# FAX

- To: 

## 6'-& NN (VI 

### <':"1 VI \ \ , 

### Fax: 

### '}..O\**ll'?1,LI~ I**  Date: 

## <J 

# 11"1 II <)/ Re: 

## C:\> (VI 

## A.

From:

## '7C::D~ 

# S'"**~,,,,** t '"" 

### fax: 202.564.8251 Phone: ,\--01..'

## Sb~ 

# .**~~** 

## S I Cc: 

### Comments:**\'~** 

## "',,t.v,)\\'" 

# rI OCSPP OPPT EAD 1200 Pennsylvania Ave. NW Washington, DC 20460 202.564.8251



### Pages: 

# 'Y

r- Urgent 

## I- For Review 

## I- Please Comme



# - Please Reply


# - Please Recycle


# - nt

### 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, PESTICIDESAND TOXIC SUBSTANCES



### Subject: Follow-upto fax transmission of4/13/12 (ePMA

### removal ofCBI claimsregarding identified studies) 

## DearMr. Merritt: This is sentto you pursuantourconversationofseveral minutes ago. In Larry Robinson's fax of April 13th 

### while there is a waiverofconfidential business information (CBI)claims in the identified studiesthere is a anassertion ofcopyrightprotections and some additional textthat provides in part ... "Reproduction, reference, summarization, retransmission orotheruse ofthis copyrighted document...includingelectronic transmission, using the internet oremail....is strictlyprohibited...." While we understand the basisforthe copyrightprotections, we have a concernthat the language in the fax mightbe interpreted to meanthat ePMA is assertingthatthe studies submitted perthe TSCA activity arenot tobemade available in an Agencye-docket (electronic docket) orbe included in EPA websites amongthe collectionofTSCAhealth and safety studies. We are thinkingthat the identified limitations noted in the fax are not intended to apply to EPA's normal procedures for the handling ofnon-CBI materials submitted by entities subjectto TSCA regulations (includingtheposting ofsuch materials ongovernmentwebsites) and submittedby such entities in orderto complywith a TSCA requirement, but are rather intended to limit entities outside the government from the unauthorized use orappropriation ofthe studies. Canyou pleaseconfirm CPMA's intent? Manythanks.
Scott M. Sherlock,Attorney Advisor Environmental AssistanceDivision Office ofPollution Prevention andToxics

### Pharma Development Corporate Toxicology 

# Hoechst Report No. 94.0363 Page 

### 1(21)

Study Title 

## Reflexblau 3G TTR

MICRONUCLEUS TEST INMALE AND FEMALE NMRI MICE AFTER ORAL ADMINISTRATION

Author Dr. W. Muller
Report completion date July 4th, 1994

Per'formina laboratory HOECHST AKTIENGESELLSCH-AFT Pharma Development Corporate Toxicology D-65926 Frankfurt am Main Laboratory Project ID: Study No. 94.0257



### DISCLAIMER This report isthe propertyof Clariamt. Its cott i confidential and notfar gem oral release. It ismet allowed to cite mod reveal the report or its costeet to a third party withoutprior written approval of Clarimalt.

### Pharma Development Corporate Toxicology 

# Hoechst Report No. 94.0363 Page 2 (21)

This report containsthe unpublished research findings of Hoechst scientists. Itshould not be published, inwhole or in part, or referredto inany publication without authorization from the company.

### Pharma Development Corporate Toxicology 

# Hoechst Report No. 94.0363 Page 3 (21)

STATEMENT OF COMPLIANCE To the best of my knowledge and belief, this study was conducted incompliance with Good Laboratory Practice regulations. No unforeseen circumstanceswere observed which might have affected the quality or integrity ofthe 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 

# Hoechst 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 ofthe bone marrow ................................................................ 11 

### 4.7 Evaluation.................................................................................... 11 4.8 Criteria for apositive response................................................................ 12 5. RESULTS.................................................................................... 12-13 6. PRELIMINARY STUDY........................................................................ 14 7. APPENDIX ................................................................................... 15- 21 7.1 Tables........................................................................................ 15-20 7.1.1 Summaryoffindings........................................................................... 15-16 7.1.2 Individual data ................................................................................. 17-20 7.2 References and guidelines...................................................................... 21Pharma Development Corporate Toxicology 

# Hoechst Report No. 94.0363 Page 6 (21) 1. SUMMARY Reflexblau 3G TTR was tested inthe 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 adose 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 inthe number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to normocytes was not changed to asignificant extent. Underthe conditions ofthe present study the results indicate that Reflexblau 3G TTR isnot mutagenic in the micronucleus test. 2. INTRODUCTION Micronuclei are small secondary nuclear structures resulting from either chromosomal breakage or malfunction of the spindle apparatus ofthe cell which regulates the distribution of chromosomes during mitosis. The micronucleus test originally described by Schmid [11 is asuitable invivo method for investigating clastogenic substances and substances which affect the mitotic spindle mechanism. Micronuclel rarely occur innormal dividing cells. The mouse has been chosen for this study since itprovides 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 ofthe Study : May 24th, 1994

Res ponsib iIit Y Head of Genetic Toxicology : Dr. W. MOLLER Head ofToxicology : 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 

# Hoechst 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,HN3SO 3 Molecular weight 638 Certificate of analysis dated March 31st, 1994 F+E Pigmente 2, Dr. A.Acs Stability and homogeneity invehicle stable for 5hours, Pharma Development Corporate Toxicology, Dr. H.- J.Pletsch, dated April 27th, 1994 Storage stability stable until April 1996, guaranteed by the sponsordated April 5th, 1994 Storage conditions dark at approximately 20 0C Appearance dark violet crystals On the day of the experiment the test substance was suspended insesame 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 inthe "Genetic Toxicology Laboratory". These instructions are inthe hands ofthe scientists and technicians performing the experiments.

