ESAC opinion on the scientific validity of the RS Comet and RSMN test methods.
Date: 2025-02-12
Institutions: Joint Research Centre
Subjects: DNA, chemical product, research method, scientific report, standard, toxic substance

ESAC Opinion 
on the 
Scientific Validity of the  
RS Comet and RSMN  
Test Methods 

ESAC Opinion No. 2025-01 
of 24 January 2025 

Clewell, R., Corsini, E., Greco, D., Kienhuis, A., Lehmann, 
D., Rodríguez, B., Segner, H., Tralau, T. 

2025 

 
  
  
 
This  document  is  a  publication  by  the  Joint  Research  Centre  (JRC),  the  European  Commission’s  science  and  knowledge 
service. It aims to provide evidence-based scientific support to the European policymaking process. The contents of this 
publication  do  not  necessarily  reflect  the  position  or  opinion  of  the  European  Commission.  Neither  the  European 
Commission nor any person acting on behalf of the Commission is responsible for the use that might be made of  this 
publication.  For  information  on  the  methodology  and  quality  underlying  the  data  used  in  this  publication  for  which  the 
source is neither Eurostat nor other Commission services, users should contact the referenced source. The designations 
employed and the presentation of material on the maps do not imply the expression of any opinion whatsoever on the 
part  of  the  European  Union  concerning  the  legal  status  of  any  country,  territory,  city  or  area  or  of  its  authorities,  or 
concerning the delimitation of its frontiers or boundaries. 

The  independent  scientific  peer  review  of  the  RS  Comet  and  RSMN  in  vitro  test  methods  described  in  this  report  was 
organised by the Joint Research Centre’s EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and 
conducted by the EURL ECVAM Scientific Advisory Committee (ESAC).  

The ESAC peer review was coordinated by João Barroso, Raffaella Corvi and Milena Mennecozzi on behalf of JRC / EURL 
ECVAM. 

Contact information 
European Commission, Joint Research Centre (JRC), Systems Toxicology Unit (F3) 
Address: Via E. Fermi 2749, I-21027 Ispra (VA), Italy 
Email: JRC-F3-ENQUIRIES@ec.europa.eu 

EU Science Hub 
https://joint-research-centre.ec.europa.eu 

JRC140306 

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ISBN 978-92-68-22510-3   doi:10.2760/2520536  

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© European Union, 2025 

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How to cite this report: European Commission, Joint Research Centre, Clewell, R., Corsini, E., Greco, D., Kienhuis, A., Lehmann, D., Rodríguez, B., 
Segner, H. and Tralau, T., ESAC Opinion on the Scientific Validity of the RS Comet and RSMN Test Methods, Mennecozzi, M., Corvi, R. and 
Barroso, J. editors, Publications Office of the European Union, Luxembourg, 2025, https://data.europa.eu/doi/10.2760/2520536, JRC140306. 
Available at: http://publications.jrc.ec.europa.eu/repository/handle/JRC140306. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EUROPEAN COMMISSION 
DIRECTORATE-GENERAL 
JOINT RESEARCH CENTRE 
Directorate F - Health and Food 
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) 

EURL ECVAM 
SCIENTIFIC 
ADVISORY 
COMMITTEE 
(ESAC) 

ESAC OPINION 

on the 

Scientific Validity of the 
RS Comet and RSMN 
Test Methods 

ESAC Opinion No. 
Relevant ESAC Request No. 
Date of Opinion 

2025-01 
2023-01 
24/01/2025 

 
 
 
 
 
 
 
 
 
Table of contents 

Abstract ................................................................................................................................................................................ 1 

ESAC Opinion ...................................................................................................................................................................... 2 

Annex 1: Composition of the ESAC and ESAC Sub-Group ........................................................................... 8 

Annex 2: ESAC Sub-Group Report on RS Comet ............................................................................................10 

Annex 3: ESAC Sub-Group Report on RSMN .....................................................................................................40 

 
 
Abstract 

ESAC,  the  EURL  ECVAM  Scientific  Advisory  Committee,  advises  EURL  ECVAM  on  scientific 
issues.  Its  main  role  is  to  conduct  independent  peer  review  of  validation  studies  of 
alternative  test  methods  and  to  assess  their  scientific  validity  for  a  given  purpose.  The 
committee  reviews  the  appropriateness  of  study  design  and  management,  the  quality  of 
results  obtained  and  the  plausibility  of  the  conclusions  drawn.  ESAC  peer  reviews  are 
formally  initiated  with  a  EURL  ECVAM  Request  for  ESAC  Advice,  which  provides  the 
necessary background for the peer-review and establishes its objectives, timelines and the 
questions to be addressed. The peer review is normally prepared by specialised ESAC Sub-
Groups.  ESAC's  advice  to  EURL  ECVAM  is  formally  provided  as  'ESAC  Opinions'  and  'Sub-
Group  Reports'  at  the  end  of  the  peer  review.  ESAC  may  also  issue  Opinions  on  other 
scientific  issues  of  relevance  to  the  work  and  mission  of  EURL  ECVAM  but  not  directly 
related to a specific alternative test method.   

The ESAC Opinion expressed in this report relates to the peer-review of the Reconstructed 
human Skin (RS) Comet and Micronucleus (RSMN) in vitro test methods for the assessment 
of genotoxicity. 

Abstract 

Page | 1 

 
 
 
 
EUROPEAN COMMISSION 
DIRECTORATE-GENERAL 
JOINT RESEARCH CENTRE 
Directorate F - Health and Food 
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) 

Ispra, 24 January 2025 

ESAC Opinion 

At its 48th meeting, held on 16-17 February 2023, the EURL ECVAM Scientific Advisory 
Committee (ESAC) (Annex 1) was formally asked by EURL ECVAM to evaluate the scientific 
validity  of  the  Reconstructed  human  Skin  (RS)  Comet  and  Micronucleus  (MN)  assays  for 
genotoxicity  testing  to  support  their  regulatory  use  and  the  development  of  OECD  Test 
Guidelines.  The  RS  Comet  and  RSMN  are  separate  in  vitro  assays  that  use  human 
reconstructed skin models and, because of their improved biological fidelity to human skin 
tissue  compared  to  traditional  submerged  monoculture  models,  are  considered  better 
models  for  predicting  human  response  to  dermal  chemical  exposure.  These  assays  were 
proposed in the Test Submissions to EURL ECVAM for use individually as follow up assays 
depending  on  the  mode(s)  of  action  represented  by  positive  results  in  the  traditional 
genotoxicity in vitro test battery to confirm or reject potential for genotoxicity following skin 
exposure. 

An  ESAC  Sub-Group  (SG)  was  established  to  assess  the  scientific  validity  of  the  RS 
Comet  and  RSMN  assays  (Annex  1).  Based  on  its  independent  assessment,  the  ESAC  SG 
delivered  a  detailed  ESAC  SG  report  for  each  method  (Annexes  2  and  3)  to  support  the 
development  of  this  opinion.  The  analysis  and  conclusions  of  the  ESAC  SG  were  based 
primarily  on  a peer-review  of  the  scientific  evidence  included  in  the  RS  Comet  and  RSMN 
files  submitted  to  EURL  ECVAM,  including  all  the  relevant  Annexes  and  supporting 
documents (i.e., the Test Submissions). The assessment also included additional resources 
and supporting information provided by the Test Submitters upon request by the ESAC SG. 

The  final  version  of  this  opinion  was  unanimously  endorsed  by  the  ESAC  by  written 

procedure on 24th January 2025. 

The ESAC recommends a slightly different test strategy than the one  proposed by the 
Test  Submitters.  The ESAC  recommends starting  with  the  RS  assay covering  the  mode  of 
action that tested positive in the standard in vitro test battery and, if a negative result is 
obtained,  performing the  second RS assay for confirmation. A positive  result in either the 
RS Comet or RSMN assay would be sufficient to conclude that the test item is genotoxic in 
the skin. 

ESAC OPINION 

Page | 2 

 
 
 
 
 
 
To  assess  the  scientific  validity  of  the  RS  Comet  and  RSMN  assays,  the  ESAC  SG 

evaluated: 

1.  RS  Comet  assay  transferability,  reproducibility,  and  its  ability  to  confirm  or  reject 
positive results in traditional in vitro Ames or mammalian cell gene mutation tests, 
2.  RSMN  assay  transferability,  reproducibility,  and  its  ability  to  confirm  or  reject 

positive results in traditional in vitro MN tests, 

3.  The  combined  use  of  RS  Comet  and  RSMN  assays  as  a  strategy  to  evaluate 

potential for dermal genotoxicity as described above. 

Based on the available information, the existing scientific literature and the experts' own 
extensive experience as detailed in the ESAC SG reports, the ESAC unanimously concluded 
the following: 

The RS Comet and RSMN assays are biologically relevant 

The  RS  Comet  and  RSMN  assays  both  rely  on  three-dimensional  (3D)  RS  models  of 
human origin. The RS Comet assay is performed in the Phenion® Full-Thickness Skin Model 
(Henkel, Düsseldorf, Germany), which is composed of mature human primary keratinocytes 
and  fibroblasts  cultured  at  the  air-liquid-interface  (ALI).  The  RSMN  assay  is  performed  in 
the  EpiDerm™  Skin  Model  (MatTek;  Ashland,  MA,  USA),  which  consists  of  highly 
differentiated  normal  human  epithelial  keratinocytes  (NHEK)  cultured  on  mesh  inserts  at 
the  ALI.  The  assays  were  designed  primarily  for  the  purpose  of  testing  dermally  applied 
cosmetic ingredients, with the readout done at the site of contact (i.e., skin). The ESAC also 
sees  value  in  potential  expanded  use  for  these  tests  for  the  evaluation  of  other  product 
categories  and  configurations  (e.g.,  pesticides,  chemical  mixtures,  final  products)  with 
dermal  exposure.  Use  of  3D  tissues  allows  for  a  more  biologically  relevant  tissue 
architecture,  which  is  more  likely  to  recapitulate  in  vivo  chemical  penetration  and 
toxicological  response.  Both  models  have  also  been  shown  to  express  xenobiotic 
metabolizing  enzymes,  which  suggests  that  these  models  have  the  potential  to  better 
mimic  in  vivo  skin  metabolism-mediated  clearance  and  bioactivation.  Thus,  the  ESAC 
considers the RS Comet and RSMN assays to offer improved biological relevance for human 
skin genotoxicity than traditional monoculture in vitro models. 

RS Comet and RSMN assays offer advantages for expanding chemical domain over 
traditional in vitro assays 

The  chemical  domain  was  not  specifically  explored  for  these  assays  in  the  two  Test 
Submissions. However, there are clear features of a 3D skin model cultured at the ALI that 
promote  an  increased  chemical  domain  compared  to  traditional  genotoxicity  tests 
performed in submerged two-dimensional (2D) monocultures. First, as previously noted, RS 
models  have  the  potential  to  account  for  in  vivo  skin  metabolism.  Second,  culturing  the 
tissues  at  ALI  allows  the  test  to  be  used  with  chemicals  and  substances  that  are  not 
generally amenable to testing in submerged in vitro systems, including chemicals that are 

ESAC OPINION 

Page | 3 

 
lipophilic  or  otherwise  poorly  dissolved  in  cell  culture  medium.  Furthermore,  complex 
mixtures and formulations in various vehicles (e.g., creams, emulsions) could potentially be 
tested in a skin model cultured at the ALI. While the Test Submissions did not specifically 
evaluate lipophilic chemicals or product formulations, and therefore no conclusions can be 
drawn  on  the  utility  of  these  assays  for  such  purposes,  the  design  of  the  assays  does 
suggest  potential  for  expanding  beyond  the  limited  chemical  domain  that  defines  all 
submerged 2D in vitro systems. 

The ESAC review would have benefited from a more robust and a priori validation 
study design 

The  ESAC  review  of  the  RS  Comet  and  RSMN  assays  relied  on  data  collected  and 
compiled  over  a  period  of  more  than  10  years  and  from  multiple  laboratories.  This 
approach  brings  inherent  challenges,  some  of  which  are  clearly  reflected  in  the  Test 
Submissions. For example, the remit of the validation studies, as well as their design, were 
subject  to  several  adjustments  over  time.  Further,  the  studies  included  in  both  Test 
Submissions  were  not  designed  to  support  the  rigorous  Within-Laboratory  Reproducibility 
(WLR),  Between-Laboratory  Reproducibility  (BLR)  and  predictive  capacity  calculations  that 
are generally performed to verify scientific validity of a proposed test method. In the RSMN 
assay submission, some test items were tested in only one laboratory. In other instances, 
chemicals  were  only  tested  in  duplicate.  In  both  the  RS  Comet  and  RSMN  assays,  the 
number of  tested chemicals was low. In general, both  Test Submissions would have been 
substantially  improved  by  developing  a  validation  study  design  a  priori,  and  consistently 
following the study design. Additionally, both studies would have benefited from testing a 
larger number of chemicals. In particular, the ESAC recommends more testing of chemicals 
that are True Positives (TP) or Misleading Positives (MP) (as only these would be tested in 
follow-up  to  in  vitro  test  battery  results),  chemicals  that  require  bioactivation,  and 
chemicals  strategically  chosen  to  maximize  the  chemical  domains  of  the  assays.  To  that 
point, the ESAC also notes that the test items were selected and classified as TP, MP and 
True Negative (TN) by subject matter experts that were not part of the validation study and 
the rationale for those decisions was not provided in the Test Submissions. Consequently, 
there  is  some  uncertainty  associated  with  these  classifications  that  the  ESAC  could  not 
resolve. 

Specific  test  conditions  or  applications  that  did  not  have  sufficient  evidence  for 
evaluation were identified and removed from consideration by the ESAC. Nevertheless, the 
RS  Comet  and  RSMN  Test  Submissions  were  clear  about  their  general  objectives  and 
contained  the  necessary  controls  and  data,  the  majority  of  which  were  published  and 
publicly available, to facilitate review by the ESAC. Further, while the design of the chemical 
test sets could be improved, the data provided in the Test Submission and published journal 
articles were sufficient for the ESAC to evaluate the scientific validity of the RS Comet and 
RSMN assays. 

ESAC OPINION 

Page | 4 

 
The Transferability, Reproducibility and, when the assays are used in combination 
as proposed by the ESAC, the Predictive Capacity of the RS Comet and RSMN are 
sufficient 

1. RS Comet assay 

Overall,  the  BLR  of  the  RS  Comet  assay  is  acceptable.  However,  the  ESAC  identified 
concerns  with  respect to the transferability and the  WLR. Only one test  item  was  used to 
test  transferability  of  the  assay  to  a  naïve  laboratory.  The  ESAC  considers  one  test  item 
insufficient  to  demonstrate  transferability  and  recommends  that  both  TP  and  TN 
compounds are included in the evaluation of transferability. Notably, most of the chemicals 
used for  WLR were only tested in two  experiments, which  does not support  evaluation  of 
WLR.  Overall,  the  data  set  suffered  from  having  a  small  number  of  tests.  The  predictive 
capacity varied widely across laboratories, with one laboratory testing too few chemicals to 
accurately evaluate the predictive capacity. The laboratory that tested the most chemicals 
(n  =  18)  demonstrated  sensitivity  and  specificity  greater  than  80%.  The  ESAC  performed 
analyses in which laboratories with insufficient tests were excluded and the BLR data was 
used  to  bolster  the  WLR  and  transferability  data.  Based  on  these  analyses,  and  the 
submitted  BLR,  the  ESAC  concludes  that  the  results  of  the  validation  study  regarding 
reproducibility, transferability and predictive capacity are adequate for the defined purpose. 

The  RS  Comet  assay  was  able  to  identify  four  out  of  seven  TP  chemicals  that  require 
bioactivation  for  genotoxicity,  demonstrating  that  this  assay  may  exhibit  metabolic 
competence needed to identify genotoxicants that require bioactivation. However, only one 
bioactive TP test item was tested in more than one laboratory, limiting the ESAC’s ability to 
evaluate  consistency  between  laboratories.  Therefore,  additional  data  should  be  collected 
to support this application of the assay. 

2. RSMN assay 

Overall,  the  predictive  capacity,  WLR  and  BLR  of  the  chemicals  tested  at  48  h  is 
adequate, with exception of the test items that require metabolic activation. Of the seven 
TP  chemicals  tested  to  evaluate  the  ability  of  the  RSMN  assay  to  identify  bioactivated 
genotoxicants, only three were tested in more than one laboratory and only one chemical 
showed a positive response in all three test laboratories. Based on the data provided, the 
ESAC concluded that the metabolic competence of the RSMN assay may not be sufficient to 
identify  bioactivated  genotoxicants,  possibly  due  to  immature  keratinocytes.  While  a 
number of peer-reviewed publications were provided  with  the  Test  Submission  to support 
the expression of native metabolizing enzymes in the EpiDerm tissue, based on the current 
validation  data  provided,  the  ESAC  cannot  draw  conclusions  on  the  utility  of  the  RSMN 
alone for testing potentially bioactivated genotoxicants. 

While the Test Submission recommended using the 72-h time point for the RSMN assay 
rather  than  the  48-h  time  point  initially  tested  by  the  validation  laboratories,  the  ESAC 
found that there were insufficient data to evaluate the 72-h time point based on the Test 

ESAC OPINION 

Page | 5 

 
Submission. However, in a recent publication (Thakkar et al., 2022)1, the added value of the 
72-h  time  point  was  demonstrated  with  a  set  of  22  fragrance  materials.  Based  on  the 
combined data, the ESAC concurs with the use of the 72-h time point for the RSMN assay 
and considers the strategy proposed appropriate. 

3. Combined RS Comet and RSMN 

While the current data was insufficient to comprehensively evaluate the utility of either 
the RS Comet or RSMN assay alone to identify bioactivated genotoxicants, the ESAC found 
that  when  results  of  the  RS  Comet  and  RSMN  assays  were  combined,  all  but  one  of  the 
seven bioactive TP test items tested in both RS Comet and RSMN were correctly classified. 
The only outlier (i.e., diaminotoluene) was negative in the RS Comet and positive in one out 
of three laboratories in the RSMN. Therefore, the ESAC concludes that the combination of 
RSMN and RS Comet is preferred for general use and particularly for identifying substances 
that require bioactivation. 

ESAC  comments  on  the  utility  of  the  RS  Comet  and  RSMN  assays  in  regulatory 
testing 

According  to  the  Test  Submitters,  the  RS  Comet  and  RSMN  assays  were  designed  to 
follow up the results of a standard in vitro genotoxicity test battery, in the context of test 
items that are primarily associated with dermal exposure. The Test Submitters also stated 
that  the  RS  assays  should  not  be  used  to  assess  genotoxicity  of  compounds  via  other 
routes of exposure (e.g. oral). The ESAC sees value in potential expanded use for these tests 
for the evaluation of other product categories and configurations (e.g., pesticides, chemical 
mixtures,  final  products).  The  ESAC  also  considers  that  these  tests  could  add  value  to  a 
WoE-based  evaluation  of  systemic  genotoxicity  provided  systemic  bioavailability  is 
considered. It should be noted that, in the opinion of the ESAC, the RS assays will not allow 
any predictions on potential systemic genotoxicity. Further, the ESAC notes that accounting 
for systemic genotoxicity will require additional considerations such as the use of liver S9 
fraction and/or data on skin absorption/penetration. In cases of significant skin absorption 
of a compound found to be positive in traditional genotoxicity assays following metabolic 
activation (by S9) but negative in the RS assays, the possibility of its liver activation into a 
genotoxic  compound  cannot  be  excluded  due  to  potential  differences  in  the  metabolic 
capacity of the skin and the liver. The use of reconstructed 3D skin models not only offers 
the  advantage  of  organ  similarity  with  regard  to  the  barrier  function  but  also  (limited) 
metabolic competence. Further, unlike other in vivo animal and non-human cell-based test 
systems,  both  RS  assays  also  offer  the  advantage  of  human  relevance.  The  ESAC 
recognizes  that  current  approaches  to  validation  of  test  methods  do  not  specifically 
acknowledge  human  relevance  and  is  of  the  opinion  that  this  is  an  important  point  that 

1 Thakkar Y, Moustakas H, Aardema M, Roy S, Pfuhler S, Api AM (2022) Use of the EpiDerm™ 3D reconstructed 
skin micronucleus assay for fragrance materials. Mutagenesis 37(2):89-111. doi: 10.1093/mutage/geab040 

ESAC OPINION 

Page | 6 

 
                                                           
should  be  given  more  weight  in  future  validations.  Hazard  classification  should  be 
straightforward for all substances identified as initial positives which are not subject to S9-
activation  as  these  will  either  be  confirmed  or  overruled  by  subsequent  testing  with  RS 
assays.  However,  for  substances  originally  requiring  S9-activation,  overruling  by  the  RS 
assays  is  less  straightforward  as  a  negative  result  obtained  using  the  RS  assays  cannot 
principally exclude the possibility of systemic activation following skin penetration. 

