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## - ntUNITED 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 transmissionof4/13/12 (ePMA

### removal ofCBIclaimsregarding identified studies) 

### DearMr. Merritt: Thisis sentto you pursuantourconversationofseveralminutes ago. In Larry Robinson'sfax of April 13th 

### while there is awaiverofconfidentialbusinessinformation (CBI)claimsin the identified studiesthere is a anassertion ofcopyrightprotections and some additional textthat providesin part ... "Reproduction,reference, summarization,retransmissionorotheruse ofthis copyrighted document.
..includingelectronictransmission,usingthe internetoremail
....is strictlyprohibited
...." Whileweunderstandthebasisforthecopyrightprotections, we have a concernthatthe language in the fax mightbeinterpreted to meanthatePMA isassertingthatthestudies submittedperthe TSCA activityarenottobemadeavailableinan Agencye-docket (electronic docket) orbe includedin EPA websitesamongthecollectionofTSCAhealthand safety studies. Wearethinkingthat the identifiedlimitations noted inthe fax arenot intendedto applyto EPA's normal proceduresforthehandlingofnon-CBI materials submittedby entities subjectto TSCA regulations(includingthepostingofsuch materials ongovernmentwebsites) and submittedby such entitiesinorderto complywithaTSCArequirement, but are rather intendedto limit entitiesoutsidethe government from the unauthorizeduseorappropriation ofthe studies. Canyou pleaseconfirm CPMA'sintent? Manythanks.

ScottM. Sherlock,Attorney Advisor Environmental AssistanceDivision OfficeofPollutionPrevention andToxics

# Hoech-st 

# I Pharma Development Report No. 92.0452 Central Toxicology page 1 (20) 

## DISCLAIMER Thisreport isthe property ofClariant. Its 

### content isconfidential and notforgeneral release. Itisnot allowed

### to citeand reveal the report orits contentto athird party without priorwritten approval ofClariant 

# p ~~Pigment 

### Blue 56 

## C

## As> 9~~w96-hours acute toxicity study inZebra fish (Brachydanio rerlo)
Authors Dr. Zak, Dr. Jung report completion date: August 03, 1992

Testing fa-ci-lity Pharma Development Toxicology Hoechst AG P.O. Box 80 03 20 6230 Frankfurt 80 Federal Republic of Germany

5tudy number 92.0298

# Hoechst Pharma Developmient Report No. 92.0452 Central Toxicology page 2 (20)



## This report contains the unpublished research findings of Hoechst scientists.
Ishould not be published, in whole or in part, or referred to in any pub- ication without authorization from the company.

# Hoechst Pharma Development Report No. 92.0452 Central Toxicology page 3 (20)

This study was conducted in compliance with the principles of good laboratory practice. No unforeseen circumstances were observed which may have affected the quality * and integrity of this study.
Study director(D.Zk

industrial Toxicology. (Dr. Jung)

# Hoechst

# I1-
Report No. :92.0452 Page 4 (20) Quality Assurance Statement Hoechst Aktiengesellschaft ---- ---- --- ---- ------- Quality Assurance(GLP) 29.07.1992 Title Pigment Blue 56 96-hours acute toxicity study in Zebra fish (Brachydanio rerio) STUDY NO. 92.0298

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 -----5.--9--

-- 9.05.1992-

18.05.1992 19.05.1992 15.05.1992 29.05.1992 27.05.1992 27.05.1992 29.07.1992 29.07.1992 29.07.1992 29.07.1992

Quality Assurance (GLP)

Mysrsider desA&.Antstal. RONl Sarr 1~ AyS~ndl VADlg3q Hlge. V'3'?2ende. Gonter met2. stebv 4=70ne' Ji'pn mann. MarlinFdfftu!. HW2 

### ~ ~ ~ ~ M Aans~ K1Hluo.HrtGrglfS1.J us Msch. Ernst Scado.. Kart-GerhrO aa cles hasn lW Utz-Hetiht Fe~i Szew GesIschalt: Frankfturt amMain - Hade1G1W~San: Franinturi amMann AbM 8 Nr 14500

