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fax: 202.564.8251 

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OCSPP OPPT EAD 

1200 Pennsylvania Ave. NW 

Washington, DC 20460 

202.564.8251 

Pages: 

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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, 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 


Hoech-st I

Pharma Development Report No. 92.0452

Central Toxicology page 1 (20)

DISCLAIMER

This report is the property of Clariant. Its

content is confidential and not for general

release. It is not allowed to cite and reveal

the report or its content to a third party

without prior written approval of Clariant

p ~~Pigment Blue 56 C As> 9~~w96-hours acute toxicity study

in Zebra 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 Devel opmient 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 des A&.Antstal. RONl Sarr 1~ AyS~ndl VADlg3q Hlge. V'3'?2ende. Gonter met2. stebv 4=70ne' Ji'pn mann. Marlin Fdfftu!. HW2 ~ K1Hluo.HrtGrglfS1. ~ ~ ~ M Aans~ J us Msch. Ernst Scado.. Kart-GerhrO cles aa hasn lW Utz-Hetiht Fe~i Szew GesIschalt: Frankfturt am Main - Hade1G1W~San: Franinturi am Mann 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 % is acceptable

even in the 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 a dark blue discoloration.

The determination of the substance concentration was observed in the 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 Ansel deunteri agen und

Profnachweise nach dew Cheikal lengesetz

Texte 16/84, Lbwneltbundesaut

and in compliance 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 in fish

Study No. 92.0298

Test substance Pigment Blue 56

Sponsor GB D, Abt. PSE

Species Zebra fish

Brachydanlo rerio (HAMI LTON- 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* / 500 mg/

Re s p ons l b 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 C40 H36 N3 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 in the 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 soluble
1121~~~ 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 ± 1 OC

Oxygen content : 2 80 % of the saturation value

Duration of lighted period :12 hours daily

Population density : _< 1 g 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 in a 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 Bevel opment 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 *sured and recorded at the inspection times. The methods used for measurement

are described in the 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 in the 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 in appearance

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 in appearance

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 in appearance

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 in appearance

and behavior 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

220 C±1 0 C
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 20.0.92n.d. 0 

92 22.0.92n.d0 

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 detectable
AnalytschesHoechst

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 Iii~ LILLJi1LI IImi~npI~I-] I L 

Rer-ort i!o. 92.0452

Paoe 20 (20)

Hoechst Aktieneselischull

6230 Fra4*turt (M) 80

Hoechst 04737

r -

Herrn Dr. Kern

Produktsi cherhei 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 in den Archiven des

finalytischel Laboratoriums gel agert

Leiter des----

ANALYTISCHEN LABORATOR] VMS - f

Dt. R. Fischbach)