Dossier: Leuco Sulfur Black 1 UUID IUC5-292149d1-a677-4fab-9692-1 fad973281 ea 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

Wilhelm Dietz I Clariant Iberica Producci6n SA. I El Prat de Uobregat (Barcelona) I Spain 

2010-07-1310:24:57 CEST 

Created the dossier 

Dossier header 

Dossier template 

Name 

Version 

REACH Registration above 1000 tonnes 

2010-02-10 

Name (given by user) Leuco Sulfur Black 1 

Dossier subject 

Name phenol 2 4-dinitro- sulfurized leu co denvatives LR ROB 00011687 TLP JR I phenol 2 4-dinitra= sulfurized Jeuco derivatives /66241-11:0 1 Clariant lb9rica 

Producci6n SA. I El Prat de Uobregat !Barcelona\ I Soain 

Submitting legal 

entity 

Clariant Iberica Producci6n SA I El Prat de Uobregat !Barcelona\ I Spain 

Douler creation date/lime 

2010-07-1310:24:57 CEST 

Type of submission 

Joint submission 

Tonnage band Over 1000 tonneslyear 

Registration dossier specific Information 

Phase-in 

coNTA\NS NO CB\ 
Legal entity: Clariant Iberica Producci6n S.A. UUID IUC5-e 16fc18b-79b0-41f2-977c-96407714ccc3 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author REACH-IT 

Date 2010-03-29 12:54:55 CEST 

Remarks 

General information 

Legal entity n- Clariant Iberica Producci6n S.A. 

Legal entity type company 

Identifiers 

Legal entity identifiers 

llclentlflartypa 

VAT 

Contact Information 

Contact address 

Address ZAl - Prat 

Address c/ Cal Coracero, 46-56 

Post.sl code 08820 

T-n El Prat de Llobregat (Barcelona) 

Country Spain 

Phone +34 93 479 8200 

Fu +34 93 479 8211 

E-mail raach.es71@clarlant.com 

Web aile http://www.clariant.es 

ESA60597473 
Contact persons 

Title Mr. 

Flnst name Product Safety 

Last name Pigments & Additives (Pfr) 

Phone +49 223 348 615 2 

E-mail SIEF.PAPfr@dariant.com 

Address lndustriestrasse 

Postal code 50351 

Town HOrth I Knapsack 

Country Germany 

Title Mr. 

Flnst name Product Safety 

Last name Pigments & Additives (Raw) 

Phone +49 619 675 783 04 

E-mail SIEF.PARaw@clariant.com 

Address Am Unisyspark 1 

Postal code 65643 

Town Sulzbach am Taunus 

Country Germany 

Title Mr. 

Flnst name Product Safety 

Last name Textile, Leather & Paper Chemicals (IT) 

Phone +39 023 631 728 4 

E-mail SIEF.TLPIT@clariant.com 
Address Via Cappelinl 

Postal code 20030 

Town 

Country 

Title 

First name 

Last name 

Phone 

E-mail 

Address 

Postal code 

Town 

Country 

Palazzolo Milanese 

Italy 

Mr. 

Product Safety 

Pigments & Additives (Dye) 

+49 619 675 782 75 

SIEF.PADye@clariant.com 

Am Unisyspark 1 

65843 

Sulzbach am Taunus 

Germany 

Mr. 

First name Product Safety 

Last name Pigments & Additives (Wad) 

Phone +49 821 479 295 1 

E-mail SIEF.PAWad@clariant.com 

Address Ludwlg-Harmann-Stralle 100 

Postal code 86366 

Town 

Country 

Tille 

Gersthofen 

Germany 

Mr. 
First name Product Safety 

Last name Textile, leather & Paper Chemicals (FR) 

Phone +33 146 969 782 

E-mail SIEF.TlPFR@clariant.com 

Address Avenue des Champs Pierreux 52 

Postal code 92000 

Town 

Country 

Title 

First name 

Last name 

Phone 

E-mail 

Add""'s 

Nan terre 

France 

Mr. 

Team 01 

REACH Center 

+39 029 918 753 8 

SIEF.MB01@Ciarlant.com 

Via lainate 26 

Postal code 20010 

Town Pogliano Milanese 

Country Italy 

Title Mr. 

First name Product Safety 

Last nama Textile, leather & Paper Chemicals (DE2) 

Phona +41 614 695 634 

E-mail SIEF.TlPDE2@clariant.com 

Add,.... Rothausstrasse 61 

Postal code 4132 

Town Muttenz 
Country Switzerland 

Title Mr. 

First name Product Sefety 

Last name Textile. leather & Paper Chemicals (GB) 

Phone +44 113 239 830 8 

E-mail SIEF.TlPGB@clariant.oom 

Address Celve~ey Lane 

Postal code l$18 4RP 

Town 

Country 

Title 

Horsfooh 

France 

Mr. 

First name Product Sefety 

Last name Textile, leather & Paper Chemicals (ES) 

Phone +34 934 798 235 

E-mail SIEF .TlPES@clariant.oom 

Address ZAl-Prat. c/Cel Coracero 46-56 

Postal code 08820 

Town El Prat de Uobregat 

Country Spain 

First name Product Sefety 

Laet name Pigments & Additives (lnt) 

Phone +49 61 96 75 76 14 5 

E-mail SIEF.PAint@clarlant.oom 
Address Am Unisyspar1< 1 

Posllllcode 65843 

Town Sulzbach am Taunus 

Country Germany 

Title Mr. 

First name Product Safety 

Last name Pigments & Additives (Azo) 

Phone +49 619 675 761 52 

E-mail SIEF.PAAzo@clariant.com 

Address Am Unisyspar1< 1 

Posllll code 65843 

Town Sulzbach am Taunus 

Country Germany 

Title Mr. 

First name Product Safety 

Last name Pigments & Additives (Hpp) 

Phone +49 619 675 761 51 

E-mail SIEF.PAHpp@clariant.com 

Address Am Unlsyspar1< 1 

Posllll code 65843 

Town Sulzbach am Tau nus 

Country Germany 

Title Mr. 
Firat n•m• Product Safety 

L.,.tn.me FUN 

Phone +49 693 051 664 3 

E-mail SIEF.FUN@clariant.com 

Address lndustrleparl< HOchst C 671 

Posl81 code 65926 

Town 

Country 

Sites 

Frankfurt am Main 

Germany 

Clariant Castellbisbal 
Site: Clariant Castellbisbal UUID IUC5-d513f60b-9cc 7 -4068-b 192-d4987bef7 e2b 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1 fad973281 ea 

Author 

Date 

Remarl<s 

mkuehn I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-04-09 14:08:56 CEST 

Site flags 

General information 

Site name Clariant Castetlblsbel 

Legal entity owner Clariant Iberica Producci6n SA I Ei Prat de Llobregat 1Barceloni!l.LSJ1lilll 

Contact address 

Address 

Postal code 

Town 

Region I State 

Country 

Poilgono lnduatrial Can Estepe sin 

08755 

Castetlbisbal 

Barcek>na 

Spain 
Substance: phenol,2,4-dinitro-,sulfurized. leuco 

derivatives LR ROB 00011687 TLP JR UUID IUC5-6927111 0-0636-4fd0-&4ac-30509507d07e 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author pbasto I Clartant Quimicos (Portugal) Lda I Le98 do Balio I Portugal 

Date 2010-07-08 15:51:48 CEST 

Remarks 

0 Related Information 

0.1 Templates 

0.2 Categories 

0.3 Mixtures 

1 General Information 

1.1 Identification 

Substance identification 

Chemtc.l name phenol,2,4-dinttro-,sulfurized,leuoo dertvatives_LR_RDB_00011687 _TLP _JR 

Public name Leuoo Sulphur Black 1 or Leuoo Sulfur Black 1 

Legal entity C!ariant fbBrjca producci6n SA I El Prat de Uobregat (Barcelona) I Spain 

Role in the supply chain 

Role: Manufadurer; Importer 

Reference substance 

phenol2 4:djoitro- su!fudzed leuco derivatjves/66241·11::0 

EC number EC name 

266-273-5 phenol, 2,4-dinltro-, sulfurized, leuco derivatives 

CAS number CAS name 

66241-11-0 
IUPACname 

Type of substance 

Composition UVCB 

Origin organic 

Contact person 

Organisation Clariantlbi>rica Producci6n S.A. 

Department Product Safety TLP 

First name Product Safety 

Last name TLP 

Phone +34 934 798 235 

E-mail SiEF .TLPES@clariant.com 

Address ZAL-Prat. c/Cal Coracaro 46-56 

Postal code 08820 

Town El Prat de Llobregat 

Region I State Barcelona 

Country Spain 

1.2 Composition 

Substance composition 

phen~.2.4-dinitro-.sulfurized. leuc:o derivatives 

Degree of purity 

ca. 52.75 % (w/w) 

Constituents 
Reference substance ohenol 2 4-dinitro· sulfurized leu co derivatives I 66241-1 1-0 

Typical 

concentration 

EC number EC name 

266-273-5 phenol, 2,4-dinitro-, sulfurized, leuco 

derivatives 

CAS CASname 

number 

66241-11..() 

IUPACname 

ca. 52.75% (wlw) 

Concentration range > 45- < 55 % (wlw) 

Remarl<a Cartfficate of analysis C43441 

Impurities 

Reference substance water I water I Water /7732-18=5 

EC number EC name 

231-791-2 water 

CAS number CAS name 

7732· 18-5 Water 

IUPAC name 

water 

Typlc.ol concenlnlllon ca. 45.75 % (wlw) 

Concenlnlllon range > 43.5- < 53.5 % (wlw) 

Reference sodium chloride I sodium chloride I Sodium ch!oride/7647-14-
substance 

EC number EC name 

231-598-3 sodium chloride 

CAS number CAS name 

7647-14-5 Sodium chloride 

IUPAC name 

sodium chloride 

Concenlnltlon range > o- < 1.5% (wlw) 

Reference 

aub&tllnce 

Concenlnltlon 

range 

sodium sulphate I disodium sulfate I Sutfyric acid sodium salt 

(1:2) /7757-82-<l 

EC number EC name 

231-820-9 sodium sulphate 

CAS number CAS name 

7757-82-<l Sulfuric acid sodium salt (1 :2) 

IUPAC name 

disodium sulfate 

> o- < 1.5% (w/w) 

Reference substance 2A:Qirlitrophenol I 2 4-dinitrophenol/ 51-28-5 

EC number EC name 

200-087-7 2,4-dinitrophenol 

CAS number CAS name 

51-28-5 

IUPAC name 

2,4-dinitrophenol 
Concentration range < 20 ppm 

Remerko Certifocate of analysis C43441 

Reference 

oubotance 

Concentration 

range 

Remarka 

1-chloro-2 4=dinitrobenzene I 1-chlorcr2 4-dlnjtrobenzene I 97-

QQ:I 

EC number EC name 

202-551-4 1-chloro-2.4-<linnrobenzene 

CAS number CAS name 

97~0-7 

IUPAC name 

1-chloro-2,4-<linitrobenzene 

< 20 ppm 

Certificate of analysis , C43441 

1.3 Identifiers 

Identifiers 

Regulatory programme identifiers 

~ j Regul.tory programmejtD J Remarka 

REACH preregistration 05-2114286843-37- IUC5-e16fc18b-79b0-4112-9ncnumber 0000 9640n14ccc3 

1.4 Analytical information 

Analytical information 

Analytical rnethodo and spectral Determination of NMR, IR, MS, HPLC/MS, UV-VIS, Physical measurements and Stability in Organic Solvents: see "CoA and 

dota spactra_200B102B09104B.pdr 

Purity of test item I water content see 'C43441_CoA.pdr (amendment to CoA) 
CoA and spectra _20081 028091 048.pdf I 187.28 KB 

Results of analysis 

Analysis type H(1)-NMR, HPLC/MS, UV-VIS and Stability in Organic Solvents 

Tested Leuko Sulfur Black 1 (press cake) 

substance 

Method used not measurable, test ttem insotubie 

Remarks Results not available, because of the insolubility of the test substanca in 

solvents used for the corresponding measurements 

Results see attached "CoA and spectra_20081028091048.pdf" 

Analysts type Water content/ Purity of the test substance (amendment to CoA) 

Tested Leuko Sulfur Black 1 (press cake) 

substalnce 

Method used Kart-Fischer-nration I Calculation 

C43441_CoA.pdf /3.98 MB 

Remarks new determination of the water content and calculation of the purity of 

the test item 

Analysis MS 

type 

Tested Leuko Sulfur Black 1 (press cake) 

substance 

Method MAL DI-MS 

used 

Remarks The test subsstance is a mixture of relative high molecular compounds with 

no defintte structure. Because of fragmentation, only fragments in the range 

of 100 to 500 Da could ba detected. 

Results see also attached "CoA and spectra_20081028091048.pdf" 

Analysis type IR 

Tested substance Leuko Sulfur Black 1 (press cake) 

Method used KBr-peUet 

Remarks The test subsstance was dried overnight 
Results see also attached "CoA and spectra_20081028091048.pdl" 

Analysis Physical measurements 

type 

Tnted Leuko Sulfur Black 1 (press cake) 

subatllnce 

Method visual method 

used 

Remarks The test substance Is insoluble in DMSO, DMSO + NH3, chlorofonn, 

methanol, water, water+ NaOH, DMF, THF, sulfuric acid and trifluoroaclic 

acid. Other solvents were not tested. 

Because of insolubi~ty of the test substance in trifluoroaclic acid, sulfuric 

acid and sodium hydroxyde, no calibration curve is available. Therefore it Is 

not possible to determine lha exact solubility in water and octanol (and lha 

log pow). 

Results sea also attached "CoA and spectra_20081028091048.pdl" 

1.5 Joint submission 

Joint submission 

General lnfonnation 

Joint submission name 

Reguletoty programme 

Lead 

Leuco_Sulphur_Biack_1 

EU: REACH 

Ciariantlb<irica Producci6n S.A I El Prat de Llobregat !Barcelona) I Spain 

1.6 Sponsors 

1.7 Suppliers 

1.8 Recipients 

1.9 Product and process oriented research and development 
2 Classification and Labelling 

2.1 GHS 

Classification and Labelling according to GHS 

General Information 

Not classified 

lmplement.tion 

State I form of the substance 

Classification 

Physical hazards 

EU 

solid 

Haz.ord Ruson for no 

statement classlflcatlon 

Explosives conclusive but not 

sullident for classification 

Flammable gases conclusive but not 

suffiCient for classification 

Flammable aerosols data lacking 

Oxidising gases data lacking 

Gaaes under pressure data lacking 

Flammable liquids data lacking 

Flammable solids conclusive but not 

suffiCient for classification 

Self-rsactlve substancaa and data lacking 

mixtures 

Pyrophortc liquids data lacking 
Pyrophorlc solids 

Self~eatlng substances and mixtures 

Subshlnces and mixtures which In 

conhlc:t with w.ler emit flammable 

goes 

Oxldlslngllqulds 

Oxidising solids 

Organic peroxides 

Corrosive to metals 

Health hazards 

Acute toxicity • oral 

Acute toxicity • dennal 

Acute toxicity • Inhalation 

Skin corrosion/Irritation 

Serious eye damaga/ eye 

Irritation 

Respiratory sansltlutlon 

Skin sansltlutlon 

Aspiration hazard 

Hazard 

-nt 

data lacking 

data lacking 

data lacking 

data lacking 

conclusive but not 

sufficient for classification 

data lacking 

data lacking 

Reason for no classification 

condustve but not sufficient for 

dasslfication 

conduslve but not sufficient for 

dasslfication 

condusive but not sufficient for 

dassification 

conduslve but not suffident for 

dasslficalion 

condusive but not sufficient for 

dasslfication 

condusive but not sufficient for 

dasslf1C8tion 

conduslve but not sufficient for 

dasslfiC8tion 

conduslve but not sufficient for 
classification 

Reproductive toxicity 

Reproductive toxicity condusive but not sufficient for 

classification 

Specific effect 

Route of exposu"' 

Effects on or via lactation conclusive but not sufficient for 

classification 

Germ cell mutagenicity 

Hazard Reason for no cluaiflcatlon 

statement 

Germ cell conclusive but not suflicient for 

mutagenicity classification 

Route of exposure 

Carcinogenicity 

Hazard Reason for no classification 

statement 

Carcinogenicity conclusive but not sufficient for 

classification 

Route of 

exposure 

Specific target organ toxicity • single 

I Hazard Reason for no classification I 
statement 

Specific target organ conclusive but not sufficient for 

toxicity - olngle classification 

Alleclldorgans 

Roull olexpooure 

Specific target organ toxicity • repeated 

HIIZIIrd Reason for no clautflcatlon 

llallment 

Specific llrget organ toxicity conclusive but not sufficient for 

-repeated classification 

Affeclld organs 

Roull olaxpooure 

Environmental hazards 

H11Z1rd Reason for no classification 

-mant 

HIIZIIrdous to the aquatic conclusive but not sufficient for 

environment classification 

HIIZIIrdous to the atmospheric data lacking 

environment 

Labelling 

Signal word No signal word 

2.2 DSD- DPD 

Classification and Labelling according 67/548/EEC (DSD) 
General Information 

Leuco Sulfur Black 1 

Not classified 

Status 67/548/EEC sett classification 

Classification 

Explosiveness 

Oxidising propertleo 

Thermal stability 

Acuta toxicity 

Acute toxicity • Irreversible damage 

after single exposurs 

Repeated dose toxicity 

lrrltallon I Corrosion 

Sensltlsatlon 

Carcinogenicity 

Mutagenicity • Genetic T oxlclty 

Toxicity to rspnoductlon ·fertility 

Classification Reason for no 

classification 

conclusive but not sufficient 

for classification 

conclusive but not suffiCient 

for classification 

conclusive but not sufficient 

for classification 

data lacking 

conclusive but not sufftcient 

for classification 

conclusive but not sufficient 

for classification 

conclusive but not suffiCient 

for classification 

conclusive but not sufficient 

for classification 

conclusive but not sufficient 

for classification 

conclusive but not sufficient 

for classification 

conclusive but not sufficient 

for classlfocation 

conclusive but not suffiCient 
for classification 

Toxicity to reproduction -

development 

condusive but not sufficient 

for classification 

Toxicity to reproduction - breastfed 

b8bin 

condusive but not sufficient 

for classification 

Environment condusive but not sufficient 

for classification 

3 Manufacture, use and exposure 

3.1 Technological process 

Technological process 

Methods of 

manufacture 

Reaction of 2,4-dinitrochlorobenzene with Inorganic alkalis and further 

reaction of obtained dinltrophenolate with reducing agents such as 

sodium sulfhydrate, sodium sulphide and sulfur. 

Reactions of hydrolisis, reduction and thlonatlon are involved in the 

manufacturing process. 

The reactions take place per batch In a dosed reactor connected to a 

water reflux with water recirculation and keeping constant the 

temperature. The gases exit is connected to a scrubber. 

The reaction is controlled by temperature, time and pressure . 

At the end the coloured pigment is isolated by acid precipitation , 

filtrated and washed. 

3.2 Estimated quantities 

Estimated quantities 

Year 2010 

Total tonnage 

Manufloctured 3000 tonneslyear 

im.,-d 

Remarks 

150 tonneslyear 
I Tonnage=3150.0 

3.3 Sites 

Sites 

Site Clariant Castellbisbal/ Castellbisbal/ Spain 

Legal entity 

owner 

Clariant Iberica Producci6n SA I El Prat de Uobregat 

!Barcelona) I Spain 

Production sHe; Use site 

3.4 Form in the supply chain 

Form in the supply chain 

Available as substance 

Substance in mixture 

Substance in mixture 

Trede name of mixture Products of Diresul selling range 

Type of mixture Liquid formulation 

Typical concanlrlltlon >= 20- <= 40 % (wlw) 

Substance in article 

3.5 Identified uses 

Identified uses 

Information on uses 
Uses by workers in Industrial settings 

IUnumber 

Identified uu name 

Process category 

Environmental release 

category 

Substance supplied to that 

uselnfonn of 

Market sector by type of 

chemical product 

Sector of end use 

Subsequent service life 

relevant for that use? 

Article category related to subeaquent service life 

IU number 

Identified use name 

Process catagory 

Environmental release 

category 

Substance supplied to that 

use In fonn of 

Market sector by type of 

Manufacture of dye preparation 

PROC 5: Mixing or blending In batch processes for 

lonnulation of preparations and articles (multistage 

and/or significant contact) 

ERC 2: Formulation of preparations 

As such 

In a mixture 

PC 34: Textile dyes, finishing and impregnating products; 

Including bleaches and other processing aids 

SU 10: Formulation [mixing] of preparations and/or re- packaging (excluding alloys) 

yes 

AC 01: Other (non intended to be released): package 

material 

2 

Manufacture of textile and paper 

PROC 6: Calendering operations 

PROC 2: Use in dosed, continuous process whh 

occasional controUed exposure 

ERC 5: Industrial use resulting In Inclusion into or onto 

a matrix 

In a mixture 
chemical product 

Sector of end use 

Subsequent service life 

relevant lor that use? 

Article cetegory related to 

subsequent service life 

PC 34: Textile dyes, finishing and impregnating 

products; inCluding bleaches and other processing aids 

PC 26: Paper and board dye, finishing and 

impregnation products: including bleaches and other 

processing aids 

SU 5: Manufacture of textiles, leather, fur 

SU 6b: Manufacture of pulp, paper and paper products 

yes 

AC 5: Fabrics, textiles and apparel 

AC 8: Paper articles 

Uses by professional workers 

IU number 

Identified use name 

Proceu category 

Environmental release category 

Subetance supplied to that 

use In form of 

Merklll sector by type of 

chemical product 

Sector of end use 

Manufacture of textile and paper 

PROC 6: Calendering operations 

PROC 2: Use in dosed, continuous process with 

occasional controlled exposure 

ERC 5: Industrial use resulting in indusk>n into or onto 

a matrix 

In a mixture 

PC 34: Textile dyes, finishing and impregnating 

products; induding bleaches and other processing aids 

PC 26; Paper and board dye, finishing and 

impregnation products: including bleaches and other 

processing aids 

SU 5: Manufacture of textiles, leather, fur 

SU 6b: Manufacture of pulp, paper and paper products 
Subuquent service life yes 

relevant lor that use? 

Article cslegoty related to AC 5: Fabrics, textiles and apparel subsequent service life 

AC 8: Papar articles 

Most common technical functions of the substance 

Technical function of substance (what It does) Colouring agents. dyes 

Remarks coloring agent 

Significant routes of exposure 

Human exposure 

Dermal 

Environmental exposure 

Water 

Pattern of exposure 

Accidental/ infrequent 

3.6 Uses advised against 

3. 7 Waste from production and use 

Waste from production and use 

Remarks Waste water does not balong to the waste. 

Other waste not Identified during production process. 

3.8 Exposure estimates 

3.9 Biocidal information 

3.10 Application for authorisation of uses 
4 Physical and chemical properties 

Endpoint summary: Physical and chemical properties 

UUID IUC5-c3ad99aa-Oc79-4 7 44-8b8d-Oc1688008384 

Dossier UUID IUC5-292149d 1-a677 -41eb-9692-1fad973281ea 

Author pbasto I Clariant Quimicos (Portugal) Lda I L~ do Balio I Portugal 

Date 2010-05-28 16:23:16 CEST 

Remarks 

Administrative Data 

Discussion 

IUCLIDPhysical -chemical data 

4.1 State of the substance at 20'C and 101 kPa 

4.2 Melting/freezing point 

4.3 Boiling point 

4.4 Relative density 

4.5 Particle size distribution 

4.6 Vapour pressure 

4. 7 Partition coefficient n-octanol/water 

4.8 Water solubility 

Reference/ Report Results 

No 

Harlan, C30920 

Harlan, C30931 

Harlan, C30942 

Waiving 

Harlan, C30953 

Harlan, C88384 

Harlan,C75560 

wet paste 

-1,7 °C 

99,9 °C 

0,5091 at 20"C 

only sold as liquid 

1,8x1o-7Pa, 250C 

impossible to 

determine 

62 mg/1 
4.10 Surface tension Harlan, C30964 negligible 

4.11 Flash point waiving not needed , Column 

2, Annex VII 

4.12 Autoflammability Harlan 0047/3108 348° C, 20 °C 

4.13 Flammability , for solids Harlan, C30986 not highly inflammable 

4.14 Explosive properties Harlan,C30997 not explosive 

4.15 Oxidizing properties Harlan, C31010 no oxidizing properties 

4.17 Stability in organic solvents and identity of relevant waiving not needed , column 2, 

degradation products Annex VII 

4.21 Dissociation constant wiaving not needed , column 2, 

Annex VII 

4.22 Viscosity Harlan, C31032 thixotrop 
4.1 Appearance/physical state/colour 

Endpoint summary: Appearance/physical state/colour 

UUID IUC5-a02788a5-13e9-4dd9-Be37 -27 eb020afflf 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author pbaslo I Clariant Quimicos (Portugal) Lda I Leo;a do Balio I Portugal 

Date 2010-04-30 15:17:22 CEST 

Remarks 

Administrative Data 

Short description of key information 

Physical state at 20 "C solid (wet paste) 

Colour : black 

Odour : odour1ess 
Endpoint study record: Appearance/physical state/colour 

UUID IUC5-580e072b-40a3-49bd-8fd4-00b856a0a7ec 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author 

Date 

Remarks 

pbasto I Clariant Quimicos (Portugal) Lda I Le<;a do Balio I Portugal 

2010-04-0811:58:38 CEST 

Administrative Data 

Purpose flag weight of evidence 

Study result type experimental result 

Reliability 1 (reliable without restriction) 

Materials and methods 

GLP compliance 

no data 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read-across) 

yes 

Test material Identity 

Identifier Identity 

CAS number 66241-11-0 

EC number 266-273-5 

Results and discussion 

Physical state at 20"C and 1013 hPa 

solid (wet paste) 
Form 

paste 

Colour 

black 

Odour 

odourless 

Substance type 

organic 

Applicant's summary and conclusion 

Conclusions 

black wet paste 
4.2 Melting point/freezing point 

Endpoint summary: Melting point/freezing point 

UUID IUC5-2c666c2d-b30c-4735-924f-!60a45fb2d87 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author pbasto I Clariant Quimicos (Portugal) Lda I Le~ do Balio I Portugal 

Dale 2010-04-1611:03:04 CEST 

Remar1<s 

Administrative Data 

Short description of key information 

The freezing point of Leuco Sulfur Black 1 was determined to be -1.7 'C ± 0.2 'C which is equal to 271 .7 K ± 0.2 K. 

Discussion 

The freezing point of Leuco Sulfur Black 1 is influenced by its high water content. It can be assumed that the measured 

value corresponds to the water fraction only. 
Endpoint study record: KEY_OECD 102_2009_Har/an_C30920_Habeck 

UUID IUC5-93aab045-c437-4355-af70-82908d9e2931 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281ea 

Author 

Date 

Remarks 

jregano I Clariant lbllfica Producci6n S.A. I El Pral de Llobregat (Barcelona) I Spain 

2010-05-1815:17:33 CEST 

Administrative Data 

Purpose flag key study; robust study summary 

Study result type 

Reliability 

experimental result Study period 2009-01-12 ti112009-01-13 

1 (reliable without restriction) 

Rationale for reliability Incl. deficiencies Guideline conform study under GLP without deviations 

Data source 

Reference 

Reference Author 

type 

study report Habeck 

c 

Data access 

Year 

2009 

data submitter Is data owner 

Data protection claimed 

yas 

Title 

Determination of the Freezing 

Point I Freezing Range 

Materials and methods 

Test guideline 

BlbNographlc Testing laboratory 

1oun::e 

Hanan Labomtories Ltd. ltingen 

(Switzerland) 

Guideline 

Report Owner 

no. company 

C30920 Clariant 

Company Report 

study no. deta 

2009-07-

03 

IDev~~n•l 
according EU Method A.1 (Melting I Freezing Temperature) 

to 

aa::ording other guideline: EEC Directive 92/69, Part A, Methods for the determination of physico-chemical properties, A.1 "Melting/Freezing lempereture", EEC 

to Publication No. L383, December 1992. 

according OECD Guideline 102 (Melting point/ Me~ing Range) 

to 

Type of method 

capillary method 

GLP compliance 

yes (ind. oertificate) 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 66241-11-0 

ECnumber 266-273-5 

Details on test matenal 

- Name of test material : Leuco Sulfur Black 1 

- Physical state: lhixotrophic, black, wet paste 

-Composition: Leuco Sulfur Black 1:52.75% (w/w) (caicuiatad by amount of by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-Dinitrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3 o/o (w/w) 

Water. 45.75 % (wiw) by Ka~-Fischer titration 

- Lot/batch No.: ESA0007595 

- Exp~ation date of the lot/batch: 2011-07-11 

- Stabll~ under test conditions: Stable under storage conditions. 

-Storage condition of test material: AI room temperatura (range of 20 ± 5 'C), light protected. 

no 

no 

no 
Results and discussions 

MeHing I freezing point 

Melt./Freez. Atm. Decomposition 

pt. pnaaure 

Decamp. Sublimation Subl. Remart.a 

temp. temp. 

1.7 '1:: ca. 100 kPa no no The study was parfonned under atmospheric pressure which was a11hat time ca. 100kPa. 

The exact value of the pressure was not recorded. 

Any other Information on results Incl. tables 

Detennination of the Freezing Point of Leuco Sulfur Black 1 (Summary) 

Freezln 

['C) 

Preliminary test Above -22 'C 

1stmain test ·1.8 

2ndmain test ·1.5 

Mean value* -1.7 

Standard deviation (±) 0.2 

*The mean freezmg po1nt was calculated from the bold values 

Applicant's summary and conclusion 

Conclusions 

Point 

The freezing point of Leuco Sulfur Black 1 was determined to be -1.7 '1:: ± 0.2 '1:: which is equal to 271 . 7 K ± 0.2 K. 

[K] 

Above 251.2 

271.4 

271.7 

271.6 

0.2 

The freezing point of Leuco SuWur Black 1 is inftuencad by Ks high water content. It can be assumed that the measured value corresponds to water fraction only. 

Executive summary 

The determination of the freezing point I freezing range of the test item was performed according to the EEC Directive 

92169, December 1992 and Council Regulation (EC) No. 44012008, A.1 "Melting/Freezing temperature" and to the OECD 

Guideline No. 102: "Melting point I melting range", adopted July 27, 1995. 
A preliminary test was performed to check the approximate freezing temperature of Leuco Sulfur Black 1. The test item 

was stored about 19 hours at -22 'C in a freezer. As the sample was frozen after this time the freezing temperature of 

Leuco Sulfur Black 1 is above -22 'C. 

During the main tests, about 1 ml of the test item placed in a small test tube were cooled down from room temperature to 

about -2 'C using a dry ice and acetone mixture. 

At -1.8 'C (1st test) and -1.5 'C (2nd test), respe ctively, the test item was frozen by visual judgement. The freezing point 

of Leuco Sulfur Black 1 was determined to be -1.7 •c ± 0.2 'C which is equal to 271.7 K ± 0.2 K. 

The freezing point of Leuco Sulfur Black 1 is influenced by its high water content. It can be assumed that the measured 

value corresponds to the water fraction only. 
4.3 Boiling point 

Endpoint summary: Boiling point 

UUID IUC5-5eef9db9-723c-42a4-81 fe-247 c 125d49ce 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author pbesto I.Ciariant Ouimicos (Portugal) Lda I Le.;a do Balio I Portugal 

Date 2010-04-28 16:28:19 CEST 

Remarks 

Administrative Data 

Short description of key information 

The boiling point of Leuco Sulfur Black 1 is determined to be 99.9 'C ± 0.1 'C which is equal to 373.1 K ± 0.1 K. 

Discussion 

The boiling point of Leuco Sulfur Black 1 is determined to be 99.9 'C ± 0.1 ·c which is equal to 373. 1 K ± 0.1 K. The 

atmospheric pressure during the measurements was 100.4 kPa (preliminary test) and 99.8 kPa (main tests). The boiling 

point of Leuco Sulfur Black 1 is influenced by its high water content. It can be assumed that the measured value 

corresponds to water only. 
Endpoint study record: KEY_OECD 103_2009_Harlan_C30931_Habeck 

UUID IUC5-55a7ec7d-25a3-41 d9-8755-183757971 a48 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

jregano I Clariant Iberica Producci6n SA I El Prat de Llobregat (Barcelona) I Spain 

2011l-05-1815:17:57 CEST 

Administrative Data 

Purpose flag key study; robust study summary 

Study result type experimental result Study period 2009-01-12 till 2009-01-13 

Reliability 1 (reliable without restriction) 

Ratlo""le for reliability Incl. deficiencies Guideline conform study under GLP without deviations 

Data source 

Reference 

Rttfwtrence Author 

type 

Year Title 

study report Habeck 2009 Determination of the BoHing Point I 

c 

Data access 

data submitter is data owner 

Data protection claimed 

yes 

Bolling Range 

Materials and methods 

Test guideline 

Bibliographic Testing laboratory 

source 

Harlan Laboretortas Ltd. ltingen 

(Switze~and) 

Guideline 

Report Owner 

no. company 

C30931 Clariant 

Company Report 

study no. date 

2009-07-

03 

I Deviational 
according EU Method A.2 (Boiling Temperature) 

to 

according OECD Guideline 103 (Boiling point/boiling range) 

to 

according other guideline: EEC Dlnective 92/69, Part A, Methods for the determination of physico-chemical properties. A.2 "Boiling temperature", EEC Publication No. 

to L383, December 1992. 

Type of method 

differential scanning catorimetry 

GLP compliance 

yes (incl. certificate) 

Test materials 

Identity of test material same as for substance defined in section 1 (If not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 66241-11-0 

EC number 266-273-5 

Details on test material 

- Name of test material : Leuco Sulfur Black 1 

- Physical state: thlxotrophlc, black, wet paste 

-Composition: Leuco Sulfur Black 1:52.75% (wlw) (calculated by amount of by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-0initrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (wlw) 

Water. 45.75 % (wlw) by Karl-Fischer titration 

- Lotlbatch No.: ESA0007595 

-Expiration date of the lotlbatch: 2011-07-11 

- Stability under test conditions: Stable under storage conditions. 

-Storage condition of test material: At room temperature (range of 20 ± 5 'C), light protected. 

no 

no 

no 
Results and discussions 

Boiling point 

Thermal Analysis: Determination of the Boiling Point of Leuco Sulfur Black 1 (Summary) 

Heating Range Heating Rate Boiling Point 

rCJ Boiling Point 

['C] ['C/min] 

PreliminaryTest 25-400 20 99.8 

1stMain Test 25-215 10 100.0 

2ndMain Test 25-215 10 99.8 

Mean of boiling point 99.9 

Standard deviation (±): 0.1 .. The mean bolimg pomt was calculated from the bold values (onset pomt of the endothermic 

reaction). 

[K] 

373.0 

373.2 

373.0 

373.1 

0.1 

The tabulated values represent rounded results obtained by calculation using the exact raw data 

In this study the DSC showed an endothermic heat effect at 99.9'C. This temperature is not the true boiling point of the 

test item as it only reflects the boiling point of the water contained by the test item. The DSC shows no further endothermic 

heat effect up to 400'C. Thus, the boiling point of the Leuco Sulfur Black 1 can be regarded as being above 400'C( see: 

Weissenfeld (2009) Vapour Pressure, Harlan Laboratories Ltd. ltingen (Switzerland), Report C30953) 

Applicant's summary and conclusion 

Conclusions 

The boiling point of Leuco Sulfur Black 1 is determined to be 99.9 'C ± 0.1 'C which is equel to 373.1 K ± 0.1 K. 

The boiling point of Leuco Sulfur Black 1 is influenced by its high water content. It can be assumed that the measured value corresponds to water fraction only. 

Executive summary 
The determination of the boiling point I boiling range of the test item was performed according to the EEC Directive 92/69 

and Council Regulation (EC) No. 440/2008, A.2 "Boiling temperature" and to the OECD Guideline No. 103: "Boiling point", 

adopted July 27, 1995. 

Preliminary Test 

The DSC-run of the preliminary test was recorded from 25 'C to 400 'C with a heating rate of 20 'C/min . An endothermic 

heat effect, the boiling, was observed at 99.8 'C. After the experiment, the sample had lost 55% of its mass and the colour 

of the sample was black. 

Main Test 

In order to determine the boiling point more precisely, further DSC-runs were recorded between 25 'C and 215 'C with a 

heating rate of 10 'C/min. During these runs, the boiling point was determined to be 100.0 'C and 99.8 'C. The samples 

lost 51% and 50% of their mass, respectively. After the measurements the sample was a black powder. 

The boiling point of Leuco Sulfur Black 1 is determined to be 99.9 'C ± 0.1 'C which is equal to 373.1 K ± 0.1 K. The 

atmospheric pressure during the measurements was 100.4 kPa (preliminary test) and 99.8 kPa (main tests). The boiling 

point of Leuco Sulfur Black 1 is influenced by its high water content. It can be assumed that the measured value 

corresponds to water only. 

Cross-reference to other study 

Weissanfefd (2009) Vapour Pressure, Harlan Laboratories Ltd. ltingen (Switzerland), Report C30953. Basis for calculation of the vapor pressure; further interpretation of the OSC 
4.4 Density 

Endpoint summary: Density 

UUID IUCS-69998325-7 dbe-40fa-a9dc-551 b451 aa275 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

pbaslo I Clarian1 Quimicos (Portugal) Lda I Le~ do Balio I Portugal 

2010-04-28 16:44:18 CEST 

Administrative Data 

Short description of key information 

The relative densily of Leuco SuHur Black 1 was detemined to be 0.5091 g/cm' (= 0.5091 · 10' kg/m') at 20.4 'C ± 0.1 'C 
Endpoint study record: KEY_OECD 109_2009_Harlan_C30942_Weissenfeld 

UUID IUC5-8818132c-510d-496d-a8be-Odf73142a 7 a 7 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remari<s 

jregano I Clariant Iberica Producci6n SA I El Prat de Llobregat (Barcalona) I Spain 

2010-05-20 16:35:17 CEST 

Administrative Data 

Purpose flag 

Study rasult type 

Reliability 

key study; robust study summary 

experimental resun Study pariod 2009-03-19 till 2009-03-30 

1 (reliable without restriction) 

Rationale for rallablllty Incl. cleflclancles Guideline conform study under GLP wnhout deviations 

Data source 

Reference 

Referance Author 

type 

study repori Weissenfeld 

M 

Data access 

data submitter is data owner 

Data protection claimed 

yes 

Year TIUe 

2009 Determination of the 

Relative Density 

Materials and methods 

Test guideline 

I Qualifier I 

Bibliographic Testing laboratory 

oource 

Harlan Laboratories Ltd. ltingen 

(Switzerland) 

Guideline 

Report Owner 

no. company 

C30942 Ctariant 

Company Report 

otudy no. date 

2009-08-

06 

I Davia!~• I 
according EU Method A.3 (Relative Density) 

to 

according OECD Guideline 109 (Density of Liquids and Solids) 

to 

according other guideline: EEC Directive 92169, Part A, Methods for the detennination of physico-chemical properties, A.3 "Relative density", EEC Publication No. 

to L383, December 1992 

Type of method 

air comparison pycnometer (for solids) 

GLP compliance 

yes (Ind. certificate) 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 86241-11-0 

EC number 266-273-5 

Details on test material 

• Name of test material : Leuco Sulfur Black 1 

- Physical state: lhlxotrophic, black, wet paste 

-Composition: Leuco Su~ur Black 1:52.75% (wlw) (calculated by amount of by-compounds) 

2,4-0initrochlorobenzene: < 20 ppm 

2,4-0initrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3 % (wlw) 

Water. 45.75 % (wlw) by Ka~-Fischer titration 

• Lot/balch No.: ESA0007595 

-Expiration dele of the lot/belch: 2011-07-11 

- Stablllty under test conditions: Stable under storage conditions. 

-Storage condition of test material: AI room temperatura (range of 20 ± 5 'C), light protected. 

no 

no 

no 
Any other infonnation on materials and methods incl. tables 

Due to the properties of the test item the handling using the pycnometer was difficult. Instead a measuring cylinder 

(volumetric flask) of known volume is filled with an accurately weighed amount of the test item. 

Test Item Treatment 

The test item was not dried before use, because the density of the whole test item mixture should be determined. 

General Test Procedure 

A volumetric flask was filled with a known amount of test item and the volume of the test item was read. This procedure 

was performed in triplicate. During the performance of the test, the temperature was kept constant and measured by a 

thermohygrograph. 

Results and discussion 

Density 

Type Denalty Temp. Remerl<a 

relative density 0.51 20.4 'C 

density 0.51 g/cm' 20.4 'C 

Any other infonnatlon on results Incl. tables 

Detennination of the Density of Leuco Sulfur Black 1 

Sample No. Temperature Sample Weight Sample Volume Density Relative 

['C) [g) [ml] [g/cm3] Density 

1 20.5 2.54327 5.0 0.5087 

2 20.5 3.48919 6.8 0.5131 
13 20.5 2.62940 

Applicant's summary and conclusion 

Conclusions 

The relative density of Leuco Sulfur Black 1 was determined to be 0(20/4) = 0.5091 

Executive summary 

5.2 0.5057 

0.5091 0.5091 

The determination of the relative density of the test item was performed in accordance with the EEC Directive 92/69 and 

Council Regulation (EC) No. 440/2008, Part A, Methods for the determination of physico-chemical properties, A.3 "Relative 

density" and the OECD Guideline for Testing of Chemicals, No. 109, "Density of Liquids and Solids". 

The density was determined at a temperature of 20.4 'C ± 0.1 'C with three independent aliquots of the test item. 

The density of the test item Leuco Sulfur Black 1 was determined to be 0.5091 g/cm3 (= 0.5091 · 103 kg/m3) at 20.4 'C ± 

0.1 'C using measuring cylinder. 

The relative density of Leuco Sulfur Black 1 was determined to be D(20/4) = 0.5091 
4.5 Particle size distribution (Granulometry) 

Endpoint study record: waiving_Particle size distribution (Granulometry) 

UUID IUC5-b830d8fa-9e6c-40b1-beb4-0e2a65a18838 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Author pbaslo I Clariant Quimioos (Portugal) Lda I Le~ do Ballo I Portugal 

Date 2010-03-23 13:25:28 GET 

Remart<s 

Administrative Data 

other justification 

JustltlcaUon for data w•lvlng waived according lo column 2 of REACH Annex VII . study nol required W the substance is sold under liquid form. 

Study result type other. 

Applicant's summary and conclusion 

Conclusions 

Substance is sold only in liquid form, particle size distribution not required . aooording to column 2 of Annex VII of REACH 

Executive summary 

Substance is sold only in liquid form, particle size distribution not required , according to column 2 of Annex VII of REACH 
4.6 Vapour pressure 

Endpoint study record: KEY_ Calculation_2009 _Harlan_ C30953_Weissenfeld 

UUID IUC5-e9b1b80d-4a9d-4858-9bb8-56e8837f19fa 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

XML Transformation V1.0 Plug-In 

2009-12-1117:14:09 GET 

Successfully migrated to IUCLID 5.2 format. 

Administrative Data 

Purpose flag 

Dllblwalvlng 

Justification for data waiving 

Study result type 

Reliability 

used for dassification 

other justification 

Calculation using Modified Watson Correlation 

estimated by calculation 

1 (reliable without restriction) 

Rationale for reliability Incl. 

deflclancles 

Using the boiling point of Leuco Sulfur Black 1 was determined by expertmentel study C30931, the vapor pressure can be calculated using 

Modified Watson Correlation 

Data source 

Reference 

Reference Author 

type 

study report Mlrjam 

Weissenfetd 

Data access 

data submitter Is data owner 

Data protection claimed 

Year Title Bibliographic source 

2009 Leuco Sulfur Black 1 - W.J. Lyman, W.F. Reahland D.H. Rosenblatt 

Calculation of the Vapor (ad); Handbook of Chemical Property Estimation 

Pressure Methods, McGraw-Hill, NY (1990) 

Testing Report Owner Company Report 

laboratory no. company study no. date 

Hanan C30953 Clartant 2009-

Laboratortas, 07-16 

Ltd 
yes 

Materials and methods 

Principles of method if other than guideline 

H the vapor pressure of Leuco Sulfur Black 1 is expected lo be so small that the value is below the measurement range of an experimental detanninalion (e.g. the gas saturation 

method), then the vapor pressure can also be calculated, using the Modified Watson Correlation. This method is described by Lyman etal. (1990). 

