Opinion on 2,2’-Methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano form) : submission III : COLIPA S79.
Date: 2018-01-30
Institutions: Directorate-General for Health and Food Safety
Subjects: analytical chemistry, chemical product, consumer protection, cosmetic product, directive (EU), skin disease

SCCS/1546/15 
Revision of 25 June 2015 

Scientific Committee on Consumer Safety 

SCCS 

OPINION ON 

2,2’-Methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-
tetramethylbutyl)phenol)  
(nano form) 

Submission III 

COLIPA n° S79 

The SCCS adopted this opinion on its 9th plenary meeting 

on 25 March 2015 

Revision of 25 June 2015  

Corrigendum: 3 September 2015, 6 October 2016 and 19 January 2018 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

About the Scientific Committees 
Three  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),  the  Scientific  Committee  on 
Health and Environmental Risks (SCHER) and the Scientific Committee on Emerging and Newly 
Identified Health Risks (SCENIHR) and are made up of external experts.   
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 
Ulrike  Bernauer,  Qasim  Chaudhry,  Pieter  Coenraads,  Gisela  Degen,  Maria  Dusinska,  Werner 
Lilienblum,  Elsa  Nielsen,  Thomas  Platzek,  Suresh  Chandra  Rastogi,  Christophe  Rousselle,  Jan 
van Benthem. 

Contact 
European Commission 
Health and Food safety 
Directorate C: Public Health  
Unit C2 – Health Information and Scientific Committees 
Office: HTC 03/073    
L-2920 Luxembourg 
SANTE-C2-SCCS@ec.europa.eu  

© European Union, 2015 

ISSN 1831-4767

ISBN 978-92-79-56139-9 

Doi: 10.2875/101475

EW-AQ-16-116-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 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

ACKNOWLEDGMENTS 

SCCS/1546/15 

SCCS members 
Dr U. Bernauer   
Dr Q. Chaudhry   
Dr M. Dusinska 
Dr W. Lilienblum 
Prof. Th. Platzek 
Dr S. Ch. Rastogi 
Dr J. van Benthem 

SCENIHR members 
Prof. P. H. M. Hoet 
Dr K. Rydzynski 

External experts 
Dr O. Bussolati 
Dr N. von Götz 
Dr S. H. Doak 
Dr T. Jung 

(chairman) 

(rapporteur) 

This opinion  has  been  subject to a commenting  period of  minimum  four weeks after  its  initial 
publication.  Comments  received  during  this  time  have  been  considered  by  the  SCCS  and 
discussed  in  the  subsequent  plenary  meeting.  Where  appropriate,  the  text  of  the  relevant 
sections of the opinion has been modified or explanations have been added. In the cases where 
the  SCCS  after  consideration  and  discussion  of  the  comments,  has  decided  to  maintain  its 
initial views, the opinion (or the section concerned) has remained unchanged. Revised opinions 
carry the date of revision. 

The  Opinion  has  been  revised  in  June  2015  and  corrected  on  3  September  2015,  6  October 
2016 and 19 January 2018. 

Keywords: SCCS,  scientific  opinion,  UV-filter,  S79,  2,2’-methylene-bis-(6-(2H-benzotriazol-2-
yl)-4-(1,1,3,3-tetramethylbutyl)phenol),  Regulation  1223/2009,  CAS  103597-45-1,  EC  403-
800-1 

Opinion  to  be  cited  as:  SCCS  (Scientific  Committee  on  Consumer  Safety),  Opinion  on  2,2’-
methylene-bis-(6(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol),  SCCS/1546/15, 
25 March 2015, revision of 25 June 2015, corrigendum of 19 January 2018 

3 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

TABLE OF CONTENTS 

ACKNOWLEDGMENTS ........................................................................................... 3 

1.

2.

3.

BACKGROUND ............................................................................................. 6

TERMS OF REFERENCE .................................................................................. 6

OPINION ..................................................................................................... 7

3.1.1. 
3.1.2. 
3.1.3. 
3.1.4. 
3.1.5. 
3.1.6. 
3.1.7. 
3.1.8. 
properties 
3.1.9. 
3.1.10. 
3.1.11. 
3.1.12. 
3.1.13. 
3.1.14. 
3.1.15. 

Chemical identity .................................................................... 7 
Physical form ......................................................................... 8 
Molecular weight .................................................................... 8 
Purity, composition and substance codes .................................... 8 
Impurities / accompanying contaminants ................................. 10 
Solubility/ dissolution ............................................................ 11 
Partition coefficient (Log Pow) ................................................ 12 
Additional physical and chemical specifications: nano specific 
12 
Additional physical and chemical specifications .......................... 15 
Microscopy .......................................................................... 16 
Crystal structure ................................................................... 17 
UV absorption ...................................................................... 17 
Zeta potential ...................................................................... 19 
Droplet size in formulations .................................................... 20 
Homogeneity and stability ...................................................... 21 
Function and uses .............................................................................. 22 

3.2. 

3.3. 

Toxicological Evaluation ...................................................................... 22 

Acute toxicity ....................................................................... 22 
Irritation and corrosivity ........................................................ 24 
Skin sensitisation .................................................................. 26 
Dermal / percutaneous absorption........................................... 27 
Repeated dose toxicity .......................................................... 31 
Mutagenicity / Genotoxicity .................................................... 36 
Carcinogenicity ..................................................................... 41 
Reproductive toxicity ............................................................. 43 
Toxicokinetics and metabolism................................................ 43 
Photo-induced toxicity ........................................................... 45 
Human data ......................................................................... 49 
Special investigations ............................................................ 52 
Safety evaluation (including calculation of the MoS) ................... 52 
Discussion ........................................................................... 54 
4. CONCLUSION ................................................................................................. 56

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. 

5. MINORITY OPINION ........................................................................................ 57

6. REFERENCES ................................................................................................. 58

4 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

Annex I ............................................................................................................ 63 

Annex II ........................................................................................................... 65 

SCCS/1546/15 

5 

 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

1.  BACKGROUND 

substance 

2,2'-methylene-bis-(6(2H-benotriazol-2-yl)-4-(l, 

The 
1,3,3- 
tetramethylbutyl)phenol)  (MBBT)  (CAS  No  103597-45-1),  Methylene  bis-benzotriazolyl 
tetramethylbutylphenol (INCI) (COLIPA S79) is currently regulated in Entry 23 Annex VI of the 
Cosmetics  Regulation  (EC)  1223/2009  with  the  maximum  concentration  of  10%  in  cosmetic 
products  such  as  sunscreens,  day  care  products  and  skin  lightening  products.  The  safety  of 
mentioned use conditions was confirmed by the Scientific Committee on Cosmetic Products and 
Non-Food (SCCNFP) in 1998 following submission I by COLIPA1 (SCCNFP/0080/98)2. 

Submission II on MBBT was submitted to the Scientific Committee on Consumer Safety (SCCS) 
in January 2013 to assess the safety of its nano form. The SCCS opinion concluded that: 

"Since no appropriate data on genotoxicity of nano form of MBBT were provided, no conclusion 
on the safety of this substance can be drawn. However regarding systemic effects there seems 
no concern for the dermal application of nano-sized MBBT" (SCCS/1460/11)3. 

In August 2014 the applicant has transmitted a new safety dossier that summarizes analytical 
and  toxicological  results  obtained  for  genotoxicity  studies  performed  with  nano  and  non-nano 
forms of MBBT. 

2.  TERMS OF REFERENCE 

In  light  of  the  new  data  provided,  does  the  SCCS  consider  Methylene  bis-benzotriazolyl 
1. 
tetramethylbutylphenol (CAS No 103597-45-1) safe when used in cosmetic products in its nano 
forms under the current use conditions up to a maximum of 10%? 

2.  Does  the  SCCS  have  any  further  scientific  concern  regard  the  use  of  Methylenebis-
benzotriazolyl tetramethylbutylphenol in cosmetic products? 

1 COLIPA - now Cosmetics Europe,- European Cosmetics Toiletry and Perfumery Association 
2 http://ec.europa.eu/health/scientific_committees/consumer_safety/opinions/sccnfp_opinions_97_04/sccp_out55_en.htm  
3 http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_129.pdf 

6 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                          
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

3.  OPINION 

3.1.  Chemical and Physical Specifications 

3.1.1.  Chemical identity 

3.1.1.1.  Primary name and/or INCI name 

2,2’-Methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) 

3.1.1.2.  Chemical names 

2,2’-Methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) 

3.1.1.3.  Trade names and abbreviations 

UV-absorbing ingredient (UV filter) 
(also referred to as MBBT) 

Nano-MBBT: 

 MBBT 
Tinosorb® MBBT 
FAT 75714B 
CGF-C002089 
 TKA 40027 
 CG 30-1881 
* 

 Tinosorb® M 
FAT 75’634 

* additional trade names are on the market 

3.1.1.4.  CAS / EC number 

CAS: 
EC: 

103597-45-1 
403-800-1  

3.1.1.5.  Structural formula 

7 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

3.1.1.6.  Empirical formula 

Formula:  C41H50O2N6 

3.1.2.  Physical form 

Yellowish powder (MBBT) 
Viscous white dispersion (nano-MBBT) 

3.1.3.  Molecular weight 

Molecular weight: 

658.89 Da 

3.1.4.  Purity, composition and substance codes 

MBBT, non-micronised: 

Method 
In  a  chemical  characterisation  study,  the  purity  and  thus  batch  composition  of  MBBT 
(FAT 75714B)  was  determined  by  means  of  HPLC-DAD  (diode-array  detection)(purity 
assessment)  and  HPLC-MS  (isomer  identification).  The  purity  was  measured  as  relative  peak 
area of the DAD signal at a wavelength of 304 nm. 

Results 
The purity of the UV-absorbing ingredient determined by chromatography is depicted below: 

Purity  of  the  UV-absorbing  ingredient  based  on  relative  peak  area  in  the  HPLC-DAD 
chromatogram 

Ingredient Concentration 

Main Peak 

Isomer Peak 

Total 

[mg/mL] 

(% area) 

(% area) 

(% area) 

Mean 

0.1 

0.4 

n.a. 

n.a.:  not applicable 

98.78 

98.59 

98.69 

1.22 

1.41 

1.32 

100 

100 

100 

Conclusion 
The purity of the UV-absorbing ingredient including main compound and isomer determined by 
chromatography was >99.9 % (area/area). 

SCCS comment 
The  SCCS  notes  that  around  1.5  %  of  the  content  has  been  identified  as  an  isomer,  full 
characterisation  of  which  has  not  been  provided.  However,  since  toxicological  studies  were 
performed with the test item containing the above mentioned isomer, the SCCS considers that 

8 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

the  toxicological  evaluation  covers  the  potential  toxicity  of  the  isomer  at  the  given 
concentration. 

MBBT, micronised: 
Tinosorb® M 
Preparation containing approx. 50 % w/w of the UV-absorbing ingredient 2,2’-Methylene-bis-
(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) 

Typical composition of the formulated UV-filter Tinosorb® M, containing micronised (ball-milled) 
MBBT as the UV-absorbing compound. 

Ingredient 

CAS Number 

Function 

Mass Fraction 

(% w/w) 

MBBT 

103597-45-1 

UV-Absorption 

46.0 – 55.0 

Decyl Glucoside 

68515-73-1 

Surfactant 

Propylene Glycol 

57-55-6 

Wetting Agent 

Xanthan Gum 

11138-66-2 

Stabiliser, Thickener 

6.0 - 10.0 

0.2 – 0.6 

0.1 – 0.5 

Water 

7732-18-5 

Vehicle 

40.0 – 42.0 

For the different batches of Tinosorb® M used in the toxicology studies, see Annex I 

14C-radio-labelled MBBT (purity 99.1 %; position of the label, see in Figure below) was mixed 
with non-radio-labelled MBBT (purity ≥ 99.5 %) and milled to produce batches of radio-labelled 
nano-MBBT.  

The UV-absorbing ingredient MBBT is micronised by means of pre-milling with a corundum disc 
mill followed by the main milling step which is conducted with a ball mill (Herzog et al., 2004a). 
This milling process is performed in deionised water using Decyl Glucoside as dispersant. After 
micronisation,  Xanthan  Gum  is  added  to  the  dispersion  in  order  to  prevent  particle 
sedimentation.  The  standard  composition  of  the  resulting  formulated  micronised  UV  filter 
Tinosorb® M is specified below: 

Physic-chemical property 

Physical state 

UV-absorbing ingredient (UV 
filter) 
solid 

pH range 

not applicable (solid) 

Odour 

characteristic odour 
organoleptic determination 

Formulated micronised UV 
filter 
liquid (aqueous dispersion) 
pH 10.5-12 at 25 °C 
determined with glass electrode 
according to DIN 19268 
trace characteristic odour 
organoleptic determination 

9 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Melting point 

195.7 °C 

Boiling point 

not applicable (solid) 

Vapour pressure 

6 x 10-13 Pa at 25°C 

Relative density 

No data 

Dynamic viscosity 

Not applicable (solid) 

Flammability 
Auto flammability 
Explosiveness 

not flammable 
No data 
not explosive 

Dry content 

No data 

not applicable (liquid) 
approximately 100 °C 
determined according to 
OECD 103 
not determined 
determined according to 
OECD 109 
1.04 – 1.12 g/cm³ at 25 °C 
200 – 1000 mPa × s at 25 °C 
determined according to 
DIN 53019 
not highly flammable 
not self-igniting 
not explosive 
55.5 – 59.5 % 
determined by means of IR 
drying of 1 g at 
135 °C for 60 minutes 

SCCS comment 
From  the  provided  description  of  the  production  process,  it  is  the  understanding  of  the  SCCS 
that  micronisation  and  addition  of  Xanthan  Gum  is  part  of  the  manufacturing  process  of  the 
nano-sized material. 
The SCCS notes that the density of MBBT (1160 kg/m³ from the CAS registry) differs from the 
density of the formulation. The dry content of the raw material should be 100% as no solvent is 
contained.  The  vapour  pressure  of  the  raw  material  is  very  low  and  thereby  the  vapour 
pressure of the emulsion is dominated by high vapour pressure components like water. While 
boiling  does  not  occur  for  MBBT,  the  formulation  boils  with  water  as  the  ingredient  with  the 
lowest boiling temperature.  

3.1.5.  Impurities / accompanying contaminants 

Impurities of the MBBT 

Method 
Typical  impurities  of MBBT  were  determined  by  means  of  HPLC-DAD  and  HPLC-MS  with  the 
concentration  limit  for  the  determination  of  impurities  set  to  0.08 %  (area/area).  This 
investigation covered known impurities and possible impurities which could come from the last 
steps  of  synthesis. 
impurities  were  assessed  by  means  of 
thermo-desorption  coupled  with  GC/MSD.  The  water  content  was  determined  by  Karl  Fischer 
titration and a metal analysis was performed by means of ICP-OES. 

In  addition,  volatile 

Results 
In this chemical characterisation study, only one relevant impurity peak was detected by HPLC 
and identified as an isomer of the UV-absorbing ingredient. The water content was determined 
to  be  <0.1 %.  All  measured  trace  metals  were  below  their  quantitation  limit.  The  volatile 
impurities Isopropanol, Dibutylamine, Xylene (mixture of ortho-, meta- and para-isomers) and 
Formaldehyde  were  identified  and shown  to have  a concentration  in  or  below  the  ppm  range. 
Additional  impurities  could  be  identified  on  a  qualitative  basis  by  means  of  NIST  library  fits 
only.  

10 

 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Impurity 

Water 

Trace metalsa 

Isopropanol 

Dibutylamine 

Xyleneb 

Formaldehyde 

Cis-3-Nonen-1-ol 

Dodecane 

9-Decyn-1-ol 

4-tert-Buthyl-o-xylene 

Concentration 

< 0.1 % 

< LOQ 

2.2 µg/g 

< 0.5 µg/gc 

6.8 µg/g 

0.93 µg/g 

n.a. 

n.a. 

n.a. 

n.a. 

a: 

Cr, Cu, Fe, Na, Pb, Sb, Se, Sn, Zn: LOQ < 10 mg/kg 
As, Cd: LOQ < 5 mg/kg 
Ni : LOQ < 3 mg/kg 
Hg: LOQ < 1 mg/kg 
mixture of ortho-, meta- and para-isomers 
value below estimated limit of detection 

b: 
c:  
n.a.:  not applicable 

Ref: ciba (2004a) 

Impurities of Tinosorb® M 

These were not separately reported. Upon request, the applicant commented: 

“The  nanomaterial  is  produced  by  the  physical  process  of  milling  technical  MBBT.  It  is  not 
heated and no chemical reaction is performed, it is not dissolved and re-crystallized again. The 
additional components (decyl glucoside, propylene glycol, xanthan gum and water) are added 
for milling purposes and to stabilize the resulting micro dispersion. They are considered to be 
formulants.  The  whole  milling  process:  dispersion  in  de-ionized  water,  adding  decyl  glucoside 
as dispersant, pre-milling in a corundum disc mill, main milling with a ball mill, stabilizing the 
microfine dispersion with xanthan gum is therefore regarded to be a formulation process. 

The  purity  of  the  UV-absorbing  ingredient  as  well  as  the  identity  and  amount  of  impurities  of 
MBBT  used  for  the  preparation  of  Tinosorb®  M  microfine  material  are  consequently  identical 
with those of MBBT in the non-micronised form.” 

3.1.6.  Solubility/ dissolution 

UV-absorbing ingredient (UV filter): 

Formulated micronised UV filter: 

extremely low solubility in water (< 5 ng/L at 25 °C) 
 increased  solubility  in  water  when  formulated  in  a  sun 
screen lotion (1.0 – 3.2 mg/L, temperature unspecified) 
 solubility in fat simulant (170 mg/100 g at 37 °C) 
in  water 
dispersible 
soluble in polar cosmetic oils at 25 °C; 

(temperature 

unspecified) 

11 

 
 
 
 
 
 
 
 
 
 
      
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

SCCS comment 
Solubility  is  given  for  the  non-micronised  form  only  and  not  for  the  nano  form.  However,  by 
definition of the physicochemical term ‘solubility’ this should not depend on the size distribution 
of the compound, if appropriate procedures are applied. It is not clear to what extent the other 
substances in the nano-formulation can influence the solubility of nano-MBBT compared to non-
nano MBBT.  

3.1.7.  Partition coefficient (Log Pow) 

UV-absorbing ingredient (UV filter): 

Formulated micronised UV filter: 

12.7  at  25 °C  (calculated  value)  determined  according 
to OECD 107 
not applicable 

SCCS comment 
Log Pow is based on distribution of solubilised substances in aqueous and organic phases and as 
such does not apply to insoluble particles. However, considering the fat solubility of MBBT, the 
SCCS considers that the partition coefficient should be similar for both the non-micronised and 
micronised forms.  

3.1.8.  Additional physical and chemical specifications: nano specific properties 

3.1.8.1.  Size and size distribution 

Methods 
The  particle  size  of  Tinosorb® M  (batch  1365CL6VY)  was  measured  with  the  fibre-optic  quasi-
elastic light scattering (FOQELS4) technique. The volume weighted particle size distribution was 
calculated from the measured scattering data, and a median value of particle size distribution 
d(0.5) was derived. With the FOQELS technique, the particle size distribution could be resolved 
to  sizes  in  the  ultrafine  range,  i.e.  well  below  100 nm.  Based  on  the  derived  particle  size, 
specific surface area and number concentration were calculated. 

The described treatment of FOQELS measurements was validated by disc centrifugation studies 
and confirmed by atomic force microscopy (Herzog et al., 2004b).  

Results 
Determination  of  the  Tinosorb® M  particle  size  distribution  by  means  of  FOQELS  technique 
revealed a d(0.5) of 150 ± 9 nm (mass based) as an average value with standard deviation of 
10 individual  measurements.  With  this  particle  size  and  based  on  a  particle  density  of 
1160 kg/m³,  a  specific  surface  area  of  34.5 m²/g  was  obtained.  The  derived  number  of 
particles in 1 cm³ at an active concentration of 50 % was 2.44 × 1020. 

Particle size, surface area and mass concentration of Tinosorb® M 

4   The  FOQELS  (fiber-optic  quasi-elastic  light  scattering)  is  a  dynamic  light  scattering  technique  that  is  based  on 
measurement  of  the  intensity  fluctuations  of  the  back-scattered  light  of  a  particle  dispersion.  For  a  detailed 
description of the methodology please refer to Herzog et al. (2004b). 

12 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                          
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Endpoint 

Particle size (d(0.5)-value) mass based 

density  

number of particles 

Datum 

150 ± 9 nm 

1160 kg/m³ 

2.44 × 1020 cm-³ 

Additional information was sent on request with regard to the particle size on a number basis. 
The sample investigated with the FOQELS method was Tinosorb M-PGL, ITEM: 0758792, which 
can be regarded as a typical production batch. 

d(0.5) = 74 sd 42 nm (number based) 

13 

 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

The  d(50)  particle  sizes  on  mass  and  on  number  basis  are  reported  below,  for  the  different 
batches in the dossier: 

Batch 
Tinosorb M Lot.01722CL4 
Tinosorb M Lot. 01635cl4 
Tinosorb M Lot.01636cl4 
Tinosorb M Lot.01637cl4 
Tinosorb M Lot.01723cl4 
Tinosorb M Lot.01724cl4 
Tinosorb M Lot. 5915252 
Tinosorb M Lot.5915253 
Tinosorb M Lot.5915254 
Tinosorb M Lot.5915255 
Tinosorb M Lot.5915257 
Tinosorb M Lot.5915258 

d50 (laserdiff.) V 
117nm 
124nm 
123nm 
125nm 
117nm 
125nm 
124nm 
126nm 
126nm 
127nm 
117nm 
126nm 

d50 (laserdiff.)N 
61nm 
62nm 
66nm 
62nm 
61nm 
66nm 
66nm 
66nm 
66nm 
66nm 
64nm 
66nm 

SCCS comment 
The d(50) particle sizes on mass and on number basis were determined by laser diffraction and 
FOQELS for a non-radiolabelled nano MBBT batch termed "cold", which was used as a reference 
for  comparison  with  the  batch  of  radiolabelled  “hot”  nano  MBBT  that  was  used  in  the 
toxicological  studies.  However,  the  resulting  values  from  laser  diffraction  and  FOQELS  are 
considerably  different.  This  might  be  due  to  the  difficulty  in  calibrating  optical  techniques  in 
view of the size dependency of their sensitivity. Herzog et al, have provided some evidence to 
show that small particles can be detected by FOQELS, which is generally considered the more 
suitable technique for smaller particle fractions. 

Sample 

d50 (Laserdiff.) V  d50 (Laserdiff.) N 

d50 (Foqels) V 

d50 (Foqels) N 

Nano MBBT cold 
(15420CL2AA) 

114nm 

84nm 

97nm 

49nm 

Overview on particle sizes for the different batches (ref submission II combined) 

Overall in Tinosorb® M batches, a mean particle size d(0.5) of 122 ± 0.005 nm (n = 32) with a 
range of 115 – 129 nm and a specific surface area of 56.0 ± 2.9 m²/g (n = 32) with a range of 
52 - 67 m²/g  was  determined.  The  MBBT  batches  displayed  a  mean  particle  d(0.5)  size  of 
207 ± 70 µm  (n = 3)  with  a  range  of  142 – 281 µm  and  a  specific  surface  area  of 
0.21 ± 0.03 m²/cm³ (n = 3) with a range of 0.19 - 0.25 m²/cm³. 

SCCS comment 
For the micronised material (Tinosorb® M), median particle size distribution measured by laser 
diffraction  method  is  reported  as  114  nm  (mass  based)  and  84  nm  (number  based).  Particle 
size  values  reported for different  batches  (section  3.1.8.1)  indicate  the  median  size  as  123  ± 
3.5 nm (mass based), and 64 ± 2.0 nm (number based). 
Median  size  values  measured  by  FOQEL,  which  is  considered  to  be  a  more  suitable  technique 
for smaller particle fractions, is reported as 150 nm (mass based) and 74 nm (number based). 
Further  characterisation  data  provided  by  the  Applicant  (see  section  3.1.9)  shows  that 
Tinosorb®  M  pH  10.5  (trade  form)  is  comprised  of  median  particle  size  of  77  nm  (number 
based). Thus, considering the batch to batch variability, the median mass-based particle size of 

14 

 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

the micronised material appears to be around 120 nm or larger when measured either by laser 
diffraction  or  the  FOQEL  method.  In  terms  of  number-based  distribution,  the  median  particle 
size  appear  to  be  around  60  nm  or  larger  when  measured  either  by  laser  diffraction  or  the 
FOQEL method.   

SCCS/1546/15 

3.1.9.  Additional physical and chemical specifications 

The submission III of July 31, 2014 provides more detailed physicochemical characterisation of 
the  MBBT  materials  used  in  genotoxicity  studies.  Chemical  composition  (w/w)  of  MBBT  in 
Tinosorb®  M  pH  10.5  and  Tinosorb  M  (not  micronised)  measured  by  UV-visible  spectrometry 
was  49.1%  and  54.7%  against  a  nominal  composition  of  50%  in  either  of  the  materials.  The 
nominal  composition  of  other  components  is  quoted  as  8%  Decyl  glucoside,  0.4%  Propylene 
glycol, 0.4% Xanthan Gum and 41.2% Water.  

Tinosorb®  M  pH  10.5 
(trade form) 

Tinosorb M (not micronised) 

Primary particle diameter (TEM): 

D50 (number%) = 77 nm 

Mean 87 nm, stdev 48 nm 

Minimum 17 nm, maximum 0.58 μm 

Fraction below 100 nm = 72% 

Primary particle diameter (TEM): 

D50 (number%) = 169 nm 

Mean 234 nm, stdev 230 nm 

Minimum 35 nm, maximum 2.8 μm 

Fraction below 100 nm = 17% 

Characterisation  of  Tinosorb®  M  pH  10.5  in  water  or  culture  medium  with  10%  (v/v)  FCS 
showed  that,  the  amount  of  particles  in  the  size  range  <100  nm  was  at  9  to  22%  in  MNT 
medium  and  11  to  19%  in  HPRT  medium  of  the  respective  concentrations  directly  after 
preparation.  For  concentrated  samples  (637.5  μg/mL),  the  particle  size  distribution  changed 
only slightly when measured 4 hours after the start of treatment. In less concentrated samples 

15 

 
 
 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

(159.4  μg/mL),  nanoparticles  were  not  detectable  after  4  hours  incubation  in  either  of  the 
media. 
The dispersion of the particles was measured in terms of area under curve (AUC) by centrifugal 
liquid sedimentation (ISO13318) and suspension laser diffraction method. Dispersability of the 
particles is quoted in terms of D50/Average Agglomeration Number (AAN). For Tinosorb® M pH 
10.5 dispersability values quoted are 93 nm / AAN ~1 (in water) and ~1.2 μm / AAN ~13 (in 
DMEM/FCS or HAM/FCS). For Tinosorb M (not micronised), the dispersability value is ~20 μm / 
AAN ~120 (in MEM/FCS or HAM/FCS). 
Potential precipitation/agglomeration of the test substance in culture media was investigated in 
the  presence  and  absence  of  S9  mix.  In  the  culture  media,  precipitates  formed  at 
concentrations  from  159.4  μg/mL  onward  in  the  absence  of  S9  mix,  and  from  39.8  μg/mL 
onward in the presence of S9 mix. At 159.4 μg/mL in culture medium with S9 mix (20% [v/v]) 
the particles in the nano cluster (<100 nm) were 28-30% before administration, and 26-28 % 
at  the  end  of  treatment  for  the  MNT  and  HPRT  medium  respectively.  Under  the  same 
experimental  conditions,  at  a  4-fold  higher  concentration,  the  fraction  of  nano-sized  particles 
was much lower: 2% before administration and 5% at the end of treatment were found in the 
HPRT medium. In the MNT medium, 1% nanomaterial was initially analysed, which after end of 
the treatment was not detectable.  
In  water  or  both  culture  media  with  10%  (v/v)  FCS,  no  particles  in  the  particle-size  cluster 
<100  nm  were  found  directly  after  preparation.  Besides,  in  culture  media  with  S9  mix  (20% 
[v/v]), the portion of particles in the nano cluster (<100 nm) was 6-26% before administration. 
Four hours after start of treatment in the presence of metabolic activation, similar amounts of 
particles  in  the  cluster  <100  nm  were  found  (between  6  and  21%)  in  the  micronucleus  test 
medium and between 10 and 41% in the HPRT medium. 
The data also indicated the formation of larger sized hetero-agglomerates with components of 
S9  mix,  but  these  were  still  well  dispersed  in  the  test  vehicle  as  the  particle  size  of  the 
dominating part of the administered dose remained below 1 μm diameter after incubation. 