Summary and Conclusions 

The  ESAC  concludes  that  the  evidence  provided  on  the  RS  Comet  assay  is 
sufficient  and  adequate  to  support  its  scientific  validity  for  use  with  chemicals 
that test positive in the Ames or mammalian cell gene mutation tests, including 
chemicals that require bioactivation. 

The ESAC further concludes that the evidence provided on the RSMN assay 72-
h  time  point  is  sufficient  and  adequate  to  support  its  scientific  validity  for  use 
with  chemicals  that  are  directly  genotoxic  and  test  positive  in  the  standard  in 
vitro  micronucleus  test  (submerged  monoculture).  However,  the  ESAC  cautions 
that insufficient data exist to evaluate the scientific validity of the test for use 
with  bioactivated  genotoxic  chemicals.  Additionally,  while  the  Test  Submission 
recommended using the 72-h time point for the RSMN assay rather than the 48-h 
time  point  initially  tested  by  the  validation  laboratories,  the  ESAC  found  that 
there  were  insufficient  data  to  evaluate  the  72-h  time  point  based  on  the  Test 
Submission.  However,  in  a  recent  publication  (Thakkar  et  al.,  2022)1,  the  added 
value  of  the  72-h  time  point  was  demonstrated  with  a  set  of  22  fragrance 
materials. Based on the combined data, the ESAC concurs with the use of the 72-
h  time  point  for  the  RSMN  assay,  and  considers  the  strategy  proposed 
appropriate. 

The ESAC concludes that these are valuable assays as part of a tiered testing 
strategy,  including  using  these  RS  assays  as  follow-up  tests  to  the  standard  in 
vitro  test  battery  for  genotoxicity  in  skin  following  dermal  application.  For 
chemicals  in  which  dermal  genotoxicity  is  the  toxicological  endpoint  of  concern 
following  application  to  the  skin,  the  ESAC  recommends  starting  with  the  RS 
assay  covering  the  mode  of  action  that  tested  positive  in  the  standard  in  vitro 
test  battery.  In  case  of  a  negative  result,  the  second  RS  assay  should  be 
performed  for  confirmation.  A  positive  result  in  either  the  RS  Comet  or  RSMN 
assay would be sufficient to conclude that the test item is genotoxic in the skin. 
The  ESAC  also  notes  the  promise  of  these  tests  to  contribute  to  an  animal-free 
New  Approach  Methodology  (NAM)-based  approach  to  systemic  genotoxicity 
following  dermal  exposure,  with  additional  development  and  as  part  of  a  WoE 
approach. 

ESAC OPINION 

Page | 7 

 
EUROPEAN COMMISSION 
DIRECTORATE-GENERAL 
JOINT RESEARCH CENTRE 
Directorate F - Health and Food 
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) 

Annex 1 

COMPOSITION OF THE ESAC 

AND ESAC SUB-GROUP

ANNEX 1 - COMPOSITION OF THE ESAC AND ESAC SUB-GROUP 

Page | 8 

 
 
 
 
 
EUROPEAN COMMISSION 
DIRECTORATE-GENERAL 
JOINT RESEARCH CENTRE 
Directorate F - Health and Food 
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) 

Composition of the ESAC and ESAC Sub-Group 

EURL ECVAM Scientific Advisory Committee (ESAC) 

•  Dr. Rebecca CLEWELL (ESAC Chair) 
•  Prof. Emanuela CORSINI 
•  Prof. Dario GRECO 
•  Dr. Sebastian HOFFMANN (excluded from review due to a conflict of interests) 
•  Dr. Anne KIENHUIS 
•  Dr. David LEHMANN 
•  Prof. Blanca RODRÍGUEZ 
•  Prof. Helmut SEGNER 
•  Dr. Tewes TRALAU 

ESAC Sub-Group 

•  Prof. Rebecca CLEWELL (Sub-Group Chair) 
•  Prof. Emanuela CORSINI 
•  Dr. Anne KIENHUIS 
•  Dr. David LEHMANN 
•  Prof. Blanca RODRÍGUEZ 
•  Prof. Helmut SEGNER 
•  Dr. Tewes TRALAU 

EURL ECVAM (Secretariat) 

•  Dr. João BARROSO (ESAC Coordinator) 
•  Dr. Raffaella CORVI 
•  Dr. Milena MENNECOZZI 
•  Prof. Maurice WHELAN (Head of Unit) 

ANNEX 1 - COMPOSITION OF THE ESAC AND ESAC SUB-GROUP 

Page | 9 

 
 
 
 
 
EUROPEAN COMMISSION 
DIRECTORATE-GENERAL 
JOINT RESEARCH CENTRE 
Directorate F - Health and Food 
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) 

Annex 2 

ESAC SUB-GROUP REPORT  

ON RS COMET 

ANNEX 2 - ESAC SUB-GROUP REPORT ON RS COMET 

Page | 10 

 
 
 
 
 
 
 
EUROPEAN COMMISSION 
DIRECTORATE-GENERAL 
JOINT RESEARCH CENTRE 
Directorate F - Health and Food 
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) 

EURL ECVAM 
SCIENTIFIC 
ADVISORY 
COMMITTEE 
(ESAC) 

ESAC SUB-GROUP REPORT 

on the 

Scientific Validity of the 
RS Comet Test Method 

Title page information 
File name 

ESAC_SG_Report_RSCOMET.docx 

Abbreviated title of ESAC 
request 

RSCOMET-RSMN 

Relating to ESAC REQUEST Nr. 

2023-01 

Request discussed through 

ESAC 48 (February 2023) 

Report to be handed over to 
ESAC Chair and EURL ECVAM 
Coordinator by 

Version tracking 

Rebecca Clewell (Sub-Group Chair) 

Date 

18/12/2023 

23/05/2024 

Version 

Author(s)  Description 

V1.0 

V2.0 

ESAC SG 

First agreed draft of ESAC SG Report  

ESAC SG 

Final approved draft of ESAC SG  Report sent to 
ESAC for endorsement 

24/01/2025 

V3.0 

ESAC 

Final version of ESAC SG Report endorsed by the 
ESAC 

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Table of Contents 

TABLE OF CONTENTS .................................................................................................................................................................. 12 

ESAC SUB-GROUP (SG) ............................................................................................................................................................... 14 

ABBREVIATIONS USED IN THE DOCUMENT ......................................................................................................................... 15 

1. STUDY OBJECTIVE AND DESIGN ........................................................................................................................................ 16 

1.1 ANALYSIS OF THE CLARITY OF THE STUDY OBJECTIVE'S DEFINITION ..................................................................................................... 16 
(a) ESAC SG summary of the study objective as outlined in the Test Submission ......................................... 16 
(b) Appraisal of clarity of study objective as outlined in the Test Submission ................................................ 16 
1.2 QUALITY OF THE BACKGROUND PROVIDED CONCERNING THE PURPOSE OF THE TEST METHOD ...................................................... 17 
(a) Analysis of the scientific rationale provided in the Test Submission .............................................................. 17 
(b) Analysis of the regulatory rationale provided in the Test Submission ........................................................... 18 
1.3 APPRAISAL OF THE APPROPRIATENESS OF THE STUDY DESIGN ................................................................................................................ 19 
1.4 APPROPRIATENESS OF THE STATISTICAL EVALUATION ................................................................................................................................ 19 

2. COLLECTION OF EXISTING DATA ........................................................................................................................................ 20 

2.1 EXISTING DATA USED AS REFERENCE DATA..................................................................................................................................................... 20 
2.2 EXISTING DATA USED AS TESTING DATA .......................................................................................................................................................... 20 
2.3 SEARCH STRATEGY FOR RETRIEVING EXISTING DATA ................................................................................................................................... 20 
2.4 SELECTION CRITERIA APPLIED TO EXISTING DATA ......................................................................................................................................... 20 

3. QUALITY ASPECTS RELATING TO DATA GENERATED DURING THE STUDY .......................................................... 21 

3.1 QUALITY ASSURANCE SYSTEMS USED WHEN GENERATING THE DATA..................................................................................................... 21 
3.2 QUALITY CHECK OF THE GENERATED DATA PRIOR TO ANALYSIS .............................................................................................................. 21 

4. QUALITY OF DATA USED FOR THE PURPOSE OF THE STUDY (EXISTING AND NEWLY GENERATED) .......... 22 

4.1 OVERALL QUALITY OF THE EVALUATED TESTING DATA (NEWLY GENERATED OR EXISTING) ............................................................ 22 
4.2 QUALITY OF THE REFERENCE DATA FOR EVALUATING RELEVANCE .......................................................................................................... 22 
4.3 SUFFICIENCY OF THE EVALUATED DATA IN VIEW OF THE STUDY OBJECTIVE ........................................................................................ 22 

5. TEST DEFINITION (MODULE 1) ............................................................................................................................................ 23 

5.1 QUALITY AND COMPLETENESS OF THE OVERALL TEST DEFINITION .......................................................................................................... 23 
5.2 QUALITY AND COMPLETENESS OF THE DOCUMENTATION CONCERNING PROTOCOLS AND PREDICTION MODELS ....................... 23 

6. TEST MATERIALS ..................................................................................................................................................................... 24 

6.1 SUFFICIENCY OF THE NUMBER OF EVALUATED TEST ITEMS IN VIEW OF THE STUDY OBJECTIVE ..................................................... 24 
6.2 REPRESENTATIVENESS OF THE TEST ITEMS WITH RESPECT TO APPLICABILITY ..................................................................................... 24 

7. WITHIN-LABORATORY REPRODUCIBILITY (WLR) (MODULE 2) ................................................................................ 25 

7.1 ASSESSMENT OF REPEATABILITY AND REPRODUCIBILITY IN THE SAME LABORATORY ....................................................................... 25 
7.2 CONCLUSION ON WITHIN-LABORATORY REPRODUCIBILITY AS ASSESSED BY THE STUDY ................................................................ 25 

8. TRANSFERABILITY (MODULE 3) .......................................................................................................................................... 26 

8.1 QUALITY OF DESIGN AND ANALYSIS OF THE TRANSFER PHASE ................................................................................................................ 26 
8.2 CONCLUSION ON TRANSFERABILITY TO A NAÏVE LABORATORY / NAÏVE LABORATORIES AS ASSESSED BY THE STUDY ........... 26 

9. BETWEEN-LABORATORY REPRODUCIBILITY (BLR) (MODULE 4) ............................................................................. 27 

9.1 ASSESSMENT OF REPRODUCIBILITY IN DIFFERENT LABORATORIES.......................................................................................................... 27 
9.2 CONCLUSION ON BETWEEN-LABORATORY REPRODUCIBILITY AS ASSESSED BY THE STUDY ........................................................... 27 

10. PREDICTIVE CAPACITY AND OVERALL RELEVANCE (MODULE 5) ......................................................................... 28 

10.1 ADEQUACY OF THE ASSESSMENT OF THE PREDICTIVE CAPACITY IN VIEW OF THE PURPOSE ........................................................ 28 
10.2 OVERALL RELEVANCE (BIOLOGICAL RELEVANCE AND ACCURACY) OF THE TEST METHOD IN VIEW OF THE PURPOSE ........... 28 

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11. APPLICABILITY DOMAIN (MODULE 6) ............................................................................................................................ 29 

11.1 APPROPRIATENESS OF STUDY DESIGN TO CONCLUDE ON APPLICABILITY DOMAIN, LIMITATIONS AND EXCLUSIONS ............ 29 
11.2 QUALITY OF THE DESCRIPTION OF APPLICABILITY DOMAIN, LIMITATIONS, EXCLUSIONS ............................................................... 30 

12. PERFORMANCE STANDARDS (MODULE 7) .................................................................................................................... 31 

12.1 ADEQUACY OF THE PROPOSED ESSENTIAL TEST METHOD COMPONENTS ......................................................................................... 31 
12.2 ADEQUACY OF THE PROPOSED REFERENCE CHEMICALS ......................................................................................................................... 31 
12.3 ADEQUACY OF THE PROPOSED PERFORMANCE TARGET VALUES ........................................................................................................... 31 

13. READINESS FOR STANDARDISED USE ........................................................................................................................... 32 

13.1 ASSESSMENT OF THE READINESS FOR REGULATORY PURPOSES ........................................................................................................... 32 
13.2 ASSESSMENT OF THE READINESS FOR OTHER USES .................................................................................................................................. 32 
13.3 CRITICAL ASPECTS IMPACTING ON STANDARDISED USE ........................................................................................................................... 33 
13.4 GAP ANALYSIS ...................................................................................................................................................................................................... 33 

14. OTHER CONSIDERATIONS .................................................................................................................................................. 34 

15. CONCLUSIONS ON THE STUDY......................................................................................................................................... 35 

15.1 ESAC SG SUMMARY OF THE RESULTS AND CONCLUSIONS OF THE STUDY ....................................................................................... 35 
15.2 EXTENT TO WHICH STUDY CONCLUSIONS ARE JUSTIFIED BY THE STUDY RESULTS ALONE ........................................................... 36 
15.3 EXTENT TO WHICH CONCLUSIONS ARE PLAUSIBLE IN THE CONTEXT OF EXISTING INFORMATION ................................................ 36 

16. RECOMMENDATIONS ........................................................................................................................................................... 37 

16.1 GENERAL RECOMMENDATIONS ......................................................................................................................................................................... 37 
16.2 SPECIFIC RECOMMENDATIONS (E.G., CONCERNING IMPROVEMENT OF PROTOCOLS) ...................................................................... 37 

17. REFERENCES ........................................................................................................................................................................... 38 

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ESAC Sub-Group (SG) 

Full title: ESAC Sub-Group on RS Comet and RSMN Assays  

Abbreviated title: ESAC SG RSCOMET-RSMN 

The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) Scientific 
Advisory Committee (ESAC) Sub-Group (SG) was established in February 2023 during the 48th plenary 
meeting of the ESAC (ESAC48) to assist in the production of an ESAC Opinion on the scientific validity 
of the Reconstructed human Skin (RS) Comet and Micronucleus (MN) in vitro test methods for the 
assessment of genotoxicity. 

This  ESAC  SG  Report  on  the  scientific  validity  of  the  RS  Comet  test  method  was  prepared  at  the 
request of EURL ECVAM by the "ESAC Sub-Group on RS Comet and RSMN Assays" (ESAC SG), which 
was charged with conducting a detailed scientific peer review of the external validation studies of the 
RS  Comet  and  RSMN in  vitro  test  methods.  The  basis  for  the  scientific  peer  review  was the  EURL 
ECVAM  Request  for  ESAC  Advice  approved  by  the  ESAC  during  the  ESAC48  plenary  meeting  of 
February 2023 (ESAC request 2023-01). 

The ESAC SG met at the Joint Research Centre (JRC) in Ispra on 22-23/06/2023 and virtually on 13 
and 26/10/2023; 8, 16, 20 and 24/11/2023; 1, 4, 11 and 18/12/2023; 10 and 22/01/2024; 5 and 
14/02/2024; 1 and 22/03/2024; 3 and 6/05/2024, to conduct its peer review. The ESAC SG Report on 
RS Comet was endorsed by the ESAC SG on 23/05/2024 and represents its consensus view. The Report 
was slightly revised and finally endorsed by the ESAC on 24/01/2025 following meetings on 8 and 
20/01/2025. 

The ESAC SG had the following members: 

•  Dr. Rebecca CLEWELL (SG Chair) 

•  Prof. Emanuela CORSINI 

•  Dr. Anne KIENHUIS 

•  Dr. David M. LEHMANN 

•  Prof. Blanca RODRÍGUEZ 

•  Prof. Helmut SEGNER 

•  Dr. Tewes TRALAU 

EURL ECVAM (Secretariat):  

•  Dr. João BARROSO (ESAC Coordinator) 

•  Dr. Raffaella CORVI 

•  Dr. Milena MENNECOZZI 

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ABBREVIATIONS USED IN THE DOCUMENT 

•  2D 
•  3D 
•  ANOVA  
•  BLR 
•  CA 
•  DSB 
•  ESAC 
•  ESAC SG 
•  EURL ECVAM   

•  JRC 
•  MN 
•  MNT 
•  MP 
•  OECD   
•  PM 
•  REACH  
•  RS 
•  SCCS 
•  SG 
•  SOP 
•  SSB 
•  TG 
•  TN 
•  TP 
•  TST 
•  Vs 
•  WLR 
•  WoE 

Two-dimensional 

Three-dimensional 

Analysis of variance 

Between-laboratory reproducibility 

Chromosomal aberration test 

Double-strand break 

EURL ECVAM Scientific Advisory Committee 

ESAC Sub-Group 

European Union Reference Laboratory for Alternatives to 
Animal Testing   
Joint Research Centre 

Micronucleus 

Micronucleus Test 

Misleading Positive 

Organisation for Economic Co-operation and Development  

Prediction Model 

Registration, Evaluation, Authorisation and Restriction of Chemicals 

Reconstructed human Skin 

Scientific Committee on Consumer Safety  

Sub-Group 

Standard Operating Procedure 

Single-strand break 

Test Guideline 

True Negative 

True Positive 

Test Submission Template (the Full Test Submission) 

Versus 

Within-laboratory reproducibility 

Weight-of-Evidence 

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1. Study objective and design 

1.1 Analysis of the clarity of the study objective's definition 

(a) ESAC SG summary of the study objective as outlined in the Test Submission 

The Reconstructed human Skin Comet (RS Comet) assay is an animal-free adaptation of the alkaline 
Comet Assay (OECD TG 489 (OECD, 2016a)) that addresses the potential of a substance to cause 
genotoxicity in the form of DNA strand breaks, which can lead to clastogenic effects, and DNA lesions 
(apurinic/apyrimidinic site) that can lead to gene mutations. The RS Comet assay has been adapted 
to  a  three-dimensional  (3D)  Reconstructed  human  Skin  (RS)  model,  namely  the  Phenion®  Full-
Thickness  Skin  Model  (Henkel,  Dusseldorf,  Germany).  The  model  is  composed  of  human  primary 
keratinocytes and fibroblasts cultured under air-liquid-interface (ALI) conditions on a bovine collagen 
scaffold. The RS Comet assay was initially developed using EpiDermTM EPI-200-MNA, the same used 
for  the  Reconstructed  human  Skin  Micronucleus  (RSMN;  see  RSMN  Sub-Group  report)  but  was 
ultimately  transitioned  to  the  Phenion®  Full-Thickness  Skin  Model,  as  the  use  of  full-thickness  RS 
resulted in fewer non-qualified experiments compared to the 3D human epidermal skin model. 

Genotoxicity  testing  usually  follows  a  tiered  approach,  with  higher  tier  testing  routinely  being 
conducted  in  vivo  as  a  follow  up  of  in  vitro  positive  results.  This  approach  creates  challenges  for 
substances  where  in  vivo  testing  is  banned,  such  as  cosmetic  ingredients.  It  also  limits  testing 
efficiency,  negatively  affects  animal  welfare,  and  fails  to  address  societal  expectations  for  more 
humane testing. 

The RS Comet Test Submission Template (TST) presents the assay as part of a tiered testing strategy 
for the evaluation of genotoxicity following dermal exposure. The respective strategy proposes the 
use of the RS Comet assay as a follow up to confirm or reject a positive Ames and/or mammalian 
cell gene mutation tests, and the RSMN assay (see Annex 3: RSMN Sub-Group Report) as a follow up 
to confirm or reject a positive in vitro micronucleus test (MNT) and/or chromosomal aberration test 
(CA). While this proposed testing battery will not inform the underlying mechanisms of genotoxicity 
or  possible  substance  or  metabolite  absorption,  it  nevertheless  covers  all  possible  types  of  DNA 
damage (i.e., gene mutations, clastogenicity, and aneugenicity). The RS Comet and the RSMN assays 
both  rely  on  3D  RS  models  with  the  proposed  strategy  aimed  at  regulatory  use  in  the  context  of 
cosmetic ingredient testing for genotoxicity associated with skin exposure. However, the tests also 
have  potential  utility  for  the  testing  of  pesticides,  substances  under  Registration,  Evaluation, 
Authorisation  and  Restriction  of  Chemicals  (REACH),  or  pharmaceutical  products,  applied  topically. 
Herein, the ESAC provides a review of both the RS Comet and RSMN TSTs. 

For  chemicals  in  which  dermal  genotoxicity  is  the  toxicological  endpoint  of  concern  following 
application to the skin, the ESAC recommends using both assays as a follow up to any positive result 
in the standard genotoxicity in vitro test battery. A positive result in either the RS Comet or RSMN 
would be sufficient to conclude that the test item is genotoxic. The ESAC recommends starting with 
the RS assay covering the mode of action that tested positive in the standard in vitro test battery and 
only performing the second RS assay in case of a negative result in the first RS assay. 