This method calculates the vapor pressure from the boiling point of the test item. 

Test materials 

Details on test material 

Identification: Leuco Sulfur Black 1 

CAS No.: 66241-11-0 

Description: Thixothrophic, black, wet paste 

Balch Number: ESA0007595 

Composition: Leuco Sulfur Black 1: 52.75% (calculated by amount of by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-Dinitrophenol: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (w/w) 

Water: 45.75% (wlw) by Karl-Fischer titration 

Stability of the Test Hem: Stable under storage condHions 

Expiry date: July 11,2011 

Storage Conditions: At room temperature (range 20 +I- 5 'C). light protected 

Results and discussions 

Vapour pressure 

< 0.0000002 Pa 

25~ 

Any other Information on results incl. tables 

The estimation of the vapor pressure of leuco Sulfur Black 1 was based on the EEC directive 92/69, Part A, Methods for 

the determination of physico-chemical properties, A.4 "Vapour Pressure", EEC Publication No. l383, December 1992 and 

the OECD Guideline No. 104, "Vapour Pressure", adopted July 27, 1995. 
Applicant's summary and conclusion 

Conclusions 

The vapor pressure of Leuco Sulfur Blacl 1 is below 

1.8 x 10 (-7) Pa at 25 •c 

Executive summary 

The boiling point of leuco Sulfur Black 1 was determined in study C30931. In this study the DSC showed an endothermic 

heat at 99.9 °C. This temperature is not the true boilng point of the test item as it only reflects the boiling point of the water 

contained . The DSC shows no further endothermic heat effect up to 400°C. Thus, the boiling point of the Leuco Sulfur 

Black 1 can be regarded as being above 4000C 

Based on this boiling point the vapor pressure of Leuco Sulfur Black 1 was calculated being below : 

1.8 x 10·7Pa at 25 oc 

calcuted using Modified Watson Correlation 
4. 7 Partition coefficient 

Endpoint summary: Partition coefficient 

UUID IUC5-2bccdaa6-24c1-4fad-94b6-5fbd39ac087b 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author pbaslo I Clariant Quimicos (Portugal) Lda I Le~ do Balio I Portugal 

Date 2010-06-1111:59:21 CEST 

Remarks 

Administrative Data 

Short description of key information 

Impossible to determine the log Pow 

Discussion 

No chromatographic method is available to estimate the solubility of Leuco Sulfur Black 1 in n-octanol _ Therefore the log 

Pow cannot be determined 
Endpoint study record: Harlan Laboratories (2010) /K2 KS! Partition coefficient_ statement 

UUID IUC5~0d7160c-5fe4-4930-89d5-7e65a039849a 

Dossier UUID IUC5-292149d1-a677-41eb-9692-1fad973281ea 

Author 

Date 

Remal't(s 

jregano I Clariant Iberica Producci6n S.A. I El Prat de Llobregat (Barcelona) I Spain 

2010-00-01 10:49:04 CEST 

Administrative Data 

Purpoaeftag key study; robust study summary 

Justification for data welvlng 

Study ruult type 

No chromatographic method is applicable to determine the solubility of Leuco Suffur Black 1 in n octanol. 

estimated by calculation Study period 

Reliability 2 (refiable with restrictions) 

April 14, 2010 

R.UOnele for reliability Incl. 

deficiencies 

Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, 

which do not affect the quality of the relevant results. 

Data source 

Reference 

Ref11rence Author 

type 

study report Dr Martin 

KOhne 

Data access 

Year 

2010 

data submitter is data owner 

Data protection claimed 

yes 

Title 

Calculation of the Partition 

Coefficient (n-Octanoll Water 

Materials and methods 

Partition coefficient type 

Bibliographic 

source 

Testlnglaboretory Report Owner Company Report 

no. company study no. date 

Ha~an Laboretorias Ltd. Zelgllweg 1 C88384 Clariant 2010-04-

4452 ltingen I Switzerland 14 
octanol-water 

Test guideline 

no guideline followed 

Type of method 

other: no method was applicable 

Principles of method If other than guideline 

The test was not feasible to complete 

GLP compliance 

no (not mandantory) 

Test materials 

Test material Identity 

Identifier Identity 

CAS number 66241-11-0 

Common name Leuco Sulfur Black 1 

Details on test material 

Identification: Leuco Sulfur Black 1 

CAS Nr.: 66241-11-0 

Description: Thlxolrophlc, black, wet presscake 

Batch Number: ESA0007595 

Compos~lon: Leuco Su~ur Black 1: 52.75% (calculated by amount of by-compounds) 

2.4-Dinltrochlorobenzene: < 20 ppm 

2.4-Dinltrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (w/w) 

Water. 45.75% (w/w) by Karl-Fischer tilration 

Stability of the test ~ern: Stable under storage conditions 

Stability of the last Hem dilution: > 4 h In water 
Expiry date: July 11, 2011 

Storage Conditions: At room temperature (range 20 +I- 5 'C). light protected 

Analytical method 

no data (It was not feasible to complete) 

Any other Information on materials and methods Incl. tables 

No data available. 

Results and discussions 

Details on results 

No chromatographic method is applicable to datermine the solubility of Leuco Sulfur Black 1 in n octanol. Therefore, no Partition coefficient could be darived. 

Any other Information on results Incl. tables 

The partition coefficient (P owl is defined as the ratio of the solubility of the test item in octanol and in water. 

No chromatographic method is applicable to determine the solubility of Leuco Sulfur Black 1 in n-octanol. 

Although it is possible to estimate the water solubility of Leuco Sulfur Black 1 (62.4 mg/L as determined in Harlan 

Laboratories Study C75560) by measurement of the Total Carbon (TC) content this analytical method is inapplicable in 

case of n-octanol as a solvent. 

Therefore it is impossible to determine the log P owof Leuco Sulfur Black I according to the guideline. 

Overall remarks, attachments 

Overall remarks 

No determination of partition coefficient by Method A8 of Commission Directive 92169/EEC (which constijutes Annex V of Council Directiva 671548/EEC) was possible. 

Applicant's summary and conclusion 

Conclusions 
No chromatographic method is available to estimate the solubility of Leuco Sulfur Black 1 in n-octanol. Therefore the log POW cannot be determined according to the guideline. 

Executive summary 

No chromatographic method is available to estimate the solubility of Leuco Sulfur Black 1 in n-octanol. Therefore the log 

P0 wcannot be determined according to the guideline. 
4.8 Water solubility 

Endpoint summary: Water solubility 

UUID IUCS-492eba04-b 1 ef-4ab4-b62b-3d9affdl54b1 

Dossier UUID IUCS-292149d 1-a677 -4feb-9692-1 fad97328 1 ea 

Author pbasto I Clariant Quimicos (Portugal) Lda I Leca do Balio I Portugal 

Date 2010-04-28 17:37:06 CEST 

Remarks 

Administrative Data 

Short description of key information 

The water solubility Is 62.4 mg/l at 20.0 'C and a pH of 7.9 

Discussion 

The water solubility of Leuco Sulfur Black 1 was determined to be 62.4 mg/L at 20.0 'C and a pH of 7.9 based on a 

measured concentration of 11.5 mg/L of carbon 
Endpoint study record: Harlan Laboratories (2010) I K1 KS/ water solubility Leuco Sulfur 

Black 1.Ciariant Products C75560 

UUID IUC5-8afebae3-bd7a-4e44-89db-43f0b4485180 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281ea 

Author 

Date 

Remarks 

jregano I Clariant Iberica Producci6n SA I El Prat de Llobregat (Barcelona) I Spain 

2010-06-01 10:38:47 CEST 

Administrative Data 

Purpose flag 

Study result type 

Reliability 

key study: robust study summary 

experimental result 

1 (reHable without restricllon) 

Study period 15-02-2010111-03-2010 

Rationale for reliability Incl. 

deficiencies 

Study conducted in compliance with egreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological 

deficiencies, which do not affect the quality of the relevant results. 

Data source 

Reference 

Reference Author 

type 

study report Dr Martin 

KOhne 

Data access 

Year 

2010 

data submitter is data owner 

Data protection claimed 

yes, but wHiing to share 

TIUe 

Leuco Sulfur Black 1: 

Determination of the Water 

Solubility 

Materials and methods 

Bibliographic 

source 

Testing laboratory Report Owner Company Report no. compeny study no. date 

Hanan Laboratories Ltd. ZelgHweg C75560 Clariant 2010-03-

1 4452 ltingen I Switzerland 11 
Test guideline 

Qualifier Guideline Dev!Mions 

according to EU Method A.6 (Water Solubility) no 

according to OECD Guideline 105 (Water Solubility) no 

Type of method 

ftask method 

GLP compliance 

yes (ind. certificate) (Date of Signature: November 12, 2008) 

Test materials 

Test material Identity 

Identifier Identity 

Common name Leuco Sulfur Black 1 

CAS number 66241-11-0 

Details on test matenal 

Identification: Leuco Sulfur Black 1 

Description: Thixotrophic, black, wet presscake 

Batch Number: ESA0007595 

Composition: Leuco Sulfur Black 1: 52.75% (calculated by amount of by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-Dinilrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (wlw) 

Water: 45.75% (w/w) by Kart-fischer titration 

Stability of the lest ~em: Stable under storage conditions 

StabHity of the test ~em dilution: > 4 h in water 

Expiry dale: July 11, 2011 

Storage Conditions: AI room temperature (range 20 +/- 5 'C), light protected 

Details on methods 
The determination was carried out using the flask melhod, Method AS Water Solubility of Commission Regulation (EC) No 440/2008 of 30 May 2008. 

Any other information on materials and methods Incl. tables 

Test Perfonnance 

Test Solutions About1.5- 1.7 g of test item were weighed into each of 6 Erlenmeyer flasks and 50 mL of double distilled water were edded to each flask. The tighUy dosed flasks were shaken at 

aboul30 'C for 24 hours, 48 hours and 72 hours, respectively. Aftarwards, the flasks were equHibrated for another 24 hours at20.0 'C. The pH was measured to be aboul7.9. 

Afterwards, the supernatant solutions were centrifuged (-2900 g for 10 minutes), filtered (0.2 ~m. Nylon) and 

5 mL of this solution was diluted up to 50 mL with water prior to analysis. The quantification was performed using TC measurement as described below. Details of sample treatment are 

given inTable 1 below. 

Analytical Method The concentration of the test item was analyzed using TC measurement. The test samples were quantified wtth reference to a calibration curve, which was generated automatically. The 

last calibration was performed on January 06, 2010. 

System Suitability check 

A system suitability check was performed before sample measurement in order to check the reliabilrty of the measurement. 

Preparation of 1,4-Benzochlnone Solutions 

75.02 mg of 1,4-banzochlnone were filled up to a volume of 1 L with MiiiK:l water to get a stock solution of nominal conoantration of 50 ppm of carbon. 2 mL of this stock solution was 

diluted to a volume of 200 mL wrth Mllli-Q water to get a solution of a nominal ooncentration of 0.5 ppm of carbon. A seoond solution of a nominal conoantration of 10 ppm of carbon was 

prepared by diluting 20 mL of tha stock solution up to 100 mL with MilK:! water.For both solutions, the TOC was measured. The system surtabillly check was sucoassfully as all TOC 

values were wHhin the specifications. 

Prepar.tlon of Saccharose SoluUona 

118.99 mg of saccharose were filled up to a volume of 1 L with Milll-0 water to get a stock solution of nominal concantretion of 50 ppm of carbon. 2 mL of this stock solution was dButed 

to a volume of 200 mL with Miii~Q water to get a solution of a nominal oonoantration of 

0.5 ppm of carbon.Afterwards, the TOC was measured. The system suitability check was sucoassfuUy as the TOC value was within the specifications. 

Data Handling 

The samples were quantified by Total Carbon (TC) measurement with referenoa to a calibration curve.The calibration curve was calculated using the software. The concentration of 

carbon of the injected samples was quantified by peak area wrth referenoa to the calibration curve. 

From the results obtained by the software the conoantration of carbon in the analyzed solutions was calculated by the following equation: 

(1) 
Where 

C1 Measured concentration of carbon in the sample solution [ppm= mgll] 

c2 Concentration of carbon in the test item solution before dilution (mgll] 

I OHution factor 

From lhe concenlration of carbon the water solubility of the test Hem was calculated by the following equation: 

(2) 

Where 

C3 = Water solubility of the test Item (mgll] 

Cc Percentage of carbon in lhe test Hem as obtained by elementar analysis 

(here: 18.5%) 

T able1 Data of the Performance of the Water Solubililv Test 

Sample Solution No. 1 2 3 4 5 6 

Weighed sample of lhe test Hem [mg] 1482.52 1674.48 1473.65 1621.87 1734.69 1563.06 

Added volume of water (ml] 50 50 50 50 50 50 

Agitation time at 30 'C (hours] 72 72 48 48 24 24 

Equilibration time at 20 'C (hours] 24 24 24 24 24 24 

pH value of test item solution 7.60 7.52 7.68 7.76 8.32 8.29 

DUution factor before quantification 10 10 10 10 10 10 
Results and discussions 

Water solubility 

62.4 mgll 

Temp. 20 'C 

pH 7.9 

Details on results 

Please see section Remarl<s on resulls including tables and figures. 

Any other Information on results Incl. tables 

The delermination of the watar solubility of Leuco Sulfur Black 1 was performed according to the Directive 92169. A.6 "Water Solubility" (1992) and the OECD guideline No. 105. "Water 

Solubility" (1995) using the flask shaking method. 

After a shaking time of 24 hours. 48 hours and 72 hours. respectively. at 30 'C. the samples were equilibrated for another 24 hours at 20 'C. Afterwards. the samples were centrifuged. 

filtered. diluted and analyzed by TC measurement. Tha water solubility of Leuco Sulfur Black 1 at 20 •c was determined to be 62.4 mg/L. Tha measured results diller by less than 15%. 

Therefore. the study is considered to be valid .The resulls of the Individual measurements of the main test are summarized in Table 2 below. The tabulated values represent rounded 

results. which were obtained by calculation using the exact raw data. 

T a ble2 R esults ofth e w ater Sol ub Ill 1ty o Leuco Su~ ur Bla cl<1 

Concentration of test item. 

Sample No. Shaklngtime at30 'C Concentration of carbon. Concentration of carbon. calculated Water solubility calculated 

analyzed calculated (rounded) 

[mg/L] (mg/L] [mg/L] (mg/L] 

1 1.094 10.94 59.14 72 

2 1.400 14.00 75.68 

3 1.147 11.47 62.00 62.35 

48 

4 1.104 11.04 59.68 so= 6.90 

5 1.143 11.43 61.78 24 

6 1.033 10.33 55.84 
The saturation mass concentration is calculated from the bold values 

SO = standard deviation 

Applicant's summary and conclusion 

Interpretation of results 

sligh~y soluble (0.1-100 mg/L} 

Conclusions 

During this study the watar solubility of Leuco Sulfur Black 1 was detennined and found to be 62.4 mg/l at 20 'C and a pH of 7.9 based on a measured cone en !ration of 11.5 mgJL of 

carbon using the flask shaking method. 

Executive summary 

During this study the water solubility of Leuco Sulfur Black 1 was determined. Vessels containing water and test Item were shaken for 24 hours, 48 hours and 72 hours, respectively, at 

30 'C. Afterwards the samplas were equilibrated for another 24 hours at20.0 'C. After dilution with water the concentrations in the aqueous solutions were determined by a TC-malhod. 

The water solubility of Leuco Sulfur Black 1 was detennlned to ba 

based on a measured concentration of 

using the flask shaking method. 

Cross-reference to other study 

System Suitability test was performed in Study C797 44 

62A mg/L at 20.0 'C and a pH of 7.9 

11.5 mgiL of carbon 
4.1 0 Surface tension 

Endpoint study record: Waiving_ Surface tension 

UUID IUC5-4c623182-907b-4871-8672-d85c85dc5463 

Dossier UUID IUC5-292149d 1-a6n -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

jregano I Clariant Iberica Producci6n S.A. I El Prat de Llobregat (Barcelona) I Spain 

2010-05-18 14:38:20 CEST 

Administrative Data 

Data waiving 

Justification for data 

waiving 

other justtficatk>n 

waived according to column 2 of REACH Annex VII: the surface activity is not a desired property of the material. Thare is no hydrophylic haad and no 

hydrophobic tail in tha stnucture of tha substanca. 
4.11 Flash point 

Endpoint study record: Waiving_F/ash point 

UUID IUCS-37401 ac0-953b-413c-9c37 -379871eee02c 

Dossier UUID IUC5-292149d1-a6n -4feb-9892-1fad973281 ea 

Author 

Dale 

Remarks 

pbasto I Clariant Ouimicos (Portugal) Lda I Le~ do Balio I Portugal 

2010-03-23 13:17:23 GET 

Administrative Data 

Data waiving study scientifically unjustified 

Justification for data waiving Waived accordig to column 2 of REACH Annex VII , test will not be preformed as the substance is a solid end does not contain any organic solvent 

Results and discussions 

Flash point 

Remarks Substance it a solid, without organic solvents 

Applicant's summary and conclusion 

Conclusions 

Solid substance Doesn't contain organic solvent 

Executive summary 

Data not required , according to column 2 of REACH Annex VII , as it is a solid substance , that doesn't contain any 

organic solvent 
4.12 Auto flammability 

Endpoint summary: Auto flammability 

UUID IUC5-a55c7ca4-20c1-4399-8355-f1cb24d69906 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281ea 

Author 

Date 

Remarks 

pbasto I Clariant Quimicos (Portugal) Lda I Lei'S do Balio I Portugal 

2010-D4-28 17:09:53 CEST 

Administrative Data 

Short description of key information 

tt has been detenmined to have an auto-ignition temperature of 348 +1- S'C. 
Endpoint study record: KEY_EU A.15_2009_Harlan_0047/310B_Tremain/Atwal 

UUID IUC5-afcfd8ff-193b-4453-932d-d 1699fd8f71a 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author 

Date 

Remarks 

jragano I Clariant Iberica Producci6n SA I El Prat de Llobragat (Barcelona) I Spain 

2010-05-1815:26:23 CEST 

Administrative Data 

Purpose !lag 

Study result type 

Reliability 

key study; robust study summary 

experimental result Study period 2009-02-11 till2009-02-17 

1 (reliable without restriction) 

Rationale for reliability Incl. deficiencies Guideline conform study under GLP without deviations 

Data source 

Reference 

Reference Author Year 

type 

study report Tremain SP, 2009 

Alwal SS 

Data access 

data submitter is data owner 

Data protection claimed 

yes 

Title 

Leuco Sulfur Black 1: Determination 

of Auto-Ignition Temperature 

(Liquids and Gases) 

Materials and methods 

Test guideline 

Bl>llographlc Tasting laboratory 

source 

Harlan Laboratories Lid. 

Shardlow (United 

Kingdom) 

Report Owner Compeny study no. 

no. company 

004713108 Clariant Harlan Laboratories 

Ltd. (Switzerland) 

C31008 

Report 

dele 

2009-

06-15 
Qualifier J Guideline lo.vtmlona 

according lEU Method A.15 (Auto-Ignition Temperature (Liquids and Gases)) (Council R&gUatlon (EC) No 44012008 of 30 May 2008, Part A: Methods for the lno to delerminatlon of physico-chemical properties.) 

GLP compliance 

yes (ind. certificate) 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 66241-11-0 

ECnumber 266-273-5 

Details on test material 

- Name of test material : Leuco Sulfur Black 1 

- Physical state: thixotrophic, black, wet paste 

- Composrtion: Leuco Sulfur Black 1:52.75% (wlw) (calculated by amount of by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-Dinilrophenote: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (wlw) 

Water: 45.75% (w/w) by Ka~-Fischer titration 

-Lot/batch No.: ESA0007595 

-Expiration date of the lot/batch: 2011-07-11 

-Stability under test conditions: Stable under storage conditions. 

-Storage condition of test material: At room temperature (range of 20 :t 5 'C), tight protected. 

Any other Information on materials and methods Incl. tables 

The auto-ignition temperature was determined by heating aliquots of the test material in a flask and observing for any 
ignition. 

Procedure 

The test flask was heated in a flask heater. Aliquots oftest material (tested as received) were dropped into the flask, which 

was observed for signs of ignition over a 300 second period. This procedure was repeated, varying the sample size, as 

necessary, until the lowest temperature at which ignition, if any, occurred within 300 seconds of insertion, was determined. 

Results and discussions 

Autoflammablllty I Self-Ignition temperature 

348't: 

at >= 101.46- <= 102.46 kPa 

Any other Information on results Incl. tables 

The test material has been determined to have an auto-ignition temperature of 348 +/- 5'C 

Results 

Test Temperature Test Weight Ignition Delay Observations 

(oCj_ Ig]_ rsl 

50 (clean flask) 0.2 >300 No Ignition 

100 (clean flask) 0.2 >300 No lanition 

150 0.2 >300 No Ignition 

200 0.2 >300 No Ignition 

250 0.2 >300 No !gnition 

300 0.2 >300 No Ignition 

350 0.2 >300 No lonition 

400 (clean flask) 0.2 >300 No Ignition 

400 (clean flask) 0.4 >300 No Ignition 
400 clean flask) 0.6 182 An oranoe flame 

396 clean flask) 0.6 74 An orange flame 

392 clean flask) 0.6 >300 No lonition 

394 clean flask 0.6 164 An orange flame 

392 clean flask) 0.8 47 An orange flame 

388 (clean flask) 0.8 163 An orange flame 

384{clean flask) 0.8 154 An orange flame 

380 (clean flask 0.8 97 An orange flame 

376 (clean flask 0.8 151 An orange flame 

372 (clean flask 0.8 86 An orang_e flame 

368 (clean flask 0.8 134 An orange flame 

360 (clean flask 0.8 97 An oranoe flame 

352 (clean flask) 0.8 >300 No Ignition 

356(clean flask 0.8 138 An orange flame 

354 (clean flask) 0.8 >300 No Ignition 

354 (clean flask) 1.0 67 An orange flame 

350 (clean flask 1.0 >300 No Ignition 

352 (clean flask) 1.0 264 An orange flame 

350 (clean flask 1.2 201 An orange flame 

348 (clean flask 1.2 >300 No Ignition 

348 (clean flask) 1.4 >300 No Ignition 

348 (clean flask) 1.6 >300 No Ignition 

348 _(clean flask 1.8 >300 No Ignition 

348 (clean flask) 2.0 75 An orange flame 

346 _(_clean flask 2.0 >300 No lonition 

346 (clean flask 2.2 >300 No Ignition 

346 (clean flask) 2.4 >300 No lonition 

346 (clean flask 2.6 >300 No Ignition 
I 346 (clean flask) 2.8 

Applicant's summary and conclusion 

Conclusions 

The test material has been determined to have an aut<Hgnition temperature of 348 +I· 5"C. 

Executive summary 

>300 I No Ignition 

The auto-ignition temperature was determined using Method A15 specified in Council Regulation (EC) No 44012008 of 30 

May 2008, Part A: Methods for the determination of physico-chemical properties. 

The test material has been determined to have an auto-ignition temperature of 348 +/- 5'C. 
4.13 Flammability 

Endpoint summary: Flammability 

UUID IUC5-c59109d0-bfe84caf-9122-5b4ccb2bf665 

Dossier UUID IUC5-292149d1-a6n 4feb-9692-1fad973281ea 

Author pbaslo I Clariant Quimioos (Portugal) Lda I Le~ do Balio I Portugal 

Date 2010-04-28 17:14:47 CEST 

Remarks 

Administrative Data 

Short description of key information 

Leuoo Sulfur Black 1 Is not "highly flammable" 

Discussion 

Leuco Sulfur Black 1 could not be ignited with a flame during the preliminary test 
Endpoint study record: KEY_2009_Harlan_C30986_Weissfeld 

UUID IUC5-<:2b41 d2a-f5a8-441d-8a12-<!65bfc89c86e 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973261 ea 

Author 

Date 

Remart<s 

jregano I Clariant Iberica Producci6n S.A. I El Prat de Llobregat (Barcelona) I Spain 

2010-05-16 12:32:03 CEST 

Administrative Data 

Purpose flag 

Study result type 

Reliability 

key study; robust study summary 

experimental result 

1 (reMable without restriction) 

Studyparlod March 24,2009- August4, 2009 

Rlllionale for reliability Incl. This study followed the procedures indicated by the following intematlonally accepted guidelines and recommendations: 

deflclanclea • EEC Directive 92169, Part A, Methods for the detennination of physico-chemical pnopartias, A.1 0 "Flammability (solids)". EEC Publication No. 

L363, December 1992. 

Data source 

Reference 

Relwence Author 

type 

study report Welssenfeid 

M 

Data access 

data submitter is data owner 

Data protection claimed 

yes, bul willing Ia shere 

Year 

2009 

• Council Regulation (EC) No. 44012006, 30 May 2008, Part A, Methods lor the detannination of physico-chemical properties, A.1 0 "Flammability 

(solids)" 

Title Bibliographic Testing laboratory Report Owner Company study Report 

source no. company no. date 

Leuco Sulfur Black 1 - Detennination of Harlan C30986 Clariant 2009-08-

the FtammabiUiy Laboralorias, Ltd 04 

Materials and methods 
Test guideline 

Guideline 

EU Method A.10 (Flammability (Solids)) 

yas (ind. certificate) 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read-across) 

yes 

Test material Identity 

Identifier Identity 

CAS number 66241-11-0 

EC number 266-273-5 

Details on test material 

Identification: Leuco Sulfur Black 1 

CAS No.:66241-11-0 

Description: Thlxothrophic, black, wet paste 

Batch Number: ESA0007595 

ComposHion: Leuco Sulfur Black 1: 52.75% (calculated by amount of by-compounds) 

2,4-0in"rochlorobenzene: < 20 ppm 

2,4-Dinitrophenol: < 20 ppm 

Su~ate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (w/W) 

Water: 45.75% (w/W) by Kar1-Fischar titration 

Stability of the Test Item: Stable under storage conditions 

Expiry data: July 11, 2011 

Storage Conditions: At room temperatura (range 20 +/- 5 'C), light protected 

Any other Information on materials and methods Incl. tables 

Preparations before the TestThe test item was homogenized by kneading and an aliquot was put onto an aluminium 

plate to form a pile with a length of 250 mm and a height of about 10 mm. 
Preliminary TestAn attempt was made to ignite the sample with the aim to determine the burning time over a distance of 

200 mm, if applicable. The flame of a gas burner was used as ignition source. The hot flame was applied for a maximum of 

two minutes. The test was conducted in a fume cupboard with constant air speed. Additionally, a draught screen was 

erected around the apparatus. 

Main TestNo further tests were performed according to the results obtained in the preliminary test. 

Results and discussions 

Solidniquid: Ignition on contact with air 

no 

Applicant's summary and conclusion 

Interpretation of results 

other: not highly flammable 

Conclusions 

Leuoo Sulfur Black 1 is not "highly flammable" according to the cr1teria of the guideline. 

Executive summary 

The determination of the flammability of the test item was performed in accordance with the EEC Directive 92/69, EEC 

Publication No. L383, December 1992 and Council Regulation (EC) No. 440/2008, 30 May 2008, Part A, Methods for the 

determination of physico-chemical properties, A. 10 "Flammability (solids)". 

In contact with the ignition source, the flame of the gas burner turned orange. Leuco Sulfur Black 1 emitted some glowing 

drops. Glowing sustained for about 1 minute but did not expand.Leuco Sulfur Black 1 could not be Ignited with a flame 

during the preliminary test (contact time of about 2 minutes). Therefore, no main test was performed. 
4.14 Explosiveness 

Endpoint summary: Explosiveness 

UUID IUC5-aa8e5b5a-7b46-4efc-bccc-<la9a 15273b5d 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author pbasto I Clarianl Quimicos (Portugal) Lda I Le99 do Balio I Portugal 

Date 2010-04-28 17:19:21 CEST 

Remarks 

Administrative Data 

Short description of key information 

Leuoo Sulfur Black 1 is not explosive 

Discussion 

Leuco Sulfur Black 1 is not a potential explosive and does not have a potential for rapid energy release. 
Endpoint study record: KEY_EC A.14_2009_Harlan_C30997 _Weissenfeld 

UUID IUC5-3776037a-1d18-4fb6-b86!>-583abca215d1 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author 

Date 

Remarks 

jregano I Clariant Iberica Producci6n S.A. I El Prat de Llobregat (Barcelona) I Spain 

201Q-05-1815:28:43 CEST 

Administrative Data 

Purpose flag 

Study result type 

key study; robust study summary 

experimental resutt Study period 2009-03-18 

Reliability 1 (reliable without restriction) 

Rationale lor reliability Incl. deficiencies Guideline conform study 

Data source 

Reference 

Reference Author Year Title Blbllogrephlc 

type source 

Testlngle-ry 

study report Weissenfeld 2009 Determination of the Hanan Laboratories Ltd. ltingen 

M 

Data access 

data submitter Is data owner 

Data protection claimed 

yes 

Explosive Properties 

Materials and methods 

Test guideline 

(Swilzerland) 

Guideline 

Report Ownar 

no. company 

C30997 Ciariant 

Company Report 

study no. data 

2009-07-

16 

I Devlet~n•l 
other guideline: UN Recommendations on the Transport of Dangerous Goods (Manual of Tests and Criteria, Fourlh revised edilioo, 2003, Appendix 6, 

"Orange Bool<" 

no (not required) 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read,.cross) 

yes 

Test material identity 

Identifier Identity 

CAS number 66241-11-0 

ECnumber 266·273--5 

Details on test material 

• Name of test material : Leuco Sulfur Black 1 

·Physical state: thixotrophic, black, -t paste 

• ComposHion: Leuco Sulfur Black 1 :52.75 % (wlw) (calculated by amount of by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4·Dinitrophenote: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (wlw) 

Water: 45.75% (wlw) by Kart-Fischer titralioo 

• Lot/batch No.: ESA0007595 

·Expiration date of the lot/batch: 2011-07·11 

• StabiiHy under test conditions: Stable under storage condilioos. 

·Storage condHion of test material: At room temperatura (range of 20 ± 5 q, light protected. 

Results and discussions 

Explosive under influence of flame 

no 

More sensitive to shock than m-dinltrobenzene 

not determined 
More sensitive to friction than m-dlnltrobenzene 

not determined 

Explosive (not specified) 

no 

Any other Information on results Incl. tables 

Calorimetric Tests The exothermic decomposition energy was determined using Differential Scanning Calorimetry in a 

closed, gold plated high pressure vessel (DSC). The calorimetric test is of special importance for the evaluation of 

compounds containing chemical groups associated with explosive properties or if no appraisal of the molecular structure 

could be performed. There was no exothermic peak found. Thus the decomposition energy does not exceed the UN limit of 

500 Jig (AHDec determined between room temperature and 500 'C ). 

Applicant's summary and conclusion 

Conclusions 

Leuco Sulfur Black 1 is not to be classified as explosive material and no experimental determination according to the EC test guideline A.14 has to be 

performed. 

Executive summary 

Reactive Groups and Oxygen Balance 

No molecular structure was given for Leuco Sulfur Black 1. Thus, the appraisal of the molecular structure and the 

calculation of the oxygen balance could not be performed. 

Calorimetric Tests 

The exothermic decomposition energy was determined using Differential Scanning Calorimetry in a closed, gold plated 

high pressure vessel (DSC). The calorimetric test is of special importance for the evaluation of compounds containing 

chemical groups associated with explosive properties or if no appraisal of the molecular structure could be performed. 

There was no exothermic peak found. Thus the decomposition energy does not exceed the UN limit of 500 Jig (AHDec 

determined between room temperature and 500 'C). 
Leuco Sulfur Black 1 is not to be classified as explosive material and no experimental determination according to the EC 

test guideline A.14 has to be performed. 

Thus it can be concluded beyond a reasonable doubt, that Leuco Sulfur Black 1 is not a potential explosive and does not 

have a potential for rapid energy release. Nevertheless, this judgment is in respect to classification; it does not necessarily 

mean that any processing hazards can be discounted. 
4.15 Oxidising properties 

Endpoint summary: Oxidising properties 

UUID IUCS-0948cf5e-1ade-4e34-b673-4087945048a4 

Dossier UUID IUCS-292149d1-a677-4feb-9692-1fad973281ea 

Author pbasto I Clariant Quimicos (Portugal) Lda I Le~ do Balio I Portugal 

Date 201CHJ4-28 17:20:40 CEST 

Remarks 

Administrative Data 

Short description of key information 

Leuco Sulfur Black 1 has no oxidizing properties. 
Endpoint study record: KEY_EU.A17_2009_Harlan_C31010_Weissenfeld 

UUID IUC5-2c948e 76-d6b1-4015-89d3-db 14c11 ba980 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarl<s 

jregano I Clariant Iberica Producci6n SA I El Prat de Llobregat (Bart:elona) I Spain 

2010-05-1815:29:24 CEST 

Administrative Data 

Purpose flag key study; robust study summary 

Study resun type 

Rllllabllhy 

experimental resu~ Study period 2009-{)4-03 till 2009-{)4-07 

1 (reHabie without restriction) 

Rillionaie for rellabllhy Incl. deficiencies Guideline conform study under GLP without deviations 

Data source 

Reference 

Referencs Author 

type 

study report Weissenfeld 

M 

Data access 

data submitter is data owner 

Data protection claimed 

yes 

Year Title 

2009 Determination of the Oxidizing 

Properties (Solids) 

Materials and methods 

Test guideline 

Bibliographic Testlng laboratory 

source 

Harlan Laboratories Ltd. 

ltingen (Switzarland) 

Guideline 

Report Owner 

no. company 

C31010 Clariant 

Company Report 

study no. data 

2()()9..()8-

06 

I Devmt~n•l 
according EU Method A.17 (Oxidising Properties (Solids)) 

to 

according other guideline: EEC Directive 92169, Part A, Methods for the determination of physico-chemical properties, A.17 "Oxidizing Properties (solids)", EEC 

to Publication No. L383, December 1992. 

GLP compliance 

yes (incl. oertlficate) 

Test materials 

Identity of test material same as for substance defined in section 1 (If not read-across) 

yes 

Test material Identity 

Identifier Identity 

CAS number 66241-11-0 

EC number 266-273-5 

Details on test material 

- Name of test meterial : Leuco Sulfur Black 1 

- Physical state: thlxotrophic, black, wet paste 

- Composition: Leuco Sulfur Black 1 :52.75 % (w/w) (calculated by amount of by-compounds) 

2,4-0initrochlorobenzena: < 20 ppm 

2,4-Dinltrophenola: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3 % (w/w) 

Water. 45.75% (w/w) by Karl-Fischer titration 

-Lot/batch No.: ESA0007595 

-Expiration date of the lot/batch: 2011-07-11 

-Stability under test conditions: Stable under storage conditions. 

-Storage condition of test material: At room temperature (range of 20 t 5 'C), light protected. 

Contact with 

powdered cellulose 

Any other information on materials and methods incl. tables 

no 

no 
The preliminary test was performed to determine if oxidizing properties were to be expected (as conduded from the 

structural formula of the test item). The preliminary test was also conducted to ascertain adequate safety precautions 

during the main study. 

A barium nitrate/cellulose mixture (at a ratio 2:1 (w/w)) was prepared with dried and sieved(< 0.125 mm) material. 

The test item is a paste containing water. As the test item should be analyzed as delivered, according to sponsor's 

request, the test item was not dried at 1 05'C prior to the test. The test item was kneaded and an aliquot was then 

immediately mixed with the cellulose (at a ratio 2:1 (w/w)) in a dosed jar. 

Both mixtures were filled into a glass funnel. The mixtures were ignited with the flame of a gas burner. The vigor and 

duration of the reactions were observed and recorded. 

Due to the unmistakable results obtained during the preliminary test, no main-test was performed to confirm the results. 

Results and discussions 

Test result 

maximum burning rate of test mixture 

Os 

maximum burning rate of reference mixture 

77s 

Any other information on results incl. tables 

During the preliminary test the Leuco Sulfur Black 1/cellulose mixture ignited poorly and the flame became orange. Without 

contact to the ignition source, the flame extinguished. Only glowing was observed for 3 minutes. The surface was 

carbonized but underneath unchanged material remained. 

Since the test item mixture did not burn without contact to the ignition source and the handling of the test item was difficult 

(contact to air should be avoided according to sponsor's request), a main test was not performed. 
The barium nitrate/cellulose mixture during the same test burned for 77 seconds with a blazing, light yellowish flame, 

emitting sparks. 

Applicant's summary and conclusion 

Interpretation of results 

no oxidising properties 

Conclusions 

Leuco Sulfur Black 1 has no oxidizing properties. 

Executive summary 

The determination of the oxidizing properties of Leuco Sulfur Black 1 was performed in accordance with the EEC Directive 

92/69, EEC Publication No. L383, December 1992 and Council Regulation (EC) No. 440/2008, Methods for the 

determination of physico-chemical properties, A.17 "Oxidizing Properties (solids)". 

During the preliminary test the Leuco Sulfur Black 1/cellulose mixture ignited poorly and the flame became orange. Without 

contact to the ignition source, the flame extinguished. Only glowing was observed for 3 minutes. The surface was 

carbonized but underneath unchanged material remained. 

The barium nitrate/cellulose mixture during the same test burned for 77 seconds with a blazing, light yellowish flame, 

emitting sparks. 

Since the test item mixture did not burn without contact to the ignition source and the handling of the test item was difficult 

(contact to air should be avoided according to sponsor's request), a main test was not performed. 

In conclusion, Leuco Sulfur Black 1 has no oxidizing properties. 
4.17 Stability in organic solvents and identity of relevant degradation products 

Endpoint study record: Waiving_ Stability in organic solvents and identity of relevant 

degradation products 

UUID IUC5-128cf15b-17 4a-4de2-ad2c-f80c6e2cfe57 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author jregano I Clariant Iberica Producci6n S.A I El Prat de Llobregat (Barcelona) I Spain 

Date 2010-05-1815:30:09 CEST 

Remarks 

Administrative Data 

Data waiving 

Justification lor data 

waiving 

study technically not feasible 

According to Reach Regulation annex Xllhe test does not need to be performed because lhe substance is not soluble in organic solvents like DMSO, 

chloroform, methanol, ethanol and 1 ,2-propylene glycol 
4.21 Dissociation constant 

Endpoint study record: Waiving 

UUID IUC5-16b2f4ad-463&-4ab6-9ad2-929f7dd73998 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

jregano I Clariant Iberica Producci6n SA I El Prat de Llobregat (Barcelona) I Spain 

201Q-05-1812:20:04 CEST 

Administrative Data 

Data waiving study technically not feasible 

Justlftcatlon for data waiving waived according to column 2 of REACH Annax IX: ~is scientifically not possible to perform the test, the analytical method is not sensitive enough. 
4.22 Viscosity 

Endpoint summary: Viscosity 

UUID IUC5-{)62973fe-8268-4048-89bc-1990d633d39a 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Author pbaslo I Clariant Quimicos (Portugal) Lda I Lei'" do Balio I Portugal 

Date 2010-04-28 17:22:28 CEST 

Remarks 

Administrative Data 

Short description of key information 

lhlxotrophic 

Discussion 

As the viscosity of the test item became lower during stirring, Leuco Sulfur Black 1 can be stated to being thixotrophic. 
Endpoint study record: KEY_OECD 114_2009_Harlan_C31032_Weissenfeld 

UUID IUC5-c371d7a 7 -a5a9-483e-a58b-e 7 e1f71920c8 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Aulhor 

Date 

Remarks 

jregano I Clarian11benca Producci6n S.A. 1 El Prat de Llobregal (Barcelona) I Spain 

2010-05-18 15:30:24 CEST 

Administrative Data 

Purpose flag key study; robust study summary 

Study result type experimental resu~ Study period 2009-03-19 

Reliability 1 (reliable without restriction) 

Data source 

Reference 

Refersnce Author 

type 

Year Title 

study report Welssenfeld 2009 Determination of the 

M 

Data access 

data submitter is data owner 

Data protection claimed 

yes 

Viscosity 

Materials and methods 

Test guideline 

Guideline 

Bibliographic Testing laboratory 

source 

Harlan Laboratories Ltd. IUngen 

(Switzerland) 

Deviations 

Report Owner 

no. compeny 

C31032 Clar1ant 

Compeny study Report 

no. 

2009-08-

06 

-

OECD Test Guideline 114 yes (Due 10 the texture of the test llem, II was not possible 10 fill an aliquot into the rotaUonal viscometer and to determine the viscosity. 
(Viscosity of Liquids) Thus, the test Item was judged visually for any changes In viscosity whie stilling.) 

other: Due to the texture of lhe test item, it was not possible to fin an aliquot into the rotational viscometer and to detennlne the viscosity. Thus, lhe test ~em was judged visually for any 

changes in viscosity while stilling. 

Principles of method If other than guideline 

Due to the texture of the test Item, it was not possible to fill an aliquot into the rotational viscometer and to determine the viscosity. Thus, the test item was judged visually for any 

changes In viscosity while stilling. 

GLP compliance 

yes (ind. certificate) 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 66241-11-0 

EC number 266-273-5 

Details on test matenal 

- Name of test material : Leuco SuWur Black 1 

- Physical stata: lhixotrophic, black, wet paste 

-Composition: Leuco Sulfur Black 1 :52.75% (wlw) (calculated by amount of by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-0inltrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (wlw) 

Water. 45.75 % (wlw) by Kari-Fischer titration 

-Lot/batch No.: ESA0007595 

-Expiration date of the lot/batch: 201 1-07-1 1 

- Stability under test conditions: Stable under storage conditions. 

-Storage condition oflest material: At room temperature (range of 20 ± 5 'C), light protected. 
Any other Information on materials and methods Incl. tables 

Principle of the Method 

The viscosity of the test item was determined by visual observation. 

Test Conditions 

The determination was carried out at room temperature. 

General Test Procedure 

About 30 g of test item were weighed into a 250 mL beaker. The test item was stirred for about one hour by a external 

stirrer with a metal blade at 150 rpm. The beaker was sealed with parafilm and the test item in the beaker was hold under 

nitrogen atmosphere. The texture of the test item was judged visually. 

Results and discussions 

Viscosity 

> 10000 mPa s (dynamic) 

Temp. 22 'C 

Remarks the viscosity oould not be exactly measured 

Any other Information on results Incl. tables 

Observation Results 

Stirring Tlme_[minJ Phy15ical state of the Test Item 

0 Coarse grained black powder 

5 Powder crushed 

10 Powder crushed 

15 Powder, substance climbs up on the beakers glass 
30 Substance black, paste-like, slobbery, oily,climbs up to 1/3 of the 

beaker's glass 

55 Substance made one big black clump 

Substance defines to be thixotrophic. 

Applicant's summary and conclusion 

Conclusions 

As the viscosity of the test ttem became lower durtng stirring. Leuco Sulfur Black 1 can be stated to being thixotrop 

Executive summary 

The determination of the viscosity of Leuco Sulfur Black 1 deviated from the OECD guideline No. 114, "Viscosity of 

Liquids" using the rotational viscometer method. Due to the texture of the test item, it was not possible to fill an aliquot into 

the rotational viscometer and to determine the viscosity. 

Thus, the test item was judged visually for any changes in viscosity while stirring. 

As the viscosity of the test item became lower during stirring, Leuco Sulfur Black 1 can be stated to being thixotrophic. 
5 Environmental fate and pathways 

Endpoint summary: Environmental fate and pathways 

UUID IUC5-fflc36e3-clbc-47c3-9ada-77ed614d43e5 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author uensenba I Clarianl Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

Date 2010-05-{)4 12:06:06 CEST 

Remarks 

Administrative Data 

Discussion 

The test item Leuco Sulfur Black 1 was investigated for its ready biodegradability in a manometric respirometry test over 

28 days according to the EU Commission Directive 92/69 EEC, C.4-D Commission Regulation (EC) No 440/2008, C.4-D 

and the OECD Guideline for Testing of Chemicals No. 301 F. 