3.1.10. Microscopy 

3.1.10.1  Scanning electron microscopy 

Method 
The  particle  size  distribution  of  Tinosorb® M  (Batch  1365CL6VY)  was  visualised  by  means  of 
scanning electron microscopy (SEM). 

Results 
A  SEM  image  of  Tinosorb® M  particles  at  50000 times  magnification  is  presented  below.  The 
particle  sizes  observed  with  this  technique  are  in  agreement  with  the  FOQELS  presented  in 
section 3.1.9 for the same batch. 

SEM picture of Tinosorb® M particles (Lot. 1365CL6VY) 

16 

 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

3.1.11. Crystal structure 

/ 

3.1.12. UV absorption 

Author: 
Title: 

Guideline: 
Batch: 
GLP: 
Reference: 

Herzog et al. (2004a) 
New UV absorbers for cosmetic sunscreens – a breakthrough for the 
photoprotection of human skin 
/ 
1365CL6VY 
/ 
Chimia 58: 554-559 

Method 
UV  spectra  were  recorded  with  the  UV-absorbing  ingredient  MBBT  (d(0.5):  169 nm  volume 
based) and with MBBT dissolved in Dioxane. 

Since  for  organic  coloured  pigments  the  UV  absorption  often  increases  with  smaller  particles 
while  the  scattering  shows  a  maximum  at  a  certain  particle  size,  the  dependence  of  UV 
attenuation  on  particle  size  was  assessed  using  MBBT  dispersions  with  different  particle  sizes 
and an UV/Vis spectrometer with integration sphere accessory.  

Results 
The UV spectra of micronised MBBT and MBBT dissolved in Dioxane are shown in Figure 1. 

Figure 3:  UV spectra of MBBT 

17 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

The  absorption  spectrum  of  MBBT  in  solution  shows  a  double-band  structure.  The  band  at 
350 nm  can  be  attributed  to  a  ππ*-charge  transfer  state.  This  is  favoured  by  the  planar 
orientation enforced by the intramolecular hydrogen bond, made possible by the hydroxy group 
in  ortho-position.  The  band  at  about  305 nm  arises  from  a  local  transition  within  the 
benzotriazole  moiety.  The  absorption  spectrum  of  micronised  particulate  MBBT  in  dispersion 
shows  distinctive  differences  to  that  in  solution,  which  are  not  explained  by  the  additional 
scattering  effect:  The  longer  wavelength  band  is  shifted  to  360 nm  and  is  followed  by  a 
shoulder at 380 nm. The short wavelength band at 305 nm shows no significant shift, but there 
is  an  additional  shoulder  at  320 nm.  These  spectral  changes  are  attributed  to  electronic 
interactions of the chromophores in the particles (Horn and Rieger,  2001) and depend on the 
particle size (Herzog et al., 2004b).  

The  specific  extinction  E(1,1)  as  a  function  of  wavelength  is  shown  in  Figure 3.4  for  different 
particle  sizes.  There  is  a  strong  effect  of  particle  size  on  the  extinction.  The  smaller  the 
particles become, the more efficient they absorb UV, due to a larger surface area and a more 
even  distribution  in  the  vehicle  as  compared  to  larger  particles.  In  terms  of  the  specific 
extinction at 360 nm, the MBBT particles at the end of the milling process show about 90 % of 
the highest possible performance.  

SCCS comment  
The  authors  of  the  dossier  did  not  specify  how  they  determine  the  ‘highest  possible 
performance’. In general and as stated in Horn and Rieger (2001), it remains a complex issue 
to relate the UV absorption of nano-emulsions to the bulk absorption properties. 

18 

0150300450600280300320340360380400Wavelength / nmE(1,1)d(0.5) = 169 nmsolution in dioxane 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Figure 4:  UV spectra of MBBT dispersions at different particle sizes 

3.1.13. Zeta potential 

Methods 
The  ζ-potential  of  Tinosorb® M  (batch  1365CL6VY)  containing  10 %  UV-absorbing  ingredient 
MBBT  in  micronised  form  was  measured  with  a  Matec MBS-8000 ESA  (electroacoustic 
spectroscopy)  system.  Titration  was  performed  with  2M  HCl  to  assess  the  ζ-potential  as  a 
function of the pH value. 

Results 
At  neutral  pH,  the  ζ-potential  was  determined  to  be  -25 mV,  which  is  advantageous  for  the 
stability of the dispersion. The ζ-potential as function of pH is shown below: 

19 

0100200300400500280300320340360380400Wavelength / nmE(1,1)0.16 µm0.22 µm0.34 µm0.78 µm4.4 µm 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

ζ-potential of Tinosorb® M particles containing 10 % MBBT

SCCS/1546/15 

40 

30 

20 

10 

0 

-10 

-20 

-30 

-40 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

Conclusion 
The  stability  of  micronised  Tinosorb® M  particles  in  dispersion  is  enhanced  by  their  negative 
ζ-potential at physiological pH. 

SCCS comment 
The  formulation  of  Tinosorb® M  contains  50%  nano-MBBT,  however  it  seems  that  the  zeta 
potential was measured at in-use concentration (10%).  

3.1.14. Droplet size in formulations 

Title: 

Reference: 
Guideline: 
GLP: 
Batch/Lot n°: 

RK11_033 Sonnenschutzspray TGV. Droplet sizing of sun sprays containing 
Tinosorb® M  (UV  Filter  –  MBBT)  using  laser  diffraction  system  (Malvern 
Mastersizer) 
RK11/033 
/ 
/ 
0004327851 

Methods 
Particle/droplet  size  measurements  were  performed  by  means  of  laser  diffraction  (Malvern 
Mastersizer)  when  spraying  UV  protective  propellant  sprays  or  pump  sprays  containing  the 
UV-absorbing  ingredient  MBBT  in  the  micronised  form  (Tinosorb® M).  The  sun  spray 
formulations listed below were sprayed into a test chamber and droplet sizes were measured at 
a distance of about 30 cm from the spray head. 

Test samples: 

1.  Formulation UV11060-1-1 (8 % MBBT)/30 % propane/butane 
2.  Formulation UV11060-1-1 (8 % MBBT)/40 % propane/butane 
3.  Formulation UV11060-2-1 (10 % MBBT)/30 % propane/butane 
4.  Formulation UV11060-2-2 (10 % MBBT)/40 % propane/butane 
5.  Formulation UV11060-1-1 (8 % MBBT)/pump spray 

20 

 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

6.  Formulation UV11060-2-2 (10 % MBBT)/pump spray 

Results 
The  mean  droplet  size  (d(0.5);  volume  distribution)  for  all  spray  formulations  (propellant 
sprays  and  pump  sprays)  was  found  to  be  >200 µm  for  all  aerosol  formulations  and  >80 µm 
for the pump spray. The droplet fraction below 10 µm was in all cases, for both types of spray, 
<1 % (volume distribution). 

SCCS comment 
The  submitted  data  are  not  considered  sufficient.  In  case  of  intended  use  in  sprayable 
applications,  appropriate  data  are  needed  on  the  physics  of  the  spray  generation  and  the 
atmospheric conditions during use (including behaviour of the aerosols in time). 

3.1.15. Homogeneity and stability 

Stability in dose formulation 

Author: 
Title: 

Reference: 
Guideline: 
GLP: 
Batch/Lot n°: 

Harlan Laboratories Ltd. (2009) 
Percutaneous penetration of 14C-FAT 75714 (MBBT) formulated as 
Tinosorb® M through pre-damaged human split-thickness skin membranes 
(in vitro) 
Study report number C27241 
OECD 428 
In compliance 
3389-279 

Methods 
In  a  percutaneous  penetration  study,  the  stability  of  14C-FAT 75714  (radiochemical  purity: 
99.1 %),  i.e.  of  the  UV-absorbing  ingredient  MBBT  in  micronised  form  (d(0.5):  89 nm), 
suspended in formulation  and water was determined at the time of application and in the skin 
rinse  fluid  (after  24h  of  exposure)  by  means  of  a  HPLC  method  with  UV  detection.  The 
radioactivity signal was monitored with a radioactivity flow monitor. 

Results 
HPLC analysis after administration on split-thickness skin membranes revealed a radiochemical 
purity of the formulated micronised test item of 98.93 %. In agreement with this outcome, the 
radiochemical  purity  of  the  test  item  in  the  skin  membrane  rinse  was  determined  to  be 
98.89 %.  Test  item  stability  in  the  dose  formulations  was  additionally  confirmed  by  the  UV-
based chromatogram at 280 nm. 

SCCS comment on physicochemical characterisation 
- Not all batches of nano-MBBT used in the toxicity studies have been characterised. 
-  The  SCCS  notes  that  around  1.5  %  of  the  content  has  been  identified  as  an  isomer,  full 
characterisation of which has not been provided. 
- Considering the fat solubility of MBBT, the SCCS considers that the partition coefficient should 
be similar for both the non-micronised and micronised forms. 
- In the majority of studies, it is not indicated whether the reported particle sizes are mass or 
number based. 

21 

 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

-  Determination  of  the  ‘highest  possible  performance’  on  UV  absorption  was  not  specified.  In 
general  and  as stated  in  Horn  and  Rieger  (2001)  it  remains  a complex  issue  to  relate  the  UV 
absorption of nano-emulsions to the bulk absorption properties. 
-  The  submitted  data  on  droplet  size  is  not  considered  sufficient.  In  case  of  intended  use  in 
sprayable applications, appropriate data are needed on the physics of the spray generation and 
the atmospheric conditions during use (including behaviour of the aerosols in time) 

SCCS/1546/15 

3.2.  Function and uses 

(CAS  No 
2,2’-methylene-bis-(6(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) 
103597-45-1),  Methylene  bis-benzotriazolyl  tetramethylbutylphenol  (INCI)    is  used  in  nano 
form as UV filter in sunscreens, day care products and skin lightening products at a maximum 
concentration of 10%. 

3.3. Toxicological Evaluation 

3.3.1.  Acute toxicity 

3.3.1.1.  Acute oral toxicity 

No studies provided with nano-sized material 

For studies with MBBT: see annex II 

3.3.1.2.  Acute dermal toxicity 

No studies provided with nano-sized material 

For studies with MBBT: see annex II 

3.3.1.3.  Acute inhalation toxicity 

Title: 
Guideline: 
GLP: 
Test item: 

Batch/lot n°: 

4-hours acute inhalation toxicity study in rats 
OECD 403 
In compliance 
FAT 75'634 (containing 50 % (w/w) MBBT); microfine material; 
d(0.5) = 128 nm 
00292CL7 

Method 
In a limit test, a group of 15 albino rats [HanRCC:WIST (SPF)] per sex was nose-only exposed 
to  a  20 %  (w/w)  aqueous  dilution  of  FAT 75'634,  i.e.  the  formulated  micronised  UV  filter 
Tinosorb® M. This tested aqueous dilution corresponds to a 10 % dilution of the UV-absorbing 
ingredient  MBBT.  The  animals  were  exposed  at  the  highest  technically  achievable  aerosol 
concentration  of  4.877 ± 0.255 mg/L  (n = 8;  determined  by  chemical  analysis)  for  a  single 
period  of  4 hours.  The  resulting  MBBT  concentration  was  0.488 mg/L.  Two  gravimetric 
measurements  of  particle  size  distribution  performed  during  the  exposure  produced  mass 

22 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

median  aerodynamic  diameters  (MMADs)  of  1.42  and  1.44 µm  with  geometric  standard 
deviations (GSDs) of 2.10 and 2.10, thus confirming that the aerosol particles were within the 
target  size  range of  1 – 4 µm  and  were  deposited in  all  regions  of  the  rat  respiratory  tract.  A 
further  group  of  15 rats  per  sex  was  exposed  to  an  aqueous  dilution  of  the  control  item, 
FAT 75’634 Placebo, at similar aerosol generation conditions. 

Each dose group was subdivided into three satellite groups of 5 rats per sex, the first satellite 
group  being  assigned  to  sacrifice  at  approximately  14 hours  post  end  of  exposure  for 
bronchoalveolar lavage fluid (BALF) and plasma sampling (day 1), the second being assigned to 
interim pathology at approximately 24 hours post end of exposure (day 2), and the third being 
assigned  to  pathology  at  14 days  post  exposure  (day 15).  Clinical  signs,  mortality  and  body 
weights were assessed periodically during the study period. The BALF examinations comprised 
total and differential cell counts and the determination of total protein, tumour necrosis factor α 
(TNFα)  and 
levels.  Pathology  examinations  comprised  complete 
macroscopic  pathology,  lung  weight  determination  and  histopathology  of  the  lungs  and 
tracheobronchial lymph nodes on days 2 and 15. 

interleukin 6  (IL-6) 

Results 
There  were  no  clinical  signs,  no  premature  deaths  and  no  toxicologically  relevant  adverse 
effects  on  body  weight noted in  this study.  At the  end  of the  observation  period (day 15), no 
organ weight changes, macroscopic and/or microscopic findings attributable to treatment with 
the placebo or test item dilutions were noted.  

Entirely  reversible  test  item-related  findings  were  noted  on  day 2  of  the  observation  period. 
These consisted of increases of total cell count (neutrophil numbers), total protein in BALF and 
of absolute and relative lung weight, as well as diffuse alveolar histiocytosis and alveolar lining 
cell  activation  in  the  treatment  group  as  compared  with  the  placebo  control  group.  These 
findings were consistent with marginally higher TNFα and IL-6 levels in BALF seen in test item 
exposed females relative to placebo control females.  

Conclusion 
Under the conditions of this study, the lethal concentration 50 (LC50) in rats was greater than 
the highest technically achievable aerosol concentration of the formulated micronised UV filter 
Tinosorb® M,  which  was  generated  from  a  20 %  aqueous  dilution.  The  LC50  of  the 
UV-absorbing ingredient MBBT in micronised form was therefore > 0.488 mg/L. 

Transient  pulmonary  effects  noted  on  day 2  of  the  observation  period  were  considered  to  be 
test  item-related  and  indicative  of  an  acute  pulmonary  clearance  reaction  associated  with  an 
inflammatory response. 

Ref.: RCC (2008) 

SCCS comment 
The  observed  effects  are  indeed  similar  to  those  observed  upon  inhalation  of  particulate 
materials  in  general  including  some  nano-particles.  The  massive  influx  of  neutrophils  is  not 
considered  a  mild  inflammation  by  the  SCCS;  however,  reversibility  of  the  inflammatory 
response was indicated by histological evaluation at day 15 after treatment.  
It was not specified whether the particle size was expressed as number based or mass based. 

Additional information: 

BALF and/or plasma results 14h post end of inhalation exposure 

23 

 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Parameter 

Summary (mean + std dev.) 

Males (N= 5) 

Placebo 

Test Item 

9.76 (1.62) 

14.37 (1.91) ** 

95.9 (1.3) 
0.0 (0.0) 
0.6 (0.6) 
0.2 (0.3) 
0.3 (0.2) 
3.0 (0.7) 
<22.4 

Total Cells 
(millions) 
% of Total cells 
Macrophages 
Eosinophils 
Lymphocytes 
Neutrophils 
Other cells 
Epithelial cells 
TNFα (pg/ml) 
IL6 (pg/ml) 
Total Protein 
(g/l) 
Plasma Protein 
(g/l) 
* / ** t-test based on pooled variance, significant at 5% or 1% level. 
a 3 animals >200, 2 at or just above LLQ 

46.4 (8.6)** 
0.1 (0.1) 
0.4 (0.4) 
50.1 (8.3)** 
0.8 (0.7) 
2.2 (0.7) 
28 (9)  
150; 111a 

165; 88 
226c 

138 (32) 

102 (30) 

61 (0.5) 

52 (17) 

Females (N= 5) 

Placebo 

8.32 (1.12) 

Test Item 
21.35 (11.27)* 

94.7 (1.2) 
0.2 (0.3) 
0.4 (0.2) 
0.8 (0.6) 
0.6 (0.2) 
3.4 (1.0) 
<22.4 
179; 104 

96 (19) 

37.4 (11.8)** 
0.0 (0.1) 
0.8 (0.7) 
59.9 (11.7)** 
0.4 (0.3) 
1.5 (0.5)** 
45 (31.52) 
236:45
72c 
154 (49) 

63 (3.0) 

63 (1.8) 

3.3.2  Irritation and corrosivity 

3.3.2.1.  Skin irritation 

No studies provided with nanosized material. 
For studies with MBBT: see annex 2 

3.3.2.2.  Eye irritation  

In vitro 

Guideline: 
Test system: 
Route: 
Substance: 
Batch: 
Purity: 
Dose: 
GLP: 
Study period: 

and 

Guideline: 
Test system: 
Route: 
Substance: 
Batch: 
Purity: 

OECD n° 437 (BCOP test) 
isolated bovine cornea 
eye 
Tinosorb® M, test substance nr. 10/0381-1 
P380; pH 10.5 
not provided 
750ul of the undiluted substance 
in compliance 
October 2010 

OECD n° 437 (BCOP test) 
isolated bovine cornea 
eye 
Tinosorb® M, test substance nr. 10/0381-1 
P380; pH 11.6 
not provided 

24 

Ref.: BASF SE (2010a) 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Dose: 
GLP: 
Study period: 

750ul of the undiluted substance 
in compliance 
October 2010 

Ref: BASF SE (2010b) 

The pH specification of the formulated micronised UV filter Tinosorb® M is pH 10.5-12 at 25 °C. 
Two in vitro studies were conducted to cover this pH specification range and to ensure that no 
treatment-related  effects  occur  at  either  extremes.  The  test  items  “Tinosorb® M  pH = 10.5” 
and “Tinosorb® M pH = 11.6” were used for these investigations.  

A  single  topical  dose  of  750 µL  of  the  undiluted  formulated  micronised  UV  filter  Tinosorb® M 
was administered to the epithelial  surface of three isolated bovine corneas. The corneas were 
exposed to the test item for 10 minutes followed by a 2-hour post incubation period. 

Corneal  opacity  was  measured  qualitatively  as  the  amount  of  light  transmission  through  the 
cornea.  Permeability  was  measured  quantitatively  as  the  amount  of  Fluorescein  dye  that 
passed across the full thickness of the cornea. Deionized water served as negative control item 
and 1 % (w/v) Sodium hydroxide served as positive control item. 

Evaluation criteria 
IVIS > 55 (risk of serious damage to eyes) 
IVIS ≤ 55 (no risk of serious damage to eyes) 

Results 
In vitro irritancy score for Tinosorb® M pH = 10.5 

Group 

Mean Opacity 

Mean Permeability 

Irritancy Score (IVIS) 

Tinosorb®
Negative control 
Positive control 

 M pH = 10.5 

    4.5 

    2.6 
148.0 

0.208 

0.001 
3.830 

    7.6 

    2.6 
205.4 

vitro irritancy score for Tinosorb® M pH = 11.6 

Group 

Mean Opacity 

Mean Permeability 

Tinosorb®
Negative control 
Positive control 

 M pH = 11.6 

    3.0 

    2.6 
148.0 

0.002 

0.001 
3.830 

Irritancy Score 
(IVIS) 

    3.0 

    2.6 
205.4 

Under  the  conditions  of  this  in  vitro  assay,  the  formulated  micronised  UV  filter  Tinosorb® M 
pH= 10.5 and pH = 11.6 did not cause serious eye damage. 

SCCS comment  
The  particle  size  distribution  of  the  material  was  not  listed  in  the  report,  and  neither  was  the 
purity (which was stated as ‘available on request’). Based on historical data, the d(0.5) value 
was estimated by the applicant as 110-130 nm (no further details provided). 
Tinsosorb ® M at pH 11.6 is a material considered to be of extreme pH, and therefore should 
be  considered  as  corrosive  a  priori  if  there  are  no  other  formulant  in  the  medium  that  can 
provide  buffering  capacity.  This  aspect  needs  to  be  taken  into  account  in  the  overall  safety 
assessment.  

25 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

In vivo 

Guideline: 
Species: 
Route: 
Group size: 
Substance: 
Batch: 
Purity: 
Dose: 
GLP: 
Study period: 

OECD n° 405 
rabbit (New Zealand white) 
eye 
3 females 
Tinosorb® M, test substance nr. 10/0381-1 
P380; pH 11.6 
not reported 
0.1 ml of the Tinosorb® M formulaton 
in compliance 
October 2010 

Three  female  New  Zealand  White  rabbits  [Crl:KBL(NWZ) SPF]  received  a  single  application  of 
0.1 mL  Tinosorb® M  pH = 11.6,  i.e.  of  the  formulated  micronised  UV  filter  Tinosorb® M  (50 % 
MBBT),  into  the  conjunctival  sac  of  the  right  eyelid.  The  treated  eyes  were  rinsed  with  warm 
tap  water  about  24 hours  after  application.  The  untreated  left  eye  of  each  animal  served  as 
negative  control.  Scoring  of  irritation  effects  was  performed  1,  24,  48  and  72 hours  after 
application  according  to  the  OECD  scoring  system  using  an  otoscope  lamp.  Viability  checks 
were  performed  on  a  regular  basis  and  body  weights  were  recorded  prior  to  test  item 
administration and after the last reading. 
Slight  conjunctival  redness  and  slight  chemosis  were  noted  in  two  of  three  animals  up  to 
24 hours  after  application.  Ocular  discharge  was  observed  in  one  animal  only  1 hour  after 
application. No corneal or iris lesions were noted in any animal at any examination timepoint. 
One  animal  was  completely  free  of  alterations.  All  ocular  reactions  were  reversible  within 
48 hours  after  application.  Mean  scores  calculated  for  each  animal  over  24,  48  and  72  hours 
were 0.0, 0.0 and 0.0 for corneal opacity, 0.0, 0.0 and 0.0 for iris lesions, 0.0, 0.3 and 0.3 for 
redness of the conjunctiva and 0.0, 0.0 and 0.0 for chemosis, respectively. 

Conclusion 
Under the conditions of this study, the formulated micronised UV filter Tinosorb® M pH = 11.6 
was slightly irritating to the rabbit eye. 

Ref: Seibersdorf (2010) 

SCCS comment  
This in vivo study was carried out for the purpose of a Japanese Quasi Drug registration.  
The particle size distribution of the material was not listed in the report, and the purity was not 
reported.  Based  on  historical  data,  the  d(0.5)  value  was  estimated  by  the  applicant  as  110-
130 nm (no further details provided). 
The  eye  irritation  observed  may  be  due  to  the  high  concentration  (6-10%)  of  decyl  glucoside 
that was present as a surfactant in the formulation. 
Tinsosorb® M at pH 11.6 is a material considered to be of extreme pH (i.e. needs considering 
corrosive  a  priori).  For  such  instances,  thorough  consideration  needs  to  be  given  before 
performing  in  vivo  testing  for  animal  welfare  reasons  as  stated  in  OECD  TG  405.  The  study 
report, however, does not indicate that this was given any consideration.  

3.3.3.  Skin sensitisation 

No studies provided with nano-sized material. 
For studies with MBBT: see annex 2 

26 

 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

3.3.4.  Dermal / percutaneous absorption 

Dermal penetration study in vitro (nano sized material) 

Guideline 
Test substance: 

Purity: 

Dose applied: 
Skin preparation: 

Skin temperature: 
Exposure period: 
Donor chamber: 
Receptor fluid: 

Control:  
Particle size:  
Skin integrity: 

Recovery: 
GLP: 

OECD 428 
A suspension of micronised 14C-FAT 75714/B (purity of radio-labelled  
item: 99.1 %; purity of non-radiolabelled item: ≥ 99.9 %), i.e. of the 
UV-absorbing ingredient MBBT, was prepared in formulation 
(batch: CE60910011) and water. The suspension was further diluted 
with water and the pH-value adjusted to pH 6.5.  
Non-radiolabelled: 99.9% 
Radiolabelled: 99.1%  
1.4 mg/cm2 in 13 µl for 24 hours 
Full thickness skin from rats and from humans (upper leg dorsal, frozen), 
removed from subcutaneous fat was used.  Sections of 200 µm thickness 
were cut off from the top by dermatome. Pieces of 1.8 x 1.8 cm were 
mounted in flow-through diffusion cells, with 0.64cm2 of skin membrane 
exposed to the donor chamber. 
ambient 
24 hours 
non-occluded 
(6 % (w/v) polyethoxyoleate (PEG 20 oleyl ether) dissolved in 
physiological saline (0.9% NaCl w/v)) 
No control was used 
d(0.5)= 90 and 99 nm (2 series of 10 measurements); mass based. 
50 µL of tritium water was applied to the skin membrane surface in 
occluded donor chamber 
99.12% (rat skin) 94.45% (human skin) 
conducted according to GLP principles but the study report was not 
signed. 

Methods 
Split thickness skin membranes of rats ([HanBrl:WIST(SPF)]; two males) and of human donors 
(Caucasian  donors;  one  male,  two  females)  were  set  up  in  flow-through  diffusion  cells,  the 
formulated  micronised  test  item  was  applied  onto  the  skin  membranes  at  a  finite  dose  of 
13 µL/cm² and the receptor fluid collected at defined time intervals over a 24-hour period. In 
accordance with the standard dermal application rate for sun creams, a single target dose level 
of  2 mg/cm²  of  MBBT  was  used,  reflecting  a  concentration  of  10 %  test  item  in  the  final 
formulation. 

Prior  to  test  item  administration,  membrane  integrity  was  checked  by  applying  50 µL  tritium 
water  (about  200000 dpm)  to  skin  membranes  mounted  in  diffusion  cells  between  the  donor 
and  receptor  chamber.  The  donor  chamber  was  covered  with  adhesive  tape  (occluded 
conditions).  The  cumulative  penetration  was  determined  over  6 hours  by  collecting  hourly 
fractions.  The  permeability  coefficient  (Kp)  of  each  skin  membrane  was  calculated  for  the 
3 - 6 hours  interval.  Rat  skin  membranes  with  Kp  > 3.5 × 10-3 cm/h  and  human  skin 
membranes with Kp > 2.5×10-3 cm/h were excluded from the subsequent experiment. 

The  formulated  test  item  was  applied  onto  skin  membranes  of  200 µm  thickness  at  a 
concentration of 70.3 mg/cm³ leading to an area concentration of 1428.5 µg/cm². For the rat 
group  7 replicates  and  for  the  human  group  5 replicates  from  2  donors  were  used  for 

27 

 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

calculation  (the  skin  of  1  donor  was  excluded  due  to  a  too  high  Kp).  During  the  24-hour 
exposure  under  non-occluded  conditions,  the  receptor  fluid  (6 %  Polyethoxyoleate  (w/v)) 
dissolved  in  physiological  saline  (0.9 %  NaCl  (w/v))  was  collected  in  hourly  intervals  between 
0-6 hours and thereafter in 2-hour intervals until the end of the experiment. At the end of the 
experiment  the  remaining  test  item  was  removed  by  rinsing  the  skin  membranes  three  times 
with  a  mild  soap  solution.  The  skin  membranes  were  removed  from  the  diffusion  cell  and 
consecutively stripped until the stratum corneum was removed from the skin membrane.  

