Opinion on HC orange No 6 (B125).
Date: 2018-06-06
Institutions: Directorate-General for Health and Food Safety
Subjects: animal experimentation, cosmetic product, environmental risk prevention, public health, scientific report, toxicology

SCCS/1579/16 
Final 

Scientific Committee on Consumer Safety 

SCCS 

OPINION ON 

HC Orange No. 6 (B125) 

The SCCS adopted this Opinion by written procedure 
on 7 November 2016 

___________________________________________________________________________________________ 

Opinion on HC Orange No. 6 (B125) 

SCCS/1579/16 

About the Scientific Committees 
Two independent non-food Scientific Committees provide the Commission with the scientific 
advice  it  needs  when  preparing  policy  and  proposals  relating  to  consumer  safety,  public 
health  and  the  environment.  The  Committees  also  draw  the  Commission's  attention  to  the 
new or emerging problems which may pose an actual or potential threat.  
They  are:  the  Scientific  Committee  on  Consumer  Safety  (SCCS)  and  the  Scientific 
Committee  on  Health,  Environmental  and  Emerging  Risks  (SCHEER)  and  are  made  up  of 
scientists appointed in their personal capacity.   

In  addition,  the  Commission  relies  upon  the  work  of  the  European  Food  Safety  Authority 
(EFSA), the European Medicines Agency (EMA), the European Centre for Disease prevention 
and Control (ECDC) and the European Chemicals Agency (ECHA).  

SCCS 
The Committee shall provide Opinions on questions concerning all types of health and safety 
risks  (notably  chemical,  biological,  mechanical  and  other  physical  risks)  of  non-food 
consumer  products  (for  example:  cosmetic  products  and  their  ingredients,  toys,  textiles, 
clothing,  personal  care  and  household  products  such  as  detergents,  etc.)  and  services  (for 
example: tattooing, artificial sun tanning, etc.). 

Scientific Committee members  
Bernauer Ulrike, Bodin Laurent, Celleno Leonardo, Chaudhry Mohammad Qasim,  Coenraads 
Pieter-Jan,  Dusinska  Maria,  Duus-Johansen  Jeanne,  Ezendam  Janine,  Gaffet  Eric,  Galli 
Corrado  Lodovico,  Granum  Brunstad  Berit,  Panteri  Eirini,  Rogiers  Vera,  Rousselle 
Christophe, Stępnik Maciej, Vanhaecke Tamara, Wijnhoven Susan 

Contact 
European Commission 
Health and Food Safety 
Directorate C: Public Health, country knowledge, crisis management 
Unit C2 – Country knowledge and Scientific Committees 
Office: HTC 03/073     
L-2920 Luxembourg 
SANTE-C2-SCCS@ec.europa.eu  

©European Union, 2016 

ISSN 1831-4767

Doi:10.2875/547862

ISBN 978-92-79-85825-3 

EW-AQ-16-018-EN-N

The  Opinions  of  the  Scientific  Committees  present  the  views  of  the  independent  scientists 
who  are  members  of  the  Committees.  They  do  not  necessarily  reflect  the  views  of  the 
European  Commission.  The  Opinions  are  published  by  the  European  Commission  in  their 
original language only. 

http://ec.europa.eu/health/scientific_committees/consumer_safety/index_en.htm 

2 

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Opinion on HC Orange No. 6 (B125) 

SCCS/1579/16 

ACKNOWLEDGMENTS 

SCCS members  
Dr U. Bernauer 
Dr L. Bodin 
Dr L. Celleno  
Prof. Q. Chaudhry 
Prof. P.J. Coenraads    
Prof. M. Dusinska 
Prof. J. Duus-Johansen 
Dr J. Ezendam 
Prof. C.L. Galli 
Dr B. Brunstad Granum  
Prof. E. Panteri  
Prof. V. Rogiers  
Dr C. Rousselle  
Dr M. Stępnik  
Prof. T. Vanhaecke  
Dr S. Wijnhoven  

(rapporteur and WG Chair) 

All Declarations of Working Group members are available on the following webpage: 
http://ec.europa.eu/health/scientific_committees/experts/declarations/sccs_en.htm  

Keywords: SCCS,  scientific  Opinion,  HC  Orange  No.  6  (B125),  Regulation  1223/2009,  CAS 
1449653-83-1  

Opinion  to  be  cited  as:  SCCS  (Scientific  Committee  on  Consumer  Safety),  Opinion  on  HC 
Orange No. 6 (B125), 7 November 2016, SCCS/1579/16 

This opinion has been subject to a commenting period of 8 weeks (from 15 November 2016 
to 10 January 2017) after its initial publication. 
There were no comments received and the final version of the opinion remained unchanged 
compared to the preliminary one.  

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Opinion on HC Orange No. 6 (B125) 

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TABLE OF CONTENTS 

1. 

2. 

3. 

BACKGROUND .............................................................................................. 5 

TERMS OF REFERENCE ................................................................................... 5 

OPINION ...................................................................................................... 6 

3.1 

Chemical and Physical Specifications ....................................................... 6 

3.1.1 
3.1.2 
3.1.3 
3.1.4 
3.1.5 
3.1.6 
3.1.7 
3.1.8 
3.1.9 

Chemical identity ..................................................................... 6 
Physical form .......................................................................... 6 
Molecular weight ..................................................................... 6 
Purity, composition and substance codes .................................... 7 
Impurities / accompanying contaminants.................................... 8 
Solubility ................................................................................ 9 
Partition coefficient (Log Pow) .................................................... 9 
Additional physical and chemical specifications ........................... 10 
Homogeneity and Stability ....................................................... 10 
Function and uses ................................................................................ 10 

Toxicological evaluation ........................................................................ 10 

3.2 

3.3 

3.3.1 
3.3.2 
3.3.3 
3.3.4 
3.3.5 
3.3.6 
3.3.7 
3.3.8 
3.3.9 
3.3.10 
3.3.11 
3.3.12 
3.3.13 
3.3.14 

Acute toxicity ......................................................................... 10 
Irritation and corrosivity .......................................................... 11 
Skin sensitisation ................................................................... 13 
Dermal / percutaneous absorption ............................................ 14 
Repeated dose toxicity ............................................................ 15 
Mutagenicity / Genotoxicity...................................................... 18 
Carcinogenicity....................................................................... 22 
Reproductive toxicity .............................................................. 22 
Toxicokinetics ........................................................................ 25 
Photo-induced toxicity ............................................................. 26 
Human data ........................................................................... 26 
Special investigations .............................................................. 26 
Safety evaluation (including calculation of the MoS) .................... 26 
Discussion ............................................................................. 26 
CONCLUSION .............................................................................................. 28 

MINORITY OPINION ...................................................................................... 28 

REFERENCES ............................................................................................... 28 

4. 

5. 

6. 

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Opinion on HC Orange No. 6 (B125) 

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1.  BACKGROUND 

Submission I on the hair dye HC Orange No. 6 (B125), with the chemical name Di-[2-[(E)-
2-[4-[bis(2-hydroxyethyl)aminophenyl]vinyl]pyridin-1-ium]-ethyl]disulphide 
dimethanesulfonate  (CAS  1449653-83-1),  was  transmitted  by  Cosmetics  Europe  in 
December 2015.  

The  new  ingredient  HC  Orange  No.  6  (B125)  is  intended  to  be  used  in  non-oxidative  hair 
colouring products at a maximum concentration of 0.5%.  

2.  TERMS OF REFERENCE 

(1) 

In light of the data provided, does the SCCS consider  HC Orange No. 6 (B125) safe 
when  used  in  non-oxidative  hair  colouring  products  at  a  maximum  concentration  of 
0.5%? 

(2) 

Does  the  SCCS  have  any  further  scientific  concerns  with  regard  to  the  use  of  HC 
Orange No. 6 (B125) in cosmetic products? 

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3.  OPINION 

3.1 

Chemical and Physical Specifications  

3.1.1  Chemical identity 

3.1.1.1  Primary name and/or INCI name 

HC Orange No. 6 

3.1.1.2  Chemical names 

Di-[2-[(E)-2-[4-[bis(2-hydroxyethyl)aminophenyl]vinyl]pyridin-1-ium]-ethyl]disulphide 
dimethanesulfonate  
CAS NAME: Pyridinium, 1,1’-(dithiodi-2,1-ethanediyl) bis[2-[(1E)-2-[4- [bis(2-hydroxyethyl) 
amino] phenyl] ethenyl]-, methanesulfonate (1:2) 

3.1.1.3  Trade names and abbreviations 

Vibracolor Copper Orange 

3.1.1.4  CAS / EC number 

CAS: 1449653-83-1 

3.1.1.5  Structural formula 

3.1.1.6  Empirical formula 

C38H48N4O4S2, 2 CH3O3S 

3.1.2  Physical form 

Red powder 

3.1.3  Molecular weight 

Molecular weight: 879.2 (688.9 + 190.2) 

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Opinion on HC Orange No. 6 (B125) 

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3.1.4  Purity, composition and substance codes  

Chemical  identification  of  HC  Orange  No.  6  was  performed  by  IR,  1H-NMR,  13C-NMR, 
fluorescence spectroscopy and mass spectrometry using electrospray ionization (ESI) in the 
alternated positive and negative ion modes, where the expected doubly-charged ion of the 
colorant  and  the  methylsulfonate  anion  were  mainly  observed  as  the  base  peaks  of  the 
corresponding spectra at m/z 344.4 and m/z 95.1 