The biochemical oxygen demand (BOD) of the test item Leuco Sulfur Black 1 in the test media was in the normal range 

found for the inoculum controls. 

Consequently, Leuco Sulfur Black 1 was not biodegradable under the test conditions within 28 days. 

In the toxicity control, containing both Leuco Sulfur Black 1 and the reference item sodium benzoate, Leuco Sulfur Black 1 

had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 142 mg/L. 

In the procedure controls, the reference item sodium benzoate was degraded by an average of 80% by Exposure Day 14, 

and reached an average biodegradation of 82% by the end of the test (Day 28), thus confirming suitability of the activated 

sludge. 
5.1 Stability 

5.1.2 Hydrolysis 

Endpoint study record: Waiving_ Hydrolysis 

UUID IUC5-84523700-e2ef-4bc1-81 d4-cd0427894ef9 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-21 14:56:46 CEST 

Administrative Data 

Data waiving 

JustlflCIItlon for data 

waiving 

study technically not feasible 

Due to the physico-<:hernical properties of the UVCB substance no analytical method is available. According to Reach Regulation Annex XI, section 2 the 

study is not required. 
5.2 Biodegradation 

5.2.1 Biodegradation in water: screening tests 

Endpoint study record: Memmert (2009) I Biodegradation In water: screening tests 

UUID IUC5-a3f5e34a-e689-4b5e-e3cd-3145768dfc6e 

Dosaler UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author 

Date 

Remarl<s 

jregano I Clariant Iberica Producci6n SA I El Prat de Llobregat (Barcelona) I Spain 

2010-05-20 16:34:30 CEST 

Administrative Data 

Purpose flag key study 

Study result type 

Reliability 

experimental resutt Study period 2009-03-23 tiH 2009-04-23 

1 (reliable without restriction) 

Rationale for reliability Incl. deflclenclaa Guideline conform study under GLP without deviations 

Data source 

Reference 

Reference Author Year 

type 

study report Mernmert 2009 u 

Data access 

data submitter is data owner 

Data protection claimed 

yes 

Title 

Ready Biodegradability of Leuco Sullur 

Black 1 in a Manometric Respirometry Test 

Materials and methods 

Bibliographic 

source 

Tasting laboratory 

Harlan Laboratories Ltd. 

ltingen (Switzerland) 

Report Owner Company 

no. company study no. 

c311n Clariant 

Report 

date 

2009-08-

19 
Test type 

ready biodegradability 

Test guideline 

Qualifier Guideline 

aCCOfding EU Method C.4-0 (Detennlnation of tha "Ready" Biodagradability- Manometric Respirometry Test) (Com. Rag. (EC) No 44012008 of 30 May 20081aying 

to down test methods pursuant to Ragulation (EC) No 1907/2006 of the European Partiament and of the Council on tha Raglstration, Evaluation, Authorisation 

and Restriction of Chemicals (REACH), C.4·D, Manometri) 

aCCOfding EU Method C.4-D (Detennination of tha "Raady" Biodagradabllity- Manometric Respirometry Test) (EU Commission Oirectiva 92169 EEC, C.4-D, Manometric 

to Respirometry Test, 1992.) 

according OECD Guideline 301 F (Ready Blodagradabiity: Manometric Respirometry Test) (OECD Guideline for Testing of Chemicals No. 301 F, Ready 

to Biodagradability: Manometric Respirometry Test, 1992.) 

GLP compliance 

yes (incl. certificate) 

Test materials 

Identity of test material same as for substance defined in section 1 (If not read-ecross) 

yes 

Test material identity 

Identifier Identity 

CAS number 66241-11-0 

ECnumber 266-273-5 

Details on test material 

- Name of test material : Lauco Sulfur Black 1 

- Physical state: thlxotrophic, black, wet paste 

-Composition: Leuco Sulfur Black 1 :52.75 % (wlw) (calculated by amount of by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-Dinitrophanole: < 20 ppm 

Suffate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3 % (wlw) 

Water. 45.75% (wlw) by Kart-Fischer titration 

Deviations 

no 

no 

no 
-Lot/batch No.: ESA0007595 

-Expiration date of the lot/batch: 2011-07-11 

- Stability under test conditions: Stable under storage conditions. 

-Storage condition of test material: AI room temperature (range of20 ± 5 'C), light protected. 

Study design 

Oxygen conditions 

aerobic 

Inoculum or test system 

activated sludge, non-adapted 

Details on Inoculum 

-Source of inoculum/activated sludge: aerobic activated sludge from a wastewater traatment plant (ARA Ergolz II, FOilinsdorf, Switzerland) treating predominantly domestic wastewater 

- LaboraiOfY culture: 

-Method of cultivation: The sludge was washed twice with tap water by centrltugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge 

suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, calculated amounts of wet sludge wera suspended In test water to 

obtain a concentration equivalent to 4 g (±10%) dry material per liter.- Storage conditions/length: During the holding period of three days prior to use, the sludge was aerated at room 

temperature. 

- Preparation of Inoculum lor exposure: Prior to use, the sludge was first thoroughly mixed and then dHuted with test water 

-Concentration of sludge: Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of1 g per liter (dry weight basis). Based on the 

determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain a final concentration of30 mg dry material per liter. 

Duration of test (contact time) 

28d 

Initial test substance concentration 

Initial cone. Ba-on 

140 mgiL test mat. 

141 mgiL tes1 mat. 

Parameter followed for biodegradation estimation 
1 02 consumption 1 

Details on study design 

Apparatus 

The test flasks (500 mL reaction vassals, labeled with the necessary information to ensure unmistakable Identification) were incubeted under continuous stining In a SAPROMA T Dt 2 

{Voith GmbH, ~nhaim, Garmany). Oxygen consumption was recorded manually by taking a daily reading et least on each working day. 

The final test volume was 250ml par test flask 

Principle: Electro-chemical analysis process: 

The biodegradation process consumes the dissolved oxygen in the test medium and generates C02. The C02 is adsofbed by soda lime, which results in a d8CI8ase of the total 

pressure in the airtight test flasks. The pressure drop Is detected and converted into an electrical signal by means of an electrode type manometer. The consumed oxygen is replaced 

by etectrolytically generated oxygen from a copper sulfate solution. 

Test duration: 28 days 

Light conditions: Dari<ness 

Test temperature: 22 'C, maintained with a built4n thermostat and checked once par week. 

pH: Prior to test start, the pH was measured In each test flask before the eddRion of the activated sludge inoculum. At tha end of incubation, tha pH was measured again in each test 

flask. 

Test water 

prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solullons: 

1) KH2P04 8.50 giL 

K2HP04 21.75 giL 

Na2HP04 x 2H20 33.40 giL 

NH4CI 0.50 giL 

The pl4 of this solution was 7.4. 

2) MgS04 x 7H20 22.50 giL 

3) CaCI2 x 2H20 36.40 giL 

4) FeCI3 x 6H20 0.25 giL, stabilized wnh one drop of concentrated HCI par liter 

To obtain the final test water, 10 mL of stock solution No. 1 and 1 mL each of stock solution Nos. 2, 3 and 4 were combined and made up to 1000 mL with purified water. The pH was 

adjusted from 7.7 to 7.4 wRh a diluted hydrochloric acid solution. 

Control and Blank System 

Inoculum blank: yey Toxicity control: yes 

Reference substance: yes 

Reference substance 

benzoic add, sodium salt 
Any other Information on materials and methods Incl. tables 

Due to the complex structure (composition) of the test item, its chemical oxygen demand (COD) was determined according 

to the EU Commission Directive 92/69/EEC, C.6 following DIN 38414-59. The COD was determined at BMG Engineering 

AG, lfangstrasse 11, 8952 Schlieren I Switzerland. The COD determination was not performed in compliance with the GLP 

regulations. Therefore, it is excluded from the Statement of Compliance in this report. However, it was performed in 

compliance with ISO/IEC 17025. 

The chemical oxygen demand (COD) of the test item Leuco Sulfur Black 1 was determined according to the EU 

Commission Directive 92/69/EEC, Part C.6 following DIN 38414-S9. The duplicate values were 0.78 and 0.81 mg 02/mg 

test item. The mean COD was 0.795 mg 02/mg test item. 

Test Concentrations 

Identification Replicate Amount of test item1 Amount of reference item 1 

No. mg/L C002 mg/L 

Test item 1 140 111 - Test item 2 141 112 - Inoculum 1 --- n.a. -- control 

Inoculum 2 --- n.a. - control 

Procedure 1 -- n.a. 100 

control 

Procedure 2 --- n.a. 100 

control 

Toxicitycontrol• 1 142 113 100 

1 The tabulated values represent rounded results obtained by calculation usmg the exact raw data. 

2Chemical oxygen demand in mg 02/L test solution. Calculation of the ThOD was not possible due to the 

Th003 

n.a. 

n.a. 

n.a. 

n.a. 

167 

167 

167 
complex structure (composition) of the test item. The COD was not determined in compliance with the GLP 

regulations and is therefore excluded from the Statement of Compliance in this report. However, the results 

will be archived under this study number. 

3Theoretical oxygen demand in mg 0211. test solution. 

• The test item can be assumed to be inhibitory if in the toxicity control less than 25% degradation (based on 

total ThOD) occurs within 14 days. 

n.a. not applicable. 

Results and discussions 

Test performance 

The percent biodegradation of the reference ~em sodium benzoate was calculated besed on the theoretical oxygen demand of 1.67 mg 02/mg. 

In the procedure controls, the reference Mm was degraded by an average of 80% by Exposure Day 14, thus confinning suMbilily of the activated sludge. At the end of the test (Day 

28), the reference ~em was degraded by an average of 82%. 

% Degradation of test substance 

Remark• 

St.dev. not applicable 

The percent biodegradation in the toxicity control, containing both the test ~em and the reference ~em, was calculated based on the COD of the test ~em and the ThOD of the reference 

item. 

In the toxicity control, the course of biodegradation over the 28-<lay exposure period was similar to the two procedure oontrols, oontainlng only the reference ~em. WMin 14 days of 

exposure, biodegredetion amounted to 50%. 

Thus, according to the test guidelines. the test Mm had no Inhibitory effect on activated sludge microorganisms at the tested concentration of 142 mg/L because biodegradation in the 

toxicity control was >25% within 14 days. 

The pH measured in all flasks at the start of the test was 7.4. At the end of exposure (Day 28), pH values of 7.1-7.8 were measured. 

BODS/ COD results 

8005/COD 

BOD5 ca. 0 mg 02/g test mat. 

Results with reference substance 

The percent biodegradation of the reference Kern sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg 02/mg. 

In the procedure controls, the reference Hem was degraded by an average of 80% by Exposure Day 14, thus confinning suilabil~ of the activated sludge. At the end of the test (Day 

28), the 

reference ~em wes degraded by an average of 82%. 
Any other Information on results Incl. tables 

Biodegradation of the Test Item 

The percent biodegradation of the test item was calculated based on the chemical oxygen demand (COD) of 0.795 mg 

02/mg test item. 

The biochemical oxygen demand (BOD) of the test item Leuco Sulfur Black 1 in the test media was in the normal range 

found for the inoculum controls throughout the study period of 28 days. 

Consequently, Leuco Sulfur Black 1 was not biodegradable under the test conditions within 28 days. 

Blod ear a dati on nt h T e es t Fl as k s 

Percentage Biodegradation1 

Test item Procedure control Toxicity 

control 

Time Replicate No. Replicate No. Replicate 

No. 

(days) 1 2 1 2 1 

0 0 0 0 0 0 

1 0 0 19 21 14 

2 -- - - - - 3 - - - - - 4 -6 -3 63 68 41 

5 -4 -3 68 72 44 

6 -4 -4 70 74 45 

7 -4 -4 72 77 47 

8 -5 -4 73 78 48 
9 - - - 10 - - -- 11 -6 -6 77 

12 -9 -8 76 

13 -9 -8 78 

14 -10 -9 79 

15 - -- - 16 - -- -- 17 - -- -- 18 -12 -11 80 

19 -13 -11 80 

20 -14 -12 80 

21 -16 -12 78 

22 -16 -12 80 

23 - - - 24 - - - 25 -18 -14 78 

26 -18 -14 80 

27 -19 -15 79 

28 -20 -15 80 

Mean (Day 28) (-17). 82 

,corrected for the mean oxygen uptake of the 1noculum controls (see Sect1on 3.5) 

- Not determined 

n.a. not applicable 

-- - - -

80 49 

81 49 

81 50 

81 50 

-- - - -

- - 84 51 

84 51 

84 51 

84 50 

84 51 

- -

- - 84 51 

84 51 

84 51 

84 51 

n.a. 

• Negative value due to higher oxygen consumption in the inoculum controls than in the test flasks with test item. 

Applicant's summary and conclusion 

Validity criteria fulfilled 

yes 

Interpretation of results 
under test conditions no biodegradation observed 

Conclusions 

Leuco Sulfur Black 1 is not biodegradable under the test cond~ions within 28 days. 

Executive summary 

The test item Leuco Sulfur Black 1 was investigated for its ready biodegradability in a manometric respirometry test over 

28 days according to the EU Commission Directive 92/69 EEC, C.4-D Commission Regulation (EC) No 440/2008, C.4-D 

and the OECD Guideline for Testing of Chemicals No. 301 F. 

The biochemical oxygen demand (BOD) of the test item Leuco Sulfur Black 1 in the test media was in the normal range 

found for the inoculum controls. 

Consequently, Leuco Sulfur Black 1 was not biodegradable under the test conditions within 28 days. 

In the toxicity control, containing both Leuco Sulfur Black 1 and the reference item sodium benzoate, Leuco Sulfur Black 1 

had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 142 mg/L. 

In the procedure controls, the reference item sodium benzoate was degraded by an average of 80% by Exposure Day 14, 

and reached an average biodegradation of 82% by the end of the test (Day 28), thus confirming suitability of the activated 

sludge. 
Endpoint study record: Waiving_ Simulation testing on ultimate degradation in test water 

UUID IUC5-3030b992-1 03b-4c44-a31 0-62e187968f4c 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad9732B 1 ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutscl11and) GmbH I Sulzbacl1 am Taunus I Germany 

2010-05-21 14:47:53 CEST 

Administrative Data 

Data waiving 

Justification for 

data waiving 

other justification 

Waiving according to "column 2" of Regulation (EC) No 190712006 (CSA doss not indicate need for further Investigations). Due to the very high molecular weight 

(>8000 DaKon) the bioavallabilily of the substance Is negligible (low potential to cross biological membranes) and no biodegradation could be expec1ed. Therefore 

addilional testing is not justified 
5.2.2 Biodegradation in water and sediment: simulation tests 

Endpoint study record: Waiving_Biodegradation in water and sediment: simulation tests 

UUID IUC5-al01798a-aa 7f-4eed-a3b4-185f56ae58f8 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1 fad973281 ea 

Author uensenba I Clariant Produkte (Deutschland) GmbH I Sul-ch am Taunus I Germany 

Date 2010-05-21 14:48:27 CEST 

Remarks 

Administrative Data 

Data w.Wing other justification 

Justification for Waiving according lo "column 2" of Regulation (EC) No 190712006 (GSA does not indicate need !Of further Investigations). Due lo lhe very high molecular weight 

data .. lvtng (>8000 Dakon) lhe bioavallability of lhe substance is nagliglble (low potential to cross biological membranes) and no biodegradation oould be expected. TherafO<e 

additional testing is not justified 
5.2.3 Biodegradation in soil 

Endpoint study record: Waiving_Biodegradation in soil 

UUID IUC5-09b6bac3-567 4-4a86-82f7 -494edffie4f8 1 

Dossier UUID IUCS-292 149d1 -a677 -4feb-9692-1fad97328 1 ea 

Author 

Date 

Remarks 

uensenba I Clarian1 Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-21 16:32:10 CEST 

Administrative Data 

Data waiving 

Justification for 

data-lvlng 

olher justification 

Waiving according 1o "column 2" of Regulation (EC) No 190712006 (CSA does not indicale need for lurlher investigations). Dua IX> lhe very high molecular weight 

(>8000 Dafton) lhe bioavallability of lhe subslence Is negligible (low potential to cross biological membranes) and no biodegradation could be expected. The<efore 

additional testing is not justified. 
5.3 Bioaccumulation 

5.3.1 Bioaccumulation: aquatic I sediment 

Endpoint study record: Waiving_ Bioaccumulation: aquatic I sediment 

UUID IUC5-e9a0ae0f-a42c-47cd-9824-47c1186fcfdf 

Dossier UUID IUC5-292149d 1-a677 -4feb-9892-1fad973281 ea 

Author 

Data 

Remart<s 

uensenba I Clarianl Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-21 14:52:55 CEST 

Administrative Data 

Dllbl waiving other justification 

Justification for Waiving according to "column 2" of Regulation (EC) No 190712006 (CSA does not indicate need for further Investigations). Due to the very high molecular weight 

data waiving (>8000 DaHon) the bioavailabiiHy of the substance Is negligible (low potential to cross biological membranes) and no bioaccumulation could be expected. 

Therefore additional testing Is not justified 
5.4 Transport and distribution 

5.4.1 Adsorption I desorption 

Endpoint study record: Waiving_ Adsorption I desorption 

UUID IUC5-25c2af70-1 080-4bd3-b58a-5d5ada3ccbf7 

Dossier UUID IUC5-2921 49d 1 -a677 -4feb-9692-1fad97328 1 ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-21 14:58:55 CEST 

Administrative Data 

Data waiving 

Justlflc:ation for data 

waiving 

sludy technically not feasible 

Due to the physico-<:hemical properties of the UVCB substance no analytical method is available. Aocording to Reach Regulation Annex XI, section 2 the 

sludy is not required. 
6 Ecotoxicological Information 

Endpoint summary: Ecotoxicologicallnformation 

UUID IUC5-3ed53458-0d75-4f7 e-bco4-aedd3d11421d 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunusl Germany 

2010-05-21 14:28:45 CEST 

Administrative Data 

PNEC aqua (freshwater) 

Justification for (no} PNEC freshwater derivation 

Derivation of PNEC is not possible. Justification: The substance was tested in concentrations in the range of its water solubility. No effects were observed in all test systems. Due to the 

high molecular weight, bloavailability of the test substance is negligible. Consequently, no LOEC, NOEC, or ECx- value can be calculeted. Since this data is a prerequisite to estimate a 

PNEC HIs not possible to derive this value. 

PNEC aqua (marine water) 

Justification for (no} PNEC marine water derivation 

Derivetion of PNEC is not possible. Justification: The substance was tested in concentrations in the range of Hs water solubility. No effects were observed in all test systems. Due to the 

high molecular weight, bloavallablHty of the test substance is negligible. Consequently, no LOEC, NOEC, or ECx- value can be calculated. Since this data is a prerequisite to estimate a 

PNEC it is not possible to derive this velue. 

PNEC aqua (intermittent releases) 

Justification for (no} PNEC Intermittent releases derivation 

Derivation of PNEC is not possible. Justification: The substance was tested In concentrations in the range of its water solubility. No effects were observed in all test systems. Due to the 

high molecular waight, bioavailabllity of the test substance is negligible. Consequentiy, no LOEC, NOEC, or ECx- value can be calculated. Since this data is a prerequisHe to estimate a 

PNEC it is not possible to derive this value. 

PNEC STP 
Justification for (no) PNEC STP derivation 

Derivation of PNEC is not possible. Justification: The substance was tested in concentrations in the range of hs water solubility. No effects were observed in aH test systems. Due to the 

high molecular weight, bloevailability of the test substance is negligible. Consequentiy, no LOEC, NOEC, or ECx- value can be calculated. Since this data is a prerequisite to estimate a 

PNEC His not possible to derive this value. 

PNEC sediment (freshwater) 

Justification for (no) PNEC sediment derivation 

Derivation of PNEC is not possible. JusHfication: The substance was tested in concentrations in the range of Hs water solubility in aquatic organisms. No effects were observed in all test 

systems. Due to the high molecular weight, bioavallabllity of the test substance is negligible and effects in other organisms could also not be expeded. Consequenity, no LOEC, NOEC, 

or ECx -value can be calculated. Since this data is a prerequisHe to estimate a PNEC it Is not possible to derive this value. 

PNEC sediment (marine water) 

Justification for (no) PNEC marine sediment derivation 

Derivation of PNEC is not possible. JusHfication: The substance was tested in ooncantrations in the range of its water solubility in aquatic organisms. No effects were observed in aH test 

systems. Due to the high molecular weight, bloevailability of the test substance Is negligible and effects in other organisms could also not be expeded. Consequenlty, no LOEC. NOEC. 

or ECx- value can be calculated. Since this data is a prerequisHe to estimate a PNEC His not possible to derive this value. 

PNEC soil 

Justification for (no) PNEC soli derivation 

Derivation of PNEC is not possible. JustifiC8tion: The substance was tested in concentrations in the range of its water solubility in aquatic organisms. No effects were observed in an test 

systems. Due to the high molecular weight, bioavallablllty of the test substance is negligible and effects in other organisms could also not be expected. Consequenlty, no LOEC, NOEC, 

or ECx- value can be calculated. Since this data is a prerequisHe to estimate a PNEC H Is not possible to derive this value. 

PNEC oral 

Justification for (no) PNEC oral derivation 

Derivation of PNEC is not possible. JusHfication: The substance was tested in ooncantrations in the range of hs water solubility in aquatic organisms. No effects were observed in an test 

systems. Due to the high molecular weight, bioavailability of the test substance is negligible and effects in other organisms could also not be expected. Consequenlty, no LOEC, NOEC, 

or ECx -value can be calculated. Since this data is a prensquisHe to estimate a PNEC it Is not possible to derive this value. 

Conclusion on classification 

Environmental classification justification 

All acute tests showed no effects and the substance has no potential forbioaccumulation. Thereforethe information is 

conclusive but not sufficient for classification. 
6.1 Aquatic toxicity 

Endpoint summary: Aquatic toxicity 

UUID IUC5-da01>46d9-4779-455a-607o-89c9c27392b2 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Author uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

Date 2010-05-11 17:25:39 CEST 

Remarks 

Administrative Data 

Discussion 

Alga 

The influence of DIRESUL BLACK ROT LIQUID on the growth of the green algae Scenedesmus subspicatus was 

investigated. The study was performed according to the EEC Directive 92/69. The test method was modified to 

differentiate between a reduced growth of algae due to real toxic effects of the test substance on the algae cells or due to 

an indirect effect. a reduced algae growth by light absorption in coloured test solutions. 

The test included two experimental parts: Experimental part A: the algae grew in test medium with dissolved dyestuff in 

Erlenmeyer flasks. Experimental part B: the algae grew in separate containers (test tube) which were surrounded by the 

black coloured solutions of the same concentration as tested in part A. 

Because of difficulties concerning the solubility of DIRESUL BLACK ROT LIQUID in different test media, several tests were 

performed. Finally, in experimental part A the following nominal concentration range was tested: 0.01 mg/1 to 200 mg/1. In 

experimental part B, the nominal concentration range of 0.01 mg/1 to 50 mg/1 was tested. 
The biological results of both experimental parts A and B were almost identical. This was shown by several calculated 

parameters, the NOEC, LOEC and the EC 50 values: 

In part A the NOEC was 1.0 mg/1, and in part B the NOEC was found to be between 1 and 5 mg/1. The LOEC was found to 

be between 1 and 5 mg/1 in part A and it was about 5 mg/1 in part B. 

The percentage inhibition of algae biomass and the algae growth rate (IJ) after 72 hours of exposure were in the same 

order of magnitude. The EC 50 values calculated for both growth parameters, showed no significant differences. 

Experimenta part A EbC 50: 

Experimental Part B 

1.84 mg/1 

5.99 mg/1 

Ere 50: 54.1 mg/1 

8.59 mg/1 

Thus, the growth inhibition of Scenedesmus subspicatug was in the same magnitude when the algae grew in test medium 

without test substance but under reduced light intensities by the filter effect (experimental part B) as in experimental part A, 

where the algae grew in test solutions without dissolved test substance. In conclusion, this modified algae test clearly 

demonstrated that the observed inhibition effect of the substance was caused only by the indirect effect, the light 

absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algae cells can be excluded up to 

a concentration of 50 mg/1. 

Fish, acute 

The acute toxicity of the test item Leuco Sulfur Black 1 to zebra fish (Brachydanio rerio) was determined in a 96-hour semistatic test with a daily test medium renewal according to the Commission Regulation (EC) No 440/2008, Part C.1 and the 

OECD Guideline for Testing of Chemicals No. 203 (1992). 

A limit test with a daily test medium renewal was performed in accordance with the test guidelines to demonstrate that the 

test item has no toxic effect on the test fish up to the loading rate of 1 00 mg/L. 
Daphnia, acute 

The acute toxicity of the test item Leuco Sulfur Black 1 to Daphnia magna was determined in a 48-hour semi-static test 

according to the EU Commission Directive 92/69/EEC, Part C.2, Commission Regulation (EC) No 440/2008, Part C.2 and 

the OECD Guideline for Testing of Chemicals, No. 202 (2004). 

A limit test with a test medium renewal after 24 hours was performed in accordance with the test guidelines to demonstrate 

that the test item has no toxic effect on the test organisms up to the solubility limit of the test item in test water. 

Due to the low water solubility of the test item, the test medium was prepared prior to the start of the test and prior to the 

test medium renewal as follows: a dispersion of the test item with the loading rate of 100 mg/L was ultrasonic treated for 15 

minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The 

undiluted filtrate of the dispersion was used as the test medium. Additionally, a control was tested in parallel. The 

preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult 

Substa Thences and Mixtures, 2000. 

No remarkable observations were made concerning the appearance of the test medium. It was clear throughout the entire 

test duration. 

No analytical work was performed due to the specific properties of the test item (insolubility in water). 

At the beginning and end of the test medium renewal periods, the pH values of the test medium were between 7.6 and 8.0. 

The dissolved oxygen concentrations in the test medium and control were at least 7.9 mgll. 

The water temperature was 20 'C during the test. 

The biological results were based on the loading rate of the test item of 100 mg/L. 
In the control and at the loading rate of 100 mg/L no immobilized daphnids were determined during the test period of 48 

hours. 

The biological test results (based on the loading rate) were as follows: 

Loading rate (mg/L) 

- 24-hour EC50: >100 

- 24-hour ECO: :;:::, 100 

- 24-hour EC100: >100 

-48 -hour EC50: >100 

- 48 -hour ECO: :;:::, 100 

- 48 -hour NOEC: :;:::, 100 

-48 -hour EC100: >100 

In conclusion, the test item Leuco Sulfur Black 1 had no acute toxic effects on Daphnia magna up to its solubility limit in the 

test water under the present test conditions. 

Toxicity to bacteria 

The inhibitory effect of the test item Leuco Sulfur Black 1 on the respiration rate of aerobic wastewater microorganisms of 

activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 

88/302/EEC, Part C.11, Commission Regulation (EC) No. 440/2008, Part C.11 and the OECD Guideline for Testing of 
Chemicals, No. 209. 

Based on the EU testing guidelines, a limit test with a nominal test concentration of 1000 mg/L was performed. 

In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16, and 50 mg/L) 

were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used. 

At the test item concentration of 1000 mg/L, the test item was not completely dissolved in the test medium. Thus, the test 

concentration was clearly above the water solubility limit of Leuco Sulfur Black 1 under the test conditions. 

The test item had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at 

a loading rate of 1000 mg/L. The saturation concentration (water solubility limit of Leuco Sulfur Black 1 under the test 

conditions) was reached and no inhibitory effect was determined after three hours contact time. 

Thus, the 3-hour NOEC (EC15) of the test item to activated sludge microorganisms was at least 1000 mg/L (i.e. the 

saturation concentration of the test item). This value might even be higher but concentrations above 1 000 mg/L were not 

tested. The 3-hour EC20, ECSO, and EC80 could not be calculated but were clearly higher than 1000 mg/L. Page 
6.1.1 Short-term toxicity to fish 

Endpoint study record: Peither (2009) I Short-term toxicity to fish 

UUID IUC5-90518ea2-2e99-4c52-b 767 -43cb6e3332d9 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-11ed973281 ea 

Author 

Date 

Remarks 

jregano I Clariantlberlca Producci6n S.A. I El Prat de Llobregat (Barcelona) I Spain 

2010-05-20 16:33:12 CEST 

Administrative Data 

Pu~ellag key study 

Study result typa 

Reliability 

experimental result Study period 2009-05-0911112009-05-15 

1 (reliable without restriction) 

Rationale for reliability Incl. deficiencies Guideline conform study under GLP without deviations 

Data source 

Reference 

Reference Author Year 

type 

study report Peither 2009 

A 

Data access 

data submitter Is data owner 

Data protection claimed 

yes 

Title 

Acute Toxicity of Leuco Sulfur Black 1 to Zebra 

Fish (Brachydanio rerlo) in a 96-Hour Test 

Materials and methods 

Test guideline 

Blbllognphlc 

aource Testing laboratory 

Harlan Laboralories Ltd. 

ltingan (Switzerland) 

Report Owner Company Report 

no. company atudy no. 

C31144 Clariant 

07 

2009-08- -
Qu .. lfler Guideline 

according EU Method C.1 (Acute Toxicity for Fish) (Convnission Regulation (EC) No 44012008 of 30 May 20081aying down test methods pursuant to Regulation (EC) 

to No 1907/2006 of the European Parliament and of tha Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), C.1 :) 

according other guideline: EU Commission Directive 92169/EEC, C.1: Acute Toxicity for Fish, 1992. 

to 

according OECD Guideline 203 (Fish, Acute T oxiclty Test) (OECD Guideline for the Testing of Chemicals, No. 203, Fish, Acute Toxiclty Test, 1992.) 

to 

GLP compliance 

yes (ind. certificate) 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read-across) 

yes 

Test material Identity 

Identifier Identity 

CAS number 66241-11-0 

ECnumber 266-273-5 

Details on test material 

- Name of test material : Leuco Sulfur Black 1 

-Physical stele: thlxotrophic, black, -t paste 

-Composition: Leuco Sulfur Black 1:52.75% (w/w) (calculated by amount of by-<:c>mpounds) 

2,4-Dinitrochlorobenzane: < 20 ppm 

2,4-Dinitrophenole: < 20 ppm 

SuWate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (w/w) 

Water: 45.75 % (w/w) by Karl-Fischer titration 

- Lot/batch No.: ESA0007595 

-Expiration date oftha lot/batch: 2011-07-11 

- Stability under test conditions: Stable under storage conditions. 

- Storage condition of test material: AI room tempsrature (ranga of 20 ± 5 'C), light protected. 

Analytical monitoring 

Devt.tlons 

no 

no 

no 
no 

Details on sampling 

No analytical work was performed due to the specific properties of the test item (insotubiity in water). 

Details on analytical methods 

No analytical work was performed due to the specific properties of the test item (insotubiHty in water). 

Vehicle 

no 

Details on test solutions 

Due to the low water solubility of the test Item. the test medium was prepared prior to the start of the test and prior to the test medium renewals as follows: a dispersion of the test item 

with the loedlng rate of 100 mgil was continuously stirred at room temperature in the derl< over 96 hours. Then, the dispersion was fiKered. The undiluted fiKrete with the maximum 

concentration of dissolved test ~em was used as the test medium. The preparation of the test medium was besed on the OECD Guidance Document on Aquatic Toxicity Testing of 

Difficutt Substances and Mixtures, 2000. 

Test organisms 

Test organisms (species) 

Brechydanio rerio (new name: Denio reno) 

Details on test organisms 

TEST ORGANISM 

- Common name: zebre fish (Brachydanio rerio) 

- Source: breeding cuKure at Hartan Laboratories 

-Length at study inHietion (length definHion, mean, range and SO): 10 fish were measured at the start of the test. The mean body length of the fish was 2.6 :t 0.05 em (Mean :t SO), 

-Weight at study initiation (mean and range, SO): 10 fish were measured at the start of the test. The mean body wet weight was 0.11 :t 0.02 g (Mean :t SO). 

- Feeding during test: not feed during test 

ACCLIMATION 

- Acclimation period: ona week 

- AccHmation condntons (same as test or not): same as test (test water and temperature) 

- Type and amount of food, Feeding frequency: During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (Tetra Min® 

Hauptlutter, supplied by TETRA-Werl<e, 49304 Melle I Germany) 

- Heatth during acciimation (any mortaiHy obsarved):During the last two weeks prior to the test, no fish died in the test fish belch and all fish were heatthy 

- No medication was applied during holding and 

accilmatization. 
Study design 

Test type 

semi-static 

Water media type 

freshwater 

Limit test 

yes 

Total exposure duration 

96h 

Post exposure observation period 

none 

Test conditions 

Hardness 

1.25 mmoUL resp. 125 mg/L as CaC03 

Test temperature 

The water temperature was 20-22 'C during the test 

pH 

The pH values in the test medium and the control ranged from 7.5 to 7. 7 

Dissolved oxygen 

The oxygen concentration was always 8.1 mgll or higher, and thus higher than 60% oxygen saturation. 

Salinity 

see table In section "Details on test condnions" 

Nominal and measured concentrations 

Nominal: 100 mg/L 
Details on test conditions 

TEST SYSTEM 

-Test vesset:One glassiest vessel with 51Hers of test medium was used for each treatment. 

-Aeration: The test vessels were slightly aerated during the test period. 

- Renewal rate of test solution (frequency/flow rate): dally renewal 

- No. of organisms per vessel: 7 

- No. of vessels per oonoentretion (replicates): 1 

-No. of vessels per control (replicates): 1 

-Biomass loading rate: 0.16g fish wet weight per Iller test medium 

TEST MEDIUM I WATER PARAMETERS 

- Source/preparation of dilution water: reconstituted test water 

Reconstituted Test Water 

Ingredients I Properties Conosntration 

mmoVlmg/L 

CaCI2 • 2H20 1.0 147 

MgS04 • 7H20 0.25 61.5 

NaHC03 0.38 32.5 

KCI 0.038 2.9 

Water Hardness 1.25 125 as CaC03 

Alkalintly 0.4 -

OTHER TEST CONDITIONS 

-Photoperiod, Light intensity: A 16-hour light to 8-hour dar!< photoperiod, with a 30-mlnute transition period was used (light intensity during the light period was approximately wHhin the 

range of 100 to 600 Lux). 

EFFECT PARAMETERS MEASURED (with observation intervals if appliosble): Mortaltly and VIsible AbnormaiHies, at 3, 24, 48. 72 and 96 hours 

TEST CONCENTRATIONS 

- Spadng fador for test conosntrations: limH test 1 OOmg/L nominal conoantrallon 

- Range finding study: The selection of the test conosntration was based on the resuhs of a range-finding test (non~LP). 

Reference substance (positive control) 

no 
Any other Information on materials and methods Incl. tables 

A semi-static test with daily test medium renewal was performed to keep the test item concentrations in the test media as 

constant as possible during the test period of 96 hours. During this semi-static test, the test fish were daily placed into a 

new test vessel with freshly prepared test medium. 

The preparation of the test medium was performed as far as possible in the dark to avoid photolytic degradation of the test 

item. 

At the start of the test and before each test medium renewal, the test medium with the loading rate of 100 mg/L was freshly 

prepared by completely dissolving 499.98, 499.91, 500.1 and 500.0 mg of the test item in 5 liter of test water, respectively 

using ultrasonic treatment for 15 minutes and stirring for 96 hours at room temperature in the dark. The long stirring period 

was chosen in consultation with the sponsor. 

The test medium was freshly prepared just before start of the test and prior to each test medium renewal. 

Results and discussions 

Effect concentrations 

Dumlon Endpoint Etr.c:tconc. Nominal/Measured Cone. based on Basis for effect Remarks (e.g. 95% CL) 

96h LCO >= 100mgll nominal test mat. mortality 

96h LC50 > 100 mgll nominal test mat. mortality 

96h LC100 > 100 mgll nominal test mat. mortaUty 

96h NOEC >= 100 mgll nominal test mat. mortaUty 

96h LOEC > 100 mgll nominal test mat. mortaUty 

Details on results 

Behavioural abnormalities: 

In the control and In the test medium with the loading rate of 100 mgll no visible abnormalities were observed at the test fish. 
Results with reference substance (positive control) 

no reference substance tested 

Reported statistics and error estimates 

no statistical analysis 

Any other lnfonnatlon on results Incl. tables 

Mort alitv and Visible Abnormalities Observed at the Test 

Loading rate Number of abnormal and dead fish I number of dead fish Type of visible 

abnormalities 

Observation time 

(mgiL) 3 hours 24 hours 48 hours 

Control 010 0/0 0/0 

100 0/0 0/0 0/0 

LC50 > 100 mg/L based on the loading rate 

FishNumber of fish tested at the s1ngle concentration and the control: 7 

Measurements I Observations 

Loading Exposure time 

rate Oh 24 h 48 h 

72 hours 

0/0 

0/0 

new old new old new old 

Appearance 100 0 0 0 0 0 0 

(1) 

pH (2) Control 7.6 7.7 7.6 7.6 7.6 7.5 

100 7.6 7.7 7.6 7.6 7.6 7.5 

Oxygen (3) Control 8.6 8.8 8.9 8.7 8.7 8.7 

100 8.1 8.8 8.3 8.8 8.5 8.6 

Temperature (4) Control 21 21 21 20 21 22 

100 21 21 21 20 21 22 

(1) Appearance of the Freshly Prepared and Old Test Med1um 

96 hours 

0/0 

0/0 

72 h 

new 

0 

7.6 

7.6 

8.5 

8.6 

21 

21 

96h 

old 

0 

7.5 

7.6 

8.6 

8.5 

22 

21 
(2) pH in the Freshly Prepared and Old Test Medium and in the Control 

(3) Oxygen Concentrations (mg/L) in the Freshly Prepared and Old Test Medium and in the Control 

(4) Temperatures ('C) in the Freshly Prepared and 0 ld Test Medium and in the Control 

Applicant's summary and conclusion 

Validity criteria fulfilled 

yes 

Conclusions 

In oonclusion, lhe test item had no acute toxic effects on the test fish up to lhe loading rate of 

100mg/L. 

Executive summary 

The acute toxicity ofthe test item Leuco Sulfur Black 1 to zebra fish (Brachydanio rerio) was determined in a 96-hour semistatic test with a daily test medium renewal according to the Commission Regulation (EC) No 440/2008, Part C. 1 and the 

OECD Guideline for Testing of Chemicals No. 203 (1992). 

A limit test with a daily test medium renewal was performed in accordance with the test guidelines to demonstrate that the 

test item has no toxic effect on the test fish up to the loading rate of 1 00 mg/L. 

Due to the low water solubility of the test item, the test medium was prepared prior to the start of the test and prior to the 

test medium renewals as follows: a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at 

room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the maximum 

concentration of dissolved test item was used as the test medium. Additionally, a control was tested in parallel. The 

preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult 

Substances and Mixtures, 2000. 

No analytical work was performed due to the specific properties of the test item (insolubility in water). 

The biological results were based on the loading rate of the test item. 
In the control and at the loading rate of the test item of 100 mg/L no mortality or other visible abnormalities were 

determined during the test period of 96 hours. 

The pH values in the test medium and the control ranged from 7.5 to 7.7. 

The oxygen concentration was always 8.1 mg/L or higher, and thus higher than 60% oxygen saturation. 

The water temperature was 20-22 'C during the test. 

The summary of the biological results is as follows: 

Loading rate (mg/L) 

- 96-hour LC50: >100 

- 96-hour LCO: ~ 100 

- 96-hour LC100: >100 

- 96-hour NOEC: ~ 1 00 

- 96-hour LOEC: >100 

In conclusion, the test item had no acute toxic effects on the test fish up to the loading rate of 100 mg/L. 
6.1.2 Long-term toxicity to fish 

Endpoint study record: Waiving_ Long-term toxicity to fish 

UUID IUC5-2398d7e 1-69ae-45c7 -93e8-1 e4440ee91 af 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1 fad973281 ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach em Taunus I Germany 

2010-05-21 14:33:09 CEST 

Administrative Data 

Datew•lvlng 

Justlfle~~tlon for 

-waiving 

olher justification 

Walving according to "column 2" of Regulation (EC) No 1007/2006 (CSA does not indicale need lor further Investigations). Due lo lhe very high molecular weight 

(>8000 Dafton) lhe bloevailability of the subslance is negligible (low polantial to cross biological membranes) and no loxic effects could be expected. Therefore 

additional testing Is nol justified 
6.1.3 Short-term toxicity to aquatic invertebrates 

Endpoint study record: Peither (2009) I Short-term toxicity to aquatic invertebrates, Daphnia 

magna 

UUID IUC5-4b26e921-<:de1-40b6-9968-b814ba3ee 187 

Dossier UUID IUC5-292149d1-a677 -41eb-9692-11ad973281 ea 

Author 

Da1e 

Remarks 

jregano 1 Clariantlbenca Producci6n S.A. I El Prat de Llobregat (Barcelona) I Spain 

2010-05-20 16:33:36 CEST 

Administrative Data 

Purpoee flag key study 

Study !HUll type 

Reliability 

experimental result Study period 2009-05-08 ti112009-05-14 

1 (reliable without restriction) 

Ra1ionale lor reliability Inc'- deficiencies Guideline conlonn study under GLP without deviations 

Data source 

Reference 

Reference Author 

type 

study report Pelther 

A 

Data access 

Yur 

2009 

data submitter is data owner 

Data protection claimed 

yes 

Title Bibliographic 

source 

Acute Toxicity of Leuco SuWur Black 1 to 

Daphnia magna in a 48-Hour Immobilization 

Test 

Testlng laboretory 

Ha~an Labora1ories Ltd. 

Hingen (Swilzeriand) 

Report Owner Company Report 

no. company study no. 

C31155 Clariant 

07 

2009-08- -
Materials and methods 

Test guideline 

Qualifier Guideline 

according EU Method C.2 (Acute Toxicity for Daphnia) (Commission Regulallon (EC) No 44012008 of 30 May 20081aying down test methods pursuant to Reguiallon 

to (EC) No 1907/2006 of the European Pa~iament and of the Council on the Registration, Evaluation, Authorisallon and Restriction of Chemicals (REACH), 

C.2,) 

according EU Method C.2 (Acute Toxicity for Daphnia) (EU Commission Directiva 92/69/EEC, C.2, Acute Toxicity for Daphnia, 1992.) 

to 

according OECD Guideline 202 (Daphnia sp. Acute immobllisation Test) (DECO Guideline for Testing of Chemicals, No. 202, Daphnia sp., Acute lmmobiUsatlon Test, 

to 2004.) 

GLP compliance 

yes (Ind. certificate) 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read-across) 

yes 

Test material Identity 

Identifier Identity 

CAS number 66241-11-0 

EC number 266-273-5 

Details on test material 

- Name of test meteriai : Lauco Sullur Black 1 

-Physical state: thixotrophic, black, wet paste 

• CornposHion: Leuco Sulfur Black 1:52.75% (w/w) (ceicuiated by amount of by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-0initrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3 % (w/w) 

Water. 45.75% (w/w) by Ka~-Fischer titration 

- Lot/batch No.: ESA0007595 

Deviations 

no 

no 

no 
- Expiration date of the lot/batch: 2011-07-11 

- StabUity under test conditions: Stable under storage conditions. 

-Storage condition of test material: At room temperature (range of 20 ± 5 'C), light protected. 

Analytical monitoring 

no 

Details on sampling 

No analytical work was perfonnad due to the specific properties of the test item (insolubility in water). 

Details on analytical methods 

No analytical work was performed due to the specific properties of the test item (insolubility in 

water). 

Vehicle 

no 

Details on test solutions 

Due to the low water solubility of the test ~em. the test medium was prepared prior to the start of the test and prior to the test medium renewals as folows: a dispersion of the test Item 

~the loading rete of 100 mg/L was continuously stinred at room temperature in the dark over 96 hours. Then, the dispersion was fi~ered. The undiluted fiHrate ~the maximum 

concentration of dissolved test ~em was used as the test medium. The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of 

Dlfficutt Substances and Mixtures, 2000. 