# Hoechst Pharma Development Report No. 92.0452 Central Toxicology page 5 (20)

INDEX QUALITY ASSURANCE UNIT DECLARATION........................ 4 1. SUMMARY............................................... 6 2. REMARKS............................................. 7 3. SURVEY................................................ 8 4. MATERIALS AND METHODS .................................. 9 4.1 Test compound.......................................... 9 * 4.2 Test organism ......................................... 10 4.3 Study groups .......................................... 11 4.4 Water for dilution..................................... 11 4,5 Test conditions ....................................... 11 4.6 Preparation of the test concentrations.................... 12 4.7 Study conduct ......................................... 12 5. RESULTS............................................... 13 5.1 Lethality........ i.................................... 13 5.2 Appearance and behavior ................................ 13 5.3 Water parameters....................................... 13 6. APPENDIX ............................................ 1 6.1 Methods for determining water parameters.................. 14 6.2 Water parameters - individual values..................... 15 6.3 Symptoms - individual values............................. 16 6.4 Analytic - report ................................... 17 19 6.5 Certificate of analysis ................................. 20

# Hoechst IB Pharma Development Report No. 92.0452 Central Toxicology page 6 (20)


### .1.SUMARY When testing the lethal effect of Pigment Blue 56 in zebra fish (Brachydanlo rerio) over a total of 96 hours according to the method described under section 4, no lethality occured at the 500 mg/i-group until 72 hours after study start. 96 hours after study start a lethality rate of 10 %was determined. The LC50 after 48 and 96 hours therefore exceeds 500 mg/i. The LCD after 96 hours was not determined, because there was a lethality rate of 10 %. Refering to the OECD Guideline a lethality rate up to 10 %isacceptable even inthe control gruop. Therefore no further concnetrations were tested. A concentration of 500 mg/i, a negative control (0 mg/i) and a control group with the emulsifying agent TWEEN 80, using the concentration 0,095 mi/i (from 

# Pnow on marked with 0*) were tested. To get a better suspension of the test substance in the test water, IWEEN 80 was used as emulsifying agent. The tested concentration showed adark blue discoloration. The determination of the substance concentration was observed inthe 500 mg/i group. All determined values were in a range of + 20 % of the theoretical value.

# Hoechst Phanna Development Report No. 92.0452 Central Toxicology page 7 (20)

2 REMARKS The acute toxicity of Pigment Blue 56 in fish was determined in accordance with procedure recommnended by the "Ad hoc Arbeitsgruppe des Umweltbundesamtes zur Entwicklung bkotoxikologischer Testverfahren in aquatischen Systemen" (dated: 01.05.84). 'Letale Wirkung beim Zebrabirbling Brachydanio rerio* (LCO, LC50, LCJOO; 48 - 96 STUNDEN) Eqpfehlung des Wweltbundesautes zur Durchfflhrung der Prfifung nach §4 Abs.1 Nr.6 der Verordnung fiber Anseldeunteriagen und Profnachweise nach dew Cheikallengesetz Texte 16/84, Lbwneltbundesaut and incompliance with the requirements of EEC-Guideline (C-Guideline C.I. Akute Toxizitit ffir Fische Richtlinie 84/449/EIG: Richtlinie der Kommission von 25. Apri1 1984 zur sechsten Anpassung der Rchtlinie 67/548/DIG des Rates zur Angleichung der Rechts- und Verwaltungsvorschriften fuir die Einstufung, Verpackung und Kennzeichnung geflhrlicher Stoffe an den technischen Fortschritt and the OECD-Guidelinle OECD-Guideline for testing of chemicals, 203 Fish, Acute Toxicity Test, Adopted: 04. April 1984

# Hoechst 

# F Pharma Development Report No. 92.0452 Central Toxicology page 8 (20)

3.SURVEY Study type acute toxicity infish Study No. 92.0298 Test substance Pigment Blue 56 Sponsor GB D,Abt. PSE Species Zebra fish Brachydanlo rerio (HAMILTON- BUCHANAN) Temperature 22 + I C Test system static Duration of study :96 hours Study start :05/18/92 Study end :05/22/92 Test concentrations :0 /0* /500mg/

Re s pons lb 1i1i t ie s Study director Dr. Zok Industrial Toxicology Dr. Jung Quality assurance Harston (Pharmacist) Analytic .Dr. Pletsch Testing facilities and Pharma Development Central Toxicology archives HOECHST AG, P.O. Box 80 03 20 6230 Frankfurt 80 Federal Republic of Germany

