-Test (two-sided) [6,7] with 5 %-level of

significance. Wilcoxon-Tests (two-sided) are performed sequentially for the ratio of polychromatic

erythrocytes for each measurement group (12h, 24h, 48h) at a multiple level of significance of 5 % [5].

Actual data were also compared with historical controls.
Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 12 (21)

4.8 Criteria for a vositive response

Both biological and statistical significances were considered together in evaluation.

A substance is considered as positive if there is a significant increase in the number of micronucleated

polychromatic erythrocytes for at least one of the time points. A test substance producing no significant

increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this

system.

5. RESULTS

Mice were treated with 2000 mg Relexblau 3G TTR per kg body weight to study the induction of micronuci in bone marrow cells.

All animals survived after application of 2000 mg per kg body weight. For only 48 hours blue colored feces

was observed as a clinical sign. Only 12 hours after application the dissection of the animals revealed a

blue colored gastro-intestinal tract.

The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The results

are summarized on page 15 and 16. Individual data of all animals in all treatment groups are presented on

page 17.-20.
Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 13 (21)

The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose group of

Reflexblau 3G TTIR was within the normal range of the negative control groups. No statistically significant

increase of micronucleated polychromatic erythrocytes has been observed. The number of norrnochromatic

erythrocytes with micronuclel did not differ from the values of the simultaneous control animals for each of

the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained

essentially unaffected by the test compound.

Cyclophosphamide, (Endoxan) induced a marked and statistically significant increase in the number of

polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity of the test

system.

Summarizing it can be stated that administration of Reflexblau 3G TTR did notl lead to a substantial

increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test.

Dr. WM.AG

Quality assurance unit HOECHST AKTIENGESELLSCHAFT 3 2A.S~ ~Pharma Development Corporate Toxicology

Dr. W~ M 1'/e

Study Director

Dr. 0. Mayer Head of Toxicology
Pharma Development Corporate ToxicologyH o c s

Report No. 94.0363.

Page 14 (21)

6. PRELIMINARY STUDY

Preliminary studies were conducted to determine the highest administrable non lethal dose level.

1st dose 2000 mg/kg body weight Reflexblau 3G TTR

Observation period April 19th - April 29th. 1994

Number of animals used 3 males and 3 females

Clinical signs feces blue colored

Lethality rate 0 out of 3 males

0 out of 3 females

Macroscopic findings no macroscopic findings were observed
Pharma Development Corporate Toxicology H oechst

Report No. 94.0363

Page 15 (21)

7. APPENDIX

7.1 Tables

7.1.1 summary of findings

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Report No. 94.0363

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Report No. 94.0363

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Pharma Development Corporate Toxicology H o eachst

Report No. 94.0363

Page 21 (21)

7.2 References and guidelines

1) Schmid, W.: The micronucleus test; Mutat. Res. 31, 9 - 15 (1975)

2) Salamone, M.; Heddle, J.; Stuart, E.; Katz, M.: Towards an improved micronucleus test;

Mutat. Res. 74. 347 -356 (1980)

3) OECD guideline for testing of chemicals 474, Genetic Toxicology,

Micronucleus test, Adopted 1983

4) EEC Directive 92/69, L 383 A, Annex B. 12., p. 154 - 156

5) Hochberg, Y.: A sharper Bonferroni procedure for multiple tests of significance;

B9iometrika 75, 800-802 (1988)

6) Hollander, M.,. Wolfe, D.A.: Nonparametric statistical methods; Wiley Series

in Probability and Mathematical Statistics; John Wiley & Sons, Inc.;New York (1973)

Excakte Verteilung ffir Rang- und Randomisierungstests im ailgemeinen c-Stichprobenproblem;

EDV in Medizin und Biologie 18; 12 - 19; Verlag Eugen Ulmer GmbH & Co., Stuttgart:

Gustav Fischer Verlag KG, Stuttgart (1987)