### Pharma Development Corporate Toxicology 

# Hoechst 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 : 7weeks 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 7days Diet : rat/mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum Water : tap water inplastic bottles, ad libitum Housing : inair-conditioned rooms inMacrolon cages Type 3(five animals percage) 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 

# Hoechst 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 forthe 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 5females 6 - 10 2 2000 10 20 5males 11 - 15 24 5females 16 - 20 4* 50 0,5 10 5males 21 - 25 24 5 females 26 - 30 1 0 0 20 5 males 61 - 65 48 5females 66 - 70 2 2000 10 20 5 males 31 -35 48 5females 36 - 40 1 0 0 20 5 males 81 - 85 12 5females 86 - 90 2 2000 10 20 5males 41 - 45 12 5females 46 - 50

*=Positive control: Endoxans (Batch No. 082527) hours p.a. =hours afteradministration

### Pharma Development Corporate Toxicology 

# Hoechst Report No. 94.0363 Page 11 (21) 4.5 Assay procedure The test substance was administered orally to the test animals inadose of 2000 mg per kg body weight. Sesame oil was administered inthe 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 adose at 50 mg per kg body weight. 4.6 Pretparation ofthe bone marrow Extraction of the bone marrow Inconformity 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. Asuspension 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 inmethanol

- 5 minutes inMay-Gr~nwalds solution

- brief rinsing twice indistilled water

- 10 minutes staining in 1part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)

- rinsing indistilledwater

- 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 inthe 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring inthe 1000 normocytes counted, were evaluated statistically. AWilcoxon-Test (one-sided) [6,7]was evaluated to check the validity of the study. This isdone 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 adifference 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 

# Hoechst Report No. 94.0363 Page 12 (21) 4.8 Criteria for avositive response Both biological and statistical significances were considered together inevaluation. A substance is considered as positive if there isa significant increase inthe number of micronucleated polychromatic erythrocytes for at least one of the time points. A test substance producing no significant increase inthe number of micronucleated polychromatic erythrocytes is considered non-mutagenic inthis system.
5. RESULTS Mice were treated with 2000 mg Relexblau 3G TTR per kg body weight to study the induction of micronuci inbone 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 inall treatment groups are presented on page 17.-20.

### Pharma Development Corporate Toxicology 

# Hoechst 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 ofthe simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by thetest compound. Cyclophosphamide, (Endoxan) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei inboth 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 isnot mutagenic inthe 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 ToxicologyPharma 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 3males and 3females Clinical signs feces blue colored Lethality rate 0out of 3males 0out of 3females Macroscopic findings no macroscopic findingswere observed

### Pharma Development Corporate Toxicology 

# Hoechst Report No. 94.0363 Page 15 (21) 7. APPENDIX 7.1 Tables 7.1.1 summary of findings

00F

IOC.

0

2 00, 00 00 0' Z 00 00 .ee00. c 0

- . M1'. One =r9n

.. nr' u ZO M
00 0 0 1.0 R0~ .R ' 0. 0

### Pharma Development Corporate Toxicology 

# Hoechst Report No. 94.0363 Page 16 (21)

a0.0 0 0 o co aO 0g0 Co0

002o 00 : Co0 0 .... ... Cco

00 0000000 0C0co C; W, ' . . *f l Ct * 0 0' Z .'-0

0--0 o=.C c

0000

::000

.. . . . . . ... :090

. . . . . . . .O . . . . . . . 101 a00 0C C 0 0

0. Co to t 1Z o0 ! 0.0 0 000 0 a a 8 w O~~. N OaNe C6 wo

I o t V

### PhraDevelopment Corporate Toxicology 

# Hoechst Report No. 94.0363 Page 17 (21) 7.1.2 Individual data

0C

C,

O- a co o co o CO Q
01MM WX00M0 . - Z 41 0'

*0w 01 C4

- MOS
z 40-

### PhraDevelopment Corporate Toxicology 

# Hoechst Report No. 94.0383 Page 18 (21)

0.0* 0 00003~00 0

0.000 O 00000 0000 

### t M 

### 2
00.~00 0- 00-- 

### ~ ~ 0-00

9. 0 M X . A,0 >-f 0 >1t.I 0 . Wr.4i M N N 1

-.- 

### ~ ~ 0. 3 .- .N. 00 W..0.00 00.00. 0 CIO

CL. 0L 0 6

0 0 hI C

OR0

### Pharma Development Corporate Toxicology 

# Hoechst Report No. 94.0363 Page 19 (21)

00. 6- , !C 61E )C :1 ; 0z

O N O . X.C flXN 0-0 00 0 00 0 03 00 0 Z Z
00000 a, I

C

- 41
0 0.MN" 0 O~ 

### ~ ~M CA CM. 4 .44 0 .. C 00 .4 -. 1.0 .1,4-IN 0 4. N.b0. 0- MVO,4 ,4 4z 4 OJC 01 11(
.

W1 X 'A z ccI.

### Pharma Development Corporate Toxicology 

# Ho

# 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 fortesting of chemicals 474, Genetic Toxicology, Micronucleus test, Adopted 1983 4) EEC Directive 92/69, L383 A,Annex B.12., p.154- 156 5) Hochberg, Y.: A sharper Bonferroni procedure for multiple tests ofsignificance; 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 imailgemeinen c-Stichprobenproblem; EDV inMedizin und Biologie 18; 12- 19; Verlag Eugen Ulmer GmbH &Co., Stuttgart: Gustav Fischer Verlag KG, Stuttgart (1987)