Radioactivity  was  measured  in  liquid  specimens  (receptor  fluid,  skin  membrane  rinse,  cell 
wash)  and  in  digested / solubilised  specimens  (tape  strips,  skin  membranes)  by  liquid 
scintillation counting (LSC) and quantified as quench-corrected disintegrations per minute. 

Skin rinse aliquots were analysed by HPLC with UV detection and radioactivity flow monitor in 
order  to  investigate  test  item  stability  over  the  exposure  period.  In  addition,  aliquots  of  the 
final suspension were analysed in a particle sizer. 

Results 
For both species 14C-FAT 75714/B amounted to more than 98.65 % of the radioactivity present 
in the skin membrane rinses. 

After  application,  an  average  of  only  0.06 %  of  the  applied  dose  penetrated  through  the  rat 
skin  membrane  within  24 hours.  The  mean  test  item  flux  could  not  be  calculated,  but  an 
estimated flux was calculated by using the values above LOQ and setting all values which were 
below  LOQ  to  the  corresponding  LOQ  and  this  flux  was  about  0.104 µg/cm²/h.  At  the  end  of 
exposure,  67.91 %  of  the  applied  dose  could  be  washed  off  from  the  skin  membranes.  After 
skin membrane rinse, 28.88 % of the dose remained in/on the skin membrane and the major 
part  of  this  remaining  test  item  was  located  in  the  stratum  corneum,  i.e.  26.86 %  of  the 
applied dose was determined in tape strips. A minor part, i.e. 2.02 % of the applied dose, was 
found in the remaining skin membrane after tape stripping. 

For  human skin  membranes,  the  test  item  penetration  closely  resembled  that  observed  in  rat 
skin  membranes.  Within  24 hours  of  exposure  only  0.01 %  of  the  applied  dose  penetrated 
totally  through  human  skin  membranes.  The  estimated  flux  was  0.086 µg/cm²/h.  Also, 
77.56 %  of  the  applied  dose  could  be  washed  off  24 hours  after  exposure  start.  In  stratum 
corneum  13.15 %  of  the  applied  dose  was  found  and  only  0.01 %  in  the  remaining  skin 
membrane  after  tape  stripping.  The  amount  of  the  test  item  in  lower  skin  layers  was 
significantly lower in human skin membranes compared to rat skin membranes.  
The mean total absorption, based on the amount penetrated through the skin membrane and 
the amount measured in the remaining skin membrane layers after tape stripping, was 2.08 % 
(n = 7)  and  0.02 %  (n = 5)  of  the  applied  dose  for  rat  and  human  skin  membranes, 
respectively.  The  calculated  absorption  values  for  human  skin  were  considered  conservative 
since most values were below LOQ.  

Percutaneous  penetration  of  the  micronised  UV-absorbing  ingredient  MBBT  through  rat  and 

human split thickness skin membranes over a 24-hour period 

Rat skin membranes 

Human skin membranes 

Applied dose 

Total applied dose 

Recovery of applied dose (mean or mean ± SD) 

1428.5 µg/cm² 

1428.5 µg/cm² 

Total absorbed dose 

- Receptor fluid 

2.08 % 
0.06 ± 0.06 % 

0.02 % 
0.01 ± < 0.01 % 

28 

 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Rat skin membranes 

Human skin membranes 

- Remaining skin membrane 

2.02 ± 1.92  % 

0.01 ± < 0.01% 

Total non-absorbed dose 

- Skin membrane rinse 
- Tape strips 
- Cell wash 

Total recovery of applied dose 

97.04 % 
67.91 ± 21.77 % 
26.86 ± 18.16 % 
2.27 ± 2.28 % 

99.12 ± 1.88 % 

94.42 % 
77.56 ± 4.84 % 
13.15 ± 6.67 % 
3.71 ± 2.20 % 

94.45 ± 5.15% 

SD: 

Standard deviation of the mean values for each 5 (human) or 7 cells (rat) 

Conclusion 
The  micronised  UV-absorbing  ingredient  MBBT  with  a  particle  size  (d(0.5))  below  100 nm 
penetrated  at  extremely  low  rates  and  to  a  very  limited  extent  through  rat  and  human  split 
thickness  skin  membranes.  The  penetration  through  rat  skin  membranes  was  higher  than 
through human skin membranes. 

Ref: RCC (2007) 

Given the fact that 5 replicates of human skin (2 donors) and 7 replicates of rat skin (2 donors) 
were assessed, the mean value + 2 SD will be used for the calculation of the Margin of Safety 
(MoS). This results in a total absorption of 0.048 % (= 0.02 + 2 × √(0.01² + 0.01²) for human 
skin and 5.922 % (= 2.08 + 2 × √(0.06² + 1.92²) for rat skin. 

SCCS comment 
-  It is noted that the recovery for the human samples is lower than for the rat samples 
-   Instead of a target dose of 2mg/ cm², only 1.4mg/ cm² was applied.  

Dermal penetration study with damaged skin in vitro (nano sized material) 

Guideline 
Test substance: 

Purity: 

Dose applied: 
Skin preparation: 

Skin temperature: 
Exposure period: 
Donor chamber: 
Receptor fluid: 

Control:  

OECD 428 
A suspension of micronized Tinosorb® M, 14C-FAT 75714/B i.e. of the UV-
absorbing ingredient MBBT, was prepared in formulation (batch: 
CE83400003) and water. The suspension was further diluted with water 
and the pH-value adjusted to pH 6.5, to a final concentration of 92.5 mg 
test item per ml suspension  
Non-radiolabelled: 100% 
Radiolabelled: 99.1% 
1.9 mg/cm2 in 13 µl for 24 hours 
Full thickness skin from humans (upper leg dorsal or abdominal area, 
frozen), removed from subcutaneous fat was used. Before sectioning, 5 
tape strips were performed.  Sections of 200 µm thickness were cut off 
from the top by dermatome. Pieces of 1.8 x 1.8 cm were mounted in 
flow-through diffusion cells, with 0.64cm2 of skin membrane exposed to 
the donor chamber. 
ambient 
24 hours 
non-occluded 
(6 % (w/v) polyethoxyoleate (PEG 20 oleyl ether) dissolved in 
physiological saline (0.9% NaCl w/v)) 
No control was used 

29 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

Particle size:  
Skin integrity: 

Recovery: 
GLP: 

d(0.5)= 87 and 91 nm (2 series of 10 measurements) 
50 µL of tritium water was applied to the skin membrane surface in 
occluded donor chamber 
100.67%  
in compliance  

Method 
The  percutaneous  penetration  of  FAT 75714  (purity:  99.1 %),  i.e.  of  the  UV-absorbing 
ingredient MBBT, formulated as micronised UV filter Tinosorb® M and suspended in formulation 
2000 UP  was  determined  in  vitro  using  split-thickness  skin  membranes  of  200 μm  thickness 
from  pre-damaged  human  cadaver  skin.  Pre-damaged  skin  membranes  were  produced  by 
stripping the membranes 5 times with an adhesive tape. 

The skin membranes (7 replicates) were set  up in flow-through diffusion cells, the formulated 
test  item  (94.186 mg/mL)  was  applied  onto  the  skin  membranes  at  a  single  dose  level  of 
1913 µg/cm²  and  the  perfusates  were  collected  at  defined  time  intervals  for  a  period  of 
24 hours. 

Results 
Within  24 hours  the  portion  of  radio-labelled  MBBT  penetrating  through  pre-damaged  human 
skin  membranes  accounted  for  0.01 %  of  the  applied  dose.  97.82 %  of  the  applied  dose  was 
removed by skin rinse and additional 2.00 % by tape stripping. In the lower skin layers about 
0.06 %  of  the  dose  was  found.  Together  with  the  amount  measured  in  the  remaining  skin 
membrane after tape stripping, the amount totally penetrated in/through pre-damaged human 
skin membrane accounted for 0.06 % of the applied dose. The mean test item flux calculated 
with LOQ values as worst case calculation was < 0.058 µg/cm²/h through pre-damaged human 
skin membranes. Recovery was 100.67 %. 

Conclusion 
The UV-absorbing ingredient MBBT formulated as micronised UV filter  Tinosorb® M penetrated 
at an extremely low rate through pre-damaged human skin membranes. 

Ref: Harlan (2009) 

SCCS comment  
The SCCS notes that there is no standard protocol for the investigation of dermal penetration 
through  damaged  skin  and  a  common  understanding  of  “damaged  skin”,  which  could  be 
wounded,  physically  damaged,  sunburnt,  etc.  In  this  particular  study,  skin  was  artificially 
damaged by tape stripping (5 tape strips). 
As a worst case approach the SCCS calculates the total absorbed dose as the receptor fluid + 
the  remaining  skin  membrane.  Given  the  fact  that  7 replicates  of  2  donors  were  used,  the 
mean value + 2 SD will be used for the calculation of the Margin of Safety (MoS), resulting in 
an absorption of 0.01+0.06 + 2x. This results in a conservative value of the total absorption of 
0.35 % (= 0.07 + 2 × √(0.01² + 0.14²) for pre-damaged human skin. 
It is not clear whether the particle size is number based or mass based.  

SCCS overall comment on dermal absorption 
Skin  penetration  of  nano-sized  MBBT  was  determined  in  vitro  in  healthy  rat  skin,  in  healthy 
human  skin,  and  in  artificially  damaged  human  skin.  Each  of  the  studies  was  not  fully  in  line 
with  the  SCCS’  basic  criteria  for  dermal  penetration  (with  respect  to  donor  numbers  and  the 
replicates  used).  Therefore  2  standard  deviations  were  added  to  the  Means  of  the 
experimentally determined absorption values to account for the uncertainties. 

30 

 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

In the studies using human intact and damaged skin, the amounts that penetrated through the 
skin  were  below  LOQ  for  many  of  the  replicates  analysed,  pointing  to  extremely  low 
penetration. 

By adding 2 standard deviations to the Mean penetration values to account for the uncertainty, 
the percentage of the amount applied that penetrated through human intact skin was 0.048 %. 
In damaged human skin, the absorption percentage was 0.35 % of the applied amount.  

In intact rat skin on the other hand, the Mean amount penetrating through the skin was 5.922 
%  of  the  applied  amount.  This  higher  value  was  used  in  MOS  calculation  as  the  most  worst 
case. Thus, the SCCS considers that the conservative approach used also covered penetration 
of nano-sized MBBT through the artificially damaged human skin. 

3.3.5.  Repeated dose toxicity 

3.3.5.1.  Repeated Dose (28 days) oral / dermal / inhalation toxicity 

Guideline/method 
Species: 
Group size: 

Test substance: 
Batch: 
Conc a.i.: 
Particle size: 

Dose applied: 

Skin area:  
Route: 
Exposure time: 
GLP: 
Date of report: 
Published: 

Exploratory study  
Rat, Wistar Han  
5 males and 5 females per dose group (2 animals per sex in a satellite 
group). 
Tinosorb® M, FAT 75’634/B  
02711CL3 
50.2% 
no data provided in the study report, the applicant indicated that the 
d(05) = 128 nm presented as additional information in Submission II    
0,  1000,  1500  and  2000  mg/kg  bw/day,  later  reduced  to  100,  400  and 
800 mg/kg bw/day, see study description below.  
~45 (males)-30 (females) cm2). 
topical application on skin of which fur was removed with animal clipper. 
6 hours per day 
in compliance  
Dec 2005 (study completed Sept 2003) 
no 

Methods 
Repeated  dermal  treatments  of  5 rats  [Crl:WI  (GLX/BRL/Han)  IGS  B0]  per  sex  per  dose  level 
with  the  test  item  FAT 75’634/B,  i.e.  with  the  formulated  micronised  UV  filter  Tinosorb® M, 
suspended in water (first treatment period: day 1 – 17; the pH was adjusted to 8.0) or in base 
ointment  (Batch  numbers  SO0616  and  SP0657;  composition  not  further  specified;  second 
treatment  period:  day  50 – 58;  no  pH  adjustment)  at  dose  levels  of  1000,  1500  or 
2000 mg/kg bw/d  (dose  volume:  2.5 mL/kg bw)  each  resulted  in  local  dermal  intolerance  and 
in clinical signs of pain.  

Based  on  this  outcome,  the  dermal  treatment  was  stopped  for  a  period  of  5 days  (day  59 –
 63).  Thereafter,  the  same  animals  received  the  test  item  suspended  in  base  ointment 
(hydrated,  hydrophilic)  at  reduced  dose  levels  of  100,  400  or  800 mg/kg bw/d  (dose 
volume 2.5 mL/kg bw)  for  29  consecutive  days  (third  treatment  period:  day 64 – 92).  A 
concurrent  control  group  of  5 rats  per  sex  was  treated  with  Tinosorb® M  Placebo  in  base 
ointment  under  the  same  experimental  conditions.  Viability,  clinical  signs,  food  consumption, 

31 

 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

body  weights,  parameters  of  clinical  laboratory  investigations  (haematology  and  clinical 
chemistry),  test  item  plasma  levels,  selected  organ  weights  and  macroscopic  and  microscopic 
changes  (skin  samples  and  other  organs)  were  assessed  and  evaluated.  The  test  substance 
was  measured  in  plasma  samples  on  day  8,  57  and  93.  Dose  formulations  were  analysed  for 
content, homogeneity and stability. 

Results 
During  the  first  and  the  second  treatment  period,  dermal  administration  of  the  test  item 
induced  signs  of  poor  local  tolerance  and  pain  (vocalisation,  hyperactivity  to  stimuli  or 
aggressiveness,  and/or  abnormal  posture)  with  dose-related  incidence  and  severity  at  1000, 
1500 and 2000 mg/kg bw/d in both water and base ointment. 

During the third treatment period, when dermally applied for 29 consecutive days at 100, 400 
or 800 mg/kg bw/d in base ointment, the test item did not induce any local or systemic clinical 
signs of toxicity. Treatment with the test item did not result in increased mortality, any change 
in body weight or food consumption, or any modification of haematology and blood chemistry 
parameters in any group during the three treatment periods. At necropsy, no test item-related 
effects on organ weights and on macroscopical endpoints were noted in any group. There were 
no histopathological findings upon examination of the skin sites and other organs related to the 
treatment  with  the  test  item.  Thus,  the  dose  level  of  800 mg/kg bw/d  was  established  as  the 
highest achievable dose level for repeated dermal treatment.  

Plasma  levels  of  the  test  substance  were  below  or  occasionally  slightly  above  the  limit  of 
quantification (5ng/ml). There was no dose relation in the plasma levels. 

Chemical  analysis  revealed  satisfactory  homogeneity  of  the  test  item  in  base  ointment  and 
demonstrated  good  agreement  between  nominal  and  analytically  determined  test  item 
concentrations  up  to  and  including  320 mg/L,  i.e.  up  to  the  dose  concentration  used  for  the 
high  dose  group.  The  stability  of  the  test  item  in  base  ointment  was  satisfactory  for  at  least 
9 days at 4 °C. 

Table 1: 

Incidence of clinical lesions recorded at the application site 

Lesion 

Control 

Dose level (mg/kg bw/d) 
Mid dose 
Low dose 

M 

F 

M 

F 

M 

F 

High dose 
F 

M 

First treatment period (vehicle: water) 
0 

1000 

1500 

2000 

Desquamation  
Scabs 
Erythema 
Second treatment period (vehicle: ointment) 

0 
0 
0 

0 
0 
0 

0 
Desquamation 
0 
Scabs  
0 
Erythema 
Third treatment period (vehicle: ointment) 

0 
0 
0 

0 

Desquamation 
Scabs 
Erythema 

M: male; F: female 

0 

0 
1/5 
0 

0 
0 
0 

0 
0 
0 

0 
0 
0 

0 
0 
0 

4/5 
0 
0 

0 
1/5 
0 

1000 

1/4 
0 
1/4 

0 
0 
0 

1500 

100 

400 

0 
0 
0 

0 
1/5 
0 

3/5 
0 
1/5 

0 
0 
1/5 

0 
0 
3/5 

0 
0 
0 

0 
2/5 
1/5 

0 
0 
0 

2000 

800 

2/5 
1/5 
1/5 

0 
1/5 
1/5 

0 
1/5 
1/5 

32 

 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Conclusion 
Under  the  conditions  of  this  dose  range-finding  study,  the  dermal  no-observed-effect-level 
(NOEL)  of  the  formulated  micronised  UV  filter  Tinosorb® M  was  determined  to  be 
800 mg/kg bw/d  in  rats,  when  applied  topically  on  the  skin  for  29  consecutive  days  in  base 
ointment.  The  observed  signs  of  poor  local  tolerance  and  pain  might  be  vehicle  related  since 
they were not confirmed in a 90-day dermal toxicity study in rats using corn oil as vehicle (RCC 
Ltd., 2002) up to the high dose level of 1000 mg/kg bw/d. 

Ref.: CIT (2005) 

SCCS comment 
At 400 and 800 mg/kg body weight minimal local skin reactions were observed, which were not 
considered  in  the  determination  of  the  NOAEL.  Therefore  the  NOAEL  of  800  mg/kg  bw 
(equivalent  to  400  mg  a.i./kg  bw)  is  based  on  lack  of  clinical  signs  when  compared  to  the 
higher  doses  that  induced  pain  and  discomfort  in  the  animals  during  the  first  and  second 
treatment cycle. Given the changes in the study protocol during the study, the results can only 
be  used  as  supportive  for  the  risk  assessment.  An  adequate  NOAEL  cannot  be  derived.  It  is 
noted that this dose range-finding toxicity was performed to provide a scientific basis for dose 
level  setting  in  a  carcinogenicity  study  by  dermal  route  in  rats.  Moreover,  a  39-week  dermal 
study in mini-pigs was provided.  
There  is  no  convincing  evidence  that  the  clinical  signs  are  vehicle  related,  since  the  90-day 
dermal study that is referred to above was not carried out using nano-sized material.  

The composition of the base ointment is not specified in the study report. No information on pH 
is given in the study report. An extreme pH cannot be excluded (see section 3.3.2.2) and might 
have been the  cause  for  signs  of pain  observed  in  the  animals.  Animal  welfare  considerations 
should have taken place before performing the study. It is not clear whether the particle size is 
number based or mass based. 

Dermal dose range finding toxicity study in mini-pigs  

Guideline/method 
Species: 
Group size: 
Test substance: 
Batch: 
Conc a.i.: 
Particle size: 

Dose applied: 

Skin area: 

Route: 
Exposure time: 

GLP: 

Exploratory study  
Göttingen mini-pigs  
one male and one female for each of the studies performed  
Tinosorb® M, FAT 75’634/B  
02711CL3 
50.2% 
no data provided in the study report, the applicant indicated that the 
d(05) = 128 nm presented as additional information in Submission II    
First study: increasing doses starting at 100, 400, 800, 1000 and 2000 
mg/kg bw/day, for three consecutive days, respectively 
Second study: dose of 2000 mg/kg bw for 14 days 
2.5 ml/kg was applied on the skin  
at least 10% of the total body area on the dorsum ranging from 371 to 
441 cm2 was clipped free of hair, of which 90% (334 to 397 cm2) was 
treated depending on the weight of the animal.  
topical application on skin  
First series: 6 hours per day for three consecutive days 
Second series: 6 hours per day for 14 consecutive days 
no  

33 

 
 
 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

Date of report: 
Published: 

7 July 2004 (study completed 1 March 2004) 
no 

Methods 
A  14-day  dermal  range-finding  study  using  one  female  and  one  male  Göttingen  mini-pig 
(Dalmose, Denmark) per study phase was performed with the test item FAT 75’634/B, i.e. with 
the  formulated  micronised  UV  filter  Tinosorb® M,  suspended  in  base  ointment.  In  study 
phase 1, progressively increasing dermal doses (100, 400, 800, 1000 or 2000 mg/kg bw; dose 
volume:  2.5 mL/kg bw)  were  administered  for  6 hours  per  day  for  3 consecutive  days, 
respectively. In study phase 2, the animals received 2000 mg/kg bw at the same dose volume 
2.5 mL/kg bw  for  6 hours  per  day  for  14 consecutive  days  based  on  the  outcome  of  study 
phase 1.  The  test  item  was  applied  unoccluded  to  each  animal  to  about  90 %  of  a  prepared 
dosing area and compared to the remaining 10 % untreated control area. 
Treatment schedule phase I. 

Viability,  clinical  signs,  food  consumption,  body  weights  and  microscopic  changes  (skin 
samples) were assessed and evaluated. 

Results 
There were no test item-related effects on viability, clinical signs, food consumption and body 
weights. No microscopic findings were noted on the skin samples from the application sites. 

Conclusion 
Under  the  experimental  conditions  of  this  study,  the  maximum  tolerated  dose  (MTD)  for 
repeated dermal treatment with the formulated micronised UV filter Tinosorb® M was above the 
high  dose  level  of  2000 mg/kg bw/d  in  Göttingen  mini-pigs.  The  dose  levels  selected  for  the 
main study were 500, 1000 and 2000 mg/kg bw/d. 

Ref: CIT (2004) 

3.3.5.2.  Sub-chronic (90 days) oral / dermal / inhalation toxicity 

Dermal repeated dose toxicity study in mini-pigs  

Guideline/method 
Species: 
Group size: 
Test substance: 
Batch: 
Conc a.i.: 
Particle size: 

Exploratory study  
Göttingen mini-pigs  
four male and four female animals per dose level  
Tinosorb® M, FAT 75’634/B  
0069CL3, and 001722CL4 
50.2% 
for batch 00696CL3 presented as additional information in Submission II 
the d(05) = 115 nm 

34 

 
 
 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

Dose applied: 

Skin area: 

Route: 
Exposure time: 
GLP: 
Date of report: 
Published: 

Batch 001722CL4 d(0.5) 117nm mass based d(0.5) 61nm number 
based. 
500, 1000, or 2000 mg/kg bw. 
2.5 ml/kg was applied on the skin  
at least 10% of the total body area on the dorsum ranging from 371 to 
441 cm² was clipped free of hair, of which 90% (334 to 397 cm2) was 
treated depending on the weight of the animal.  
topical application on skin  
6 hours per day for a period of 39 weeks 
in compliance 
10 February 2006 (study completed 24 January 2005) 
no 

Methods 
Three  groups  of  4 Göttingen  mini-pigs  (Dalmose,  Denmark)  per  sex  per  dose  level  received 
dermal  applications  of  FAT 75’634,  i.e.  of  the  formulated  micronised  UV  filter  Tinosorb® M, 
suspended  in  a  hydrophilic  ointment  at  500,  1000  or  2000 mg/kg bw/d  (dose  volume: 
2.5 mL/kg bw) for 39 weeks on closely clipped dermal skin (approx. 10 % of the body surface 
area).  The  corresponding  dose  levels  expressed  in  topical  dosage  equivalents  are  depicted  in 
Table 5.7. 

Table 2:  Topical daily dosage equivalents 

Tinosorb® M 
(mg/kg bw) 
500 
1000 
2000 

(mg/kg bw) 
250 
500 
1000 

MBBTa 
(%)b  
10 
20 
40 

a:  UV-absorbing ingredient in micronised form 
b:  based on a dose volume of 2.5 mL/kg bw active ingredient 

mg/cm² 
7 
14 
28 

A  concurrent  control  group  of  4 animals  per  sex  received  Tinosorb® M  Placebo  in  hydrophilic 
ointment at a concentration equivalent to the concentration of this component in the high dose 
group. 

Viability,  clinical  signs, 
food  consumption,  body  weights,  ophthalmoscopic  endpoints, 
parameters of clinical laboratory investigations (haematology, clinical chemistry and urinalysis), 
test  item  plasma  levels  (week 13,  26  and  end  of  study  before  daily  dosing),  selected  organ 
weights  and  macroscopic  and  microscopic  changes  (skin  samples)  were  assessed  and 
evaluated. Dose formulations were analysed for content, homogeneity and stability. 

Results 
Chemical  analysis  of  dose  formulations  met  the  acceptance  criteria  for  stability,  homogeneity 
and  concentrations.  No  test  item-related  effects  were  noted  on  viability,  clinical  signs,  food 
consumption,  body  weights  and  on  haematology,  clinical  chemistry  and  urinalysis  parameters 
as  well  as  organ  weights.  There  were  no  test  item-related  findings  at  ophthalmological 
examination. No test item-related macroscopic and microscopic findings were noted in organs 
and tissues including treated skin samples. 

In  high  dose  animals  treated  at  2000 mg/kg bw/d,  detectable  plasma  concentrations  below  
30 ng/mL  of  MBBT  (micronised  form)  were  observed  in  one  male  and  one  female  only.  Test 

35 

 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

item  concentrations  in  plasma  were  below  the  limit  of  quantitation  (< 5 ng/mL)  in  all  other 
samples taken during the study. 

SCCS/1546/15 

Conclusion 
Under the experimental conditions of this study, no target organ toxicity was identified. Topical 
application of the test item did not induce a significant systemic exposure. 
The no-observed-adverse-effect-level (NOAEL) for repeated dermal treatment of mini-pigs with 
the  UV-absorbing  ingredient  MBBT  and  the  formulated  micronised  UV  filter  Tinosorb® M  was 
1000 mg/kg bw/d and 2000 mg/kg bw/d, respectively. 

MBBT  is  not  subject  to  classification  for  specific  target  organ  toxicity  according  to  Regulation 
(EC) No. 1272/2008. 

Ref: CIT (2006a) 

SCCS comment 
Some absorption was noted in two animals (30ng/mL), while in all other animals (n=22) levels 
were below detection limit of 5 ng/mL. 

3.3.5.3.  Chronic (> 12 months) toxicity 

/ 

3.3.6.  Mutagenicity / Genotoxicity 

3.3.6.1.  Mutagenicity / Genotoxicity in vitro 

In vitro mammalian cells 

Micronucleus test in V79 cells 

Guideline: 
Cells: 
Replicates: 
Test substance: 
Vehicle: 
Treatment 

GLP: 
Reference: 

OECD 487  
V79 cells 
Triplicate cultures   
Tinosorb® M pH= 10,5 (49.1% MBBT in aqueous solution) 
Ultrapure water 
4h treatment; harvest time 24 or 44 hours after the beginning of treatment 
Or 30h treatment; harvest time 46h after the beginning of treatment 
In compliance 
Study report number 33M0407/10M204 BASF SE (2014) 

Methods 
The  cytokinesis  blocked  micronucleus  assay  was  conducted  in  V79  cells.  The  test  item 
Tinosorb® M pH= 10,5 (49.1% MBBT in aqueous solution) was evaluated in three independent 
experiments  using  either  4h  or  30h  exposure  durations.  Experiments  were  conducted  in  the 
absence  and  presence  of  metabolic  activation  (S9-mix  prepared  from  the  liver  of  rats  given 
phenobarbital- and β-naphthoflavone). Following a range finding cytotoxicity test, the following 
concentrations were selected for the micronucleus assay: 

36 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

  1st Experiment- 

o  4  hours  exposure;  24  hours  harvest  time;  without  S9  mix:  0;  343.8;  687.5; 

1375.0; 2750.0; 5500.0; 10200.0 μg/mL 

o  4 hours exposure, 24 hours harvest time, with S9 mix: 0; 343.8; 687.5; 1375.0; 

2750.0; 5500.0; 10200.0 μg/mL 

  2nd Experiment 

o  30 hours exposure, 46 hours harvest time, without S9 mix: 0; 23.4; 46.9; 93.8; 

187.5; 375.0; 750.0; 1500.0; 3000.0 μg/mL 

o  4 hours exposure, 44 hours harvest time, with S9 mix: 0; 375.0; 750.0; 1500.0; 

3000.0; 6000.0; 10200.0μg/mL 

The 2nd experiment in the absence and presence of S9 mix was not scorable for the detection 
of  micronucleated  cells  due  to  poor  slide  quality.  Thus,  this  experiment  was  repeated  and 
designated as the 3rd experiment. 