Table  Summarising  Composition  of  Vibracolor  Copper  Orange  for  the  batches  014L001, 
015D001 and Op. 07/13: 

014 L 001  
97.4% w/w  

015 D 001  
91.8% w/w 
(1)  

Op. 07/13  
93.0% w/w (1)  

0.01 (area%)   0.036 (2)  

0.0146 (2)  

0.40 (area%)   0.73 (3)  

0.45 (3)  

0.7 (area%)  

1.7 (1)  

0.45  

0.41  

265nm 
2.8  
(area%) 
Not performed  

450nm  
2.5  
(area%) 

0.9  
(estimated 
value  
El. Ana.)  
23.0 

4.3  
(K.F. Method)  

0.8 (mean value)  
(K.F. Method)  

19.5 

21.0 

- 

- 

0.05 

0.11 

< 0.1 

Not Performed 

<50 

<50 

<50 

Conform 

HPLC determination 

R0062327A (Aldehyde)  
(area% or % w/w)  
R0070527B  
(area% or % w/w)  
Sum of other impurities > 
0.1%  
(area% or % w/w)  
Sum of other impurities < 
0.1%  
(area%)  
Water content (% w/w)  

Methanesulfonate ion 
(Theoretical value: 21.6 %w/w) 
(% w/w) 
Acetate ion (% w/w)  

Chloride ion (% w/w) 

Elemental analysis 

Nitrosamines (μg/kg) 
Total N-Nitroso compounds 
(expressed as NO) 
Residual solvents (μg/g) (4)  
Ethanol  

Isopropanol  

<1000  
(Detected)  
4800  

1400  

- (5)  

4500  

<1000  
(Detected)  
conform  
- 
97.5  

conform  
<0.1  
100.3  

Heavy metals  
Ashes (% w/w)  
Total (% w/w)  
(1) Determined against Vibracolor Copper Orange (R0068893B) 014 L 001 (97.4% pure) 
(2) Against batch R0062327A 001P 001 reference standard (99.4% pure) 
(3) Against batch R0070527B 002 L 001 reference standard (96.7% pure) 
(4) Residual solvents content: All solvents used in the process were checked. Only ethanol and isopropanol  
    were detected with content above 0.1% (w/w). 
(5) Solvent not used in this batch 

conform  
- 
99.1  

SCCS comment 
The  applicant  should  provide  analytical  data  on  the  method  used  to  quantify 
methanesulfonate ion. 

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3.1.5  Impurities / accompanying contaminants 

Several  impurities  were  detected  in  the  three  batches.  Two  of  them  were  identified  as 
R0062327A  (Rt  =  12.0min)  and  R0070527B  (Rt  =  17.4min).  A  quantification  of  the 
impurities  with  a  level  greater  than  0.1%  (area%)  were  performed  against  the  primary 
reference standard Vibracolor Copper Orange batch 014 L 001.  
Potential  impurities  for  Vibracolor  Copper  Orange  can  be  starting  material,  synthesis 
intermediaries or by-products. Sum of the impurities above 0.1% (area %) content: About 
1 to 3 %.  

Total Ion Current profile of the impurities present in the batch #5 of the Vibracolor Copper 
Orange colorant using the Total Ion Current detection (FT-ICR) 

Characterisation of the structure of the impurities present in the two batches 014 L 001 and 
015  D  001  of  the  Vibracolor  Copper  Orange  colorant  at  a  level  greater  than  0.1%,  as 
determined from the UV profile performed  by  HPLC/ESI/MS and HPLC/ESI/MSn, the  results 
of which are presented in the table below. 

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Sum of other impurities content in: 
- Vibracolor Copper Orange batch 014 L 001: 0.7% (Area%) 
- Vibracolor Copper Orange batch 015 D 001: 1.7% w/w 
Total N-Nitroso compounds content (expressed as NO): < 50 μg/kg 

SCCS comment  
Information  regarding  hazard  classification  for  the  impurities  R0062327A  (Aldehyde)  and 
R0070527B  should  be  provided.  Any  impurity  belonging  to  CMR  (carcinogenic,  mutagenic 
and reproduction toxic) classes must be quantified. 
Since  Vibracolor  Copper  Orange  is  in  the  form  of  a  methanesulfonate  salt,  any  potential 
methane  sulfonate  impurities  such  as  ethyl  methanesulfonate  should  be  reported  and 
quantified. 

3.1.6  Solubility 

The solubility of HC Orange No. 6 was evaluated at 23oC according to the EP 5.07/5.11 
method.  

Water: between 100 and 500g/L (freely soluble), pH = 6.8. 
Absolute Ethanol: less than 0.1g/L (practically insoluble) 
DMSO: between 0.1 and 1g/L (very slightly soluble) 
Corn Oil: less than 0.1g/L (practically insoluble) 

3.1.7  Partition coefficient (Log Pow) 

Log POW: < -2 (batch 014 L001) 
Theoretical  value:  -2.7  (ClogP  SYBIL,  v.5.2)  -  error  code  10,  valid  estimate  for  difficult 
structure. 

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3.1.8  Additional physical and chemical specifications 

Melting point: batch 014 L 001:106°C (ΔH = 4J/g) and 182°C (ΔH = 63J/g) 

     batch 015 D 001: 104°C (ΔH = 28J/g) and 151°C (ΔH = 41J/g) 

Boiling point:        / 
Flash point:          / 
Vapour pressure:  / 
Density:              / 
Viscosity:            / 
pKa: 1.8 to 3.5 (25°C and ionic strength 0.15M)- experimental(GLpKa Sirius)   
Refractive index: / 
pH at 23±2°C: 7.9 ± 0.2 
Half-wave oxidation potential at pH 9.9 (voltametry): 595mV/ECS 
Surface tension in water (air/water interface): Measured at 1% weight on Vibracolor Copper 
Orange batch 015 D 001 at 23±2°C and after an equilibrium time equal to 15 min: 
The batch solution has no effect on water surface tension (γ= 72.8±0.3 mN/m). 
Critical Micellar Concentration is not detected. 
UV-Vis  spectrum  (200nm-800  nm):  λmax  at  442  and  277  nm  in  water/acetonitrile 
95:5(v/v) 

3.1.9  Homogeneity and Stability 

According to the analytical certificate, the compound is considered to be stable when stored 
at room temperature, away from light and shielded from humidity. 

SCCS comment 
Stability  of  HC  Orange  No  6  at  concentration  of  0.5%  and  under  use  conditions  should  be 
provided. 

Function and uses  

3.2 
The  ingredient  HC  Orange  No.  6  is  intended  to  be  used  in  non-oxidative  hair  colouring 
products at a maximum concentration of 0.5%. 

3.3 

Toxicological evaluation 

3.3.1  Acute toxicity 

3.3.1.1  Acute oral toxicity 

No acute oral toxicity study for HC Orange No. 6 was submitted. 

In  the  14-day  dose-range  finding  study  in  rats,  HC  Orange  No  6  did  not  induce  deaths  at 
dose  levels  up  to  1000  mg/kg/day.  Based  on  these  results,  it  can  be  concluded  that  oral 
exposure to HC Orange No. 6 does not induce acute toxicity by the oral pathway.   

3.3.1.2  Acute dermal toxicity 

No data submitted. 

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3.3.1.3  Acute inhalation toxicity 

No data submitted. 

3.3.1.4  Acute intraperitoneal toxicity 

No data submitted. 

3.3.2  Irritation and corrosivity 

3.3.2.1  Skin irritation 

Guideline: 

Test system: 

 In vitro EpiskinSM Skin Irritation Test, ECVAM validated protocol (ESAC 
statement 2007) 
 Reconstructed  Human  Epidermis  Model  EpiskinSM  (small  model, 
0,38cm2) 
3 different tissue batches 
R0068893B (HC Orange No. 6) 
R0068893B 015 D001 
91.8% 
neat substance 
10 ± 2mg 
15 minutes 