# Hoechst H Pharma Development Report No. 92.0452 Central Toxicology page 9 (20)

4. MATERIALS AND METHOQDS 4.1 Test compound Trade name :Pigment Blue 56 Synonyms :Reflex-Blau 3G Chemical name Benzenesulfonic acid, 2-methyl-4-[[4-[[4-[(3- methylphenyl)amino]phenyl][4-[(3-methylphenyl) iiino-2,5-cyclohexadien-1-yl idene]methyl] phenyl 

### ]amino]

- * 

### Empirial formula C 40 H 36 N 3 S03 Product No. :DRAC 585 CAS No. :006417-46-5 Category :blue pigment to improve black color Composition :approx. 1.5 % Methyldiphenylamine approx. 3,5 %productmsixture of complex comnposition Batch No. :21/92 Certificate of analysis :No. 4737, dated June 11, 1992 Sample received :03/27/92 Storage :in fume cupboard at approx. 20 OC inthe dark Stability under storage conditions :02197 Appearance :brownblue brazing approx. 25 %fluid paste Molecular weight :638 g/mol Melting point :no exotherm decomposition up to 400 OC Shaking weight 0.7 kg/i pH in water 4 -5 Biodegradability approx. 90 % biodegradability Aqueous solubility soluble1121~~~ TILI.~1KIIf~f '7117111'i 1 "1' 

# Ho-echst Pharma Development Report No. 92.0452 Central Toxicology page 10 (20)

4.2 Test oroanism Species Zebra fish Brachydanio rerlo (HAMILTONW-BUCHANAN) Breeding station Dept. of Toxicology (Hoechst AG) Date of hatching 09/22/91 Delivery date 06/23/92 The fish were kept for at least 14 days before the start of the study in water for dilution (see point 4.4) under the following conditions: Temperature : 22 ± 1OC Oxygen content : 2 80 % of the saturation value Duration of lighted period :12 hours daily Population density : _< 1g fish/l water Feeding :twice daily ad libitum Feed :Tetra Min, Tetra Werke, FMelle The body length of 10 representative fish from each batch were measured. Body length (cm) Batch no. Date n Variation range x s 13/91/H]2a/L6 05/22/92 10 2.7 - 3.5 3.1 0.24

# Hoechst
Pharma Development Report No. 92.0452 Central Toxicology page 11 (20)

4.3 Study groups There were 10 fish in each group. As a result of the dose-finding studies the following concentration was tested.

Start of Study Test chamber No. Concentration (mg/i) 

### 05/18/92 7/ 8 /6 

### 0/0-/500

4.4 Water for dilution Reconstituted water, composition according to ISO/DIS 7346/] was used as water for dilution. Preparation was carried out in a unit consisting of two Hostalenl- lined steel vessels each with a capacity of 1700 litres. The dilution water prepared as described and was aerated until oxygen saturation. The pH was measured before the use of each study batch and was 7.9 - 8.0. 4.5 Test conditions The study was conducted ina static system. The test chambers, which were calibrated to 10 liters, were made out of glass (length 30 cm, width 22 cm, height 24 cm) and stood in a water bath made out of INostalit ZA with a Plexiglass 

# p viewing panel. The temperature of the water bath was regulated by a thermostat to 22 + I OC. The chambers were illuminated from above from 06.00 a.m. to 06.00 p.m.. The test chambers were not aerated during the course of the study.

# Hoechst 

# I
Pharma Bevelopment R~eport Noc. 92.0452 Central Toxicology page 12 (20)
4.6 Preparation of the test concentrations The test substance was made into a paste using 0.095 ml Tween 80 /1 as a solubilizing agent. Water for dilution was added and the mixture homogenized using an Ultra-Turrax and Ultrasonic Bath and poured into the chamber under stirring with a glass rod. The chamber content was then stirred for approximately 2 hours with a KPG-stirrer and a glass rod. The test batch was present as a dark blue discoloration. Ndo separation and no formation of substance deposits on the bottom of the chamber were observed. In order to determine the substance concentration, water samples were taken from the middle of the test chamber at study start, and after 48 and 96 hours using a 

# p screw-neck glass bottle. For the methods and individual results of analysis see page 17 -19. Nominal

. Actial (sng/1) x ONg/lI) 500 486.1 97.2 The mean analytical results were in a range of + 20 Z of the nominal concentration. 4.7 Study conduct After the test concentrations had been prepared and water parameters recorded, 10 randomized fish were assigned to each test and control chamber. The fish received no feed for the entire study period. Inspection of the fish took place after 2 - 4 , 24, 48, 72 and 96 hours and involved recording the lethality and visible changes in appearance and behavior. Dead fish were removed from the chambers. Fish were considered dead when there was a lack of opercular movement and no response to slight mechanical stimulus. The water parameters were also mea*suredand recorded at the inspection times. The methods used for measurement are described inthe appendix (page 14).