  3rd Experiment 

o  30  hours  exposure,  46  hours  harvest  time,  without  S9  mix:  0;  46.9;  93.8; 

187.5; 375.0; 750.0; 1500.0; 3000.0 μg/mL 

o  4 hours exposure, 44 hours harvest time, with S9 mix: 0; 375.0; 750.0; 1500.0; 

3000.0; 6000.0; 10200.0 μg/mL 

In  all  experiments,  following  the  exposure  period,  medium  was  removed  and  replaced  with 
fresh medium containing cytochalasin B until harvest. Following harvest at least 1000 cells for 
each culture were analysed for micronuclei, i.e. 2000 cells for each test group. Positive controls 
were  ethyl  methanesulfonate  (300,  400,  500  μg/mL)  without  S9  mix  and  cyclophosphamide 
(0.5, 1.0 μg/mL) with S9 mix. 

Results 
No  biologically  relevant  increase  in  the  number  of  micronucleated  cells  was  observed  either 
with or without S9 mix, regardless of dose applied or exposure time. Cytotoxicity was however 
observed with increasing doses, with excessive cell death at top doses of 1500 and 3000 μg/mL 
following  30h  exposure/16h  recovery  without  S9;  5500.0  and  10200.0  μg/mL  following  4h 
exposure / 24h recovery without S9; 10200.0 μg/mL following 4h exposure/24h recovery, and 
4h exposure/44h recovery with S9. 

Conclusion 
The study authors concluded that under the conditions of the study, Tinosorb® M pH= 10.5 did 
not  produce  micronuclei  in  cultured  V79  cells  either  in  the  absence  or  presence  of  metabolic 
activation and was therefore considered to have no clastogenic or aneugenic potential. 

Guideline: 
Cells: 
Replicates: 
Test substance: 
Vehicle: 
Treatment 

GLP: 
Reference: 

OECD 487  
V79 cells 
Duplicate cultures   
Tinosorb® M (not micronized) (54.7% MBBT in aqueous solution) 
Ultapure water 
4h treatment; harvest time 24 or 44 hours after the beginning of treatment 
Or 30h treatment; harvest time 46h after the beginning of treatment 
In compliance 
Study report number 33M0045/13M030 BASF SE (2014) 

37 

 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Methods 
The  cytokinesis  blocked  micronucleus  assay  was  conducted  in  V79  cells.  The  test  item 
Tinosorb®  M  (not  micronised)  (54.7%  MBBT  in  aqueous  solution)  was  evaluated  in  two 
independent  experiments  using  either  4h  or  30h  exposure  durations.  Experiments  were 
conducted in the absence and presence of metabolic activation (S9-mix prepared from the liver 
of rats given phenobarbital- and β-naphthoflavone). Following a range-finding cytotoxicity test, 
the following concentrations were selected for the micronucleus assay: 

  1st Experiment- 

o  4  hours  exposure;  24  hours  harvest  time;  without  S9  mix:  0;  171.9;  343.8; 

687.5; 1375.0; 2750.0; 5500.0 μg/mL 

o  4 hours exposure, 24 hours harvest time, with S9 mix: 0; 171.9; 343.8; 687.5; 

1375.0; 2750.0; 5500.0 μg/mL 

  2nd Experiment 

o  30  hours  exposure,  46  hours  harvest  time,  without  S9  mix:  0;  46.9;  93.8; 

187.5; 375.0; 750.0; 1500.0; 3000.0; 6000.0 μg/mL 

o  4 hours exposure, 44 hours harvest time, with S9 mix: 0; 375.0; 750.0; 1500.0; 

3000.0; 6000.0 μg/mL 

In  all  experiments,  following  the  exposure  period,  medium  was  removed  and  replaced  with 
fresh medium containing cytochalasin B until harvest. Following harvest at least 1000 cells for 
each culture were analysed for micronuclei, i.e. 2000 cells for each test group. Positive controls 
were  ethyl  methanesulfonate  (300,  400,  500  μg/mL)  without  S9  mix  and  cyclophosphamide 
(0.5, 1.0 μg/mL) with S9 mix. 

Results 
No  biologically  relevant  increase  in  the  number  of  micronucleated  cells  was  observed  either 
with or without S9 mix, regardless of dose applied or exposure time. Cytotoxicity was however 
observed  with  increasing  doses,  with  excessive  cell  death  at  top  doses  from  750  μg/mL 
following 30h exposure/16h recovery without S9; from 22750  μg/mL following 4h exposure / 
24h recovery without S9; at 5500 μg/mL and 6000 μg/mL following 4h exposure/24h recovery 
and 4h exposure/44h recovery with S9 respectively. 

Conclusion 
The  study  authors  concluded  that  under  the  conditions  of  the  study,  Tinosorb®  M  (not 
micronized)  (54.7%  MBBT  in  aqueous  solution)  did  not  produce  micronuclei  in  cultured  V79 
cells either in the absence or presence of metabolic activation and was therefore considered to 
have no clastogenic or aneugenic potential. 

Hrpt Forward Mutation Assay in CHO cells 

Guideline: 
Cells: 
Replicates: 
Test substance: 
Vehicle: 
Treatment 
GLP: 
Reference: 

OECD 476  
Chinese Hamster Ovary (CHO) cells 
Triplicate cultures   
Tinosorb® M pH= 10,5 (49.1% MBBT in aqueous solution) 
Ultapure water 
4 h or 24h treatment 
In compliance 
Study report number 50M0407/10M208 BASF SE (2014) 

38 

 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Methods 
The hprt locus for 6-thioguanine (6-TG) resistance in the CHO cell line was used. The test item 
Tinosorb® M pH= 10,5 (49.1% MBBT in aqueous solution) was evaluated in three independent 
experiments using either 4h or 24h exposure durations. Treatment and were conducted in the 
absence  and  presence  of  metabolic  activation  (S9-mix  prepared  from  the  liver  of  rats  given 
phenobarbital- and β-naphthoflavone). Following a range-finding toxicity test based on cloning 
efficiency, the following concentrations were selected for assessment: 

  1st Experiment- 

o  4 hours exposure without S9 mix: 0; 43.0; 85.9; 171.9; 343.5; 687.5; 1375.0; 

2750.0; 5500.0 μg/mL 

o  4  hours  exposure  with  S9  mix:  0;  43.0;  85.9;  171.9;  343.5;  687.5;  1375.0; 

2750.0; 5500.0 μg/mL 

  2nd Experiment 

o  24  hours  exposure  without  S9  mix:  0;  23.4;  46.9;  93.8;  187.5;  375.0;  750.0; 

1500.0; 3000.0 μg/mL 

o  4  hours  exposure  with  S9  mix:  0;  93.8;  187.5;  375.0;  750.0;  1500.0;  3000.0 

μg/mL 

In the 2nd experiment in the presence of metabolic activation, the required clear cytotoxicity of 
10  -  20%  relative  survival  (relative  cloning  efficiency)  was  not  obtained  up  to  the  highest 
applied  concentration.  For  this  reason  a  repeat  experiment,  designated  3rd  experiment,  was 
performed. 

  3rd Experiment 

o  4 hours exposure with S9 mix: 0; 187.5; 375.0; 750.0; 1500.0; 3000.0; 6000.0 

μg/mL 

Positive  controls  were  ethyl  methanesulfonate  (300  μg/mL)  without  metabolic  activation  and 
7.12-Dimethylbenz[a]anthracene  (1.25  μg/mL)  in  the  presence  of  a  metabolic  activation 
system. Cells were suspended in culture medium and exposed to various concentrations of the 
test item, solvent or positive controls. After the treatment period the cells were re-suspended 
in culture medium for 6-8days. At the end of the expression period, cultures were then plated 
for  6-TG  resistance  for  7days.  Colonies  of  each  test  group  were  fixed  with  methanol,  stained 
with Giemsa and counted. 

Results 
No biologically relevant increase in the number of mutant colonies was observed either with or 
without  S9  mix,  regardless  of  the  dose  applied  or  exposure  time.  Cytotoxicity  was  however 
observed at the highest doses applied in all experiments. Substantial toxicity occurred at 2750 
and  5500  μg/mL  following  4h  exposure  without  S9;  1500  and  3000  μg/mL  following  24h 
exposure without S9; 2750, 3000, 5500 and 6000 μg/mL following 4h exposure with S9. 

Conclusion 
The study authors concluded that under the conditions of the study, Tinosorb® M pH= 10,5 did 
not induce any biologically relevant increase in mutation frequency in cultured CHO cells either 
in  the  absence  or  presence  of  metabolic  activation.  It  was  therefore  considered  to  have  no 
mutagenic potential. 

39 

 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Guideline: 
Cells: 
Replicates: 
Test substance: 
Vehicle: 
Treatment 
GLP: 
Reference: 

OECD 476  
Chinese Hamster Ovary (CHO) cells 
Triplicate cultures   
Tinosorb® M (not micronized) (54.7% MBBT in aqueous solution) 
Ultapure water 
4 h or 24h treatment 
In compliance 
Study report number 0M0045/13M020 BASF SE (2014) 

Methods 
The hprt locus for 6-thioguanine (6-TG) resistance in the CHO cell line was used. The test item 
Tinosorb®  M  (not  micronized)  (54.7%  MBBT  in  aqueous  solution)  was  evaluated  in  three 
independent  experiments  using  either  4h  or  24h  exposure  durations.  Treatments  were 
conducted in the absence and presence of metabolic activation (S9-mix prepared from the liver 
of  rats  given  phenobarbital-  and  β-naphthoflavone).  Following  a  range-finding  toxicity  test 
based on cloning efficiency, the following concentrations were selected for assessment: 

  1st Experiment- 

o  4 hours exposure without S9 mix: 0; 43.0; 85.9; 171.9; 343.8; 687.5; 1375.0; 

2750.0; 5500.0 μg/mL 

o  4  hours  exposure  with  S9  mix:  0;  43.0;  85.9;  171.9;  343.8;  687.5;  1375.0; 

2750.0; 5500.0 μg/mL 

  2nd Experiment 

o  24  hours  exposure  without  S9  mix:  0;  23.4;  46.9;  93.8;  187.5;  375.0;  750.0; 

1500.0; 3000.0 μg/mL 

o  4  hours  exposure  with  S9  mix:  0;  93.8;  187.5;  375.0;  750.0;  1500.0;  3000.0 

μg/mL   

In  the  2nd  experiment  the  4h  exposure  in  the  presence  of  metabolic  activation  did  not  fulfil 
guideline  recommendations.  For  this  reason  a  repeat  experiment,  designated  as  the  3rd 
experiment, was performed. 

  3rd Experiment 

o  4  hours  exposure  with  S9  mix:  0;  46.9;  93.8;  187.5;  375.0;  750.0;  1500.0; 

3000.0 μg/mL 

Positive  controls  were  ethyl  methanesulfonate  (300  μg/mL)  without  metabolic  activation  and 
7.12-Dimethylbenz[a]anthracene  (1.25  μg/mL)  in  the  presence  of  a  metabolic  activation 
system. Cells were suspended in culture medium and exposed to various concentrations of the 
test item, solvent or positive controls. After the treatment period the cells were re-suspended 
in culture medium for 6-8 days. At the end of the expression period, cultures were then plated 
for 6-TG resistance for 7 days. Colonies of each test group were fixed with methanol, stained 
with Giemsa and counted. 

Results 
No biologically relevant increase in the number of mutant colonies was observed either with or 
without  S9  mix,  regardless  of  the  dose  applied  or  exposure  time.  Cytotoxicity  was  however 
observed at the highest doses applied in all experiments. Substantial toxicity occurred at 2750 
and  5500  μg/mL  following  4h  exposure  without  S9;  1500  and  3000  μg/mL  following  24h 
exposure without S9; 2750, 3000, and 5500 μg/mL following 4h exposure with S9. 

40 

 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Conclusion 
The  study  authors  concluded  that  under  the  conditions  of  the  study,  Tinosorb®  M  (not 
micronised) (54.7% MBBT in aqueous solution) did not induce any biologically relevant increase 
in  mutation  frequency  in  cultured  CHO  cells  either  in  the  absence  or  presence  of  metabolic 
activation. It was therefore considered to have no mutagenic potential. 

Discussion on Mutagenicity / Genotoxicity 
Additional in vitro genotoxicity studies conducted on both nano-sized and non-nanosized MBBT 
in  a  formulation  comparable  to  Tinosorb®  M  indicated  that  both  substances  did  not  induce 
chromosomal  aberrations  or  mutagenicity  under  the  described  experimental  conditions.  The 
following  statement  is  made:  “According  to  the  additional  investigations  on  particle  size 
distribution  in  culture  media  performed  during  the  pre-  and  main  tests  of  the  comparative  in 
vitro  studies  listed  above,  the  exposure  of  the  respective  cells  in  culture  to  the  substance  in 
nanosized  form  has  been  confirmed.  The  newly  derived  in  vitro  data  represent  models  for  a 
direct  interaction  between  the  test  substance  and  target  cells  and  the  observed  cytoxicity 
provide evidence for a direct interaction between test substance and the cells investigated.” 

SCCS comment  
It  is  noted  that  both  Tinosorb®  M  (not  micronised)  (54.7%  MBBT  in  aqueous  solution)  and 
Tinosorb®  M  pH=  10,5  do  not  induce  significant  increases  in  genotoxicity  in  either  the 
cytokinesis  blocked  micronucleus  assay  or  hrpt  mutation  assay.  The  experiments  were 
conducted  in  the  presence  and  absence  of  S9,  and  at  multiple  exposure  time  points.  In  all 
cases, no evidence of gross chromosomal damage or mutagenicity was detected. However, no 
direct evidence of cell uptake of the substance has been provided. 

The applicants make a statement to indicate that evaluation of agglomeration and presence of 
cytotoxicity  provides  evidence  for  a  direct  interaction  between  the  test  substance  and  cells. 
However, the experiments on particle size distribution in culture media simply characterise the 
level  of  agglomeration  in  the  test  systems.    It  does  not  demonstrate  direct  exposure  to  the 
cells  or  direct  physical  interaction  between  the  test  agent  and  the  target  cells.  Cytotoxicity 
induced  by  nanomaterials  is  also  not  a  direct  indicator  of  cell  uptake  as  it  can  be  induced 
through  a  variety  of  other  mechanisms.  The  applicants  have  therefore  not  provided  evidence 
for cell uptake and thus direct exposure is not confirmed. 

3.3.6.2  Mutagenicity/Genotoxicity in vivo 

No studies provided with nano-sized material. 
For studies with MBBT: see Annex II 

3.3.7.  Carcinogenicity 

Rat skin carcinogenicity 

Guideline/method:  OECD 451 (1981) 
Species/strain:  
Group size:  
Test substance: 

Wistar Han rats 
50 animals per sex and dose 
FAT 75’634/B (Tinosorb M; microfine material; d(0.5) = 128 nm. Contains 
approximately 50% active ingredient Tinosorb MBBT (FAT 75'714). 

41 

 
 
 
 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

Batch: 
Concentrations: 

Exposure: 
Vehicle: 
Negative control: 

Positive controls: 
GLP: 
Study period: 

02711CL3 
100, 400 or 800 mg/kg/day under a constant dosage-volume of 2.5 ml/kg 
bw/day (Correspond to 50, 200, or 400 mg/kg bw/day Tinosorb MBBT 
(active ingredient) 
Daily by cutaneous application for at least 104 weeks. 
Base ointment, hydrated, hydrophilic  
No treatment group and one placebo group (Containing all the substances 
of the formulation without the active component MBBT) 
None 
In compliance  
2003 -2006. 

Method 
Groups  of  50  male  and  50  female  Wistar  Han  rats  (7  weeks  old)  received  the  test  item,  FAT 
75'634/B,  daily  by  cutaneous  application  at  the  dose-level  of  100,  400  or  800  mg/kg  bw/day 
(corresponding to 50, 200 or 400 mg/kg bw/day of the active ingredient Tinosorb MBBT (FAT 
75'714))  for  at  least  104  weeks.  The  quantity  of  dosage  form  applied  to  each  animal  was 
adjusted  according  to  the  most  recently  recorded  body  weight.  A  constant  dosage-volume  of 
2.5  ml/kg  bw/day  was  used.  An  additional  group  of  50  males  and  50  females  received  the 
placebo  diluted  in  the  vehicle  under  the  same  experimental  conditions  and  acted  as  negative 
control group. A further group, also of 50 males and 50 females, was not treated and acted as 
an untreated group.  

The  animals  were  checked  daily  for  mortality,  clinical  signs  and  possible  signs  of  irritation.  A 
detailed clinical examination was performed weekly. The animals were palpated every 2 weeks 
after  6  months  of  treatment  in  order  to  monitor  the  onset  and  progression  of  any  palpable 
masses. Body weight and food consumption were recorded at designed intervals (weekly during 
the first 13 weeks of treatment and then monthly). 

Results 
The  study  authors  stated  that  there  were  no  test  item-related  effects  on  survival  rate  during 
the study. The body weight changes and food consumption were unaffected by treatment with 
the test item at any dose-level. 

Scabs were seen at the application site at a higher incidence and severity in males treated at 
100  and  400  mg/kg  bw/day  and  in  both  males  and  females  given  800  mg/kg  bw/day  with 
dose-relationship. 

The  clinical  signs  observed  mainly  occurred  late  during  the  treatment  period  and  were 
associated with the age and general condition of the animals. The incidence, nature and time of 
onset  of  the  clinical  signs  seen  in  treated  group  animals  were  similar  to  those  observed  in 
control animals. 

The frequency of palpable masses (0 to 12% in males; 22 to 38% in females) and their time of 
onset  (weeks  27  to  104)  were  similar  in  control  and  treated  groups.  For  females,  a  greater 
number of dose-related palpable masses were observed. Since no treatment-related neoplastic 
findings were noted, these incidences were considered as fortuitous. 

There were no hematological or blood biochemical differences which could be attributed to the 
treatment with the test item. 

42 

 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

The  test  item  was  not  detectable  in  vehicle  control  animals.  In  test-treated  groups,  the  test 
item was detectable in a few occasions without sex- or dose-relationship and at minimal levels 
(6.0  to  47.9  ng/ml).  Consequently,  it  was  considered  that  the  systemic  exposure  via  the 
cutaneous route was minimal. 

No  treatment-related  macroscopic  changes  were  noted.  The  incidence  and  distribution  of  the 
neoplastic  and  non-neoplastic  findings  corresponded  to  the  usual  background  pathology 
observed  in  aging  Wistar  rats  kept  under  laboratory  conditions  and  did  not  suggest  any 
relationship to treatment. 

Conclusion 
Under  the  conditions  of  this  study,  the  formulated  micronised  UV  filter  Tinosorb® M  was  not 
carcinogenic when applied by the dermal route at 100, 400 or 800 mg/kg bw/day. Accordingly, 
the UV-absorbing test item ingredient MBBT in its micronised form did not reveal carcinogenic 
properties at 50, 200 or 400 mg/kg bw/day.  

Ref.: CIT (2006b) 

SCCS comment 
An  increased  frequency  of  malignant  tumours  with  metastasis  was  found  in  the  low  dose  and 
high  dose  male  rats,  but  not  in  the  middle  dose  or  in  the  female  rats  in  a  two-year  dermal 
painting study with FAT 75’634/B; microfine material (d(0.5) = 128 nm) containing 50% MBBT. 
However,  little  information  is  available  in  relation  to  the  response  of  carcinogenic  substances 
after dermal application of Wistar rats in the performing laboratory and no positive control are 
available.  Moreover,  due  to  the  abnormal  dose  response  results  and  effects  observed  only  in 
males it is difficult to draw any conclusion from the study with regard to potential carcinogenic 
effects. 
The SCCS considers the study of no value.  

3.3.8.  Reproductive toxicity 

No studies provided with nanosized material. 
For studies with MBBT: see Annex II 

3.3.8.1.  Reproduction toxicity 

No studies provided with nanosized material. 
For studies with MBBT: see Annex II 

3.3.8.2.  Teratogenicity 

No studies provided with nanosized material. 
For studies with MBBT: see Annex II 

3.3.9.  Toxicokinetics and metabolism 

Title: 

Methylene-bis-benzotriazolyl 
(CGF-C2089): 
Metabolic fate following oral administration or  in vivo dermal application in 
the rat 

tetramethylbutyl 

phenol 

43 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Guideline: 

OECD 417  

GLP: 

In compliance 

Test item: 

Methylene-bis-benzotriazolyl tetramethylbutyl phenol (CGF-C2089)), non-
micronized material 

Batch/lot n°: 

001719BO 

Reference: 

CTL (2002a) 

Methods 

For assessment of the metabolic fate of the test item CGF-C2089 (purity: 99.6 %), i.e. of 
the UV-absorbing ingredient MBBT in micronised form, 4 Wistar derived rats [Alpk:APrSD] 
per sex were given a single oral dose of 50 mg/kg bw [14C]-MBBT (radiochemical purity: 
99.1 %). The excretion of radioactivity in urine and faeces was monitored via metabolism 
cages  for  3  days  after  dosing.  After  this  period,  the  rats  were  killed  and  residual 
radioactivity  was  measured  in  blood,  selected  tissues  and  the  remaining  carcasses.  An 
additional group of 9 rats per sex received a single oral dose of 50 mg/kg bw [14C]-MBBT 
and  radioactivity  was  measured  in  blood  and  plasma  over  a  24-hour  time  course  after 
dosing. 

Radioactivity  in  the  samples  was  determined  by  liquid  scintillation  counting.  Analysis  of 
metabolites was performed by HPLC-MS (Ion trap mass spectrometer). 

The  dose  formulations  comprised  unlabelled  and  radiolabelled  test  item  suspended  in 
0.5% (w/v) CMC in 0.1% (w/v) aqueous Tween 80. Dose formulations were analysed for 
radioactivity  content  by  liquid  scintillation  counting.  The  particle  size  of  the  milled  test 
item was determined by scanning electron microscopy (SEM) to be in the range of 300 to 
2000 nm. 

Results 

Following  a  single  oral  dose  of  50 mg/kg  bw  [14C]-MBBT (50.4 mg/kg bw based on analytical 
results),  urinary  excretion  accounted  for  a  mean  total  of  <0.01%  of  the  dose  for  both 
males  and  females,  and  faecal  excretion  accounted  for  mean  totals  of  96  and  97%  for 
males  and  females,  respectively.  Only  one  component,  identified  as  the  parent  MBBT, was 
found  in  the  faecal  extracts.  Residues  in  tissues  were  very  low  (<0.01  %  of  the  dose).  The 
radioactivity  remaining  in  the  residual  carcass  accounted  for  <0.07  %  of  the  dose  for  males 
and  <0.08  %  for  females.  The  concentration  of  radioactivity  in  blood  and  plasma  was  below 
the  limit  of  detection  (LOD  <0.035  µg/g  in  blood;  LOD  <  0.016  µg/g  in  plasma)  at  all  time 
points  up  to  24  hours  after  dosing  and  the  area  under  the  curve  (AUC)  could  thus  not  be 
calculated. Cage washes amounted to <0.01 and <0.02 for male and female rats respectively. 

Conclusion 

The  study  indicated  negligible  systemic  availability  of  the  UV-absorbing  ingredient  MBBT  in 
micronised  form  after  oral  administration.  MBBT  was  quantitatively  and  rapidly  excreted  as 
parent compound  via the faeces. 

44 

 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

3.3.10. Photo-induced toxicity 

3.3.10.1.  Phototoxicity / photoirritation and photosensitisation 

No studies provided with nanosized material. 
For studies with MBBT: see Annex II 

3.3.10.2.  Phototoxicity / photomutagenicity / photoclastogenicity 

For studies with MBBT: see Annex II 

Photo-co-carcinogenicity (microfine material) 

Title: 

Guideline: 
GLP: 
Test item: 

Batch/lot n°: 

Thirteen-week topical range-finding study of Tinosorb® M, Tinosorb® MBBT 
and Tinosorb® S5 in hairless mice, with or without simulated sunlight 
/ 
In compliance 
Tinosorb® M; microfine material; d(0.5) = 127 nm or 115 nm 
Tinosorb® MBBT (non-micronised) 
05122CL2M, 00696CL3 (Tinosorb® M) 
02347CN2, 15420CL2 (Tinosorb® MBBT) 

Methods 
Twenty-two groups of 6 albino hairless mice [Crl:SKH1-hrBR] per sex were assigned to groups 
receiving no treatment, treatments of only UV irradiation at 2 levels (600 and 1200 RBU/week), 
vehicle (base ointment) without or with UV irradiation at 2 levels (600 and 1200 RBU/week), or 
the UV absorbing ingredient MBBT or the formulated micronised UV filter  Tinosorb® M in base 
ointment both without or with UV irradiation at 1 level (1200 RBU/week). The treatments with 
vehicle  and  test  items  were  administered  topically  (100 µL/mouse)  once  daily  and  then  the 
appropriate  groups  of  mice  were  irradiated  once  daily,  5 days  per  week,  for  13  weeks. 
Formulations  were  applied  to  the  back  and  sides  (approximately  25 cm²)  of  the  mice  before 
daily  UV  irradiation  exposures  on  Monday,  Wednesday  and  Friday  and  after  UV  irradiation 
exposures on Tuesday and Thursday.  

The  test  item  purity  was  99.8 %  for  MBBT  (non-micronised)  and  50.7 %  for  Tinosorb® M. 
(microfine). 

Dosage equivalents of MBBT (considered as 100 % pure) and Tinosorb® M (considered as 50 % 

pure) 

Test item concentration 

Dermal dose level 

(mg/g) 

25 

50 

100 

200 

(%) 

2.5 

5.0 

10.0 

20.0 

(mg/cm²) 

(mg/kg bw/d) 

0.1 

0.2 

0.4 

0.8 

80 

160 

325 

650 

5 Information on Tinosorb S is not provided in the present document. 

45 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                          
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

The lower UV irradiation level was 600 RBU (Robertson-Berger units) per week and the higher 
UV irradiation level was 1200 RBU per week6.  

All  mice  were  observed  for  viability  and  clinical  signs.  Each  mouse  was  graded  for  skin 
reactions  on  a  weekly  basis.  Skinfold  thickness  was  measured  periodically  at  three  sites  per 
mouse.  Body  weights  were  recorded  weekly.  At  the  end  of  the  treatment  period  all  study 
animals  were  sacrificed  and  examined  macroscopically.  Dose  formulations  used  in  this  study 
were  analysed  by  HPLC  with  UV  detection  in  order  to  verify  adequate  concentration, 
homogeneity and stability. 

Results 
In male and female mice no adverse cutaneous or other clinical responses occurred that were 
related  to  administration  of  any  of  the  formulations  of  the  test  items  MBBT  or  Tinosorb® M. 
Erythema was reduced in some groups of male mice administered test article formulations and 
exposed  to  UV  irradiation,  as  compared  with  the  groups  administered  the  vehicle  formulation 
and  exposed  to  UV  irradiation.  A  similar  pattern  of  findings  of  mean  skinfold  thickness  also 
occurred,  and  significant  reductions  in  mean  skinfold  thickness  occurred  in  some  groups  of 
mice administered the test item formulations and exposed to UV irradiation, as compared with 
the groups only administered the vehicle formulation and exposed to UV irradiation. 

The  few  incidences  of  mortality  (1 male  in  the  vehicle  control  group  and  1 male  treated  with 
MBBT at 50 mg/g and 1200 RBU/week) observed during the course of the study were unrelated 
to  the  treatment  with  the  test  items  MBBT  and  Tinosorb® M.  There  were  no  test  item-related 
effects  on  body  weights.  Chemical  analyses  confirmed  acceptable  concentration,  homogeneity 
and stability of MBBT in the vehicle. 

the  given  experimental  conditions,  clinical  observations 

Conclusion 
including  cutaneous 
Under 
observations,  skinfold  thickness  and  body  weight  did  not  preclude  the  use  of  any  of  the 
concentrations  of  the  UV-absorbing  ingredient  MBBT  or  the  formulated  micronised  UV  filter 
Tinosorb® M (2.5 to 20 %) in a long-term study. Adverse effects of these formulations did not 
occur on observed parameters over those seen in the groups of mice administered the vehicle 
formulation and either not exposed or exposed to UVR. 