(b) Appraisal of clarity of study objective as outlined in the Test Submission 

The review of the RS Comet assay by the ESAC relies on data collected and compiled over a period of 
more than 10 years. This approach brings inherent challenges, some of which are clearly reflected in 
the TST. For example, the remit of the validation study, as well as its design, were subject to several 
adjustments over time (see below). 

The  Test  Submitters  were  able  to  retrospectively  address  most  of  these  issues,  although  some 
uncertainties remain about the reasoning behind some of the test substance selections as well as the 
justification  for  some  of  the  protocol  adjustments.  Regarding  the  chemical  space  covered  in  the 
validation studies, the TST would have benefitted from additional details related to the underlying 

ANNEX 2 - ESAC SUB-GROUP REPORT ON RS COMET 

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mechanisms, substance class representation, and stability of substances selected for the validation 
study.  For  example,  only  a  small  number  of  substances  included  in  the  validation  study  require 
bioactivation  (i.e.,  7  of  which  3  were  also  evaluated  during  assay  development  and  optimization). 
Furthermore, the strategy for chemicals selection has not been clearly described or justified relative 
to the context of use described in the TST or in the supporting documents. To this point, there appears 
to be a disconnect between the proposed purpose of the assay (i.e., confirmation of positive results 
obtained in the standard in vitro genotoxicity battery) and the chemicals selection strategy. A balanced 
set of true positive (TP), true negative (TN), and misleading positive (MP) substances would seem to 
be more appropriate for a standalone use of the methods rather than the proposed use of confirming 
positive results. These limitations are addressed in later sections of this report (see subsections 6.2 
and 11.2). 

Nevertheless, the TST is clear about its general objective and contains the necessary controls and 
data,  the  majority  of  which  is  published  and  publicly  available,  to  facilitate  review  by  the  ESAC. 
Further, while a more context-specific test item set would be preferred, the substances tested during 
the validation study  included  TP, TN, and  MP.  Likewise, metabolic competency was tested using a 
selection of chemicals that require bioactivation.  Thus, the TST contains sufficient information for 
evaluation  of  the  RS  Comet  assay  and  clearly  outlines  the  potential  regulatory  benefit  of  its 
application. 

1.2 Quality of the background provided concerning the purpose of the test method 

The  TST  adequately  describes  the  purpose  of the  assay  (i.e.,  follow-up test  for  substances testing 
positive in the Ames and/or mammalian cell gene mutation tests, and for which genotoxicity was the 
toxicological endpoint of concern following topical application to the skin). The method design concept 
involved  merging  conventional  genotoxicity  parameters  with  existing  3D,  RS  technology.  This 
integration led to the development of the "3D" RS Comet assay, discussed in more detail here, and an 
RSMN assay (see Annex 3: RSMN Sub-Group Report). The combined use of these assays is considered 
suitable  for  investigating  positive  results  obtained  from  standard  two-dimensional  (2D)  in  vitro 
genotoxicity assays (i.e., Ames test, mammalian cell gene mutation test, MNT and CA). Depending on 
the outcomes of the standard in vitro battery, which typically covers all genotoxicity endpoints (i.e., 
gene  mutation,  clastogenicity,  and  aneugenicity),  a  choice  would  be  made  between  the  RS  Comet 
assay or RSMN assay (or both). 

In  recent  decades,  the  use  of  animal  testing  for  chemicals  has  come  under  increased  scrutiny, 
prompting a global shift away from such studies due to concerns for animal welfare. Reflecting this, 
the  European  Union  implemented  the  7th  Amendment  to  the  Cosmetics  Directive  of  the  European 
Commission in 2003, prohibiting the use of in vivo follow-up testing for cosmetic ingredients since 
March 2009. In the context of a 'test battery,' this implies that a positive outcome from an in vitro 
standard  genotoxicity  assay  would  render  the  ingredient  unsuitable  for  use  in  cosmetic  products. 
Contemporary OECD genotoxicity Test Guidelines (TGs), such as OECD TGs 474 (OECD, 2016b), 488 
(OECD, 2022), and 489 (OECD, 2016a), now underscore the importance of considering the intended 
or expected route of human exposure. Additionally, there is a growing emphasis on testing the 'site-
of-contact,' as outlined in OECD TGs 488 (OECD, 2022) and 489 (OECD, 2016a). This is particularly 
relevant  for  ingredients  in  cosmetics,  household  products,  pesticides,  where  the  primary  site  of 
exposure is often the skin. All these points support the development and application of the RS Comet 
and RSMN assays. 

(a) Analysis of the scientific rationale provided in the Test Submission 

While the RS Comet assay TST provides a cursory overview of the scientific rationale for test method 
development, the submission relies heavily on information previously published in scientific journals 
by the Test Submitters and the corresponding standard operating procedure (SOP) for the method. 
This format for a TST poses substantial challenges for reviewers because many information sources 

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must be reviewed to properly assess the test method under consideration. Future submissions would 
benefit from including a  summary  of  key information on  scientific  rationale,  regulatory  need,  and 
assay performance in the main TST document. Nonetheless, the submission package, which includes 
the  TST  and  several  informative  publications,  provide  the  necessary  scientific  rationale  for  the 
validation of this assay. 

The neutral Comet assay is a single cell gel electrophoresis assay used to detect DNA double strand 
breaks (DSBs). The alkaline version of the Comet assay can detect both single strand breaks (SSBs) 
and DSBs. Thus, the alkaline Comet assay is a more comprehensive measure of chemical-induced 
DNA damage. Strand breaks may result from direct interaction of the test chemical with DNA, as well 
as incomplete DNA repair or can occur at alkali labile sites. In its alkaline version, the Comet assay 
covers DNA damage which may lead to clastogenic damage and potentially gene mutation lesions. 
Notably,  and  although  mutagenicity  might  be  the  result  of  a  variety  of  mechanisms,  the  alkaline 
Comet Assay is reported to have a high predictive capacity for this endpoint (Kirkland et al., 2008). 

Apart from its ease of use, all versions of the Comet assay have the inherent technical advantage of 
straightforward adaptability to a variety models (e.g., in vivo, in vitro). Plus, the rodent in vivo version 
(OECD TG 489 (OECD, 2016a)) has regulatory recognition. Adapting the Comet assay for use with 
human-derived 3D skin models for in vitro genotoxicity testing therefore, not only appears to be a 
straightforward choice for overcoming any species-specific differences but is also logical in terms of 
potential regulatory applicability as a component of tiered testing. The published literature included 
in  the  TST  makes  a  compelling  case  for  the  RS  Comet  assay  being  sufficiently  metabolically 
competent and thus a biologically fit for purpose assay - for non-systemic dermal genotoxicity testing. 
Furthermore, the air-liquid-interface (ALI) culture conditions of the RS Comet assay facilitates testing 
of a wide range of substances, including lipophilic substances at dose ranges relevant for topical skin 
exposure or application. 

(b) Analysis of the regulatory rationale provided in the Test Submission 

The regulatory rationale is described clearly in the TST. The need to develop reliable in vitro tests for 
genotoxicity  testing  associated  with  skin  exposure  was  predominantly  triggered  by  the  European 
animal  testing  ban  for  cosmetics  in  conjunction  with  inherent  challenges  of  alternative  testing 
systems, one being the limited ability of existing cell-based in vitro methods to deliver sufficiently 
reliable predictions (e.g., elevated number of false positives). In addition, existing cell-based methods 
(i.e., MNT, CA, Ames test, and mammalian cell gene mutation tests) utilize rat liver S9 fraction to test 
for  bioactivation  of  chemicals,  which  has  an  obvious  limitation  in  the  assessment  of  human  skin 
genotoxicity. Citations provided in the TST (Pfuhler et al., 2020; Wiegand et al., 2014) along with those 
identified by the ESAC (Bataillon et al., 2019; Pfuhler et al., 2021) discuss the difference in metabolic 
enzyme expression in skin vs. liver and in rat vs. human, which provides a strong argument for the 
development a metabolically competent skin genotoxicity assay for assessment of human risk. These 
citations also provide evidence that the RS Comet assay uses a metabolically competent human skin 
model. 

The RS Comet assay is intended to be used within regulatory genotoxicity hazard identification testing 
strategies to follow up positive results from the classical in vitro test battery, which is used as a first 
step. It is proposed to be used instead of in vivo genotoxicity test methods (OECD TG 489 (OECD, 
2016a) in the context of test items that are causing genotoxicity in the form of DNA strand breaks in 
vitro and are primarily associated with dermal exposure. 

The TST makes a clear case for the performance of the test method and for the potential use of the 
RS  Comet  assay  as  a  standalone  test  for  detecting  genotoxicity  associated  with  skin  exposure. 
However,  as  presented,  the  TST  does  not  address  concerns  associated  with  potential  future 
applications. For example, the ESAC sees the potential for using the combination of the RS Comet and 
RSMN assays as a substitute for in vivo genotoxicity assays and questions whether this use scenario 
will  sufficiently  address  potential  mutagenicity,  or  systemic  effects  from  skin  penetration  and 
absorption. Importantly, the testing strategy is only being evaluated by the ESAC for the context of 

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use, in which the RS Comet assay is used in combination with a RSMN evaluation for in vitro skin 
genotoxicity testing following topical exposure (see section 13). 

1.3 Appraisal of the appropriateness of the study design 

The TST compiles data from several published studies, thereafter, compiled to support the validation 
of the RS Comet assay. This, together with the protracted timeline associated with completing the 
validation  study,  introduces  challenges  such  as  protocol  modifications,  change  of  participating 
laboratories, and varying numbers of technical and biological replicates. While a more targeted and 
concise approach  would  have been preferable, the overall design seems  adequate  with respect to 
critical  parameters,  including  study  design,  laboratory  independence,  blinded  sampling  and 
independent result evaluation, all being sufficiently fulfilled. Moreover, the submitted SOP provides 
clear guidance on experimental design, result evaluation and substance calls. 

1.4 Appropriateness of the statistical evaluation 

The statistical methods used in the evaluation of the validation study data are standard, well-vetted 
methodologies and therefore appropriate for the purpose of evaluating the assay. However, no power 
calculation  to  support  the  number  of  chemicals  necessary  to  assess  the  Within-Laboratory 
Reproducibility (WLR) or the Between-Laboratory Reproducibility (BLR) has been performed. 

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2. Collection of existing data 

2.1 Existing data used as reference data 

The in vitro test battery data and in vivo reference data were collected from the literature and the 
EURL ECVAM Genotoxicity and Carcinogenicity Consolidated Database (Kirkland et al., 2016; Corvi et 
al., 2018; Madia et al., 2020). 

The  RS  Comet  assay  data  that  were  used  for  the validation/prediction  model (PM)  were  produced 
during Phases 1 and 2 of the validation study. The results of Phase 1 were published by Reisinger et 
al. (2018). The results of Phase 2 were published by Pfuhler et al. (2020). There are earlier publications 
which present data from the RS Comet assay development (e.g., Reus et al., 2013), but these data 
were not used for the validation/PM. No data from other sources were used in the validation report. 

2.2 Existing data used as testing data 

Not applicable. All data were the result of prospective testing. 

2.3 Search strategy for retrieving existing data 

Genotoxicity data were accessed from published literature and the EURL ECVAM database (Kirkland 
et al., 2016; Corvi et al., 2018; Madia et al., 2020). However, the search strategy was not specified in 
the TST. 

2.4 Selection criteria applied to existing data 

The selection criteria were based on the EURL ECVAM Genotoxicity and Carcinogenicity Consolidated 
Database. 

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3. Quality aspects relating to data generated during the study 

3.1 Quality assurance systems used when generating the data 

It was stated in subsection 3.5 of the TST that the acquisition of the data for the assessment of the 
reproducibility and predictive capacity did not follow any formal quality assurance system. Full Good 
Laboratory Practice (GLP) compliance is not a requirement for test method development or validation. 
The Test Submitters agreed to follow the safeguards recommended by Balls et al. (1995). The ESAC 
agrees with the quality assurance measures taken in this validation study. 

3.2 Quality check of the generated data prior to analysis 

As noted in the TST, acquisition of the data for the assessment of the reproducibility and predictive 
capacity  did  not  follow  any  formal  quality  assurance  system.  However,  several  safeguards 
recommended by Balls et al. (1995) were applied, which the ESAC considers acceptable. 

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4. Quality of data used for the purpose of the study (existing 
and newly generated) 

4.1 Overall quality of the evaluated testing data (newly generated or existing) 

The quality of the data provided is high. Data are properly reported and appropriate quality criteria 
for analysing the data were provided. All underlying data used for analyses were provided with the 
TST. 

4.2 Quality of the reference data for evaluating relevance1 

The Test Submitters  used reference  chemicals supported  by  the literature and documented in the 
EURL ECVAM Genotoxicity and Carcinogenicity Consolidated Database. 

4.3 Sufficiency of the evaluated data in view of the study objective 

The strategy for test chemicals selection has not been clearly described or justified relative to the 
context of use described in the TST or the supporting documents. A balanced set of TP, TN, and MP 
would seem to be more appropriate for a standalone use of the method rather than confirmation of 
positives  obtained  using  traditional  genotoxicity  assays  as  was  the  proposed  context  of  use. 
Nonetheless, the ESAC considers the quality of the RS Comet dataset sufficient to draw conclusions 
on assay validity (see subsections 4.1 and 4.2). 

1 OECD guidance document Nr. 34 on validation defines relevance as follows: "Description of relationship of the 
test to the effect of interest and whether it is meaningful and useful for a particular purpose. It is the extent to 
which  the  test  correctly  measures  or  predicts  the  biological  effect  of  interest.  Relevance  incorporates 
consideration of accuracy (concordance) of a test method." 

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5. Test definition (Module 1)  

5.1 Quality and completeness of the overall test definition 

Information provided regarding the biological and mechanistic relevance of the method is considered 
sufficient. When using the RS Comet assay, test items are assessed in a commercially available 3D 
RS  model  (Phenion®  Full-Thickness  Skin  Model).  The  RS  Comet  assay  is  biologically  relevant  for 
investigating DNA damage in the skin after topical exposure. 

A  detailed  SOP  was  provided  by  the  Test  Submitters  (see  Attachment  1.1  provided  with  the  TST) 
together  with  supporting  published  papers,  clearly  describing  the  test  system  used,  the  endpoint 
measured, the quality criteria applied to the test system, the acceptance criteria applied to the results, 
and the positive and negative controls. Over the years, several modifications of the protocol have 
been  introduced  to  optimise  it,  including  single  cell  isolation,  microscopical  analysis,  experimental 
design,  PM,  cytotoxicity  assessment  and  DNA  damage  read-outs.  The  protocol  recommended  for 
future use is the version reported in Attachment 1.1 provided with the TST. The positive control in 
standard  experiments  is  methyl  methane  sulfonate  at  a  concentration  of  5  μg/cm2,  dissolved  in 
acetone.  In  experiments  using  the  DNA  repair  inhibitor  aphidicolin,  which  are  required  to  confirm 
negative results (Brinkmann et al., 2013; Reisinger et al., 2018), the positive control is benzo(a)pyrene 
at a concentration of 12.5 μg/cm2, dissolved in acetone. The RS Comet assay measures three readouts: 
one related to DNA damage and two cytotoxicity readouts (intracellular ATP and adenylate kinase 
leakage) after topical exposure over a period of 48 h. Test item concentrations exceeding those cut-
off values for toxicity are not assessed for DNA damage. 

For data analysis and data interpretation (paragraph 2.1.6, point of the TST), the Test Submitters have 
considered the procedure as applied for the in vivo study (OECD TG 489 (OECD 2016a)). Although two 
different PMs were explored during the validation study (Reisenger et al., 2018; Pfuhler et al., 2020), 
the data presented in the TST were analysed with one PM (PM1: an ANOVA followed by the one-sided 
Dunnett test) since it was stated by the Test Submitters that both PMs performed comparably well. 

5.2 Quality and completeness of the documentation concerning protocols and prediction 
models 

The SOP of the RS Comet assay evolved over several years and is supported by several publications. 
The SOP is well-described, including a description of the purpose, materials, study design, and assay 
procedure. It includes a clear data evaluation section which describes data processing, validity criteria, 
and evaluation of the results. 

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6. Test materials 

6.1 Sufficiency of the number of evaluated test items in view of the study objective 

The main selection criterion for the initial selection of chemicals appears to be based on matched 
data from in vivo rodent skin exposure studies. This approach yielded a limited list of test chemicals 
that was later expanded to enlarge the substance pool for statistical and validation purposes, as well 
as to include more data rich substances. Attachment 9 provided with the TST included information on 
basic physicochemical properties, structural information, and a full list of test substances. There were 
no statistical tests conducted at study initiation to justify the number of chemicals selected for the 
validation  study.  Nevertheless,  the  number  of  chemicals  tested  seems  sufficient  to  assess  the 
capacity of the method to detect expected  modes of  action  and  to evaluate  reproducibility  of the 
assay. The resulting list is fit for purpose in terms of the number of substances tested for assessing 
WLR and BLR. The ESAC noted that only a limited number of chemicals was selected for evaluating 
metabolic activation. 

6.2 Representativeness of the test items with respect to applicability 

The test items were selected for the validation study according to their description as TN, TP, and MP. 
The ESAC considers the test items sufficiently representative for the purpose of the study. Information 
was also provided on the genotoxic mode of action (e.g., alkylating agent). It should be noted that the 
TST  does  not  make  any  statements  related  to  defining  the  applicability  domain  or  the 
representativeness of substance class/underlying mechanisms and coverage of technical parameters 
such as lipophilicity/solubility and stability. 

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7. Within-laboratory reproducibility (WLR) (Module 2) 

7.1 Assessment of repeatability and reproducibility in the same laboratory 

The  WLR  of  the  RS  Comet  assay  was  assessed  during  two  phases  of  the  validation  study  and  is 
described in two separate publications (Reisinger et al., 2018; Pfuhler et al., 2020). 

A total of 24 test items (8 from Phase 1 and 16 from Phase 2) were used to demonstrate WLR. While 
the number seems reasonable, no power calculation to determine the number of chemicals necessary 
to assess the WLR or the BLR has been performed. In addition, none of the 5 laboratories participating 
in the validation study tested all 24 chemicals (see Table 1). 

The test items used in the validation study are listed in Attachment 4 provided with the TST together 
with  their  CAS  numbers,  commercial  source,  purity,  physical  form,  and  relevant  physical/chemical 
properties. For those items, a range of toxic effects (toxic, non-toxic) and various modes of action are 
reported by the Test Submitters. 

Results for WLR (excluding experiments which were deemed invalid due to protocol validity criteria, 
not expert judgement as described in Attachment 4 provided with the TST) are as follows with the 
caveat that most of calculations are based on data from only 2 experiments per test item (see Table 
1). 

Table 1. Reproducibility within each laboratory over time (WLR) in Phases 1 and 2 

Discordant 

Concordant 

Total 

% 

Lab A 

Lab B 

Lab C 

Lab D 

Lab E 

All labs 

0 

1 

0 

1 

0 

2 

7 

7 

3 

3 

3 

7 

8 

3 

4 

3 

23 

25 

100 

87 

100 

75 

100 

91 

Some  potential  sources  of  variability  (e.g.,  test  substance  precipitation,  background  levels,  dose 
selection) were described by the Test Submitters, but measures to control them were not proposed. 
Outlying values for cadmium chloride, 7,12-Dimethylbenz[a]-anthracene, and 2-acetylaminofluorene 
were identified from the results obtained in the WLR study. Reasons for these outlying values were 
discussed in the TST. 

7.2 Conclusion on within-laboratory reproducibility as assessed by the study 

Typically, three independent experiments are required for the assessment of WLR. For the RS Comet 
assay, most test items used by the Test Submitters to assess WLR have only been evaluated in two 
experiments. Only three chemicals had three replicates (i.e., 7,12-dimethylbenz[a]-anthracene and Di-
(2-ethylhexyl)phthalate in Lab A and cyclohexanone in Lab B). Results are concordant in those three 
cases and exhibit high WLR based on three experiments. Given these results, the ESAC considers there 
is some uncertainty in the conclusion of the WLR based on two experiments only. 

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8. Transferability (Module 3)  

8.1 Quality of design and analysis of the transfer phase 

A detailed SOP has been provided as Attachment 1.1 to the TST. The SOP submitted clearly describes 
the  test  system  used,  the  endpoint  measured,  the  quality  criteria  applied  to  the  test  system,  the 
acceptance criteria applied to the results, and the positive and negative controls. 