10µl of 50mg/ml aqueous solution of Sodium Dodecyl Sulfate 
10µl of PBS+ 

Replicates:  
Test substance:   
Test batch: 
Purity: 
Test item:   
Dose level:  
Treatment period:  
Post-treatment incubation time: 42 hours 
Positive control:  
Negative control:  
Direct interaction with MTT: negative 
Colouring of tissue:   positive 
GLP:   
Study period:  

in compliance 
October- November 2013 

The  test  item,  positive  and  negative  controls  were  tested  in  triplicate.  One  additional 
negative control which followed the same treatment as the negative control except the MTT 
incubation  period  was  added.  Three  additional  tissues  were  used  and  followed  the  same 
treatment  with  the  test  item  as  the  other  tissues,  except  for  the  MTT  incubation  period. 
These tissues were used as additional specific controls in order to quantify the non-specific 
colour due to the colouring interactions of HC  Orange No. 6 with the tissue. 10 ± 2 mg of 
the test substance and 10 µl of the different controls were applied onto the epidermis. After 
a 15-minute treatment period at room temperature, tissues were rinsed with PBS+ and the 
epidermis  was  transferred  in  2  ml/well  of  fresh  maintenance  medium  for  42  ±  1  hours  at 
37°C, 5% CO2 and 95% humidity. Maintenance culture media were kept frozen at -20°C for 
further  IL-1α measurements. After the 42-hour incubation period, each epidermis unit was 
transferred to another  12-well plate containing 2 ml/well of dye  solution (0.30 mg/ml MTT 
in  assay  medium),  except  for  the  negative  control  and  the  test  item-treated  epidermis 
without  MTT,  which  were  transferred  in  another  12-well  plate  containing  2  ml/well  fresh 
assay medium. After a 3-hour ± 15-minute incubation period at 37.0°C, 5.0% CO2 and 95% 
humidity, a biopsy of the entire epidermis was taken. For all tissues with the test item, the 
superficial  epidermis  layer  (containing  most  of  the  remaining  colour)  was  removed  and 
discarded. The epidermis was separated from the collagen matrix and both were transferred 
into  a  tube  containing  500  µl  acidified  isopropanol.  Formazan  crystals  were  extracted  and 
after  homogenisation  the  optical  density  was  measured  at  570  nm  versus  acidified 
isopropanol as blank and the % cell viability was calculated. 
IL-1α  released  in  the  culture  medium  was  determined  by  a  classic  quantitative  sandwich 
immunoassay  technique.  Monoclonal  specific  IL-1  antibodies  were  pre-coated  onto 

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microplates. 200 µl of standards or samples were added in the wells enabling IL-1 to bind 
to immobilised antibody. After washing, an enzyme-linked polyclonal antibody specific to Il-
1α  was  added  to  the  wells.  A  substrate  solution  was  added  and  the  intensity  of  the  colour 
developed was measured at 450 nm. 

Results 
The mean viability value for undiluted HC Orange No. 6 was 89.2 ± 3.6 % and the mean IL-
1α release was 10.3 ± 3.1 pg/mL. 

Conclusion 
Under the conditions of this study, undiluted HC Orange No. 6 is predicted to be non-irritant 
to the skin. 

Ref.: 3 

3.3.2.2  Mucous membrane irritation / Eye irritation 

OECD 437 (September 2009) 
Bovine cornea 
6 corneas per condition 
R0068893B (HC Orange No. 6) 
R0068893B 015 D001 
91.8 % 
20% (w/w) in 0.9% (w/v) NaCl 

Guideline:  
Test material: 
Replicates: 
Test item: 
Test batch: 
Purity: 
Test item: 
Treatment period:  4 hours ± 10 minutes 
Positive control: 
Negative control:  0.9% (w/v) NaCl 
GLP: 
Study period:  

in compliance 
May 2013 

20% (w/v) Imidazole in 0.9% (w/v) NaCl 

Bovine eyes (from cattle aged less than 12 months) were collected at slaughterhouses and 
prepared within 4 hours of collection. Too big eyes or defective corneas were rejected. 750 
± 8 µl of test item diluted at 20% (w/w) in 0.9% (w/v) NaCl was applied onto the cornea 
(category: solid non surfactant). The test item remained in contact with the isolated cornea 
for  4  hours  ±  10  minutes.  Six  corneas  per  condition  were  used.  At  the  end  of  the  contact 
period, the corneas were rinsed and prepared for measurement of opacity (changes in light 
transmission).  Three  of  these  corneas  were  further  used  to  measure  the  permeability 
(evaluation of transfer of 5 mg/ml fluorescein through the cornea by measuring the optical 
density at 490 nm of the media in the ocular posterior compartment). The remaining three 
corneas  were  kept  for  histological  analysis.  The  IVIS  score,  which  is  the  combination  of 
opacity  and  permeability,  was  then  calculated.  Negative  and  positive  control  substances 
were tested according to the same experimental conditions. 

Results 
The  IVIS  score  obtained  for  HC  Orange  No.  6  diluted  at  20%  (w/w) in  NaCl 0.9%  after  4-
hour contact was 10.3 ± 1.5; IVIS for the positive control was calculated to be 184.8 ± 4.4. 
Corneas stained orange upon application of the test item. Histological examination revealed 
changes in the epithelium characterised by minimal to slight erosion/ulceration, minimal to 
slight  cell  dissociation  and  minimal  to  mild  cell  alteration.  There  were  no  changes  in  the 
corneal stroma or endothelium. 

Conclusion 
Under the conditions of this study, HC Orange  No. 6 diluted at 20% (w/w) in physiological 
saline  was  concluded  to  be  neither  corrosive  nor  severely  irritant  to  eyes.  Additionally, 
histological evaluation of the cornea suggested only minimal to slight eye irritation potential 
for HC Orange No. 6 at the concentration tested. 

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SCCS comment 
Because  colouring  of  the  cornea  has  occurred,  interference  with  both  the  opacity  and  the 
permeability measurement endpoints of the BCOP assay are to be expected, which may lead 
to false estimates of the eye irritation potential.  Under the conditions of this study, an eye 
irritation potential of HC Orange No. 6 at 20% (w/w) cannot be excluded. Considering that 
the  maximum  intended  concentration  of  HC  Orange  No.  6  in  a  hair  dye  product  is  0.5% 
(w/w), it can be assumed that eye irritation will be of limited concern. 

Ref.: 11 

3.3.3  Skin sensitisation 

Local Lymph Node Assay (LLNA) 

OECD 429 

4 mice per group (main study); 2 mice per group (pre-test) 

Guideline:   
Species/strain:   Female CBA/J mice 
Group size: 
Test substance:  R0068893B 
014 L 001   
Batch: 
> 95% (HPLC, UV detection) 
Purity:  
Vehicle:  
Dimethyl sulfoxide  (DMSO) 
Concentration:   0.5, 1, 5, 10 or 25% (w/v)  
Positive control: alpha-hexylcinnamaldehyde (HCA) at 25% (v/v) in Acetone/Olive Oil (4:1;  

GLP:  
Study period:   May – June 2010 

AOO).  
In compliance 

In  a  pre-test  it  was  shown  that  the  highest  technically  achievable  concentration  in  DMSO 
(25%) did not induce any clinical signs of toxicity and no increase in ear thickness.  In the 
main LLNA study, mice were topically treated with HC Orange No. 6 at 0.5, 1, 5, 10 or 25% 
(w/v) in DMSO. The negative control group received the vehicle alone.  
The  test  item  or  vehicle  was  applied  to  the  ears  on  three  consecutive  days.  On  day  five, 
mice  were  sacrificed  and  auricular  lymph  nodes  were  sampled,  pooled  per  group,  and  the 
proliferation  of  lymphocytes  was  evaluated  by  measuring  the  incorporation  of  [3H]-
methylthymidine.  

Results 
There were no lymphoproliferative responses, with SI values of 0.43, 0.67, 1.48, 2.13 and 
2.41  obtained  at  HC  Orange  No.  6  concentrations  of  0.5%,  1%,  5%,  10%  and  25%, 
respectively. 

Conclusion 
Under  the  conditions  of  this  study,  HC  Orange  No.  6  did  not  induce  delayed  contact 
hypersensitivity and was therefore considered to be devoid of sensitising potential.  

SCCS comment 
HC Orange red No. 6 induced a dose-dependent increase of lymphocyte proliferation. For all 
tested  concentrations,  the  SI  values  were  below  3.  Under  the  conditions  of  this  study,  HC 
Orange red No. 6 is not considered a skin sensitiser. 

Ref: 12  

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3.3.4  Dermal / percutaneous absorption 

In vitro percutaneous absorption under non-oxidative conditions 

OECD TG 428 (2004) 
Frozen human dermatomed skin (400 µm) 

Guideline:   
Test system:  
Membrane integrity:  Checked by electrical resistance, at least 10 k 
Replicates:  
Test substance:   
Batch: 
Purity: 
Test item: 

12 intact skin samples (4 donors) 
[vinyl–2,2’-14C]-R0068893B 
CFQ41583 
>97% 
 Hair  dye  formulation  (P1104361,  batch  number  LG  03/11/12) 
containing 0.5% w/w R0068893B 
20 mg/cm2 of the test formulation (100 µg/cm2 R0068893B) 
2.54 cm2 
20 minutes 
24 hours 
Degassed phosphate buffered saline (PBS) 

Dose applied: 
Exposure area:   
Exposure period: 
Sampling period: 
Receptor fluid: 
Solubility in receptor fluid: Ultra-pure water: between 100 and 500 g/L 
Absolute ethanol:  
Mass balance analysis: Provided 
Tape stripping:   
Method of Analysis:  Liquid scintillation counting (LSC) 
GLP:   
Study period: 

In compliance 
18 March - 16 April 2013 

less than 100 g/L 

Yes (20) 