Test organisms 

Test organisms (species) 

Daphnia magna 

Details on test organisms 

TEST ORGANISM 

- Common name: Daphnia magna 

Tha study was perfonnad with young daphnlds of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the 

University of Sheffield I UK in 1992. Since that time, the clone has baen bred at Harlan Laboratories in reconst~uted water of the quality idantical to the water quality used In the tests (in 

respect to pH, main ions. and total hardness) and under temperature and light conditions identical to those of the tests. 

At the start of the tes~ the organisms used In the test were 6-24 hours old and were not first brood progeny. 

Tth asphnlds were not fed during the test 

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium 

dichromate is tested as a positive control twice a year. The resutt of the latest positive control test 

In Februar 2009 (46-hour ECSO: 0.71 mg/L, study C36915) Indicated that the sensttivity of the 
test organisms was within the internal historical range (48-hour EC50 from 1 996 to 2009: 

0.53-1.1 mg/l). 

Study design 

Test type 

semi.static 

Water media type 

freshwater 

Limit test 

yes 

Total exposure duration 

48h 

Post exposure observation period 

none 

Test conditions 

Hardness 

2.5 mrnoi/L rasp. 250mg/L as CsC03 

Test temperature 

Water temperature: 20'C 

pH 

7.6 - 8.0 (In test medium and control) 

Dissolved oxygen 

7.9-8.3 mg/l (in test medium and control) 

Salinity 

sae table in saction "Details on test conditions• 
Nominal and measured concenlnltlons 

nominal 1 OOmg/L 

Details on test conditions 

TEST SYSTEM 

-Test vessel: The test was perfonned In 100-ml glass beakers filled with 50 ml of lest medium. The test vessels ware covered with glass plates to reduce the loss of water by 

evaporation and to avoid the entry of dust Into the solutions. 

-Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. During the lest period, the test water was not aerated. 

- Renewal rate of test solution (fraquencylflow rate): daily ren-al 

- No. of organisms per vessel: 20 

-No. of vessels per concentration (replicates): 

- No. of vessels per control (replicates): 1 

TEST MEDIUM I WATER PARAMETERS 

- Source/preparation of dilution water: reconstitutes test water 

Reconstituted Test Water 

Ingredients I Properties Concentration 

mmolll mg/L 

CaCI2 • 2H20 2.0 294 

MgS04 • 7H20 0.5 123 

NaHC03 0.75 65 

KCI 0.075 5.8 

Water Hardness 2.5 250 as CaC03 

Alkalinity 0.8-

The ratio of Ca:Mg and Na:K was 4:1 and 10:1, raspectively, based on molarily 

OTHER TEST CONDITIONS 

A 16-hour light to 8-hour dark cycle with a 3Q.minute transHion period was used. Light Intensity during the light period was approximately be-n 520 and 680 Lux. 

EFFECT PARAMETERS MEASURED (with observation intervals W applicable): The daphnids were observed for immobility after 24 and 48 hours of exposure (daphnids not being able 

to swim within 15 seconds after gentle agHation of the test beaker are considered to be Immobilized). 

TEST CONCENTRATIONS 

-Spacing factor for test concentrations: limit test, 100mg/L nominal 

- Range finding study: The limit test was besed on the results of a rangefinding test (non-GLP). 

Reference substance (positive control) 

yes (potassium dichromate, sae •results with reference substance) 
Any other information on materials and methods incl. tables 

Study Design 

A semi-static test with test medium renewal after 24 hours was performed to keep the concentration of the test item in the 

test medium as constant as possible during the test period of 48 hours. After 24 hours, the test organisms were placed in 

clean test vessels with freshly prepared test medium of the corresponding concentration. 

Due to the low water solubility of the test item, dispersions with the loading rate of 100 mg/L were prepared before start of 

the test and before the renewal of the test medium by dispersing 100.3 and 100.0 mg of the test item in 1000 ml of test 

water, respectively using ultrasonic treatment for 15 minutes and intense stirring. No auxiliary solvent or emulsifier was 

used. 

The dispersions were stirred by a magnetic stirrer at room temperature in the dark over 96 hours to dissolve a maximum 

amount of the test item in the respective dispersion. The long stirring period was chosen in consultation with the sponsor. 

After the 96-hour stirring period, the dispersions of the test item were filtered through a membrane filter (Schleicher & 

Schuell, Type NC45, pore size 0.45 IJm). The undiluted filtrates containing the maximum concentration of dissolved test 

item were used as test medium at the start of the test (= addition of daphnids) and at the test medium renewal. 

The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult 

Substances and Mixtures, 2000 

Results and discussions 

Effect concentrations 

Duntlon Endpoint Effec:tconc. Nominal/Measured Cone. based on Baals lor effec:t Remarks (e.g. 95% CL) 

48 h ECO >= 100 mgll nominal last mat. mobility 
48h ECSO > 100 mg/L nominal test mat. mobility 

48h EC100 > 100 mgiL nominal tastmat. mobility 

48h NOEC >= 100 mg/L nominal tastmat. mobility 

Details on results 

• Behavioural abnormalities: none 

• In the control and at the loading rate of 100 mgiL no immobilized daphnids were determined during the last period of 48 hours. 

Results with reference substance 

Reference Substance 

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate Is tested as a positive oontrot twice a year. The resuH of the latest positive 

control test In Februar 2009 (48-hour ECSO: 0. 71 mg/L, study C36915) indicatad that the sensHivity of the test organisms was wHhin the Internal historical range (48-hour ECSO from 

1996to2009: 0.53-1.1 mg/L). 

Reported statistics end error estimates 

no statistical analysis 

Any other Information on results Incl. tables 

Effect of Leuco Sulfur Black 1 on the Mobilltv ofDaDhnla magna 

Loading No. of daphnlds Immobilized daphnlds Immobilized daphnlds 

rate tested after 24 hours after 48 hours 

(mall) 

No. % No. % 

Control 20 0 0 0 0 

100 20 0 0 0 0 

Measurements I Observations 

Loading Exposure time 

rate Oh I 24h I 48h 

new I old I new I old 
Appearance (1) Control 0 0 0 0 

100 0 0 0 0 

pH (2) Control 7.8 7.8 8.0 7.9 

100 7.6 7.6 8.0 8.0 

Oxygen (3) Control 8.3 8.4 7.9 8.0 

100 8.0 8.1 8.0 8.1 

Temperature (4) Control 20 20 20 20 

100 20 20 20 20 

(1) Appearance of the Freshly Prepared and Old Test Medtum 

(2) pH in the Freshly Prepared and Old Test Medium and in the Control 

(3) Oxygen Concentrations (mg/L) in the Freshly Prepared and Old Test Medium and in the Control 

(4) Temperatures ('C) in the Freshly Prepared and 0 ld Test Medium and in the Control 

Applicant's summary and conclusion 

Validity criteria fulfilled 

yes (In lhe control no immobilized daphnids were determined during the test period of 48 hours; Oxygen content >7.9mg/L.) 

Conclusions 

In conclusion, lhe test item Leuco Sulfur Black 1 had no acute toxic effecls on Daphnia magna up to Its solubility limll in lhe test water under lhe present test conditions (loading rate 

1 OOmg/L nominal). 

Executive summary 

The acute toxicity of the test item Leuco Sulfur Black 1 to Daphnia magna was determined in a 48-hour semi-static test 

according to the EU Commission Directive 92/69/EEC, Part C.2, Commission Regulation (EC) No 440/2008, Part C.2 and 

the OECD Guideline for Testing of Chemicals, No. 202 (2004). 

A limit test with a test medium renewal after 24 hours was performed in accordance with the test guidelines to demonstrate 

that the test item has no toxic effect on the test organisms up to the solubility limit of the test item in test water. 

Due to the low water solubility of the test item, the test medium was prepared prior to the start of the test and prior to the 

test medium renewal as follows: a dispersion of the test item with the loading rate of 100 mg/L was ultrasonic treated for 15 
minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The 

undiluted filtrate ofthe dispersion was used as the test medium. Additionally, a control was tested in parallel. The 

preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult 

Substa Thences and Mixtures, 2000. 

No remarkable observations were made concerning the appearance of the test medium. It was clear throughout the entire 

test duration. 

No analytical work was performed due to the specific properties of the test item (insolubility in water). 

At the beginning and end of the test medium renewal periods, the pH values of the test medium were between 7.6 and 8.0. 

The dissolved oxygen concentrations in the test medium and control were at least 7.9 mg/L. 

The water temperature was 20 'C during the test. 

The biological results were based on the loading rate of the test item of 100 mg/L. 

In the control and at the loading rate of 100 mg/L no immobilized daphnids were determined during the test period of 48 

hours. 

The biological test results (based on the loading rate) were as follows: 

Loading rate (mg/L) 

- 24-hour EC50: >100 

- 24-hour ECO: ? 100 
- 24-hour EC100: >100 

-48 -hour EC50: >100 

-48-hourECO: ~100 

-48 -hour NOEC: ~100 

-48 -hour EC100: >100 

In conclusion, the test item Leuco Sulfur Black 1 had no acute toxic effects on Daphnia magna up to its solubility limit in the 

test water under the present test conditions. 
6.1.4 Long-term toxicity to aquatic invertebrates 

Endpoint study record: Waiving_ Long-term toxicity to aquatic invertebrates 

UUID IUC5-fa6ba43d-cc0c-46a4-bdec-3c12f05eafa4 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutsdlland) GmbH I Sulzbach am Taunus I Germany 

2010-{)5-21 14:33:51 CEST 

Administrative Data 

Data w•lvlng 

Justification for 

ut.-lvlng 

other justification 

Waiving according to "column 2" of Regulation (EC) No 1907/2006 (CSA does not indicale need for further investigations). Due to the very high molecular weight 

(>8000 DeHon) the bloevailabilily of the subslence is negligible (low polential to cross biological membranes) and no toxic effects could be expected. Therefore 

addltionallesting is nol juslified 
6.1.5 Toxicity to aquatic algae and cyanobacteria 

Endpoint study record: Key _201_1993_RCC_333977 

UUID IUC5-73675ea2-8fc8-4fc9-a942-2603464c8c95 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

uensenba I Clartant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-t812:12:38 CEST 

Administrative Data 

Purpose flag 

Study result type 

Reliability 

key study 

experimental result 

1 (reliable without restriction) 

Rationale for reliability Incl. deficiencies Study performed according to OECD-Guideline and GLP 

Data source 

Reference 

Re,.,..nce Author 

type 

study report RCC 

Data access 

Year 

1993 

data submitter is data owner 

Data protection claimed 

yes. but wHIIng to share 

Title 

Acute Toxicity of Diresul Black ROT Liquid to 

scenedesmus subspicatus 

Materials and methods 

Test guideline 

Bibliographic 

source 

Testing laboratory 

RCC- Umweltchemie 

AG ltingen 

Report Owner Company Report 

no. company study no. elate 

333977 Clariant 
Guideline 

OECD Guideline 201 (Alga, Growth Inhibition Test) 

Method was modified to differentiate between a reduced growth of algae due to real toxic effects of the test substance or due to an indirect effect, a reduced algae growth by light 

absoprtion in coloured test solutions. 

GLP compliance 

yes (incl. certificate) 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read-across) 

no 

Test material identity 

ldenttfler Identity 

other. Leuco Sulfur Black 1 formulation see confidential details on test material 

Confidential details on test material 

Leuco Sulfur Black 1: 30.2 % Sodium hydroxide: 2.7% 

Sodium sulfide: 3.6 % 

Inorganic sodium salts: 3.9 % water. 59.6% 

Analytical monitoring 

yes 

Details on analytical methods 

Apparatus: Perkin-Elmer Lambda 15 UVIVis spectrophotometer 

Wavelength: 580 nm 

Vehicle 

no 
Test organisms 

Test organisms (species) 

Scenedesmus subspicatus (new neme: Desmodesmus subspicatus) 

Study design 

Test type 

static 

Water media type 

freshwater 

Umlttest 

no 

Total exposure duration 

72h 

Test conditions 

pH 

IV. the start of the tests, the pH ranged from 8.1 to 9.5. 

IV. the end of the tests, the pH ranged from 8.1 to 8.9. 

Nominal and measured concentrllt/ons 

200 mg DIRESUL BLACK ROT LIQUID were dissolved in test medium and filled to a volume of 200 mi. From this stock solution further dilutions in test medium were prepared and the 

respective amount of algae added. The finel nomine! test concentrations were: 50, 5, 1, 0.5, 0.1 and 0.01 mg/1. 

DIRESUL BLACK ROT LIQUID was found to be stable under the test conditions. The concentration in all lasts ranged from 85.5% to 107.7% of the nominal concentration. 

Reference substance (positive control) 

yes (potassium dichromate) 

Results and discussions 

Effect concentrations 
72h ECSO 5.99 mg/L nominal test mat. biomass Experimental Part B 

72h ECSO 1.84 mg/L nominal test mat. biomass Experimental Part A 

72h ECSO 54.1 mg/L nominal test mat. growth rate Experimental Part A 

72h ECSO 8.59 mg/L nominal test mat. growth rate Experimental Part B 

72 h NOEC 1-Smg/L nominal test mat. biomass Experimental Part B 

72 h LOEC ca. 5 mg/L nominal test mat. biomass Experimental Part B 

72 h NOEC 1 mg/L nominal test mat. biomass Experimental Part A 

72 h LOEC 1-5mgll. nominal test mat. biomass Experimental Part A 

Details on results 

The test included two experimental parts: 

Expertmantal pert A: the algae grew In test medium w~h dissolved dyestuff in Erlenmeyer flasks. Experimental pert B: the algae grew in separate containers (test tube) which were surrounded by the black coloured solutions of the same concentration as tested In pert A. 

Applicant's summary and conclusion 

Conclusions 

The growth Inhibition of Scenedesmus subspicatus was In the same magnitude when the algae grew In test medium ~t test substance but under reduced light intensities by the 

filter effect (experimental part B) as in experimental part A, where the algae grew in test solutions -• dissolved lest substance. In conduslon, this modified algae test clearly 

demonstrated that the obsarved lnhi~n elfed of the substence was caused only by the indlrad effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test 

substence on the algae ceHs can be exduded up to a concentration of 50 mg/1. 

Executive summary 

The influence of DIRESUL BLACK ROT LIQUID on the growth of the green algae Scenedesmus subspicatus was 

investigated. The study was performed according to the EEC Directive 92/69. The test method was modified to 

differentiate between a reduced growth of algae due to real toxic effects of the test substance on the algae cells or due to 

an indirect effect, a reduced algae growth by light absorption in coloured test solutions. 

The test included two experimental parts: Experimental part A: the algae grew in test medium with dissolved dyestuff in 

Erlenmeyer flasks. Experimental part B: the algae grew in separate containers (test tube) which were surrounded by the 

black coloured solutions of the same concentration as tested in part A. 
The biological results of both experimental parts A and B were almost identical. This was shown by several calculated 

parameters, the NOEC, LOEC and the EC 50 values: 

In part A the NOEC was 1.0 mg/1, and in part B the NOEC was found to be between 1 and 5 mg/1. The LOEC was found to 

be between 1 and 5 mg/1 in part A and it was about 5 mg/1 in part B. 

The percentage inhibition of algae biomass and the algae growth rate (IJ) after 72 hours of exposure were in the same 

order of magnitude. The EC 50 values calculated for both growth parameters, showed no significant differences. 

Experimental Part A EbC 50: 

Experimental Part B 

1.84 mg/1 

5.99 mg/1 

Er C 50: 54.1 mg/1 

8.59 mg/1 

Thus, the growth inhibition of Scenedesmus subspicatus was in the same magnitude when the algae grew in test medium 

without test substance but under reduced light intensities by the filter effect (experimental part B) as in experimental part A, 

where the algae grew in test solutions without dissolved test substance. In conclusion, this modified algae test clearly 

demonstrated that the observed inhibition effect of the substance was caused only by the indirect effect, the light 

absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algae cells can be excluded up to 

a concentration of 50 mg/1. 
6.1.7 Toxicity to microorganisms 

Endpoint study record: Memmert (2009) I Toxicity to microorganisms 

UUID IUCS-9961 e854-53954e6b-a2b4-31634 7d06ad2 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

jregano I Clariantlbl!rica Producd6n S.A. I El Prat da Llobregat (Barcelona) I Spain 

2010-QS-20 16:34:03 CEST 

Administrative Data 

Purpose flag 

Study result type 

Reliability 

key study 

experimental resu~ 

1 (reliable without restriction) 

Rationale lor reliability Incl. deflclellCiea Guideline conform study under GLP without deviations 

Data source 

Reference 

Re...,..nce Author Year 

type 

study report Memmert 2009 

u 

Data access 

data submitter is data owner 

Data protection claimed 

yes 

Title 

Toxicity of Leuco Sulfur Black 1 to Activated Sludge in a Respiration Inhibition 

Test 

Materials and methods 

Bibliographic 

source 

Teatlnglabonltory Report 

no. 

Harlan Laboratories Ltd. C31166 

llingen (Switzerland) 

Owner Company Report 

company study no. date 

Clarlant 2009-oS19 
Test guideline 

Owl IIIIer Guideline 

according EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test) (Com. Reg. (EC) No 44012008 of 30 May 2008 laying down test methods 

to pwsuant to Reg. (EC) No 1907/2008 of the European Parliament and of the Council on the Reglstra~on. Evaluation, Authorisation and Restriction of 

Chemicals (REACH), Part C.11, Activated S) 

according EU Method C .11 (Biodegradation: Activated Sludge Respiration Inhibition Test) (EU Commission Directive 881302/EEC, Part C.11, Activated Sludga 

to Respiration Inhibition Tast, 1988) 

according OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test) (OECD Guideline for Testing of Chemicals, No. 209, Activated Sludge, Respiration 

to Inhibition Test, 1984.) 

GLP compliance 

yes (incl. cartificate) 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read-.cross) 

yes 

Test material Identity 

ldentlfter Identity 

CASnumbar 66241-t1-0 

ECnumbar 266-273-5 

Details on test material 

- Name of test material : Leuco Sulfur Black 1 

- Physical state: thlxotrophic, black, wet paste 

-Composition: Leuco SuWur Black t :52.75% (w/w) (calculated by amount of by-compounds) 

2,4-0initrochlorobanzene: < 20 ppm 

2,4-Dinltrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3 % (w/w) 

Water: 45.75% (w/w) by Karl-Fischer titration 

- Lot/batch No.: ESA0007595 

-Expiration date of the lot/batch: 2011-07-11 

- Stabiity under test conditions: Stable under storage conditions. 

Deviations 

no 

no 

no 
- Storage condition of test material: AI room lemperature (range of 20 ± 5 'C), light protected. 

Analytical monitoring 

not required 

Details on sampling 

For measurement of the respiration rate a wall-mixed sample of each test medium was poured into a BOO-flask after three hours incubation time, and was not further aerated. 

Details on analytical methods 

For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then the 

dissolved oxygen concentration was measured with an oxygen electrode f"NTW TriOxmatic® 300 and an oxygen meter WTW Oxi 539, Wlssenschaftlich-Technische Wer1<staetten 

WTW, Weilheim/Germany), end was continuously recorded. During measurement, the samples wera continuously stirred on a magnetic stirrer. The oxygen consumplion rale (in mg 02 L-1 minute-1) was delennined from the linear part of the respiration curve in the range 6.5-2.5 mg 02/L. In case of very rapid oxygen consumption, the range used was below the limits 

indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was detennined over a period of at least ten minutes. 

Vehicle 

no 

Details on test solutions 

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for dlfficuK test substances) 

The final nominal concentration tested was 1000 mg/1.. 

Based on the resuils of a ~xparimenl (without GLP), no stock solution could be prapared due to the low water solubility of the test "em under the pre-test cond~ns. 

Therefore, 500.0 mg of the test hem was weighed by means of an analytical balance and transferred to the designated test vessel with 284 mL tap water. 

The test Item was mixed into tha tap water by uKrasonic treatment for fifteen minutes and intense stinring for 24 hours at room temperature in the dari< to dissolve a maximum amount of 

the lest hem and/or disperse " as homogeneously as possible. No emulsifiers or solvents were used. 

After stirring, the appearance of the lest medium was evaluated, and the test concentration was dearly above the water solubility li~ of Leuco Sulfur Black 1 under the test cond"ions. 

Then, 16 mL synthetic sewage feed and 200 mL activated sludge inoculum were added 

Test organisms 

Test organisms (species) 

activaled sludge of a predominantly domestic sewage 

Details on Inoculum 

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARAErgotz II, Fullinsdiorf I Switzerland) treating predominantly domestic wastewater. The 

sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, 
thereafter dried and the ratio of wet to dry weight was calculated. 

Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per l~er. During the holding period of three days 

prior to use, the sludge was fed daily with 50 ml synthetic sewage feed" per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of 

the activated sludge was measured again In the inoculum used for the test. The pH of the activated sludge Inoculum was adjusted from 8. 7 to 7.3 with a diluted sulfuric acid solution. 

• Synthetic sewage feed: 

16g peptone 

11 g meat extract 

3 g urea 0.7 g NaCI 

0.4 g CaCI2 • 2H20 

0.2 g MgS04 • 7H20 

2.8 gK2HP04 

filled up to a final volume of 1 liter with deionized water 

Study design 

Test type 

static 

Water media type 

freshwater 

Limit test 

yes 

Total exposure duration 

3h 

Post exposure observation period 

none 

Test conditions 

Hardness 

not dertermined 

Test temperature 

20'C 
pH 

see table "Remarks on results" 

Dissolved oxygen 

see table *Remarks on results" 

Salinity 

synthetic waste water 

Nominal and measured concentrations 

1000mg/L 

Details on test conditions 

TEST SYSTEM 

- Test vessel: 2000mt glass beakers 

- Aeration: During the Incubation period of 3 hours the single test medium and the controls were rontinuously aarated by Intense st~ring on magnetic stirrers to avoid possible foaming 

and/or stripping of the test Hem. 

- No. of vessels per concentration (replicates): 1 

-No. of vessels per control (replicates): 2 

TEST MEDIUM I WATER PARAMETERS 

-The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergotz II, FOUinsdorf I Swilz-nd) traating predominantly domestic wastewater. The 

sludge was washed twice with tap water by centrifugation and the supernatant liquid phese was decanted. A homogenized aliquot of the final sludge suspension was weighed, 

thereafter dried and the ratio of wet to dry weight was calculated. 

- Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concantration equivalent to 3 g dry material per IHer. During the holding period of three days 

prior to use, the sludge was fed daily with 50 ml synthatic sewage feed• per IHer and was kept at room temperature under rontinuous aaratlon until use. Bafore use, the dry welght of 

the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludige inoculum was edjusted from 8.7 to 7.3 with a diluted sulfuric acid solution. 

• Synthetic sewage feed: 

16 g peptone 

11 g meat extract 

3 g urea 

0.7 g NaCI 

0.4 g CaCI2 • 2H20 

0.2 g MgS04 • 7H20 

2.8 gK2HP04 

filled up to a final volume of 1 liter with deionized water 
EFFECT PARAMETERS MEASURED (with observation intervals Wapplicable): respiration rate 

TEST CONCENTRATIONS 

1 OOOmg/L nominal 

Reference substance (positive control) 

yes (3,5-<llchlorophenol) 

Any other lnfonnatlon on materials and methods Incl. tables 

At the start of the test (after the stirring period of 24 hours), synthetic sewage feed and activated sludge inoculum were 

added. The inoculum had a sludge concentration of 2. 7 g/L dry weight (corresponding to about 1.1 g dry material per liter 

test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, 

secondly to the test solutions of the reference item, thirdly to the test solution of the test item, and finally to the second 

control. 

During the incubation period of 3 hours the single test medium and the controls were continuously aerated by intense 

stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item. The concentration of dissolved 

oxygen did not drop below 2.5 mg/L during the incubation period. Just before measurement of the respiration rates the 

dissolved oxygen concentrations were at least 8.5 mg/L. The temperature in the test media, measured in one control, was 

20 'C at the start and at the end of the incubation period. 

Results and discussions 

Effect concentrations 

Remarks (e.g. 95% CL) 

M. the test Item concentration of 1000 mgll, the test ttem was not completely dissolved In the test medium. Thus, the test concentration was clearly above the water solubility limit of 

Leuco SuWur Black 1 under the test conditions. 

Results with reference substance (positive control) 

The 3-hour EC50 of the reference ~em 3,5-<llchlorophenol (positive control) was calculated to be 18 mgll (the 95% confidence limits could not be calculated due to mathematical 
reasons). The :Hlour EC50 is within the guideline-recommended range of 5-30 mgll, confirming suitability of the activated sludge used. 

Reported statistics and error estimates 

no statistical analysis done 

Any other Information on results Incl. tables 

Influence of Leuco Sulfur Black 1 (Test Item) and 3,5-Dichlorophenol (Reference Item) on the Oxygen 

Consumption of Activated Sludge 

Vessel Nominal Oxygen Inhibition pH Oxygen 

no. concentration consumption values Concentration 

of test rate (mg 02/L) 

chemical 

(mg/L) (mg 02 L-1 (%) start* end* start* end* 

min-1) 

Control 

A 0 1.415 7.5 8.4 7.9 8.5 

B 0 1.423 7.9 8.3 7.9 8.9 

Mean 1.419 

Deviation (%) 0.6 

Test Item 

5 I 1000 1.397 1.6 8.1 8.2 7.9 8.7 

Reference Item 

1 5 1.520 -7.1 7.8 8.2 7.9 8.9 

2 16 0.734 48.3 8.0 8.3 8.0 8.8 

3 50 0.204 85.6 8.0 8.4 8.2 9.2 

• Start and end of the 3-hour mcubatlon penod 

-%inhibition: increased oxygen consumption rate relative to control 

Applicant's summary and conclusion 
Validity criteria fulfilled 

yes 

Conclusions 

The 3-hour NOEC (EC15) of Leuco Sulfur Black 1 to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher but concentrations abova 1000 mg/L 

were not tested. 

Executive summary 

The inhibitory effect of the test item Leuco Sulfur Black 1 on the respiration rate of aerobic wastewater microorganisms of 

activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 

88/302/EEC, Part C.11, Commission Regulation (EC) No. 440/2008, Part C.11 and the OECD Guideline for Testing of 

Chemicals, No. 209. 

Based on the EU testing guidelines, a limit test with a nominal test concentration of 1000 mg/L was performed. 

In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16, and 50 mg/L) 

were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used. 

At the test item concentration of 1000 mgll, the test item was not completely dissolved in the test medium. Thus, the test 

concentration was clearly above the water solubility limit of Leuco Sulfur Black 1 under the test conditions. 

The test item had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at 

a loading rate of 1000 mg/L. The saturation concentration (water solubility limit of Leuco Sulfur Black 1 under the test 

conditions) was reached and no inhibitory effect was determined after three hours contact time. 

Thus, the 3-hour NOEC (EC15) of the test item to activated sludge microorganisms was at least 1000 mg/L (i.e. the 

saturation concentration of the test item). This value might even be higher but concentrations above 1000 mg/L were not 

tested. The 3-hour EC20, EC50, and EC80 could not be calculated but were clearly higher than 1000 mg/L. Page 
6.2 Sediment toxicity 

Endpoint study record: Waiving_ Long term testing Sediment toxicity 

UUID IUC5-2ced649d-4c42-41 Of-a7ce-{; 14d2506104a 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1 fad973281 ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-21 16:20:09 CEST 

Administrative Data 

Data waiving 

Justification for 

data waiving 

other justification 

Waiving according to "column 2" of Regulation (EC) No 190712006 (CSA does not indicate need for further Investigations). Due to the very high molecular weight 

(>8000 Dation) the bloevallabiiHy of the substance is negligible (low potential to cross biological membranes) and no toxic effects could be expected. Therefore 

additional testing is not justified 
6.3 Terrestrial toxicity 

6.3.1 Toxicity to soil macroorganisms except arthropods 

Endpoint study record: Waiving_ Short term toxicity to soil macroorganlsms except 

arthropods 

UUID IUC5-aafbb6a9-8c65-416d-a 161-35e 7ec89e6e1 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author uensenba I Clarianl Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

Date 2010-05-2116:21:59 CEST 

Remarks 

Administrative Data 

D81a waiving other justification 

Justification for Waiving according to "column 2" of Regulation (EC) No 190712006 (CSA does nollndicale need for further investigations). Due lo the very high molecular weight 

data waiving (>6000 DaHon) the bioavailability of the substance is negligible (low potential to cross biological membranes) and no toxic effects could be expected. Therefore 

additional tasting is not justified 
Endpoint study record: Waiving_ Long term toxicity to soil macroorganisms except 

arthropods 

UUID IUC5-19196dal>-9944-4cec-8531-e6234 741 ab34 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author 

Date 

Remartc.s 

uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-{)5-21 16:24:01 CEST 

Administrative Data 

Data waiving 

Juatlflcatlon for 

data waiving 

olher justification 

Waiving accon:ling 1o "column 2' of Regulation (EC) No 190712006 (CSA does nol indicate nead for lurlher Investigations). Due lo lhe very high molecular weighl 

(>8000 DaMon) lhe bioavailabillly of the substance is negligible (low potentiallo cross biological membranes) and no loxic allecls could be expected. Therefore 

addHional testing Is nol justified 
6.3.3 Toxicity to terrestrial plants 

Endpoint study record: Waiving_ Short-term toxicity to terrestrial plants 

UUID IUCS-28691 b63-da93-4d15-a3df-f0fd4fcd43a6 

Dossier UUID IUCS-292149d 1-a677 -4feb-9692-1fad973261 ea 

Author 

Date 

Remarks 

uensenba I Clarlant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-21 16:2627 CEST 

Administrative Data 

Data waiving 

Justification for 

--lYing 

other justification 

Waiving according to "column 2" of Regulation (EC) No 190712006 (CSA does not indicate need for further Investigations). Due to the very high molecular weight 

(>8000 Dalon) the bloevallability of the subBiance Is negligible (low potential to cross biological membranes) end no toxic effects could be expected. Therefore 

additional testing Is noljustifled 
Endpoint study record: Waiving_ Long term toxicity to terrestrial plants 

UUID IUC5-d4fc96dc-5719-4 787 -a3db-18d850f1e51a 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author 

Date 

Remart<s 

uensenba I Clariant Produkte (Deulschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-21 16:27:46 CEST 

Administrative Data 

Data w•lvlng 

JustlliCIIUon for 

data-lvlng 

other justification 

Waiving acx:ording to "column Z' of Regulation (EC) No 1907/2006 (CSA does not indicate need for further investigations). Due to the very high molecular weight 

(>8000 Dakon) the bioavailability of the substance Is negligible (low potential to cross biological membranes) and no toxic effects could be expected. Therefore 

additional testing is not justified 
6.3.4 Toxicity to soil microorganisms 

Endpoint study record: Waiving_ Toxicity to soil microorganisms 

UUID IUC5-a35b509b-8257 -4e91-8b57 -Q88e9fcaf22c 

Dossier UUID IUC5-292149d1-a6n -4feb-9692-1 fad973281 ea 

Author 

Date 

Remarks 

uensenba I Clarlant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-21 16:28:01 CEST 

Administrative Data 

Dahl waiving 

Justification for 

-waiving 

other jusliflcation 

Waiving according to "column 2" of Regulation (EC) No 1907/2006 (CSA does nol indicate need for further Investigations). Due to the very high molecular weight 

(>8000 Dalon) the bloavaolability of the substance is negliglbla (low potential to cross biological membranes) and no toxic effects could be expected. Therefore 

additional testing is not justified 
6.3.5 Toxicity to birds 

Endpoint study record: Waiving_ Toxicity to birds 

UUID IUC5-149aa32c-5909-4821-b6a2-2f1605c38b2f 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-21 16:29:42 CEST 

Administrative Data 

Data waiving 

Justification for 

data waiving 

other justification 

Waiving according to "column 2" of Regulation (EC) No 190712006 (GSA doas not indicate need lor further investigations). Due to the very high molecular weight 

(>8000 Dakon) the bloevailabilky of the substance is negligible (low potential to cross biological membranes) and no toxic effects could be expected. Therefore 

additional testing is not justified 
7 Toxicological information 

Endpoint summary: Toxicological information 

UUID IUCS-1 a26f429-de64-4dfb-b484-e7a7ba13a7e6 

Dossier UUID IUC5-292149d1-a6n -4feb-9692-11ad97328 1 ea 

Author 

Date 

Remarks 

uensenba I Clarlant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-21 15:25:38 CEST 

Administrative Data 

Workers 

Acute I short-term exposure - systemic effects 

Dermal DN(M)EL 

Justification for (no) DN(M)EL derivation I applied assessment factors 

Leuco Sulfur Black 1 is not dassified lor acute systemic effects. Thus, the derivation of the DNEL is not required (see also "discussion"). 

Inhalation DN(M)EL 

Justification for (no) DN(M)EL derivation I applied assessment factors 

Leuco Sulfur Black 1 is not dassified lor acute systemic effects. Thus, the derivation of the DNEL is not required (see also "discussion"). 

Acute I short-term exposure • local effects 

Dermal DN(M)EL 

Justification for (no) DN(M)EL derivation I applied assessment factors 

Leuco Sulfur Black 1 is not dassified lor acute systemic effects. Thus, the derivation of the DNEL Is not required (see also "discussion"). 

Inhalation DN(M)EL 

Justification for (no) DN(M)EL derivation I applied assessment factors 
Leuco SuHur Blacl< 1 is not classified for acute systemic effects. Thus, the derivation of the DNEL is not required (see also "discussion"). 

Long-tenn exposure - systemic effects 

Dermal DN(M)EL 

DNEL (Derived No Effect Level) 25 mglkg bwlday 

Aueument 40 DOH NOAEL Moat repeated dose toxicity 

bctor descriptor Hnsltlve 

starting endpoint 

point 

Justification for (no) DN(M)EL derivation I applied assessment factors 

Inhalation DN(M)EL 

DNEL (Derived No Effect Level) 176 mglm' 

A ... ssment 10 DOH NOAEC Most repeated dose toxicity 

factor descriptor Hnslllve 

starting endpoint 

point 

Justification for (no) DN(M)EL derivation I applied assessment factors 

see •discussion" 

Long-tenn exposure • local effects 

Dermal DN(M)EL 

Justification for (no) DN(M)EL derivation I applied assessment factors 

Leuco Sulfur Blacl< 1 is not classified for acute systemic effects. Thus, the derivation of the DNEL is not required (see also "discussion"). 

Inhalation DN(M)EL 

Justification for (no) DN(M)EL derivation /applied assessment factors 

Leuco SuHur Blacl< 1 is not classified for acute systemic effects. Thus, the derivation of the DNEL is not required (see also "discussion"). 

Discussion 
Acute/short term exposure - systemic and local effects 

Leuco Sulfur Black 1 did not induce any toxic effects in the acute oral and dermal and oral repeated dose toxicity study. It 

did not induce skin irritation in the acute dermal toxicity study in rats, induced only minimal signs of irritation to the rabbit 

eye shortly after instillation without any relevance for classification and was not sensitising in the LLNA. 

As Leu co Sulfur Black 1 did not show acute systemic nor acute local toxicity, the derivation of acute DNELs for systemic 

and local effects is not required. This is in accordance with the ECHA guidance document on information requirements and 

chemical safety assessment, chapter R.8. 

Long-term exposure - systemic effects 

In a repeated dose toxicity study with reproduction/developmental toxicity screening according to OECD guideline 422 and 

GLP Leuco Sulfur Black 1 was administered by gavage to groups of 10 rats per sex and dose at 0, 100, 300 and 1000 

mg/kg bw/d. Male rats were treated for 29 days, female rats for 14 days prior to pairing, though the pairing and gestation 

periods until the F1 generation had reached day 4 postpartum. 

The only effect seen was a dark discolouration of faeces in all high dose animals starting on day 3 of the study and lasting 

until termination. Discolouration of organs or urine or substance deposits in organs were not found. This is strongly 

indicates that systemic absorption did not occur. 

Based on the results obtained in this study a NOAEL of 1000 mg/kg bw/d was derived. 

Leuco Sulfur Black 1 is an UVCB substance with a mean molecular weight in the range of 8000 to 9000 Da and a numberaverage molecular weight (Mn) of about 3000 Da. It is very poor1y or even not soluble in common polar and non-polar 

solvents such as water (solubility of 62.4 mg/L), DMSO, methanol, DMF, THF, chloroform and not soluble in acids like 

sulfuric acid and trifluoroacetic acid. Due to these substance characteristics it is very unlikely that Leuco Sulfur Black 1 can 

cross any biological membrane. 

In summary, no evidence of toxicity was seen in the OECD 422 guideline study. The outcome of the study and the 

substance characteristics show that an absorption of toxicologically significant amounts of Leuco Sulfur Black 1 

subsequent to oral, dermal or inhalation exposure is negligible. 

Nevertheless, a DNEL for the dermal and inhalation exposure route is derived assuming absorption of 5 % for all exposure 
routes. 

Dermal exposure to Leu co Sulfur Black 1 and the formulated Sulfur dye preparation can occur at the workplace. Inhalation 

exposure to Leuco Sulfur Black 1 is not possible due to pasty consistence and can only occur when the substance is 

formulated in aqueous media. 

The NOAEL of 1000 mglkg bw/d from the OECD guideline 422 study in rats is used as starting points for the establishment 

of the DNELs. 

The oral NOAEL reflects also the dermal NOAEL because no difference in absorption is expected between both routes. 

For the derivation of the worker inhalation DNEL (assuming a daily exposure for 8 hours), the oral NOAEL is converted into 

a repeated inhalation NOAEC according to the following formula: 

inhalation NOAEC = 

oral NOAEL * 1/sRV(rat) * ABSoral-raUABSinh-human * sRV(human)JwRV(human) 

with: 

oral NOAEL = 1000 mg/kg bw/d 

sRV(rat) = standard respiratory volume (rat) = 0.38 m3/kg (8 h) 

ABSoral-raUABSinh-human = 1 

sRV(human)/wRV(human) = 6.7 m3/10m3 

Thus, the inhalation NOAEC is 1763 mg/m3 for the worker. 

The following assessment factors are used for the derivation of the long-term systemic DNELs: 

lnterspecies (allometric scaling rat-> human (not to be applied for inhalation exposure): 4 

(no additional factor for remaining differences is needed because of the substance characteristics (see above)) 

lntraspecies: 5 (worker) 

Exposure duration: 2 (subacute to chronic) 

(the factor of 2 instead of 6 is used as there was no effect seen in the OECD guideline 422 study justifying a higher factor) 

Dose-response: 1 

Quality of database: 1 
The resulting overall assessment factors and DNELs are: 

worker (dermal exposure) 

overall assessment factor: 4 x 5 x 2 x 1 x 1 = 40 

DNEL: 1000 mg/kg bw/d /40 = 25 mg/kg bw/d 

worker (inhalation exposure): 

overall assessment factor: 5 x 2 x 1 x 1 = 1 0 

DNEL: 1763 mg/kg bw/d /10 = 176 mg/kg bw/d 

Long-term exposure - local effects 

Leuco Sulfur Black 1 does not induce any local toxic effects. Moreover, no evidence of toxicity was seen in any of the 

available tests. Thus, the derivation of a long-term DNEL for local effects is not required. This is in line with the ECHA 

guidance document on information requirements and chemical safety assessment, chapter R.B. 

General population 

Acute I short-term exposure - systemic effects 

Dermal DN(M)EL 

Justification for (no) DN(M)EL derivation I applied assessment factors 

Leuco Sulfur Black 1 is not classified lor acute systemic effects. Thus. the derivation of the DNEL Is not required (sse also "discussion"). 

Inhalation DN(M)EL 

Justification for (no) DN(M)EL derivation I applied assessment factors 

Leuco Sulfur Black 1 Is not classified for acute systemic effects. Thus, the derivation of the DNEL Is not required (sse also "discussion"). 

Oral DN(M)EL 

Justification for (no) DN(M)EL derivation I applied assessment factors 

Leuco Sulfur Black 1 is not classified for acute systemic effects. Thus. the derivation of the DNEL is not required (sse also "discussion"). 

Acute I short-term exposure -local effects 
Dennal DN(M)EL 

Justification for (no) DN(M)EL derivation I applied assessment factors 

Leuoo Sulfur Black 1 is not dassified for acute systemic effects. Thus, the derivation of the DNEL Is not required (see also "discussion"). 

Inhalation DN(M)EL 

Justlflcation for (no) DN(M)EL derivation I applied assessment factors 

Leuoo Sulfur Black 1 is not dassified for acute systemic effects. Thus, the derivation of the DNEL is not required (see also "discussion"). 

Long-term exposure - systemic effects 

Dennal DN(M)EL 

DNEL (Derived No Effect Level) 12.5 mg/kg bw/day 

AOHaarnent 80 Dose NOAEL Moat repeated dose toxicity 

factor descriptor sensitive 

startlng endpoint 

point 

Justification for (no) DN(M)EL derivation I applied assessment factors 

see "discussion" 

Inhalation DN(M)EL 

DNEL (Derived No Effect Level) 43.5 mglm' 

Assessment 20 Dose NOAEC Moat repeated dose toxicity 

factor descriptor sensitive 

startlng endpoint 

point 

Justification for (no) DN(M)EL derivation I applied assessment factors 

see "discussion" 

Oral DN(M)EL 

DNEL (Derived No Effect Level) 12.5 mglkg bw/day 

AOHaarnent 80 Dose NOAEL Moat repeated dose toxicity 

factor descriptor sensitive 
starting endpoint 

point 

Justification for (no) DN(M)EL derivation I applied assessment factors 

see "discussion" 

Long-tenn exposure • local effects 

Dennal DN(M)EL 

Justification for (no) DN(M)EL derivation I applied assessment factors 

Leuco Sulfur Black 1 is not dasslfied for acute systemic affects. Thus, the derivation of lhe DNEL Is not required (see also 'discussion'). 

Inhalation DN(M)EL 

Justification for (no) DN(M)EL derivation I applied assessment factors 

Leuco Sulfur Black 1 is not dassified for acute systemic effects. Thus, the derivation of the DNEL is not required (see also 'discussion'). 

Discussion 

Acute/short term exposure -systemic and local effects 

Leuco Sulfur Black 1 did not induce any toxic effects in the acute oral and dermal and oral repeated dose toxicity study. It 

did not induce skin irritation in the acute dermal toxicity study in rats, induced only minimal signs of irritation to the rabbit 

eye shortly after instillation without any relevance for classification and was not sensitising in the LLNA. 

As Leuco Sulfur Black 1 did not show acute systemic nor acute local toxicity, the derivation of acute DNELs for systemic 

and local effects is not required. This is in accordance with the ECHA guidance document on information requirements and 

chemical safety assessment, chapter R.8. 

Long-term exposure - systemic effects 

No evidence of toxicity was seen in the OECD 422 guideline study (NOAEL of 1000 mg/kg bw/d). The outcome of the 

study and the substance characteristics show that an absorption of toxicologically significant amounts of 

Leuco Sulfur Black 1 subsequent to oral, dermal or inhalation exposure is negligible. (See also discussion under DNEL 

derivation for the worker.) 
Leucosulfur Black 1 is only used professionally and not included in articles intended to release the substance. 

Nevertheless, a DNEL for the oral, dermal and inhalation exposure route is derived assuming absorption of 5 %for all 

exposure routes. 

The NOAEL of 1000 mglkg bw/d from the OECD guideline 422 study in rats is used as starting points for the establishment 

of the DNELs. 

The oral NOAEL reflects also the dermal NOAEL because no difference in absorption is expected between both routes. 

For the derivation of the consumer inhalation DNEL (assuming a daily exposure for 24 hours), the oral NOAEL is converted 

into a repeated inhalation NOAEC according to the following formula: 

inhalation NOAEC = 

oral NOAEL * 1/sRV(rat) * ABSoral-rat/ABSinh-human 

with: 

oral NOAEL = 1 000 mg/kg bw/d 

sRV(rat) = standard respiratory volume (rat)= 1.15 m3

/kg (24 h) 

ABSoral-rat/ABSinh-human = 1 

Thus, the inhalation NOAEC is 870 mg/m3 for the consumer. 