Transferability  was  carried  out  using  only  one  test  item:  methyl  methane  sulfonate,  the  positive 
control. All the laboratories participating in the validation study demonstrated successful transfer of 
the  experimental  procedures  of  the  RS  Comet  assay  in  two  parallel  preceding  projects  using  this 
chemical. The physical and chemical properties of methyl methane sulfonate and test results for this 
chemical were reported in Attachment 9 provided with the TST. 

From the information provided by the Test Submitters, it appears that the RS Comet assay can be 
transferred to laboratories with competencies in the alkaline Comet assay in accordance with OECD 
TG 489 (OECD, 2016a).  The Test Submitters claimed  that standard technical laboratory  skills and 
expertise in interpreting results of genotoxicity test methods are sufficient to conduct the RS Comet 
assay. No specific training is, therefore, recommended by the Test Submitters. However, it is clear 
from the associated publications (e.g., Pfuhler et al., 2020) that expert judgement is a critical aspect 
of results interpretation when using the RS Comet assay. For that reason, the ESAC recommends the 
use of this assay in laboratories experienced in the evaluation of Comet assay data (in vivo or in vitro) 
or to gain experience through testing of compounds with known activity in the Comet assay prior to 
testing unknown chemicals (i.e., Proficiency testing). 

Recommendations  are  provided  for  critical  aspects  of  the  SOP  (e.g.,  reagent  purity,  protecting 
Phenion® Full-Thickness Skin Model from direct UV light). Five consecutive experiments with methyl 
methane  sulfonate  or  benzo(a)pyrene  are  suggested  for  a  naïve  laboratory  with  no  previous 
experience in RS Comet assay. 

Successful training should be demonstrated by testing at least one positive or, preferably, two positive 
and  one  negative  test  items  not  previously  tested,  following  the  entire  protocol,  i.e.,  from  the 
determination  of  solubility  to  the  final  judgement  of  the  result.  The  Test  Submitters  recommend 
selecting the test item(s) for training from the list of those tested in the validation exercise. 

8.2  Conclusion  on  transferability  to  a  naïve  laboratory  /  naïve  laboratories  as 
assessed by the study 

The  SOP  supporting  the  transfer  to  laboratories  with  competencies  in  alkaline  Comet  Assay  is  in 
accordance  with  OECD  TG  489  (OECD,  2016a).  Transferability  was  assessed  with  one  test  item 
(methyl methane sulfonate, the positive control). All five laboratories participating in the validation 
study  demonstrated  successful  transfer  in  previous  studies.  The  Test  Submitters  recommend 
demonstrating successful transfer of the test to all new laboratories by testing more than one positive 
test item with five consecutive experiments (or preferably one positive and one negative test item). 
The  five  experiments  must  meet  the  test  validity  criteria  and  must  show  clear  increases  in  DNA 
migration. 

The ESAC’s opinion is that one test item is not enough to demonstrate transferability. More than one 
test item is required to increase confidence in the method, and this should include two positive and 
one negative test items. 

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9. Between-laboratory reproducibility (BLR) (Module 4) 

9.1 Assessment of reproducibility in different laboratories 

The  BLR  was  assessed  testing  8  test  items  (i.e.,  cadmium  chloride,  N-ethyl-N-nitrourea,  7,12-
Dimethylbenz[a]-anthracene, propyl gallate, eugenol, Di-(2-ethylhexyl)phthalate, cyclohexanone, and 
mitomycin  C)  in  five  laboratories.  See  Attachments  8  and  9  provided  with  the  TST  for  additional 
information. 

The BLR of the RS Comet assay was assessed by the Test Submitters and reported to be equal to 
87.5% (consistent with information presented in Attachment 8 provided with the TST). 

Cadmium  chloride  did  not  produce  reproducible  positive  responses.  Given  the  well-known 
biochemistry of cadmium chloride (i.e., non-specific binding), it is not uncommon to obtain variable 
results. 

Seven test items had concordant calls in three laboratories and one test item, cadmium chloride, was 
positive in two laboratories and negative in one. This resulted in a BLR estimate of 7/8 = 87.5%. The 
results are summarised in Attachment 8 provided with the TST. 

Potential  experimental  sources  of  variability  that  can  result  in  reduced  BLR  primarily  relate  to 
experiments  with  borderline  results  and  differences  in  dose  selection  as  well  as  differences  in 
chemical-cytotoxicity  levels  between  laboratories.  These  factors  may  also  explain  why  some 
substances  are  positive  in  main  experiments  performed  in  some  laboratories  and  positive  in 
experiments where aphidicolin was included to increase sensitivity in other laboratories, as observed 
for 7,12-dimethylbenz[a]-anthracene in the validation study. 

9.2 Conclusion on between-laboratory reproducibility as assessed by the study 

Typically, three independent laboratories are required for the assessment of BLR. In the RS Comet 
assay, five laboratories tested 8 test items (with at least 3 laboratories out of the 5 involved in each). 
Concordant results were obtained with 7 test items in three laboratories, whereas cadmium chloride 
provided  discordant  results  in  one  laboratory  versus  the  other  two,  with  no  obvious  explanation 
provided. Based on these results, BLR was high (i.e., 87.5%). 

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10. Predictive capacity and overall relevance (Module 5) 

10.1 Adequacy of the assessment of the predictive capacity in view of the 
purpose 

Based on the materials provided by the Test Submitters, it is unclear if there is a Lead Lab. 

Overall, a limited number of chemicals (i.e., 32 test items) were used to assess the predictive capacity 
of  the  RS  Comet  assay  (Attachment  9  provided  with  the  TST).  Information  on  mode  of  action, 
reference  in  vitro  and  in  vivo  genotoxicity  data,  as  well  as  carcinogenicity  data  are  reported  in 
Attachment 10 of the TST. The 32 chemicals comprised 8 liquids and 24 solids, which all had a low 
to very low vapour pressure. The chemicals were, except for cadmium chloride, organics representing 
various chemical classes and featuring numerous functional groups. To explore the potential of the 
test  method  to  detect  pro-mutagens,  3  pro-mutagenic  chemicals  (i.e.,  benzo(a)pyrene,  2-
acetylaminofluorene  (Lab  B),  and  2,4-diaminotoluene  (Lab  B))  were  used  in  the  development  and 
optimisation  of  RS  Comet  assay.  However,  7  pro-mutagenic  chemicals  were  tested  during  the 
validation  study  (cyclophosphamide  (Lab  B),  benzo(a)pyrene  (Lab  A),  7,12-dimethylbenz[a]-
anthracene (Labs A, B, and C), 2-acetylaminofluorene (Lab B), 2,4-diaminotoluene (Lab B), 2-amino-
3-methylimidazo[4,5-f]quinolone (Lab A), and 4-chloroaniline (Lab B)). Of the 15 test items used as 
TP, 7 test items needed metabolic activation to show positive results (Attachment 10 provided with 
the  TST).  Chemicals  were  initially  selected  by  external  experts  from  a  master  list  prepared  by 
Cosmetics Europe. As discussed above, test items cover different modes of action and a range of 
toxic effects. 

The  predictive  capacity  of  the  RS  Comet  assay  was  calculated  based  on  3  different  scenarios 
(paragraph  2.5.2  of  the  TST).  Data  from  5  laboratories  were  combined,  with  a  maximum  of  18 
chemicals tested in a single laboratory (Lab B). In the validation study, 8 test items (5 false negative 
and 3 false positive or equivocal prediction) were not correctly predicted in at least one laboratory. 

Based on the PMs, e.g., for scenario A, sensitivity among the different laboratories ranged from 25% 
(Lab D) to 100% (Labs C and E) and specificity from 86% (Lab A) to 100% (Labs C, D, and E), with an 
overall accuracy (mean) > 83%. The low sensitivity of Lab D is due to the limited number of chemicals 
tested (n = 2). For Lab B, which tested the highest number of chemicals (i.e., 18): sensitivity was 80%, 
specificity 87%. The low sensitivity in Lab D may indicate a possible problem in the implementation 
of the method. The ESAC notes that only one chemical was used in the transfer phase, which may be 
not sufficient. 

Only one TP that needed metabolic activation (i.e., DMBA) showed a consistent positive study outcome 
in three different laboratories. All other metabolic active TPs were tested in only one laboratory and 
showed either a positive study outcome (i.e., 2-AAF, BaP, and CP) or a negative study outcome (i.e., 
2,4-DAT, 4-Chloroaniline, and IQ). These results suggest that not enough data is provided to conclude 
on the applicability of the RS Comet assay to test metabolic active genotoxic compounds.  

The RS Comet assay, with its promising predictive capabilities, has potential utility for identification 
of genotoxic hazard. To that point, Lab B, which tested 18 chemicals, demonstrated a sensitivity and 
specificity  higher  that  80%.  However,  the  validation  study  suffers  from  the  lack  of  an  a  priori 
validation design that could have improved the quality of the study and the evaluation of predictive 
capacity. 

10.2 Overall relevance (biological relevance and accuracy) of the test method in 
view of the purpose 

The overall relevance and accuracy of the RS Comet assay as a confirmation of a positive result in 
the Ames and/or mammalian cell gene mutation tests following dermal exposure is adequate. 

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11. Applicability domain (Module 6) 

11.1 Appropriateness of study design to conclude on applicability domain, 
limitations and exclusions 

For  organizational  purposes,  the  ESAC  discusses  the  strengths  and  limitations  of  three  different 
aspects of the applicability domain below. 

Biological domain: As proposed in the TST, the RS Comet assay would be used for screening chemicals 
for potential genotoxicity resulting from dermal application of the chemical. The test was designed 
primarily for the purpose of screening cosmetic ingredients and is therefore focused on the relevant 
portal of entry (i.e., skin). Use of 3D RS allows for a more biologically relevant tissue architecture, 
which is more likely to recapitulate in vivo chemical penetration and toxicological response. Based on 
the  evidence  provided  in  the  TST,  the  ESAC  concurs  that  the  assay  is  useful  for  the  purpose  of 
identifying  chemicals  with  the  potential  to  cause  genotoxicity  following  dermal  application  –  in  a 
tiered testing paradigm that includes the standard in vitro genotoxicity assays and the RSMN assay 
as  described  in  section  1.  However,  for  the  broader  purpose  of  human  health  risk  assessment, 
additional evidence would be required to make a regulatory decision, as this assay lacks the ability 
to account for potential systemic effects from dermal exposure. 

Chemical domain: The applicability domain of the test items was not specifically explored in the TST. 
However, there are clear advantages to the use of a 3D RS model that is cultured at the ALI compared 
to traditional genotoxicity assays performed in submerged 2D monocultures, particularly in terms of 
the  potential  to  increase  the  chemical  applicability  domain.  The  RS  Comet  assay  utilizes  a  full-
thickness skin model that also has the potential to be useful for identifying metabolically activated 
chemicals in a human-relevant context. This attribute is a significant advantage over immortalized or 
cancer cell line-based assays currently used in the traditional 2D submerged assays that rely on rat 
liver S9 fraction to approximate human skin metabolism. 

the 

remaining 

Evidence  of  metabolic  competence  and  ability  of  the  RS  Comet  assay  to  identify  bioactivated 
genotoxicants is provided in the TST and referenced publications (e.g., Reisinger et al., 2018; Pfuhler 
et al., 2020; Brinkmann et al., 2013; Reus et al., 2013). Out of 16 test items used as TP chemicals, 7 
that  require  bioactivation  were  evaluated  during  the  validation  study.  Of  those,  4  were  correctly 
identified  as mutagenic  (i.e.,  benzo(a)pyrene,  cyclophosphamide,  7,12-Dimethylbenz[a]-anthracene, 
and  2-acetylaminofluorene)  whereas 
(i.e.,  2-amino-3-
methylimidazo[4,5-f]quinoline, 2,4-Diaminotoluene, and 4-chloroaniline). However, it should be noted 
that  benzo(a)pyrene  (Brinkmann  et  al.,  2013)  and  2,4-diaminotoluene  (Reus  et  al.,  2013)  were 
correctly  classified  during  assay  development  and  optimization  while  the  results  for  2-
acetylaminofluorene  were  not  published  or  included  in  the  TST.  In  addition,  because  the  model 
involves culturing at the ALI, a broader universe of chemicals and substances that are not generally 
well-tolerated  by  submerged  in  vitro  assays  can  be  tested  (e.g.,  lipophilic  and  otherwise  poorly 
dissolved in cell culture medium). Furthermore, complex mixtures and formulations in various vehicles 
(creams, emulsions, etc.) could potentially be tested in a skin model cultured at the ALI. Since the TST 
did not specifically evaluate lipophilic chemicals or product formulations, no conclusions can be drawn 
on the utility of this assay for such purposes. However, the design of the assay suggests the potential 
to  expand  beyond  the  limited  chemical  space  evaluated  in  the  validation  study,  which  defines  all 
submerged 2D in vitro systems. 

three  were  not 

Mechanistic  domain:  Regarding  the  chemical  space  covered  by  the  chemicals  evaluated  in  the 
validation  study,  the  TST  would  have  benefitted  from  additional  details  regarding  underlying 
mechanisms, substance class representation, and test chemical stability. Hence, there remain some 
uncertainties regarding the reasoning of some of the substance selections. A recently developed AOP 
for genotoxicity (Sasaki et al., 2020) provides a streamlined, science-backed series of key events for 
various mechanisms of chemical-induced genotoxicity and would be a valuable resource for better 
defining the domain of applicability for this assay in terms of mode of action. 

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In conclusion, only a very limited number of chemicals have been tested (i.e., n = 32) and information 
related to the genotoxic mode of action provided in the TST was also limited. For that reason, the 
applicability domain of the RS Comet assay will need to be evaluated further along with its potential 
for broader use. 

11.2 Quality of the description of applicability domain, limitations, exclusions 

This study did not attempt to define the applicability domain in terms of chemical space. As such, this 
cannot be evaluated by the ESAC. 

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12. Performance standards (Module 7) 

12.1 Adequacy of the proposed Essential Test Method Components 

Not applicable. 

12.2 Adequacy of the proposed Reference Chemicals 

Not applicable. 

12.3 Adequacy of the proposed performance target values 

Not applicable. 

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13. Readiness for standardised use 

13.1 Assessment of the readiness for regulatory purposes 

The RS Comet assay should be considered together with the RSMN assay, as they are intended to be 
used within tiered regulatory genotoxicity hazard identification testing strategies to follow up positive 
results from the classical in vitro test battery. The RS Comet assay is proposed to be used instead of 
in vivo genotoxicity test methods (e.g., OECD TG 489 (OECD, 2016a)), in the context of test items that 
are causing DNA strand breaks in vitro and are primarily associated with the dermal exposure route. 
In combination with the RSMN assay, they cover all genotoxicity endpoints that usually need to be 
addressed for regulatory purposes (gene mutation, clastogenicity, and aneugenicity). 

The approach proposed by the Test Submitters involves conducting the RS Comet assay as a follow-
up to a positive Ames and/or mammalian cell gene mutation tests and a negative result in the RS 
Comet assay would support overriding the initial positive result. Similarly, a positive in vitro MNT or 
CA would trigger a follow-up with the RSMN assay, and a negative result in the RSMN would support 
overriding  the  initial  positive  result.  According  to  the  Test  Submitters,  in  cases  where  both  Ames/ 
mammalian  cell  gene  mutation  tests  and  MNT/CA  studies  yield  positive  results,  follow-up  testing 
should be conducted with both the RS Comet and RSMN assays, and a positive follow-up result in 
either the RS Comet assay or the RSMN assay would lead to the conclusion of a genotoxicity hazard 
associated with topical exposure. 

As a precautionary note, test items requiring metabolic activation may be classified as false negatives 
based on RS genotoxicity tests alone. This is particularly crucial when these compounds are accessible 
for systemic absorption. Considering the skin’s lower metabolic capacity, initial results might appear 
negative. However, once absorbed, chemicals could undergo systemic bioactivation, thereby elevating 
the risk of neoplasia, which is an unacceptable outcome. 

The 12th Revision of the Scientific Committee on Consumer Safety (SCCS) Notes of Guidance for the 
Testing  of  Cosmetic  Ingredients  and  Their  Safety  Evaluation,  concluded that  (Pfuhler  et  al.,  2020; 
Pfuhler et al., 2021; SCCS, 2023): 

a.  3D  tissue  models  simulate  in  vivo-like  conditions  including  cell  viability,  proliferation, 
differentiation,  morphology,  gene  and  protein  expression.  These  models  can  complement 
classical 2D cell culture-based assays; 

b.  3D tissue-based genotoxicity assays can be used as 2nd tier assays to follow-up on positive 

results from standard in vitro assays; 

c.  For adoption of a tissue model as a 2nd tier assay, ability to detect the full range of genotoxic 
damage (leading to mutagenicity, clastogenicity, aneugenicity) should be demonstrated; 

d.  The 72-h protocol for the RSMN has higher sensitivity than the 48-h protocol; 
e.  The RS Comet assay (Pfuhler et al., 2020) and RSMN (Pfuhler et al., 2021) assays are now 
sufficiently validated to move towards individual OECD TGs, but an independent peer review 
of the validation study is still needed. 

13.2 Assessment of the readiness for other uses 

For the intended purpose, no mechanistic limitations of the RS Comet assay are known. Since only a 
very limited number of chemicals have been tested (n = 32), its applicability for screening purposes 
will need to be evaluated further. The applicability to mixtures and final formulations has not been 
demonstrated yet. 

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13.3 Critical aspects impacting on standardised use 

In the validation study, which was conducted over a protracted period of time, the composition of the 
participating laboratories changed. While results are promising, the low sensitivity (i.e., 25%) obtained 
in Lab D should not be minimised as it may indicate possible problems in the implementation of the 
method. In the transfer phase only one chemical was used, which may be not sufficient. 

The  method  has  been  optimized  and  it  offers  several  advantages  compared  to  traditional  2D 
submerged  assays  and  no  critical  deficiencies  have  been  identified.  The  only  technical  limitation 
identified  relates  to  test  item  solubility.  The  suitability  of  solvents  other  than  acetone  and  70% 
ethanol, has not been tested. 

13.4 Gap analysis 

Even if different chemical classes have been tested, due to a small number of chemicals tested (n = 
32), the applicability domain of the RS Comet assay will need to be evaluated further. 

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14. Other considerations 
The ESAC sees value in potential expanded use for this method and the combination with the RSMN 
for the evaluation of other product categories and configurations (e.g., pesticides, chemical mixtures, 
final products), provided systemic bioavailability is considered. 

The  RS  Comet  and  RSMN  assays  were  designed  for  the  follow  up  of  a  positive  Ames  and/or 
mammalian  cell  gene  mutation  tests,  and/or  a  positive  MNT  and/or  CA  performed  as  part  of  the 
traditional in vitro genotoxicity test battery, respectively. As such, the assays can aid with weight-of-
evidence  (WoE)-based  regulatory  decision-making  for  potentially  genotoxic  substances  with  skin 
contact. In this context, the use of 3D RS models not only offers the advantage of organ similarity 
with regard to barrier function but also (limited) metabolic competence. Unlike other test systems, 
however, the models also provide human relevance. It should be noted that, as presented by the Test 
Submitters, the system will not allow any predictions on potential systemic genotoxicity. Accounting 
for systemic genotoxicity will require additional considerations such as the use of liver S9 fraction 
and/or  data  on  skin  absorption/penetration.  In  cases  of  significant  skin  absorption  of  a  compound 
found  to  be  positive  in  traditional  genotoxicity  assays  following  metabolic  activation  (by  S9)  but 
negative in the RS assays, the possibility of its liver activation into a genotoxic compound cannot be 
excluded  due  to  potential  differences  in  the  metabolic  capacity  of  the  skin  and  the  liver.  Hazard 
classification should be straight forward for all substances identified as initial positives which are not 
subject to S9-activation as these will either be confirmed or overruled by subsequent testing with RS 
assays. However, for substances originally requiring S9-activation, overruling in the RS assays is less 
straight  forward  as  a  negative  result  obtained  using  the  RS  assays  cannot  principally  exclude  the 
possibility of systemic activation following skin penetration. Therefore, such substances will have to 
undergo further testing followed by WoE analysis. 

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15. Conclusions on the study 

15.1 ESAC SG summary of the results and conclusions of the study 

General comments 

The ESAC agrees with the proposed use of the RS Comet assay as a confirmatory assay to follow up 
positive  in  vitro  Ames  and/or  mammalian  cell  gene  mutation  test  findings  following  dermal 
application for potential genotoxic chemicals. 

The RS Comet is not currently proposed as a stand-alone assay for systemic genotoxicity. If it were 
to be considered for use in systemic genotoxicity assessment, data derived from the assay would 
need to be considered together with other data in a WoE approach. 

The ESAC had numerous discussions on the role of this assay in a WoE approach. A demonstration of 
some of the considerations that the ESAC believes are important are discussed in section 14 above. 
These considerations include, but are not limited to, the degree of metabolic activation and potential 
systemic availability. 