Human skin samples were obtained from a tissue bank. Each membrane was stored frozen 
at approximately -20°C, on aluminium foil until required for use. Skin membranes were cut 
from the samples at a thickness setting of 400 μm using an electric dermatome. 
Prior to dosing, the membrane integrity was checked by measuring electrical resistance. The 
doses were applied to the surface of 12 intact skin membranes (from 4 human donors) at a 
rate of 20 mg/cm2, corresponding to a nominal concentration of 100 μg/cm2 of R0068893B. 
At the end of the 20-minute exposure period, the skin surface was washed with water (10 x 
1270 μL), and then by 2% sodium dodecyl sulphate (SDS) in water (1 x 1270 μL). Between 
each set of washes, the washing fluid was aspired three times. The skin surface was washed 
further with water (10 x 1270 μL), after which the skin surface was dried with cotton wool 
swabs.  Following  decontamination,  the  cells  were  returned  to  the  water  bath  for  the 
remainder  of the 24-hour run time. Following this, a 24-hour mass balance procedure was 
performed. The skin surface was washed with sponges soaked in 3% Teepol L® in water and 
further  sponges  pre-wetted  with  water.  The  stratum  corneum  was  removed  by  a  tape-
stripping process, removing up to a maximum of 20  strips from  each  skin membrane. The 
flange skin was cut away from the dermis and the epidermis on the remaining skin disc and 
the epidermis was separated from the dermis using a heat separation technique. 
The  penetration  process  was  monitored  using  [14C]-radiolabelled  R0068893B,  which  was 
incorporated  into  the  formulation,  prior  to  application.  The  ‘Hair  dye  formulation’  was 
analysed  by  HPLC  to  assess  radiochemical  purity  and  concentration.  The  distribution  of 
R0068893B  within  the  test  system  was  measured  and  a  24-hour  penetration  profile  was 
determined  by  collecting  receptor  fluid  samples  20  minutes,  1,  2,  4,  8,  12,  and  24  hours 
following application. The samples were analysed by liquid scintillation counting (LSC). 

Results 
The  HPLC  method  used  was  found  to  be  suitably  sensitive  to  confirm  the  radiochemical 
purity  of  [14C]-R0068893B  when  formulated  in  the  ‘Hair  dye  formulation’.  HPLC  analysis 
confirmed the stability of the test materials for a period exceeding the length of the study. 
Three of the 12 dosed cells either had a mass balance outside the range of 85-115% (SCCS, 
2010) or a penetration profile that indicated that the membrane had become damaged over 

14 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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the course of the 24-hour run and were therefore rejected and not included in the mean ± 
SD. 
Mean  recovery  of  the  applied  test  material  was  good  at  91.6%,  with  individual  cell  values 
ranging from 86.0% to 100% (n=9). 
Table 1 shows the distribution of R0068893B in the test system. 

Table 1: Summary of R00668893B distribution in the test system 

% of applied dose 

Test compartment 
n = 9 

μg R0068893B 
per cm² 
Mean  SD 
0.062 
106 
0.190 
0.022 
0.049 
0.001 
0.006 
0.001 
106 
0.045  0.078 
106 

SD 
0.040 
Donor chamber 
5.18 
Skin wash at 20 minutes 
0.131 
Skin wash at 24 hours 
0.011 
Stratum corneum 
0.069 
Living epidermis 
0.001 
Dermis 
0.004 
Flange 
0.0004 
Receptor fluid 
5.20 
Total non-absorbed 
0.067 
Systemically available 
Total recovered 
5.25 
Total non-absorbed = Sum of donor chamber, skin wash, flange and stratum corneum 
Systemically available = Sum of remaining skin, dermis and receptor fluid 
Stratum corneum = Amount in tape strips 
Remaining skin = Tissue remaining after tape stripping 

Mean 
0.054 
91.3 
0.164 
0.019 
0.042 
0.001 
0.005 
0.0009 
91.6 
0.039 
91.6 

0.046 
6.00 
0.152 
0.013 
0.080 
0.001 
0.004 
0.0004 
6.03 

6.08 

Conclusion 
The  results  obtained  in  this  study  indicate  that  R0068893B  present  at  0.5%  in  a  hair  dye 
formulation  penetrated  through  human  dermatomed  skin  at  an  extremely  slow  rate.  The 
extent  of  R0068893B  penetrating  through  human  skin  to  the  receptor  fluid  amounted  to 
only 0.0009% (0.001 ± 0.0004 μg/cm2) of the applied dose, after 24 hours. 
The mean total systemically available dose of R0068893B was 0.039% of the applied dose 
(corresponding to 0.045 ± 0.078 μg/cm2). 

SCCS comment 
SCCS  considers  that  the  mean  +  2SD  i.e.  0.201  μg/cm2  should  be  used  for  the  MoS 
calculation because of the short exposure time. 

Ref.: 4,5 

3.3.5  Repeated dose toxicity 

3.3.5.1  Repeated Dose (14 days) oral toxicity: dose finding for 90-day study 

Guideline:   
Species/strain:   
Group size: 
Test substance:   
Batch: 
Purity: 
Vehicle: 
Dose levels: 

/ 
Rat / Wistar Hannover 
5 animals/sex/group 
R0068893B 
R0068893B 015 D001 
91.8 ± 1% 
deionised water 
0, 100, 300 and 1000 mg/kg bw/day 

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Dose volume: 
Route: 
Administration:   
GLP:  
Study period: 

4 ml/kg 
oral 
by gavage 
in compliance 
September 2011 – May 2013 

The study was performed to obtain preliminary information on the major toxic effects of the 
test item in order to select appropriate dose levels for further sub-chronic toxicity studies. 
Test substance was administered by oral gavage to male and female rats (5/sex/group) for 
14  days  at  doses  of  0,  100,  300  and  1000  mg/kg  bw/day.  The  doses  were  based  on  a 
previous study where 1000 mg/kg bw/day did not produce excessive toxic effects. 

Results 
The test item did not cause any mortality or clinical signs of toxicity during the study. 
No statistically significant effects on body weight were observed in either sex. However, the 
body weight gains over the 14-day treatment period were lower in both males (-19.1%) and 
females (-34.4%) at 1000 mg/kg bw/day (statistically significant in females only). 

In  females  at  1000  mg/kg  bw/day,  there  were  statistically  higher  values  of  food 
consumption, but no other changes (e.g. clinical signs) were found. The higher consumption 
values for this dose group were punctual and not dose-related. 

With  regard  to  clinical  pathology,  in  females  dose-related  higher  cholesterol  levels  at  100, 
300  and  1000  mg/kg  bw/day  compared  to  the  control  group  were  observed.  No  additional 
alterations were observed except for some isolated, small or not dose-related differences.  

Statistically significantly higher adrenal gland weights (absolute and relative) were observed 
at  1000  mg/kg  bw/day  in  males.  However,  the  adrenal  glands  were  not  examined 
microscopically  and  the  significance  of  this  finding  could  not  be  determined.  Nothing  was 
observed during macroscopic and microscopic examinations, except for stomach  erosion in 
one male at 1000 mg/kg bw/day. 

Conclusion 
Only  small  effects  were  observed  at  1000  mg/kg  bw/day  in  both  male  and  female  rats. 
Thus, the doses recommended for the 13-week repeated dose oral toxicity study were 100, 
300 and 1000 mg/kg bw/day. 

Ref: 6 

3.3.5.2  Sub-chronic (90 days) toxicity (oral) 

Guideline: 
Species/strain: 
Group size: 
Test substance: 
Batch: 
Purity: 
Vehicle: 
Dose levels: 
Dose volume: 
Route: 
Administration: 
GLP: 
Study period: 
The  test  substance  was  administered  by  oral  gavage  to  male  and  female  rats 
(10/sex/group) at dose levels of 0, 100, 300, 1000 mg/kg bw/day once daily for 90 days, in 

OECD TG 408 (1998) 
Rats Wistar Hannover 
10 animals/sex/group 
R0068893B 
R0068893B 015 D 001 (provided by Sponsor) 
91.8±1% 
deionized water 
0, 100, 300, 1000 mg/kg bw/day 
4 ml/kg bw/day 
oral 
gavage 
in compliance 
21 December 2011 – 4 April 2014 

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a constant dosage volume of 4 ml/kg. The dose levels were based on the findings in the 14-
day dose-range finding study described above. 

Results 
The stability of the substance was confirmed with the certificate of analysis provided by the 
sponsor.  The  stability  results  for  the  test  substance  in  the  lowest  and  highest  dosing 
formulations  were  satisfactory  for  7  days.  Thus,  test  solutions  were  prepared  weekly  and 
stored at 2-8 °C prior to use under inert gas and protected from light. Test solutions were 
stirred continuously during administration to maintain homogeneity. 

No deaths occurred during the study. No treatment-related clinical findings were observed, 
including during the ophthalmological examination. 

There  was  a  treatment-related  decrease  in  body  weight  and  body  weight  gain  at  1000 
mg/kg bw/day when compared to controls (body weight gain: -18% in males and -19 % in 
females).  There  was  also  a  decrease  of  lower  magnitude  in  males  only  at  100  mg/kg 
bw/day.  This  finding  was  considered  to  be  a  chance  event  due  to  the  absence  of  a  dose-
response relationship and because it was observed in males only.  

There were statistically significant changes in food consumption in both males and females 
in  treated  rats  compared  to  control  groups  at  some  time  points.  However,  these  changes 
were small in magnitude, not dose proportional, and did not statistically affect overall food 
consumption,  except  in  males  at  100  mg/kg  bw/day.  These  findings  were  therefore 
considered unrelated to the administration of the test item. 