The following assessment factors are used for the derivation of the long-term systemic DNELs: 

lnterspecies (allometric scaling rat-> human (not to be applied for inhalation exposure): 4 

(no additional factor for remaining differences is needed because of the substance characteristics (see above)) 

lntraspecies: 10 (consumer) 

Exposure duration: 2 (subacute to chronic) 

(the factor of 2 instead of 6 is used as there was no effect seen in the OECD guideline 422 study justifying a higher factor) 

Dose-response: 1 

Quality of database: 1 
The resulting overall assessment factors and DNELs are: 

consumer (oral exposure) 

overall assessment factor: 4 x 1 0 x 2 x 1 x 1 = 80 

DNEL: 1000 mg/kg bw/d /80 = 12.5 mglkg bw/d 

consumer (dermal exposure) 

overall assessment factor: 4 x 1 0 x 2 x 1 x 1 = 80 

DNEL: 1000 mg/kg bw/d /80 = 12.5 mglkg bw/d 

consumer (inhalation exposure): 

overall assessment factor: 1 0 x 2 x 1 x 1 = 20 

DNEL: 870 mg/kg bw/d I 20 = 43.5 mg/m3 

Long-term exposure - local effects 

Leuco Sulfur Black 1 does not induce any local toxic effects. Moreover, no evidence of toxicity was seen in any of the 

available tests. Thus, the derivation of a long-term DNEL for local effects is not required. This is in line with the ECHA 

guidance document on information requirements and chemical safety assessment, chapter R.8. 
7.1 Toxicokinetics. metabolism and distribution 

Endpoint summary: Toxlcokinetics, metabolism and distribution 

UUID IUC5-e019da60·99cd-4350-99ca-82e9faf3be4 7 

Dossier UUID IUC5-292149d 1-asn -4feb-9692-1fad973281ea 

Author lhr I SCC GmbH I Wendelsheim I Germany 

Date 2010-05-2617:11:52 CEST 

Remarl<s 

Administrative Data 

Discussion 

The sulfur dye Leuco Sulfur Black 1 is a black pasty UVCB substance with a mean molecular weight in the range of 8000 

to 9000 Da and a number-average molecular weight (Mn) of about 3000 Da. It is very poorly or even not soluble in 

common polar and non-polar solvents such as water (solubility of 62.4 mg/L), DMSO, methanol, DMF, THF, chloroform 

and not soluble in acids like sulfuric acid and tritluoroacetic acid. The vapour pressure of the pure substance, calculated 

from the boiling point of > 400 'C, is 1.8 x 1 OE-5 hPa at 25 'C. 

Based on these properties it is very unlikely that Leuco Sulfur Black 1 can cross any biological membrane. Thus, 

absorption of toxicologically significant amounts of Leuco Sulfur Black 1 following oral, dermal or inhalation exposure can 

be excluded. This is substantiated by the results obtained in toxicity and ecotoxicity studies, where no substance related 

local and/or systemic effects indicative of toxicity were seen in all tests, induding acute animal and acute ecotoxicity 

studies, sensitization, repeated dose toxicity and mutagenicity tests. 

In the acute oral toxicity study black stained faeces was observed for maximally four days starting the day after 

administration of the substance at 2000 mglkg bw. Dark discoloured faeces were also seen in the repeated dose toxicity 

study (OECD 422) at 1000 mg/kg bw/d, starting on day 3 of the study and lasting throughout the administration period until 

termination. The discolouration of faeces was the only effect observed in both studies and can be attributed to the strong 
staining properties of the substance. This indicates that Leuco Sulfur Black 1 is directly excreted and that absorption in the 

gastrointestinal tract does not occur following oral administration, especially since organs or urine were not discoloured 

and signs of substance deposits were not found in histopathological analysis of the organs. 

Based on the substance properties and as absorption via any route is negligible, metabolism and distribution in the body is 

also negligible. After oral administration Leuco Sulphur Black 1 is excreted via faeces as demonstrated in the acute oral as 

well as repeated dose toxicity study (OECD 422). 
7.2 Acute Toxicity 

Endpoint summary: Acute Toxicity 

UUID IUC5-<:e8d63d8-f0f4-4df6-ab3a-ee2829d2e160 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author lhr I SCC GmbH I Wendelsheim I Germany 

Date 2010-05-26 16:22:28 CEST 

Remarks 

Administrative Data 

Short description of key information 

The LD50 oral and dermal in the rat is above 2000 mg/kg bw. Leuco SuWur Black 1 did neither cause lethality nor clinical signs indicative of toxicity in both arute toxicity studies. 

Discussion 

The acute toxicity of Leuco Sulfur Black 1 by oral application was investigated according to the OECD Guideline 423 under 

GLP. 

Female rats were treated by oral gavage administration at a dosage of 2319 mg active ingredient I kg bw (corresponding to 

4396 mg test item I kg bw). 

No deaths occurred during the study. Except for black stained faeces from test day 2 up to test day 5 at the latest, no 

clinical signs were noted in all animals during the observation time. The body weight of the animals was within the range 

commonly recorded for this strain and age. No macroscopic findings were observed at necropsy. The LD50 is greater than 

2000 mglkg bw. 

The acute toxicity by dermal application of Leuco Sulfur Black 1 was investigated according to OECD Guideline 402 under 

GLP. 
Five male and five female rats were treated at 2319 mg active ingredient/kg (corresponding to 4396 mg test item I kg bw) 

by dermal application. The test item was formulated in purified water and administered at a volume dosage of 6 mUkg. 

The application period was 24 hours. 

No deaths occurred during the study. No clinical signs were observed during the course of the study. 

Due to moderate black staining of the treated skin after the removal of the application patch, the assessment of erythema 

was first prevented on test day 2 in all animals. From test day 3 to test day 7 at the latest, a slight black staining was still 

present. Erythema was assessable, but not noted in all animals from test day 3 to the end of the study. No oedema was 

observed throughout the whole observation period. Scaling was observed in one male on test day 4. 

The body weight of the animals was within the range commonly recorded for this strain and age. No macroscopic findings 

were observed at necropsy. The LD50 is greater than 2000 mg/kg bw. 

An acute inhalation toxicity study has not been performed because exposure of humans via inhalation is not likely due to 

the physical state (wet paste) and vapour pressure (calculated as 1.8 x 10E-5 hPa at 25 'C) of the sub stance. 

Justification for classification or non-classification 

Based on the available data Leuco Sulfur Black 1 is not subject to classification and labelling according to Directive 

67/548/EEC and Regulation 1272/2008/EC. 
7 .2.1 Acute toxicity: oral 

Endpoint study record: Simon (2009) I key (class method) 

UUID IUC5-cdf11 aa3-76c1-4e25-9054-e23a5a96f2o4 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

lhr I SCC GmbH I Wendelsheim I Germany 

2010-05-26 16:20:05 CEST 

Administrative Data 

Purpc>HIIag 

Study result type 

Reliability 

key study; robust study summery; used for dasslfication; used for MSDS 

experimental result Study period 2009-01-20 til 2009-02-12 

1 (reliable without restriction) 

Rationale for rallablllty Incl. deficiencies Guideline-conform study under GLP wrthout deviations 

Data source 

Reference 

Rafentnce Author 

type 

Year Title 

study report Simon 2009 Acute Oral Toxicity Study 

c 

Data access 

data submitter Is data owner 

Data protection claimed 

yes 

in Rats 

Materials and methods 

Test type 

BlbUographlc Testing labonltory 

source 

Harlan Laboratories Ltd .• FOHinsdorf I 

Switzerland 

Report o..- Compeny study Report 

no. compeny no. 

C31043 Clariant 

24 

2009-08- -
acute toxic class method 

Umlttest 

yes 

Test guideline 

Qwollfier Guideline 

according to OECO Guideline 423 (Acute Oral toxicity- Acute Toxic Class Method) 

according to EU Method 8.1 tris (Acute Oral Toxicity -Acute Toxic Class Method) 

GLP compliance 

yes (incl. osrtificate) 

Test materials 

Deviation• 

no 

no 

Identity of test material same as for substance defined In section 1 (If not read-across) 

yes 

Test material Identity 

Identifier Identity 

CAS number 66241-11-Q 

ECnumber 266-273-5 

Details on test material 

- Name of test material : Leuco Sulfur Black 1 

- Physical state: thixotrophic, black, wet paste 

- Compos~ion: Leuco Sulfur Black 1:52.75% (w/w) (calculated by amount of by-compounds) 

2.4-Dimrochlorobenzene: < 20 ppm 

2,4-0initrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3 % (w/w) 

Water: 45.75% (w/w) by Kar1-Fischer titration 

- Lot/batch No.: ESA0007595 

-Expiration date of the lot/batch: 2011-Q7-11 

- Stabil~ under test conditions: Stable under storage conditions. 

-Storage condition of test material: At room temperature (range of 20 ± 5 'C), light protected. 
Test animals 

Species 

rat 

Strain 

Wistar 

Sex 

female 

Details on test animals and environmental conditions 

TEST ANIMALS 

-Source: Harlan Laboratories Ltd., FOIIinsdorf I Switzerland 

-AGe at study in~iation: 11 weeks 

- Weight at study Initiation: 175.0 g - 197.7 g 

-Fasting period before study: lasted for approximately 15 to16 hours (access to water was permitted) 

- Housing: In groups of three In Makrolon type-4 cages w~ wire mesh tops and standard softwood bedding 

-Diet (e.g. ad libitum): Pelleted standard Provimi Kllba 3433 rat/mouse maintenance diet, ad libldum 

-Water (e.g. ed libitum): Commun~ tap water from FOIIInsdolf ad libitum 

-Acclimation period: 2009~1-20 to 2009~1-26 (females, 2000 mglkg), 2009~1-22 to 2009-01-28 (females, 2000 mglkg) 

ENVIRONMENTAL CONDITIONS 

- Temperature (t:}: 22 ± 3 t: -Humidity(%): 30-70% 

- I'Jr changes (per hr): 10 - 15 

-Photoperiod (hrs dark I hrs light): 12112 

Administration I exposure 

Route of administration 

oral:gavege 

Vehicle 

water 
Details on oral exposure 

VEHICLE 

-Concentration in vehicle: 0.439 g substance/mL vehicle 

- Amount of vehicle (W gavage): 10 mllkg 

- Justification for choice of vehicle: found to be a suitable vehicle 

- Purity: purified water 

MAXIMUM DOSE VOLUME APPLIED: 10 mllkg bw 

DOSAGE PREPARATION (ff unusual): prior to dosing 

CLASS METHOD (ff applicable) 

-Rationale for the selection of the starting dose: no justification (no data available) 

Doses 

2319 mg active ingredient/kg bwequals 4396 mg test item I kg bw (Lueco Sulfur Black 1: 52.75% w/w) 

No. of animals per sex per dose 

3 females at 2319 mglkg bw 

3 females at2319 mglkg bw 

Control animals 

no 

Details on study design 

- Duration of observation period following administration: 14 days 

-Frequency of observations and weighing: 

Viability I Mortality: Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1 and twice dally during 

days 2-15. 

Body Weights: On test days 1 (prior to administration), 8 and 15. 

- Necropsy of survivors performed: yes 

-Other examinations performed: clinical signs (Daily during the acclimatization period, during the first30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test 

day 1. depending on the occurrence of clinical signs of toxicity. Once daily during days 2-15. 

Statistics 

No statistical analysis was usad. 
Any other information on materials and methods Incl. tables 

nment 

Femalee 

3 

Results and discussions 

Effect levels 

No deaths occurred during the study. 

Clinical signs 

For the first group of animals treated at 2319 mglkg, no clinical signs were observed in any of the animals on test 1. On test day 2, all three females were noted with black-stained feces 

and the cage of the animals was changed to fadlitate further assessment. All animals were free of any signs from test day 3 until the end of the study on test day 15. The second group 

of animals treated at 2319 mglkg were free of clinical signs on test day 1 as well. On test day 2 up to test day 5 all animals were observed with black-stained feces. On each of these 4 

days the animals were moved to new ceges. All animals were free of any clinical signs from test day 6 until the end of the study. 

Body-lght 

The body weight of the animals was within the renge commonly recorded for this strain and age. 

Gross pathology 

No macroscopic findings were observed at necropsy. 

Other findings 

None 

Any other Information on results Incl. tables 

Table 2: Number of animals dead (and with evident toxicity] [and time range within which mortality occurred] 
Dose Mortality Time range of deaths 

lmalkabwl I# deadltotall (hours} 

2319 0/3 n.a. 

2319 013 n.a. 

Applicant's summary and conclusion 

Interpretation of results 

not classified 

Criteria used for Interpretation of results 

EU 

Conclusions 

Number with evident 

toxlclty(#ltotal) 

013 

013 

The median lethal dose LDSO (tamale rat)of Leuco Sulfur Black 1 after single oral administration to female rats. observed over a period of 14 days is greater than 2000 mglkg body 

weight 

Executive summary 

The acute toxicity of Leuco Sulfur Black 1 by oral application was investigated according to the OECD Guideline 423 under 

GLP. 

Two groups, each of three female HanRcc:WIST (SPF) rats, were treated by oral gavage administration at a dosage of 

2319 mg active ingredient I kg body weight (corresponding to 4396 mg test item I kg body weight). The test item was 

formulated in purified water according to the content of the active ingredient at a concentration of 0.439 glml and 

administered at a dosing volume of 10 mllkg. 

No deaths occurred during the study. Except for black stained feces from test day 2 up to test day 5 at the latest, no 

clinical signs were noted in all animals during the observation time. The body weight of the animals was within the range 

commonly recorded for this strain and age. No macroscopic findings were observed at necropsy. LD50 was greater than 

2319 mglkg bw. 
7.2.2 Acute toxicity: inhalation 

Endpoint study record: waiving 

UUID IUC5-fa34d 11 O-e335-4b41-be 7b-26db4554 7 cd2 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1 fad973281 ea 

Author 

Date 

Remarks 

uensenbe I Clartant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-llS-21 13:21:33 CEST 

Administrative Data 

Dats waiving other justification 

Justification for In accordance with Regulation (EC) No 190712006 Annex VIII, column 2, an acute inhalation toxicity study has not to be performed because exposure of humans 

data waiving via inhalation is not likely taking into account the vapour pressure of the substance and the possibility of exposure to aerosols, particles or droplets of an lnhalable 

size. The dermal route has been studied in addition to the oral route because skin contact In production and/or use is likely. 
7 .2.3 Acute toxicity: dermal 

Endpoint study record: Arcelin (2009) I acute dermal toxicity 

UUID IUC5-986964c8-cbf4-4b59-8183-60f9b94f8559 

Dossier UUID IUC5-292149d 1-a6n -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

thr I SCC GmbH I Wendelsheim I Germany 

2010-05-26 16:25:27 CEST 

Administrative Data 

Purpose Illig 

Study result type 

Relleblllty 

key study; robust study summary; usad for dasslfication; usad for MSDS 

expertmenlal resun Study period 2009-01-20 tiH 2009-02-10 

1 (reliable without reslriction) 

R.tlon11le for ralleblllty Incl. deficlencleo Guideline-conform study under GLP without deviations 

Data source 

Reference 

Referance Author Year 

type 

study report Aroelin 2009 

G 

Data access 

data submitter Is data owner 

Data protection claimed 

yes 

Title 

Acute Dermal Toxidty 

Study In Rats 

Materials and methods 

Test type 

Bibliographic Testing leboralory 

source 

Harlan Laboratories Ltd .• FOIIinsdorf I 

Switzerland 

Report Owner Company study Report 

no. company no. date 

C31054 Clariant 2009-09-

18 
standard acute melhod 

Umlttest 

yes 

Test guideline 

Qualifier Guideline 

according to EU Method B.3 (Acute Toxicity (Dennal)) 

according to OECD Guideline 402 (Acute Dermal Toxicity) 

GLP compliance 

yes (incl. certificate) 

Test materials 

Devlotlono 

no 

no 

Identity of test material same as for substance defined In section 1 (If not read-across) 

yes 

Test material Identity 

Identifier Identity 

CAS number 66241-11~ 

ECnumber 266-273-5 

Details on test material 

• Name of test material : Lauco Sulfur Black 1 

• Physical state: lhixotrophic, black, wet paste 

• ComposKion: Leuco Sulfur Black 1:52.75% (w/w) (calculated by amount of by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-DinKrophenola: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3 % (w/w) 

Water: 45.75% (w/w) by Karl..flscher titration 

• Lot/batch No.: ESA0007595 

·Expiration date of the lot/batch: 2011~7-11 

• StabiKy under test conditions: Stable under storage conditions. 

• Storage condition of test material: At room temperature (range of 20 ± 5 'C), light protected. 
Test animals 

Species 

rat 

Strain 

Wistar 

Sex 

male/female 

Details on tast animals and environmental conditions 

TEST ANIMALS 

- Source: Harlan Laboratories ltd., FOIIinsdorf I Switzerland 

-Age at study initiation: Males: 9 weeks; females: 11 weeks 

-Weight at study initiation: 182.9 g- 267.1 g 

- Fasting period befora study: no data 

- Housing: Individually In Makrolon type-3 cages wtth standard softwood bedding 

- Diet (e.g. ad libttum): Pelleted standard Provimi Kliba 3433 rat/mouse maintanance diet, ad libidum 

-Water (e.g. ad libttum): Community tap water from FOIIinsdorf ed libitum 

-Acclimation period: 2009.01-20 to 2009-Q1-26 

ENVIRONMENTAL CONDITIONS 

- Temperature ('C): 22 ± 3 'C 

- Humidtty (% ): 30 - 70% 

- Air changes (per hr): 10 • 15 

- Photoperiod (hrs dark I hrs light): 12 112 

Administration I exposure 

Type of coverage 

semiocclusive 

Vehicle 

water 

Details on dermal exposure 

TEST SITE 
- Area of exposure: bad< 

- % coverage: 10 % of the total body surface 

-Type of wrap n used: not specified 

REMOVAL OF TEST SUBSTANCE 

-Washing (if done): flushed wi1h lukewarm tap water and dapped off with disposable paper towels. 

- Time after stert of exposure: 24 hrs 

TEST MATERIAL 

- Arnount(s) applied (volume or weight with un~): 2319mglkg body weight active ingredient (corresponding to 4396 mglkg of the test item; based on the amount of 52.75% (wlw) Leuco 

Sulfur Black 1] or 6 ml application volume/kg body weight 

-Concentration (ff solution): 0.73 g/ml 

- Constant volume or concentration used: yes -For solids, paste formed: yes 

VEHICLE 

- Purity: purified water 

Duration of exposure 

24 hrs 

Doses 

Males and Females: 2319 mglkg bw active ingredient (corresponding to 4396 mglkg of the test ~em: based on the amount of 52.75% (wlw) Leuco Sulfur Blad< 1) 

Nola: Tha dose levels are In terms of tha active ingredient (Leu co Suller Bled< 1: 52.75 % wlw) in the test ~em. 

No. of animals per sex per dose 

5 malas, 5 females 

Control animals 

no 

Details on study design 

-Duration of observation period following edministralion: 14 days 

- Frequency of observations and weighing: 

Viability I Mortal~: Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1 and twice daHy during 

days 2-15. 
Body Weights: On test days1 (prior to administration), 8 and 15. 

- Necropsy of survivors performed: yes 

- Other examinations performed: 

dinical signs (Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1. Once daHy during days 2- 15.) 

Local Dermal Signs: Once daily during days 2 (following dressing removal) through day 15 

Statistics 

No statistical analysis was usad. 

Any other Information on materials and methods Incl. tables 

Table 1: Animal assignment 

Dose (mglkg Males Females 

bwl 

2319 mo/ko* 5 - 2319 mg/kg* - 5 

*2319 mg/kg active 1ngred1ent (corresponding to 4396 mg/kg ofthe test item; based on the amount of 52.75% (w/w) Leuco 

Sulfur Black 1 ) 

Results and discussions 

Effect levels 

No daalhs occurred during the study. 

Clinical signs 

No clinical signs were observed during the course of the study. 

Body weight 
The body weight of the animals was within the range oommonly recorded for this strain and age. 

Gross pathology 

No maaosoopic findings were recorded at necropsy. 

Other findings 

Local Dermal Signs 

Due to moderate black staining of the traaled skin after the removal of the application patch, the assessment of erythema was first pravenled on test day 2 in all animals. From test day 

3 to test day 7 at the lates~ a slight black staining was still present. Erythema was assessable, but not noted In all animals from test day 3 to the end of the study. No oedema was 

observed throughout the whole oberservation period. SceHng was observed In one male on test day 4. 

Any other Information on results Incl. tables 

Table 2: Number of animals dead [and with evident toxicity] [and time range wHhln which mortality occurred] 

Dose Cone. MortaiHy (I dead/total) Time Number with evident 

range of toxlclty(lltotal) 

(mglkg In vehicle deaths 

bw) (%1* Male Female Combined I hours I Male Female Combined 

Control Control n.a. n.a. n.a .. n.a .. n.a .. n.a .. n.a .. 

2319* 0.73g/ml 0/5 - n.a. n.a. 0/5 - n.a. 

2319* 0.73g/ml - 0/5 n.a. n.a. - 0/5 n.a. 

n.a .. not applicable 

*2319 mg/kg active Ingredient (corresponding to 4396 mgJkg of the test item; based on the amount of 52.75% (w/w) Leuco Sulfur Black 1) 

Table 3: Clinical/local signs (Males) 

Dose Ani- Signs Testdavs 

mg/kg mal Sex 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 

bw No. 0.5* 1* 2* 3* 5* 

231ga 1 M 

No .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r 

clinical 
signs 

Scaling .r 

Black 2 1 1 1 1 1 stainina 

Erythema § § § § § # 0 0 0 0 0 0 0 0 0 0 0 0 0 

Oedema § § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Eschars § § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

2 M No 

clinical .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r 

signs 

Black 2 1 1 1 staining 

Erythema § § § § § # 0 0 0 0 0 0 0 0 0 0 0 0 0 

Oedema § § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Eschars § § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

3 M No 

clinical .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r 

signs 

Black 2 1 1 1 1 1 staining 

Erythema § § § § § # 0 0 0 0 0 0 0 0 0 0 0 0 0 

Oedema § § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Eschars § § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

4 M 

No .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r 

clinical 
sians 

Black 2 1 1 1 1 1 stain ina 

Erythema § § § § § # 0 0 0 0 0 0 

Oedema § § § § § 0 0 0 0 0 0 0 

Eschars § § § § § 0 0 0 0 0 0 0 

5 M No 

clinical .r .r .r .r .r .r .r .r .r .r .r .r 

signs 

Black 2 1 1 1 1 1 staining 

Erythema § § § § § # 0 0 0 0 0 0 

Oedema § § § § § 0 0 0 0 0 0 0 

Eschars § § § § § 0 0 0 0 0 0 0 

Key: 0 no findings, 1 slight, 2 moderate,/" noted 

* Examinations were performed within the first 30 minutes and 1, 2, 3 and 5 hours after treatment. 

§Assessment of dermal findings was not possible underneath the application patch 

#Assessment of erythema was not possible due to black staining of the skin 

0 

0 

0 

.r 

0 

0 

0 

82319 mglkg active Ingredient (corresponding to 4396 mglkg of the test Kem; baaed on the amount of 52.75% (wlw) Leuco SuWur Black 1) 

no dlnlcal signs were evident in any animal during the acdimatization period. 

Overall remarks, attachments 

Overall remarks 

Table 4: Clinical/local signs (Females) 

0 0 0 0 0 0 

0 0 0 0 0 0 

0 0 0 0 0 0 

.r .r .r .r .r .r 

0 0 0 0 0 0 

0 0 0 0 0 0 

0 0 0 0 0 0 

2 3 4 5 6 7 8 9 10 11 12 13 14 15 
bw No. 0.5* 1* 2* 3* 5* 

231ga 6 F No 

clinical .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r 

signs 

Black 2 1 1 1 1 1 staining 

Erythema § § § § § # 0 0 0 0 0 0 0 0 0 0 0 0 0 

Oedema § § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Eschars 0 § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

7 F No 

clinical .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r 

signs 

Black 2 1 1 1 staining 

Erythema § § § § § # 0 0 0 0 0 0 0 0 0 0 0 0 0 

Oedema § § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Eschars § § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

No 

8 F clinical .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r 

signs 

Black 2 1 1 1 staining 

Erythema § § § § § # 0 0 0 0 0 0 0 0 0 0 0 0 0 

Oedema § § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Eschars § § § § § 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

9 F 

No .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r .r 

clinical 
sians 

Black 2 1 1 1 1 1 staining 

Erythema § § § § § # 0 0 0 0 0 0 

Oedema § § § § § 0 0 0 0 0 0 0 

Eschars § § § § § 0 0 0 0 0 0 0 

10 F No 

clinical .r .r .r .r .r .r .r .r .r .r .r .r 

signs 

Black 2 1 1 1 1 1 staining 

Erythema § § § § § # 0 0 0 0 0 0 

Oedema § § § § § 0 0 0 0 0 0 0 

Eschars § § § § § 0 0 0 0 0 0 0 

Key: 0 no findings, 1 slight, 2 moderate, noted 

• Examinations were performed within the first 30 minutes and 1, 2, 3 and 5 hours after treatment. 

§Assessment of dermal findings was not possible underneath the application patch 

..Assessment of erythema was not possible due to black staining of the skin 

0 

0 

0 

.r 

0 

0 

0 

82319 mglkg active ingr>ldient (corresponding to 4396 mglkg of the test ttem; based on the amount of 52.75% (wlw) Leuco Sulfur Black 1) 

no clintcal signs were evident in any animal during the acdlmatization period. 

Applicant's summary and conclusion 

Interpretation of results 

notdasslfled 

Criteria used for Interpretation of results 

EU 

Conclusions 

0 0 0 0 0 0 

0 0 0 0 0 0 

0 0 0 0 0 0 

.r .r .r .r .r .r 

0 0 0 0 0 0 

0 0 0 0 0 0 

0 0 0 0 0 0 
The median lethal dose L050 (rat) of Leuco Sulfur Black 1 after single dermal adminislration to rats of both sexes, observed over a period of 14 days is greater than 2000 mglkg body 

weight 

Executive summary 

The acute toxicity by dermal application of LEuco Sulfur Black 1. was investigated according to OECD Guideline 402 under 

GLP. 

Five male and five female HanRcc:WIST (SPF) rats were treated with Leuco Sulfur Black 1 at 2319 mg active ingredienUkg 

(corresponding to 4396 mg test item/kg) by dermal application. The test item was formulated in purified water according to 

the content of the active ingredient at a concentration of 0. 73 g/mL and administered at a volume dosage of 6 mL/kg. The 

application period was 24 hours. 

No deaths occurred during the study. No dinical signs were observed during the course of the study. 

Due to moderate black staining of the treated skin after the removal of the application patch, the assessment of erythema 

was first prevented on test day 2 in all animals. From test day 3 to test day 7 at the latest, a slight black staining was still 

present. Erythema was assessable, but not noted in all animals from test day 3 to the end of the study. No oedema was 

observed throughout the whole oberservation period. Scaling was observed in one male on test day 4. 

The body weight of the animals was within the range commonly recorded for this strain and age. No macroscopic findings 

were observed at necropsy. 
7.3 Irritation I corrosion 

Endpoint summary: Irritation I corrosion 

UUID IUC5-50fa682b-20ac-4b90-87a3-d06b3088e470 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281 ea 

Author thr I SCC GmbH I Wendelsheim I Germany 

Date 2010-05-26 16:52:29 CEST 

Remarks 

Administrative Data 

Short description of key information 

Leuco Sulfur Black 1 ceused none to very mild signs of skin or eye irritation In the rat or rabbh, respectively, without any relevance for dasslficetion and labeHing. 

Key value for chemical safety assessment 

Skin Irritation I corrosion 

not Irritating 

Eye Irritation 

not irritating 

Respiratory Irritation 

not irritating 

Discussion 

The skin irritation potential of Leuco Sulphur Black 1 was assessed in the acute dermal toxicity study according to OECD 

Guideline 402 under GLP. 
Five male and five female rats were treated at 2319 mg active ingredient/kg (corresponding to 4396 mg test item I kg bw) 

by dermal application. The test item was formulated in purified water and administered at a volume dosage of 6 mUkg. 

The application period was 24 hours. 

No deaths occurred during the study. No clinical signs were observed during the course of the study. 

Due to moderate black staining of the treated skin after the removal of the application patch, the assessment of erythema 

was first prevented on test day 2 in all animals. From test day 3 to test day 7 at the latest, a slight black staining was still 

present. Erythema was assessable, but not noted in all animals from test day 3 to the end of the study. No oedema was 

observed throughout the whole observation period. Scaling was observed in one male on test day 4. 

The primary eye irritation potential of Leuco Sulphur Black 1 was investigated according to OECD test guideline No. 405 

and GLP. The test item was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White 

rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test item instillation. 

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal 

opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris 

were 0.00 for all three animals. The individual mean scores for all three animals for the conjunctival reddening and swelling 

were 0.00 and 0.00, respectively. 

The instillation of Leuco Sulfur Black 1 into the eye resulted in mild, early-onset and transient ocular changes, such as 

reddening and swelling of the conjunctivae, redness of the sclera as well as discharge. These effects were reversible and 

were no longer evident 24 hours after treatment in all animals. Black test item remnants were observed in the eye or 

conjunctival sac of all three animals at the 1-hour reading. No abnormal findings were observed in the cornea or iris of any 

animal at any of the examinations. 

Thus, the test item did not induce significant or irreversible damage to the rabbit eye. 

Based on the lacking skin and eye irritation potential, the high boiling point and consequently very low vapro pressure, a 

respiratory irritation can be excluded. 

Justification for classification or non-classification 

Based on the available data Leuco Sulfur Black 1 is not subject to classification and labelling according to Directive 

67/548/EEC and Regulation 1272/2008/EC. 
7 .3.1 Skin irritation I corrosion 

Endpoint study record: waiving_ Skin irritation I corrosion 

UUID IUC5-49607fb8-499b-48f2-94ed-debdl2ee647b 

Dossier UUID IUC5-292149d 1-a6IT -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-QS-21 13:27:28 CEST 

Administrative Data 

Data w•lvlng other justifocalion 

Justlllcallon for data In accordance with Regulation (EC) No 190712006 Annex VIII, column 2, a skin irritation study does not need to be conducted because the acule toxicity study 

w.lvtng by the dermal route did not Indicate skin irritation at a dose level even higher than the limit dose of 2 000 mglkg bw. 
7 .3.2 Eye irritation 

Endpoint study record: Arcelin (2009) Eye irritation (in vivo) 

UUID IUCS-341 a0a6c-347c-4c5a-95e 7 -ba9a6fa01 e2c 

Dossier UUID IUC5-292149d1-a6n -4feb-9692·1 fad973281 ea 

Author 

Date 

Remarks 

lhr I SCC GmbH I Wendelsheim I Germany 

2010-05-26 16:27:16 CEST 

Administrative Data 

Purposeft.lg key sludy 

Study reault type 

Reliability 

experimental resun Study period 2009-02-18 tin 2009-03-02 

1 (reUeble wilhout restriction) 

Rationale for reliability Incl. deficiencies Guideline conform study under GLP wilhout deviations 

Data source 

Reference 

Reference Author 

type 

Year Title 

sludy report Arcelin 2009 Primary Eye Irritation Sludy 

G 

Data access 

data submitter is data owner 

Data protection claimed 

yes 

in Rabbits 

Materials and methods 

Type of method 

Bibliographic Testing laboratory 

source 

Hanan Laboralories Ltd. FOIIinsdorf 

(Swilzeriand) 

Report Owner Company study Report 

no. company no. data 

C31065 Clarianl 2009-08-

20 
in vivo 

Test guideline 

Qualifier Guideline Deviation a 

according lo EU Method B.5 (Acule Toxicily: Eye IITitation I Corrosion) no 

according lo OECD Guideline 405 (Acule Eye IITitation I Conosion) no 

GLP compliance 

yes (ind. certificate) 

Identity of test material same as for substance defined in section 1 (If not read-ecross) 

yes 

Test materials 

Test material Identity 

Identifier Identity 

CAS number 66241-11-0 

EC number 266-273-5 

Details on test material 

- Name of lest material : Leuco Sulfur Black 1 

-Physical stale: lhixotrophlc, black, wet paste 

- ComposHion: Leuco Sulfur Black 1 :52.75% (w/w) (calculated by amount of by-rompounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2.4-Dinitrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3 % (w/w} 

Water: 45.75% (w/w) by Karl-Fischer titration 

-Lot/batch No.: ESA0007595 

-Expiration date of the lot/batch: 2011-07-11 

- StebiiHy under test conditions: Stable under storage condHions. 

-Storage condition of test material: At room temperature (range of 20 ± 5 'C), light protected. 

Test animals 
Species 

rabbit 

Strain 

New Zealand Whhe 

Details on test animals and environmental conditions 

TEST ANIMALS: Young AduH Naw Zaaland WhKe RabbK, SPF 

-Source: Ha~an Laboratories B.V. 5960 AD Horst I The Nethe~ands 

-Age at study initiation: 15 weeks (male), 15- 16 weaks (females) 

-Weight at study initiation: 2608 g (male), 2729-2892 g (females) 

- Housing: Individually In stainless steel cages equipped with feed hoppars and drinking water bowls. Wood blocks and haysticks for gnawing. 

-Diet (e.g. ad libKum): Pelleted standard Provimi Kllba 3418 rabbit maintenance diet ad libKum 

-Water (e.g. ad libitum): Community tap water from FOIIinsdorf ed libitum 

- Acclimation : Under laboratory oonditions after health examination. Only animals without any visual signs of illness were used for the study 

-Randomization: Selected by hand at time of delivery. No computer generatad randomization program. 

ENVIRONMENTAL CONDITIONS 

-Temperature (q: 17-23 'C 

-Humidity(%): between 30-70% (values above 70% during daaning process possible) 

-Air changes (per hr): Air-mnditioned with 10-15 air changes per hour 

-Photoperiod (hrs dart< I hrs ~ght): automaticaUy controlled light cyde of 12 hours light and 12 hours dart<, music during the daytime light period. 

Test system 

Vehicle 

unchanged (no vehide) 

Amount/concentration applied 

TEST MATERIAL 

- Amount(s) applied (volume or weight with un~): 0.1 gl animal (undiluted) 
VEHICLE: none 

Duration of treatment I exposure 

The eyes of all animals were rinsed with sterile physiological saline (B. Braun, Emmenbrllci<e. Swltzet1and; Lot-No. 7 443A 191. expiry date 30-Sep-2009) after the 1-hour observation 

because black test Item remnants (partides) were observed at this observation time point. 

Observation period 

72h 

Number of animals 

Number of Animals per Test: 3 (Animals of both sexes were used) 

Control animals 

other: left eye of each animal treated, right eye served for reference control 

Details on study design 

REMOVAL OF TEST SUBSTANCE 

The eyes of all animals were rinsed with sterile physiological saline (B. Braun, Emmenbroci<e, Swltzet1and; Lot-No. 7 443A 191. expiry date 3()-Sep-2009) after the 1-hour observation 

because blaci< test ~em remnants (particles) were observed at this observation time point. 

SCORING SYSTEM: 

The eye reectlons were assessed according to the numerical scoring system listed In the 

Commission Regulation (EC) No. 440/2008, B.5, at approximately 1, 24, 48 and 72 hours after 

administration. Sderel reddening and ocular discharge were also assessed. 

TOOL USED TO ASSESS SCORE: 

Eye examinations were made with a Varte Cliptrix diagnostic-jamp 

Any other Information on materials and methods Incl. tables 

The pH of the test item was measured before the start of the study in a 1% dilution in purified water and was found to be 6. 

The eyes of the animals were examined one day prior to test item administration. Animals with overt signs of ocular injury 

or irritation which may have interfered with the interpretation of the results were not used in the test. 

On the day of treatment, 0.1 g of Leuco Sulfur Black 1 was placed in the conjunctival sac of the left eye of each animal 

after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to 
prevent loss of test item. The right eye remained untreated and served as the reference control. 

As an irritant effect seemed to be unlikely, but could not be fully excluded, a single animal (one female) was treated first. 

As neither a corrosive effect nor a severe irritant effect was observed after the 1- and 24-hour examinations, the test was 

completed using the two remaining animals 

The eyes of all animals were rinsed with strile physiological saline (B. Braun, EmmenbrOcke, Switzerland; Lot-No. 

7443A191, expiry date 30-Sep-2009) after the 1-hour observation because black test item remnants (particles) were 

observed at this observation time point. 

Observations 

Viability I Mortality: Daily from acclimatization of the animals to the termination of test. 

Clinical Signs (systemic): Daily from acclimatization of the animals to the termination of test. 

Body Weights: At start of acclimatization, on the day of application and at termination of observation. 

Necropsy 

No necropsy was performed on the animals sacrificed at termination of observation. 

The animals were killed by intravenous injection of pentobarbitone into the ear vein at a dose of at least 1 mUkg body 

weight (162 mg sodium pentobarbitone/kg body weight) and discarded. 

Results and discussions 

Overall Irritation I corrosion results 

lrrlhlllon p•rameter 

overall irritation score 
overall irritation seo<e mean 48hours 

overall irritation seo<e mean 72 hours 

Irritant/corrosive response data 

no irritation ellects obseJVed 

Other effects 

Viability I Mortality: No mortality occurred. 

0 0 

0 0 

Clinical Signs: No clinical signs were obseiVed during the course of the study. 

Body Weights: The body weights of all rabbits were considered to be within the nonnal range of variability. 

Pathology, Macroscopic Findings: No necropsy was performed at tha end of the study. 

Any other information on results incl. tables 

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal 

opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris 

were 0.00 for all three animals. The individual mean scores for all three animals for the conjunctival reddening and swelling 

were 0.00 and 0.00, respectively. 

No abnormal findings were observed in the cornea or iris of any animal at any of the measurement intervals. Slight 

conjunctival reddening and swelling, slight scleral reddening, as well as slight ocular discharge was noted in all three 

animals at the 1-hour reading. 

No abnormal findings were observed in the treated eye of any animal 72 hours after treatment, the end of the observation 

period for all animals. 

Coloration: No staining produced by the test item of the treated eye was observed. 

Test Item Remnants: Black test item remnants were evident in the eye or conjunctival sac of all three animals at the 1-

hour reading. 
Corrosion: No corrosion of the cornea was observed at any of the reading times. 

E I . S :ye mtat1on cores 

Animal Sex Evaluation Corneal Area of Corneal Iris Conjunctivae 

Number Interval* Ooacitv Ooacitv 

Redness Chemosis Sclera 

19 M 0 0 0 1 1 1 

20 F 1 hour 0 0 0 1 1 1 

21 F 0 0 0 1 1 1 

19 M 0 0 0 0 0 0 

20 F 24 hours 0 0 0 0 0 0 

21 F 0 0 0 0 0 0 

19 M 0 0 0 0 0 0 

20 F 48 hours 0 0 0 0 0 0 

21 F 0 0 0 0 0 0 

19 M 0 0 0 0 0 0 

20 F 72 hours 0 0 0 0 0 0 

21 F 0 0 0 0 0 0 . ExaminatiOns were performed at the specified times after InstillatiOn of the test item . 

EY'e Irritation Scores - Mean Values after 24 48 and 72 Hours 

Animal Sex Corneal N Iris N Conjunctivae 

Number Opacity Redness N Chemosis N 

19 M 0.00 3 0.00 3 0.00 3 0.00 3 

20 F 0.00 3 0.00 3 0.00 3 0.00 3 

21 F 0.00 3 0.00 3 0.00 3 0.00 3 

Evaluated Intervals Corneal Opacity Con unctivae 
24hours 

48 hours Not Irritating 

72 hours 

Applicant's summary and conclusion 

Interpretation of results 

not Irritating 

Criteria used for Interpretation of results 

EU 

Conclusions 

Redness Chemosis 

Not Irritating Not Irritating Not Irritating 

Based upon the referred classification (Commission Directive 2001159/EC of August 06, 2001 ), Leuco Sulfur Black 1 is considered to be 'not irritating' to the rabbit eye. 

Executive summary 

The primary eye irritation potential of Leuco Sulfur Black 1 was investigated according to OECD test guideline No. 405 and 

Commission Regulation (EC) No. 440/2008, B.5. The test item was applied by instillation of 0.1 g into the left eye of each 

of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 

hours after test item instillation. 

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal 

opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris 

were 0.00 for all three animals. The individual mean scores for all three animals for the conjunctival reddening and swelling 

were 0.00 and 0.00, respectively. 

The instillation of Leuco Sulfur Black 1 into the eye resulted in mild, early-onset and transient ocular changes, such as 

reddening and swelling of the conjunctivae, redness of the sclera as well as discharge. These effects were reversible and 

were no longer evident 24 hours after treatment in all animals. Black test item remnants were observed in the eye or 

conjunctival sac of all three animals at the 1-hour reading. No abnormal findings were observed in the cornea or iris of any 
animal at any of the examinations. No corrosion was observed at any of the measuring intervals. 

No clinical signs were observed. 

Thus, the test item did not induce significant or irreversible damage to the rabbit eye. 

Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 06, 2001 ), Leuco Sulfur Black 

1 is considered to be "not irritating" to the rabbit eye. 
7.4 Sensitisation 

Endpoint summary: Sensitisation 

UUID IUC5-5039afad-2a 7e-4143-1!483-350bl9bc60cd 

Dossier UUID IUC5-292149d 1-a6n -4feb-9692-1fad973281 ea 

Author thr I SCC GmbH I Wendelsheim I Germany 

Date 2010-05-26 16:47:38 CEST 

Remarks 

Administrative Data 

Skin sensitisation 

Short description of key information 

Leu co Sulfur Black 1 did not cause skin sensitisetion in the LLNA In mice. 

Key value for chemical safety assessment 

Skin sensitisation 

not sensitising 

Discussion 

The skin sensitisation potential of Leu co Sulfur Black 1 was investigated in the LLNA according to OECD test guideline No. 

429 and GLP. Three groups each of four female mice were treated daily with the test item at concentrations of 5, 10 and 

25% (w/w) in dimethylformamide by topical application to the dorsum of each ear lobe (left and right) for three consecutive 

days. A control group of four mice was treated with the vehicle (dimethylformamide) only. Five days after the first topical 

application the mice were injected 3H-methyl thymidine. Approximately five hours later, the mice were sacrificed, the 

draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared 

from pooled lymph nodes and the proliferative capacity of pooled lymph node cells was determined by the incorporation of 
3H-methyl thymidine measured in a scintillation counter. 

All treated animals survived the scheduled study period and no signs of toxicity were observed. 

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentration resulted in 3-fold or 

greater increase in incorporation of3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). 

The estimated concentration of test item required to produce a S.l. of 3 is referred to as the EC3 value. 

In this study Stimulation Indices of 1.42, 1.12 and 1.61 were determined with the test item at concentrations of 5, 10 and 

25% in dimethylforrnamide. 

The EC3 value could not be calculated, since none of the tested concentrations induced a S.l. greater than 3. Thus, the 

test item was not a skin sensitizer. 

Respiratory sensitisation 

Discussion 

At present there is no accepted experimental test system available to evaluate this endpoint Based on the substance 

properties (mean MW >8000 Da and very poor solubility in water), the fact that no toxicity was seen in any of the available 

studies and that there are no human data available indicative of a respiratory sensitisation potential, it is concluded that 

Leuco Sulfur Black 1 does not raise any concern regarding respiratory sensitisation. 