Ref.: CR-DDS (2003) 

Title: 

Reference: 
Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

Twelve-month  topical  study  to  determine  the  influence  of  Tinosorb® M 
(FAT 75’634) on photocarcinogenesis in hairless mice 
Study report number 203-007 
/ 
In compliance 
Tinosorb® M (FAT 75’634); microfine material; d(0.5) = 115 nm 
00696CL3 

Methods 
The  potential  of  the  formulated  micronised  UV  filter  Tinosorb® M,  containing  50 %  of  the 
UV-absorbing  ingredient  MBBT,  to  influence  the  development  or  growth  of  skin  tumours  in 
hairless  mice  [Crl:SKH1-hrBR]  exposed  to  UV  irradiation  was  determined.  A  total  of 
5 experimental  groups  with  each  36 mice  per  sex  were  treated  on  5 days/week  for 

6   The  RBU  (Robertson-Berger  unit)  is  a  measure  of  biological  effectiveness  of  UV  irradiation.  A  dose  of  400 RBU 

approximates one minimal erythemal dose in previously untanned human skin. 

46 

 
 
 
 
 
 
 
 
                                          
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

40 consecutive  weeks.  The  experimental  design  included  2 calibration  groups  receiving  only 
UV irradiation at 600 or 1200 RBU (Robertson-Berger units) per week, 1 vehicle control group 
receiving  base  ointment  and  600 RBU/week  and  2 groups  receiving  MBBT  (applied  as 
Tinosorb® M) in base ointment at 50 or 200 mg/g and 1200 RBU/week. The dosage equivalents 
are shown below: 

SCCS/1546/15 

Dosage equivalents of Tinosorb® M 

Test item concentration 

Dermal dose level 

(mg/g) 

50 

200 

(%) 

5 

20 

(mg/cm²) 

(mg/kg bw/d) 

0.2 

0.8 

160 

650 

Formulations  (100 µL/mouse)  were  applied  topically  to  the  back  and  sides  (approximately 
25 cm²)  of the  mice  before  daily  UV irradiation  exposures  on  Monday,  Wednesday  and  Friday 
and 1 hour after UV irradiation exposures on Tuesday and Thursday. 

The  study  design  specified  termination  12 weeks  after  the  last  UV irradiation  dose,  or  when 
animal  tumour  burden  criteria  required  an  earlier  termination  of  a  study  group.  The  study 
determined  the  increase  in  UV irradiation-induced  skin  tumours,  assessed  as  the  prevalence 
and the time to onset of tumours. The animals were observed for external changes in skin and 
their  tumours  charted,  measured,  and  tabulated  weekly.  Body  weights  were  recorded  on  a 
weekly basis. A record of gross changes at necropsy, and tissue or lesion histopathology were 
not conducted for this study. 

Results 
Non-tumour endpoints 

There  were  no  test  item-related  adverse  effects  on  survival,  clinical  signs  including  local  skin 
reactions, body weights and necropsy observations. A protective effect of  Tinosorb® M and its 
UV-absorbing  ingredient  MBBT  was  indicated  by  a  reduced  mortality  in  the  exposed  groups 
compared to the high UV irradiation control group (1200 RBU/week). 

Skin reactions occurred in both male and female mice and included erythema, oedema, flaking, 
thickening,  wrinkling,  residue,  scab(s)  and  white  raised  area(s).  None  of  these  findings  were 
considered adverse responses to administration of the Tinosorb® M formulations. In male mice, 
the incidence and severity of skin reactions were comparable in all mice exposed to the high UV 
irradiation dose (mice administered the 50 or 200 mg/g formulation and the high UV irradiation 
calibration  group)  except  that  the  occurrences  of  flaking  grade  2  (distinct  scales)  were 
increased  in  mice  administered  the  Tinosorb® M  formulations,  as  compared  with  the  UV 
irradiation calibration group. In female mice, the incidence and severity of skin reactions were 
comparable  in  all  groups  of  mice  exposed  to  the  high  UV  irradiation  dose  except  that  the 
occurrences  of  oedema  grade 1,  flaking  grade  1  and  flaking  grade  2  were  increased  in  mice 
administered  the  50 mg/g  Tinosorb® M  formulation,  as  compared  with  the  vehicle  formulation 
group. 

Time to tumour 

A  dose-dependent  protective  effect  of  Tinosorb® M  on  the  time  for  development  of  the  first 
tumour (>1 mm) was detected. Unbiased median time data are summarised below: 

Unbiased median week to tumours with a size of ≥ 1 mm 

47 

 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Tinosorb®

 M (mg/g) 

UV (RBU/week) 

Vehicle 

600 

50 

1200 

Time to tumour (weeks) 

Males 

Females 

Sexes combined 

34.5 

33.0 

33.5 

26.0 

27.0 

26.0 

200 

1200 

28.0 

28.0 

28.0 

n.a. 

600 

37.0 

35.5 

37.0 

n.a 

1200 

22.5 

22.5 

22.5 

n.a.:  

not applicable, no treatment with test item or vehicle, exposure to UV irradiation only. 

For the onsets of tumours with a size above 1 mm in comparison with the high UV irradiation 
calibration  group,  administration  of  50  or  200 mg/g  Tinosorb® M  delayed  the  development  of 
skin tumours. 

Sacrifice of groups 
Sacrifice  of  groups,  based  on  tumour  burden  criteria,  occurred  in  week 36  for  the 
1200 RBU/week calibration group, in week 40 for the Tinosorb® M low dose group, in week 45 
for the Tinosorb® M high dose, in week 49 for the vehicle control group and in week 52 for the 
600 RBU/week calibration group. 

Tumour yield 
The  average  number  of  tumours  per  surviving  mouse  (tumour  yield)  was  highest  in  the 
1200 RBU/week  calibration  group  followed  by  the  vehicle  control  group,  the  low  dose  group 
receiving  Tinosorb® M  at  50 mg/g  and  1200 RBU/week,  the  600 RBU/week  calibration  group, 
and the high dose group receiving Tinosorb® M at 200 mg/g and 1200 RBU/week. The vehicle 
enhanced the tumour yield compared to the low UV irradiation calibration group. Furthermore, 
Tinosorb® M decreased the tumour yield compared to the high UV irradiation calibration group 
(1200 RBU/week).  

Tumour yield of tumours with a size of ≥ 1 mm 

Tinosorb®

 M (mg/g) 

Vehicle 

UV (RBU/week) 

Treatment week (#) 

600 

49 

50 

1200 

36 

Average number of tumours per surviving mouse 

Males 

Females 

Sexes combined 

5.59 

5.89 

5.74 

5.12 

4.42 

4.73 

200 

1200 

36 

3.47 

2.91 

3.19 

n.a.a 

600 

49 

4.48 

4.00 

4.26 

n.aa 

1200 

36 

6.06 

6.32 

6.20 

a:  n.a.; not applicable, no treatment with test item or vehicle, exposure to UV irradiation only. 

Group comparisons of tumour onset (Peto analysis) 
The  comparison  of  tumour  relative  risk  by  Peto  analysis  revealed  that  50  or  200 mg/g 
Tinosorb® M  in  base  ointment  significantly  delayed  (p < 0.001)  the  development  of  skin 
tumours in both sexes combined and in separate groups of male and female mice as compared 

48 

 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

to the 1200 RBU calibration group. Additionally, the dose-related effectiveness of Tinosorb® M 
was seen in the significantly delayed (p < 0.01 or p < 0.001) skin tumour onset for 200 mg/g 
as compared with the 50 mg/g formulation. 

Tumour potency factors (TPFs) 
The estimated tumour potency factors (TPFs) are summarised below. The TPF results show that 
the  vehicle  slightly  enhanced  a  photocarcinogenic  response,  that  MBBT / Tinosorb® M 
formulations  effected  a  dose-related  reduction  of  TFP  compared  to  the  1200 RBU  calibration 
group, that dermal treatment with the test item did not adversely affect the photocarcinogenic 
response  and  reduced  the  potential  of  the  high  UV  irradiation  dose  to  induce  tumours  in  a 
dose-dependent manner. 

Tumour potency factors (TPFs) for tumours with a size of ≥ 1 mm 

MBBT (mg/g) 

Vehicle 

UV (RBU/week) 

600 

TPFs 

Males 

Females 

Sexes combined 

1.10 

1.12 

1.14 

50 

1200 

1.63 

1.52 

1.64 

200 

1200 

1.47 

1.43 

1.47 

n.a. 

600 

1 

1 

1 

n.a 

1200 

2 

2 

2 

Conclusion 
MBBT  applied  as  Tinosorb® M  (microfine  material)  in  base  ointment  formulations  showed  a 
dose-dependent reduction in UV irradiation-induced skin tumour development compared to UV 
irradiation  alone  as  indicated  by  the  increased  time  to  first  tumour,  delayed  mortality,  lower 
tumour  yield  and  reduced  tumour  potency  factor.  Based  on  these  results,  the  UV-absorbing 
ingredient MBBT applied in the formulated micronised UV filter Tinosorb® M shows a protective 
effect for photo-co-carcinogenesis. 

Ref.: CR-DDS (2005e) 

3.3.11. Human data 

3.3.11.1.  Human skin distribution and penetration in vitro 

3.3.11.2.  Phototoxicity and photoallergenicity in vivo 

Title: 
Reference: 
Guideline: 
GCP: 
Test item: 
Batch/lot n°: 

Evaluation of phototoxicity in humans 
HTR project number 100239A 
/ 
In compliance 
CGF-C 2089 (microfine material) 
Not indicated 

Methods 
A total of 30 subjects were enrolled for the in vivo phototoxicity patch test on humans, of which 
28 subjects (4 males, 24 females) completed all study phases.  

49 

 
 
 
 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

The test item CGF-C 2089 (5 %, purity not indicated), i.e. the UV-absorbing ingredient MBBT in 
micronised  form,  formulated  as  micronised  UV  filter  Tinosorb® M  (10 %)  in  white  cream  base 
common to cosmetic lotions was topically applied to 28 human volunteers. Each 200 µL of the 
test item, vehicle control (white cream base with placebo), and saline were topically applied to 
separate sites on each volunteer on one side of the spine. Duplicate applications were made on 
the  opposite  side  of  the  spine.  The  treatment  sites  were  covered  with  an  occlusive  dressing. 
After 24 hours of exposure, the patches and excess test material from the left paraspinal region 
were  removed.  The  test  sites  were  then  exposed  to  16 J/cm²  UVA  irradiation  followed  by 
exposure to 0.75 times the volunteer’s MED (minimal erythemal dose) of UVB irradiation. The 
patches  from  the  non-irradiated  test  sites  on  the  right  paraspinal  region  were  then  removed 
and served as controls to assess non-phototoxic reactions. Skin reactions were assessed 1, 24, 
48, and 72 hours following irradiation and patch removal. 

Results 
The irradiated test item-treated sites exhibited lower skin reactions than the irradiated vehicle 
control and saline treatment sites. Only very few dermal responses with a score of grade 1 or 
higher  were  found  during  the  study,  leading  to  the  conclusion  that  the  test  articles  are 
non-irritating. 

Conclusion 
Under  the  test  conditions  put  forth  in  this  in  vivo  study,  the  UV-absorbing  ingredient  MBBT 
(CGF-C 2089;  5%)  formulated  as  micronised  UV  filter  in  Tinosorb® M  (10 %)  did  not  show 
evidence  of  eliciting  a  phototoxic  response  in  human  subjects.  MBBT  in  micronised  form  was 
not an irritant to human skin. 

Ref.: Hill Top Research (1998a) 

Title: 
Reference: 
Guideline: 
GCP: 
Test item: 
Batch/lot n°: 

Evaluation of human photoallergy by repeated insult patch test 
HTR Project No. 100239B 
/ 
In compliance 
CGF-C 2089 (microfine material) 
Not indicated 

Methods 
A  total  of  35 subjects  were  enrolled  for  the  in  vivo  human  repeated  insult  patch  test  (HRIPT) 
adapted according to Kaidbey and Kligman (1980), of which 26 subjects (3 males, 23 females) 
completed all study phases.  

Induction period (weeks 1 – 3) 

For  induction,  200 µL  of  the  test  item  CGF-C 2089  (5  %,  purity  not  indicated),  i.e.  the 
UV-absorbing  ingredient  MBBT  in  micronised  form,  formulated  as  micronised  UV  filter 
Tinosorb® M (10 %) in white cream base, vehicle control (white cream base with placebo) and 
saline  were  applied  six  times  (twice  per  week)  to  separate  skin  application  sites  of  the 
volunteers. The application sites were covered with an occlusive dressing for 24 hours. Within 
10 minutes  after  removal  of  the  patches,  the  application  sites  were  exposed  to  UVB/UVA 
irradiation at twice the MED (minimal erythemal dose).  

Challenge period (week 6) 

After a rest period (weeks 4 and 5), a challenge experiment was carried out. Duplicate topical 
applications of 200 µL of the test item, vehicle control, and saline were made to naive sites on 
both  sides  of  each  volunteer’s  spine.  The  test  sites  were  covered  with  an  occlusive  dressing. 

50 

 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

After  24 hours  of  exposure,  the  patches  and  excess  test  material  from  one  side  of  the  spine 
were  removed.  The  test  sites  were  then  exposed  to  16 J/cm²  UVA  irradiation  followed  by 
exposure to 0.75 times the volunteer’s MED of UVB irradiation. 

Skin  reactions  were  assessed  1,  24,  48  and  72 hours  following  irradiation  and  patch  removal 
using erythema scores from 0 (no visible reaction) to 3 (strong reaction). 

Results 
The irradiated test item–treated sites exhibited lower skin reactions than the irradiated vehicle 
control and saline treatment sites. 

Conclusion 
Under the test conditions, neither the UV-absorbing ingredient MBBT (5 %) nor the formulated 
micronised UV filter Tinosorb® M (10 %) were photosensitisers or sensitisers to human skin. 

Ref.: Hill Top Research (1998b) 

3.3.11.3.  Human patch test 

Title: 
Reference: 
Guideline: 
GCP: 
Test item: 
Batch/lot n°: 

Human patch tests of Tinosorb® M (substance) 
/ 
/ 
/ 
Tinosorb® M (20% aqueous solution), microfine material 
000620B1 

Methods 
A total of 40 subjects (13 males and 27 females) were enrolled for an in vivo human patch test. 
0.015 mL  of  a  20 %  aqueous  solution  of  the  formulated  micronised  UV  filter  Tinosorb® M 
(controls: purified water and 20 % Tinosorb® M with water instead of MBBT) was used in 8-mm 
Finchamber for 24 hours occlusively. Skin reactions were observed 1 and 24 hours after patch 
removal and scored and evaluated according to Japanese Criteria7. 

Results 
Slight erythema were observed in 3 subjects and evident erythema in 1 subject at 1 hour after 
patch  removal,  but  the  reaction  disappeared  within  1,  2  and  4 days  after  patch  removal.  For 
the  control  treatments,  slight  erythema  were  observed  in  5 subjects  and  evident  erythema  in 
1 subject. All skin reactions were fully reversible at the dose and control patch sites. 

The calculated skin stimulation index of Tinosorb® M was 6.25. The indices for the base solution 
and purified water were 6.25 and 8.75, respectively. 

Conclusion 
Under  the  conditions  of  this  study  and  considering  the  underlying  evaluation  scale,  the 
formulated  micronised  UV  filter  Tinosorb® M  in  water  showed  no  potency  of  causing  reactions 
on human skin. 

Ref.: Kawai Institute of Dermatology (2001a) 

7   Kawamura et al. (1970) Nichihi-kaishi 80 No.5, 301; Nasu (1985). Hifu (skin) 27 No.4, 793-803 

51 

 
 
 
 
 
 
 
 
 
 
 
 
 
                                          
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

SCCS comment 
Neither guidelines nor GLP have been reported. These tests can serve as supporting evidence, 
however they are of limited valued. 

Title: 
Reference: 
Guideline: 
GCP: 
Test item: 
Batch/lot n°: 

Human patch tests of Tinosorb® M containing sunscreen (product) 
/ 
/ 
/ 
20 % Tinosorb® M containing sunscreen, microfine material 
/ 

Methods 
A total of 40 subjects (13 males and 27 females) were enrolled for an in vivo human patch test. 
0.015 mL  of  a  sunscreen  formulation  containing  20 %  of  the  formulated  micronised  UV  filter 
Tinosorb® M  (control:  Tinosorb® M-free  sunscreen  formulation)  was  used  in  8-mm  Finn 
Chamber  for  24 hours  occlusively.  Skin  reactions  were  observed  1  and  24 hours  after  patch 
removal and scored and evaluated according to Japanese Criteria7. 

Results 
Slight  erythema  was  observed  in  1 subject  at  1 hour  after  patch  removal,  but  the  reaction 
disappeared  within  24 hours  after  patch  removal.  For  the  control  treatments,  slight  erythema 
was  observed  in  2 subjects.  The  skin  reactions  were  fully  reversible  at  the  dose  and  control 
patch sites. 

The calculated skin stimulation index of the test item formulation was 1.25. The index for the 
control sunscreen formulation was 2.5. 

Conclusion 
Under  the  conditions  of  this  study  and  considering  the  underlying  evaluation  scale,  the 
formulated  micronised  UV  filter  Tinosorb® M  in  sunscreen  showed  no  potency  of  causing 
reactions on human skin. 

Ref.: Kawai Institute of Dermatology (2001b) 

3.3.12. Special investigations 

No data submitted 

3.3.13. Safety evaluation (including calculation of the MoS) 

Margin of Safety (MoS) as provided in the dossier 

While microfine MBBT (d(0.5) = 95 nm) is not considered to be absorbed across human skin or 
absorbed systemically to a relevant extent, the calculation of a Margin of Safety (MoS) for the 
dermal route of exposure is provided in the table below. Taking the standardised approach to 
risk characterisation, the systemic estimated human exposure that could be assumed to result 
from 10 % microfine MBBT in cosmetic products applied  to normal skin is used. The resulting 
systemic exposure dose in humans (SED Human) is then compared with the systemic exposure 
dose  in  rats  (SED  Rat)  estimated  from  the  subchronic  dermal  repeated  dose  toxicity  study  in 

52 

 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

rats  using  the  in  vivo  NOAEL  and  the  dermal  absorption  rate  determined  in  the  in  vitro 
penetration study with rat skin. 

SCCS/1546/15 

MoS calculations for microfine MBBT based on human skin in vitro study results on normal skin, 

as provided by the applicant. 

Parameter 

Adult body weight 

Body surface area 

Sunscreen applied (if at 1 mg/cm²) 

Microfine MBBT applied (10 %) 

Microfine MBBT d(0.5) = 95 nm 

60 kg 

17500 cm² 

18 g 

1800 mg 

Skin absorption (human), (RCC Ltd., 2007)a 

0.048 % of applied dose 

SED Human 

NOAEL 
(Rat 13-week dermal study; RCC Ltd., 2002)b 

0.0144 mg/kg bw/d 

1000 mg/kg bw/d 

Skin absorption (rat), (RCC Ltd., 2007)a 

5.922 % of applied dose 

SED Rat 

MoS (SED Rat / SED Human) 

SED: 
Systemic exposure dose 
NOAEL:  No-observed-adverse-effect-level 
MoS:  Margin of safety 
a: 
b: 

2 SD were added to the mean absorption value  
in this study MBBT was not in nanosized form. 

59.2 mg/kg bw/d 

4112 

Based  on  the  calculations  presented  above,  the  MoS for MBBT  with a d(0.5) = 95 nm  is  4112 
based on a comparison of the internal dose between rat and man. 

It  should  be  noted  that  the  above  calculations  are  highly  conservative  with  regard  to  the 
human  skin  absorption  value  used.  Dermal  absorption  values  were  mainly  below  the  limit  of 
quantification  and  the  majority  of  the  dose  was  recovered  from  the  skin  compartment,  i.e. 
stratum corneum, rather than the receptor fluid. 

SCCS comment  
The  MOS  calculation  is  based  on  read-across:  no  repeated  dose  toxicity  study  with  the  nano-
sized material is available in rats.  
A 39-week dermal toxicity study is available  that was conducted on mini-pigs  and carried out 
with  the  nano  MBBT,  from  which  it  can  be  concluded  that  no  effects  are  observed  until  the 
highest dose level tested (1000 mg a.i./kgbw/day). Since there are no dermal penetration data 
available  for  mini-pig  skin,  a  MoS  cannot  be  calculated.  However,  in  a  weight  of  evidence 
approach, it can be concluded that based on the low absorption in humans (dermal penetration 
values derived are conservative because of the low levels), and the absence of effects in a 39-
week  dermal  study  in  the  mini-pig  up  to  the  highest  dose  tested  (1000mg  a.i./kg  bw/day), 
there  seems  to  be  no  concern  for  the  dermal  application  of  nano-sized  MBBT  (up  to  10%  in 
sunscreen formulations) with regard to systemic effects. 

53 

 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

3.3.14. Discussion 

It should be noted that the risk assessment for S79 is based on mass-dose metrics, and not on 
particle  number  or  surface  area.  At  present,  the  most  adequate  dose  metric  for  hazard  and 
exposure  characterisation  for  nanoparticles  is  still  under  discussion.  However,  as  cosmetic 
ingredients  are  usually  assessed  and  regulated  based  on  the  concentration  in  the  finished 
product, this dose metric is maintained in this Opinion. 

Physicochemical properties 
This  assessment  only  refers  to  the  materials  described  in  the  present  Opinion,  including  the 
specific  preparation  procedure  reported  for  the  nano-sized  material.   MBBT  'as  manufactured' 
has been characterised by a considerable number of physicochemical techniques.  The solubility 
of  MBBT,  an  organic  UV absorber  in  water  is  very  low,  while  there  is  a  considerable  solubility 
reported in the lipid phase. The manufacturing of nano-MBBT by ball milling leads to a pre-final 
solid-in-liquid emulsion which has been characterised for the size distribution by Dynamic Light 
Scattering  (DLS),  Fiber-Optic  Quasi-Elastic  Light  Scattering  (FOQELS),  CLS  disc  centrifugation 
and  Transmission  Electron  Microscopy  (TEM).  Despite  this,  a  weakness  of  the  present 
submission is that the data raise questions in regard to the potential cut-off in the study being 
at  particle  sizes  of  less  than  50  nm.   Evidence  has  been  provided  that  the  radioactive  batch 
needed  for  toxicological  studies  shows  the  same  physicochemical  characteristics  as  the  non-
radioactive  material.  This  type  of  nano-MBBT  has  been  used  in  the  consequent  toxicological 
studies, and has been documented to be constant in its batch to batch variation on the basis of 
two  datasets  taken  for  materials  manufactured  according  to  the  same  recipes  some  years 
apart. 

Irritation /sensitisation 
In a BCOP study, nanosized MBBT (in the Tinosorb formulation, pH 11.6 and pH 10.5; particle 
size not reported in study report, but according to the applicant (d(0.5) = 110-130nm) did not 
cause serious eye damage. 
In an eye irritation study in rabbits, the formulated micronised UV filter Tinosorb® M pH = 11.6 
was slightly irritating to the eye. 
No  skin  irritation  study  was  performed  with  nanosized  MBBT.  Non-nano-sized  MBBT  was  not 
irritating  in  the  rabbit  skin  irritation  study.  In  a  14-day  cumulative  primary  irritation  study  in 
guinea  pigs,  MBBT  in  non-nano-sized  form  was  not  a  skin  irritant  at  concentrations  of  15  or 
40%.  MBBT  was  considered  to  be  slightly  irritant  at  65%  with  a  Primary  Irritation  Index  of 
0.04. 
No sensitisation study was carried out using nano-sized MBBT. 
In  a  maximisation  study  in  guinea  pigs,  none  of  the  20  animals  exposed  to  non-nanosized 
MBBT  showed  a  dermal  reaction  after  the  challenge.  A  few  case  reports  indicate  possible 
allergic  reactions  to  MBBT  (Gonzalez-Perez,Contact  DERM  2007);  however,  it  was  also 
suggested that this might be due to the composition of the preparation with decyl  glucoside as 
allergen (ref Andrade Contact Dermatitis 2010; O Connell et al Contact Dermatitis 2011). 

Dermal absorption 
Skin  penetration  of  nano-sized  MBBT  was  determined  in  vitro  in  healthy  rat  skin,  in  healthy 
human  skin,  and  in  artificially  damaged  human  skin.  Each  of  the  studies  was  not  fully  in  line 
with  the  SCCS’  basic  criteria  for  dermal  penetration  (with  respect  to  donor  numbers  and  the 
replicates  used).  Therefore  2  standard  deviations  were  added  to  the  Means  of  the 

54 

 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

experimentally  determined  absorption  values  to  account  for  the  uncertainties.  In  the  studies 
using  human  intact  and  damaged  skin,  the  amounts  that  penetrated  through  the  skin  were 
below LOQ for many of the replicates analysed pointing to extremely low penetration. 

By adding 2 standard deviations to the Mean penetration values to account for the uncertainty, 
the percentage of the amount applied that penetrated through human intact skin was 0.048 %. 
In damaged human skin, the absorption percentage was 0.35 % of the applied amount.  

In intact rat skin on the other hand, the Mean amount penetrating through the skin was 5.922 
%  of  the  applied  amount.  This  higher  value  was  used  in  MOS  calculation  as  the  worst  case. 
Thus,  the  SCCS  considers  that  the  conservative  approach  used  also  covered  penetration  of 
nano-sized MBBT through the artificially damaged human skin. 

Following  a  6-hour  topical  exposure,  the  in  vivo  dermal  absorption  of  the  non-nanosized 
UV-absorbing ingredient MBBT from a 0.2 and a 10 % formulation was very low and accounted 
for  less  than  0.8 %  and  0.4 %  of  the  dose,  respectively,  over  5 days.  No  systemically 
measurable concentrations were detected. 

Inhalation 
The  submitted  data  on  exposure  to  spray  applications  is  not  sufficient.  Appropriate  data  are 
needed  on  the  physics  of  the  spray  generation  and  the  atmospheric  conditions  during  use 
(including behaviour of the aerosols in time). 
In  an  acute  inhalation  study,  the  massive  influx  of  neutrophils  and  the  decrease  of 
macrophages  cannot  be  considered  to  be  merely  a  mild  inflammation  after  inhalation  of 
particles.  
No  information  is  available  on  the  effects  of  S79  after  repeated  exposure  to  lower  doses  via 
inhalation.  From  other  inhalation  studies  using  nano-materials,  it  is  known  that  the  nano-
material  is  not  cleared  from  the  lung  in  the  same  way  as  is  the  case  for  ‘conventional’ 
substances. In addition, the effect of the formulation on the behaviour of the S79 particles in 
the human lung is not known.   
Therefore,  at  the  moment,  it  cannot  be  concluded  that  the  use  of  10%  S79  in  spray 
formulations is safe, because of the uncertainties associated with the repeated exposure of the 
lung  to  low  doses  of  S79  and  the  limited  information  on  actual  exposure  during  use  of  spray 
applications. 
In  view  of  the  noted  effects  in  the  lung,  to  ensure  the  safe  use  in  spray  application  the 
following  information  would  be  needed:  a  28-day  repeated  dose  inhalation  study,  including 
tissue  distribution  and  systemic  toxicity,  determination  of  an  LOAEL  and  NOAEL  and  possible 
recovery. However, due to the Cosmetics regulation animal studies are not allowed anymore. 