The ESAC sees value in potential expanded use for this method and the combination with the RSMN 
assay. See other considerations (section 14) for additional discussion. 

Chemicals selection 

The strategy for chemicals selection has not been clearly described or justified relative to the context 
of  use  described  in  the  TST  or  in  the  supporting  documents.  To  this  point,  there  appears  to  be  a 
disconnect between the proposed purpose of the assay (i.e., confirmation of positive results obtained 
in the standard in vitro genotoxicity battery) and the chemicals selection strategy. A balanced set of 
TP, TN, and MP substances would seem to be more appropriate for a standalone use of the methods 
rather than the proposed use of confirming positive results. The ESAC also notes that the test items 
were selected and classified as TP, TN, and MP by subject matter experts that were not part of the 
validation study and that the rational for those decisions was not provided in the TST. Consequently, 
there is some uncertainty associated with these classifications that the ESAC could not resolve. 

Validation study design 

The TST compiles data from several published studies, thereafter, compiled to support the validation 
of the RS Comet assay. This, together with the protracted timeline for completing the validation study, 
introduce challenges such as protocol modifications, change of participating laboratories, and varying 
numbers of technical and biological replicates that were tested in the participating laboratories. While 
a more targeted and concise approach to validation would have been preferable, the overall design 
seems adequate with respect to critical parameters such as study design, laboratory independence, 
blinded  sampling,  and  independent  result  evaluation.  Moreover,  the  submitted  SOP  provides  clear 
guidance on experimental design, result evaluation and substance calls. 

Conclusions on the chemical space of the RS Comet assay cannot be made due to the limited number 
of chemicals tested and since the test substance applicability domain was not specifically explored 
in the TST. For example, the TST did not specifically evaluate lipophilic chemicals. It is noted that the 
RS Comet assay has the potential to be useful for identifying metabolically activated chemicals in a 
human relevant context because three of five chemicals requiring metabolic activation were correctly 
identified as genotoxic by the RS Comet assay (Reisenger et al., 2018; Pfuhler et al., 2020). While that 
is the case, it would have been preferrable to have tested the seven TP test items requiring metabolic 
activation  in  more  than  one  laboratory  to  show  consistency  between  laboratories.  With  the  data 
currently  provided,  ESAC  cannot  conclude  on  the  potential  of  the  RS  Comet  assay  to  identify 
metabolically activated chemicals. 

ANNEX 2 - ESAC SUB-GROUP REPORT ON RS COMET 

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Overall,  the  BLR  is  acceptable.  However,  the  ESAC  has  some  concerns  with  respect  to  the 
transferability and the WLR. The ESAC’s opinion is that one test item is not enough to demonstrate 
transferability. More than one test item is required to increase confidence in the method, and this 
should include two positive and one negative test items. With the evidence for BLR, there may be 
enough evidence for transferability. Furthermore, because there were only two experiments, there is 
some uncertainty in the conclusion of the WLR. The overall relevance and accuracy of the assay as a 
confirmation of a positive result in the Ames or mammalian cell gene mutation tests is adequate. 

Despite  these  limitations,  the  ESAC  concludes  that  the  results  of  the  validation  study  regarding 
reproducibility, transferability and predictive capacity are adequate for the defined purpose. 

Biological, chemical and mechanistic domain of the assay 

Compared  to  traditional  genotoxicity  assays  performed  in  submerged  2D monocultures,  there  are 
clear advantages to the use of the RS Comet assay, which is composed of a 3D skin model cultured 
at the ALI. Because the model possesses in vivo-like tissue structure, more types of chemicals can be 
tested. While that is the case, additional studies are needed to fully define the applicability domain, 
including the metabolic competence of the RS Comet assay by testing more TP test items that require 
metabolic  activation  and  testing  them  in  more  than  one  laboratory.  Based  on  the  data  available, 
neither  the  RSMN  assay  (metabolic  competence  may  be  limited  due  to  maturation  state  of 
keratinocytes) nor the RS Comet assay (not enough data to conclude on potential to correctly classify 
metabolic active test items) demonstrated success identifying metabolically activated genotoxicants. 
However, the combination of the RS Comet and RSMN assays correctly identified six out of seven TP 
test items that require bioactivation (strategic combination of 1). The only outlier (i.e., diaminotoluene) 
was  negative  in  the  RS  Comet  and  positive  in  1  out  of  3  laboratories  in  the  RSMN  (strategic 
combination of 0.33). Therefore, also for bioactive chemicals, using the combination of the RSMN and 
RS Comet assays is preferred. 

15.2 Extent to which study conclusions are justified by the study results alone 

Data presented in this study were published in peer reviewed publications. The RS Comet assay was 
designed primarily  for  the  purpose  of  screening  cosmetic ingredients  and  is therefore  focused  on 
portal  of  entry,  specifically  skin.  However,  laboratory  transferability  was,  depending  upon  the 
laboratory, demonstrated using only one chemical during the transfer phase. Consequently, there is 
some doubt about whether the method was successfully transferred to all participating laboratories, 
which  may  affect  the  reproducibility  and predictive capacity  of the model.  Rather  than  testing  all 
chemicals  in  three  laboratories,  the  Test  Submitters  applied  a  lean  design  that  included  more 
laboratories  and  the  testing  of  relatively  few  chemicals  in  each  laboratory.  Consequently,  the 
uncertainty around the values obtained from any one laboratory is high. However, when all data are 
combined the calculated WLR is 91% and the study conclusions are supported by the data. Similarly, 
with only a limited number of chemicals included in the design, BLR of 87.5% was achieved. Despite 
some deficiencies in the study design (as noted above), the ESAC concludes that WLR and BLR are 
sufficient and justified by the study results. 

15.3  Extent  to  which  conclusions  are  plausible  in  the  context  of  existing 
information 

All published data generated using the RS Comet assay have been included in the TST. Additionally, 
the ESAC is aware  that the  RS Comet assay has been used to generate data used to support the 
regulatory assessment of cosmetic ingredients. 

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16. Recommendations 

16.1 General recommendations 

The validation study would have benefited from applying a more comprehensive, a priori validation 
strategy. The ESAC sees validation as a process of continuous confidence building rather than a single 
ring trial study. To that point, the ESAC does not see a problem with how this study was conducted 
(i.e., multiple steps). It is, however, critical that studies like the one evaluated here are coherent across 
study phases to promote confidence in the data. Specific recommendations related to the validation 
and application of the RS Comet assay include: 

•  Apply a more mechanistically driven approach for chemicals selection. To ensure sufficient 
coverage of the chemical space for the intended purpose of the method, it would have been 
helpful  if the  chemicals  selected  for  the validation were  coordinated  at  study  initiation  to 
evaluate, for example, metabolism and in vitro biokinetics. 

•  The ESAC suggests demonstrating method transferability by testing one negative and two 

positive test items (as opposed to relying solely on a single test item). 

•  Predictive  capacity  was  determined  using  a  relatively  small  set  of  chemicals  (i.e.,  32). 

Including more chemicals in the validation would have been preferred. 

•  As  a  best  practice,  three  replicate  experiments  within  a  laboratory  should  be  used  for  a 

validation study to assess WLR. 

•  As a best practice, at least three laboratories should be used for a validation study to assess 

BLR. 

•  Due  to the limited  number  of  test items  evaluated,  additional  studies  are  needed  to  fully 
define the applicability domain. To that point, the ESAC recommends further evaluating the 
utility of the RS Comet assay, including more compounds that require bioactivation. This could 
be accomplished by using more publicly available data as it becomes available. 

•  The suitability of solvents for use in the RS Comet assay other than acetone and 70% ethanol 
should be investigated and guidance for selection of solvents for specific test items should 
be included in the SOP. 

•  Future applications: 

o  The assay is fit for the application described in the TST. Because the method provides 
valuable information for compounds applied topically to the skin in general, the use 
application could potentially be broadened and the ESAC would recommend validation 
for other topically applied product classes such as mixtures, and formulations. 
o  Due to differences in skin and liver metabolism, the ESAC expresses some concern 
related  to  the  possibility that  a topically  applied chemical that  penetrates the skin 
may become systemically available and could be bioactivated by the liver. End users 
cannot  always  assume  that  a  compound  applied  topically  to  the  skin  will  not  be 
systemically available and metabolized by the liver. Hazard classification should be 
straight forward for all substances identified as initial positives which are not subject 
to  S9-activation  as  these  positive  results  will  be  either  confirmed  or  overruled  by 
subsequent  RS-testing.  However,  for  substances  originally  requiring  S9-activation, 
overruling by the RS assays is less straight forward as a negative RS-result cannot 
principally  exclude the  possibility of systemic activation  following  skin  penetration. 
Therefore,  such  substances  will  have  to  undergo  further  testing  followed  by  WoE 
analysis (see other considerations, section 14). 

16.2 Specific recommendations (e.g., concerning improvement of protocols) 

Not applicable. 

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17. References 

•  Balls M, Blaauboer BJ, Fentem J, Bruner L, Combes RD, Ekwall B, Fielder RJ, Guillouzo A, Lewis 
RW, Lovell DP, Reinhardt CA, Repetto G, Sladowski D, Spielmann H, Zucco F (1995) Practical 
aspects  of  the  validation  of  toxicity  test  procedures:  the  report  and  recommendations  of 
ECVAM workshop 5. Altern Lab Anim. 23(1):129-147. doi: 10.1177/026119299502300116. 
https://journals.sagepub.com/doi/10.1177/026119299502300116. 

•  Bataillon M, Lelièvre D, Chapuis A, Thillou F, Autourde JB, Durand S, Boyera N, Rigaudeau AS, 
Besné  I,  Pellevoisin  C  (2019)  Characterization  of  a  new  reconstructed  full  thickness  skin 
model, T-Skin™, and its application for investigations of anti-aging compounds. Int J Mol Sci 
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•  Brinkmann  J,  Stolpmann  K,  Trappe  S,  Otter  T,  Genkinger  D,  Bock  U,  Liebsch  M,  Henkler  F, 
Hutzler  C,  Luch  A  (2013)  Metabolically  competent  human  skin  models:  activation  and 
genotoxicity of benzo[a]pyrene. Toxicol Sci 131(2):351-9. doi: 10.1093/toxsci/kfs316. 
https://academic.oup.com/toxsci/article/131/2/351/1643026?login=true. 

•  Corvi R, Madia F (2018) EURL ECVAM genotoxicity and carcinogenicity consolidated database 
of Ames positive chemicals. European Commission, Joint Research Centre (JRC) [Dataset] 
PID: http://data.europa.eu/89h/jrc-eurl-ecvam-genotoxicity-carcinogenicity-ames. 

•  Kirkland D, Speit G (2008) Evaluation of the ability of a battery of three in vitro genotoxicity 
tests  to  discriminate  rodent  carcinogens  and  non-carcinogens  III.  Appropriate  follow-up 
testing in vivo. Mutat Res 654(2):114-32. doi: 10.1016/j.mrgentox.2008.05.002. 
https://www.sciencedirect.com/science/article/pii/S1383571805000835?via%3Dihub. 

•  Kirkland D, Kasper P, Martus HJ, Mueller L, van Benthem J, Madia F, Corvi R (2016) Updated 
recommended  lists  of  genotoxic  and  non-genotoxic  chemicals  for  assessment  of  the 
performance  of  new  or 
improved  genotoxicity  tests.  Mutat  Res  795:7-30.  doi: 
10.1016/j.mrgentox.2015.10.006. 

•  Madia F, Corvi R (2020) EURL ECVAM genotoxicity and carcinogenicity consolidated database 
of Ames negative chemicals. European Commission, Joint Research Centre (JRC) [Dataset] 
PID: http://data.europa.eu/89h/38701804-bc00-43c1-8af1-fe2d5265e8d7. 

•  OECD (2016a) TG 489: In Vivo Mammalian Alkaline Comet Assay, adopted 29 July 2016. 

https://www.oecd-ilibrary.org/environment/test-no-489-in-vivo-mammalian-alkaline-comet-
assay_9789264264885-en. 

•  OECD (2016b) TG 474: Mammalian Erythrocyte Micronucleus Test, adopted 29 July 2016. 

https://www.oecd-ilibrary.org/environment/test-no-474-mammalian-erythrocyte-
micronucleus-test_9789264264762-en. 

•  OECD  (2022)  TG  488:  Transgenic  Rodent  Somatic  and  Germ  Cell  Gene  Mutation  Assays, 

adopted 30 June 2022. 
https://www.oecd-ilibrary.org/environment/test-no-488-transgenic-rodent-somatic-and-
germ-cell-gene-mutation-assays_9789264203907-en. 

•  Pfuhler  S,  van  Benthem  J,  Curren  R,  Doak  SH,  Dusinska  M,  Hayashi  M,  Heflich  RH,  Kidd  D, 
Kirkland D, Luan Y, Ouedraogo G, Reisinger K, Sofuni T, van Acker F, Yang Y, Corvi R (2020) 
Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and 
way forward. Report of the working group from the 7th International Workshop on Genotoxicity 
Testing  (IWGT).  Mutat  Res  Genet  Toxicol  Environ  Mutagen  850-851:503135.  doi: 
10.1016/j.mrgentox.2020.503135. 
https://www.sciencedirect.com/science/article/pii/S138357182030005X?via%3Dihub. 

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•  Pfuhler S, Pirow R, Downs TR, Haase A, Hewitt N, Luch A, Merkel M, Petrick C, Said A, Schäfer-
Korting M, Reisinger K (2021) Validation of the 3D reconstructed human skin Comet assay, 
an animal-free alternative for following-up positive results from standard in vitro genotoxicity 
assays. Mutagenesis 36(1):19-35. doi: 10.1093/mutage/geaa009. 
https://academic.oup.com/mutage/article/36/1/19/5802010. 

•  Reisinger K, Blatz V, Brinkmann J, Downs TR, Fischer A, Henkler F, Hoffmann S, Krul C, Liebsch 
M, Luch A, Pirow R, Reus AA, Schulz M, Pfuhler S (2018) Validation of the 3D Skin Comet assay 
using full thickness skin models: Transferability and reproducibility. Mutat Res Genet Toxicol 
Environ Mutagen 827:27-41. doi: 10.1016/j.mrgentox.2018.01.003. 
https://www.sciencedirect.com/science/article/pii/S1383571817302176?via%3Dihub. 

•  Reus AA, Reisinger K, Downs TR, Carr GJ, Zeller A, Corvi R, Krul CA, Pfuhler S (2013) Comet 
assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-
laboratory 
reproducibility  with  coded  chemicals.  Mutagenesis  28(6):709-20.  doi: 
10.1093/mutage/get051. 
https://pubmed.ncbi.nlm.nih.gov/24150594/. 

•  Sasaki  JC,  Allemang  A,  Bryce  SM,  Custer  L,  Dearfield  KL,  Dietz  Y,  Elhajouji  A,  Escobar  PA, 
Fornace AJ Jr, Froetschl R, Galloway S, Hemmann U, Hendriks G, Li HH, Luijten M, Ouedraogo 
G,  Peel  L,  Pfuhler  S,  Roberts  DJ,  Thybaud  V,  van  Benthem  J,  Yauk  CL,  Schuler  M  (2020) 
Application of the adverse outcome pathway framework to genotoxic modes of action. Environ 
Mol Mutagen 61(1):114-134. doi: 10.1002/em.22339. 
https://onlinelibrary.wiley.com/doi/10.1002/em.22339. 

•  SCCS (2023) Scientific Committee on Consumer Safety. Notes of Guidance for the Testing of 
Cosmetic Ingredients and Their Safety Evaluation - 12th revision, from pages 74-75. Adopted 
May 15, 2023. SCCS/1647/22. 
https://health.ec.europa.eu/publications/sccs-notes-guidance-testing-cosmetic-ingredients-
and-their-safety-evaluation-12th-revision_en. 

•  Wiegand, C., Hewitt, N. J., Merk, H. F. and Reisinger, K (2014) Dermal xenobiotic metabolism: 
a comparison between native human skin, four in vitro skin test systems and a liver system. 
Skin Pharmacol Physiol 27:263–275. doi: 10.1159/000358272. 
https://karger.com/spp/article/27/5/263/295819/Dermal-Xenobiotic-Metabolism-A-
Comparison-between. 

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EUROPEAN COMMISSION 
DIRECTORATE-GENERAL 
JOINT RESEARCH CENTRE 
Directorate F - Health and Food 
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) 

Annex 3 

ESAC SUB-GROUP REPORT  

ON RSMN 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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EUROPEAN COMMISSION 
DIRECTORATE-GENERAL 
JOINT RESEARCH CENTRE 
Directorate F - Health and Food 
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) 

EURL ECVAM 
SCIENTIFIC 
ADVISORY 
COMMITTEE 
(ESAC) 

ESAC SUB-GROUP REPORT 

on the 

Scientific Validity of the 
RSMN Test Method 

Title page information 
File name 

ESAC_SG_Report_RSMN.docx 

Abbreviated title of ESAC 
request 

RSCOMET-RSMN 

Relating to ESAC REQUEST Nr. 

2023-01 

Request discussed through 

ESAC 48 (February 2023) 

Report to be handed over to 
ESAC Chair and EURL ECVAM 
Coordinator by 

Version tracking 

Rebecca Clewell (Sub-Group Chair) 

Date 

18/12/2023 

23/05/2024 

Version 

Author(s)  Description 

V1.0 

V2.0 

ESAC SG 

First agreed draft of ESAC SG Report  

ESAC SG 

Final approved draft of ESAC SG  Report sent to 
ESAC for endorsement 

24/01/2025 

V3.0 

ESAC 

Final version of ESAC SG Report endorsed by the 
ESAC 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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Table of Contents 

TABLE OF CONTENTS .................................................................................................................................................................. 42 

ESAC SUB-GROUP (SG) ............................................................................................................................................................... 44 

ABBREVIATIONS USED IN THE DOCUMENT ......................................................................................................................... 45 

1. STUDY OBJECTIVE AND DESIGN ........................................................................................................................................ 46 

1.1 ANALYSIS OF THE CLARITY OF THE STUDY OBJECTIVE'S DEFINITION ..................................................................................................... 46 
(a) ESAC SG summary of the study objective as outlined in the Test Submission ......................................... 46 
(b) Appraisal of clarity of study objective as outlined in the Test Submission ................................................ 46 
1.2 QUALITY OF THE BACKGROUND PROVIDED CONCERNING THE PURPOSE OF THE TEST METHOD ...................................................... 47 
(a) Analysis of the scientific rationale provided in the Test Submission .............................................................. 47 
(b) Analysis of the regulatory rationale provided in the Test Submission ........................................................... 48 
1.3 APPRAISAL OF THE APPROPRIATENESS OF THE STUDY DESIGN ................................................................................................................ 49 
1.4 APPROPRIATENESS OF THE STATISTICAL EVALUATION ................................................................................................................................ 49 

2. COLLECTION OF EXISTING DATA ........................................................................................................................................ 50 

2.1 EXISTING DATA USED AS REFERENCE DATA..................................................................................................................................................... 50 
2.2 EXISTING DATA USED AS TESTING DATA .......................................................................................................................................................... 50 
2.3 SEARCH STRATEGY FOR RETRIEVING EXISTING DATA ................................................................................................................................... 50 
2.4 SELECTION CRITERIA APPLIED TO EXISTING DATA ......................................................................................................................................... 50 

3. QUALITY ASPECTS RELATING TO DATA GENERATED DURING THE STUDY .......................................................... 51 

3.1 QUALITY ASSURANCE SYSTEMS USED WHEN GENERATING THE DATA..................................................................................................... 51 
3.2 QUALITY CHECK OF THE GENERATED DATA PRIOR TO ANALYSIS .............................................................................................................. 51 

4. QUALITY OF DATA USED FOR THE PURPOSE OF THE STUDY (EXISTING AND NEWLY GENERATED) .......... 52 

4.1 OVERALL QUALITY OF THE EVALUATED TESTING DATA (NEWLY GENERATED OR EXISTING) ............................................................ 52 
4.2 QUALITY OF THE REFERENCE DATA FOR EVALUATING RELEVANCE .......................................................................................................... 52 
4.3 SUFFICIENCY OF THE EVALUATED DATA IN VIEW OF THE STUDY OBJECTIVE ........................................................................................ 52 

5. TEST DEFINITION (MODULE 1) ............................................................................................................................................ 53 

5.1 QUALITY AND COMPLETENESS OF THE OVERALL TEST DEFINITION .......................................................................................................... 53 
5.2 QUALITY AND COMPLETENESS OF THE DOCUMENTATION CONCERNING PROTOCOLS AND PREDICTION MODELS ....................... 53 

6. TEST MATERIALS ..................................................................................................................................................................... 54 