With regard to the functional observational battery, a statistically significantly lower number 
of  times  passing  through  the  centre  and  higher  freezing  times  were  noted  in  1000 
mg/kg/day males during the Open Field Evaluation. Furthermore, lower mean hindlimb foot 
splay  measurements  at  heights  of  40  cm  and  30  cm,  were  observed  in  males  at  300  and 
1000 mg/kg bw/day, respectively. In females at 1000 mg/kg/day, the mean righting reflex 
time  at  30  cm  height  was  minimally  higher  when  compared  with  the  control  group.  These 
changes were isolated, of minimal magnitude, lacked consistency within a sex and/or were 
observed in a single sex. Finally, all values remained within the historical control range, and 
were then considered as normal biological variations unrelated to  the administration of the 
test item. 

No  prominent  alterations  were  found  in  the  haematological  parameters  in  treated  rats 
compared to the controls, except for lower Activated Partial Thromboplastin Time observed 
in male rats at all dose levels. These effects on Activated Partial Thromboplastin Time were 
statistically  significant  at  1000  mg/kg  bw/day.  However,  these  differences  from  controls 
were  principally  due  to  incidentally  high  values  in  control  males.  Since  values  observed  in 
test  item-treated  groups  remained  within  the  historical  control  data  range  for  rats  of  this 
strain and age, they were considered to be the result of normal biological variation. 

With  regard  to  clinical  chemistry  parameters,  few  statistically  significant  or  otherwise 
notable  differences  between  control  and  test  item-dosed  groups  were  observed.  Most 
changes  were  consistent  with  normal  biological  variation  for  this  strain  of  rats  at  this  age, 
were  of  minor  magnitude,  inconsistent  between  sexes,  and  unrelated  to  dose.  Thus,  they 
were considered to be incidental and unrelated to administration of test item.  

For the urinalysis parameters there were no changes of toxicological significance in treated 
groups  compared  to  the  controls.  At  1000  mg/kg  bw/day,  urine  was  orange-coloured  at 
1000 mg/kg/day, which was considered to be related to the colour of  the test item and/or 
its  metabolites.  Therefore,  these  changes  were  considered  to  be  without  toxicological 
significance. 
lsolated,  statistically  significant  changes  in  organ  weights  occurred  in  the  treated  rats. 
However,  these  changes  were  considered  unrelated  to  treatment  because  most  of  the 

17 

 
 
 
 
 
 
 
 
 
 
 
 
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differences  were  of  small  magnitude  and  not  dose  related,  limited  to  a  single  sex  or  not 
observed  in  relative  weights  (to  brain  weight).  The  increases  in  relative  (to  body  weight) 
brain  and  testis  weights  observed  in  males  at  1000  mg/kg  bw/day  compared  to  controls, 
were  considered  to  be  secondary  to  the  body  weight  changes.  Furthermore,  the  absolute 
and  relative  (to  organ  weight)  heart  weights  were  slightly  to  moderately  higher  in  both 
sexes  at  1000  mg/kg/day.  However,  in  the  absence  of  associated  macroscopic  or 
microscopic  changes  and  of  similar  changes  in  relative  (to  brain  weight)  weights,  these 
changes were considered to be of no toxicological significance. At 1000 mg/kg bw/day, the 
kidneys weights in females displayed consistent changes, but they were of small magnitude 
and were considered to be of no toxicological significance.  

A  few  macroscopic  lesions  were  observed,  involving  both  the  control  and  the  300  mg/kg 
bw/day group that were considered unrelated to treatment. 
Scattered  microscopic  lesions  were  seen  in  control  and  treated  rats  at  very  low  incidence, 
but none of the findings was considered related to the administration of the test item. 

Conclusion 
According  to  the  applicant,  the  NOAEL  was  considered  to  be  300  mg/kg  bw/day  for  both 
males and females. 

SCCS comment 
The SCCS agrees with the NOAEL of 300 mg/kg bw/day.  

Ref.: 7 

3.3.5.3  Chronic (> 12 months) toxicity 

3.3.6  Mutagenicity / Genotoxicity 

3.3.6.1  Mutagenicity / Genotoxicity in vitro 

Bacterial Reverse Mutation Test 

OECD TG 471 (1997) 

R0068893B 014 L 001 (red powder) 

Guideline:   
Species/Strain:  Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA 102  
Replicates:  
triplicates plates in two separate experiments  
Test substance:  R0068893B (hair-dye B125 - HC Orange No 6) 
Batch: 
Purity:               98.4% (UV detector) 
Solvent:            water (at concentrations up to at least 62.50 mg/ml) 
Positive controls: yes 
Concentrations:  experiment I: 0, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate without and 
with  S9-mix.  experiment  II:  0,  160,  300,  625,  1250,  2500  and  5000 
µg/plate with and without with S9-mix 
direct  plate  incorporation  incubated  for  3  days  protected  from  light  with 
and without S9-mix 
in compliance 
21 November 2013 - 12 February 2014 

GLP:  
Study period: 
Results 
Experiment  1  treatments  of  all  the  tester  strains  were  performed  in  the  absence  and 
presence of S-9, using final concentrations of R0068893B at 5, 16, 50, 160, 500, 1600 and 
5000  μg/plate,  plus  vehicle  and  positive  controls.  Following  these  treatments,  evidence  of 
toxicity  in  the  form  of  a  slight  thinning  of  the  background  bacterial  lawn  was  observed  at 

Treatment: 

18 

 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
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Opinion on HC Orange No. 6 (B125) 

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5000 μg/plate in strains TA98, TA1537 and TA102 in the presence of S-9. A reduction in the 
number  of  revertant  colonies  was  also  observed  at  5000  μg/plate  in  strain  TA100  in  the 
absence of S-9. 

Experiment 2 treatments of narrowed concentration intervals were employed covering the 
range  160-5000  μg/plate,  in  order  to  examine  more  closely  those  concentrations  of 
R0068893B approaching the maximum test concentration. In addition, all treatments in the 
presence of S-9 were further modified by the inclusion of a pre-incubation step, in order to 
increase the range of mutagenic chemicals that could be detected using this assay system. 
Following these treatments, evidence of toxicity was observed at 5000 μg/plate in all strains 
in the absence of S-9 and at 2500 and/or 5000 μg/plate in all strains in the presence of S-9. 
In  addition,  a  reduction  in  revertant  numbers  was  observed  at  300  μg/plate  in  strain 
TA1537 in the absence  of S-9, but this was only observed at a single concentration at the 
lower  end  of  the  range.  Negative  and  positive  controls  were  in  accordance  with  the  OECD 
guideline. 

Conclusion 
R0068893B  did  not  induce  gene  mutants  in  five  histidine-requiring  strains  (TA98,  TA100, 
TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions 
of this study. These conditions included treatments at concentrations up to 5000 μg/plate in 
the absence and in the presence of a rat liver metabolic activation system (S-9). 

Ref.: 8 

In vitro Mammalian Cell Gene Mutation Test (Hprt-locus) 

Guideline:   
Cells:  
Replicates:  
Test substance:   
Batch:  
Purity:  
Solvent:  
Positive controls:  
Concentrations:  

Treatment:  

GLP:   
Study period:  

OECD TG 476 (1997) 
L5178Y tk+/- (3.7.2C) mouse lymphoma cells 
duplicate cultures in two independent experiments 
R0068893B (hair-dye B125 - HC Orange No 6) 
R0068893B 014 L 001 (red powder) 
>95% (UV detector) 
DMSO/Water 
yes 
experiment I: 0, 660. 1200, 1400, 1600, 1800 and 2000 μg/mL 
without S9-mix; 0, 100, 200, 300, 400, 500, 600, 800 and 1000 
μg/mL with S9-mix 
experiment II 0, 500. 750, 1000, 1250, 1500, 1750 and 2000 μg/mL 
without S9-mix; 0, 200, 400, 500, and 700 μg/mL with S9-mix 
experiment III: 0, 500. 1000, 2000, 3000, 4000, 4500 and 5000 
μg/mL without S9-mix 
experiment IV: 0, 750. 1500, 2250, 3000, 4000, 4500 and 5000 
μg/mL without S9-mix 
experiment I: 3 h treatment both with and without S9-mix; 
expression period 7 days 
experiment II: 3 h treatment both with and without S9-mix;  
expression period 7 days 
experiment III: 3 h treatment without S9-mix; expression period 7 
days 
experiment IV: 3 h treatment without S9-mix; expression period 7 
days 
in compliance 
5 November 2010 – 30 May 2011 

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Results 
In  experiment  1,  six  concentrations,  ranging  from  660  to  2000  μg/mL  in  the  absence  of  a 
rat liver metabolic activation system S-9 and eight from 100 to 1000 μg/mL in the presence 
of  S-9,  were  tested.  Post-treatment  precipitate  was  observed  at  1000  μg/mL  in  the 
presence  of  S-9,  but  there  was  again  no  post-treatment  precipitate in  the  absence  of  S-9. 
Seven  days  after  treatment  the  highest  concentrations  analysed  to  determine  viability  and 
6-thioguanine (6TG) resistance were 2000 μg/mL in the absence of S-9 and 1000 μg/mL in 
the presence of S-9, which gave 74% and 90% of relative survival (RS), respectively. 
In experiment 2, seven concentrations, ranging from 500 to 2000 μg/mL in the absence of 
S-9 and four concentrations, ranging  from 200 to 700 μg/mL in the presence of S-9, were 
tested. Post-treatment precipitate was observed at 700 μg/mL and above in the presence of 
S-9  but  there  was  again  no  post-treatment  precipitate  in  the  absence  of  S-9.  Seven  days 
after  treatment  the  highest  concentrations  analysed  to  determine  viability  and  6TG 
resistance were 2000 μg/mL in the absence of S-9 and 700 μg/mL in the presence of S-9, 
which gave 82% and 89% RS, respectively. 
In experiment 3, seven concentrations, ranging from 500 to 5000 μg/mL, were tested in the 
absence  of  S-9.  Seven  days  after  treatment  the  highest  concentration  analysed  to 
determine viability and 6TG resistance was 5000 μg/mL in the absence of S-9, which gave 
25% RS. 
In experiment 4, seven concentrations, ranging from 750 to 5000 μg/mL, were tested in the 
absence of S-9. Seven days after treatment the highest concentration selected to determine 
viability and 6TG resistance was 5000 μg/mL in the absence of S-9, which gave 58% RS. 