Repeated dose toxicity 
In  a  dermal  repeated  dose  range-finding  study  in  rats,  some  clinical  effects  (vocalization  and 
pain) were noted at dose levels of  500 mg a.i./kg bw/day and higher when exposed to nano-
sized  MBBT.  These,  or  other  effects  were  not  seen  in  the  39  week  min-  pig  study,  where  the 
animals were exposed to dose levels up to 1000 mg a.i./kg bw/day.  
No  effects  were  noted  in  rats  in  dermal  or  oral  repeated  dose  toxicity  studies  with  non-
nanosized MBBT.  

55 

 
 
 
 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

Genotoxicity 
The evidence provided in the dossier showed that MBBT in non-micronised form did not induce 
DNA repair in liver cells of treated rats. Also, MBBT in the non-micronised form did not induce 
damage to the chromosomes or the mitotic apparatus of mouse bone marrow cells. However, 
no  evidence  is  provided  to  show  that  the  test  substance  reached  the  target  cells,  and/or  was 
taken up by these cells. These studies therefore do not allow a firm conclusion to be drawn on 
the  potential  genotoxic  hazard  of  the  nanosized  form  of  the  compound  because  it  was  not 
demonstrated whether the compound reached the target organs/cells.  
The  in  vitro  cytokinesis  blocked  micronucleus  assay  and  HPRT  forward  mutation  assay  was 
conducted  on  both  Tinosorb®  M  (not  micronised)  (54.7%  MBBT  in  aqueous  solution)  and 
Tinosorb®  M  pH=  10.5.  No  evidence  of  chromosomal  damage  or  mutagenicity  was  observed 
indicating that there was no evidence of genotoxicity. Although these data are accepted by the 
SCCS, there remains doubt over the negative results as no direct evidence of cell uptake of the 
substance has been provided.  

Toxicokinetics 
Although  dermal  uptake  is  very  low,  as  shown  in  the  skin  penetration  studies,  potential 
bioaccumulation after repeated skin applications should be considered. 

Carcinogenicity 
An  increased  frequency  of  malignant  tumours  with  metastasis  was  found  in  the  low  dose  and 
high dose in male rats, but not in the middle dose or in the female rats in a two year dermal 
painting study with FAT 75’634/B; nanosized MBBT (doses: 50, 200, 500 mg a.i./kg bw/day). 
However,  since  little  information  is  available  in  relation  to  the  response  of  carcinogenic 
substances after dermal application of Wistar rats in the performing laboratory and no positive 
control  is  available,  it  is  difficult  to  draw  any  conclusion  from  the  study  in  regard  to  the 
potential carcinogenic effects.   

4. CONCLUSION 

1. 
In  light  of  the  new  data  provided,  does  the  SCCS  consider  Methylene  bis-benzotriazolyl 
tetramethylbutylphenol (CAS No 103597-45-1) safe when used in cosmetic products in its nano 
forms under the current use conditions up to a maximum of 10%? 

The calculation for margin of safety in this Opinion is based on a 39-week dermal toxicity study 
in the mini-pig, as no repeated dose toxicity study with the nano-sized material is available in 
rats. Also, dermal penetration data are not available for mini-pig skin. The SCCS has based this 
Opinion  on  the  overall  weight  of  evidence  that  suggests  a  very  low  absorption  of  MBBT  in 
human  intact  skin  and  artificially  damaged  skin  (top  skin  layers  removed),  and  the  lack  of 
adverse  effects  in  mini-pigs  up  to  the  highest  dose  tested  (1000mg  a.i./kg  bw/day)  over  39 
weeks.  These  together  indicate  that  dermal  application  of  nano-sized  MBBT  with  regard  to 
systemic  effects  is  not  a  safety  concern.  The  SCCS  has  therefore  concluded  that  the  use  of 
MBBT 
in 
nano-structured  form  with  the  following  characteristics  as  a UV-filter at  a concentration  up  to 
10% in dermally applied cosmetic products is considered to not pose a risk of adverse effects in 
humans after application on healthy, intact skin and also damaged skin.  

[2,2’-methylene-bis-(6(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol)] 

  The material has a purity of ≥98.5% with the isomer faction not exceeding 1.5%, and 

the impurity profile not significantly different from that indicated in section 3.1.5. 

56 

 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

  The  material  has  a  median  particle  size  (d0.5)  of  120  nm  or  larger  in  terms  of  mass 
distribution,  and/or  60  nm  or  larger  in  terms  of  number  size  distribution  (by  laser 
diffraction).  

  The  material  complies  with  other  physicochemical  specifications  of  the  evaluated 
material as listed under section 3.1 of this opinion in terms of chemical identity, physical 
form, chemical composition, solubility, zeta potential, etc. 

  Compared  to  human  intact  skin  or  artificially  damaged  skin  (top  skin  layers removed), 
the Mean amount penetrating through intact rat skin was considerably higher (5.922 % 
of  the  applied  amount  versus  0.048  %  in  intact  human  skin  and  0.35  %  in  artificially 
damaged human skin). The higher value for rat skin was used in MOS calculation as the 
worst  case.  Thus,  the  conservative  approach  used  is  considered  to  also  cover  the 
damaged human skin that is devoid of top layers.  

In  view  of  the  limited  available  information  on  inhalation  toxicity,  which  indicates  severe 
inflammatory  effects  of  microfine  MBBT  in  the  respiratory  tract,  caution  is  warranted  against 
the use of the material in applications that could lead to exposure of the consumer’s lungs by 
inhalation.  This  Opinion  therefore  does  not  apply  to  such  applications  that  might  lead  to 
exposure of the consumer’s lungs to MBBT nanoparticles by inhalation. 

It should also be noted that this Opinion is based on the currently available scientific evidence, 
which shows an overall very low dermal absorption of MBBT in nano- or larger particle forms. If 
any new evidence emerges in the future to show that the nano-form of MBBT used in cosmetic 
products  can  penetrate  skin  in  any  significant  amounts  to  reach  viable  cells,  the  SCCS  may 
consider revising this assessment.  

2.  Does  the  SCCS  have  any  further  scientific  concern  regard  the  use  of  Methylenebis-
benzotriazolyl tetramethylbutylphenol in cosmetic products? 

The in vitro genotoxicity assessment of MBBT was negative in two different test systems. These 
tests were appropriately applied and demonstrate that there was no evidence for chromosomal 
damage  or  mutagenicity  when  mammalian  cells  were  exposed  to  both  non-micronised  and 
nano-forms of MBBT. Although these test data are accepted by the SCCS, no experimental data 
on uptake/internalisation of the particle by cells has been provided.  

In addition, the SCCS has the following concerns: 
-  In  the  study  in  rats,  clinical  effects  (pain  and  vocalisation)  after  dermal  application  were 
noted at concentrations of 20% (500mg a.i. /kg bw/d and higher). In the carcinogenicity study, 
scabs  were  seen  at  a  dose  level  of  100  mg  a.i./kg/bw/day  and  higher.  It  is  worthwhile  to 
monitor possible irritation effects via the cosmetovigilance programs.  
-  Given  the  physicochemical  properties  (high  lipophilicity)  of  the  substance,  potential 
bioaccumulation in selected tissues is of concern, especially over long-term use.  

This opinion does not address the effects of MBBT on the environment. 

5. MINORITY OPINION 

/ 

57 

 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

6. REFERENCES 

Submission I, 1998 

1.  Müller K., Report on UV-Vis absorption spectra, Test No. 102409, Ciba-Geigy Ltd., 1991 
2.  Hartmann H.R., Acute oral toxicity in the rat, Test No. 914048 Ciba-Geigy Ltd, 1991 
3.  Hartmann H.R., Acute dermal toxicity in the rat, Test No. 914049Ciba-Geigy Ltd. 1991 
4.  Hagemann Ch., Acute eye irritation/corrosion study in the rabbit, Test No. 914051, Ciba-

Geigy Ltd., 1991 

5.  Hagemann  Ch.,  Acute  dermal  irritation/corrosion  study  in  the  rabbit,  Test  No.  914050, 

Ciba-Geigy Ltd., 1991 

6.  Hagemann  Ch.,  Skin  sensitisation  test  in  the  Guinea  Pig,  Test  No.  914052,  Ciba-Geigy 

7. 

Ltd., 1991 
Allard G., Subchronic 90-day oral toxicity (gavage) study with CGF- C002089, Project No. 
653995, RCC, 1998 

8.  Ogorek  B.,  Salmonella  and  Escherischia/liver-microsome  test,  Test  No.  914047,  Ciba-

Geigy Ltd., 1991 

9.  Wollny H.E., Photomutagenicity in a salmonella typhimurium and escherichia coli reverse 

mutation assay, Project No. 689365, RCC, 1998 

10.  Ogorek  B.,  Cytogenetic  test  on  Chinese  Hamster  Cells  in  vitro,  Test  No.  914116,Ciba-

Geigy Ltd., 1991 

11.  Arcelin  G.,  Determination  of  phototoxicity  with  CGF-C002089  in  Albino  Guinea  Pigs, 

Project No. 653973, RCC, 1997 

12.  Arcelin  G.,  Determination  of  photoallergenicity  with  CGF-C002089  in  Albino  Guinea  Pigs, 

Project No. 653984, RCC, 1997, RCC, 1997 

13.  Watkinson  A.,  In  vitro  human  skin  distribution  of  the  UV  absorber  CGF-C002089,  Study 

No. CSC/1 f/97, An-eX analytical services Ltd., 1998 

14.  Watkinson  A.,  In  vitro  human  skin  penetration  of  the  UV  absorber  CGF-C002089,  Study 

No.CSC/1e/97 An-eX analytical services Ltd., 1998 

15.  Watkinson A., In vitro human skin penetration and distribution of the UV absorber CGF-

C002089, Report No. CSC/9/97, An-eX analytical services Ltd., 1998 

16.  Bronaugh, R.L.  and  Stewart,  R. F.,  Methods  for  in-vitro percutaneous  absorption  studies 

III: Hydrophobic compounds. J. Pharm. Sci., 73, 1255-1258 (1984) 

17.  Herzog  B.,  Determination  of  photostability  of  CGF-C002089,  Ciba  Spezialitätenchemie 

Grenzach GmbH, 1998 

Submission I, addendum 1998 

1. 

2. 

3. 

4. 

Acklin  (1991)  Report  on  melting  point,  Test  Number  102504,  Ciba-Geigy  Ltd,  Basle, 
Switzerland 
Crameri (1991) Report on water solubility, Test number 103055, Ciba-Geigy Ltd., Basle, 
Switzerland 
Jäkel  (1991)  Report  on  partition  coefficient,  Test  number  AD-91/3T,  Ciba-Geigy  Ltd., 
Basle, Switzerland 
Jost  (1991)  Report  on  fat  solubility,  Test  number  102410,  Ciba-Geigy  Ltd.,  Basle, 
Switzerland 

5.  Geoffroy  (1991)  Determination  of  vapour  pressure,  Test  number  AD  91/3T,  Ciba-  Geigy 

Ltd, Basle, Switzerland 

58 

 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

6.  Hadju  (1991)  Report  on  flammability  of  solids,  Test  number  AD-91/3T.FLA,  Ciba-  Geigy 

7. 

Ltd., Basle, Switzerland 
Schürch (1991) Report on explosive properties, Test number AD-91 / 3T.EXP, Ciba-Geigy 
Ltd., Basle, Switzerland 

8.  Hajdu  (1991)  Report  on  oxidizing  properties,  Test  number  AD-91/3T.OPR,  Ciba-  Geigy 

Ltd., Basle, Switzerland 

9.  Müller  K.  (1991)  Report  on  UV-Vis  absorption  spectra, Test  number  102409,  Ciba-Geigy 

Ltd., Basle, Switzerland 

10.  Becker (1998) Prenatal toxicity study with CGF-C002089 in the rat, RCC project 680771, 

RCC, Itingen, Switzerland 

11.  Parisse  (1998)  Evaluation  of  phototoxicity  in  humans,  HTR  project  no.  100239A,  Hilltop 

Research Inc., New Jersey, USA 

12.  Parisse  (1998)  Evaluation  of  human  photoallergy  by  repeated  insult  patch  test,  HTR 

project no. 100239B, Hilltop Research Inc., New Jersey, USA 

13.  Czich  (1998)  Chromosome  aberration  assay  in  vitro:  Photomutagenicity  in  Chinese 
hamster  V79  cells  with  CGF-C002089,  RCC-CCR  project  615905,  RCC,  Itingen, 
Switzerland 

14.  Arcelin G. (1997) Determination of photoallergenicity with CGF-C002089 in Albino Guinea 

Pigs, Project No. 653984, RCC, Itingen, Switzerland 

Submission II, 2011 

1.  BASF SE (2010a). Tinosorb® MpH = 10.5: Bovine corneal opacity and permeability test 

(BCOP test). GLP study report number 63V0407/10A158. 

2.  BASF SE (2010b). Tinosorb® M pH= 11.6: Bovine corneal opacity and permeability test 

(BCOP test). GLP study report number 63V0381/10A157. 

3.  BASF  SE  (2011).  RK11_033  Sonnenschutzspray  TGV.  Droplet  sizing  of  sunsprays 
containing  Tinosorb®  M  (UV  Filter-  MBBT)  using  laser  diffraction  system  (Malvern 
Mastersizer). Study report number RK11/033. 

4.  Ciba-Geigy Ltd. (1991a). Report on water solubility. GLP study report number 103055. 
Ciba-Geigy Ltd. (1991b). Report on fat solubility. GLP study report number 102410. 
5.  Ciba-Geigy Ltd. (1991c). Repeated dose oral toxicity in rats. 14-Day range finding test. 

Study report number 914053. 

6.  Ciba-Geigy  Ltd.  (1992).  28  Days  subacute,  oral  toxicity  study  in  rats  (gavage).  GLP 

study report number 914054. 

7.  Ciba  Specialty  Chemicals  Inc.  (2004a).  Chemical  characterisation  of  Tinosorb®  MBBT 

(FAT 75714). GLP study report number 04.156. 

8.  Ciba Specialty Chemicals Inc. (2004b). Löslichkeit von Tinosorb® MBBT und Tinosorb® S 

in Wasser aus einer Sonnencreme (German). Study report number 04.075. 

9.  CIT (2002). Bone marrow micronucleus test by intraperitoneal route in mice. GLP study 

rep01t number 24890 MAS. 

10. CIT  (2004).  Two-phase  dose  range-finding  toxicity  study  by  cutaneous  route  in 

Gottingen minipigs. Study report number 25380 TSN. 

11. CIT (2005). Dose range-finding toxicity study by cutaneous route in rats. Study report 

number 25602 TSR. 

12. CIT  (2006a).  39-Week  toxicity  study  by  cutaneous  route  in  Gottingen  minipigs.  GLP 

study report number 25381 TCN. 

13. CIT (2006b). Carcinogenicity study by cutaneous route in rats. GLP study report number 

25379 TCR. 

59 

 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

14. CR-DDS  (2003).  Thirteen-week  topical  range-finding  study  of  Tinosorb®  M,  Tinosorb® 
MBBT  and  Tinosorb®  S  in  hairless  mice,  with  or  without  simulated  sunlight.  GLP  study 
report 203-006 

15. CR-DDS  (2005a).  Oral  (gavage)  fertility  and  general  reproduction  toxicity  study  of 

Tinosorb® MBBT (FAT 75'714) in rats. GLP study report number 203-009. 

16. CR-DDS  (2005b).  Oral  (gavage)  developmental  and  perinatal  I  postnatal  reproduction 
toxicity study of Tinosorb® MBBT (FAT 75'714) in rats, including a postnatal behavioural 
I functional evaluation. Study report number 203-011. 

17. CR-DDS  (2005c).  Oral  (stomach  tube)  dosage-range  developmental  toxicity  study  of 

Tinosorb® MBBT (FAT 75'714) in rabbits. GLP study report number 203-0lOP. 

18. CR-DDS (2005d). Oral (stomach tube) developmental toxicity study of Tinosorb® MBBT 

(FAT 75'714) in rabbits. GLP study report number 203-010. 

19. CR-DDS (2005e). Twelve-month topical study to determine the influence of Tinosorb® M 
(FAT  75'634)  on  photocarcinogenesis  in  hairless  mice.  GLP  study  report  number  203-
007. 

20. CTL  (2001a).  CGF-C2089  and  CGF-C1607:  Androgen  competitive  binding  assay.  Study 

report number 024530. 

21. CTL  (2001b).  CGF-C2089  and  CGF-C1607:  Estrogen  competitive  binding  assay.  Study 

report number 024529. 

22. CTL  (2002a).  Methylene-bis-benzotriazolyl  tetramethylbutyl  phenol  (CGF-C2089): 
Metabolic fate following oral administration or in vivo dermal application in the rat. GLP 
study repot1 number UR0687. 

23. CTL  (2002b).  CGF-C2089:  Uterotrophic  assay    in  immature  rats.  GLP  study  report 

number ZR1599. 

24. Harlan  Laboratories  Ltd.  (2009).  Percutaneous  penetration  of  14C-FAT  7514  (MBBT) 
formulated  as  Tinosorb®  M  through  pre-damaged  human  split-thickness  skin 
membranes (in vitro). GLP study report number C27241. 

25. Herzog  B,  HUglin  D,  Borsos  E,  Stehlin  A  and  Luther  H  (2004a).  New  UV  absorbers  for 
cosmetic  sunscreens  -  a  breakthrough  for  the  photoprotection  of  human  skin.  Chimia 
58: 554- 559. 

26. Herzog B, Katzenstein A, Quass K, Stehlin A and Luther H (2004b).  Physical properties 
of organic particulate UV-absorbers used in sunscreens. I. Determination of particle size 
with  fiber-optic  quasi-elastic  light  scattering  (FOQELS),  disc  centrifugation,  and  laser 
diffractometry. J. Colloid Interface Sci.271: 136- 144. 

27. Hill  Top  Research  Inc.  (1998a).  Evaluation  of  phototoxicity  in  humans.  GCP  report 

number 100239A. 

28. Hill  Top  Research  Inc.  (1998b).  Evaluation  of  human  photoallergy  by  repeated  insult 

patch test. GCP report number 1002398. 

29. Institut  Pasteur  de  Lille  (2004).  In  vivo  I  in  vitro  unscheduled  DNA  synthesis  (UDS) 
assay in rat hepatocytes with FAT75'7141B. GLP study report number FSR-IPL 040022 
30. Kawai Institute of Dermatology (2001a). Human patch tests of Tinosorb® M (substance). 

No report number. 

31. Kawai  Institute  of  Dermatology (2001b).  Human  patch tests  of  Tinosorb®  M  containing 

sunscreen (product). No report number. 

32. RCC Ltd. (1998a). Dose range-finding prenatal toxicity study with CGF-C002089 in the 

rat. Study report number 680760. 

33. RCC Ltd. (1998b). Prenatal toxicity study with CGF-C002089 in the rat. GLP study report 

number 680771. 

34. RCC Ltd. (1998c). Subchronic 90-day oral toxicity (gavage) study with CGF-C002089 in 

the rat. GLP study report number 653995. 

35. RCC  Ltd.  (2001).  CGF-C2089:  14-Day  dose    range-finding  dermal  toxicity  (semi-

occlusive) study in the Wistar rat. Study report number 795014. 

60 

 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

36. RCC  Ltd.  (2002).  CGF-C2089:  90-Day  dermal  toxicity  (semi-occlusive)  study  in  the 

Wistar rat. GLP study report number 795025. 

37. RCC  Ltd.  (2007).  14C-FAT75714/B  (MBBT):  Percutaneous  penetration  of  14C-
FAT75714/B  (MBBT)  formulated  as  Tinosorb®  M  through  rat  and  human  split  thickness 
skin membranes (in vitro). GLP study report number B23635. 

38. RCC Ltd. (2008). FAT 75'634: 4-Hour acute inhalation toxicity study in rats. GLP study 

report number B25007. 

39. Seibersdorf  Labor  GmbH  (2010).  "Tinosorb®  M  pH  =  11.6":  Acute  eye  irritation  I 

corrosion in rabbits. GLP study report number SL-LT-358/10. 

40. Springborn Laboratories Inc. (200 I). A 14-day cumulative primary skin irritation study 

in guinea pigs with CGF-C2089. GLP study report number 3466.24 

References provided upon request 

1.  Ashby  J,  Tinwell  H,  Soames  AR  and  Foster  JR  (1999).  Induction  of  hyperplasia  and 
increased DNA content in the uterus of immature rats exposed to Coumestrol. Environ. 
Health Perspect. 107: 819-822. 

2.  Ashby  J  and  Lefevre  PA  (2001).  Replacement  of  surgical  castration  by  GnRH  inhibition 

for rat prostate androgen receptor preparations. J Appl. Toxicol. 21(5): 353-354. 

3.  Ashby  J,  Odum  J,  Paton  D,  Lefevre  PA,  Beresford  N  and  Sumpter  JP  (2000).  Re-
evaluation  of  the  first  synthetic  estrogen,  1-keto-1,2,3,4-tetrahydrophenanthrene  and 
bisphenol  A,  using  both  the  ovariectomised  rat  model  used  in  1933  and  additional 
assays. Toxicol. Lett. 115:231-238. 

4.  Ashby  J,  Tinwell  H,  Plautz  .T,  Twomey  K  and  Lefevre  PA  (2001).  Lack  of  binding  to 
isolated estrogen or androgen receptors, and inactivity in the immature rat uterotrophic 
assay,  of  the  ultraviolet  sunscreen  filters  Tinosorb®  M-active  and  Tinosorb®  S.  Regul. 
Toxicol. Pharmacal. 34: 287-291. 

5.  Berset G, Gonzenbach H, Christ R, Martin R, Deflandre A, Mascotte RE, Jolley JD, Lowell 
W, Pelzer Rand  Stiehm T (1996). Proposed protocol for detern1ination of photostability. 
Part I: cosmetic UV filters. Int. J Cosmet. Sci. 18(4): 167- 177. 

6.  Horn  D  and  Rieger  J  (2001).  Organische  Nanopartikel  in  wassriger  Phase  -  Theorie, 

Experiment und Anwendung (German). Angew. Chern. 113: 4460- 4492. 

7.  Peto R, Pike MC, Day NE, Gray RG, Lee PN, Parish S, Peto J, Richards S and Wahrendorf 
J  (1980).  Guidelines  for  simple,  sensitive  significance  tests  for  carcinogenic  effects  in 
long  term animal experiments. IARC Monorgr. Eva!. Carcinog. Risk Chern. Hum. Suppl. 
2:311-426 

8.  Shelby  MD,  Newbould  RR,  Tully  DB,  Chae  K  and  Davis  VL  (1996).  Assessing 
environmental  chemicals  for  estrogenicity  using  a  combination  of  in  vivo  and  in  vitro 
assays. Environ. Health Perspect. 104: 1296- 1300 

Submission III, 2014 

1.  BASF  SE  (2014);  Tinosorb®M  (not  micronized),  Determination  of  test  item  size 
distribution  in  the  genotoxicity  testing  medium  before  and  after  incubation;  Study 
number 13E04243 and 13E4532  

2.  BASF SE (2014); In Vitro Micronucleus Assay In V79 Cells (Cytokinesis Block Method). 

Study report number 33M0407/10M204.  

3.  BASF SE (2014); In Vitro Gene Mutation Test In CHO Cells (HPRT Locus Assay). Study 

report number 50M0407/10M208.  

61 

 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

4.  BASF SE (2014); In Vitro Micronucleus Assay In V79 Cells (Cytokinesis Block Method). 

Study report number 33M0045/13M030.  

5.  BASF SE (2014); In Vitro Gene Mutation Test In CHO Cells (HPRT Locus Assay). Study 

report number 0M0045/13M020.  

SCCS/1546/15 

62 

 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Annex I 

Overview on particle size distributions (mass based) of the different batches: 

Date of 
analysis 

Tinosorb® M 

Batch 

Particle size (µm) 

d(0.5) 

d(0.9) 

Surface area 
(m²/g) 

24-May-2002 

005839D2 

06-Oct-2002 

003736D2 

05122CL2 

10-Jan-2003 

00007CL3 

00696CL3 

02711CL3 

02-Jul-2003 

08706CL3 

12-Sep-2003 

11327CL3 

12-Jan-2004 

00023CL4 

14-Jun-2004 

07132CL4 

03-Nov-2004 

13115CL4 

06-Jan-2005 

00069CL5 

19-Apr-2005 

04673CL5 

22-Jan-2006 

14628CL5 

14-Feb-2006 

01408CL6 

19-May-2006 

05806CL6 

05-Dec-2006 

12623CL6 

03-Jan-2007 

13636CL6 

00292CL7 

11-Jul-2007 

11145CL7 

08-Jan-2008 

00071CL8 

04-Jul-2008 

06908CL8 

21-Dec-2008 

13536CL8 

03-Jan-2009 

13557CL8 

0.265 

0.297 

0.241 

0.218 

0.214 

0.233 

0.230 

0.219 

0.257 

0.239 

0.231 

0.217 

0.241 

0.229 

0.221 

0.228 

0.245 

0.225 

0.247 

0.243 

0.243 

0.227 

0.213 

0.215 

0.127 

0.128 

0.127 

0.118 

0.115 

0.128 

0.125 

0.118 

0.125 

0.118 

0.120 

0.117 

0.127 

0.120 

0.119 

0.121 

0.129 

0.119 

0.128 

0.124 

0.129 

0.124 

0.118 

0.119 

63 

54 

53 

54 

58 

59 

54 

55 

58 

57 

56 

58 

58 

58 

57 

57 

67 

53 

57 

53 

54 

53 

55 

57 

57 

 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Date of 
analysis 

Tinosorb® M 

Batch 

Particle size (µm) 

d(0.5) 

d(0.9) 

Surface area 
(m²/g) 

05-Jul-2009 

03844CL9 

0.117 

04-Dec-2009 

0003961345 

0.124 

13-Jan-2010 

0004044844 

0.117 

09-Jul-2010 

0004499156 

0.117 

19-Dec-2010 

0004901430 

0.128 

03-Jan-2011 

0004901438 

0.127 

29-Jun-2011 

0005307759 

0.128 

30-Aug-2011 

0005423704 

0.118 

MBBT 

21-Nov-2006 

02174CN6 

142.44 

21-Nov-2006 

04296CN6 

195.82 

21-Nov-2006 

00140CN6 

281.34 

0.217 

0.231 

0.215 

0.219 

0.241 

0.238 

0.242 

0.223 

484.30 

511.94 

683.71 

58 

55 

52 

58 

53 

54 

53 

58 

approx. 0.192a 

approx. 0.192a 

approx. 0.248a 

64 

 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Studies performed with non-nanosized MBBT  

Annex II 

The summaries of these studies have been copied from submission II. For submission 
I,  only  a  short  summary  of  the  end-point  is  presented.  The  study  reports  were 
assessed  in  more  detail  when  necessary  for  the  safety  assessment  of  the  nanosized 
material (in that case a comment from the SCCS was added) 

A.  Acute toxicity 

A.1.  Acute oral toxicity 

For Tinosorb as test article TKA 40027 (Batch No: EN 302690.12, purity 97.7%) an acute oral 
toxicity  study  was  performed  according  to  OECD  401  in  male  (n=5)  and  female  (n=5)  albino 
rats. One single oral dose of 2000 mg/kg was administered orally by gavage. No deaths were 
observed and for both sexes the LD50 was considered greater than 2000 mg/kg body weight. 
At  the  dose  of  2000  mg/kg  body  weight  some  indications  for  toxicity  were  observed  i.e. 
piloerection, hunched posture and dyspnoea. 

Ref.: 2 (subm I) 

A.2.  Acute dermal toxicity 

No studies were provided with Tinosorb M. For Tinosorb as test article TKA 40027  (Batch No: 
EN 302690.12, purity 97.7%) an acute dermal toxicity study was performed according to OECD 
402 in male (n=5) and female (n=5) albino rats. One single dose of 2000 mg/kg body weight 
was administered on the back of the animals on a shaved area. No deaths were observed and 
for both sexes the LD50 after dermal administration was considered greater than 2000 mg/kg 
body weight. 