# Hoechst
Phanna Development Report No. 92.0452 Central Toxicology page 13 (20)



### 5.REULTS 5.1 Lethality In this 96-hours acute toxicity study of Pigment Blue 56 in zebra fish (Brachydanio renao) no lethality occured at the 500 mg/l-gruop until 72 hours after study start. 96 hours after study start a lethality rate of 10 %was determined. The LC50 after 48 and 96 hours therefore exceeds 500 mg/I. The LCO after 96 hours was not determined, because there was a lethality rate of 10 %. Refering to the OECD Guideline a lethality rate up to 10 %is acceptable even inthe control gruop. Therefore no further concentrations were tested. 05.2 Apoearance and behavior Recording of syiitoiis was difficult, because of the dark blue discoloration of the test water caused by the test substance. There were no changes inappearance and behavior in comparison to the control-group. 5.3 Water Darameters The following ranges for the concentration groups and the control groups were obtained for the whole study period; Parameter Concentration group Controls

PH (1) 7.8 - 8.1 7.9 - 8.2 oxygen content (mg/l) 5.8 - 8.9 7.2 - 9.2 * Temperature(OC) 21.8 - 21.9 21.6 - 21.9 The individual values are given in the appendix (page 15). Dr. Zok/AD Audited by Pharma Development Toxicology Quality assurance HOECHST AKTIENGESELLSCHAFT
Dr. Zok Study director

Dr. Jung Industrial Toxicology

# Hoechst Pharma Development Report No. 92.0452 Central Toxicology page 14 (20)

6.1 Methods for determining water Parameters Parameter Unit Method Temperature VC electronic measurement using oxygen meter OXI 196 (WTW) and oxygen electrode EO 196-1.5 with in-built temperature sensor (Wissenschaftl ich Technische Werkstitten, Weilheim) 

# p Oxygen content mg/l electronic measurement using oxygen meter OXI 196 (WTW) and oxygen electrode EO 196-1.5 (Wissenschaftlich Technische Werkst~tten, Weilheim) PH 1electronic measurement using pH meter PH 763 (Knick) and combined electrode (Schott-Gerite GmbH, Hofheim)

# Hoechst Pharma Development Report No. 92.0452 Central Toxicology page 15 (20)
6.2 water piaeers- individual values
Concen- Test Study PH (1) tration chamber start before hours after study start (wig/i) No. (date) start 0 2-4 24 48 72 96

0o 05/18/92 7.9 8.2 8.1 8.1 8.1 8.0 8.1 0* 8 05/18/92 8.0 8.2 8.2 8.0 8.1 7.9 8.0 500 6 05/18/92 8.0 8.1 8.1 7.9 8.0 7.8 7.9 6.2.2 Oxygen content Concen- Test Study 0 content (mg/l) tration chamber start before hours after study start (mg/i) No. (date) start 0 2-4 24 48 72 96

0 7 05/18/92 9.8 9.2 8.7 8.1 7.8 7.7 7.8 0.8 05/18/92 9.8 9.1 8.5 7.8 7.6 7.3 7.2 500 6 05/18/92 9.8 8.9 8.3 7.8 7.7 6.6 5.8 Oxygen saturation (100 %)at 8.8 8.8 8.8 8.8 8.8 8.8 8.8 22 OC and localized air pressure * 6.2.3 Teumperature Concen- Test Study Temperature (CC) tration chamber start before hours after study start (mg/i) No. (date) start 0 2-4 24 48 72 96

0 7 05/18/92 21.8 21.6 21.8 21.8 21.8 21.9 21.8 0.8 05/18/92 21.5 21.8 21.9 21.9 21.9 21.9 21.9 500 6 05/18/92 21.3 21.9 21.9 21.8 21.8 21.9 21.8

# Hoechst Pharnia Development Report No. 92.0452 Central Toxicology page 16 (20)
6.3 Sygmptoms
- individual values 0 Mg/l (Control group) hours after study start -2-4 24 48 72 96 Lethality (absolute) 0/10 