6.1 SUFFICIENCY OF THE NUMBER OF EVALUATED TEST ITEMS IN VIEW OF THE STUDY OBJECTIVE ..................................................... 54 
6.2 REPRESENTATIVENESS OF THE TEST ITEMS WITH RESPECT TO APPLICABILITY ..................................................................................... 54 

7. WITHIN-LABORATORY REPRODUCIBILITY (WLR) (MODULE 2) ................................................................................ 55 

7.1 ASSESSMENT OF REPEATABILITY AND REPRODUCIBILITY IN THE SAME LABORATORY ....................................................................... 55 
7.2 CONCLUSION ON WITHIN-LABORATORY REPRODUCIBILITY AS ASSESSED BY THE STUDY ................................................................ 57 

8. TRANSFERABILITY (MODULE 3) .......................................................................................................................................... 58 

8.1 QUALITY OF DESIGN AND ANALYSIS OF THE TRANSFER PHASE ................................................................................................................ 58 
8.2 CONCLUSION ON TRANSFERABILITY TO A NAÏVE LABORATORY / NAÏVE LABORATORIES AS ASSESSED BY THE STUDY ........... 58 

9. BETWEEN-LABORATORY REPRODUCIBILITY (BLR) (MODULE 4) ............................................................................. 59 

9.1 ASSESSMENT OF REPRODUCIBILITY IN DIFFERENT LABORATORIES.......................................................................................................... 59 
9.2 CONCLUSION ON BETWEEN-LABORATORY REPRODUCIBILITY AS ASSESSED BY THE STUDY ........................................................... 59 

10. PREDICTIVE CAPACITY AND OVERALL RELEVANCE (MODULE 5) ......................................................................... 60 

10.1 ADEQUACY OF THE ASSESSMENT OF THE PREDICTIVE CAPACITY IN VIEW OF THE PURPOSE ........................................................ 60 
10.2 OVERALL RELEVANCE (BIOLOGICAL RELEVANCE AND ACCURACY) OF THE TEST METHOD IN VIEW OF THE PURPOSE ........... 61 

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11. APPLICABILITY DOMAIN (MODULE 6) ............................................................................................................................ 62 

11.1 APPROPRIATENESS OF STUDY DESIGN TO CONCLUDE ON APPLICABILITY DOMAIN, LIMITATIONS AND EXCLUSIONS ............ 62 
11.2 QUALITY OF THE DESCRIPTION OF APPLICABILITY DOMAIN, LIMITATIONS, EXCLUSIONS ............................................................... 63 

12. PERFORMANCE STANDARDS (MODULE 7) .................................................................................................................... 64 

12.1 ADEQUACY OF THE PROPOSED ESSENTIAL TEST METHOD COMPONENTS ......................................................................................... 64 
12.2 ADEQUACY OF THE PROPOSED REFERENCE CHEMICALS ......................................................................................................................... 64 
12.3 ADEQUACY OF THE PROPOSED PERFORMANCE TARGET VALUES ........................................................................................................... 64 

13. READINESS FOR STANDARDISED USE ........................................................................................................................... 65 

13.1 ASSESSMENT OF THE READINESS FOR REGULATORY PURPOSES ........................................................................................................... 65 
13.2 ASSESSMENT OF THE READINESS FOR OTHER USES .................................................................................................................................. 65 
13.3 CRITICAL ASPECTS IMPACTING ON STANDARDISED USE ........................................................................................................................... 66 
13.4 GAP ANALYSIS ...................................................................................................................................................................................................... 66 

14. OTHER CONSIDERATIONS .................................................................................................................................................. 67 

15. CONCLUSIONS ON THE STUDY......................................................................................................................................... 68 

15.1 ESAC SG SUMMARY OF THE RESULTS AND CONCLUSIONS OF THE STUDY ....................................................................................... 68 
15.2 EXTENT TO WHICH STUDY CONCLUSIONS ARE JUSTIFIED BY THE STUDY RESULTS ALONE ........................................................... 70 
15.3 EXTENT TO WHICH CONCLUSIONS ARE PLAUSIBLE IN THE CONTEXT OF EXISTING INFORMATION ................................................ 70 

16. RECOMMENDATIONS ........................................................................................................................................................... 71 

16.1 GENERAL RECOMMENDATIONS ......................................................................................................................................................................... 71 
16.2 SPECIFIC RECOMMENDATIONS (E.G., CONCERNING IMPROVEMENT OF PROTOCOLS) ...................................................................... 71 

17. REFERENCES ........................................................................................................................................................................... 72 

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ESAC Sub-Group (SG) 

Full title: ESAC Sub-Group on RS Comet and RSMN Assays 

Abbreviated title: ESAC SG RSCOMET-RSMN 

The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) Scientific 
Advisory Committee (ESAC) Sub-Group (SG) was established in February 2023 during the 48th plenary 
meeting of the ESAC (ESAC48) to assist in the production of an ESAC Opinion on the scientific validity 
of the Reconstructed human Skin (RS) Comet and Micronucleus (MN) in vitro test methods for the 
assessment of genotoxicity. 

This ESAC SG Report on the scientific validity of the RSMN test method was prepared at the request 
of  EURL  ECVAM  by  the  "ESAC  Sub-Group  on  RS  Comet  and  RSMN  Assays"  (ESAC  SG),  which  was 
charged with conducting a detailed scientific peer review of the external validation studies of the RS 
Comet and RSMN in vitro test methods. The basis for the scientific peer review was the EURL ECVAM 
Request for ESAC Advice approved by the ESAC during the ESAC48 plenary meeting of February 2023 
(ESAC request 2023-01). 

The ESAC SG met at the Joint Research Centre (JRC) in Ispra on 22-23/06/2023 and virtually on 13 
and 26/10/2023; 8, 16, 20 and 24/11/2023; 1, 4, 11 and 18/12/2023; 10 and 22/01/2024; 5 and 
14/02/2024; 1 and 22/03/2024; 3 and 6/05/2024, to conduct its peer review. The ESAC SG Report on 
RSMN was endorsed by the ESAC SG on 23/05/2024 and represents its consensus view. The Report 
was slightly revised and finally endorsed by the ESAC on 24/01/2025 following meetings on 8 and 
20/01/2025. 

The ESAC SG had the following members: 

•  Dr. Rebecca CLEWELL (SG Chair) 

•  Prof. Emanuela CORSINI 

•  Dr. Anne KIENHUIS 

•  Dr. David M. LEHMANN 

•  Prof. Blanca RODRÍGUEZ 

•  Prof. Helmut SEGNER 

•  Dr. Tewes TRALAU 

EURL ECVAM (Secretariat):  

•  Dr. João BARROSO (ESAC Coordinator) 

•  Dr. Raffaella CORVI 

•  Dr. Milena MENNECOZZI 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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ABBREVIATIONS USED IN THE DOCUMENT 

•  2D 
•  3D 
•  BLR 
•  CA 
•  ESAC 
•  ESAC SG 
•  EURL ECVAM 

Two-dimensional 

Three-dimensional 

Between-laboratory reproducibility 

Chromosomal aberration test 

EURL ECVAM Scientific Advisory Committee 

ESAC Sub-Group 

European Union Reference Laboratory for Alternatives to 
Animal Testing 

•  JRC 
•  MN 
•  MNT 
•  MP 
•  OECD   
•  REACH  
•  RhE 
•  RS 
•  SCCS 
•  SG 
•  SOP 
•  TG 
•  TN 
•  TP 
•  TST 
•  Vs 
•  WLR 
•  WoE 

Joint Research Centre 

Micronucleus 

Micronucleus Test 

Misleading Positive 

Organisation for Economic Co-operation and Development 

Registration, Evaluation, Authorisation and Restriction of Chemicals 

Reconstructed human Epidermis 

Reconstructed human Skin 

Scientific Committee on Consumer Safety 

Sub-Group 

Standard Operating Procedure 

Test Guideline 

True Negative 

True Positive 

Test Submission Template (the Full Test Submission) 

Versus 

Within-laboratory reproducibility 

Weight-of-Evidence 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

Page | 45 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1. Study objective and design 

1.1  Analysis of the clarity of the study objective's definition 

(a) ESAC SG summary of the study objective as outlined in the Test Submission 

The Reconstructed human Skin Micronucleus (RSMN) assay is an in vitro genotoxicity assay for the 
detection  of  micronuclei.  Micronuclei  may  contain  chromosome  fragments  produced  from  DNA 
breakage  (clastogens)  or  whole  chromosomes  produced  by  disruption  of  the  mitotic  apparatus 
(aneugens). Unless fully repaired, the respective DNA damage might either result in DNA mutation or 
chromosomal aberrations, and ultimately, carcinogenicity. Clastogenicity and aneugenicity are crucial 
endpoints for the toxicological safety assessment of substances. 

Genotoxicity  testing  usually  follows  a  tiered  approach  with  higher  tier  testing  routinely  being 
conducted  in  vivo  as  a  follow  up  of  in  vitro  positive  results.  This  approach  creates  challenges  for 
substances  where  in  vivo  testing  is  banned,  such  as  cosmetic  ingredients.  It  also  limits  testing 
efficiency,  negatively  affects  animal  welfare,  and  fails  to  address  societal  expectations  for  more 
humane testing. The RSMN assay is an adaptation of the in vivo and the in vitro micronucleus tests 
(MNTs),  which  have  OECD  Test  Guidelines  (TGs)  (OECD  TG  474  (OECD,  2016a)  and  OECD  TG  487 
(OECD, 2023)). The RSMN assay transfers the micronucleus detection method to three-dimensional 
(3D) Reconstructed human Skin (RS) models (i.e., EpiDermTM Reconstructed human Epidermis (RhE) 
model). 

The RSMN Test Submission Template (TST) presents the assay as part of a tiered testing strategy for 
the evaluation of genotoxicity following dermal exposure. The respective strategy proposes the use 
of the RSMN assay as a follow up to confirm or reject a positive in vitro MNT and/or chromosomal 
aberration test (CA), and the RS Comet (see Annex 2: RS Comet Sub-Group Report) as a follow up to 
confirm or reject a positive Ames and/or mammalian cell gene mutation tests. While the proposed 
testing battery will not inform the underlying mechanisms of genotoxicity or possible substance or 
metabolite absorption, it nevertheless covers all possible types of DNA damage (i.e., gene mutations, 
clastogencity and aneugenicity). The RSMN and RS Comet assays both rely on 3D RS models with the 
proposed  strategy  aimed  at  regulatory  use  in  the  context  of  cosmetic  ingredient  testing  for 
genotoxicity  associated  with  skin  exposure.  However,  the  tests  also  have  potential  utility  for  the 
testing  of  pesticides,  substances  under  Registration,  Evaluation,  Authorisation  and  Restriction  of 
Chemicals (REACH), or dermally applied pharmaceutical products, applied topically. Herein, the ESAC 
provides a review of both the RS Comet and RSMN TSTs. 

For  chemicals  in  which  dermal  genotoxicity  is  the  toxicological  endpoint  of  concern  following 
application to the skin, the ESAC recommends using both assays as a follow up to any positive result 
in the standard genotoxicity in vitro test battery. A positive result in either the RSMN or RS Comet 
would be sufficient to conclude that the test item is genotoxic. The ESAC recommends starting with 
the RS assay covering the mode of action that tested positive in the standard in vitro test battery and 
only performing the second RS assay in case of a negative result in the first RS assay. 

(b) Appraisal of clarity of study objective as outlined in the Test Submission 

The review of the RSMN assay by the ESAC relies on data collected and compiled over a period of 
more than 10 years. This approach brings inherent challenges, some of which are clearly reflected in 
the TST. For example, the remit of the validation study, as well as its design, were subject to several 
adjustments over time (see below). 

The  Test  Submitters  were  able  to  retrospectively  address  most  of  these  issues,  although  some 
uncertainties remain about the reasoning behind some of the test substance selections as well as the 
justification  for  some  of  the  protocol  adjustments.  Regarding  the  chemical  space  covered  in  the 
validation studies, the TST would have benefitted from additional details related to the underlying 
mechanisms, substance class representation, and stability of substances selected for the validation 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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study.  For  example,  only  a  small  number  of  chemicals  included  in  the  validation  study  require 
bioactivation  (i.e.,  7  evaluated  in  Pfuhler  et  al.,  2021).  Data  on  2  chemicals  requiring  metabolic 
activation, benzo[a]pyrene (BaP) and cyclophosphamide (CPA), not considered in this validation study, 
were evaluated by Kidd et al. (2021). Furthermore, the strategy for chemicals selection has not been 
clearly described or justified relative to the context of use described in the TST or in the supporting 
documents.  To  this  point, there  appears  to be  a  disconnect  between the  proposed  purpose  of  the 
assay (i.e., confirmation of positive results obtained in the standard in vitro genotoxicity battery) and 
the  chemicals  selection  strategy.  A  balanced  set  of  true  positive  (TP),  true  negative  (TN),  and 
misleading positive (MP) substances would seem to be more appropriate for a standalone use of the 
methods rather than the proposed use of confirming positive results. These limitations are addressed 
in later sections of this report (see subsections 6.2 and 11.2). 

Nevertheless, the TST is clear about its general objective and contains the necessary controls and 
data,  the  majority  of  which  is  published  and  publicly  available,  to  facilitate  review  by  the  ESAC. 
Further, while a more context-specific test item set would be preferred, the substances tested during 
the validation study included  TP,  TN,  and MP.  Likewise, metabolic competency was tested using a 
selection of chemicals that  require bioactivation. Thus, the  TST contains sufficient information  for 
evaluation of the RSMN assay and clearly outlines the potential regulatory benefit of its application. 

1.2 Quality of the background provided concerning the purpose of the test method 

The  TST  adequately  describes  the  purpose  of the  assay  (i.e.,  follow-up test  for  substances testing 
positive in the in vitro MNT and/or in vitro CA, and for which genotoxicity was the toxicological endpoint 
of  concern  following  application  to  the  skin).  The  method  design  concept  involved  merging 
conventional  genotoxicity  parameters  with  existing  3D,  RS  technology.  This  integration  led  to  the 
development of the "3D" RSMN assay, discussed in more detail here, and an RS Comet assay (see 
Annex 2: RS Comet Sub-Group Report). The combined use of these assays is considered suitable for 
investigating  positive  results  obtained  from  standard  two-dimensional  (2D)  in  vitro  genotoxicity 
assays (i.e., Ames test, mammalian cell gene mutation test, MNT and CA). Depending on the outcomes 
of the standard in vitro battery, which typically covers all genotoxicity endpoints (i.e., gene mutation, 
clastogenicity,  and  aneugenicity),  a  choice  would  be  made  between  the  RSMN  assay  or  RS  Comet 
assay (or both). 

In  recent  decades,  the  use  of  animal  testing  for  chemicals  has  come  under  increased  scrutiny, 
prompting a global shift away from such studies due to concerns for animal welfare. Reflecting this, 
the  European  Union  implemented  the  7th  Amendment  to  the  Cosmetics  Directive  of  the  European 
Commission in 2003, prohibiting the use of in vivo follow-up testing for cosmetic ingredients since 
March 2009. In the context of a 'test battery,' this implies that a positive outcome from an in vitro 
standard  genotoxicity  assay  would  render  the  ingredient  unsuitable  for  use  in  cosmetic  products. 
Contemporary OECD genotoxicity TGs, such as OECD TGs 474 (OECD, 2016a), 488 (OECD, 2022), and 
489 (OECD, 2016b), now underscore the importance of considering the intended or expected route of 
human exposure. Additionally, there is a growing emphasis on testing the 'site-of-contact,' as outlined 
in OECD TGs 488 (OECD, 2022) and 489 (OECD, 2016b). This is particularly relevant for ingredients in 
cosmetics, household products, pesticides, where the primary site of exposure is often the skin. All 
these points support the development and application of the RSMN and RS Comet assays. 

(a) Analysis of the scientific rationale provided in the Test Submission 

While the RSMN assay TST provides a cursory overview of the scientific rationale for test method 
development, the submission relies heavily on information previously published in scientific journals 
by the Test Submitters and the corresponding standard operating procedure (SOP) for the method. 
This format for a TST poses substantial challenges for reviewers because many information sources 
must be reviewed to properly assess the test method under consideration. Future submissions would 
benefit from including a summary of  key  information on scientific  rationale,  regulatory  need,  and 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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assay performance in the main TST document. Nonetheless, the submission package, which includes 
the  TST  and  several  informative  publications,  provide  the  necessary  scientific  rationale  for  the 
validation of this assay. 

The MNT is designed to identify small, erratic (third) nuclei that are formed during the anaphase of 
mitosis or meiosis. Micronuclei are cytoplasmic bodies containing a portion of acentric chromosome 
or a whole chromosome which was not carried to the opposite poles during the anaphase. Micronuclei 
that  remain  after  cell  division  are  an  indicator  of  permanent  chromosomal  damage.  Micronuclei 
containing fragments of chromosomes indicate that the cell has undergone clastogenic DNA damage. 
Micronuclei containing whole chromosomes indicate that the cell has undergone aneugenic events. 
The MNT is used as a tool for genotoxicity assessment of various chemicals (Sommer et al., 2020). 

Because the MNT requires cell division to occur, the assay can only be performed in dividing cells. In 
vivo, the MNT is usually performed on bone marrow. This comes with the advantage of a metabolically 
active  model  organism  but  requires  analytical  proof  of  internal  exposure  for  the  results  to  be 
toxicologically relevant. In contrast, traditional in vitro assays will always be subject to direct exposure 
but  usually  rely  on  rapidly  dividing  immortalized  or  cancer  cell  lines.  These  systems  only  feature 
limited metabolic competence which hence usually needs to be provided externally, for example by 
means of addition of or preincubation with S9-extract. The latter can be partially overcome by using 
more complex models, such  as 3D human skin  models.  While  not  fully comparable, these models 
usually possess increased metabolic competence and do so with direct human relevance. However, 
initial efforts to transition to the full thickness skin models were unsuccessful since it contains mature 
keratinocytes, which are in a senescent state not suitable for micronucleus evaluation. Thus, the RhE 
model  was  ultimately  proposed  for  use,  as  it  still  has  proliferating  cells,  unlike  full thickness skin 
models. However, it is important to note that use of this model with immature keratinocytes may 
limit the utility of this assay for testing metabolically activated chemicals, as immature keratinocytes 
do not express the full complement or capacity of metabolic enzymes. 

The MNT has become a gold standard for rodent in vivo testing of chromosomal abnormalities (OECD 
TG  474  (OECD,  2016a))  and  has  long-standing  regulatory  recognition.  The  use  of  the  MNT  in 
conjunction with human-derived 3D skin models for in vitro genotoxicity testing therefore not only 
appears a straightforward choice for overcoming any species-specific effects but is also logical in 
terms of potential regulatory applicability as a component of tiered testing. Apart from the results of 
the validation trials, the TST draws support from its extensive literature references. Altogether this 
makes a solid case for the RSMN assay being a reasonable test for non-systemic dermal genotoxicity 
testing. Furthermore, the air-liquid-interface (ALI) culture conditions of the  RSMN assay facilitates 
testing  of  a  wide  range  of  substances, including  lipophilic  substances  at  dose  ranges  relevant  for 
topical exposure or application. 

(b) Analysis of the regulatory rationale provided in the Test Submission 

The regulatory rationale is described clearly in the TST. The need to develop reliable in vitro tests for 
genotoxicity  testing  associated  with  skin  exposure  was  predominantly  triggered  by  the  European 
animal  testing  ban  for  cosmetics  in  conjunction  with  inherent  challenges  of  alternative  testing 
systems, one being the limited ability of existing cell-based in vitro methods to deliver sufficiently 
reliable predictions (e.g., elevated number of false positives). In addition, existing cell-based methods 
(i.e., MNT, CA, Ames test, and mammalian cell gene mutation tests) utilize rat liver S9 fraction to test 
for  bioactivation  of  chemicals,  which  has  an  obvious  limitation  in  the  assessment  of  human  skin 
genotoxicity. Citations provided in the TST (Pfuhler et al., 2020; Pfuhler et al., 2021) along with those 
identified by the ESAC (Bataillon et al., 2019; Wiegand et al., 2014) discuss the difference in metabolic 
enzyme expression in skin vs. liver and in rat vs. human, which provides a strong argument for the 
development a metabolically competent skin genotoxicity assay for assessment of human risk. These 
citations  also  provide  evidence  that  the  RSMN  assay  uses  a  metabolically  competent  human  skin 
model. 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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The RSMN assay is intended to be used within regulatory genotoxicity hazard identification testing 
strategies to follow up positive results from the classical in vitro test battery, which is used as a first 
step. It is proposed to be used instead of in vivo genotoxicity test methods (i.e., OECD TG 474 (OECD, 
2016a)  and  TG  475  (OECD,  2016c))  in  the  context  of  test  items  that  are  causing  clastogenic 
(chromosomal breaks and translocations) or aneugenic (abnormal number of chromosomes) effects 
in vitro and are primarily associated with dermal exposure. 