In experiments 1 and 2, no statistically significant increases in mutant frequency (MF) were 
observed  following  treatment  with  R0068893B  at  any  concentration  tested  in  the  absence 
and  presence  of  S-9  and  there  were  no  significant  linear  trends.  In  experiment  3,  a 
significant increase in MF was observed at 4500  μg/mL and a statistically significant linear 
trend  was  also  observed.  However,  the  MF  value  was  less  than  three  times  the  historical 
control and no such increase in MF was observed in higher concentration (5000 μg/mL) and 
the effect was not reproduced in experiment 4. This isolated observation was therefore not 
considered biologically relevant. 

Conclusion 
R0068893B  (hair-dye  B125  -  HC  Orange  No  6)  did  not  induce  gene  mutations  at  the  Hprt 
locus in L5178Y mouse lymphoma cells when tested under the  conditions employed in this 
study.  These  conditions  included  treatments  up  to  the  limit  of  solubility  in  culture  medium 
(up  to  5000  μg/mL  -  the  maximum  recommended  concentration  according  to  the  current 
regulatory guidelines) in the absence of S-9. 

Ref.: 9 

SCCS comment: 
Only 3 h exposure was used. 

Micronucleus Test in Human Lymphocytes 

Guideline:   
Cells:  
Replicates:  
Test substance:   
Batch:  
Purity:  
Solvent:  
Positive controls:  
Concentrations and treatment:  

OECD TG 487 (2010) 
human lymphocytes 
duplicate cultures in 2 independent experiments 
R0068893B 
R0068893B 014 L 001 (red powder) 
>95% (UV detector) 
DMSO/Water 
yes 

experiment I: vehicle DMSO (limited solubility) 

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3 h treatment + 21 recovery 0, 200, 400, 600, 800, 1000, 1200, 
1400, 1500, 1750 and 2000 μg/ml without S9-mix  
3 h treatment + 21 recovery 0, 200, 400, 600, 800, 1000, 1200, 
1400, 1500 and 1750 with S9-mix 
24 h treatment + 0 recovery 0, 200, 400, 600, 800, 1000, 1200, 
1400, 1500 and 1750 without S9-mix 
experiment II: vehicle water (enabling higher concentrations to be 
tested) 3 h treatment + 21 recovery 0, 250, 500, 1000, 2000, 3000, 
4000 and 5000 μg/ml without S9-mix  
24 h treatment + 0 recovery 0, 250, 500, 1000, 2000, 3000, 4000 
and 5000 μg/ml without S9-mix 
Treatment: experiment I and II: 3 + 21 hour treatment both with 
and without S9-mix; test article added at 48 h following culture 
initiation (stimulation by PHA). Cultures were sampled 3 or 24 h after 
the beginning of the treatment. (i.e. 72 h after culture initiation) 
in compliance 
27 January 2011 – 24 March 2014 

GLP:   
Study period:  

Results 
R0068893B was tested in an in vitro micronucleus assay using duplicate human lymphocyte 
cultures  prepared  from  the  pooled  blood  of  two  male  donors  in  two  sets  of  experiments. 
Treatments  covering  a  broad  range  of  concentrations  were  performed  both  in  the  absence 
and  presence  of  metabolic  activation  (S-9)  from  Aroclor  1254-induced  animals.  The 
dimethyl  sulphoxide  (DMSO)  was  used  as  the  vehicle.  The  highest  concentrations  used  in 
Experiments  1  and  2  were  either  5000  μg/mL  or  were  limited  by  solubility  in  culture 
medium and were determined following Range-Finder Experiments 1 and 2. The test article 
concentrations  for  micronucleus  analysis  were  selected  by  evaluating  the  effect  of 
R0068893B  on  the  replication  index  (RI).  In  the  Micronucleus  Experiments,  micronuclei 
were analysed at three or four concentrations, limited by the observation of post-treatment 
precipitate under each treatment condition.  

Treatment  of  cells with R0068893B (hair-dye  B125 - HC Orange  No  6) in the absence and 
presence  of  S-9  in  Experiment  1  in  DMSO  and  in  the  absence  of  S-9  in  Experiment  2  in 
water resulted in frequencies of binucleated cells with micronuclei (MNBN) that were similar 
to (and not significantly different from) those observed in concurrent vehicle controls at all 
concentrations  analysed.  The  MNBN  cell  frequencies  of  all  R0068893B  treated  cultures 
under all treatment conditions in Experiments 1 and 2 fell within the normal ranges. 

Conclusion 
R0068893B (hair-dye B125 - HC Orange No 6) did not induce micronuclei in cultured human 
peripheral  blood  lymphocytes  in  the  absence  and  presence  of  S-9.  The  maximum 
concentration  analysed  under  all  treatment  conditions  was  either  5000  μg/mL  (the 
maximum  concentration  recommended  by  the  test  guidelines)  or  was  limited  by  the 
solubility of the test article in culture medium. 

Ref.: 10 

SCCS overall comment on mutagenicity 
Under the experimental conditions used, HC Orange No 6 did not show gene mutations and 
did  not  induce  an  increase  in  micronucleated  cells  and,  consequently,  is  not  considered  to 
be mutagenic, clastogenic and/or aneugenic in human lymphocytes. 

21 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Opinion on HC Orange No. 6 (B125) 

SCCS/1579/16 

3.3.6.2  Mutagenicity / Genotoxicity in vivo 

3.3.7  Carcinogenicity 

3.3.8  Reproductive toxicity 

3.3.8.1  Two generation reproduction toxicity 

3.3.8.2  Other data on fertility and reproduction toxicity 

3.3.8.3  Developmental Toxicity 

Prenatal Developmental Toxicity Study in Rats: dose-range finding study 

10 females/group 

OECD TG 414 (2001) 
Guideline:   
Species/strain:  Rat, Wistar Hannover 
Group size: 
Test substance:  R0068893B 
Batch: 
Purity: 
Vehicle: 
Dose levels: 
Dose volume:  4 mL/kg bw 
Route: 
Administration:  Gavage 
GLP:  
Study period: 

Oral 

R0068893B 015 D 001 
91.8±1 % 
deionized water 
0, 100, 300 and 1000 mg/kg bw/day 

In compliance 
5 October 2011- 16 October 2013 

The  test  solutions  (at  100,  300  and  1000  mg/kg  bw/day)  or  deionized  water  were 
administered  to  the  mated  females  rats  daily  (control  group-  8  animals,  group  100 
mg/kg/day9 animals, group 300 mg/kg/day-9 animals, group 1000 mg/kg/day-6 animals), 
by  gavage,  from  implantation  until  one  day  prior  to  the  scheduled  caesarean  section  (i.e. 
from day 6 to day 19 of gestation). A constant dose-volume of 4 mL/kg was used.  

Results 
No chemical analysis of the test item solutions was carried out. The stability, homogeneity 
and concentration of the test solutions were not determined for this study. 
No mortality or clinical symptoms were observed in any of the groups. 
No  biologically  or  statistically  significant  differences  in  body  weight,  body  weight  gain  or 
food consumption were observed between the exposed and the control group. 
A small but not statistically and biologically significant decrease in corrected maternal body 
weight was observed in all treated dams. 
No  biologically  relevant  changes  were  observed  in  reproduction  parameters  between  the 
control  and  the  exposed  dams.  There  were  no  changes  observed  at  necropsy  in  either 
treated or control dams. 
Foetal  evaluation  revealed  no  changes  in  body  weights  and  placental weights  between  the 
exposed  and  control  groups.  Similarly  no  external  alterations  were  noted  in  foetuses  from 
the exposed or control dams. 

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Conclusion 
Based on the results the authors of the report recommended the following doses of the test 
item  for  the  prenatal  developmental  oral  toxicity  study  in  rats:  100,  300  and  1000 
mg/kg/day. 