Justification for classification or non-classification 

Based on the available data Leuco Sulfur Black 1 is not subject to classification and labelling according to Directive 

67/548/EECand Regulation 1272/2008/EC. 
7 .4.1 Skin sensitisation 

Endpoint study record: LLNA 

UUID IUC5-e412fb2 1 -5b5c-4bbe-9b09-b92dclb4 1 9a6 

Dossier UUID IUCS-2921 49d 1 -a677 -4feb-9692-1 lad97328 1 ea 

Author 

Dale 

Remarks 

lhr I SCC GmbH I Wendelsheim I Germany 

2010-{)5-26 16:33:34 CEST 

Administrative Data 

Purpose flag key study; robust study summary; used for dassification; used for MSDS 

Study result type expertrnental resuH 

Reliability 1 (reliable without restriction) 

Data source 

Reference 

Reference Author Year 

type 

study report Vogel 2009 

J 

Data access 

data submitter is data owner 

Data protection claimed 

yes 

Tille 

Local Lymph Node Assay 

(LLNA) in Mice 

Materials and methods 

Type of method 

tnvivo 

Bibliographic Testing 18boralory 

source 

Hanan CCR GmbH. Rossdorf 

(Grmany) 

Report no. Owner Company study Report 

company no. dele 

1234901 Clariant 2009-{)9. 

(C31100) 17 
Type of study 

Mouse locallymphnode assay (LLNA) 

Test guideline 

Guideline 

OECD Guideline 429 (Sidn Sensttisation: Local Lymph Node Assay) 

yes (ind. cet11ficate) 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Details on test material 

Identification: Leuco Sulfur Black 1 

CAS No.: 66241-11-0 

Description: Thlxolhrophic, black, wet paste 

Batch No.: ESA0007595 

Composition: Leuco Sulfur Black 1: 52.75% (calculated by amount of by-compounds) 

2,4-Dinttrochlorobenzene: < 20 ppm 

2,4-Dinttrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3 % (wlw) 

Water: 45.75 % (wlw) by Ka~-Fischar titration 

Stebittty of the test rtern Stable under s1orage condttions 

Stability in solvent: > 4 h in water 

Storage: />J room temperature (range 20 ± 5 'C), light protected 

Expira1ion Date: July 11, 2011 

Test animals 

Species 

mouse 

Strain 
CBA 

Sex 

female 

Details on test animals and environmental conditions 

TEST ANIMALS 

• Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst I The Netherlands 

• Age at study initiation: 8 • 12 weeks 

• Weight at study initiation: 15 • 25 g 

• Housing:single caging 

• Diet (e.g. ad libi1um): pelleted standard dial (Harlan Laboratories GmbH, 33178 Borchen), ad Hbidum 

• Water (e.g. ad libitum): tap water, (Gemeindewerl<e, 64380 Rossdorf), ad libitum 

• Acctlmatlon period: At least 5 days prior to the start of dosing under test condHions after heaHh examination. Only animals without any visible signs of Illness will be used for the study 

ENVIRONMENTAL CONDITIONS 

• Temperature ('C): 22 + 2'C 

• HumidHy (% ): 45-65% 

• Photoperiod (hrs dar!< I hrs light): artificial light 6:00 a.m. • 6:00 p.m. 

Test system 

LLNA 

Vehicle 

dirnethytformamida 

Concentration 

5, 1 0, and 25% 

No. of animals per dose 

4 

Details on study design (LLNA) 

To determine the highest non-irritant test concentration. a pre-test was performed in two animals. Two mice were treated with concentrations of 10 and 25% each on three consecutive 

days. 

In the pr&-tast clinical signs were recorded within 1 hour and 24 ± 5 hours after each application as well as on day 7. At the tasted concentrations lheanimats did not show any signs of 

irritatlon or systemic toxicHy. 

The 1ast Hem in the main study was assayed at 5, 10 and 25%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local 

imtatlon. No severe imtant effects were tolerated choosing the test concentrations. 
Positive control substance(s) 

hexyl dnnamic aldehyde (CAS No 101~) (In acetone:olive oil (4+1) using CBAICeOtaHsd mice in June 2009) 

Statistics 

The mean values and standard deviations were calculated in the body weight tables. 

Any other lnfonnatlon on materials and methods Incl. tables 

Interpretation of Raw Data 

The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio oA-iTdR incorporated 

into lymph node cells of test lymph nodes relative to that recorded for oontrollymph nodes (Stimulation Index; S.l.). Befora DPM/node values were determined, mean scinUIIetionbeckground DPM was subtracted from test and control raw data. A test Item is regarded as a sensltiser In the LLNA ff the following criteria are fuffllted: First, that exposure to at least 

one concentration of the test Item resuned In an incorporation oFHTdR at least 3-fotd or greater than that recorded in oontrol mice, as indicated by the stimulation Index. 

Second, that the data are compatible with a oonventional dose response, anhough allowance must be made (especially at high topical concantrations) for enher local toxicity or 

Immunological suppression. 

Observations 

In adld~ion to the sensHising reactions the following observations and data were recorded during the test end observation period: 

MortaiHy I ViebiiHy:once daily (week day) from experimental start to necropsy. 

Body weights:prior to tha first application and prior to treatment wlthltiTdR. 

CHnical signs (local/systemic): In the pre-test clinical signs were recorded within 1 hour and 24±4hours after each application as well as on 

day 7. In the main experiment clinical signs were recorded within 1 hour and 24±4 hours after the first and second application (days 1 

and 2) as well as on the day of preparation (day 6).EspeciallylhetreatrnentsHeswereobservedcaretully. 

Results and discussion 

Positive control results 

Experiment performend in January 2009, 5, 10, and 25% alpha-Hexylcinnamaldehyde yielded a S.l. of 1.49, 4.17, and 4.90, respectively. The EC3 value calculated was 7.8% 

LLNA 

Disintegrations per minute (DPM) 

see table below 
Stimulation Index 

1.4 (at 5 %). 1.1 (at 10 %), 1.6 (at 25 %) 

Any other Information on results Incl. tables 

Test item Calculation Result 

concentration Group Measurement 

number of DPM per 

% (w/w) DPM DPM-BGa) lymph nodes lymph nodeb) S.l. 

-- BGI 25 -- --- -- - -- BGII 39 -- --- -- -- 0 1 2735 2703 8 337.9 1.00 

5 2 3869 3837 8 479.6 1.42 

10 3 3067 3035 8 379.4 1.12 

25 4 4386 4354 8 544.3 1.61 BG = Background (1 ml 5% trichloroacetic acid) In duplicate 1-Control - Group 2-4--Test Group S.I.-Stlmulation - Index 

•l=The mean value was taken from the figures BG I and BG ubl=Since the lymph nodes of the animals of a doss group were pooled, DPM/node was determined by dividing the 

measured value by the number of lymph nodes pooled 

The EC3 value can not be calculated, since all S.l.'s are below 3. 

Viability I Mortality 

No deaths occurred during the study period. 

Clinical Signs 

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. 

Body Weights 
The body weight of the animals, recordedprior to the first application and prior to treatment with3HTdR, was within the 

range commonly recorded for animals of this strain and age. 

Applicant's summary and conclusion 

Interpretation of results 

not sensitising 

Conclusions 

The test item Leuco Sulfur Black 1 was not a skin sensiliser under the described conditions. 

Executive summary 

The present study (Vogel2009) analyses the sensilislng potential ofleuco Sulfur Black 1 

Three groups each of four female mice ware treated daily with the lest Hem at concentrations of 5, 10 and 25% (w/w) In dimethylformamlde by topical applicetion to tha dorsum of each 

ear lobe (left and right) lor three consecutive days. A control group of four mice was treated with the vehicle (dimathylformamlde) only. Five days after the first lopical application the 

mioa were injected intravenously inlo alall vein with redio-labetled thymidine eH-methyi thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the 

draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node oalls were prepared from pooled lymph nodes. which were subsequenUy washed 

and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node oalls was determined by the incorporation offi-methyl thymidine measured in absclntHiation counter. 

All treated animals survived the scheduled study period and no signs of toxicity were observed. 

A test Hem is regarded as a sensHiser in the LLNA if the exposure 1o one or more test concentration resuHed in 3-fold or greater increase in incorporation ofiHTdR compared with 

concurrent controls, as indiceled by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.l. of 3 is refemed lo as the EC3 value. 

In this study Stimulation Indices of 1.42, 1.12 and 1.61 were delermlned with the test item at concentrations of 5, 10 and 25% in dlmethylformamide. 

The EC3 value could not be calculated, since none of tha tested concentrations induced a S.l. greater than 3. 

The test Hem Leuco Sulfur Black 1 was not a skin sensiliserunder the described condHions. 
7.5 Repeated dose toxicity 

Endpoint summary: Repeated dose toxicity 

UUID IUC5-07 4041 c6-f34c-4c11-9b 71-6382281125b6 

Dossier UUID IUC5-292149d 1-a6n -4feb-9692-11ad973281 ea 

Author thr I SCC GmbH I Wendelsheim I Germany 

Date 2010-05-26 17:13:00 CEST 

Remarks 

Administrative Data 

Short description of key information 

Leuco Sulfur Black 1 did not cause any toxic affects in the repeated dose toxicity study with reproduction/developmental toxicity screening in rats (OECD 422). The limK dose of 1000 

rng/kg bwld represents the NOAEL. 

Key value for chemical safety assessment 

Repeated dose toxicity: oral 

Effect NOAEL 1000 mg/kg bwlday 

level 

Tnt subacute 

type 

Species rat 

Discussion 

In a repeated dose toxicity study according to OECD guideline 422 and GLP Leuco Sulfur Black 1 was administered by 

gavage to groups of 10 rats per sex and dose at 0, 100, 300 and 1000 mg/kg bw/d. Male rats were treated for 29 days, 

female rats for 14 days prior to pairing, though the pairing and gestation periods until the F1 generation had reached day 4 
postpartum. The test item was administered in water as vehicle at a dose volume of 10 mllkg bw/d. Control animals were 

doses with the vehicle alone. 

Observation and examinations consisted of mortality, clinical signs, detailed clinical observations, food consumption, body 

weight, functional observation battery, clinical biochemistry, haematology, urinalysis, macroscopical examination at 

necropsy, organ weights and histopathological examination of organs. The only effect seen was a dark discolouration of 

faeces in all high dose animals starting on day 3 of the study and lasting until termination. Based on this finding a NOAEL 

of 1000 mg/kg bw/d was derived. 

The discolouration of faeces was the only effect observed in this study and also in the acute oral toxicity study. Organs or 

urine were not discoloured and signs of substance deposits were not found in histopathological analysis of the organs. 

This indicates that Leuco Sulfur Black 1 is directly excreted and that absorption in the gastrointestinal tract does not occur 

following oral administration. 

The mean molecular weight and number-average molecular weight (Mn) of Leuco Sulfur Black 1 is in the range of 8000-

9000 Da and about 3000 Da, respectively.lt is very poorly or even not soluble in common polar and non-polar solvents 

such as water (solubility of 62.4 mg/L), DMSO, methanol, DMF, THF, chloroform and not soluble in acid likes sulfuric acid 

and trifluoroacetic acid. The vapour pressure of the pure substance, calculated from the boiling point of> 400 'C, is 1.8 x 

1 OE-5 hPa at 25 'C. These substance properties supported by the results of oral toxicity studies demonstrate that the 

absorption of Leuco Sulfur Black 1 via the oral, dermal or inhalation route is negligible and, consequently, that systemic 

bioavailability is also negligible. Based on the substance properties and the fact that no toxicity was seen in any of the 

available toxicity studies it is concluded that exposure to Leuco Sulfur Black 1 does not raise any health concern. In line 

with Regulation (EC) No 1907/2006 Annex XI and taking into account the REACH paradigm that new tests on vertebrates 

shall only be conducted or proposed as a last resort when all other data sources have been exhausted, further animals 

studies to examine the repeated dose toxicity of Leuco Sulfur Black 1 are not necessary and should not be performed. 

Justification for classification or non-classification 

Based on the available data Leuco Sulfur Black 1 is not subject to classification and labelling according to Directive 

67/548/EECand Regulation 1272/2008/EC. 
7 .5.1 Repeated dose toxicity: oral 

Endpoint study record: Repeated dose toxicity: ora/.001 OECD 422 Leuco Sulfur Black 1 

C31087 

UUID IUC5-d08b5!2b-3ffd-40b5-8bde-37609a519786 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1 fad973281 ea 

Author 

Date 

Remarks 

thr I SCC GmbH I Wendelsheim I Germany 

2010-05-26 16:34:35 CEST 

Administrative Data 

Purpoae flag 

Study result type 

key study; robust study summary 

experimental resutt 

Reliability 1 (reliable without restriction) 

Study period 

Rationale for reliability This study has been performed in compliance wllh the: 

incl. deflcienclea 

Between 20 May 2009 and 23 March 2010 

Swiss Ordinance relating to Good laboratory Practice adopted May 18th, 2005 (SR 813.112.1 }. This Ordinance is based on the OECD Principles of Good 

Laboratory Practice, as revised In 1997 and adopted on November 26th, 1997 by decision of the OECD Council [C (97)1861Final}. 

Data source 

Reference 

Reference Author 

type 

study report Ceccatelli 

R 

These principles are compatible with Good laboratory Practice regulations specified by regulatory authorities throughout the European Convnunity. the 

Unned States (EPA and FDA). and Japan (MHLW, MAFF and METI). 

There were no circumstances thet may have affected the qualny or Integrity of the data. 

Year Title Blbllogr8phlc Tntlng labo1'8tory Report Owner Compeny Report 

source no. cornpeny study no. date 

2010 Combined Repeated Dose Toxicity Study with the Harten laboratories Ud., C31087 Ciariant 201(). 

Reproduction/Developmental Toxicity Screening WOiferstrasse 4 4414 03-23 

Test In the Han Wlstar Rat Filillnsdort, Switzertand 
Data access 

data submitter is data owner 

Cross-reference to same study 

Toxicity to reproduction 

T ocicity to reproduction 

Toxicity to reproduction.001 OECD 422 in Rat Main Study 

Materials and methods 

Test type 

combined repeated doss and reproduction I developmantal screening 

Limit test 

yes 

Test guideline 

Qualifier Guideline 

according to OECD Guideline 422 (Combined Repsated Doss Toxicity Study wi1h the Reproduction I Developmental Toxicity Screening Test) 

according to other guideline: EPA OPPTS 670.3650 (Combined Repeetad Dose Toxicity Study with the Reproduction/Developmental Toxidty Screening Test) 

GLP compliance 

yes (ind. certificate) 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read;~~cross) 

yes 

Test material Identity 

Identifier Identity 

Common name Leuco Sulfur Black 1 

CAS number 66241-11-0 

Details on test material 

Devlatlone 

no 

no 
Desaiplion: Thixolrophic, black, wet paste 

Batch Number: ESA0007595 

Composition: Leuco Sulfur Black 1: 52.75% (calculated by amount of by-compounds) 

2,4-0initrochlorobenzene: < 20 ppm 

2,4-0initrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (w/w) 

Water. 45.75% (w/w) by Kari Fischer titration 

S1abiity of Test Item: Stable under storage conditions 

Stabiity of Test Item In Vehide: Seven days 

Expiry Date (Retest Date): 11-Jul-2011 

Storage Conditions: At room temperature (20 ± 5 'C) , light proteded 

Sefety Precautions: Routine hygienic procedures (gloves, goggles, face mask). 

Test animals 

Species 

rat 

Strain 

Wistar 

Sex 

male/female 

Details on test animals and environmental conditions 

Animals: Rat, HanRcc: WIST(SPF) 

Rationale: Recognized by inlemationel guidelines as a recommended test system. 

Breeder. Harian Laboratories Ltd., Laboratory Animal Services, WOifenrtrasse 4, 4414 FOIIinsdorf, Swltzeriand 

Number of Animals: 40 males (10 per group), 40 females (10 per group) 

Age (at Start of Tnsatment): 11 weel<s 

Body Weight Range (at Start of Tnsatment): Males: 290 to 324 g, Females: 284 to 212 g 

ldentiflcalion of Pnsntal Animals: Cege card and individual animal number (ear tattoo). 

Identification of Pups: On day 1 postpartum, pups were individually tattooed with Indian ink. 

Randomization: Computer-generated random algorithm. In addition body weights (recorded on lhe day of allocation) were taken into consideration in order to ensuns similar mean body 

weights In ali groups. 

Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness wens used for the study. 

Conditions: Standard laboratory conditions. Air-<:anditioned with 1 0 - 15 air changes per hour, continuously monitonsd environmental conditions (temp. range: 22 ± 3 'C; relative 

humidity range: 37.5- 80.9%). Thens was 12 hour ftuorescentlighl/12-hour darl< cyde wilh music during the light period. 

Administration I exposure 
Route of administration 

oral: gavage 

Vehicle 

water 

Details on oral exposure 

Dose Formulations 

The dose formulations were prepared weekly using the test Item as supplied by the Sponsor. 

Leuco Sulfur Black 1 was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous 

suspanslon was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations ware prepared lor each concentration. 

Homogeneity of the test Hem in the vehicle was maintained during the dally administration period using a magnetic stirrer. 

Storage of Dose Formulations 

Dose formulations were stored at room temperature (20 ± 5 q in brown glass beakers. 

Based upon the results of stabiHty analyses performed within the Harian Laboratories study no. C31076, dose formulations were stable lor at least one week. 

Treatment 

Method: Oral, by gavage 

Rationale lor Method: Administration by gavage Is a common and accepted route of exposure for studies of this type. 

Target Dose Levels: 

Group 1: 0 mg/l<g bwlday (control group) 

Group 2: 100 mglkg bw/day 

Group 3: 300 mg/l<g bwlday 

Group 4: 1000 mg/l<g bw/day 

Rationale for Dose Level Selection: The dose levels were selectad based on a previous dose range finding toxicity study in Han Wister Rats, Harian Laboratories Study C31076, using 

dose levels of 116, 348 and 1180 mglkglday adjustad for 52.75% Leuco Sulfur Black 1 dye stuff in the product and corresponding to 220, 680 and 2200 mglkg bwlday of product. The 

NOAEL of this study resultad to be 1160 mg dye stuff /kg bw/day or 2200 mg product/kg bwlday. 

Dose Volume: 10 mL/kg body weight 

Analytical verification of doses or concentrations 

yes 

Details on analytical verification of doses or concentrations 

Analysis of Dose Formulations 

On the first treatment day duplicate samples from the control group as well as three duplicate samples (top, middle and bottom) of about 2 g of each concentration ware taken prior to 

dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stabHity (4 hrs and 7 days). During the last 
week of the treatment. samples ware taken from the middle to confll'm concentration. The allquots for analysis of dosa formulations were trensferred Into tightly closed vessels protected 

against oxygen. One set of samples was delivered at ambient temperature to Dr. K. Morgenthel (Harlan Laboratories Ltd., ltingen I Switzerland) and stored thera at mom temperature 

(20 ± 5 'C) until analysis. The remaining set of sa mples was stored at the facUlty at mom temperature (20 ± 5 'C) in cese of repeated analysis. 

Analysis of homogeneity and concentration was performed gravimetrically by weighing the residue attar drying by lyophilization. Stablllly of doss formulations was confinned by IRspectroscopy. 

The application formulations investigated during the study were found to comprise Leu co Sulfur Black 1 in the range of 81.7% to 102.5%. The homogeneous distribution of Lauco Sulfur 

Black 1 in the preparations was approved beceuse Individual recoveries found did not deviate"""" than 14.2% (<15%) from the corresponding mean. In addition, the test Rem was 

found to be stable in applicetion formulations when kept for 4 hours and for 7 days under storage condRions due to slrrilar IR spectra compered to the reference spectrum obtained from 

the test Hem. Thus, identity of the test itam in applicetion fonnulations was confinned. In conclusion, the resuHs indicate the accurate use of the tast item Leuco Sulfur Black 1 and highly 

purified water as vehicle during this study. 

Duration of treatment I exposure 

Males: 29 days 

Females: Approximately 7 weeks 

Frequency of treatment 

Daily 

Doses/concentrations 

100 mglkg bw/day 

Baals actual Ingested 

300 mglkg bw/day 

Baals actual ingested 

1000 mglkg bw/day 

S.als actual ingested 

No. of animals per sex per dose 

10 

Control animals 

yes. concurrent vehicle 

Detlllls on study design 

STUDY SCHEDULE 

Males 
Acclimatization: 7 days 

First Test Hem Administration : Day 1 of pre-pairing 

Pre-Pairing: 14 days 

Pairing: 14 days maximum 

Treabnent Ends: On day before sacrifice 

Necropsy: After treatment of at least 28 days, when no longer needed for assessment of repro-ductive effects 

Females 

Acclimatization: 7 days 

First Test Hem Administration: Day 1 of pre-pairing 

Pre-Pairing: 14 days 

Pairing: 14 days maximum 

Gestation: Approximately 21 days 

Treabnent Ends: On day 4 post pariUm 

Necropsy:On day 5 postpartum (pups on day 4 post partum) 

Examinations 

Observations and examinations performed and frequency 

VIABILITY I MORTALITY 

Twloe daily 

CLINICAL SIGNS 

Dally cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of diflicuH or prolonged parturition, and 

behavioral abnormalitias In nesting and nursing. 

FOOD CONSUMPTION 

Males: Weekly during pre-pairing and aHer pairing periods 

Females: Pre-pairing period days 1 -8 and 8- 14; gestation days 0-7, 7- 14 and 14- 21 post coitum, and days 1 -4 postpartum. 

No food consumption was recorded during the pairing period. 

BODY WEIGHTS 

Recorded daily from treatment start to day of necropsy. 

DETAILED CLINICAL OBSERVATIONS 

Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the horne cage. Animals were observed for the 

following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory 

pattern). Changes in gail, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported. 

FUNCTIONAL OBSERVATION BATERY 

At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed wHh five P generation males and 

five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following: 

- Cege-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behavior (e.g. circling, stereotypy) and posture as well as resistance to removal 

-Hand-held observations: palpebral closure, pinna raftex, lacrimation, pupH siza, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to 
handling. 

-Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, ga~ evaluation, quantity of urine and fecal 

pellets voided. 

-Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing abil~ (Preyar's reflex), urine or feces, soiling, 

ganerel abnormalities, posture. 

-Measurements 1 Counts: hind limb I fore limb grip strength, landing foot splay, ractal temperature. 

Any ebnonnal findings were recorded and, where appropriate, graded in severity. 

Additionally, locomotor activ~ was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activ~ of the anlmals 

(besed on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the totals~ over 30 minutes were reporied. 

CUNICAL LABORATORY INVESTIGATIONS 

Blood and urine samples were obtained on the day of the scheduled necropsy from 5 males from each group. Males were kept in metabolism cage and fasted overnight (but allowed 

acoass to water ad Nbitum) for approximately 18 hours. 

Blood samples from 5 lactating females from each group were obtained on day 5 post partum. The females ware fasted for approximately 18-20 hours before blood sampling but 

allowed acoass to water ad libitum. 

Blood samples wera drawn sublingually from all animals under light isoflurane anesthesia. The samples were collected eariy in the working day to reduca biological variation caused by 

circadian rhythms. 

HEMATOLOGY 

The following hematology parameters were determined: 

Complete Blood een Count 

Erythrocyte count 

Reticutocyte Count 

Hemoglobin 

Hematoait 

Mean corpuscular volume 

Red cell volume distribution width 

Mean corpuscular hemoglobin 

Mean corpuscular hemoglobin concentration 

Hemoglobin concentration distribution width 

Leukocyte count, total 

Differential leukocyte count 

Platelet count 

Coagulation: 

Prothrombin time (= Thromboplastin time) 

Activated partial Thromboplastin time 

CUNICAL BIOCHEMISTRY 

The following clinical biochemistry parameters were determined: 

Glucose 

Urea 

Creatinine 

Bilirubin, total 
Cholesterol, total 

Triglycerides 

Aspartate aminolransferase 

Alanine aminotransferase 

Alkaline phosphatase 

Gamma-glutamy14ranslerase 

Bile acids 

Sodium 

Potasalum 

Chloride 

Calcium 

Phosphorus 

Protein, total 

Albumin 

Globulin 

Albumin/Globulin ratio 

URINE ANALYSIS 

The following urine parameters were detennined: 

Relative density 

colour 

Appearance 

pH 

Protein 

Glucose 

NKrile 

Ketones 

Urobilinogen 

Bilirubin 

Erytrocytes 

Leukocytes 

Volume (18 hours) 

Sacrifice and pathology 

TERMINATION OF THE STUDY 

Pups were sacrificed on day 4 postpartum. 

NECROPSY 

All animals wera saaificed by an injection of sodium pentobarbital. Dead pups, except those excessively cannibalized, were examined macroscopically. All pups were examined 

macroscopically for any structural changes, eKher at the scheduled necropsy or during the study n death occurred. 

ORGAN WEIGHTS 

The testes and epididymides of aU parantal males were weighed as pairs. 

In addition, from 5 malas and females killed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, es appropriate, 
and lhelr wet weight taken. 

Adrenal glands (weighed as pairs) 

Brain 

Heart 

Kidneys (weighed as pairs) 

Liver 

Thymus 

Spleen 

TISSUE PRESERVATION 

Tha following tissues from aN parental males were preserved in neutral phosphate buffered 4% formaldehyde solution: 

Prostate 

Seminal vesicles with coagulating gland 

Testes (in Bouin's fixative) 

Epididymides (in Bouin's fixative) 

Tha following tissues from an parental females were preserved in neutral phosphate buffered 4% formaldehyde solution: 

Ovaries 

In addition, from the five males and females par group selected for organ weights and from all animals found dead or kHied in extremis, the following tissues were preserved In neutral 

phosphate buffered 4% formaldehyde solution: 

Gross lesions 

Brain 

Spinal chord 

SmaH and large intestines (incl. Payer's patches) 

Stomach 

Liver 

Kidneys 

Adrenals 

Spleen 

Heart 

Thymus 

Thyroids, and parathyroids ff possible 

Trachea and lungs (preserved by inflation with fixative and then immersion) 

Uterus (with vagina) 

Urinary bladder 

Lymph nodes (mesenterial, mandibular) 

Peripheral nerve (sciatic) 

Bone marrow 

HISTOTECHNIQUE 

All organ and tissue samples to be examined by the principal investigator were processed, embedlded and cut at an approximate thickness of 2 - 4 micrometers and stained with 

hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. 

HISTOPATHOLOGY 
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and higiHiose groups were examined by the principal investigator. The same applied to all 

occurring gross lesions. 

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell sbuclu111. 

Histological examination of ovaries was canied out on females that did not give birth. 

Statistics 

The following statistical methods were used to analyze food consumption, body weights, organ weight, locomotor activity and macroscopical findings and reproduction data: 

• Means end standard deviations of various data were calculated. 

• The Dunnell-test (many to one 1-test) based on a pooled variance estimate was applied H the variables could be assumed to follow a normal distribution for the comparison of lhe 

treated groups and the control groups for each sex. 

·The Steel-test (many-ooe renk test) was applied Instead of the Dunnell·tasl when the data could not be assumed to follow a normal distribution. 

• Fishers exact-test was applied H the variables could be dichotomized wllhoutloss of information. 

Results and discussions 

Effect levels 

Endpoint Effect Bued Sex 

lev., on 

Basis for effect level/ Remark• 

NOAEL >= 1000 test male/female With the exception for a dark discoloration of feces observed In all animals treated wtth the test ttem at the dose level of 1000 mglkg body 

mat. 

Results of examinations 

Clinical signs and mortality 

weight/day, no other test tterJHelaled effects were observed during the study. The discoloration of feces was considered to be due to the 

staining property of the test ttem and not to be adverse. 

yes (dark dlsoololl!tion of feces at the dose level of 1000 mg/kg bw/day) 

Body -lght and -lght gain 

no effects 

Food consumption and compound Intake (If feeding study) 

no effects 

Food efficiency 

not examined 

Water consumption and compound Intake (If drinking water study) 
not examined 

Ophthalmoscopic examination 

not examined 

Haematology 

no effects 

Clinical chemistry 

no effects 

Urinalysis 

no effaciS 

Neurobehaviour 

no effects 

Organ weights 

no effects 

Gross pathology 

no effects 

Histopathology: neoplastic 

no eflects 

Details on results 

CLINICAL SIGNS AND MORTALITY 

BODY WEIGHT AND WEIGHT GAIN 

FOOD CONSUMPTION AND COMPOUND INTAKE (K feeding study) 

FOOD EFFICIENCY 

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study) 

OPHTHALMOSCOPIC EXAMINATION 
HAEMATOLOGY 

CLINICAL CHEMISTRY 

URINALYSIS 

NEUROBEHAVIOUR 

ORGAN WEIGHTS 

GROSS PATHOLOGY 

HISTOPATHOLOGY: NON-NEOPLASTIC 

HISTOPATHOLOGY: NEOPLASTIC (if applicable) 

HISTORICAL CONTROL DATA (if applicable) 

OTHER FINDINGS 

Any other Information on results Incl. tables 

IN LIVE OBSERVATIONS 

Detailed Clinical Observations (Dally) 

All animals survived until the scheduled necropsy. 

In group 4, dark discoloration offeces was observed in males and females starting on day 3 of the pre-pairing period onwards. This was likely due 

to the staining property of the test item at higher concentration. 

In groups 2 and 3, no clinical signs were observed for the entire duration of the study. 

(See attached Tables on pp. 1 to 3) 

Detailed Clinical Observations (Weekly) 

No findings were observed during the weekly examination. 

Functional ObservatlonaiBattery 
None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect. 

The frequency of rearing number and incidence of urine puddles during the open field observation did not give indication of any test item-related 

effect. Observational as spontaneous vocalization when the rat was removed from the cage was considered to be of incidental nature. 

Mean values of grip strength (fore- and hind paws) and landing foot splay gave no indication of any test item-related effect. 

Body temperature in males was statistically significantly lower in group 4 compared to the control group (37.9 'C compared to 38.7 'C in the control 

group). However, this value was within the range of the historical control data and therefore was not considered to be a test item-related effect. 

(See attached Tables on pp. 4 to 7 and attached Historical Control Data on pp. 3 to 14) 

Locomotor Activity 

Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor and did not give any indication of a test item-related 

effect. 

Food Consumption - Males - Pre-pairing and After Pairing Periods 

Mean food consumption was not considered to be affected by the treatment with the test item. 

During the pre-pairing period, the lower food consumption observed in groups 2 and 3 (-6.2 and -4.6% compared to the control group) was 

considered to be incidental since this reduction did not follow a dosEKiependent pattern and in group 4 mean food consumption was similar to the 

control (-0.4% compared to the control). 

During the after pairing period, mean food consumption compared to the control was: +2.2, -1.1 and +1.5%, in groups 2, 3 and 4, respectively 

(See attached Tables on pp. 8 to 11) 

Body Weights- Males - Pre-pairing, Pairing and After Pairing Periods 

Mean body weight and mean body weight gain were not affected by the treatment with the test item for the whole duration of the study. The 

statistically significantly lower body weight gain observed in group 2 between day 4 and 7 was considered to be incidental since there was no dosedependency. 

In order of ascending dose level, body weight gain was: during the pre-pairing period +13.0%, +10.9%, +11.5 and +11.9%, during the pairing period 

+4.9%, +5.7%, +4.4% and +4.7%, and during the after pairing period +4.1%, +5.6%, +4.8% and +4.5%, respectively. 

(See attached Tables on pp. 12 to 20) 
Food Consumption - Females - Pre-pairing, Gestation and Lactation Periods 

No test item-related effects were observed in mean food consumption for the entire duration of the study. 

Mean food consumption in groups 2, 3 and 4, compared to the control group, was: during the pre-pairing period +5.0%, +2.2% and +2.2%, during 

the gestation period +0.4%, -2.1% and -0.4%, and during the lactation period +11.3%, -3.1% and -3.7%, respectively. 

(See attached Tables on pp. 21 to 26) 

Body Weights - Females -Pre-pairing, Pairing, Gestation and Lactation Periods 

No test item-related effects were noted in mean body weight and mean body weight gain of females during the whole study at any dose level. 

In order of ascending dose level, body weight gain was: during the pre-pairing period +7.5%, +8.3%, +9.3% and +8.5, during the gestation period 

+63.5%, +63.3%, +60.2% and +60.5%, and during the lactation +3.1%, +6.2%, +4.6 and +2.7%, respectively. 

(See attached tables on pp. 27 to 39) 

CLINICAL LABORATORY INVESTIGATION 

Hematology 

The assessment of the hematology data did not reveal any test item-related effects in males and females. 

Clinical Biochemistry 

Males: 

In group 4, the concentration of potassium was statistically significantly higher (+17.8% compared to the control). Since the mean value was within 

the range of the historical control data, this was not considered to be a test item-related effect. 

In groups 2 and 3 no changes were noted. 

Females: 

The assessment of the dinical biochenistry data did not reveal any test item-related effects in females. 

(See attached Tables on pp. 40 to 41 and attached Historical Control Data on pp. 1 to 2) 

Urine analysis 

The assessment of the urine analysis data did not reveal any test item-related effects in males and females. 
TERMINAL FINDINGS 

Organ Weights 

No test item-related effects were noted in absolute and relative weight of organs in males and females. All the statistically significant changes were 

within the range of the historical control data or did not follow a dose-dependent pattern. 

(See attached Tables on pp. 44 to 49 and attached Historical Control Data on pp. 39 to 62) 

Macroscopical Findings 

The findings noted during the macroscopical examination did not give indication of any test item-related effect. 

Males: 

In group 4, spleen with a constriction was noted in male no. 34. No other findings were noted. 

Females: 

In group 4, female no. 72 was noted to have the liver caudal lobe adherent to the lateral lobe. 

In groups 2 and 3 no findings were noted. 

In group 1, female no. 43 was noted to have tan or dark red or red brown discolored nodules on the left uterine hom. Same female had only two 

implantation sites and did not deliver any pups. 

Histopathology Findings 

In group 4, in the lungs of animal no. 75 some alveolar macrophages (moderate severity degree) were found to contain dark pigment. This finding 

was possibly related to accidental aspiration of dose solution. All further lesions recorded during the microscopic investigation were within the range 

of background alterations that may be recorded in this type of study, and in rats of this strain and age. 

In group 1, in one infertile female (number 43) a deciduoma was noted. This lesion was deemed to have contributed to infertility. 

Qualitative staging of thePAS-stained testes was conducted and there were no abnormal lesions encountered during sperm staging regarding 

completeness of stages and maturation of cell populations. Individual lesions recorded were within the range of background alterations that may be 

recorded in this type of study, in rats of this strain and age. 

Applicant's summary and conclusion 
Conclusions 

This study is a valid investigation of the toxicological effects resuHing from repeated oral-gavage administration of the test item Leuco Sulfur Black 1 (CAS#66241-11-0) to rats over 

approximately 28 days. Leuco Sulfur Black 1 was admlnisterad in highly purified water as vehicle at dosages of 100,300, end 1000 mglkg body weight/day, and controls received the 

vehicle only. Leuco Sulfur Black 1 was administered to male rats for 29 days and to female rats lor 14 days prior to pairing, through the pairing and gestation periods unlit the F1 

generation reached day 4 post partum. 

Administrations at 1000 mglkg body weight/day caused a dark discoloration of feces In all animals after the onsat of the treatment until the end of sllldy. This was Nkely due to the 

staining property of the test item and not considered to be adversa. 

Food consumption and body weight ware not affected for the entire duration of the sllldy. The evaluation of the functional battery observations and the assassment of clinical laboratory 

Investigations parameters did not reveal any test Hem-related effect. 

All the changes notad in the organ weights were within the bacl<ground of the biological variation. 

No adversa findings were noted during macroscopical and histopathology examinations. 

Based on thesa resuhs a general NOAEL (No Observed Adverse Effect Level) was established at 1000 mglkg body weight/day. 

Executive summary 

The purpose of this sllldy was to generate pretimlnary information on the possible health hazards likely to arise from repaatad exposure over a relatively limited pariod of time. In 

addHion H provided information on possible eflecls on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus 

andparlllrltion. 

This sllldy should provide information to assess the need to conduct furlher investigations and may provide guidance in the design of subsequent studies. 

Dose levels were seleclad in agreement wHh the Sponsor, based on the resuhs of a dose range-finding sllldy (Harlan Laboratories Sllldy C31076). 

Leuco Sulfur Black 1 was administered to male rats lor 29 days and to female rats for 14 days prior to pairing, through the pairing and gestation parlods until the F1 generation reachad 

day 4 post parlllm. 

The following doss levels were applied: 

Group 1: 

Group2: 

Group3: 

Group4: 

0 mglkg body weight/day (control group) 

100 mglkg body weight/day 

300 mglkg body weight/day 

1000 mglkg body weight/day 

A standard dose volume of 10 mUkg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water). 

The following results were obtained: 
Gene,.l T olerllblllty 

All the animals survived until the scheduled necropsy. AI t 000 mglkg body weightiday, a dati< discoloration of feces was obsarved in all animals starting on day 3 of the pra-pairing 

period onwards. 

Functional Observetlon.ol 8ettery 

No test item-related effects ware noted. 

Food Consumption 

Food consumption was not affected by the treatment with the test ~em in males and females. 

Body Weights 

No test Item-related effects ware noted in body weight and body weight gain in males and females for the entire duration of the study. 

Clinical Laboratory Investigations 

The assessment of the clinical laboratory investigations parameters did not give indication of any test ijem-related effect. 

Reproduction and BIHdlng Oats Mean precoital time, fartll~ and gestation indax and conception rate were not affected by the treatment with the test Ham. 

Implantation rate and post implatation loss ware also not alfected by the treatment with the test item. 

Orvan Weights 

Weight of organs was not alfected by the treatment with the test ijem. All the alterations noted ware within the range of historical control data. 

Macroac:oplclll Findings and Hlatopathologlcal Eumln.otlons 

No adverse effects of the test Hem were noted during the histopathological examination. 

Conclusion 

Basad on thesa resulte a general NOAEL (No Obsarved Adverse Effect Level) was established at 1000 mglkg body weight/day. 
Endpoint study record: waiving_Repeated dose toxicity 

UUID IUC5-e66359e9-ad84-4e86-a489-5011b522b4bc 

Dossier UUID IUC5-292149d1-a677-4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-21 13:43:48 CEST 

Administrative Data 

Datllwalvlng 

Justification 

forwaiving 

study scientifically unjustified 

In accordance with Regulation (EC) No 190712006 Annex XI, a subchronic toxicity study is scientificaHy not justified and, thus, should not be conducted because of the 

following: 

Leuco Sulfur Black 1 has a mean molecular weight in the range of 8000 to 9000 Da and a number-average molecular weight (Mn) of about3000 Da. It is very poorly 

or even no1 soluble in common polar and non-polar solvents such as water (solubility of 62.4 mg/L), DMSO, methanol, DMF, THF, chloroform and no1 soluble In acids 

Hke sulfuric acid and trlftuoroacelic acid. The vapour pressure of the pure substance, calculated from the boiling point of> 400 'C, is 1.8 x 10E-5 hPa at25 'C. 

Based on these properties II is very unlikely that Leuco Sulfur Black 1 can cross any biological membrane. Thus, an absorption of toxicologically significant amounts of 

Leuco Sulfur Black 1 following oral, dermal or inhalation exposuns can be excluded. This Is substantiated by the results obtained in toxicity and ecotoxicity studies, 

whans no substance related local and/or systemic effects indicative of toxicity wens seen in all tests, including acute animal and acute ecotoxicity studies, sensKization, 

repeated dose toxicity and mutegenlcKy tests. 

In the acute oral toxicity study black stained faeces was observed for maximally four days starling the day after administration of the substance al2000 mgl1<g bw. 

Dart< discoloured faeces wens also seen in the repeated dose toxicKy study (OECD 422) at1000 mgl1<g bwld, starling on day 3 of the study and lasting throughout the 

administration period until termination. The dlscolouration of faeces was tha only effect observed in both studies and can be attributed to the strong staining properties 

of the substance. This indicates that Leuco Sulfur Black 1 is directly excreted and that absorption in tha gastrointestinal tract does not occur following oral 

administration, especially since organs or urine wens not discoloured and signs of substance deposits were nof found in the histopathological analysis of the organs. 

In conclusion, the available toxicity and ecotoxicity data together with the intrinsic substance properties are sufficient to demonstrate that exposure to Leuco Sulfur 

Black 1 does not cause any haaRh concern. 
7 .5.2 Repeated dose toxicity: dermal 

Endpoint study record: waiving_Repeated dose toxicity: derma/.001 

UUID IUC5-6e0385ef-4a64-48c4-9680-a0774b47064c 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1 fad973281 ea 

Author thr I SCC GmbH I Wendelsheim I Germany 

Date 2010-05-26 16:35:53 CEST 

Remarks 

Administrative Data 

Data wslvlng siUdy scientifically unjustified 

Justification for data wslvlng see dela waiving under repealed dose loxicity: orel (CSR section 5.6.1.1) 
7 .5.3 Repeated dose toxicity: inhalation 

Endpoint study record: waiving_ Repeated dose toxicity: inhalation.001 

UUID IUC5-7e173fd7 -29cf-4 711-ba 10-1 e 736b54465d 

Dossier UUID IUC5-292149d1-a6n -4feb-9692-1fad973281 ea 

Author lhr I SCC GmbH I Wendelsheim I Germany 

Date 2010-05-26 16:36:42 CEST 

Remarks 

Administrative Data 

Data waiving sludy scientifically unjuslifoed 

Justlllcatlon for data waiving see dala waiving under repeated dose toxicity: oral (CSR section 5.6.1.1) 
7.6 Genetic toxicity 

Endpoint summary: Genetic toxicity 

UUID IUC5-455d61 b4-0863-40ee-bcd0-03c1 ae1 075b 7 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1 fad973281 ea 

Author lhr I SCC GmbH I Wendelsheim I Germany 

Date 2010-05-26 16:46:32 CEST 

Remarks 

Administrative Data 

Short description of key information 

Leuco Sulfur Black 1 was neither mutagenic In lhe Ames lest nor in the HPRT assay on V79 calls and was non-dastogenic In the in vitro chromosome aberration assay using V79 cells. 

All assays were performed with and without metabolic activation sys1am. 

Key value for chemical safety assessment 

Genetic toxicity 

negative 

Discussion 

An aqueous Leuco Sulfur Black 1 formulation containing less than typical sulfur dye concentration (30.2% instead of 52.75 

%) was tested in the bacterial reverse mutation assay with Salmonella typhimurium strains TA1535, TA1537, TA1538, 

TA98 and TA 100 using the plate incorporation method at five dose levels, in triplicate, both with and without a metabolic 

activation system (59 mix from induced rat liver). Two independent experiments were conducted. The assay was 

performed according to OECD test guideline No. 471 and GLP. The dose range was 312.5 to 5000 ~g/plate. Distilled water 

was used as solvent 
The concurrent positive controls gave increases in revertants within the expected ranges. 

The test item did not cause toxicity. Precipitations occurred at and above 6251Jg test item/plate. No significant increase in 

the number of revertants was recorded for any of the bacterial strains with any dose of the test item either with or without 

metabolic activation. 

In this assay Leuco Sulfur Black 1 precipitated at and above test item concentrations of 625 IJg/plate, demonstrating that 

the highest possible Leuco Sulfur Black 1 concentrations which can be studied in the Ames test were achieved. Thus, the 

assay is fully valid for the assessment of the mutagenic potential of Leu co Sulfur Black 1. 

Leuco Sulfur Black 1 was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the 

Chinese hamster according to OECD test guideline No. 476 and GLP. The study was performed in two independent 

experiments. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second 

experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. 

Precipitation of the test item was observed at 1251Jg/ml and above with and at 250 IJg/ml and above without metabolic 

activation. 

Relevant cytotoxic effects defined as a relative cloning efficiency I below 50% in both parallel cultures occurred at 500 

IJg/mL in both experiments without metabolic activation. In the presence of metabolic activation cytotoxic effects as 

described above were noted at 125 IJg/ml and above in the first experiment. 

The positive controls ethylmethanesulfonate and DMBA showed a distinct increase in induced mutant colonies. 

No relevant and reproducible increase in mutant colony numbers/10E6cells was observed in the main experiments up to 

the maximum concentration. All mutant frequencies remained well within the historical range of solvent controls. No 

significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any 

of the experimental groups. 