Ref.: 3 (subm I) 

B. 

Irritation and corrosivity 

B.1.  Skin irritation 

Skin irritation in rabbits 

For Tinosorb as test article TKA 40027 (Batch No: EN 302690.12, purity 97.7%) a skin irritation 
test was performed according to OECD 404 in male New Zealand white rabbits (n=3).  A gauze 
patch of approximately 12-16 cm2 was applied to shaved skin (36 cm2 of skin was shaved). The 
gauze patch contained 0.5 g of the test article. Skin reactions were evaluated at 1, 24, 48, and 
72 hours after removal of the skin patch. 
Grade 1 erythema and oedema were observed in all animals one hour after treatment. Grade 1 
erythema was observed in one animal up to 48 hours after treatment. 

Ref.: 5 (subm I) 

65 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

Skin irritation in guinea pigs after repeated exposure 

SCCS/1546/15 

Title: 

Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

A 14-day cumulative primary skin irritation study in guinea pigs with 
CGF-C2089 
/ 
In compliance 
CGF-C2089 (non-micronized) 
001719B0 

Methods 
The test item CGF-C2089, i.e. the UV absorbing ingredient MBBT in non-micronised form, was 
administered  for  14  consecutive  days  on  the  skin  of  Hartley-derived  albino  guinea  pigs.  Six 
animals per dose level received a 0.1 or 0.2 mL dermal application of the test item suspended 
on 0.5% (w/v) CMC in 0.1% (w/v) Tween 80 at 15, 40, or 65% (w/w). After approximately 24 
hours,  residual  test  item  was  removed.  This  procedure  was  repeated  daily  for  a  total  of  14 
exposure periods. Application sites were scored for signs of dermal irritation for up to 72 hours 
following the final test item removal on day 14. Viability, clinical signs of systemic toxicity and 
body  weights  were  assessed  periodically  during  the  study  period.  Dose  formulations  were 
analysed  for  concentration  verification  during  the  study  period.  Dose  formulations  were 
analysed for concentration by means of a HPLC method. 

Results 
There were no test item related effects on viability, clinical signs of systemic toxicity and body 
weights. 
Exposure  to  15%  formulations  of  MBBT  resulted  very  slight  erythema  (1/6  test  sites  on  two 
occasions, day 3 and day 8). No other signs of dermal irritation were noted at both dose levels 
after the last exposure on day 14. 

Exposure  to  40%  formulations  of  MBBT  resulted  in  very  slight  erythema  on  4/6  test  sites 
sporadically during the day 1-14 day exposure period. No dermal irritation was observed after 
day 11 of the exposure period of 14 days. 

Exposure to 65% formulation of MBBT produced very slight and intermittent erythema on 3/6 
test sites during the exposure period. During the 1-72 hours scoring interval after the final test 
item removal, very slight erythema was noted in 1/6 animals. 

The  very  slight  irritation  was  sporadic  across  groups  and  may  have  been  associated  with  the 
physical  removal  of  the  test  item  daily  as  there  was  no  irritation  after  the  final  test  article 
removal with the exception of one test site which was 1 hour after the final test article removal.  

The  achieved test  item  concentrations  in  the dose  formulations  were  acceptable  for the  study 
purpose. 

Conclusion 
Under  the  conditions  of  this study,  the  UV-absorbing  ingredient  MBBT  in  non-micronised  form 
was no skin irritant at concentrations of 15 or 40%. MBBT was considered to be a slight irritant 
at  65%  with  a  Primary  Irritation  Index  of  0.04  by  the  study  director,  however,  the  sporadic 
signs of very slight erythema noted in this study were not unambiguously test item related. It 
is  likely  that  findings  were  more  associated  with  the  daily  physical  removal  of  the  test 
substance. 

Ref.: 41 (subm II) 

66 

 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

SCCS comment 
In  an  ISO  standard  a  Primary  Irritation  Index  or  PII  score  between  0  -  0.4  in  rabbits  is 
considered  to  be  in  a  negligible  response  category.  The  PII  score  is  rather  low  and  may  be 
considered  negligible.  As  indicated,  repeated  test  item  removal  may  have  added  to  the  low 
irritation observed. 

B.2  Sensitisation 

In  a  Maximisation  test,  Tinosorb  (non-nanosized,  TKA  40027,  batch  no  EN  302690.12,  purity 
97.9%)  was  tested  in  10  male  and  10  female  guinea  pigs.  The  induction  consisted  of  three 
pairs  of  intradermal  injections  (0.1ml  adjuvant,  5%  test  article  in  arachide  oil  and  5%  test 
article  in  an  adjuvant  saline  mixture)  and  an  epidermal  application  one  week  later  (30%  test 
article)  under  occlusion  for  48h).  In  week  5,  the  animals  were  challenged  via  a  dermal 
application  to  10%  of  the  test  substance.  None  of  the  animals  of  the  test  group  showed  skin 
reactions 24 and 48 hours after removing the challenge application. 
Ref Subm I (Hageman 1991) 

C.  Dermal / percutaneous absorption 

Title: 

Guideline: 
GLP: 
Test item: 

Batch/lot n°: 

tetramethylbutyl 

Methylene-bis-benzotriazolyl 
(CGF-C2089): 
Metabolic fate following oral administration or  in vivo dermal application in 
the rat 
OECD 427 
In compliance 
Methylene-bis-benzotriazolyl tetramethylbutyl phenol (CGF-C2089)), non-
micronized material 
001719BO 

phenol 

Methods 
For  the  assessment  of  percutaneous  absorption  in  vivo,  two  doses  were  prepared  in  the 
commercial  formulation,  which  were  equivalent  to  concentrations  of  0.2 %  and  10 %  of 
CGF-C2089,  i.e.  of  the  UV-absorbing  ingredient  MBBT.  The  formulation  comprised  unlabelled 
and  14C-radiolabelled test  item  homogeneously dispersed  in  the  vehicle  (formulation,  Xanthan 
gum, Propylene glycol and water) such that a dose of a set volume (100 µL/rat) was equivalent 
to the nominal dose level of 0.2 or 10 mg/rat. 

In  each  case,  unlabelled  test  item  (purity:  99.6 %)  and  14C-radiolabelled  test  item 
(radiochemical purity: 99.1 %) were mixed and milled to a particle size comparable to that of 
the  commercial  formulation,  nominally  200 nm.  The  particle  size  of  the  milled  test  substance 
was determined by scanning electron microscopy (SEM) to be in the range 300 and 2000 nm, 
with a typical particle size of approximately 1000 nm. 

The dermal absorption in 32 male rats [Alpk:APfSD, Wistar derived] was investigated following 
a single application of nominally 0.2 or 10 mg of the formulated active ingredient to 10 cm² of 
skin.  After  dosing,  the  application  sites  were  protected,  but  not  occluded,  using  O-rings 
incorporating  a  nylon  gauze  cover.  A  strip  of  non-occlusive  elasticized  bandage  was  wrapped 
around the rat and over the application devices to help to hold them in place. Rats were housed 
individually in metabolism cages for the collection of urine and faeces. After a 6-hour exposure, 
the  first  two  groups  were  terminated  and  the  application  sites  of  all  the  remaining  rats  were 
washed to remove the unabsorbed dose. Urine, faeces and cage wash were collected from each 
cage  after  the  6-hour  skin  wash,  and  then  at  daily  intervals  after  dosing  for  the  duration  of 

67 

 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

each  experiment.  Groups  of  4 rats  were  terminated  at  6,  24,  72  and  120 hours  after  dosing. 
Under  anaesthesia,  the  skin  was  washed  to  remove  unabsorbed  residual  test  item  before 
exsanguination.  The  application  site  skin  was  then  tape-stripped  to  remove  the  stratum 
corneum.  The  dose  formulations  and  all  samples,  including  selected  tissues  and  residual 
carcasses  were  analyzed 
liquid  scintillation  counting. 
Disintegration  per  minute  (dpm)  values  were  calculated  using  the  appropriate  quench 
correction data. 

for  radioactivity  by  means  of 

Results 
The  homogeneity  of  the  radiolabelled  test  item  in  both  dose  formulations  was  satisfactory 
throughout the periods of dosing. The test item was stable in both dose formulations for longer 
than their period of use in the study. 

Following  dermal  exposure  to  the  0.2 %  formulation  for  6 hours,  approximately  97 %  of  the 
applied  radioactivity  was  removed  from  the  skin  surface  by  aqueous  washing.  Approximately 
0.7 %  (0.4 %  was  found  in  the  stratum  corneum)  of  the  dose  remained  associated  with  the 
application  site  and  some  of  this  was  available  for  absorption.  However,  the  area  under  the 
curve (AUC) could not be calculated because of the not detectable radiolabel in the blood. The 
residue associated with the application site remained low, and declined at later timepoints. The 
amount of dose absorbed remained similar at 0.2 - 0.8 % after 6, 24, 72 and 120 hours. 

Following  dermal  exposure  to  the  10 %  formulation  for  6 hours,  approximately  98 %  of  the 
applied radioactivity was washed from the skin surface. Approximately 0.2 % (0.1 % was found 
in the stratum corneum) of the dose remained associated with the application site following the 
6-hour  skin-wash  and  some  of  this  was  available  for  absorption.  The  residue  associated  with 
the  application  site  remained  similar  at  later  time-points.  The  amount  of  dose  absorbed 
remained similar at 0.2 - 0.4 % after 6, 24, 72 and 120 hours. 

Percutaneous penetration of the UV-absorbing ingredient MBBT through rat skin in vivo over a 
5-day period 

Time after application 

6 hours 

24 hours 

72 hours 

120 hours 

Recovery of applied dose for the 0.2 % formulation (% or % ± SD) 

Total absorbed dosea 
Total non-absorbed doseb 

< 0.34 

0.80 ± 1.20 

0.27 ± 0.05 

< 0.53 

97.98 ± 1.59 

98.62 ± 3.12 

97.70 ± 1.95 

99.07 ± 2.38 

Total recovery 

98.32 ± 1.72 

99.42 ± 2.02 

97.97 ± 1.99 

99.60 ± 2.29 

Recovery of applied dose for the 10 % formulation (% or % ± SD) 

Total absorbed dosea 
Total non-absorbed doseb 

< 0.21 

< 0.41 

< 0.18 

0.34 ± 0.17 

97.63 ± 4.63 

98.06 ± 4.25 

99.86 ± 4.24 

101.08 ± 0.63 

Total recovery 

97.84 ± 4.68 

98.46 ± 3.77 

100.03 ± 4.22 

101.42 ± 0.55 

a:  Sum of radioactivity recovered in urine, faeces cage wash, bandage, tissues, GI tract with contents and carcass; 

given as percentage of applied dose 

b:  Sum  of  radioactivity  recovered  in  6-hour  skin  wash  and/or  terminal  skin  wash  and  stratum  corneum,  skin 

application site, covers and O-rings; given as percentage of applied dose 

SD:  Standard deviation of the mean value for 3 or 4 animals 

68 

 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

Conclusion 

Following  a  6-hour  topical  exposure,  the  in  vivo  dermal  absorption  of  the  micronised 
UV-absorbing ingredient MBBT from a 0.2 and a 10 % formulation was very low and accounted 
for not more than 0.8 % and 0.4 % of the dose, respectively, over 5 days. The topically applied 
micronised  UV-absorbing 
ingredient  MBBT  did  not  achieve  systemically  measurable 
concentrations and was thus not bioavailable. 

Ref.: CTL (2002a) 

D.  Repeated dose toxicity 

D.1.  Repeated Dose (28 days) oral / dermal / inhalation toxicity 

Title: 

Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

14-Day dose range-finding dermal toxicity (semi-occlusive) study in the 
Wistar rat 
Comparable to OECD 410 
In compliance 
CGF-C2089 (non-micronised) 
001719B0 

Methods 
The  test  item  CGF-C2089  (purity:  >99 %),  i.e.  the  UV-absorbing  ingredient  MBBT  in 
non-micronised  form,  suspended  in  0.5 %  (w/v)  CMC  in  0.1 %  (w/v)  aqueous  Tween 80  was 
administered  daily  (semi-occlusive)  to  the  skin  of  14 rats  [HanIbm:WIST  (SPF)]  per  sex  per 
dose level at 100, 300 or 1000 mg/kg bw for at least 14 days. A concurrent untreated control 
group  and  a  concurrent  vehicle  control  group  of  each  14 rats  per  sex  were  included  in  this 
study. 

Clinical signs, food consumption and body weights were recorded periodically. After treatment 
on  days 1  and  14,  blood  samples  were  withdrawn  for  plasma  analyses.  At  the  end  of  the 
treatment period, all animals were sacrificed and examined for gross lesions. Organ weights of 
selected organs were recorded. Concentration, homogeneity and stability of dose formulations 
were determined. 

Results 
The  dermal  administration  did  not  yield  evidence  for  test  item-related  effects  upon  viability, 
clinical  signs  of  systemic  toxicity,  food  consumption,  body  weights,  organ  weights  and 
incidence  or severity  of  gross  lesions.  Local  effects  were  seen  in  3 male  rats  only  (very  slight 
skin flaking in 1 animal of the 300 mg/kg bw/d group and in 2 animals of the 1000 mg/kg bw/d 
group  (transient))  and  were  considered  to  be  treatment-  but  not  test  item-related.  Mean 
plasma levels of the test item were below the limit of quantitation (1 ng/mL). Analysis of dose 
formulations revealed decreasing recoveries with increasing test item concentration. Based on 
this  outcome  and  in  order  to  improve  test  item  suspension,  it  was  decided  to  use  corn  oil  as 
vehicle for the subsequent 90-day dermal study in rats (RCC Ltd., 2002). 

Conclusion 
Under  the  conditions  of  this  study,  1000 mg/kg bw/d  of  the  UV-absorbing  ingredient  MBBT  in 
non-micronised form was established as the no-observed-effect-level (NOEL). Thus, the dermal 
no-observed-adverse-effect-level (NOAEL) was > 1000 mg/kg bw/d. 

Ref.: RCC (2001) 

69 

 
 
 
 
 
 
 
 
  
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

D.2.  Sub-chronic (90 days) oral / dermal / inhalation toxicity 

Title: 
Guideline: 
GLP: 
Test item: 
Batch/lot n°/ 

CGF-C2089: 90-Day dermal toxicity (semi-occlusive) study in the Wistar rat 
OECD 411 
In compliance 
CGF-C2089 (non-micronised) 
0017198B0 

Methods 
The  test  item  CGF  C2089  (purity:  >99 %),  i.e.  the  UV-absorbing  ingredient  MBBT  in 
non-micronised  form,  suspended  in  corn  oil  was  administered  for  6 hours  per  day  (semi-
occlusive)  to  five  groups  of  10 rats  per  sex  per  dose  level  at  0  (untreated),  0  (vehicle),  150, 
450 or 1000 mg/kg bw for 91/92 days. The untreated control, the vehicle control and the high 
dose groups each contained an additional 10 rats per sex that were allowed to recover for a 28-
week period after the last exposure. 

Clinical  signs,  food  consumption,  body  weights,  ophthalmoscopic  endpoints,  haematology, 
clinical  chemistry  and  urinalysis  parameters,  test  item  and  hormone  plasma  levels,  estrous 
cycles,  selected  organ  weights  and  macroscopic  as  well  as  histopathologic  findings  were 
assessed  and  evaluated.  Dose  formulations  were  analysed  for  content,  homogeneity  and 
stability.  For  plasma  level  determinations  on  days 1,  15  and  28  (all  allocated  animals)  and 
day 57  (control  animals  only)  as  well  as  day  64  (treated  animals  only),  blood  was  collected 
from 3 satellite animals/sex/dose level for each timepoint once prior to dosing and at 0.5, 1, 2, 
6  and  24 hours  after  treatment.  Plasma  levels  were  then  quantified  by  means  of  a  LC/MS 
method. 

Results 
Chemical analysis of dose formulations confirmed appropriate dosing.  

Test  item-related  deaths  (1 animal  each  in  the  0  (control),  150  and 1000 mg/kg bw/d  groups 
were  killed  in  extremis)  and  signs  of  clinical  toxicity  did  not  occur.  Signs  of  local  irritation 
(transient  flaking / erythema)  reported  were  comparable  between  vehicle  control  and  dose 
groups  and  considered  to  be  related  to  the  mechanical  effect  of  the  daily  removal  of  the 
wrapping.  No  test  item-related  effects  were  noted  on  food  consumption,  body  weights, 
ophthalmoscopic  endpoints,  body  weight,  haematology,  clinical  biochemistry  and  urinalysis, 
oestrus  cyclicity,  hormone  levels  (evaluated  for  testosterone,  oestradiol,  thyroid  stimulating 
hormone,  follicle  stimulating  hormone  and  luteinising  hormone),  macroscopic  findings  and 
organ  weights.  Microscopic  changes  were  limited  to  the  skin  of  the  dose  site  and  included 
hyperkeratosis and acanthosis at incidences similar across the study groups. 

Evaluation of test item plasma levels on day 1 showed no evidence for dermal absorption. On 
treatment  days 15  and  28,  sample  contamination  was  noted  in  animals  at  all  dose  levels. 
Values  were  reported  but  not  considered  valid  for  assessment.  On  treatment  day 57,  minor 
traces  of  the  test  item  were  detected  in  three  control  males  and  one  vehicle  control  male, 
whereas all control females were without findings. Analytical quantification of plasma samples 
collected  after  64 days  from  test  item-treated  animals  revealed  varying  levels  in  plasma, 
neither  dose-dependent  nor  consistent,  and  this  finding  was  considered  as  contamination-
related. 

70 

 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Conclusion 

Under  the  conditions  of  this  study,  no  target  organ  was  identified  following  repeated  dermal 
treatment of rats with the UV-absorbing ingredient MBBT in non-micronised form. The dermal 
no-observed-effect-level  (NOEL)  was  established  at  the  high  dose  level  of  1000 mg/kg bw/d. 
The corresponding dermal no-observed-adverse-effect-level (NOAEL) was > 1000 mg/kg bw/d.  

Ref.: RCC (2002) 

E.  Mutagenicity/Genotoxicity in vivo 

Title: 
Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

Bone marrow micronucleus test by intraperitoneal route in mice 
OECD 474 
In compliance 
CGF-C2089 (non-micronised) 
001719B0 

Methods 
Three groups of 5 mice per sex [Swiss Ico:OF1(IOPS Caw)] received a first and, 24 hours later, 
a  second  intraperitoneal  treatment  with  CGF-C2089  (purity:  99.6 %),  i.e.  with  the 
UV-absorbing ingredient MBBT in non-micronised form, suspended in corn oil at dose levels of 
500,  1000  or  2000 mg/kg bw  (dose  volume:  10 mL/kg bw).  A  concurrent  control  group  of 
5 mice per sex were treated in the same manner with the vehicle only. A positive control group 
of  5 mice  per  sex  received  a  single  oral  gavage  of  Cyclophosphamide  at  a  dose  level  of 
50 mg/kg bw. 

All treatment and negative control group animals were killed 24 hours after the last treatment 
and  the  positive  control  group  animals  were  killed  24 hours  after  the  single  treatment. 
Following  preparation  of  bone  marrow  smears,  the  number  of  micronucleated  polychromatic 
erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes (PE) for each animal. The 
PE  and  normochromatic  (NE)  erythrocyte  ratio  was  established  by  scoring  a  total  of 
1000 erythrocytes (PE + NE). 

Results 
The mean values of MPE and the PE/NE ratio in the test item-treated groups were equivalent to 
those  of  the  vehicle  control  group  and  no  statistically  significant  difference  was  noted.  The 
mean values of MPE and the PE/NE ratio for the vehicle and positive controls  were consistent 
with  the  laboratory’s  historical  data.  The  positive  control  item  Cyclophosphamide  induced  a 
highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the 
test system. 

No clinical signs, mortalities have been observed. 

Cytogenetic effects of the UV-absorbing ingredient MBBT 

Group 

Dose level 

MPE/1000PE 

PE/NE 

(mg/kg bw/d) 

(mean ± SD) 

(mean ± SD) 

Vehicle controla 

Test item 

0 
500 
1000 

0.5 ± 0.5 
0.6 ± 1.3 
0.6 ± 0.9 

0.3 ± 0.2 
0.2 ± 0.1 
0.2 ± 0.1 

Males 

71 

 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Group 

Dose level 

MPE/1000PE 

PE/NE 

Positive controlb 

Vehicle controla 

Test item 

(mg/kg bw/d) 
2000 
50 

0 
500 
1000 
2000 
50 

Females 

(mean ± SD) 
0.2 ± 0.4 
25.2 ± 4.6* 

0.4 ± 0.9 
0.3 ± 0.4 
0.4 ± 0.7 
0.5 ± 0.9 
12.7 ± 1.8* 

(mean ± SD) 
0.2 ± 0.1 
0.4 ± 0.1 

0.4 ± 0.1 
0.3 ± 0.1 
0.3 ± 0.1 
0.4 ± 0.1 
0.8 ± 0.4 

Positive controlb 
Corn oil 
Cyclophosphamide 

a: 
b: 
MPE:  Micronucleated polychromatic erythrocytes 
PE: 
NE:  Normochromatic erythrocytes 
SD:  Standard deviation 
*: 

Polychromatic erythrocytes 

p < 0.001 (based on the 2 x 2 contingency table for MPE) 

Conclusion 
Under  the  conditions  of  this study,  the  UV-absorbing  ingredient  MBBT  in  non-micronised  form 
did  not  induce  damage  to  the  chromosomes  or  the  mitotic  apparatus  of  mouse  bone  marrow 
cells. 

Ref.: CIT (2002) 

SCCS comment 
The  SCCS  does  not  agree  to  the  conclusion  on  the  non-micronised  form.  This  study  does  not 
allow us to draw conclusions on a potential genotoxic hazard of the non- micronised form of the 
compound as there is no indication of exposure.  

Title: 

Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

In vivo / in vitro unscheduled DNA synthesis (UDS) assay in rat hepatocytes 
with FAT 75’714/B 
OECD 486 
In compliance 
FAT 75’714/B 
15420CL2 , non-micronised 

Method 
Three  male  Fischer  rats  (IFFA  CREDO  origin)  per  dose  (1000  or  2000 mg/kg bw)  and  per 
expression  time  (12-16 hours  in  assay 1,  2-4 hours  in  assay 2)  were  treated  by  oral  gavage 
with  FAT 75’714/B  (purity:  99.8 %),  i.e. the  UV-absorbing  ingredient  MBBT  in non-micronised 
form,  suspended  in  0.5 %  CMC  for  the  unscheduled  DNA  synthesis  (UDS)  test  (dose  volume: 
10 mL/kg bw). At each expression time, assigned animals were killed and liver cells prepared. 
Hepatocyte  cultures  were  then  radiolabelled  with  3H-Thymidine,  prepared  for  autoradiography 
and  grains  counted  using  an  image  analysis  system.  Concurrent  negative  (0.5 %  CMC)  and 
positive  controls  (2-Acetamidofluorene  at  25 mg/kg bw  for  assay 1;  Dimethylhydrazine  at 
10 mg/kg bw for assay 2) were included in this study. 

Results 
Over the two experiments at the two doses tested, the net nuclear grain counts were below the 
threshold  value  for  a  positive  response.  Furthermore,  the  frequency  of  cells  in  S-phase  was 
low.  Overall,  treatment  with  the  test  item  did  not  induce  a proliferative  effect  in  rat  liver  and 

72 

 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

did  not  reveal  genotoxic  activity.  Treatment  with  the  positive  control  items  resulted  in  the 
appropriate response over background. 

SCCS/1546/15 

Unscheduled DNA synthesis in rat hepatocytes 

Group 

NNG Count 

Cells in repair  
NNG Count ≥ 5 

% cells in repair 
NNG Count ≥ 5 

(mean ± SD) 

(mean ± SD) 

(mean ± SD) 

Assay 1: 12-16 hour expression time 

Vehicle controla 

-1.98 ± 3.60 

1000 mg/kg bw 

-2.13 ± 3.66 

2000 mg/kg bw 

-1.84 ± 3.78 

Positive controlb 

5.77 ± 4.87 

6.00 ± 1.06 

6.62 ± 0.02 

6.17 ± 0.89 

8.90 ± 0.65 

Assay 2: 2-4 hour expression time 

Vehicle controla 

-2.63 ± 4.20 

1000 mg/kg bw 

-3.16 ± 4.14 

2000 mg/kg bw 

-4.03 ± 4.42 

5.41 ± 0.20 

6.08 ± 0.91 

5.85 ± 0.35 

1.19 ± 0.67 

1.56 ± 1.05 

2.18 ± 0.51 

59.2 ± 3.31 

1.96 ± 0.89 

1.53 ± 0.19 

0.71 ± 0.53 

Positive controlc 

14.61 ± 6.10 

15.32 ± 0.88 

94.26 ± 12.23 

a:  0.5 % CMC 
b:  2-Acetamidofluorene (2-AAF) 
c:  Dimethylhydrazine 
NNG: 

Net nuclear grain 

Conclusion 
Under  the  experimental  conditions  employed,  the  UV-absorbing 
non-micronised form did not induce DNA repair in liver cells of treated rats.  

ingredient  MBBT 

in 

Ref.: Institut Pasteur de Lille (2004) 

SCCS comment 
The  SCCS  does  not  agree  to  the  conclusion  on  the  non-micronised  form.  This  study  does  not 
allow us to draw conclusions on a potential genotoxic hazard of the non-micronised form of the 
compound since it is not demonstrated that the testing compound has reached the target. 

F.   Endocrine effects 

Title: 
Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

Methods 

CGF-C2089 and CGF-C16078: Androgen competitive binding assay 
Not indicated 
No 
CGF-C2089 (non-micronised) 
CTL test substance Y11707/001 

8  

Information on CGF-C1607 is not provided in the present document 

73 

 
 
 
 
 
 
 
 
 
 
 
                                          
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

The androgen competitive binding assay was conducted according to Ashby and Lefevre (2001) 
using  cytosol  isolated  from  the  prostates  of  8 week  old  male  rats  [Alpk:APfSD]  treated 
24 hours previously with the GnRH-anatagonist Antarelix to mimic surgical castration. Aliquots 
of prostate cytosol were incubated with duplicate dose levels of the test item in 10 µL Dimethyl 
sulfoxide  (DMSO)  and  3H-R1881  (17α-Methyl-3H-trienolone)  for  17 hours  at  4 °C.  The 
receptor-ligand complex was precipitated and the radioactivity determined by liquid scintillation 
counting.  Ten-fold  dilutions  of  R1881  (Methyl-trienolone)  and  testosterone  (positive  control) 
and  the  test  item  CGF-C2089  (5 x 10-10  to  5 x 10-4 mol/L  in  DMSO),  i.e.  the  UV-absorbing 
ingredient  MBBT  in  non-micronised  form  were  assayed.  Two  separate  experiments  were 
conducted,  each  with  duplicate  dose  levels  of  test  item  and  of  vehicle  alone  to  ascertain  a 
100 % binding value. 

Results 
MBBT  failed  to  compete  with  R1881  in  the  androgen  competitive  binding  assay.  Testosterone 
and R1881 induced the appropriate response. 

Conclusion 
Under  the  conditions  of  this  in  vitro  study,  the  UV-absorbing  ingredient  MBBT  displayed  no 
intrinsic androgenic activity. 