Ref: 1 

Prenatal Developmental Toxicity Study in Rats 

25 females/group 

Guideline:   
OECD TG 414 (2001) 
Species/strain:  Rat, Wistar Hannover 
Group size: 
Test substance:  R0068893B 
Batch: 
Purity: 
Vehicle: 
Dose levels: 
Dose volume:  4 mL/kg bw 
Route: 
Administration:  Gavage 
GLP:  
Study period: 

Oral 

R00688938 015 0 001 
91.8±1 % 
deionized water 
0, 100, 300 and 1000 mg/kg bw/day 

In compliance 
24 November 2011- 2 February 2015 

The dose levels were based on the findings in the 14-day dose-range finding study. 
The  test  solutions  (at  100,  300  and  1000  mg/kg  bw/day)  or  deionized  water  were 
administered to the mated females rats daily (25/group), by gavage, from implantation until 
one day prior to the scheduled caesarean section (i.e. from day 6 to day 19 of gestation). A 
constant dose-volume of 4 mL/kg was used. Animals that were not pregnant were excluded 
from calculations regarding mean maternal bodyweights, food consumption and gestational 
parameter (n= 19, 19, 20 and 23, for 0, 100, 300 and 1000  mg/kg bw/day, respectively). 
ln  addition,  one  rat  from  the  control  group  with  total  resorptions  was  excluded  from  the 
calculation  of  mean  body  weight,  body  weight  gain,  food  consumption,  corrected  maternal 
body weight and uterine weight. Furthermore, the proportion of live foetuses was calculated 
excluding this dam with total resorptions.  

Results 
The stability of the substance was confirmed with the certificate of analysis provided by the 
sponsor.  Test  solutions  were  prepared  at  the  lowest  and  highest  concentrations.  Duplicate 
samples  were  taken  on  the  day  of  preparation  and  after  4  and  7  days  storage  at  +2°C  to 
+8°C. The concentration of the test item in control and test solutions was determined at the 
beginning  and  at  the  end  of  the  treatment  period.  The  acceptance  criterion  with  regard  to 
both stability and concentration at each time-point was that mean concentration was ±10% 
of  the  nominal  value.  All  values  were  within the  acceptable  range.  The  test  solutions  were 
prepared daily and administered within 2 hours after preparation. 

No mortality was observed in any group during the study. 

Body weight and body weight gain measurements showed a decrease in body weight gain in 
dams given 1000 mg/kg/day between days 6 and 12 (23%), with subsequent recovery. This 
resulted  in  a  slight  decrease  in  overall  body  weight  gain  (-7.1%  and  -18.8%  without  and 
with  correction  for  uterus  weight,  respectively).  Although  the  changes  did  not  reach 
statistical  significance,  according  to  the  authors  of  the  study  report  they  were  likely  to  be 
treatment related and an indication of slight maternal toxicity. 

Corrected  body  weight  gain  measurements  revealed  a  higher  mean  unopened  uterine 
weight  in  the  dams  exposed  to  100  and  300  mg/kg/day  compared  to  the  control  group 
(+16.4% and +17.6%, respectively). These differences were not statistically significant and 

23 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Opinion on HC Orange No. 6 (B125) 

SCCS/1579/16 

were considered by the authors of the report to be related to the higher number of foetuses 
in  both  groups,  therefore  being  not  related  to  the  treatment.  Corrected  maternal  body 
weight  gain  was  lower  in  dams  exposed  to  300  and  1000  mg/kg/day  (-20.9%  mid  and  -
18.8% high dose, when compared to the controls). According to the authors of the report, 
the changes were likely to be treatment related and were  considered  to be of toxicological 
significance,  despite  the  fact  that  they  did  not  achieve  statistical  significance  or  show  any 
dose-related trend. 

Food  consumption  measurements  did  not  reveal  any  biologically  or  statistically  significant 
changes between the groups. 

No  significant  effects  were  observed  in  the  gestational  parameters  or  maternal  necropsy 
findings in any treated group when compared to the control group. 

No  changes  in  mean  placenta  weight  were  observed  when  compared  to  control  values. 
Although  mean  foetal  body  weight  was  slightly  and  not  significantly  lower  at  1000 
mg/kg/day  in  comparison  to  the  control  values,  the  authors  of  the  report  considered  the 
effect to be attributed to dosing with R0068893B. 
External examinations of the foetuses, except one foetus excluded from the analysis due to 
spontaneous,  non-test-item  related  malformations,  did  not  reveal  any  variations  or 
malformations in control or exposed dams. 
No soft tissue malformations were observed in treated or control foetuses. 
The  total  number  of  soft  tissue  variations  was  significantly  higher  in  foetuses  from  dams 
exposed to 1000 mg/kg/day compared to the  control group.  This was primarily due to the 
higher  incidence  of  bilateral  dilatation  of  the  renal  pelvis,  which  was  significantly  different 
from controls on a foetal (+9.7%) and litter (+36.4%) basis. Since these changes occurred 
in  the  highest  dose  group  and  because  the  incidence  values  were  outside  of  the  historical 
control  range,  they  were  considered  by  the  authors  of  the  report  to  be  related  to  the 
administration of R00688938 and to be secondary to the maternal toxicity observed at 1000 
mg/kg/day. 
The  few  skeletal  malformations  observed  in  treated  and  control  foetuses  were  of  the  type 
and incidence commonly observed in rats of this strain and were evenly distributed among 
groups.  Therefore  they  were  considered  by  the  authors  of  the  report  to  be  unrelated  to 
exposure to R00688938. 

Applicant's  summary  of  foetal  skeletal  variations  and  retardations  observed  in  the  study  is 
presented below: 

Foetal skeletal variations: 

-  The  litter  incidence  of  supernumerary  ribs  was  significantly  higher  in  all  treated 
groups,  while  the  foetal  incidence  was  significantly  higher  only  at  1000  mg/kg/day. 
The foetal incidence remained within historical control data range, although for litters 
it was outside this range. Despite the differences  observed in litter incidence, these 
changes  did  not  show  any  dose-relationship  and  the  associated  changes  in  foetal 
incidence were of low magnitude; 

-  Foetal and/or litter incidences of some other  skeletal variations were  lower than for 
controls in some or all groups given R006688938 (e.g. bipartite sternebrae, enlarged 
fontanel). According to the authors of the report, these changes were considered to 
be  unrelated  to  exposure  to  R00688938  and  of  no  biological/toxicological 
significance; 

-  A  higher  incidence  of  dumbbell-shaped  sternebrae  was  observed  in  foetuses  from 
dams  exposed  to  300  and  1000  mg/kg/day.  Similar  changes  in  incidences  of 
dumbbell-shaped  thoracic  vertebrae  were  observed  in  foetuses  at  1000  mg/kg/day. 
These  incidences  at  1000  mg/kg/day  were  significantly  increased  over  control 
values, except for the foetal incidence of dumbbell-shaped sternebrae; 

-  The  incidence  values  of  dumbbell-shaped  thoracic  vertebrae  were  within  historical 
control  range  and  these  findings  were  thus  considered  to  be  unrelated  to  the 

24 

 
 
 
 
 
 
 
 
 
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Opinion on HC Orange No. 6 (B125) 

SCCS/1579/16 

administration  of  R00688938.  ln  contrast,  the  increased  incidences  of  dumbbell-
shaped  sternebrae  at  300  and  1000  mg/kg/day  were  considered  treatment  related 
since the incidence values observed are outside the historical control range. 

Foetal skeletal retardations: 

-  The incidences of incomplete ossification of interparietal bone in foetuses from dams 
exposed  to  300  and  1000  mg/kg/day  were  significantly  higher  than  in  the  control 
group. According to the authors of the report, these high incidences were considered 
to  be  unrelated  to  treatment  since  they  remained  within  historical  control  data 
range; 

-  A  higher  foetal  incidence  of  unossified  metacarpals  was  noted  at  300  and  1000 
mg/kg/day,  statistically  significant  at  1000  mg/kg/day.  This  finding  was  considered 
by  the  authors  of  the  report  to  be  treatment  related  at  1000  mg/kg/day  since  the 
incidence value observed was outside historical control range; 

-  At  300  mg/kg/day,  higher  incidences  of  unossified  caudal  vertebrae,  incomplete 
parietal bone ossification and incomplete ossification of sacral vertebrae were noted, 
whereas  the  incidence  of  incomplete  supraoccipital  bone  ossification  was  higher  at 
100  and  300  mg/kg/day,  compared  to  the  control  group.  These  changes  reached 
statistical  significance  but  were  considered  by  the  authors  of  the  report  to  be 
unrelated to treatment in the absence of dose-related relationship, specifically in the 
absence of similar changes at the highest dose level of 1000 mg/kg/day. 

Conclusion 
Based  on  the  above  results,  the  authors  of  the  study  report  established  the  No  Observed 
Adverse Effect Level (NOAEL) for maternal toxicity of R0068893B at 100 mg/kg/day and the 
NOAEL for foetal developmental toxicity at 300 mg/kg/day.  

Ref.: 2 

SCCS comment 
The study showed that the corrected maternal body weight gain was lower in dams exposed 
to  300  and  1000  mg/kg/day  (-20.9%  mid  and  -18.8%  high  dose,  when  compared  to  the 
controls).  SCCS  agrees  that  the  changes  could  be  treatment  related  and  should  be 
considered to be of toxicological significance, despite the fact they did not achieve statistical 
significance or show any dose-related trend. Therefore the NOAEL for maternal toxicity can 
be established at 100 mg/kg. 
The SCCS agrees that the test item R00688938 did not show any teratogenic potential. 
Most foetal skeletal variations were observed at 1000 mg/kg/day and their incidences were 
outside  of  historical  control  range.  Moreover,  they  were  associated  with  other  foetus 
developmental  disturbances  (e.g.  decreased  body  weight).  Some  foetal  skeletal  variations 
which  were  observed  at  100  and  300  mg/kg  were  most  likely  caused  by  retarded 
ossification  due  to  slight  maternal  toxicity  at  these  doses.  Therefore,  they  should  not  be 
treated  as  toxicologically  relevant,  even  if  they  were  statistically  significant.  The  SCCS 
agrees  that  the  NOAEL  value  for  developmental  toxicity  can  be  established  at  300 
mg/kg/day.  