In conclusion, Leuco Sulfur Black was not mutagenic in theHPRTassay with and without metabolic activation. 

Leuco Sulfur Black 1, suspended in deionised water, was assessed for its potential to induce structural chromosome 

aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments according to OECD test guideline 

No. 473 and GLP. 

In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated for structural 

chromosome aberrations. Cells were exposed to the test item for 4 hours with and without S9 in experiment I and again for 

4 hours with S9 and continuously for 18 hours in experiment II. Dose selection for the cytogenetic experiments was 
performed considering the toxicity data and the occurrence of precipitation. The chosen treatment concentrations were 

between 2.3 and 300 ~g/ml (with and without S9). The evaluated concentrations were 19.5, 39.1 and 78.1 ~g/ml in 

experiment I and 37.5, 75 and 150 ~g/ml in experiment II. 

In the absence and presence of S9 mix no cytotoxicity was observed up to the highest evaluable concentration. Due to 

strong test item precipitation concentrations higher than those examined could not be evaluated for cytogenetic damage. 

No clastogenicity was observed at the concentrations evaluated either with or without metabolic activation. However, in 

experiment II in the presence of S9 mix a single statistically significant increase in the number of aberrant cells excluding 

gaps (3.0 %) was observed at the highest evaluable concentration (150 ~g/ml). This value was dearly in the laboratory's 

historical solvent control data range (0.0- 4.0 % aberrant cells excluding gaps) and is therefore regarded as biologically 

irrelevant. 

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the 

control cultures. 

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells 

with structural chromosome aberrations. 

In conclusion, the test item was not clastogenic in this in vitro chromosome aberration assay. 

Justification for classification or non-classification 

The available data demonstrate that Leuco Sulfur Black 1 has no genotoxic potential. Thus, it is not subject to classification 

and labelling according to Directive 67/548/EECand Regulation 1272/2008/EC. 
7 .6.1 Genetic toxicity in vitro 

Endpoint study record: key_ 471_Safepharm_1991 

UUID IUC5-ac176bd8-fc03-4e62-a18e-3a2266ab21eb 

Oosaler UUID IUC5-292149d1-a677-4feb-9692-1fad973281ea 

Author 

Date 

Remarks 

thr I SCC GmbH I Wendelsheim I Germany 

2010-05-26 16:44:32 CEST 

Administrative Data 

Purpose flag key study 

Study F8sult type experlmentel resun 

Reliability 1 (reiable without restriction) 

Data source 

Reference 

Reference Author Veer Title 

type 

study report Jenkinson 1991 Leuco sulphur black 1: reverese mutation 

PC assay "Ames test"' using salmonella 

typhimurium 

Data access 

data submrtter is data owner 

Materials and methods 

Type of genotoxicity 

gene mutation 

Type of study 

Bibliographic 

oource Teeling laboratory R_.t Owner Company Report no. company study no. data 

Safepharm Laboratories 4711657 Clarlant 1991-02-

Ltd. Derby, DE1 2BT, UK 14 
bacterial reverse mutation assay (e.g. Ames test) 

Test guideline 

Guideline 

DECO Guideline 471 (Bacterial Reverse Mutation Assay) 

yes (Ind. certificate) 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

no 

Test material identity 

Identifier 

other: Leuco Sulfur Black 1 formulation see confidential details on - material 

Confidential details on test material 

Leuco Sulfur Black 1: 30.2 % Sodium hydroxide: 2.7% 

Sodium sulfide: 3.6 % Inorganic sodium salts: 3.9 % 

water: 59.6% 

Method 

Species/strain 

Species/strain other: TA1535, TA1537, TA1538, TA98, TA100 

Additional not applicable 

atreln 

ch•racterlatlca 

Met.obollc with and w~hout 

IIC!Iv•Uon 

Met.lbollc S9 mix derived form Aroclor 1254 inducad rat livers 

•ctlvatlon 

ayatem 
Test concentrations 

0, 8, 40, 200, 1000 and 5000 ~g/plate (experiment I, with and without S9 mix) 

0, 312.5, 625, 1250, 2500 and 5000 ~glplate (experiment II, with and without S9 mix) 

Vehicle 

sterile distilled water 

Controls 

Negative no 

controls 

Solvent I yes (distilled water) 

vehicle 

controls 

True no 

negative 

controls 

Positive yes 

controls 

Positive other. -S9 mix: ENNG (TAtOO, TA1535), 9-AA (TA1537), 4-NOPD (TA1538), 4-NQO ( TA98): +S9 mix: 2-AA (TA1535), BP (al other strains) 

control 

substance 

Details on test system and conditions 

Five concentrations of the test Hem ware assayed in triplicate against each tester strain, using the direct plate incorporation method. 

0.1 mL of the diluted test Hem or negative control solution was added to 2.0 ml of trace supplemented top agar at 45'1:: In sterile tubes. A 0.1 ml aliquot of bacterial suspension and 0.5 

ml of the S9 liver microsome mix or buffer was added. The mix was overlaid onto sterile plates of Vogei-Brunner agar. After approximately 48 hours at 37"C the plates ware scored for 

revertant colonies and thinning of bad<ground lawn 

Evaluation criteria 

For a substance considered to be positive, H should have induced a dose-related and statistically significsnl increase In the mutation rate in one or more strains. To be considered 

nagative the numbr of reveriatns compared to spontaneous reveriants should be less than twofold at each dose level. 

Statistics 

All data are statistically analysed using the methods recommendad by UKEMS (Kirkland OJ (ed) Statistical evaluation of mutagenicHy test data. Reprot- part Ill (1989)- Cambridge 

universHy press) 

Results and discussions 
Test results 

Species/strain other. TA1535, TA1537, TA1538, TA98, TA100 

Metabolk: with and without 

IICIIvatlon 

Test al strains/cell types tested 

system 

Genotoxlclty negative 

Cyt-xlclty no, but tested up to precipitating concentrations 

Vehicle yes 

controls 

valid 

Negatlve not applicable 

controls 

valid 

Positive yes 

controls 

vaHd 

Add/tiona/Information on results 

No toxicity was exhibited. Precipitation was observed with doses at and above 625 ~glplate. this did not interfere with the scoring of revertant colonies. No significant increases in the 

numbers of revertant colonies were recorded for any of the strains of Selmonelly used at any dose level etther wtth or without S9. 

Applicant's summary and conclusion 

Executive summary 

An aqueous Leuco Sulfur Black 1 formulation containing less than typical sulfur dye concentration (30.2% instead of 52.75 

%) was tested in the bacterial reverse mutation assay with Salmonella typhimurium strains TA1535, TA1537, TA1538, 

TA98 and TA 100 using the plate incorporation method at five dose levels, in triplicate, both with and without a metabolic 

activation system (S9 mix from induced rat liver). Two independent experiments were conducted. The assay was 

performed according to OECD test guideline No. 471 and GLP. The dose range was 312.5 to 5000 IJg/plate. Distilled water 

was used as solvent. 

The concurrent positive controls gave increases in revertants within the expected ranges. 
The test item did not cause toxicity. Precipitations occurred at and above 625 j..lg test item/plate. No significant increase in 

the number of revertants was recorded for any of the bacterial strains with any dose of the test item either with or without 

metabolic activation. 

In this assay Leuco Sulfur Black 1 precipitated at and above test item concentrations of 625 1Jg/plate, demonstrating that 

the highest possible Leuco Sulfur Black 1 concentrations which can be studied in the Ames test were achieved. Thus, the 

assay is fully valid for the assessment of the mutagenic potential of Leuco Sulfur Black 1. 
Endpoint study record: 1234903_HPRT 

UUID IUC5-7f0c301 e-409c-4381>-a151-edcd5b837397 

Dossier UUID IUC5-292149c1-a677-4feb-9692-1fad973281ea 

Author 

Date 

Remarks 

thr I SCC GmbH I Wendelsheim I Germany 

2010-05-26 16:43:42 CEST 

Administrative Data 

Purpose flag 

Study result type 

Reliability 

key study; robust study summary 

experimental resuH 

1 (reliable without restriction) 

Rationale for reliability Incl. deflclencleo according to OECD 4 76 

Data source 

Reference 

Reference Author Year 

type 

study report Wollny 2009 

H-E 

Data access 

data submitter is data owner 

Data protection claimed 

yes. but wUiing to share 

Title 

Gene Mutation Assay In Chinese Hemster 

V79 Cells in vitro ty79 I HPRT) 

Materials and methods 

Type of genotoxlclty 

gene mutation 

BlbHognphlc T•tlng laboratory Report Owner Compeny Report 

source no. compeny study no. date 

Her1an Cytolest Cell C31133 Clarient 1234903 2009-00- Research GmbH 07 
Type of study 

mammaUan cell gene mutation assay 

Test guideline 

Guideline 

OECD Guideline 4 76 (In vitro Mammalian Cell Gene Mutation Test) 

yes (Ind. certificate) 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read..across) 

yes 

Details on test material 

ldenlification: Leuco Sulfur Black 1 

CAS No.: 66241-11-0 

Description: Thixotrophlc, blacl<, wet paste 

Belch No.: ESA0007595 

Composition: Leuco Sulfur Blacl< 1: 52.75 % (calculated by amount of by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-0ini1rophenole: < 20 ppm 

Sulfale: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (w/w) 

Water: 45.75% (w/w) by Kar1-Fischer titration 

Molecular Weight: Not applicable, because test Kern is an UVCB substance 

Stablity of Test nem: Stable under storage oondKions 

Storage: At room temperatura (range 20 +1- 5"C), I ight protected 

Expiry Date: July 11, 2011 

On the day of the experiment (immediately before treatment), the test Kern was suspended in deionised water. The final concentration of daionised water in the culture medium was 10 

%vlv. 

Method 

Target gene 

HPRT 
Species/strain 

Species/strain Chinese hamster lung fibroblasts (V79) 

Details -Type and identity of media: MEM 

on - Periodically checked for Mycoplasma contamination: yes 

rnamrnallen - Periodically checked for karyotype stability: yas 

cell - Periodically "cleansed" against high spontaneous background: yes 

lines 

(If 

-llceble) 

Metabolic 

activation 

with and whhout 

-bollc Rat liver S9 

activation 

sysam 

Test concentrations 

Experiment 1: 

wfthoul S9 mix: 31.3; 62.5; 125.0; 250.0; and 500.0 ~g/ml 

wfth S9 mix: 31.3; 62.5; 125.0; 250.0; and 500.0 ~g/ml 

Experiment II: 

without S9 mix: 31.3; 62.5; 125.0; 250.0; and 500.0 ~g/ml 

with S9 mix: 31.3; 62.5; 125.0; 250.0; and 500.0 ~g/ml 

Vehicle 

- Vehide(s)lsolvent(s) used: water 

-Justification for choice of solvent/vehicle: solub!Rty properties 

Controls 

Negative no 

controls 

Solvent/ yes 

vehicle 

controls 

True no 

negllllve 

controls 

Poeltlve yes 

controls 
Positive ethytmethanesulphonate 

control 

oubslllnce 

Remarko without metabolic activation 

N-tlve no 

controll 

Solvent I yas 

vehicle 

controls 

True no negative 

controll 

Poolllve yes 

controls 

Positive 7 ,12-dimelhytbenzanthracene 

control 

oubslllnce 

Rem.orko with metabolic activation 

Details on test system and conditions 

METHOD OF APPLICATION: in medium 

DURATION 

-Exposure duration: experiment 1: 4 hours with and without metabolic activation 

experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation 

-Expression time (cells in growth medium): 72 hours 

- Selection time (if incubation with a saleclion agent): 1 0 days 

SELECTION AGENT (mutation assays): Thioguanine 

NUMBER OF REPLICATIONS: 2 

NUMBER OF CELLS EVALUATED: >1.5x10exp.6 

DETERMINATION OF CYTOTOXICITY 

-Method: cloning efficiency 
Evaluation criteria 

A test ttem producing neither a concentration-related Increase of the mutant frequency nor a reproducible positive response at any of the test points Is considered to be non-mutagenic 

in this system. 

A mutagenic response is described as follows: 

The test ttem Is classified as mutagenic n tt Induces reproducibly with one of the concen"'lnltlons a mutation frequency that Is three times higher than the spontaneous mutation 

fre""quency in the experiment. 

The test ttem Is desslfied as mutagenic If there is a reproducible concentration-related Increase of the mutation frequency. Such evaluation may be considenad also in tha case lhet a 

threefold increase of the mutant frequency Is not observed. 

In a case by case evaluation this decision depends on the level of the correspon"'Cling solvent control data. 

Stet/sties 

Linear regression analysis (least squares) . 

Results and discussions 

Test results 

Speclealatraln Chinese hamster lung fibroblasts (V79) 

Metabolic with and without 

actiYIIIlon 

Genotoxiclly negative 

Cytotoxicity yes at 500 ~g/ml (-59, both experiments), and>= 125 ~g/ml (+S9, 1st experiment) 

Vehicle yes 

controls 

valid 

Positive yes 

controls 

valid 

Additional lnfonnation on results 

TEST-SPECIFIC CONFOUNDING FACTORS 

- Effects of pH: not effected 

- Effects of osmolaltty: not Increased 

- Water solubility: soluble 

-Precipitation: precipttatlon at 250 ~g/ml and above after 4 hours treatment without metabolic activation. at 125 ~g/ml and above after 4 hours treatment with metabolic activation. at 

125 ~g/ml and above after 24 hours treatment without metabolic activation. 

-Other confounding effects: none 
RANGE-FINDING/SCREENING STUDIES: 

The concentration range of the pre-lest was llm~ed by the solubil~ of the test item in water and aqueous media. Test ttem concentrations between 3.9 and 500 ~glmL were used to 

evaluate toxicity In the presence (4 h treatment) and absence (4 hand 24 h treat-ment) of metabolic activation. Relevant cytotoxic effects were noted at250 and 500 ~glmL without 

metabolic activation. In the presence of metabolic activation no cytotoxic~ oc-rurred up to the rneximum concentration. 

The last medium of the pre-experiment was checked lor precipitation at the end of each treatment period (4 or 24 hours) just prior to removal of the test ~em. Precipitation of the test 

ttem visible to the unakled eye occurred in at 125 ~glmL and above lolowing 4 hour treatment- and -out metabolic activation. Following continuous traatrnent lor 24 hours wtlhout 

metabolic activation precipitation occurred at250 ~glmL and above. 

COMPARISON WITH HISTORICAL CONTROL DATA: controls -in historical range 

ADDITIONAL INFORMATION ON CYTOTOXICITY: 

Relevant cytotoxic effects defined as a relative doning efficiency I below 50% In both parallel cultures occurred at 500 ~glmL in both experiments wtthout metabolic activation. In the 

presence of metabolic activation cytotoxic effeds as described above were noted at 125 ~glmL and above in the first experiment. 

Any other Information on results Incl. tables 

Summary Table 

relative relative mutant relative relative mutant 

cone. ~g 59 doning doning colonies/ induction doning doning colonies/ induction 

permL mix efflciency I efficiency II 1o"ce11s lador efficiency I effidency II 106cells factor 

% % % % 

column 3 4 5 6 8 9 10 

Experiment I, 4 h treatment culture I culture II 

Solvent control with water 100.0 100.0 25.8 1.0 100.0 100.0 9.4 1.0 

Positive control with EMS 150.0 116.4 85.6 133.3 5.2 102.3 79.9 110.0 11.7 

Test Item 15.6 97.1 cuhure was not continued' 84.6 culture was not continued" 

Test item 31.3 71.3 105.4 14.5 0.6 90.1 82.9 15.8 1.7 

Testttem 62.5 59.3 105.4 12.0 0.5 98.2 85.8 27.7 3.0 

Testttem 125.0 59.4 103.2 11.9 0.5 70.8 76.4 28.7 3.1 

Test~em 250(p) 44.2 102.1 15.7 0.6 84.2 84.5 24.2 2.6 
TestKem SOO(p) 29.3 81.3 18.4 0.7 37.0 60.3 23.9 2.5 

Solvent control wilh water 100.0 100.0 29.1 1.0 100.0 100.0 18.1 1.0 

Positive control wilh DMBA 1.1 45.9 92.6 891.7 30.6 40.6 71.2 1421.6 78.6 

Test ~em 15.6 67.0 cunure was not contin~ 76.3 culture was not contin~ 

Test ~em 31.3 70.8 85.1 13.3 0.5 67.6 90.6 17.8 1.0 

Test ~em 62.5 50.5 83.7 19.5 0.7 39.0 90.3 18.5 1.0 

Test ~em 125 (p) 44.0 95.1 13.5 0.5 36.2 88.9 10.6 0.6 

Test ~em 250 (p) 32.8 83.3 22.6 0.8 24.1 93.9 21.9 1.2 

Test~em 500(p) 39.9 107.4 23.3 0.8 24.8 72.5 18.5 1.0 

Experiment II, 24 h treatment culture I cunurell 

Solvent control wilh water 100.0 100.0 17.8 1.0 100.0 100.0 15.8 1.0 

Positive control wKh EMS 75.0 107.3 79.4 255.7 14.4 98.0 86.7 383.9 24.2 

Test Hem 15.6 109.5 cunure was not contin~ 105.4 rutture was not contlnued11 

Test Ham 31.3 109.4 83.2 22.9 1.3 111.6 85.9 20.1 1.3 

Test item 62.5 94.9 62.9 19.1 1.1 96.3 71.5 35.7 2.3 

TestKem 125.0 102.3 73.8 23.1 1.3 100.5 91.5 38.1 2.4 

Tes!Kam 250 (p) 62.2 77.6 18.4 1.0 76.8 88.3 32.5 2.1 

TestKam SOO(p) 16.9 83.2 12.4 0.7 9.4 70.1 19.0 1.2 

Experiment II, 4 h treatment 

Solvent control wilh water 100.0 100.0 15.2 1.0 100.0 100.0 11.1 1.0 

PO&Kive control wilh DMBA 1.1 36.5 88.1 744.4 48.9 24.2 91.0 841.6 75.6 

Test ~em 15.6 106.3 cunure was not contin~ 82.8 cullure was not continued~ 

Test Hem 31.3 + 94.5 90.1 20.7 1.4 78.9 119.6 12.2 1.1 

TestKem 62.5 + 82.2 85.1 15.3 1.0 77.1 100.3 18.0 1.6 

Test Hem 125 (p) 78.5 92.8 12.6 0.8 68.2 79.7 23.3 2.1 
Test Kern 250 (p) + 79.9 89.8 16.9 1.1 54.2 61.8 

Test Kern 500 (p) 74.4 95.0 21.4 1.4 54.9 88.4 

# culture was not continued since a minimum of only four analysable concentrations is required 

P = precipitation 

Applicant's summary and conclusion 

Conclusions 

Conclusion: 

22.3 

12.1 

2.0 

1.1 

In conclusion it can be stated that under the ex~perimental conditions reported the test nem did not Induce gene mutations at the HPRT locus In V79 a>lls. Therefore, Leuco Su~ur 

Blacl< 1 is considered to be non-mutagenic in this HPRT assay. 

Executive summary 

The test item Leuco Sulfur Black 1 was assessed for its potential to induce gene mutations at the HPRT locus using V79 

cells of the Chinese hamster. 

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment 

the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a 

treatment time of 4 hours with and 24 hours without metabolic activation. 

Precipitation of the test item was observed at 125 J.lg/ml and above with and at 250 J.lg/ml and above without metabolic 

activation. 

Relevant cytotoxic effects defined as a relative cloning efficiency I below 50% in both parallel cultures occurred at 500 

J.lg/ml in both experiments without metabolic activation. In the presence of metabolic activation cytotoxic effects as 

described above were noted at 125 J.lg/ml and above in the first experiment. 

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the 

maximum concentration. All mutant frequencies remained well within the historical range of solvent controls. The induction 

factor was equal to or exceeded slightly the threshold of three times the corresponding solvent control in the second 

culture of experiment I without metabolic activation at 62.5 and 125.0 J.lg/ml (induction factor of 3.0 and 3.1, respectively). 

This marginal effect was judged as biologically irrelevant since it was based upon the rather low solvent control of 9.4 

mutant colonies/1 o6cells. Furthermore, the increase was not reproduced in the parallel culture under identical conditions 

and not dose dependent as indicated by a lacking statistical significance 
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant 

frequencies using SYSTATastatistics software. No significant dose dependent trend of the mutation frequency indicated by 

a probability value of <0.05 was determined in any of the experimental groups. 

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.4 up to 29.1 

mutant colonies per 1 o6cells; the range of the groups treated with the test item was from 11.9 up to 38.1 mutants per 

106cells. 

Ethylmethanesulfonate (0.15 mg/ml in experiment I and 0.075 mg/ml in experiment II) and DMBA (1.1 IJg/mL) were used 

as positive controls and showed a distinct increase in induced mutant colonies. 
Endpoint study record: 1234902_CA79 

UUID IUC5-8c54462c-9cd7-434c-be03-8d6abcd1da8a 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-11ad973281 ea 

Author 

Date 

Remart<a 

thr I SCC GmbH I Wendelsheim I Germany 

2010-05-26 16:45:11 CEST 

Administrative Data 

Purpose flag 

Study result type 

key study; robust study summary 

experimental resutt 

RellabiiHy 1 (reliable without restriction) 

Rllllonale for reliability Incl. deficiencies According to OECD guideline 4 73 

Data source 

Reference 

Referenca Author Year 

type 

study report HaiiC 2009 

Data access 

data submitter is data owner 

Data protection claimed 

yes. but willing to share 

Title 

In Vitro Chromosome Aberration Test in 

Chinese Hamster V79 Cells with Leuco Sulfur 

Black 1 

Materials and methods 

Type of genotoxicity 

Bibliographic Testing laboratory Report Owner Company Report 

sou rca no. company study no. date 

Harlan Cytotest Cell Research C31122 Clariant 1234902 20CMHl7-

GmbH. Rossdorf (Garmany) 07 
chromosome aberration 

Type of study 

in vitro mammalian chromosome aberration test 

Test guideline 

Guideline 

OECD Guideline 473 (In llilro Mammalian Chromosome Aberration Test) 

yes (inct. certificate) 

Test materials 

Identity of test material same as for substance defined In section 1 (If not read-across) 

yes 

Method 

Target gene 

Chromosome aberration 

Species/strain 

Species/strain Chinese hamster lung fibroblasts (V79) 

Details 

on mammalian 

cell 

lines 

(If 

-llcloble) 

Met8bollc 

actiYatlon 

Thawed stock cukures were propagated at 37 'C in 8 0 em' plastic flasks (Greiner, 72632 Fricl<enhausen, Germany). About 5 x 105 cells per flask were seeded into 15 

mL of MEM (Minimal Essential Medium; Invitrogen Gibco, 76131 Ka~sruhe, Germany) supplemented with 10% fetal calf serum (FCS; Invitrogen Glbco, 76131 

Ka~ruhe, Germany). Additionally, the medium was supplemented with 1 % 100x Penicilin/ Streptomycin solution (10.000 Unlls/ml PenicHiin, 10 mg/ml Streptomycin; 

PAA Laboretories GmbH, 35091 COibe, Germany) and 1 %Amphotericin B-solution (250 ~g/ml, PAA Laboretories GmbH, 35091 COibe, Germany). The cells were 

sub-cultured twice weekly. The cell cullures were incubated at 37 'C In a humidified atmosphere with 1 .5 %carbon dioxide (98.5% air). 

-Periodically checl<ed for Mycoplasma contamination: yes/no 

- Periodically checked for karyotype stability: yes/no 

with and without 

-bollc rat liver S9 

8CIIY811on 

system 

Test concentrations 
Without S9 mix: 

19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250,2500 and 5000 ~glml 

2.3, 4.7, 9.4, 18.8, 37.5, 75, 150 and 300 ~glml 

WothS9mlx: 

19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500and 5000 ~glml 

2.3, 4.7, 9.4, 18.8, 37.5, 75, 150 and 300 ~glml 

Vehicle 

- Vehicle(s)/solvent(s) used: deionlsed water 

-Justification for choice of solwmt/vehicle: solubi6ty (OECD Guideline 473) 

Controls 

~no 

controls 

Solvent I yes (deionised water) 

vehicle 

controls 

True no 

negative 

controls 

Positive yes 

controls 

Positive ethylmethanesulphonate (cylophosphamide) 

control 

subelllnce 

RIII1Uirks in tha absence or presence of S9 

Details on test system and conditions 

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 

hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test Kern. 

METHOD OF APPLICATION; in minimal essentila medium 

DURATION 

- Exposure duration: 4 and 18 hours 

-Expression time (cells in growth medium): 18 hours 

-Fixation time (start of exposure up to fixation or harvest of cells): 18 hours 

SELECTION AGENT (mutation assays): no selection agent 
SPINDLE INHIBITOR (cytogenetic assays): colcemid 

STAIN (for cytogenetic assays): Giemsa 

NUMBER OF REPLICATIONS: 2 parallel cuKures in each experimental group 

NUMBER OF CELLS EVALUATED: 100/cuKure 

DETERMINATION OF CYTOTOXICITY 

-Method: mHotic index; cloning efficiency; relative total growth 

OTHER EXAMINATIONS: 

- Determination of polyploidy: no evidence of an increase in the rate of polyploid cells 

- Detennination of endorepUcation: no evidence in the rate of endomitotic cells 

OTHER: none 

Evaluation criteria 

Evaluation of the cultures was performed (according to standard protocol of the "ArbeHsgruppa der lndustrie, Cytogenetik") using NIKON miaoscopas with 1 OOx objectives. Breaks, 

fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps-recorded as -II but not inciuded in the calculation 

of the aberration rates. 100 -• spread metaphases par cuKure -rescored for cytogenetic demage on coded slides. 

Only metaphases with characteristic chromosome numbers of 22 ± 1 were induded in the analysis. To describe a cytoloxic eflect the mHotic index (% cells in mitosis) was determined. 

In addition, the number of polyploid cells In 500 metaphases par cuKure was determined (% polyploid metaphases; in the cased this aneuploid cell line polyploid means a near 

tetraploid karyotype). 

Statistics 

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). 

Any other Information on materials and methods Incl. tables 

Phenobarbitallb-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared 

from 8- 12 weeks old maleWistar rats (HsdCpb:WU, Harlan Laboratories GmbH, 33178 Borchen, Germany)weight approx. 

220 - 320 g induced by intraperitoneal applications of 80 mg/kg b.w. Phenobarbital (Desitin; 22335 Hamburg, Germany) 

and by peroral administrations of80 mglkgb.w.b-Naphthoflavone(Sigma-Aidrich Chemie GmbH, 82024 Taufkirchen, 

Germany) each, on three consecutivedays. The livers were prepared 24 hours after the last treatment. The S9 fractions 
were produced by dilution of the liver homogenate with a KCI solution (1 :3 parts) followed by centrifugation at 9000 g. 

Aliquots of the supernatant were frozen and stored in ampoules at --80 'C. Small numbers of the ampoules were kept at20 'C for up to one week. 

The protein concentration was 35.0 mg/ml (Lot no. 2111 08). 

Results and discussions 

Test results 

SpeciH/atrllln Chinese hamster lung fibroblasts (V79) 

Metabolic with and without 

IICitvllllon 

T•t 

system 

Genotoxtclty 

C~xlclty 

Vehicle 

controls 

valid 

an strains/cell types tested 

negative 

no. but tested up to precipitating concentrations 

yes 

Positive yes 

controls 

valid 

Additional Information on results 

The test item Leuoo Sulfur Black 1, suspended in deionised water, was assessed for its potential to induce structural duomosome aberrations in V79 cells of the Chinese hamster in 

vitro in the absence and presence of metabolic activation by 59 mix. 

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 

hours with 59 mix and 18 hours without 59 mix. The chromosomes were prepared 18 hours after start of treatment with the-item. 

In each experimental group two perellel cukures were set up. 100 metaphases per cukure were evaluated for structure! chromosome aberrations. 

In both experiments no relevant influence of the test item on pH value or osmolarky was observed (Exp. 1: solvent control286 mOsm, pH 7.4 versus 285 mOsm and pH 7.4 at 5000 

~g/ml: Exp. II: solvent control284 mOsm, pH 7.4 versus 290 mOsm and pH 7.4 at 300 ~glml). 

Precipitation of the test item in cukure medium was observed after treatment with 78.1 ~g/ml and above In the absence and presence of 59 mix in Experiment I and with 150 ~g/ml and 

above In the absence and presence of 59 mix in Experiment II. 

No clear toxic effacts indicated by reduced mHotic Indices or reduced cell numbers were observed after treatment with the test Hem (see Table 2, 3 and 6, pages 25 to 27 and 30). Due 

to strong test kern precipitation concentr&tions higher than those examined could not be evaluated for cytogenetic damage. 

In both experiments in the absence and presence of 59 mix no biologlcaUy relevant increase In the number of cells carrying structural chromosome aberrations was observed (see 

Table 4, 5, 7 and 8, pages 28, 29, 31 and 32). The aberration rates of the cells after treatment with the- Hem (0.0 • 3.0 %aberrant cells. excluding geps) were dose to the range of 
the solvent control values (0.5- 2.0 % aberrant ceHs. excluding gaps) and within the range of the laboratOf}''s historical solvent control data (0.0- 4.0 %aberrant cells, excluding gaps). 

A single significant inaease was observed In Experiment II in the prasence of 59 mix after treatment with 150 ~g/ml. Although this increase of 3.0 % aberrant cells excluding gaps was 

statistically significant compared to the low response (0.5% aberrant cells) in the solvent control data, the response is within the historical control data range (0.0- 4.0% aberrant cells, 

excluding gaps). Therefore, this observation has to be regarded as biologlcaly Irrelevant. 

No evidence of an increase in polyploid melaphases was noticed after treatment with the test item as compared to the controls. 

In both experiments, either EMS (400 or 1000 ~g/ml) or CPA (1.4 ~glmL) were used as positive controls and showed distinct inaeases in the number of cells with structural 

chromosome aberrations. 

In conclusion, H can be stated that under the experimental condHions reported, the test item Leuco Sulfur Black 1 did not induce structural chromosome aberrations in V79 cells 

(Chinese hamster eel line) when tested up to the highest evaluable concentrations. 

Any other Information on results lncl- tables 

Summary of results of the chromosome aberration study with Leuco Sulfur Black 1 

Exp. Preparation Test item Cell numbers Mitotic indices Aberrant celts 

interval concentration in% in% in% 

in !Jg/ml of control of control incl. gaps• exd. gaps• with 

excha es 

exposure period 4 hrs without 59 mix 

18 hrs Solvent control1 100.0 100.0 1.5 1.0 0.0 

Positive control2 n.t. 89.5 14.0 14.05 4.0 

19.5 85.0 87.5 1.5 1.5 1.0 

39.1 110.8 95.0 2.5 2.5 0.5 

78.1p 100.2 107.0 2.0 2.0 0.0 

Exposure period 18 hrs without 59 mix 

18 hrs Solvent control1 100.0 100.0 1.0 1.0 0.0 

Positive control3 n.t. 70.5 13.0 11.55 3.0 

37.5 97.5 96.9 0.0 0.0 0.0 

75.0 82.2 90.3 2.5 2.0 0.0 

15o.oP 77.0 112.8 1.5 1.5 0.5 

Exposure period 4 hrs with 59 mix 

18 hrs Solvent control1 100.0 100.0 2.0 2.0 0.0 
Positive controt4 n.t. 89.2 10.5 10.01 

19.5 79.4 113.5 2.0 1.0 

39.1 113.8 120.0 0.0 0.0 

78.1p 90.3 93.0 2.5 2.0 

18hrs Solvent control1 100.0 100.0 0.5 0.5 

Positive control4 n.t. 87.0 15.5 13.55 

37.5 96.4 104.0 2.5 2.5 

75.0 93.1 106.0 0.5 0.5 

1so.oP 84.1 113.0 3.5 3.01 

Including cells carrying exchanges 

n.t. Not tested 

p Precipitation occurred 

s Aberration frequency statistically significant higher than corresponding control values 

Deionised water 10.0% (v/v) 

2 EMS1000.0 IJg/ml 

EMS400.0 IJg/mL 

4 CPA 1.4 j.Jg/ml 

Applicant's summary and conclusion 

Conclusions 

2.5 

0.0 

0.0 

0.0 

0.0 

2.5 

1.0 

0.0 

0.5 
In conclusion, ~can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 eels (Chinese hamster cell Hne) in 

vitro. 

Theralore, Leuco Sulfur Black 1 is considerad to be non-dastogenic in this chromosome aberration test in the absence and presence of metabolic activatiOn, when -ed up to the 

highest evaluable concentrations. 

Executive summary 

The test item Leuco Sulfur Black 1, suspended in deionised water, was assessed for its potential to induce structural chromosome aberrations 

in V79cells of the Chinese hamsterin vitroin two independent experiments. 

In each experimental group two paraUel cullunss were set up. 100 metaphases per cuKure wens evaluated lor structural chromosome aberrations. 

The highest applied concentration in lhe pre-test on toxicity (5000 ~g/ml) wes chosen with respeclto lhe curnsnl OECD Guideline 473. 

Dose seleclion for the cytogenetic experiments was performed considering the toxicity dataand lhe occurnsnce of precipitation. The chosen traatment concentrations were between 2.3 

and 300 ~g/ml (with and without S9). 

In lhe absence and pnssence of S9 mix no cytotoxicity was observed up to lhe highest evaluable concentration. Due to strong last ~ern pnscip- concentrations higher lhan lhose 

examined could not be evaluated for cytogenetic damege. 

No clastogenicily was observed atlhe concentrations evaluated eKher with or wKhoul metabolic activation. However, in Experiment II in the presence of S9 mix a single statisticaHy 

significant increase in the number of aberrant cells excluding gaps (3.0 %) was observed atlhe highest evaluable concentration (150 ~ghnl). This valua was clearly in lhe laboratory's 

historical solvent control data range (0.0- 4.0% aberrant cells excluding gaps) and lslhensfore nsgarded as biologically Irrelevant. 

No evidence of an increase in polyploid metaphases was ~ after traatrnenl with the test Kem as comparad to the control cullunss. 

Appropriate mutagens were used as positive controls. They induced statisticaly significant increases (p < 0.05) in cells with structural chromosome aberrations. 
7. 7 Carcinogenicity 

Endpoint summary: Carcinogenicity 

UUID IUC5-9303f711-1 e0d-4db9-9899-c0cb658f3d71 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Author thr I SCC GmbH I Wendelsheim I Germany 

Date 2010-05-26 16:48:23 CEST 

Remarks 

Administrative Data 

Discussion 

There is no carcinogenicity study available. However, there is no concern regarding carcinogenicity because Leuco Sulfur Black I is 

not genotoxic and the mean molecular weight of 8000 - 9000 Da does not indicate significant absorption. 

Justification for classification or non-classification 

Based on the available information Leuco Sulfur Black 1 is not subject to classification and labelling according to Directive 

67/548/EECand Regulation 127212008/EC. 
7.8 Toxicity to reproduction 

Endpoint summary: Toxicity to reproduction 

UUID IUC5-d90b0564-9095-4680-af3b-faed4el68e8e 

Dossier UUID IUCS-2921 49d 1-a6n -4feb-9692-1fad973281 ea 

Author lhr I SCC GmbH I Wendelsheim I Germany 

Da1e 2010-05-26 16:49:10 CEST 

Remarks 

Administrative Data 

Effects on fertility 

Short description of key information 

Leuao Sulfur Black 1 did not cause any toxic effects in lhe repeated dose toxicity study wilh reproduction/developmental toxicity saeening in rats (OECD 422). The llmH dose of 1000 

IJ19/kg bwld was derived as NOAEL for general parental toxicity and reproductive/developmental toxicity. 

Key value for chemical safety assessment 

Effect NOAEL 1000 mg/kg bwlday 

level 

lor 

fertility 

(01"111 

exposurs) 

Discussion 

In a repeated dose toxicity study with reproduction/developmental toxicity screening according to OECD guideline 422 and 

GLP Leuco Sulfur Black 1 was administered by gavage to groups of 10 rats per sex and dose at 0, 100, 300 and 1000 

mg/kg bw/d. Male rats were treated for 29 days, female rats for 14 days prior to pairing, though the pairing and gestation 

periods until the F1 generation had reached day 4 postpartum. The test item was administered in water as vehicle at a 
dose volume of 10 ml/kg bw/d. Control animals were doses with the vehicle alone. 

Observation and examinations consisted of mortality, clinical signs, detailed clinical observations, food consumption, body 

weight, functional observation battery, clinical biochemistry, haematology, urinalysis, macroscopical examination at 

necropsy, organ weights and histopathological examination of organs. Reproductive parameters examined were 

examination of reproductive organs with special emphasis on the stages of spermatogenesis and histopathology of 

interstitial cell structure, mating performance and fertility, duration of gestation, implantation sites, pre- and postimplantation loss, corpora lutea count. Parameters relevant for developmental toxicity consisted of litter size, number of live 

and dead pups, sex ratio, pup weight at day 4 post partum, macroscopical examination of pups at termination. 

The only effect seen was a dark discolouration of faeces in all high dose animals starting on day 3 of the study and lasting 

until termination. Based on this finding a NOAEL of 1000 rng/kg bw/d was derived for general toxicity as well as for 

reproductive and developmental toxicity. 

The discolouration of faeces was the only effect observed in this study and also in the acute oral toxicity study. Organs or 

urine were not discoloured and signs of substance deposits were not found in histopathological analysis of the organs. 

This indicates that Leuco Sulfur Black 1 is directly excreted and that absorption in the gastrointestinal tract does not occur 

following oral administration. 

The mean molecular weight and number-average molecular weight (Mn) of Leuco Sulfur Black 1 is in the range of 8000-

9000 Da and about 3000 Da, respectively. It is very poorly or even not soluble in common polar and non-polar solvents 

such as water (solubility of 62.4 mg/L), DMSO, methanol, DMF, THF, chloroform and not soluble in acid likes sulfuric acid 

and trifluoroacetic acid. The vapour pressure of the pure substance, calculated from the boiling point of > 400 'C, is 1.8 x 

10E-5 hPa at 25 'C. These substance properties supported by the results of oral toxicity studies demonstrate that the 

absorption of Leuco Sulfur Black 1 via the oral, dermal or inhalation route and, consequently, systemic bioavailability is 

negligible. 

Based on the substance properties and the fact that no toxicity was seen in any of the tests it is concluded that exposure to 

Leuco Sulfur Black I does not raise any health concern. In line with Regulation (EC) No 1907/2006 Annex XI and taking into account 

the REACH paradigm that new tests on vertebrates shall only be conducted or proposed as a last resort when all other data sources 

have been exhausted, further animals studies to examine the reproductive and developmental toxicity of Leuco Sulfur Black 1 are not 
necessary and should not be performed. 

Developmental toxicity I teratogeniticy 

Short description of key information 

Leuco Sulfur Black 1 did not cause any toxic effects in the repeated dose toxicity study with reproduction/developmental toxicity saeening In rets (OECD 422). The limH dose of 1000 

rnglkg bw/d was derived as NOAEL for general perental toxicity and reproductive/developmental toxidty. 

Discussion 

see "effects on fertility" 

Justification for classification or non-classification 

Based on the available data Leuco Sulfur Black 1 is not subject to classification and labelling according to Directive 

67/548/EECand Regulation 1272/2008/EC. 
7 .8.1 Toxicity to reproduction 

Endpoint study record: Toxicity to reproduction.001 OECD 422 Leuco Sulfur Black 1 C31087 

UUID IUC5-82c86a1a-8126-46be-b0df-<lc7ab37c29c5 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Garmany 

2010-05-21 14:10:47 CEST 

Administrative Data 

Purpose ftag 

Study result type 

RetlabiiHy 

key study; robust study summary 

experimental result 

1 (relable without restriction) 

Study period 

Rationale for rallabiiHy This study has been performed in compliance with the: 

Incl. deficiencies 

Between 20 May 2009 and 23 March 2010 

Swiss Ordinance relating to Good Laboratory Practice adopted May 18th, 2005 (SR 813.112.1). This Ordinance is based on the OECD Principles of Good 

Laboralory Practice, as revised in 1997 and adopted on November 26th, 1997 by decision oftha OECD Councii(C (97)186/Final]. 

Data source 

Reference 

Rafentnce Author 

type 

study report Ceccatalli 

R 

Data access 

These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the 

United States (EPA and FDA), and Japan (MHLW, MAFF and METI). 

There were no circumstances the! may have affected the quality or integrity of the data. 

Year Title Bibliographic Testing laboratory Report OWner Company Report 

source no. company study no. date 

2010 Combined Repeatad Dose Toxicity Study with the Harlan Laboratories ltd., C31087 Clarlant 2011).. 

Reproduction/Developmental Toxicity Screening WOI!erstrasse 4 4414 03-22 

Test in the Han Wistar Ret FOIIinsdorf, Switzeriand 
data submitter is data owner 

Materials and methods 

Test type 

saeening 

Limit test 

no 

Test guideline 

Qualifier Guideline 

according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with tha Reproduction I Developmental Toxicity Screening Test) 

according to othar guideline: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with tha Reproduction/Developmental Toxicity Screening Test) 

GLP compliance 

yes (Ind. certificate) 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

Common name Leuco Sulfur Black 1 

CAS number 66241-11-0 

Deteils on test material 

Description: Thixotrophic, black, wet peste 

Batch Number: ESA0007585 

Composition: Leuco SuWur Black 1: 52.75% (calculated by amount of by-<:ompounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-Dinitrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Devlmlons 

no 

no 
Sodium: 1.3% (wlw) 

Water. 45.75% (wlw) by Karl Fischer titration 

Stability of Test Hem: Stable under storage conditions 

Stability of Test Item in Vehide: Seven days 

Expiry Date (Retest Date): 11-Jul-2011 

Storage Conditions: At room temperature (20 :t 5 'C) • light proteded 

Safety Preeautions: Routine hygienic procedures (gloves, goggles, face mask). 

Test animals 

Species 

rat 

Strain 

Wistar 

Sex 

male/female 

Details on test animals and environmental conditions 

Animals: Rat, HanRcc: WIST(SPF) 

Rationale: Recognized by international guidallnes as a recommended test system. 

Breeder. Harlan Laboratories Ltd., Laboratory Animal Services, WOiferstrassa 4, 4414 FOIIInsdorf, Switzerland 

Number of Animals: 40 males (1 0 per group), 40 females (1 0 per group) 

Age (at Start of Treatment): 11 weeks 

Body Weight Range (at Start of Treatment): Males: 290 to 324 g. Females: 284 to 212 g 

Identification of Prental Animals: Cage card and individual animal number (ear tattoo). 

Identification of Pups: On day 1 post partum. pups were Individually tattooed with Indian ink. 

Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body 

weights In all groups. 

Acclimatization: Under test condftions after health examination. Only animals without any visible signs of Illness were used for the study. 

Conditions: Standard laboratory conditions. Air-conditioned with 10- 15 air changes per hour. continuously monitored environmental conditions (temp. range: 22 :t 3 'C; relative 

humidfty range: 37.5- 60.9%). There was 12 hour fluorescent light 112-hour dark cyde with music durlng the light period. 

Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and stenllzed standard softwood bedding ('Lignocei'J. Rettenmaier & SOhne GmbH & CoKG, 73494 

Rosanberg I Ge111111ny, Imported by Provimi Klibe SA, 4303 Kalseraugst I SWitzerland). During the pre-pairing period, cages with males were interspersed amongst those holding 

females to promote the davelopment of regular eslrus cydes. 

Oiet: PeHetad standard Kiiba Nafeg 3433 rodant maintenance diet (Provimi Kllba SA. 4303 Kaiseraugst I Switzerland) was available ad libftum (batch no. 15109). 

Water: Communfty tap-water from FOIIinsdorf was available ad iibftum In water bottles. 

Administration I exposure 

Route of administration 
oral: gavage 

Vehicle 

water 

Details on exposure 

Dose Fonnulations 

The dose formulatlons were prepared weekly using the test item as supplied by the Sponsor. 

Leuco Sulfur Black 1 was weighed Into a glass beaker on a tered precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous 

suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. 

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. 