The TST makes a clear case for the performance of the test method and for the potential use of the 
RSMN assay as a standalone assay for detecting genotoxicity associated with skin exposure. However, 
as presented, the TST does not address concerns associated with potential future applications. For 
example, the ESAC sees the potential for using the combination of the RSMN and RS Comet assays 
as a substitute for in vivo genotoxicity assays and questions whether this use scenario will sufficiently 
address potential mutagenicity, or systemic effects from skin penetration and absorption. Importantly, 
the testing strategy is only being evaluated by the ESAC for the context of use, in which the RSMN 
assay is used in combination with the RS Comet for in vitro skin genotoxicity testing following topical 
exposure (see section 13). 

1.3 Appraisal of the appropriateness of the study design 

The TST compiles data from several published studies, thereafter, compiled to support the validation 
of  the  RSMN  assay.  This,  together  with  the  protracted  timeline  associated  with  completing  the 
validation  study,  introduces  challenges  such  as  protocol  modifications,  change  of  participating 
laboratories, and varying numbers of technical and biological replicates. While a more targeted and 
concise approach  would  have been preferable, the overall design seems  adequate  with  respect to 
critical  parameters,  including  study  design,  laboratory  independence,  blinded  sampling  and 
independent result evaluation, all being sufficiently fulfilled. Moreover, the submitted SOP provides 
clear guidance on experimental design, result evaluation and substance calls. 

1.4 Appropriateness of the statistical evaluation 

The statistical methods used in the evaluation of the validation study data are standard, well-vetted 
methodologies and therefore appropriate for the purpose of evaluating the assay. However, no power 
calculation  to  support  the  number  of  chemicals  necessary  to  assess  the  Within-Laboratory 
Reproducibility (WLR) or the Between-Laboratory Reproducibility (BLR) has been performed. 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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2. Collection of existing data 

2.1 Existing data used as reference data 

The in vitro test battery data and in vivo reference data were collected from the literature and the 
EURL ECVAM Genotoxicity and Carcinogenicity Consolidated Database (Kirkland et al., 2016; Corvi et 
al., 2018; Madia et al., 2020). 

The RSMN assay data that are used for the validation/prediction model were produced during Phases 
1 and 2 of the validation study. The results of Phase 1 were published by Aardema et al. (2010). The 
results of Phase 2 were published by Pfuhler et al. (2021). There are earlier publications which present 
data from the RSMN assay development (Curren et al., 2006; Mun et al., 2009) and that assess the 
transferability  of  the  assay  (Hu  et  al.,  2009),  but  they  were  not  used  for  the validation/prediction 
model. In addition, Kidd et al. (2021) conducted a study to validate the RSMN assay for use in their 
laboratory. No data from other sources were used in the validation report. 

2.2 Existing data used as testing data 

Not applicable. All data were the result of prospective testing. 

2.3 Search strategy for retrieving existing data 

Genotoxicity data were accessed from published literature and the EURL ECVAM database (Kirkland 
et al., 2016; Corvi et al., 2018; Madia et al., 2020). However, the search strategy was not specified in 
the TST. 

2.4 Selection criteria applied to existing data 

The selection criteria were based on the EURL ECVAM Genotoxicity and Carcinogenicity Consolidated 
Database. 

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3. Quality aspects relating to data generated during the study 

3.1 Quality assurance systems used when generating the data 

It was stated in subsection 3.5 of the TST that the acquisition of the data for the assessment of the 
reproducibility and predictive capacity did not follow any formal quality assurance system. Full Good 
Laboratory Practice (GLP) compliance is not a requirement for test method development or validation. 
The Test Submitters agreed to follow the safeguards recommended by Balls et al. (1995). The ESAC 
agrees with the quality assurance measures taken in this validation study. 

3.2 Quality check of the generated data prior to analysis 

As noted in the TST, acquisition of the data for the assessment of the reproducibility and predictive 
capacity  did  not  follow  any  formal  quality  assurance  system.  However,  several  safeguards 
recommended by Balls et al. (1995) were applied, which the ESAC considers acceptable. 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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4. Quality of data used for the purpose of the study (existing 
and newly generated) 

4.1 Overall quality of the evaluated testing data (newly generated or existing) 

The quality of the data provided is high. Data are properly reported and appropriate quality criteria 
for analysing the data were provided. All underlying data used for analyses were provided with the 
TST. 

4.2 Quality of the reference data for evaluating relevance1 

The Test Submitters used chemicals supported by the literature and documented in the EURL ECVAM 
Genotoxicity and Carcinogenicity Consolidated Database. 

4.3 Sufficiency of the evaluated data in view of the study objective 

The strategy for test chemicals selection has not been clearly described or justified relative to the 
context of use described in the TST or the supporting documents. A balanced set of TP, TN, and MP 
would seem to be more appropriate for a standalone use of the method rather than confirmation of 
positives  obtained  using  traditional  genotoxicity  assays  as  was  the  proposed  context  of  use. 
Nonetheless, the ESAC considers the quality of the RSMN dataset sufficient to draw conclusions on 
assay validity (see subsections 4.1 and 4.2). 

1 OECD guidance document Nr. 34 on validation defines relevance as follows: "Description of relationship of the 
test to the effect of interest and whether it is meaningful and useful for a particular purpose. It is the extent to 
which  the  test  correctly  measures  or  predicts  the  biological  effect  of  interest.  Relevance  incorporates 
consideration of accuracy (concordance) of a test method." 

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5. Test definition (Module 1) 

5.1 Quality and completeness of the overall test definition 

Information provided regarding the biological and mechanistic relevance of the method is considered 
sufficient. When using the RSMN assay, test items are assessed in a commercially available 3D RS 
model (EpiDermTM RhE model; MatTek; Ashland, MA, USA). The RSMN assay is biologically relevant for 
investigating DNA damage in the skin after topical exposure. 

A  detailed  SOP  was  provided  by  the  Test  Submitters  (see  Attachment  1.1  provided  with  the  TST) 
together  with  supporting  published  papers,  clearly  describing  the  test  system  used,  the  endpoint 
measured, the quality criteria applied to the test system, the acceptance criteria applied to the results, 
and the positive and negative controls. Over the years, several modifications of the protocol have 
been  introduced  to  optimise  it,  including  preparation  and  application  of  test  items,  aspects  of 
experimental design, and other methodological considerations. The protocol recommended for future 
use foresees starting with a 72-h experiment, instead of a 48-h experiment to reduce the workload 
per substance, while maintaining the predictive capacity. 

Genotoxicity potential is determined by measuring the frequency of micronucleated binucleated cells 
in the test item treated cultures compared to the solvent control for statistical significance and/or 
biological relevant increase (tissues with less than 60% (55±5%) survival are considered toxic and 
are not scored for micronuclei). To measure chromosomal damage, keratinocytes are isolated from 
the skin tissues and binucleated cells with micronuclei are scored, after acridine-orange staining using 
a fluorescence microscope. The percent binucleation is determined based on at least a 500-cell count. 
The validity criteria and assessment are clearly described in the SOP. Mitomycin C (3 μg/ml) is used 
as positive control. Negative controls include untreated tissues and a solvent control. Acetone or 70% 
ethanol (in water) are typically used as solvents. 

5.2 Quality and completeness of the documentation concerning protocols and prediction 
models 

The SOP of the RSMN assay evolved over several years and is supported by several publications. The 
SOP  is  well-described,  including  a  description  of  the  purpose,  materials,  study  design,  and  assay 
procedure  along  with  a  description  of  how  to  terminate  experiments  and  stain  and  score  slides. 
Detailed data analysis instructions are also provided, including validity criteria. 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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6. Test materials 

6.1 Sufficiency of the number of evaluated test items in view of the study objective 

The main selection criterion for the initial selection of chemicals appears to be based on matched 
data from in vivo rodent skin exposure studies. This approach yielded a limited list of test chemicals 
that was later expanded to enlarge the substance pool for statistical and validation purposes, as well 
as to include more data rich substances. No specific criteria to select test items specifically for WLR 
were  applied.  Several  Attachments  (A2;  A4,  A5;  A8,  A10,  A14)  provided  with  the  TST  included 
information on mode of action and the list of test substances used in the different phases of the 
validation study. There were no statistical tests conducted at study initiation to justify the number of 
chemicals  selected  for  the  validation  study.  Nevertheless,  the  number  of  chemicals  tested  seems 
sufficient to assess the capacity of the method to detect expected modes of action and to evaluate 
reproducibility of the assay. The resulting list is fit for purpose in terms of the number of substances 
tested  for  assessing WLR and  BLR.  The  ESAC  noted  that  only  a limited  number  of  chemicals  was 
selected for evaluating metabolic activation. 

6.2 Representativeness of the test items with respect to applicability 

The test items were selected for the validation study according to their description as TN, TP, and MP. 
The ESAC considers the test items sufficiently representative for the purpose of the study. Information 
was also provided on the genotoxic mode of action (e.g., alkylating agent). It should be noted that the 
TST  does  not  make  any  statements  related  to  defining  the  applicability  domain  or  the 
representativeness of substance class/underlying mechanisms and coverage of technical parameters 
such as lipophilicity/solubility and stability. 

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7. Within-laboratory reproducibility (WLR) (Module 2) 

7.1 Assessment of repeatability and reproducibility in the same laboratory 

In the TST, it is stated that the validation study was not specifically designed for the WLR assessment, 
and for most cases only two experiments per test item are available, making it difficult to assess the 
WLR. The test items informing the WLR are identified in Attachment 4 of the TST. 

No specific criteria to select test items specifically for WLR assessment were applied. The WLR was 
addressed by considering the test items for which at least two valid 48-h experiments and/or at least 
two valid 72-h experiments were available. As this was the case for 38 of the total 43 test items, 
they cover almost the entire validation set of the RSMN assay as explored so far. The number of test 
items is consistent with the number typically used in a validation study. 

Results from two of the laboratories were consistent but only tested a small number of chemicals. 
Because the number of chemicals evaluated was so small, the ESAC also looked across laboratories 
and observed that results for Labs B and D were generally consistent while results for Lab A were 
inconsistent.  Results  produced  by  Lab  C  were  generally  consistent,  but  the  number  of  test  items 
evaluated was very limited and, for that reason, it is not possible to evaluate WLR for this laboratory. 
Based on data reported in Attachment 4, the ESAC re-calculated the reproducibility of results obtained 
at 48 h or 72 h separately (Tables 1 and 2). 

Table 1. Reproducibility within each laboratory over time (WLR) in Phases 1 and 2 at 48 h 

Discordant  

Concordant 

Total 

% 

Lab A 

Lab B 

Lab C 

Lab D 

All Labs 

6 

2 

0 

1 

9 

13 

18 

6 

13 

50 

19 

20 

6 

14 

59 

Average 

68% 

90 

100 

93 

85 

88 

Note: Calculations are based on data obtained from at least two experiments. 

The average of the WLR at 48 h based on data obtained from at least 2 experiments among the 
four laboratories is 88%. 

Table 2. Reproducibility within each laboratory over time (WLR) in Phases 1 and 2 at 72 h 

Discordant 

Concordant 

Total 

Lab A 

Lab B 

Lab C 

Lab D 

All Labs 

1 

1 

3 

0 

5 

3 

3 

0 

0 

6 

4 

4 

3 

0 

11 

Average 

% 

75 

75 

0 

- 

55 

51 

Note: Calculations are based on data obtained from at least two experiments. 
The average of the WLR at 72 h based on data obtained from at least 2 experiments among the 
four laboratories is 51%. 

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The tables below are based on data obtained from at least 3 experiments, which are required for 
the assessment of WLR, at 48 h and 72 h (Tables 3 and 4). 

Table 3. Reproducibility within each laboratory at 48 h based on data from 3 experiments 

Discordant 

Concordant 

Total 

Lab A 

Lab B 

Lab C 

Lab D 

All Labs 

6 

1 

0 

1 

8 

2 

1 

0 

4 

7 

8 

2 

0 

5 

15 

Average 

% 

25 

50 

- 

80 

47 

51 

Table 4. Reproducibility within each laboratory at 72 h based on data from 3 experiments 

Discordant 

Concordant 

Total 

Lab A 

Lab B 

Lab C 

Lab D 

All Labs 

0 

1 

0 

0 

1 

1 

0 

0 

0 

1 

1 

1 

0 

0 

2 

Average 

% 

100 

0 

- 

- 

50 

50 

Based on three independent experiments for one test item (note the individual laboratories evaluated 
1  –  8  test  items),  the  average  WLR  for  48  h  and  72  h  is  51%  (25-80%)  and  50%  (0-100%), 
respectively. Both of these values are well-below the 80% value considered acceptable for WLR. For 
the majority of test items, only two experiments are available. To better understand the robustness 
of results for the same test item across laboratories, the concordance of experiments for the same 
test items tested in the different laboratories was calculated for both 48 h and 72 h (Tables 5 and 
6). Based on these calculations, concordance for 48 h and 72 h is 72% and 33%, respectively. 

Table 5. Concordance of experiments for the same chemicals tested in the different 
laboratories at 48 h 

Discordant 

Concordant 

Total 

All Labs 

5 

13 

18 

% 

72 

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Table 6. Concordance of experiments for the same chemicals tested in the different 
laboratories at 72 h 

Discordant 

Concordant 

Total 

All Labs 

2 

1 

3 

% 

33 

7.2 Conclusion on within-laboratory reproducibility as assessed by the study 

Typically,  three  independent  experiments  are  required  for  the  assessment  of  WLR.  For  the  RSMN 
assay, most test items used by the Test Submitters to assess WLR have only been evaluated in two 
experiments. The ESAC analysed the data for those test items that had three or more experiments at 
either 48 h (Table 3) or 72 h (Table 4). For Lab A in which 8 test items were evaluated at 48 h, 6 are 
discordant. In Lab B, 1 of 2 test items are discordant at 48 h. No test items were evaluated in Lab C. 
Finally, in Lab D, 1 of 5 test items is discordant at 48 h. Based on three independent experiments for 
one test item, the average WLR for 48 h and 72 h is 51% (25-80%) and 50% (0-100%), respectively. 
Both values are well-below the 80% value considered acceptable for WLR. When calculated based on 
three  independent  experiments,  concordance  is  very  poor,  especially  in  Lab  A.  Concordance  of 
experiments for the same test items evaluated in the different laboratories at 48 h is 72% (Table 5), 
which is still below the target of 80% for concordance. Data at 72 h are insufficient to draw any 
conclusions  on the  WLR.  Consequently,  there is  a  high  degree  of  uncertainty for  WLR  calculations 
based  on  the  results  of  only  two  experiments  because  there  is  a  high  probability  that  a  third 
experiment would be discordant. To better understand these results, the ESAC sought input from the 
Test  Submitters.  The  Test Submitters  stated that there  are  no  obvious  reasons  for  most  of  these 
discordances but described several factors related to standardisation of the protocol that may impact 
WLR (see TST). 

While the WLR based on data obtained from at least two experiments among the four laboratories is 
acceptable  (68-100%),  the  WLR  based  on  three  independent  experiments  is  not  (25-80%).  Thus, 
based on the available data, ESAC cannot conclude on the WLR. However, it is important to remember 
that the RSMN assay will be used in a tiered approach and in practice the yes/no classification will be 
based on the majority of calls. For example, two positive experiments will be sufficient to classify the 
test  item  as  positive  for  genotoxicity,  the  results  of  the  third  experiment  will  not  change  the 
classification. For that reason, the WLR of the RSMN is acceptable. In addition, a recent study (Thakkar 
et  al.,  2022)  evaluating  22  fragrance  materials  in  the  RSMN  assay  provided  valuable  insight  and 
enhanced confidence in the assay. All things considered, the proposed strategy, consisting of an initial 
assay utilizing a 2-day dosing regimen (48-h treatment), followed by a confirmatory assay using a 
3-day dosing regimen (72-h treatment) if the initial result is negative, is deemed appropriate. 

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8. Transferability (Module 3) 

8.1 Quality of design and analysis of the transfer phase 

Successful transfer of the RSMN assay was demonstrated in the publications by Hu et al. (2009) and 
Aardema et al. (2010). The design and organization of these demonstrations are summarized below. 

Preliminary transfer - Early in the development of the RSMN assay, it was successfully transferred 
using a protocol similar to the SOP used during the validation study from the developing laboratory 
(i.e., Institute for In Vitro Sciences, Inc. (IIVS)) to two American laboratories (Procter and Gamble and 
MatTek).  Five  test  items,  three  positives  and  two  negatives,  were  correctly  predicted  by  all  three 
laboratories  during  transfer.  Two  additional  negative  test  items  were  tested  by  only  two  of  the 
laboratories and were also correctly predicted (Hu et al., 2009). See Attachment 5 of the TST for the 
specific test items used. 

Validation  study  -  The  RSMN  was  formerly  transferred  to  naïve  labs  as  part  of  Phase  1  of  the 
validation study using a protocol detailed in Dahl et al. (2011). This protocol was “basically the same 
as used in Phase 2 of the validation study. However, relative viable cell count was not yet used as a 
cytotoxicity readout and a 72-h time point was not used to follow up results of the 48-h time point.” 
Using  this  protocol,  the  RSMN  assay  was  also  successfully  transferred  to  two  naïve  European 
laboratories (i.e., L’Oréal and Henkel). Successful transfer was demonstrated using over-sea shipped 
tissues  testing  the  two  positive  test  items  vinblastine  sulfate  and  MMC.  The  results  have  been 
reported by Aardema et al. (2010). 

Establishment  of  the  SOP for  conducting the RSMN assay - The  Test Submitters  provided an SOP 
(Attachment 1 of the TST) to follow when using the RSMN assay moving forward. The SOP clearly 
describes the test system used, the endpoint measured, the quality criteria applied to the test system, 
the acceptance criteria applied to the results, and the positive and negative controls. This SOP was 
used in the later phases of the validation study, but not in the transferability phase. 

The ESAC wants to draw attention to the following points: to conduct the RSMN assay, laboratories 
must  possess  standard  technical  laboratory  skills  and  the  ability  to  follow  OECD  TGs  as  well  as 
expertise in interpreting data and results of genotoxicity tests. Experience with 3D tissue handling and 
with defining the dose range for in vitro genotoxicity tests is particularly important. 

As noted in the TST, successful training should be demonstrated by testing at least one positive, or 
preferably one positive and one negative item, following the entire protocol from determination of 
solubility  to  the  final  call.  Test  items  should  be  selected  from  the  list  of  items  tested  during  the 
validation exercise. Note that additional experiments are required to establish a reliable laboratory-
specific historical control range. 

8.2  Conclusion  on  transferability  to  a  naïve  laboratory  /  naïve  laboratories  as 
assessed by the study 

Transferability studies are limited and were conducted using protocols that were ultimately converted 
into an SOP; however, they are sufficient to demonstrate transferability of the RSMN assay to naïve 
laboratories. 

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9. Between-laboratory reproducibility (BLR) (Module 4) 

9.1 Assessment of reproducibility in different laboratories 

The test items informing BLR are identified in Attachment 8 of the TST. BLR was addressed in both 
phases  of  the  validation  study.  In  Phase  I,  chemicals  were  selected  by  an  independent  chemicals 
selection  expert  team  (Aardema  et  al.,  2010).  In  Phase  2,  they  were  initially  selected  by  external 
experts (i.e., R. Corvi and D. Kirkland) from a chemical master list prepared for Cosmetics Europe. In 
total, 22 test items informed the BLR. Chemicals were selected to obtain a balanced selection with 
respect to the test items reference result (11 TP, 4 MP, and 7 TN) and to address various modes of 
action.  The  substances  were  mainly  organic  representing  various  chemical  classes  and  many 
functional groups. In addition, two metal salts were included. 

Three of the 22 test items were also used during the development of the RSMN assay (MMC by Curren 
et al., 2006; n-ethyl-n-nitrosourea and 4-nitrophenol by Mun et al., 2009). In addition, some test items 
were also used for the assessment of the transferability (MMC by Hu et al., 2006 and Aardema et al., 
2010; methyl methanesulfonate and 4-nitrophenol by Hu et al., 2009). 

Sixteen test items had concordant calls and 4 test items (cadmium chloride, 2,4-diaminotoluene, 5-
fluorouracil, phenantrene) had discordant calls in two to four laboratories. The remaining two test 
items  (i.e.,  resorcinol,  tolbutamide)  were  negative in  one  or  two  laboratories and  equivocal in  one 
laboratory. 

The overall BLR calculated by the Test Submitters was 16/22 = 73%. The ESAC noted that this is 
slightly below the 80%, the value considered acceptable for BLR. 