3.3.9  Toxicokinetics 

No data 

3.3.9.1  Toxicokinetics in laboratory animals 

25 

 
 
 
 
 
 
 
 
 
 
 
 
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Opinion on HC Orange No. 6 (B125) 

SCCS/1579/16 

3.3.9.2  Toxicokinetics in humans 

3.3.10  Photo-induced toxicity 

3.3.10.1   Phototoxicity / photo-irritation and photosensitisation 

/ 

3.3.10.2   Photomutagenicity / photoclastogenicity 

/ 

3.3.11  Human data 

/ 

3.3.12  Special investigations 

/ 

3.3.13  Safety evaluation (including calculation of the MoS) 

CALCULATION OF THE MARGIN OF SAFETY 

(non-oxidative conditions) 
(on-head concentration 0,5 %) 

Absorption through the skin  
Skin Area surface 
Dermal absorption per treatment 
Typical body weight of human 
Systemic exposure dose (SED)  
No observed adverse effect level 
(prenatal, developmental, oral, rat) 
Bioavailability 50%* 

A 
SAS 
SAS x A x 0.001 

=  0.201 µg/cm² 
=  580 cm2 
=  0.12 mg 
=  60 kg 

SAS x A x 0.001/60  =  1.94x10-3 mg/kg bw 
NOAEL 

=  100 mg/kg bw/d 

=  50 mg/kg bw/d 

Margin of Safety 

adjusted NOAEL/SED  =  25773 

* standard procedure according to the SCCS's Notes of Guidance for the testing of cosmetic ingredients and their 
safety evaluation. 

3.3.14  Discussion 

Physicochemical properties 
The  SCCS  notes  the  presence  of  ethanol  in  the  batches  used  for  the  chemical  analysis.  
Since  Vibracolor  Copper  Orange  is  in  the  form  of  a  methanesulfonate  salt,  any  potential 
methane sulfonate impurities such as ethyl methanesulfonate (which may be formed in the 
presence of ethanol) should be reported and quantified. 

26 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Opinion on HC Orange No. 6 (B125) 

SCCS/1579/16 

General toxicity 
No  acute  oral  toxicity  study  with  R0068893B was  submitted  but  repeated  dose  studies  did 
not show the potential for acute toxicity.  
Daily  administration  of  100,  300  and  1000  mg/kg  bw/day  R0068893B  by  oral  gavage  to 
male and female Wistar Hannover rats for 90 days resulted in a treatment-related decrease 
in  body  weight  and  body  weight  gain  at  1000  mg/kg  bw/day  when  compared  to  controls 
(body  weight  gain:  -18%  in  males  and  -19  %  in  females).  Therefore  the  NOAEL  for  this 
study can be established at 300 mg/kg. 

Developmental toxicity 
Some foetal skeletal variations which were observed at 100 and 300 mg/kg/day were most 
likely  caused  by  retarded  ossification  due  to  slight  maternal  toxicity  at  these  doses.  The 
SCCS concluded that the NOAEL value for developmental toxicity can be established at 300 
mg/kg/day. 
The study showed that the corrected maternal body weight gain was lower in dams exposed 
to  300  and  1000  mg/kg/day  (-20.9%  mid  and  -18.8%  high  dose,  when  compared  to  the 
controls).  SSCS  agree  that  the  changes  could  be  treatment  related  and  should  be 
considered to be of toxicological significance, despite the fact they did not achieve statistical 
significance or show any dose-related trend. Therefore the NOAEL for maternal toxicity can 
be established at 100 mg/kg. 

Irritation/sensitisation 
The  in  vitro  tests  did  not  indicate  skin  irritancy.  While  an  eye  irritation  potential  at  20% 
cannot be excluded, it can be assumed that this will be of no concern  in view of the 0.5% 
intended use concentration. 
The in vivo tests did not indicate a skin-sensitising potential. 

Dermal absorption  
The  submitted  documents  indicate  a  very  low  rate  of  dermal  penetration.  Because  of  the 
relatively  short  exposure  time  in  the  test  system,  the  mean  +  2  SD  is  used  for  the 
calculation of the MoS. 

Mutagenicity 
The  genotoxicity  of  HC  Orange  No.  6  was  investigated  in  the  three  endpoints  of 
genotoxicity:  gene  mutations,  structural  chromosome  aberrations  and  aneuploidy.  Di-[2-
[(E)-2-[4-[bis(2-hydroxyethyl)aminophenyl]vinyl]pyridin-1-ium]-ethyl]disulfide 
dimethanesulfonate  did not induce gene mutants in five strains of Salmonella typhimurium 
(TA98,  TA100,  TA1535,  TA1537  and  TA102)  up  to  concentration  5000  μg/plate  in  the 
absence and in the presence of a rat liver metabolic activation system (S-9 mix), it did not 
induce gene mutants at the Hprt locus in  L5178Y mouse lymphoma cells in the absence or 
presence  of  S-9  mix  up  to  5000  μg/mL  and  did  not  inducemicronuclei  in  cultured  human 
peripheral blood lymphocytes either in the absence or the presence of S-9 mix.  
Thus, Di-[2-[(E)-2-[4-[bis(2-hydroxyethyl)aminophenyl]vinyl]pyridin-1-ium]-ethyl] disulfide 
dimethanesulfonate  can  be  considered  to  have  no  genotoxic  potential  and  additional  tests 
are unnecessary. 

27 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Opinion on HC Orange No. 6 (B125) 

SCCS/1579/16 

4.  CONCLUSION 

(1) 

In light of the data provided, does the SCCS consider HC Orange No. 6 (B125) safe 
when used in non-oxidative hair colouring products at a maximum concentration of 
0.5%? 

The SCCS considers HC Orange No. 6 (B125) safe when used in non-oxidative hair colouring 
products at a maximum concentration of 0.5%. 

(2)  Does  the  SCCS  have  any  further  scientific  concerns  with  regard  to  the  use  of  HC 

Orange No. 6 (B125) in cosmetic products? 

The SCCS has concerns regarding a potential presence of  methanesulfonates impurities, in 
particular ethyl methanesulfonate. Information on these impurities has not been provided.  

5.  MINORITY OPINION 

/ 

6.  REFERENCES 

1.  Betancourt  Martell  A.  (2013).  Preliminary  Prenatal  Developmental  Oral  Toxicity 

Study in Rats for R0068893B. Bioagri 9050.314.004.11  

2.  Betancourt  Martell  A.  (2015).  Prenatal  Developmental  Oral  Toxicity  Study  with 

R0068893B in Wistar Rats. Bioagri 9050.314.005.11  

3.  Chagneau C. (2013). Primary Cutaneous Tolerance. Prediction of the acute irritant 

potential  on  human  reconstructed  epidermis  EpiskinSM  model.  Episkin  Study  No. 

13-BPL-0120  

4.  Davies  D.J.  (2013a).  In  Vitro  Penetration  of  [14C]-R0068893B  through  Human 

Dermatomed Skin. DTL Study Reference Number: JV2262-REG  

5.  Davies D.J. (2013b). Final Report – Report Amendment 1 – In Vitro Penetration of 

[14C]-R0068893B  through  Human  Dermatomed  Skin.  DTL  Study  Reference 

Number: JV2262-REG  

6.  Gomez  J.R.  (2013).  14-Day  Oral  Toxicity  Study  in  rats  for  R0068893B.  Bioagri 

9050.306.004.11  

7.  Gomez  J.R.  (2014).  Repeated  Dose  13-week  Oral  Toxicity  Study  with  R0068893B 

in Wistar Rats. Bioagri 9050.307.002.11  

8.  Lloyd  M.  (2014a).  R0068893B:  Bacterial  reverse  mutation  assay.  Covance  Study 

No. 8295094  

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Opinion on HC Orange No. 6 (B125) 

SCCS/1579/16 

9.  Lloyd  M.  (2014b).  Mutation  at  the  hprt  locus  of  mouse  lymphoma  L5178Y  cells 

(MLA) using the MicrotitreR fluctuation technique. Covance Study No. 8238619  

10. Lloyd  M.  (2011c).  Induction  of  micronuclei  in  cultured  human  peripheral  blood 

lymphocytes. Covance Study No. 8242983  

11. Maillet  S.  (2013).  Ocular  primary  irritation  B.C.O.P.  –  Study  Performed  on  the 

Isolated  Bovine  Cornea.  Measurement  of  the  Bovine  Corneal  Opacity  and 

Permeability  (according  to  the  OECD  protocol  n°437  of  7  September  2009). 

Histology. IEC Report No 130622RD1 

12. Tiwari  V.K.  (2011).  Skin  Sensitization  Study  of  R0068893B  by  Local  Lymph  Node 

Assay in Mice. JRF Study No. 10176 

29