Storaga of Dose Fonnulations 

Dose formulations were stored at room temperature (20 t 5 'C) in brown glass beakers. 

Based upon the results of stablfity analysas performad within the Harlan Laboratories study no. C31076, dose formulations were stable for at least one week. 

Treatment 

Method: Oral, by gavage 

Rationale for Method: Administration by gavaga is a common and accepted route of exposure for studies of this type. 

Target Dose Levels: 

Group 1: 0 rngJl<g bwlday (control group) 

Group 2: 100 mglkg bwlday 

Group 3: 300 mglkg bw/day 

Group 4: 1000 mg/kg bwlday 

Rationale for Dose lave! Selection: The dose levels were selected based on a previous dose renga finding toxicity study in Han Wistar Rats, Harlan Laboratories Study C31076, using 

dose levels of 116, 348 and 1160 rngJl<glday adjusted for 52.75% Leuco Sulfur Black 1 dye stuff in the product and corresponding to 220, 680 and 2200 mglkg bwlday of product. The 

NOAEL of this study resulted to ba 1160 mg dye stuff /kg bw/day or 2200 mg product/kg bwlday. 

Dose Voluma: 10 mllkg body weight 

Details on mating procedure 

During the pairing period, females were housed with sexually mature males (1 :1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until 

evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually W: -the daily vaginal smaar was sperm positive, or 

-a copulation plug was observed. 

The day of mating was designated day 0 post coitum. 

All dams wera allowed to give birth and rear their Hlters (F1 pups) up to day 4 postpartum. Day 0 was designated as the day on which a female hed delivered all her pups. 
Analytical verification of doses or concentrations 

yes 

Details on analytical verification of doses or concentrations 

Analysis of Dose Formulations 

On lhe first treatment day duplicate samples from lhe control group as well as three duplicate samples (top, middle and bottom) of aboul2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from lhe middle only to conflnn stability (4 tvs and 7 days). During lhe last 

week of lhe treatment, samples were taken from lhe middle to confirm concentration. The aliquots for analysis of dose formulations were transferred into tightly closed vessels protected 

against oxygen. One set of samples was delivered at ambient temperature to Dr. K. Morgenlhel (Harten Laboratories Ltd., ltingen I Switzerland) and slorad lhere at room temperature 

(20 ± 5 'C) until analysis. The remaining sat of sa mples was stored at lhe facility at room temperature (20 ± 5 'C) in case of repeated analysis. 

Analysis of homogeneity and concentration was performed gravimetrically by weighing lhe residue after drying by lyophilization. Stability of dosa formulations was confinned by IRspectroscopy. 

The application formulations investigated during lha study were found to comprise Leuco Sulfur Black 1 In lhe range of 81.7% to 102.5%. The homogeneous distribution of Leuco Sulfur 

Black 1 in lhe preparations was approved because individual recoveries found did not daviate more lhan 14.2% (<15%) from lha corresponding mean. In addilion,lhe test item was 

found to be stable in epplicetion formulations when kepi for 4 hours and for 7 days under storage condKions due to similar IR spectra compared to lhe reference spedrum obtained from 

lhe test Hem. Thus, idantity of lhe test Kem In application formulations was confinned. In conclusion, lhe results indicate lhe accurate use of lha test item Leuco Sulfur Black 1 end highly 

purified water as vehicle during lhis study. 

Duration of treatment I exposure 

Males: 29 days 

Females: Approximately 7 weeks 

Frequency of treatment 

DeUy 

Details on study schedule 

Males 

Acclimatization: 7 days 

First Test Item Administration : Day 1 of pre-pairing 

Pre-Pairing: 14 days 

Pairing: 14 days maximum 

Treatment Ends: On day before sacrifice 

Necropsy: After treatment of at least 28 days, when no longer needed for assessment of repro-ductive effects 

Females 

Acclimatization: 7 days 

First Test Item Administration: Day 1 of pre-pairing 

Pre-Pairing: 14 days 
Pairing: 14 days maximum 

Gestation: Approximately 21 days 

Treatment Ends: On day 4 postpartum 

Necropsy:On day 5 postpartum (pups on day 4 post par1um) 

Doses I concentrations 

100 rng/kg bw/day 

Baals actual ingested 

300 mg/kg bw/day 

Baals actual ingested 

1000 mg/kg bw/day 

Baals actual ingested 

No. of animals per sex per dose 

10 

Control animals 

yes, concurrent vehide 

Further details on study design 

-Dose salectlon rationale: 

-Rationale for animal assignment (if not random): 

-Other: 

Positive control 

Not requirad. 

Examinations 

Parental animals: Observations and examinations 

VIABILITY I MORTALITY 

Twice daily 

CLINICAL SIGNS 

Dally cage-side dlnical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were obsarved for signs of difficutt or prolonged parturition, and 

behavioral abnormalities in nesting and nursing. 
FOOD CONSUMPTION 

Males: Weekly during pre-pairing and after pairing periods 

Females: Pre-pairing period days 1 -8 and 8 -14; gestation days 0-7, 7- 14 and 14- 21 post coilum, and days 1 -4 post parium. 

No food consumption was recorded during the pairing period. 

BODY WEIGHTS 

Recorded daily from treatment start to day of necropsy. 

DETAILED CLINICAL OBSERVATIONS 

Once prior to the first administration of the test Item and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the 

following: changes in skin, fur, eyes, mucous membranes, ocrurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory 

panem). Changes in gall, posture and response to handling as well as the prasence of clonic or tonic movements, stereotypies or bizarre behavior were also reported. 

FUNCTIONAL OBSERVATION BATERY 

At ana time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 postpartum) relevant parameters were performed wllh five P generation males and 

five P generation females fmm each group. This FOB assessment was conducted following the daily dose administretion. Animals were observed for the following: 

-Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnonnai behavior (e.g. drciing, stereotypy) and posture as well as resistance to removal 

-Hand-held observations: palpebral dosure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tons, extensor thrust response, righting reflex and reaction to 

handling. 

-Open field observations: level of ambulatory activlly induding rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal 

pellets voided. 

- Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, musde movements, eyes, hearing abilily (Preyer's reflex), urine or feces, soling, 

generei abnormalities, posture. 

-Measurements 1 Counts: hind limb I fore limb grip strength, landing foot splay, redal temperature. 

Any abnormal findings were recorded and, where appropriate, graded in severity. 

Addllionaily, locomotor activily was measured quantitatively for the same animals. Activity was measured with an Activlly Monitor AMS-0151 (FMI, Germany). Activily of the animals 

(based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported. 

CLINICAL LABORATORY INVESTIGATIONS 

Blood and urine samples were obtained on the day of the scheduled necropsy from 5 males fmm each group. Males were kept in metabolism cage and fasted overnight (but allowed 

access to water ad libllum) for approximately 18 hours. 

Blood samples from 51actatlng females fmm each group were obtained on day 5 post parium. The females were tested for approximately 18-20 hours before blood sampling but 

allowed access to water ad libitum. 

Blood samples were drawn sublingually from all animals under Nght isoflurane anesthesia. The samples were colieded early in the working day to reduce biological variation caused by 

circadian rhythms. 

HEMATOLOGY 

The following hematology parameters were determined: 

Complete Blood Cell Count: 

Erythrocyte count 

Reticulocyte Count 

Hemoglobin 

Hematocrit 

Mean corpuscular volume 
Red cell volume distribution width 

Mean corpuscular hemoglobin 

Mean corpuscular hemoglobin concentration 

Hamoglobin concentration dislribution wldlh 

Leukocyte count, lolal 

Differential leukocyte count 

Plalelet count 

Coagulation: 

Prothrombin time(= Thromboplastin time) 

Activaled partial Thromboplastin time 

CLINICAL BIOCHEMISTRY 

The following dinical biochemistry paramelers were determined: 

Glucose 

Urea 

Creatinine 

Bilirubin, tolal 

Cholesterol, total 

T riglycerides 

Aspartate aminotransferase 

Alanine aminotransferase 

Alkaline phosphalase 

Gamma-glulamyl-transferase 

Bile acids 

Sodium 

Potassium 

Chloride 

calcium 

Phosphorus 

Protein, tolal 

Albumin 

Globulin 

Albumin/Globulin ratio 

URINE ANALYSIS 

The following urine parameters were determined: 

Relative density 

colour 

Appearance 

pH 

Protein 

Glucose 

Nitrite 

Ketones 
Urobilinogen 

Bilirubin 

Erytrocytes 

Leukocytes 

Volume (18 hours) 

Estrous cyclicity (Parental animals) 

Not reported. 

Sperm parameters (Parental animals) 

tes1es weight, epididymes weight, stages of spermatogenesis 

Litter observations 

The litters were examined for litter size, live births, sbll births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identificatiion) 

on days 0 (n possible), 1 and 4 postpartum. 

Postmortem examinations (Parental animals) 

TERMINATION OF THE STUDY 

Males were sacrificed after treatment of 29 days, when no longer needed for the assessmen1 of reproductive effects. Dams were sacrificed on day 5 postpartum. When birth did not 

occur on the expected date (day 21 post coltum), the dam was secrificed on day 26 post collum. 

NECROPSY 

All animals were sacrificed by an injecbon of sodium pentobarbital. All P generabon animals were exsanguinated. All parent animals were examined macroscopically for any structural 

changes, efther at the scheduled necropsy or during the study n death occurred. For the parent animals, special attention was directed at the organs of the reproducbve system. 

The number of implantation sftes and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed In a solution of ammonium sulfide to visualize 

possible hemorrhagic areas of implantation sites. 

ORGAN WEIGHTS 

The testes and epididymides of aM parental males were weighed as pairs. 

In addftion, from 5 mates and females killed at the end of the study which were seiected from each group, the following organs were trimmed from any adherent tissue, as appropriate, 

and their wet weight taken. 

Adrenal glands (weighed as pairs) 

Brain 

Heart 

Kidneys (weighed as pairs) 

Liver 

Thymus 

Spleen 

TISSUE PRESERVATION 
The following tissues from al parental males were preserved in neutral phosphate buffered 4% formaldehyde solution: 

Prostate 

Seminal vesicles with coagulating gland 

Testes (in Bouin's fixative) 

Epididymides (in Bouin's fixative) 

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution: 

Ovaries 

In addition, from the five males and females per group selected for organ weights and from all animals fOL<Id deed or killed in extremis, the following tissues were preserved in neutral 

phosphate buffered 4% formaldehyde solution: 

Gross lesions 

Brain 

Spinal chonl 

SmaN and large intestines (incl. Payer's patches) 

Stomach 

Liver 

Kidneys 

Adrenals 

Spleen 

Heart 

Thymus 

Thyroids, and parathyroids W possible 

Trachea and lungs (preserved by inflation with fixative and then immersion) 

Uterus (with vagina) 

Urinary bladder 

Lymph nodes (mesentarial, mandibular) 

Paripheral nerve (sciatic) 

Bone marrow 

HISTOTECHNIQUE 

All 0f11811 and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with 

hematoxylin and eosin. Addltlonaly, the testis was stained by PAS-hematoxylin. 

HISTOPATHOLOGY 

Slides of all organs and tissues cotlectad at terminal sacrifice from tha animals of the control and high-dose groups were examined by the principal investigator. The same applied to all 

occurring gross lesions. 

Special emphasis was made on tha stages of spermatogenesis and histopathology of Interstitial cell structure. 

Histological examination of ovaries was carried out on females that did not give birth. 

Postmortem examinations (Offspring) 
TERMINATION OF THE STUDY 

Pups were sacrificed on day 4 post pariUm. 

NECROPSY 

All animals were seafficed by an Injection of sodium pentobarbital. Dead pups, except lhose excessively cannibalized, -re examined macroscopically. All pups were examined 

macroscopically for any structural changes, either at lhe scheduled necropsy or during the study W death occurred. 

Statistics 

The following statistical melhods -reused to analyze food consumption, body -lghts, organ -ight,locomotor activity and macroscopical findings and reproduction data: 

-Means and standard deviations of various data were calculated. 

-The Dunnell-test (many to one t-test) based on a pooled variance estimate was applied ~ lhe variables could ba assumed to follow a normal distribution for lhe comparison of lhe 

treated groups and lhe control groups for each sex. 

-The Steel-test (many-one rank test) was applied instead of lhe Dunnell-test when the data could not be assumed to follow a no""al distribution. 

- Fisher's exact-test was applied ~ lhe variables could ba dichotomized without loss of information. 

Reproductive Indices 

From lhe on~ine recorded reproduction data, the following parameters were calculated: fertility indices, mean precoltal time, post-implantation losses, mean litter size, pup sex ratios. 

Offspring viability Indices 

From lhe on4ine recorded reproduction data. the following parameters were calculated: deed I ive pups at first litter check, postnatal loss (up to day 4 post parium). 

Results and discussions 

Effect levels 

Endpoint Generation Sex Effect level 

NOAEL p male/female >= 1000 

mg/kg 

bw/day 

Based Baals for effect level/ Remarks 

on 

With the exception for a darl< discoloration of feces obse!Ved In all animals treated with lhe test Hem at the dose level of 

1000 mglkg body -ightlday, no olher test item-related ellects -re observed during lhe study. The discoloration of 

feces was considered to ba due to the staining property of lhe test Hem and not to be adverse. 

Results of exam.nations: parental animals 

Clinical signs (parental animals) 

yes 

Body weight and food consumption (parental animals) 

no-cts 
Reproductive function: estrous cycle (parental animals) 

not examined 

Reproductive function: sperm measures (parental animals) 

not examined 

Reproductive performance (parental animals) 

no elfecls 

Organ weights (parental animals) 

no elfecls 

Gross pathology (parental animals) 

no effects 

Histopathology (parental animals) 

no elfecls 

Details on results (parental animals) 

PARENT ANIMALS 

Delalled Clinical Observations (Dally) 

All animals survived until the scheduled necropsy. 

In group 4, dar1< discoloration of feces was observed in males and females slarting on day 3 of the pre-pairing period onwards. This was likely due to the slainlng property of the test 

llem al higher concentration. 

In groups 2 and 3, no clinical signs were observed for the entire duration of the study. 

(See attached Tables on pp. t to 3) 

Delalled Clinical Observations (Weekly) 

No findings were obsarved during the weekly examination. 

Functional Observational Battery 

None of the parameters under investigation during the functional observational battery gave an indication of a last ileiTHBiated effect. 

The frequency of rearing number and incidence of urine puddles during the opan fiald observation did not give indicalion of any test llern-related effect. Observational as spontaneous 

vocalization when the rat was removed from the cage was considered to be of incidenlal nature. 
Mean values of grip strength (fore- and hind paws) and landing foot splay gave no indication of any test Kern-related effect. 

Body temperature in males was statistically significantly lower in group 4 compared to the control group (37.9 'C compared to 38.7 'C in the control grou p). However, this value was 

within the range of the historical control data and therefore was not considered to be a test Ham-related effect. 

(See attached Tables on pp. 4 to 7 and attached Historical Control Data on pp. 3 to 14) 

Looomotor Activity 

Looomotor activtly was assessed quantitatively in terms of low beam counts in activtly monitor and did not give any indication of a test item-related effect. 

Food Consumption - Males - Pre-pairing and After Pairing Periods 

Mean food consumption was not considered to be affected by the treatment with the test Kern. 

During the pre-pairing period, the lower food consumption observed in groups 2 and 3 (-6.2 and -4.6% compared to the control group) was considered to be incidental since this 

reduction did not follow a dOSIHlependent pattam and in group 4 mean food consumption was similar to the control (-0.4% compared to the control). 

During the after pairing period, mean food consumption compared to the control was: +2.2, -1.1 and +1.5%, in groups 2, 3 and 4, respectively 

(See attached T abies on pp. 8 to 11) 

Body Weights - Males- Pre-pairing, Pairing and After Pairing Periods 

Mean body weight and mean body weight gain were not effected by the treatment with the test Hem for the whole duration of the study. The stetistically significantly lower body weight 

gain observed in group 2 between day 4 and 7 was considered to be incidental since there was no dose-dependency. 

In order of ascending dose level, body weight gain was: during the pre-pairing period +13.0%, +10.9%, +11.5 and +11.9%, during the pairing period +4.9%, +5.7%, +4.4% and +4.7%, 

and during the after pairing period +4.1%, +5.6%, +4.8% and +4.5%, respectively. 

(See attached Tables on pp. 12 to 20) 

Food Consumption -Females- Pre-pairing, Gestation and Lactation Pariods 

No test Hem-related effects were observed In mean food consumption for the entire duration of the study. 

Mean food consumption In groups 2, 3 and 4, compared to the control group, was: during the pre-pairing period +5.0%, +2.2% end +2.2%, during the gestation pariod +0.4%, -2.1% and 

-0.4%, and during the lactation period +11.3%, -3.1% and -3.7%, respectively. 

(See attached Tables on pp. 21 to 26) 

Body Weights- Females -Pre-pairing, Pairing, Gestation and Lactation Periods 

No test item-related effects were noted In mean body weight and mean body weight gain of females during the whole study at any dose level. 

in order of ascending dose level, body weight gain was: during the pre-pairing pariod +7.5%, +8.3%, +9.3% and +8.5, during the gestation period +63.5%, +63.3%, +60.2% and +60.5%, 

and during the lectation +3.1%, +62%, +4.6 and +2.7%, respectively. 

(See attached tables on pp. 27 to 39) 
CLINICAL LABORATORY INVESTIGATION 

Hematology 

The assessment of the hematology data did not reveal any test Item-related effects in males and females. 

CMnical Biochemistry 

Males: 

In group 4,the concentration of potassium was statistically significantly higher (+17.8% compared to the control). Since the mean value was within the range of the historical control 

data, this was not considered to be a test Hem-relatad effect. 

In groups 2 and 3 no changes were noted. 

Females: 

The assessment of the clinical biochenlstry data did not reveal any test ltem-relatad affects In females. 

(See attached Tables on pp. 40 to 41 and attached Historical Control Data on pp. 1 to 2) 

Urine analysis 

The assessment of the urine analysis data did not reveal any test item-related effects in males and females. 

REPRODUCTIONANDBREEDINGDATA- PARENT ANIMALS 

Please see section "Any otherinformation on results including tables• for the table "Summary of Performance - P Animals Breeding for F1 LHters" 

Mating Performance and Fertility 

All females malad within the first pairing period. The median and mean precoltal times were unaffectad by treatment with the test item. Mean precoital times were 2.6, 3.1, 2.8 and 3. 7 

days in groups 1, 2. 3 and 4, respectively. The median precoital time was 3, 4, 3 and 4 days in order of ascending dose level. 

Due to one female (no. 58) in group2 which was not pregnant, the fertRily Index and conception rate were 100% in groups 1, 3 and 4, and 90% in group 2. 

In group 1, one female (no. 43) did not deliver any pup and had only two Implantation sites. Therefore, the gestation index was 90% in group 1, and 100% in groups 2, 3 and 4. 

Duration of Gestation 

The mean duration of gestation was unaffected by the exposure to the test item. Mean duration of gestation was 21.4, 21.8, 21.5 and 21.4 days, in order of ascending dose level. 

Corpora Lutea Count 

Maan number of corpora lutea per dam (determined at necropsy) was similar In all groups (16.9, 18.3, 17.2 and 16.4 in order of ascending dose level) and gave no indication of a test 

ltem-relatad effect. 

Implantation Rate and Post-implantation Loss 

The mean number of implantations per dam was not affected by the treatment with the test Item. 
In groups 3 and 4, tolal post~mplantation loss was statistically significantly higher but within the range of the historical control data, therefore was not considered to be test item-related. 

The mean numbers of implantations per litter were 15.0, 16.1, 14.3 and 14.6 in order of ascending dose level. The mean incidence of post-implantation loss as a percentage of tolal 

implantations was 1.5, 4.8, 8.4 end 6.2%, in order of ascending dose level. 

(See attached Tables on pp. 42 to 43 and attached Historical Control Data on pp. 15 to 38) 

Utter Size at First Litter Check 

At first litter check, mean litter size was not effected by the treatment with the lest ~em. 

Due to the higher number post~mplantation loss which occurred in groups 3 and 4, the birth index was statisticeHy significantly lower but wtthin the range of the historical control data. 

Birth index was 98.5, 95.2, 91.6 and 93.8% and mean litter size at first litter check was 14.8, 15.3, 13.1 and 13.7, In order of ascending dose level. 

Postnatal Loss Days 0 - 4 Post Parium 

Postnatal loss up to day 4 p.p. was not affected by the treatment wtth the test Item at any dose level. 

Mean postnatal loss was 0.3, 0.0, 0.0 and 0.1, corresponding to a viabll~ index of 97.7, 100.0, 100.0 and 99.3% in groups 1, 2, 3 and 4, respectively. 

TERMINAL FINDINGS 

Organ Weights 

No test Item-related effects were noted in absolute and relative weight of organs in males and females. All the statistically significant changes were w~in the range of the historical 

control data or did not follow a dose-dependent psttem. 

(See attached Tables on pp. 44 to 49 and attached Historical Control Data on pp. 39 to 62) 

Maaosoopical Findings 

The findings noted during the macroscopical examination did not give indication of any test ltem-<etated effect. 

Males: 

In group 4, spleen~ a constriction was noted in male no. 34. No other findings were noted. 

Females: 

In group 4, female no. 72 was noted to have the liver caudal lobe adherent to the lateral lobe. 

In groups 2 and 3 no findings were noted. 

In group 1, female no. 43 was noted to have tan or dark red or red brown discolored nodules on the left uterine horn. Same female had only two Implantation sites and did not deliver 

any pups. 

Histopsthology Findings 

In group 4, In the lungs of animal no. 75 some alveolar macrophages (moderate sev~ degree) were found to contain dark pigment. This finding was possibly related to accidental 

aspiration of dose solution. All further lesions recorded during the microscopic investigation were ~in the range of background alterations that may be recorded in this type of study, 

and in rats of this strain and age. 
In group 1, in one Infertile female (number 43) a deciduoma was noted. This lesion was deemed to have contribu1ed to infertitty. 

Qualitative staging of thePAS-stained testes was conducted and there were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of 

cell populations. Individual lesions recorded were within the range of background aHerations that may be recorded In this type of study, in rats of this strain and ega. 

Results of examinations: offspring 

VIability (offspring) 

no effects 

Clinical signs (offspring) 

no effects 

Body weight (offspring) 

no effects 

Saxual maturation (offspring) 

not examined 

Organ weights (offspring) 

not examined 

Gross pathology (offspring) 

no effects 

Histopathology (offspring) 

not examined 

Details on results (offspring) 

LIITER DATA- F1 PUPS 

External Examination at First Utter Check and during Lactation 

No abnormal findings were noted at first litter check or during the first 4 days postpartum. 

In group 1, two male pups from sama litter and one male pup from another litter were noted to have no milk in tha stomach at first litter check. 

Sex Retios 

Sex ratios at first litter check and on day 4 postpartum were unaffected by exposure to the test item. 
The proportion of males on day 4 post parium was 48, 49, 44 and 50%, in order of ascending doss level. 

Body Weights to Day 4 Post Parium 

Mean pup weights on day 1 post parium were unaffected by treatment with the test Hem. On day 1 post parium mean pup weights were6.0,6.1, 6.4and6.0 gin order of ascending dose 

level. 

During the lactation period (to day 4 post parium), mean pup weight gain was not affected by the treatmentwHh lhe test Hem (+41.7, +39.3, +39.1 and 43.3%, in order of ascending 

doss level). Mean pup weights on day 4 post parium were 8.5, 8.5, 8.9 and 8.6 g in order of ascending doss level. 

Macroscopical Findings 

No abnormal findings ware noted during lhe macroscopical examination of pups. 

Any other lnfonnatlon on results incl. tables 

Summary of Perfonnance • P Animals Breeding for F1 Litters 

Group 

(mglkg{dayl 

Female numbers 

Number of females paired 

Number of females mated 

Number of non pregnant females (A) 

Numbers of females, 

which did not deliver any pups (B) 

Number of females which reared their 

pups until day 4 post partum 

(A) Female No. 58 was not pregnant. 

(0) 

41-50 

10 

10 

0 

9 

(B) Female No. 43 had only two implantation sites. 

Overall remarks, attachments 

Overall remarks 

2 

(100) 

51-60 

10 

10 

1 

0 

9 

3 4 

(300) (1000) 

61-70 71-80 

10 10 

10 10 

0 0 

0 0 

10 10 

Relevant reproduction parameters (number of corpora lutea, implantation sites, pre- and post implantation losses, number 

of pups) were not affected by the treatment with the test item. The litter size at first litter check and on day 4 postpartum 

was also not affected. 
Applicant's summary and conclusion 

Conclusions 

This study is a valid investigation of the loxicological effecls resulting from repealed oral-gavage admlnislralion of the test item leuco Sulfur Black 1 (CAS#66241-11-0) to rats over 

approximately 28 days. Leuco Sulfur Black 1 was administered In highly purified waler es vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the 

vehicle only. Leuco SUlfur Black 1 was administered 1o male rets for 29 days and to female rats for 14 days prior lo pairing, through the pairing and geslelion periods until the Fl 

generation reached day 4 post par1um. 

Administrations at 1000 mglkg body weight/dey caused a dark discoloration of feces in all animals after the onset of the treatment untH the and of study. This was likely due to the 

staining property of the test Kern and not considered lo be adverse. 

Food consumption and body weight were not affected for the entire duration of the study. The evaluation of the functional battery observations and the assessment of clinical laboratory 

investigations parameters did not reveal any test Kern-related effect. 

Relevant reproduction parameters (number of corpora lulea, implantation sKes, pre- and post Implantation losses, number of pups) were not affected by the treatment with the lest Kern. 

The Iiiier size at first litter check and on dey 4 postpartum was also not affected. 

All the changes noted in the organ weights were within the background of the biological variation. 

No adverse findings were noted during macroscopical and histopathology examinations. 

Based on these results a general NOAEL (No Observed Adverse Effect Level) was established at 1000 mglkg body weight/day. 

The NOEL (No Observed Effect Level) for reproduction/developmentalloxicKy was considered to be 1000 mgJkg/dey. 

Executive summary 

The purpose of this study was to generate pretiminary information on the possible heaKh hazards likely to arise from repeated exposure over a relatively limKed period of time. In 

addition K provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus 

and parturition. 

This study should provide information 1o assess the need lo conduct furlhar investigations and may provide guidance in the design of subsequent studies. 

Dose levels were selected in agreement with the Sponsor, based on the results of a dose range-finding study (Harlan Laboratories Study C31076). 

Leuco Sulfur Black 1 was administered to male rats for 29 days and to female rats for 14 days prior lo pairing, through the pairing and gestation periods until the Ft generation reached 

day 4 post partum. 

The following dose levels were applied: 

Group!: 

Group2: 

0 mglkg body weight/day (control group) 

100 mglkg body weight/day 
Group3: 

Group4: 300 mg/kg body weighUday 

1000 mg/kg body weighUday 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosad wHh lha vehicle alone (highly purified water). 

The following resuHs were obtained: 

PARENT ANIMALS 

Genenol T-.blllty Alllha animals survived untillha schaduled necropsy. AI. 1000 mglkg body welghUday, a dark discoloration of feces was observed in all animals starting on day 3 of lha pre-pairing 

period onwards. 

FunctloiUII Ob•rvatlon81 Battery No test item-related effects were noted. 

Food Consumption 

Food consumption was not afleded by the treatment wHh the test Hem in males and females. 

Body Weights 

No test Hem-related effects were noted in body weight and body weight gain in males and females for lha entire duration of the study. 

Clinical Laboratory lnvestlgatlions 

The assessment of the clinical laboratory investigations parameters did not give indication of any test Item-related efled. 

Reproductlion and a..-lng Data 

Mean pracoital time, fertility end gestation index and concaplion rate were not afleded by tha treatment with the test Hem. 

Implantation rate and post implatation loss were also not affected by lha treatment with lha test Hem. 

Organ Weights 

Weight of organs was not affeded by the treatment with lha test Hem. Ail lha alterations noted were within lha range of historical control data. 

Macroscopical Findings and Histopathological Examinations 

No adverse effects of the test item were noted during the histopathological examination. 

UTTER DATA- F1 PUPS 

Findings at Firat Uttar Check and during Lactation 

The mean number of pups at firstiHter check and on day 4 post parium were not affected by the treatment with lha test item. The sex ratio was also not efleded. No abnormal pup was 

noted at any dose level. 

Pup Weights to Day 4 Post Parium 

Mean pup weight gain to dey 4 post parium was not aflectd by the treatment with the lest item. 

Macroscopical Findings 

AI. necropsy of pups, lhare were no abnormal findings. 
Conclusion 

Based on these results a general NOAEL (No Observed Adverse Effect Level) was established al1000 mg/kg body weight/day. 

The NOEL (No Observed Effect Level) for raproductionldevelopmental toxicity was considerad to be 1000 mg/kg body weight/day. 
Endpoint study record: waiving_ Toxicity to reproduction.001 

UUID IUC5-89c0c88b-a9a4-4963-9b56-b0b1 Oc4 76117 

Doss Ia< UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarl<s 

uensenba I Clarlant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2011J-05-2114:11:40 CEST 

Administrative Data 

Date w.Jvlng 

JustlllcMion 

for data 

-lvlng 

study scientifiCally unjustified 

In accondance with Regulation (EC} No 1907/2006 Annex XI, a one or two generation reproductive toxicity study Is scientifically not justified and, thus, should not be 

conducted because of the following: 

Leuco Sulfur Blacl< 1 has a mean molecular weight in the range of 8000 to 9000 Da and a number-average molecular weight (Mn) of about 3000 Da. his very poorly 

or even not soluble in common polar and non-polar solvents suches water (solubillly of 62.4 mgll), DMSO, methanol, DMF, THF, chloroform and not soluble In acids 

tike suWuric acid and trifluoroacetic acid. The vapour prassure of the pure substance, calculated from the bolllng point of > 400 't:, Is 1.8 x 1 OE-5 hPa at 25 'C. 

Basad on these properties it Is very unlikely that Leuco Sulfur Black 1 can cross any biological membrane. Thus, an absorption of toxicologically significant amounts of 

Leuco Sulfur Blacl< 1 following orel, dermal or Inhalation exposure can be exdudad. This is substantiatad by the results obtalnad In toxicity and ecoloxicity studies, 

where no substance reiatad local and/or systemic effects Indicative of toxicity were seen in all tests, including acute animal and acute ecoloxicity studies, sensitization, 

genoloxiclly tests and a repaatad dose toxicity study wtth reproductiveldevolpmental toxicity screening. 

In the acute oral toxicity study black stainad faeces was observed for maximally four days starting the dey after administration of the substance at 2000 mglkg bw. 

Dart< discoloured faeces were also seen in the repaatad dose toxiclly study with reproductive/devolpmental toxicity screening (OECD 422) at 1000 mglkg bwld, 

starting on day 3 of the study and lasting throughout the administration pariod until termination. The discolouration of faeces was the only effect observad and can be 

attributad to the strong staining properties of the substance. This indicates that Leuco Sulfur Blacl< 1 is directly excreted and that absorption in the gastrointestinal tract 

does not occur following oral administration, espacially since organs or urine wera not discoloured and signs of substance deposits were nol found In the 

histopathological analysis of the orgens. 

In conciuslon, the available toxicity and ecotoxiclly data together with the intrinsic substance properties are sufficient to demonstrate that exposure to Leuco Sulfur 

Blacl< 1 does nol cause any heahh concern. 
7 .8.2 Developmental toxicity I teratogenicity 

Endpoint study record: waiving_ Developmental toxicity I teratogenicity.001 

UUID IUC5-cd2b3712-<>4<:6-41b4-b396-7ce7bdcaa2d5 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

uensenba I Clariant Produkte (Deutschland) GmbH I Sulzbach am Taunus I Germany 

2010-05-2114:12:26 CEST 

Administrative Data 

Date w•lvlng 

Justification 

for date 

-lvlng 

study scientifically unjustified 

In accordance with Regulation (EC) No 1907/2006 Annex XI, a teratogenicily study is scientifically not justified and, thus, should not be conducted because of the 

following: 

Leuco Sulfur Black 1 has a mean molecular weight in the range of 8000 to 9000 Da and a number-average molecular weight (Mn) of about 3000 Da. It is very poorly 

or even not soluble in common polar and non-polar solvents such as water (solubility of 62.4 mg/L), DMSO, methanol, DMF, THF, chloroform and not soluble In acids 

~ke sulfuric acid and 1riftuoroacetic acid. The vapour pressure of the pure substance, calculated from the bolllng point of> 400 'C, is 1 .8 x 1 OE-5 hPa at 25 'C. Based on these properties~ Is very unlikely that Leuco Sulfur Black 1 can cross any biologk:el membrane. Thus, absorption of toxicologically significant amounts of 

Leuco Sulfur Black 1 following oral, dermal or Inhalation exposure Is negHgible. This is substantiated by the resuhs obtained in toxicity and ecotoxicily studies, where 

no substance related local and/or systemic effects indicative of toxicity were -n In all tests, including acute animal and acute ecotoxlcily studies, sensitlzatlon, 

genotoxicity tests and a repeated dose toxicity study with reprodudlveldevolpmental toxicity screening. 

In the acute oral toxlcily study black stained faeces was observed lor maximally four days starting the day alfer administration of the substance at 2000 mg/kg bw. 

Dark discoloured faeces were also seen in the repeated dose toxicity study with reproductlveldevolpmental toxlcily screening (OECD 422) at 1000 mglkg bw/d, 

starting on day 3 of the study and lasting throughout the administration period until termination. The discolouration of faeces was the only ailed observed and can be 

attributed to the strong staining properties of the substance. This indicates that Leuco Sulfur Black 1 Is dlrectiy excreted and that absorption In the gastrointestinal tract 

does not occur following oral administration, especially since organs or urine were not discoloured and signs of substance deposits were not found in the 

histopathological analysis of tha organs. 

In conclusion, the available toxicity and ecotoxicity data together with the intrinsic substance properties are sufficient to demonstrete that exposure to Leuco Sulfur 

Black 1 does not cause any health concern. 
7.9 Specific investigations 

7 .9.1 Neurotoxicity 

Endpoint summary: Neurotoxicity 

UUID IUC5-a04392ee-dbfd-4d39-95ec-aef5eb8bf91 7 

Dossier UUID IUC5-292149d 1 -a677 -4feb-9692-1fad97328 1 ea 

Author thr I SCC GmbH I Wendelsheim I Germany 

Date 2010-05-26 16:49:43 CEST 

Remarks 

Administrative Data 

Discussion 

There are no studies available specifically addressing this endpoint However, based on the substance properties (mean 

MW >8000 Da and very poor solubility in water) and the fact that no toxicity was seen in any of the available, it is 

concluded that Leuco Sulfur Black 1 does not raise any concern regarding this endpoint 

Justification for classification or non-classification 

Based on the available data Leuco Sulfur Black 1 is not subject to classification and labelling_ 
7 .9.2 lmmunotoxicity 

Endpoint summary: lmmunotoxicity 

UUID IUC5-351fad61-1 d5a4814-a2fb-b9ec0c7209e6 

Dossier UUID IUC5-292149d1-a6n -4feb-9692-1fad973281 ea 

Author lhr I SCC GmbH I Wendelsheim I Germany 

Date 2010-05-26 16:51:40 CEST 

Remarks 

Administrative Data 

Discussion 

There are no studies available specifically addressing this endpoint. However, based on the substance properties (mean 

MW >8000 Da and very poor solubility in water) and the fact that no toxicity was seen in any of the available, it is 

concluded that Leuco Sulfur Black 1 does not raise any concern regarding this endpoint. 

Justification for classification or non-classification 

Based on the available data Leuco Sulfur Black 1 is not subject to classification and labelling. 
11 Guidance on safe use 

Additional information: Guidance on safe·use.001 

UUID IUCS-be2cb819-599a-41 05-b877 -c0ea861 c5267 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

pbasto I Clariant Quimicos (Portugal) Lda /lei"' do Balio I Portugal 

2010-06-11 10:40:39 CEST 

Administrative Data 

Flrst..,ld measures 

If someone exposad to the product feels unwell seek medical assistance 

In case of contact with skin wash immediaUy with water 

Fire-fighting measures 

Suitable exlinguishing media water spray jel, dry powder , carbon dioxide 

Acddent.l release measures 

Take up mechanically . 

Do not allow to entry to drains, water courses or soil. 

Dispose the absorbed material in accordance with local ragulalions 

Handling and storege 

Handling: 

Observe the usual precautions for handling chemicals 

keep away from food. 

Wash hand before breaks and after work. 

Storage 

Stable under normal conditions 

Keep container tighUy closed, dry in a cool area 
Avoid impact , friction and accumulationof eleclm5tactic charges 

Keep awy from ignition sources. 

Shlbtllty and ""'ctlvlty 

Stable under usual conditions 

Disposal considerations 

Dispose according to local regulations for chemicel produdS 
13 Assessment Reports 

Additional information: CSR 

UUID IUC5-0d168ec0-7fc5-4eeb-bd1 a-177f823bbf5d 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

jregano I Clariant lbertca Producci6n SA I El Prat de Llobregat (Barcelona) I Spein 

2010-06-23 08:51:01 CEST 

Administrative Data 

Type of report 

REACH Chemical safely report (CSR) 

Document 

LSBI<1_CSR_23 06 2010.pdf /284.52 KB (application/pdf) 
Reference substance: sodium sulphate UUIO ECB5-<>db493fc-4080-4e80-8761-<>c15 1 506fddf 

Dossier UUIO IUC5-292149d 1 -a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

mkuehn I Clariant Produkte (Deulschland) GmbH I Sulzbach am Taunus I Germany 

2010-06-Q1 15:53:58 CEST 

General information 

Reference substance n•m• sodium sulphate 

EC inventory 

EC number 23H!20-9 CAS number 7757-82-8 

EC n•me sodium sulphate 

Molecular fonmula H204S.2Na 

Reference substance information 

CAS information 

CAS number 7757-82-8 

CAS name Sulfuric acid sodium salt (1 :2) 

IUPAC name 

disodium sulfate 

Synonyms 

Name Sulfuric acid disodium satt 

Name Sulfuric acid disodium satt 

Group I category information 

DSL Category: lnorganics 

Molecular and structural information 

Molecular fonmul• H204S.2Na 
Molecul•r -lght range 142.0421 

SMILES nolatlon [Na+].[Na+].[O.]S(=O)(=OMO-J 

InC hi lnChi=1/2Na.H204S/c;; 1-5(2,3)4/h;;(H2,1,2,3,4 )/q:z-+1 ;/p-2 

Slructural fonnula 
Reference substance: 2.4-dinitrophenol UUID ECB5-16d37b76-0b88-40cb-8cc5-07c7d66002ef 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973261 ea 

Author 

Date 

XML Transformation V1.0 Plug-In 

2007-05-10 11 :00:00 CEST 

Remarks Successfully migrated lo IUCLID 5.2 formal. 

General information 

Reter.nce oubagnce name 2,4-dinltrophenol 

EC inventory 

ECnumber 

EC name 

200-067-7 CAS number 51-26-5 

2,4-dinitrophenol 

-cular formula C6H4N205 

Reference substance information 

CAS Information 

CAS number 51-26-5 

IUPAC name 

2,4-dinrtrophenol 

Synonyms 

Name Phenol, 2,4-dinrtroName Phenol, 2,4-dlnitroGroup I category Information 

DSL Category: Organics 

Molecular and structural information 

-cular formula C6H4N205 

Molecular -lght range 184.1064 
SMILES notation 

In Chi 

SlrUclur•l formula 

Oc1ccc(cc1 [N+}(=O)(O-})[N+](=O)[O-} 

lnChi=1/C6H4N205/c9-6-2-1-4(7(1 0)11 )3-5(6)8(12)131h1-3,9H 

OH 

NQ 
Reference substance: water UUID ECB5-910811d3-a683-4371-b479-777b663a18c5 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

XML Transformation V1.0 Plug-In 

2010-04-{19 16:35:06 CEST 

Successfully migrated to IUCLID 5.2 format 

General information 

Reference •ubstance name water 

EC inventory 

EC number 

EC name 

231-791-2 CAS number 7732-18-5 

water 

Molecular formula H20 

Reference substance information 

EU:REACH 

CAS Information 

CAS number 7732-18-5 

CAS name Water 

IUPAC name 

water 

Synonyms 

Name Water 

Name Water 

Group I category information 

DSL Category: lnorganics 

Molecular and structural information 
EU:REACH 

Molecular formula H20 

Molecular -lght range 18.0153 

SMILES notation 0 

lnChl lnChi=1/H20/h1H2 

Structural formula 
Reference substance: sodium chloride UUID ECB5-fdal2f4f-2a29-4181-ae 78-5b5622d4912b 

Dossier UUID IUC5-292149d 1-a6n -4feb-9692-1 fad973281 ea 

Author 

Date 

Remarks 

XML Transformation V1.0 Plug-In 

2010-02-23 13:59:30 CET 

Successfully migrated to IUCLID 5.2 format. 

General information 

R.,.,..nce substance name sodium chloride 

EC inventory 

EC numbsr 

EC name 

231-598-3 CAS numbsr 7647-14-5 

sodium chloride 

Molecular formula GINa 

Reference substance information 

CAS Information 

CAS number 7647-14-5 

CAS name Sodium chloride 

IUPAC name 

sodium chloride 

Synonyms 

Name Sodium chloride (NaCI) 

Name Sodium chloride (NaCI) 

Group I category Information 

DSL Category: lnorganics 

Molecular and structural information 

Molecular formula GINa 
Molecul•r -lght f8nge 58.4428 

SMILES no18tlon (Na+].[CI-) 

InCh I lnChi=1/CIH.Nalh1H~q;+1/p-1 

Structuf81 formula 

Cl 
Reference substance: 1-chloro-2.4-dinitrobenzene UUID ECB5-f3f3978e-cfca-442d-bd31-9e04ac3b4354 

Dossier UUID IUC5-292149d1-a677 -4feb-9692-1fad973281 ea 

Author 

Date 

Remarks 

XML Transformation V1.0 Plug-In 

2007-05-1011:00:00 CEST 

Successfully migrated lo IUCLID 5.2 format. 

General information 

Reference substance name 1-chloro-2,4-dlnnrobenzene 

EC inventory 

EC number 

EC name 

202-551-4 CAS number 97-00-7 

1-chloro-2,4-dinilrobenzene 

Molecular formula C6H3CIN204 

Reference substance information 

CAS Information 

CAS number 97-00-7 

IUPAC name 

1-chloro-2,4-dlnitrobenzene 

Synonyms 

N"""' 1-chloro-2,4-dinnrobenzene 

Name Benzene, 1-chloro-2,4-dinitroName Benzene, 1-chloro-2,4-diMroGroup I category information 

DSL Celegory: Organics 

USEPA Category: Neutral Organics;Polynilroaromatics 

Molecular and structural information 
Molecular formula C6H3CIN204 

Molecular -lght range 202.552 

SMILES notation [O-)[N+K=O)c1ccc(CI)c(c1MN•](=OMO-] 

InCh I InCh I= 1/C6H3CIN204/c7 -5-2-1-4(8(10)11 )~(5)9(12)13/h 1-3H 

Structural formula 

Cl 

NO? 
Reference substance: phenol.2.4-dinitro-,sulfurized, leuco derivatives UUID IUC5~a653db5-12844 7 db-9384-05f22219el4d 

Dossier UUID IUC5-292149d 1-a677 -4feb-9692-1 fad973281 ea 

Author 

Date 

Remarks 

XML Transfonnation V1.0 Plug-In 

2010-01-15 08:28:39 CET 

Successfully migrated to IUCLID 5.2 format. 

General information 

Reference substance name phenol,2,4-<linitro-,sulfurized, leuco derivatives 

EC inventory 

EC number 266-273-5 CAS number 66241-11-0 

EC name phenol, 2.4-<linitro-, sulfurized, leuco derivatives 

Reference substance information 

CAS information 

CAS number 66241-11-0 

Molecular and structural information 

Molecular weight renge > 8000-- < 9000 

Remarks It is a UVCB , ~does not have a defined molecular formula because ~was not possible to establish a defined chemical structure 
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