Ref.: CTL (2001a) 

Title: 
Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

CGF-C2089 and CGF-C16079: Estrogen competitive binding assay 
Not indicated 
No 
CGF-C2089 (non-micronised) 
CTL test substance Y11707/001 

Methods 
The estrogen competitive binding assay was conducted using the cytosol isolated from the uteri 
of  21-25 day  old  female  rats  [Alpk:APfSD]  as  described  by  Ashby  et al.  (1999,  2000),  using 
methods  based  upon  Shelby  et al.  (1996).  Uterine  cytosol  was  incubated  with  duplicate  dose 
levels of the test item in Dimethyl sulfoxide (DMSO) and 3H-17β-Estradiol at 4 °C for 17 hours. 
The  receptor-ligand  was  precipitated  with  Hydroxyl  apatite,  washed  and  then  suspended  in 
Optiphase  for  the  determination  of  radioactivity  in  a  liquid  scintillation  counter.  Ten-fold 
dilutions  of  17β-Estradiol,  the  anti-oestrogen  Faslodex  (positive  control)  and  the  test  item 
CGF-C2089  (purity  not  indicated),  i.e.  the  UV-absorbing  ingredient  MBBT  in  non-micronised 
form  (5 x 10-10  to  5 x 10-4 mol/L  in  DMSO)  were  assayed.  Two  experiments  were  conducted, 
each  with  duplicate  dose  levels  of  the  test  item  and  of  vehicle  alone  to  ascertain  a  100 % 
binding value. 

Results 
MBBT  failed  to  compete  with  3H-17β-Estradiol  in  the  estrogen  competitive  binding  assay. 
17β-Estradiol and Faslodex induced the appropriate response.  

Conclusion 
Under the conditions of this in vitro study, the UV-absorbing ingredient MBBT in non-micronised 
form displayed no intrinsic estrogenic activity. 

Ref.: CTL (2001b) 

9  

Information on CGF-C1607 is not provided in the present document. 

74 

 
 
 
 
 
 
 
 
 
 
                                          
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Title: 
Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

CGF-C2089: Uterotrophic assay in immature rats 
Not indicated 
In compliance 
CGF-C2089 (non-micronised) 
003542B0 

Methods 
An  uterotrophic  assay  was  performed  using  19-20 days  old  rats  [Alpk:APfSD].  The  test  item 
CGF-C2089  (purity:  ≥ 98 %),  i.e.  the  UV-absorbing  ingredient  MBBT  in  non-micronised  form, 
suspended in Arachis oil was administrated to groups of 10 female rats at daily dose levels of 0 
(vehicle  control),  250,  500  or  1000 mg/kg bw  by  single  subcutaneous  injection  on 
3 consecutive days. An additional group of rats was treated daily under the same experimental 
conditions  with  the  positive  control  item  17β-Estradiol  benzoate  at  0.4 mg/kg bw  on 
3 consecutive days.  

Clinical signs and body weights were recorded daily during the study period. All animals were 
sacrificed  24 hours  after  the  last  administration.  The  uteri  were  trimmed  free  of  fat,  gently 
blotted  and  weighed.  Dose  formulations  were  analysed  for  concentration  and  homogeneity 
using a HPLC method with UV detection. 

Results 
No signs of test item-related toxicity were noted in this study. The test item was inactive in the 
immature  rat  uterotrophic assay  at  all  dose  levels  tested.  The  positive control  item  induced  a 
distinct  increase  in  blotted  uterus  weights  (±  adjustment  for  body  weight)  thus  confirming 
sensitivity of the test system. Based on chemical analysis results, the dose formulations were 
acceptable for use in this study. 

Conclusion 
Under the conditions of this in vivo study, the UV-absorbing ingredient MBBT in non-micronised 
form displayed no uterotrophic potency. 

Ref.: CTL (2002b) 

G.   Reproduction toxicity 

Title: 

Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

Oral (gavage) fertility and general reproduction toxicity study of 
Tinosorb® MBBT (FAT 75’714) in rats 
ICH Harmonised Tripartite Guideline, Stages A and B 
In compliance 
Tinosorb® MBBT (FAT 75’714) (non-micronised) 
15420CL2 

Methods 
The  test  item  Tinosorb® MBBT  (purity:  99.8 %),  i.e.  the  UV-absorbing  ingredient  MBBT  in 
non-micronised  form,  suspended  in  0.5 %  (w/v)  CMC  in  0.1 %  (w/v)  aqueous  Tween 80  was 
administered  by  daily  single  oral  gavage  to  25 rats  [Crl:CD(SD)IGS  BR  VAF/Plus]  per  sex  per 
dose  level  at  100,  300  or  1000 mg/kg bw  (dose  volume:  10 mL/kg bw).  Concurrent  control 
animals  (25 rats  per  sex)  were  treated  with  the  vehicle  only  under  the  same  experimental 
conditions. 

75 

 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

Male  rats  were  treated  daily  beginning  28 days  before  cohabitation,  through  cohabitation 
(maximum of 16 days), and continuing through the day before sacrifice, which was the end of 
the  cohabitation  period.  Female  rats  were  dosed  once  daily  beginning  15 days  before 
cohabitation, during cohabitation and continuing through day 7 of gestation. 

Viability,  clinical  signs,  food  consumption  and  body  weights  were  determined  periodically. 
Oestrous cycling was assessed during the in-life period. Males were sacrificed at the end of the 
cohabitation  period  and  the  reproductive  organs  weighed  (testes,  epididymides,  seminal 
vesicles),  and  sperm  evaluated  for  concentration  and  motility.  Females  were  sacrificed  on 
gestational day 13 and examined to determine the number of corpora lutea, implantation sites, 
and of viable and non-viable embryos. Placentae were examined for size, colour and shape. 

Dose formulations were analyzed for concentration, homogeneity and stability. 

Results 
No test item-related effects on viability, clinical signs, body weight, body weight development 
and  food  consumption  were  noted  in  male  and  female  rats  at  the  dose  levels  tested.  No 
necropsy  observations  were  found  to  be  test-article  related.  There  were  no  test  item-related 
adverse effects on mating, fertility and other reproductive parameters such as oestrous cycling 
or  male  reproductive  organ  weights  and  sperm  concentration  and  motility.  In  females,  no 
loss, 
Caesarean-sectioning  parameters 
implantations,  appearance  of  placentae  and  viable  versus  non-viable  embryos)  were  affected 
by test item dosages as high as 1000 mg/kg bw/d. 

(numbers  of  corpora 

lutea,  preimplantation 

Conclusions 
Under  the  conditions  of  this  study,  the  oral  no-observed-effect-level  (NOEL)  for  parental  and 
reproductive toxicity of the UV-absorbing ingredient MBBT (non-micronised) was set at the high 
dose  level  of  1000 mg/kg bw/d.  Based  on  the  study  results,  MBBT  does  not  display  adverse 
effects on reproduction parameters and taking all other data on reprotoxicity into account, it is 
thus  not  subject  to  classification  as  reproductive  toxicant  according  to  Regulation  (EC) 
No. 1272/2008.  

Ref.: CR-DDS (2005a) 

Title: 

Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

Oral (gavage) developmental and perinatal / postnatal reproduction toxicity 
study of Tinosorb® MBBT (FAT 75’714) in rats, including a postnatal 
behavioural / functional evaluation 
ICH Harmonised Tripartite Guideline, Stages C through F 
In compliance 
Tinosorb® MBBT (FAT 75’714) (non-micronised) 
15420CL2 

Methods 
The  test  item  Tinosorb® MBBT  (purity:  99.8 %),  i.e.  the  UV-absorbing  ingredient  MBBT  in 
non-micronised  form,  suspended  in  0.5 %  (w/v)  CMC  in  0.1 %  (w/v)  aqueous  Tween 80  was 
administered by oral gavage to 24 pregnant female rats [Crl:CD(SD)IGS BR VAF/Plus] per dose 
level  at  100,  300  or  1000 mg/kg bw/d  (dose  volume:  10 mL/kg bw)  from  day 6  of  gestation 
through  day 20  of  lactation  or  day  24  of  gestation  (rats  that  did  not  deliver  a  litter).  A 
concurrent control group of 24 pregnant female rats received the vehicle only under the same 
experimental conditions. 

F0 generation rats were observed for viability, clinical signs, body weight development and food 
consumption.  Maternal  behaviour  was  assessed  on  days  1,  4,  7,  14  and  21  of  the  lactation 

76 

 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

period.  After  completion  of  the  21-day  postpartum  period,  the  F0  generation  animals  were 
sacrificed  and  a  gross  necropsy  of  the  thoracic,  abdominal  and  pelvic  viscera  was  performed. 
The number and distribution of implantation sites was recorded. 

Each  litter  was  evaluated  for  viability.  The  pups  in  each  litter  were  counted  and  clinical 
observations  as  well  as  body  weights  were  recorded.  Reflex  and  physical  developmental 
measures  (surface  righting,  eye  opening,  acoustic  startle,  air  righting,  and  pupil  constriction) 
were  also  evaluated  during  the  pre-weaning  period.  Pups  culled  on  day 28  post-partum  were 
sacrificed and a gross necropsy was performed; in addition, the brain was cross-sectioned and 
examined for hydrocephaly. 

F1  generation  rats  selected  for  continued  observation  were  examined  for  clinical  signs  during 
the post weaning period. Body weights and food consumption data except during cohabitation 
were  determined.  The  rats  were  evaluated  for  sexual  maturation,  motor  activity  and  for 
performance  in  a  watermaze  test.  At  approximately  90 days  of  age,  the  F1  generation  rats 
were  assigned  to  cohabitation.  Male  rats  were  sacrificed  after  completion  of  the  cohabitation 
period;  a  gross  necropsy,  assessment  of  reproductive  organ  weights,  microscopical 
examination and a sperm evaluation were performed. Female rats were sacrificed on day 21 of 
gestation,  Caesarean-sectioned,  and  a  gross  necropsy  was  performed.  Foetuses  weight,  sex 
and  gross  external  alterations  were  evaluated.  Dose  formulations  were  analysed  for 
concentration, homogeneity and stability. 

Results 
There  were  no  indications  of  test  item-related  general  toxicity  or  adverse  effects  on 
reproduction in the F0 and F1 generation rats or development in the F1 generation rats and the 
F2 generation foetuses. 

All  F0  generation  female  rats  survived  until  scheduled  sacrifice.  None  of  the  clinical  signs  or 
gross lesions observed at necropsy of these rats was considered test item-related. There were 
no  dose-dependent  changes  in  body  weight,  body  weight  development  and  food  consumption 
throughout  gestation  and  lactation.  All  pregnant  rats  delivered  litters  and  had  surviving  pups 
when  the  litters  were  weaned  on  day 21  of  lactation.  With  few  exceptions  considered  as 
incidental  findings  within  historical  control  ranges,  i.e.  a slight  decrease  in  the  lactation  index 
due to mainly a single litter in the high dose group and an increased percentage of high dose 
male pups per litter, all natural delivery and litter observations (including evaluations of reflex 
and physical development) were comparable among the four dose groups.  

There were no test item-related deaths among the F1 generation male and female rats. Clinical 
signs,  body  weight,  body  weight  gain  and  food  consumption  as  well  as  anatomical  indices  of 
sexual maturation and performance in a motor activity test and watermaze task of these rats 
were  not  affected  by  maternal  administration  of  the  test  item.  There  were  no  statistically 
significant or biologically important effects on the mating and fertility parameters. All necropsy 
observations  in  the  F1  generation  rats  were  considered  unrelated  to  maternal  dosages. 
Treatment  with  the  test  item  did  not  induce  changes  in  male  reproductive  organ  weights  or 
sperm motility and density. Microscopic examination of reproductive organs did not reveal test 
item-related  alterations.  No  Caesarean-sectioning  or  litter  parameters  were  affected  by  the 
maternal  dosages  of  the  test  item,  and  all  F2  foetuses  appeared  normal  at  gross  external 
examination.  

The concentration, homogeneity and stability of the dose formulations were acceptable. 

77 

 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

Conclusion 
Under  the  experimental  conditions  employed,  the  oral  no-observed-effect-level  (NOEL)  for 
general  toxicity  and  for  adverse  effects  on  development  and  reproduction  in  rats  of  the 
UV-absorbing ingredient MBBT in non-micronised form was established at the high dose level of 
1000  mg/kg  bw/d.  Therefore  and  taking  all  other  data  on  reprotoxicity  into  account,  MBBT  is 
not  subject  to  classification  as  reproductive  toxicant  according  to  Regulation  (EC) 
No. 1272/2008. 

Ref.: CR-DDS (2005b) 

H.  Developmental toxicity 

H.1.  Developmental toxicity in rats 

Title: 
Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

Dose range-finding prenatal toxicity study with CGF-C002089 in the rat 
Not indicated 
No 
CGF-C002089 (non-micronised) 
507514.52 

Methods 
Groups  of  5 pregnant  female  rats  [HanIbm:WIST  (SPF)]  per  dose  level  were  treated  with  the 
test  item  CGF-C002089  (purity:  99.9 %),  i.e.  with  the  UV-absorbing  ingredient  MBBT  in 
non-micronised  form,  in  0.5 %  (w/v)  CMC  in  0.1 %  (w/v)  aqueous  Tween 80  by  daily  oral 
gavage  from  days 6  to  17  of  gestation  at  100,  300  or  1000 mg/kg bw  (dose  volume: 
10 mL/kg bw).  Additional  5 female  pregnant  rats  received  vehicle  only  and  served  as 
concurrent  control  group.  All  animals  were  sacrificed  on  day 21  of  gestation  and  the  foetuses 
removed by Caesarean section. 

Viability,  clinical  signs,  food  consumption  and  body  weights  were  recorded  periodically  during 
the  study  period.  Post  mortem  examinations  included  macroscopic  examination  of  dams  and 
the assessment of the number of corpora lutea. Foetuses were sexed, weighed and examined 
for  gross  external  abnormalities.  Sex  ratios,  pre-implantation  loss  and  post-implantation  loss 
were calculated. 

Results 
Test  item-related  effects  on  survival,  clinical  appearance,  food  consumption,  body  weights  or 
macroscopic findings did not occur in any dam. No test item-related reproductive effects (mean 
numbers  of  corpora  lutea  and  implantation  sites,  and  percent  of  pre-  and  post-implantation 
loss) were noted. Test item-related foetal effects (external abnormalities, sex ratios and body 
weights) did not occur. 

Conclusion 
Under the condition of this range-finding study, the maternal and developmental no-observed- 
effect-level (NOEL) was 1000 mg/kg bw/d. The corresponding no-observed-adverse-effect-level 
(NOAEL) for maternal and developmental effects was > 1000 mg/kg bw/d. Identical dose levels 
were selected for the main study. 

Ref.: RCC (1998a) 

78 

 
 
 
 
 
 
 
 
 
 
 
 
 
Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

SCCS/1546/15 

Title: 
Guideline: 
GLP: 
Test item: 
Batch/lot n°: 

Prenatal toxicity study with CGF-C002089 in the rat 
OECD 414 
In compliance 
CGF-C002089 (non-micronised) 
507514.52 

Methods 
The  test  item  CGF-C002089  (purity:  99.9 %),  i.e.  the  UV-absorbing  ingredient  MBBT  in  non-
micronised  form,  suspended  in  0.5 %  (w/v)  CMC  in  0.1 %  (w/v)  aqueous  Tween 80  was 
administered by single daily oral gavage to 22 pregnant female rats [HanIbm:WIST (SPF)] per 
dose  group  from  days  6  to  17  of  gestation  at  100,  300  or  1000 mg/kg bw  (dose  volume: 
10 mL/kg bw). A concurrent control group of 22 female rats was treated with the vehicle only 
under  the  same  experimental  conditions.  Animals  were  sacrificed  on  day 21  of  gestation  and 
the foetuses removed by Caesarian section. 

Viability,  clinical  signs,  food  consumption  and  body  weights  were  recorded  periodically  during 
the  study  period.  Post  mortem  examinations  included  macroscopic  examination  of  dams  and 
the assessment of the number of corpora lutea. Foetuses were sexed, weighed and examined 
for  gross  external  abnormalities.  Viscera,  brains  and  skeletons  of  foetuses  were  examined  for 
abnormalities and variations. Sex ratios, pre-implantation loss and post-implantation loss were 
calculated. Dose formulations were analysed for content, homogeneity and stability. 

Results 
Test  item-related  effects  on  survival,  clinical  signs,  food  consumption,  body  weights  or 
macroscopic findings did not occur in any dam. No test item-related reproductive effects (mean 
number of implantation sites, mean post-implantation loss, and mean number of foetuses per 
dam)  were  noted.  Test  item-related  foetal  effects  did  not  occur  in  the  parameters  external, 
visceral, and skeletal abnormalities; sex ratios; body weights; and stage of development. The 
few observed significant differences in the various maternal or foetal parameters assessed were 
not  considered  test  item-related  since  they  were  within  the  range  of  normal  biological 
variability  for  the  strain  and  age  of  rat  used  and/or  did  not  exhibit  a  dose-response.  The 
concentration,  homogeneity  and  stability  of  the  dose  formulations  were  acceptable  in  this 
study. 

Conclusion 
Under  the  condition  of  this  study,  the  maternal  and  developmental  no-observed-effect-level 
(NOEL) of the UV-absorbing ingredient MBBT in non-micronised form was the high dose level of 
1000 mg/kg bw/d.  The  corresponding  no-observed-adverse-effect-level  (NOAEL)  for  maternal 
and  developmental  effects  was  > 1000 mg/kg bw/d.  Based  on  this  outcome  and  taking  all 
other  data  on  repro-toxicity  into  account,  MBBT  does  not  display  adverse  effects  on 
development  in  rats  and  is  not  subject  to  classification  as  reproductive  toxicant  according  to 
Regulation (EC) No. 1272/2008. 

Ref.: RCC (1998b) 

H.2  Developmental toxicity in rabbits 

Title: 

Guideline: 
GLP: 

Oral (stomach tube) dosage-range developmental toxicity study of 
Tinosorb® MBBT (FAT 75’714) in rabbits 
Not indicated 
In compliance 

79 

 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

Test item: 
Batch/lot n°: 

Tinosorb® MBBT (FAT 75’714) (non-micronised) 
15420CL2 

Methods 
Suspensions  of  the  test  item  Tinosorb® MBBT  (purity:  99.8  %),  i.e.  of  the  UV-absorbing 
ingredient  MBBT  in  non-micronised  form,  in  0.5 %  (w/v)  CMC  in  0.1 %  (w/v)  aqueous 
Tween 80, were administered by oral gavage once daily to 6 timed-mated female New Zealand 
White rabbits [Hra:NWZ(SPF)] per dose level on days 6 through 19 of gestation at 250, 500 or 
1000 mg/kg bw (dose volume: 10 mL/kg bw). Concurrent control animals received the vehicle 
only under the same experimental conditions.  

Viability,  clinical  signs,  food  consumption  and  body  weights  were  recorded  periodically.  All 
surviving  rabbits  were  sacrificed  on  day 29  and  examined  for  the  number  and  distribution  of 
corpora  lutea,  implantation  sites  and  uterine  contents.  A  gross  necropsy  of  the  thoracic, 
abdominal  and  pelvic  viscera  was  performed.  Gravid  uterine  weights  were  recorded.  Foetuses 
were weighed and examined for gross external alterations and sex. 

Results 
One  mid-dose  doe  was  sacrificed  on  day 23  after  an  abortion  that  was  not  related  to  the 
treatment  with  the  test  item.  All  remaining  rabbits  survived  until  scheduled  sacrifice.  In  the 
dams,  no  test  item-related  changes  were  noted  in  clinical  signs,  food  consumption,  body 
weights  and  gross  lesions.  Caesarean  sectioning,  litter  observations  and  the  foetal  gross 
observations were all within the ranges observed historically. 

Conclusion 
A no-observed-effect-level (NOEL) was not derived by the study author. Based on the results of 
this  study,  the  oral  NOEL  for  maternal  and  developmental  toxicity  of  the  UV-absorbing 
ingredient  MBBT  in  non-micronised  form  was  at  the  high  dose  level  of  1000 mg/kg bw/d. 
Dosages of 100, 300 and 1000 mg/kg bw/d were selected for the main developmental toxicity 
study with MBBT in rabbits. 

Ref.: CR-DDS (2005c) 

Title: 

Guideline: 

GLP: 
Test item: 
Batch/lot n°: 

Oral (stomach tube) developmental toxicity study of Tinosorb® MBBT 
(FAT 75’714) in rabbits 
ICH Harmonised Tripartite Guideline: Detection of toxicity to reproduction of 
medical products 
In compliance 
Tinosorb® MBBT (FAT 75’714) (non-micronised)) 
15420CL2 

Methods 
The test item Tinosorb® MBBT (purity: 99.8%), i.e. the UV-absorbing ingredient MBBT in non-
micronised  form,  suspended  in  0.5 %  (w/v)  CMC  in  0.1 %  (w/v)  aqueous  Tween  80,  was 
administered by oral gavage to 20 female pregnant rabbits [Hra:NWZ(SPF)] per dose group on 
days 6  through  19  of  gestation  at  dosages  of  100,  300  or  1000 mg/kg bw/d  (dose  volume: 
10 mL/kg bw). 

Viability,  clinical  observations,  food  consumption  and  body  weights were  assessed  periodically 
during  the  study  period.  Rabbits  were  sacrificed  on  day 29,  Caesarean-sectioned  and  a  gross 
necropsy  of  the  thoracic,  abdominal  and  pelvic  viscera  was  performed.  The  number  and 

80 

 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

distribution of corpora lutea were recorded. The uterus of each rabbit was excised, weighed and 
examined for pregnancy, number and distribution of implantation sites, live and dead foetuses 
and early and late resorptions.  Placentae were examined for size, colour and shape. Foetuses 
were  weighed,  sexed  and  examined  for  gross  lesions.  Cavitated  organs  were  evaluated  in  all 
foetuses. The brain was examined  in situ. All  foetuses were examined for skeletal alterations. 
Dose formulations used in this study were analysed for content, homogeneity and stability. 

Results 
Viability, clinical observations, food consumption and body weights in the does were unaffected 
by  the  treatment  with  the  test  item  at  all  dose  levels.  No  external,  soft  tissue  or  skeletal 
alterations  (malformations  or  variations)  in  foetuses  were  caused  by  the  test  item  up  to  and 
including  the  high  dose  level  of  1000 mg/kg bw/d.  All  litter  parameters  remained  unaffected 
and clinical observations were considered unrelated to the test item. 

Conclusion 
Under  the  conditions  of  this  study,  the  oral  no-observed-effect-level  (NOEL)  for  maternal  and 
developmental  toxicity  of  the  UV-absorbing  ingredient  MBBT  in  non-micronised  form  was 
established at 1000 mg/kg bw/d. Thus, MBBT does not display adverse effects on development 
in  rabbits  and  taking  all  other  data  on  reprotoxicity  into  account,  it  is  not  subject  to 
classification as reproductive toxicant according to Regulation (EC) No. 1272/2008. 

In  Submission  1,  information  on  photosensitization  and phototoxicity  of  MBBT  was  submitted. 
The text below is directly copied from submission I 

Ref.: CR-DDS (2005d) 

Photosensitisation 

Species 
Albino  Dunkin  Hartley  Guinea  Pig,  HsdPoc:  DH,  SPF,  body  weight  range  at  start  of 
acclimatization 282-407 g; age at start of acclimatization 5-7 weeks. 

Number of animals 
The study was performed with 30 male animals. The animals were distributed as follows: Ten 
animals for the control group and 20 animals for the test group. 

Substance: 
• Test article: CGF-C002089 
• Batch No.: 507514.52 
• Purity: 99.9% 

Test procedure 

Induction (test days 1-10) 
On test day 1 the nuchal skin area of the test group was shaved and a test site of 6-8 cm2 was 
defined by four 0.1 ml injections intradermally of Freund's Complete Adjuvant and physiological 
saline  1:1  into  the  corners.  0.1  ml  of  the  75%  test  article  dilution  in  PEG  400  were  then 
epicutaneously applied to a skin area of 8 cm². 
The test site was exposed to 1.8 J/cm2  UV-B irradiation and 1 OJ/cm2 UV-A. 
The  mixture  of  Freund's  Complete  Adjuvant  and  physiological  saline  was  injected  only  once. 
The  remaining  procedure  (topical  application  of  the  test  article  followed  by  irradiation)  was 
repeated four times within 2 weeks (days 1, 3, 6, 8 and 10). At day 1 of induction the control 

81 

 
 
 
 
 
 
 
 
 
 
 
 
SCCS/1546/15 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

animals  received  4  injections  intradermally  of  Freund's  Complete  Adjuvant  and  physiological 
saline 1:1 (0.1 ml) into the corners of the test site of 6-8 cm2. No additional applications were 
performed during induction. The animals remained untreated. 

Challenge 
On  day  22  concentrations  of  75%,  50%,  25%  and  15%  of  the  test  article  in  PEG  400  were 
applied  to  the  left  flank  at  a  dose  of  0.025  ml/2  cm2.•  The  left  flank  was  then  exposed  to  1 
OJ/cm2 UV-A irradiation only. After irradiation of the left flank, the right flank was treated with 
the  test  article  accordingly  without  irradiation.  The  control  animals  were  treated  in  the  same 
way as described above. 

Light source 

Induction 
UV-A 320-400 nm 10J/cm² 
UV-B 280-320 nm 1.8J/cm² 

Challenge 
UV-A 320-400 nm 10J/cm² 
Philips Actinic "TLD" Lamps (36W/08) 
Philips UV-B-Sunlamp TL 20W/12 
Philips Actinic "TLD" Lamps (36W/08) 
The duration of the exposure was regulated by a time control device. 

Observation times 
Each animal was assessed for reactions at 24, 48 and 72 hours after exposure. 

Main findings 
Under the experimental conditions employed, none of the animals of the test group showed 
skin reactions 24, 48 or 72 hours after removing of the challenge application. 

Sensitisation rate: 0 out of 20 treated animals 

Conclusion 
The  test  substance  possesses  no  photoallergenic  and  allergenic  potential  in  the  guinea  pig 
under  the  study  conditions.  The  results  presented  for  the  control  group  may  lead  to  the 
conclusion that the test substance showed no irritation potency nor phototoxic potency. 

Phototoxicity 
Method: 

Species: 
Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF, body weight range at start of 
acclimatisation 296-385 g; age at start of acclimatization 5-7 weeks. 
Number of animals: The study was performed with 15 male animals. The animals were 
distributed as follows: Five animals for the control group and 1 0 animals for the test group. 

Substance: 
• Test article: CGF-C002089
• Batch No.: 507514.52
• Purity: 99.9%

82 

Revision of opinion on 2,2'-methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol) (nano)  
and corrigendum 
_______________________________________________________________________________________________ 

The  test  article  was  homogeneously  mixed  with  PEG  400  as  vehicle.  0.025  ml/  2  cm²  of  the 
test  article  suspension  were  applied  at  concentrations  of  25%  and  15%.  The  remaining  test 
article concentrations of 75% and 50% were applied with a spatula in order to saturate the test 
sites. The test sites on all animals were pretreated approximately 30 to 50 minutes prior to the 
test article application with 2% DMSO diluted with ethanol (0.025 ml/cm²) to enhance the skin 
penetration of the test article. 

SCCS/1546/15 

Light source: 
• Philips Actinic "TLD" Lamps (36W/08)
• Energy 1 x1 Oexp. 4 Ergs/cm²/sec
• Spectrum 320-400nm
• Irradiation dose 20J/cm² UV -A

The  different  test  article  concentrations  were  applied  to  the  left  flank  of  the  animals.  Thirty 
minutes  after  application  of  the  test  article  the  left  flank  of  the  animals  was  exposed  to  non-
erythematogenic UV-A irradiation (20J/cm²). 
After  irradiation  the  same  treatment  was  performed  on  the  right  flank  but  the sites remained 
unexposed to light after treatment and served as control  sites. Control animals  were exposed 
to UVA similarly, except they were treated with the solvent (PEG 400) only. 

Observation times 
The animals were examined 24, 48 and 72 hours after application of the test article for signs of 
erythema and oedema. 

Main findings 
No phototoxic reactions were observed after test article administration in PEG-400. 

Conclusion 
Due to the results determined it is concluded that under the conditions used, the test substance 
does not exhibit phototoxic potential in guinea pigs of this strains and age 

83