### 0110 0/10 0/10 0/10 No changes inappearance and behavior 10/10 10/10 10/10 10/10 10/10

Solvent control with 0.095 ml Tween 80/1 h - - - - - - - - - - - - - - - - - hours after study start 2-4 24 48 72 96 lethality (absolute) 0/10 0/10 0/10 0/10 0/10 No changes inappearance and behavior 10/10 10/10 10/10 10/10 10/10

Soo ma/l hours after study start- 2-4 24 48 72 96 lethality (absolute) 0/10 0/10 0/10 0/10 1/10 recording of symptoms was difficult, because of the blue discoloration x x x X x No changes inappearance andbehavior a a a a a

o - one fish s - some fish a - all fish

# Hoechst Analytisches Laboratorium Report No. 92.0452 Toxikologie-Alalytic Page 17 (20)

Report Analytical dietermination of concentrations of test substanc in vate 1. Project 1.1 Fish, Acute Toxicity Test 1.2 Project No.: 92.0298 2. Test Substanc 2.1 common name :Pigment Blue 56 2.2 Chemical name 2.3 Batch No.: 21/92 2.4 Code No.: HOE CG 0423 OD ZD. . 0001 2.5 Purity : will be delivered later 2.6 water solubility (informal value) :>500 Mg/L (1h) >500 mg/L (24h) 3. Analytical Method 3.1 The determination of the concentrations was performed by photometrical detection ( k -603nM) after HPLC-separation on a reversed phase column. 3.2 Sample preparation 3.3 Evaluation of the results: The evaluation has been performed by the method of the external standard. 4. Samplina 4.1 Representative sample of total water amount 10 L :20 ml 4.2 Appearance of sample : blue solution~ 4.3 Temperature of water samples during the toxicity testing 220C±1 0C

# Hoechst Analytisches ILaboratorium Report No. 92.0452 ToiooieAayi Page 18 (20)

Found concentration (mg/L) Sample Nominal Date of 

### (mean value) No. mg/L Sampling Oh 48h 96h 90 0 18.05.92 n.d. 91 0 20.0.92n.d. 92 0 22.0.92n.d

99 0 18.05.92 n.d.

100 20.05.92 n.d. 101 0* 22.05.92 n.d.

93 500 18.05.92 482.5 94 500 20.05.92 486.4 95 500 22.05.92 489.5

5. Remarks: n.d. = not detectableAnalytschesHoechst AnltshsLaboratoriun Report No. 92.0452 Toxikologie-Analytik Page 19 (20)

6, General Testing Facility: Hoechst AG Anallytisches Laboratorium Toxikologie Analytik (Aupenstelle Kastengrund) Postfach 800 320 6230 Frankfurt/H. 80 Seonsor/Archive: Hoechst AG Pharma Entwicklung Zentrale Toxikologie Gewerbetaxikologie Kasterigrund Postfach 800 320 6230 Frankfurt/m.80 Monitorina Scientist Dr.Jung Quality assurance unit (GLp) Ap S.J. Harston, Hoechst AG 

# I"audited by1 quality assurance" 

### Date: 

## ?Oki'C Project Staff J.Hager
Date 

## ?, 

## 41 Report issued by ;~I Dr.H.-J.Pletsch

### ~~~~JL1LL~~1-19LI-U I L Iii~ LILLJi1LI IImi~npI~I-]

Rer-ort i!o. 92.0452 Paoe 20 (20)

Hoechst Aktieneselischull 6230Fra4*turt (M) 80 

# Hoechst 04737

r - Herrn Dr. Kern Produktsicherhei t/Ofologie e10.9 GBDAn.ltisehes Laboratorhjm Datum Produkt: pigm~ent Blue 56 (Peflex-Blau 3t)jAXcx CC,0 Chargen-Nr.: 21/92

4". W: 1aAW"Xk HOE CG 0423 00 Z024 0001 Untersuchungsbetufld w(HCE CG 0423

.. 0001) = 24,2 % (rn/rn) Caus dew N-Gehalt der Elernentaranalyse) 

# p

- Stabilit~t (Angabe des IHerstellers): 5 Jabre bei Rauteiperatur Verfalldatun des Analysenzertifikates: Februar 1997 Nel:enkompoleftel und Anmerkungen siehe Anlage RUckstellwluster werden inden Archiven des finalytischel Laboratoriums gelagert Leiter des---- ANALYTISCHEN LABORATOR]VMS

- f Dt. R. Fischbach)