The Test Submitters identified experiments with borderline results and differences in dose selection 
as well as cytotoxicity between laboratories as potential experimental sources of variability that can 
result in reduced BLR. 

9.2 Conclusion on between-laboratory reproducibility as assessed by the study 

Typically, three independent laboratories are required for the assessment of BLR. For the RSMN assay, 
15 test items had a call (i.e., negative or positive result) in at least three laboratories for the 48-h 
time point. The remaining seven test items had a call from only two laboratories. Only one test item 
had data for the 72-h time point in three laboratories. Therefore, the ESAC analysed the data for test 
items that gave results in three or more laboratories for only the 48-h time point. This results in 15 
test items of which 11 were reproducible between laboratories and 4 were not, resulting in a similar 
overall BLR as reported by the Test Submitters. 

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10. Predictive capacity and overall relevance (Module 5) 

10.1 Adequacy of the assessment of the predictive capacity in view of the 
purpose 

Overall, a limited number of chemicals (i.e., 43 test items) was used to assess the predictive capacity 
of the RSMN assay (Attachment 9 provided with the TST). Information on mode of action, reference 
in vitro and in vivo genotoxicity data, as well as carcinogenicity data are reported in Attachment 10 
of the TST. For two TP test items listed in Attachment 10, there is no indication of in vivo genotoxicity 
data (i.e., cyclopenta[c,d]pyrene and 4-vinyl-1-cyclohexene diepoxide). The chemicals were, with two 
exceptions (i.e., cadmium chloride and potassium bromate), organics representing various chemical 
classes and featuring numerous functional groups. 

Test items were selected to cover a variety of modes of action and to include three categories: TN 
(10 test items) and TP (21 test items), with concordant in vitro and in vivo data, and MP (12 test 
items) for which positive in vitro findings were reported but not confirmed in in vivo studies. Two of 
the test items used  for  the  optimisation of the test method were also used  in the assessment of 
predictivity.  Of  the  21  TP  test  items,  7  needed  metabolic  activation  to  show  positive  results 
(Attachment 10 provided with the TST). 

The  predictive  capacity  of  the  RSMN  assay  was  analysed  using  two  different approaches  and  the 
related results were reported in the TST as well as in Attachment 9 provided by the Test Submitters. 
Three out of seven TP that require metabolic activation were tested in more than one laboratory and 
allowed  for  between  laboratory  concordance  assessment  (i.e.,  2-AAF,  2,4-DAT,  and  4-vinyl-1-
cyclohexene  diepoxide).  Only  one  test  item  (i.e.,  4-vinyl-1-cyclohexene)  showed  a  positive  study 
outcome in all three laboratories. Two metabolically active test items (i.e., CPPE and diethylstilbestrol) 
were only tested in one laboratory and showed a positive study outcome. Another two pro-mutagens 
(i.e., 2-AAF and DMBA) were not identified as TP (tested in three and one lab(s), respectively). These 
results indicate that metabolic activation may not be sufficient in the RS model to form micronuclei. 
Other false negative test items or those not consistently classified among the different laboratories 
were cytosine arabinoside, cadmium chloride, 5-fluorouracil. Among the MP, curcumin and eugenol 
were classified as positive (false positive), while all the other MP were correctly classified as negative. 
Among the TN, diclofenac and phenanthrene were classified as positive (false positive). 

False  predictions  have  been  identified  and  extensively  discussed  in  paragraph  2.5.3  of  the  TST. 
Thirteen test items were not correctly predicted in at least one laboratory. In about half of the cases, 
a possible explanation for misclassification has been suggested by the Test Submitters. 

In a publication made later available by the Test Submitters (Thakkar et al., 2022), additional MP test 
items were tested, and all were properly classified as negative by the RSMN, further supporting the 
high  specificity  of  the  assay.  However,  the  sensitivity  of  the  RSMN  in  some  of  the  participating 
laboratories (i.e., Lab B and Lab D) was very low: 8/13 (61.5%) for Lab B and 2/4 (50%) in Lab D, 
which is of concern as this indicates a possible high rate of false negative results. This together with 
the  low  predictive  capacity  toward  chemicals  requiring  metabolic  activation  points  out  to  some 
limitations of the assay that requires caution in the classification. Among the TP test items with a 
univocal positive/negative classification, the sensitivity over test items is 14/21 (67%). Considering 
the risk associated with incorrect classification, before concluding on the negativity of a test item, the 
ESAC recommends using the RSMN in combination with the RS Comet assay and together with any 
other additional information before concluding on the negativity. 

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10.2 Overall relevance (biological relevance and accuracy) of the test method in 
view of the purpose 

Even if generated by using a limited number of chemicals, conclusions on the use of the RSMN assay 
as a ‘surrogate’ of the usual in vivo follow-up for in vitro positive test items are supported by the 
available data, with some limitations. The incorrect predictions need be viewed in the context of the 
proposed  application  of  the  RSMN  assay, i.e.,  as  one  of  three  higher  tier test methods (RS  Comet 
assay and Hen’s Egg Test for Micronucleus Induction) to follow-up potentially incorrect positive results 
from the standard in vitro test battery. 

Overall, the false negative chemicals were 33.3% (7 out of 21), and the false positive 27.3% (6 out 
of 22), with a clear improvement concerning the specificity compared to the standard in vitro MNT, 
which has a specificity of approximately 30% (Kirkland et al., 2005). In addition, among the 12 MP 
test items included (positive in vitro and negative in vivo), 10 were correctly classified as negative 
and only two were judged positive (i.e., curcumin and eugenol). This provides an acceptable rational 
of the use of the RSMN assay as a follow up from the standard in vitro test battery positive results 
as a surrogate of in vivo methods for dermal applied compounds. The latest being particularly relevant 
for those regions in which cosmetic ingredients cannot be tested in vivo, e.g., the EU. However, the 
sensitivity of 67% (14/21) requires caution in the interpretation as TP maybe wrongly classified as 
negative. 

While the 72 h has been used to increase the sensitivity, it is not clear how the specificity would be 
affected because only a few TN and MP test items have been tested at 72 h. However, in a recent 
publication  (Thakkar  et  al.,  2022),  the  added  value  of  the  72-h  time  point  has  been  clearly 
demonstrated. In this paper, a set of 22 fragrance materials were evaluated in the RSMN assay. For 
these materials, in vitro as well as in vivo MNT data are also available. The results of the RSMN assay 
were in 100% agreement with results from the in vivo MNT. In the dose range-finding assay and the 
first main study, tissues were treated with a 2-day dosing regimen (48-h harvest). In the confirmatory 
assay, tissues were treated with a 3-day dosing regimen (72-h harvest). Each chemical was tested in 
triplicate, using tissues generated from the same batch/skin donor and results from both 48 h and 
72 h are shown. Thus, these results are useful and increase the confidence on the RSMN assay. The 
strategy used  (the  current  study  design for the RSMN assay involves  an initial  assay using 2-day 
dosing regimen, and if the result is negative then a follow-up confirmatory assay using 3-day dosing 
regimen is conducted) is proper. 

The Test Submitters stated that in the bridging study one third of all 48-h TN and MP test items (6/18) 
were tested at 72 h and all six were negative. The Test Submitters therefore do not expect a different 
response between the TN at 48 h and 72 h. The ESAC can only partially concur with this statement 
as only 4 test items were negative in all experiments performed in a laboratory. 

Another  limitation  identified  is  the  predictive  capacity  toward  test  items  requiring  metabolic 
activation, as only one out of seven TP metabolic active test item could consistently be identified in 
more than one laboratory. 

Overall, the predictive capacity and overall relevance of the chemicals tested at 48 h, with exception 
of the ones that need metabolic activation, is adequate even if not all laboratories were performing 
equally well. For the chemicals tested at 72 h, conclusions cannot be made due to the limited amount 
of data available. 

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11. Applicability domain (Module 6) 

11.1 Appropriateness of study design to conclude on applicability domain, 
limitations and exclusions 

For  organizational  purposes,  the  ESAC  discusses  the  strengths  and  limitations  of  three  different 
aspects of the applicability domain below. 

Biological domain: As proposed in the TST, the RSMN assay would be used for screening chemicals 
for potential genotoxicity resulting from dermal application of the chemical. The test was designed 
primarily for the purpose of screening cosmetic ingredients and is therefore focused on the relevant 
portal of entry (i.e., skin). Use of 3D RS allows for a more biologically relevant tissue architecture, 
which is more likely to recapitulate in vivo chemical penetration and toxicological response. Based on 
the  evidence  provided  in  the  TST,  the  ESAC  concurs  that  the  assay  is  useful  for  the  purpose  of 
identifying  chemicals  with  the  potential  to  cause  genotoxicity  following  dermal  application  –  in  a 
tiered  testing  paradigm  that  includes  the  standard  in  vitro  genotoxicity  assays  and  the  RS  Comet 
assay as described in section 1. However, for the broader purpose of human health risk assessment, 
additional evidence would be required to make a regulatory decision, as this assay lacks the ability 
to account for potential systemic effects from dermal exposure. 

Chemical domain: The applicability domain of the test items was not specifically explored in the TST. 
However, there are clear advantages to the use of a 3D RS model that is cultured at the ALI compared 
to traditional genotoxicity assays performed in submerged 2D monocultures, particularly in terms of 
the potential to increase the chemical applicability domain. In the case of the RSMN assay, the use of 
EpiDerm™  RhE  tissues  also  has  the  potential  to  be  useful  for  identifying  metabolically  activated 
chemicals in a human relevant context. This attribute is a significant advantage over immortalized or 
cancer cell line-based assays currently used in the traditional 2D submerged assays that rely on rat 
liver S9 fraction to approximate human skin metabolism. 

Evidence  of  metabolic  competence  and  ability  of  the  RSMN  assay  to  identify  bioactivated 
genotoxicants is provided in the TST and referenced publications. Out of 15 test items used as TP 
chemicals,  4  bioactivated  genotoxicants  were  evaluated  during  the  validation  study.  In  addition, 
because the model involves culturing at the ALI, a broader universe of chemicals and substances that 
are  not  generally  well-tolerated  by  submerged  in  vitro  assays  can  be  tested  (e.g.,  lipophilic  and 
otherwise poorly dissolved in cell culture medium). Furthermore, complex mixtures and formulations 
in various vehicles (creams, emulsions, etc.) could potentially be tested in a skin model cultured at 
the ALI. Since the TST did not specifically evaluate lipophilic chemicals or product formulations, no 
conclusions can be drawn on the utility of this assay for such purposes. However, the design of the 
assay suggests the potential to expand beyond the limited chemical space evaluated in the validation 
study, which defines all submerged 2D in vitro systems. 

Mechanistic  domain:  Regarding  the  chemical  space  covered  by  the  chemicals  evaluated  in  the 
validation  study,  the  TST  would  have  benefitted  from  additional  details  regarding  underlying 
mechanisms, substance class representation, and test chemical stability. Hence, there remain some 
uncertainties regarding the reasoning of some of the substance selections. A recently developed AOP 
for genotoxicity (Sasaki et al., 2020) provides a streamlined, science-backed series of key events for 
various mechanisms of chemical-induced genotoxicity and would be a valuable resource for better 
defining the domain of applicability for this assay in terms of mode of action. 

Unlike the RS Comet, the RSMN assay is not expected to have fully mature keratinocytes because it 
utilizes the EpiDerm™ RhE model. As such, it is unlikely that the RSMN possesses maximal metabolic 
competence. To that point, most of the pro-mutagens (e.g., DMBA, 2-AAF, CPPE, 2,4-DAT) included in 
the validation study to explore the potential of the RSMN assay to detect substances that need to be 
metabolically activated to show their mutagenic potential tested negative in at least one laboratory. 
Thus, caution must be used when interpreting negative results obtained with test items that require 
metabolic activation. 

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Overall, the predictive capacity, WLR, and BLR of the test items evaluated at 48 h is adequate, except 
for  test  items  that  require  metabolic  activation.  In  addition,  of  the  seven  TP  test  items  tested  to 
evaluate the ability of the RSMN test to identify bioactivated genotoxicants, only three were tested in 
more  than  one  laboratory  and  only  one  test  item  showed  a  positive  response  in  all  three  test 
laboratories.  Based  on  the  data  provided  in  the  TST,  the  ESAC  concluded  that  the  metabolic 
competence of the RSMN may not be sufficient, possibly due to use of the EpiDerm™ RhE model that 
utilizes immature keratinocytes. While a number of peer-reviewed publications provided with the TST 
showed evidence for the existence of native metabolizing enzymes in the EpiDerm™ RhE tissues, the 
ESAC cannot draw conclusions on the utility of the RSMN alone for testing potentially bioactivated 
genotoxins with the data in-hand. 

However, all seven bioactive TP test items, except one, that were tested in both RS Comet and RSMN 
were correctly classified in at least one assay. The only outlier (i.e., diaminotoluene) is still classified 
correctly with a strategic combination score of 0.33 instead of 0. These results support the use of the 
tiered testing paradigm proposed in the TST because it is possible to correctly identify test items that 
require bioactivation when using the RSMN and RS Comet assays in combination. Therefore, the ESAC 
concludes  that  the  combination  of  RSMN  and  RS  Comet  is  preferred  for  general  use  and  for 
substances that require bioactivation. 

11.2 Quality of the description of applicability domain, limitations, exclusions 

This study did not attempt to define the applicability domain in terms of chemical space. As such, this 
cannot be evaluated by the ESAC. 

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12. Performance standards (Module 7) 

12.1 Adequacy of the proposed Essential Test Method Components 

Not applicable. 

12.2 Adequacy of the proposed Reference Chemicals 

Not applicable. 

12.3 Adequacy of the proposed performance target values 

Not applicable. 

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13. Readiness for standardised use 

13.1 Assessment of the readiness for regulatory purposes 

The RSMN assay should be considered together with the RS Comet assay, as they are intended to be 
used within tiered regulatory genotoxicity hazard identification testing strategies to follow up positive 
results from the classical in vitro test battery. The RSMN assay is proposed to be used instead of in 
vivo genotoxicity test methods (i.e., OECD TG 474 (OECD, 2016a) and TG 475 (OECD, 2016c)), in the 
context  of  test  items  that  are  causing  clastogenic  (chromosomal  breaks  and  translocations)  or 
aneugenic (abnormal number of chromosomes) effects in vitro and are primarily associated with the 
dermal exposure route. In combination with the RS Comet assay, they cover all genotoxicity endpoints 
that usually need to be addressed for regulatory purposes (i.e., gene mutation, clastogenicity, and 
aneugenicity). 

The approach proposed by the Test Submitters involves conducting the RSMN assay as a follow-up 
to a positive in vitro MNT or CA, and a negative result in the RSMN assay would support overriding 
the initial positive result. Similarly, a positive Ames and/or mammalian cell gene mutation tests would 
trigger a follow-up with  the  RS  Comet  assay,  and a negative result in  the  RS Comet assay  would 
support overriding the initial positive result. According to the Test Submitters, in cases where both 
Ames/mammalian  cell  gene  mutation  tests  and  MNT/CA  studies  yield  positive  results,  follow-up 
testing should be conducted with both the RS Comet and RSMN assays, and a positive follow-up result 
in either the RS Comet assay or the RSMN assay would lead to the conclusion of a genotoxicity hazard 
associated with topical exposure. 

As a precautionary note, test items requiring metabolic activation may be classified as false negatives 
based on RS genotoxicity tests alone. This is particularly crucial when these compounds are accessible 
for systemic absorption. Considering the skin's lower metabolic capacity, initial results might appear 
negative. However, once absorbed, chemicals could undergo systemic bioactivation, thereby elevating 
the risk of neoplasia, which is an unacceptable outcome. 

The 12th Revision of the Scientific Committee on Consumer Safety (SCCS) Notes of Guidance for the 
Testing  of  Cosmetic  Ingredients  and  Their  Safety  Evaluation,  concluded that (Pfuhler  et  al.,  2020; 
Pfuhler et al., 2021; SCCS, 2023): 

a.  3D  tissue  models  simulate  in  vivo-like  conditions  including  cell  viability,  proliferation, 
differentiation,  morphology,  gene  and  protein  expression.  These  models  can  complement 
classical 2D cell culture-based assays; 

b.  3D tissue-based genotoxicity assays can be used as 2nd tier assays to follow-up on positive 

results from standard in vitro assays; 

c.  For adoption of a tissue model as a 2nd tier assay, ability to detect the full range of genotoxic 
damage (leading to mutagenicity, clastogenicity, aneugenicity) should be demonstrated; 

d.  The 72-h protocol for the RSMN has higher sensitivity than the 48-h protocol; 
e.  The RS Comet assay (Pfuhler et al., 2020) and RSMN (Pfuhler et al., 2021) assays are now 
sufficiently validated to move towards individual OECD TGs, but an independent peer review 
of the validation study is still needed. 

13.2 Assessment of the readiness for other uses 

For the intended purpose, no mechanistic limitations of the RSMN assay are known, possibly except 
for metabolic activation. Since only a very limited number of chemicals have been tested (n = 43), its 
applicability for screening purposes will need to be evaluated further. The applicability to mixtures 
and final formulations has not been demonstrated yet. 

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13.3 Critical aspects impacting on standardised use 

In the validation study, which was conducted over a protracted period of time, the composition of the 
participating  laboratories  changed.  Still,  the  method  has  been  optimized  and  offers  several 
advantages compared to traditional cell cultures. No critical aspects impacting standardized use have 
been identified. The only technical limitation identified relates to test item solubility. The suitability 
of solvents other than acetone and 70% ethanol, has not been tested. Finally, the Test Submitters 
recommend only using the 72-h time point. More experiments should be performed at the 72-h time 
point for the ESAC to be fully confident that 72 h is the only time point required. 

13.4 Gap analysis 

Even if different chemical classes have been tested, due to a small number of test items (n = 43), 
the applicability of RSMN assay will need to be evaluated further. Test items that require metabolic 
activation may give false negative results. 

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14. Other considerations 
The  ESAC  sees  value in  potential  expanded  use  for  this method  and  the  combination  with the  RS 
Comet for the evaluation of other product categories and configurations (e.g., pesticides, chemical 
mixtures, final products), provided systemic bioavailability is considered. 

The RSMN and RS Comet assays were designed for the follow up of a positive MNT and/or CA, and/or 
a positive Ames and/or mammalian cell gene mutation tests performed as part of the traditional in 
vitro  genotoxicity  test  battery,  respectively.  As  such,  the  assays  can  aid  with  weight-of-evidence 
(WoE)-based  regulatory  decision-making  for  potentially  genotoxic  substances with  skin  contact.  In 
this context, the use of 3D RS models not only offers the advantage of organ similarity with regard 
to barrier function but also (limited) metabolic competence. Unlike other test systems, however, the 
models also provide human relevance. It should be noted that, as presented by the Test Submitters, 
the system will not allow any predictions on potential systemic genotoxicity. Accounting for systemic 
genotoxicity will require additional considerations such as the use of liver S9 fraction and/or data on 
skin absorption/penetration. In cases of significant skin absorption of a compound found to be positive 
in traditional genotoxicity assays following metabolic activation (by S9) but negative in the RS assays, 
the possibility of its liver activation into a genotoxic compound cannot be excluded due to potential 
differences in the metabolic capacity of the skin and the liver. Hazard classification should be straight 
forward  for  all  substances  identified  as  initial  positives  which  are  not  subject  to  S9-activation  as 
these  will  either  be  confirmed  or  overruled  by  subsequent  testing  with  RS  assays.  However,  for 
substances originally requiring S9-activation, overruling by the RS assays is less straight forward as 
a negative result obtained using the RS assays cannot principally exclude the possibility of systemic 
activation following skin penetration. Therefore, such substances will have to undergo further testing 
followed by WoE analysis. 

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15. Conclusions on the study 

15.1 ESAC SG summary of the results and conclusions of the study 

General comments 

The ESAC agrees with the proposed use of the RSMN assay as a confirmatory assay to follow up 
positive  in  vitro  MNT  and/or  CA  findings  following  dermal  application  for  potential  genotoxic 
chemicals. 

The RSMN assay is not currently proposed as a stand-alone assay for systemic genotoxicity. If it were 
to be considered for use in systemic genotoxicity assessment, data derived from the assay would 
need to be considered together with other data in a WoE approach. 

The ESAC had numerous discussions on the role of this assay in a WoE approach. A demonstration of 
some of the considerations that the ESAC believes are important are discussed in section 14 above. 
These considerations include, but are not limited to, the degree of metabolic activation and potential 
systemic availability. 

The  ESAC  sees value in  potential  expanded  use  for  this method  and  the  combination  with the  RS 
Comet assay. See other considerations (section 14) for additional discussion. 

Chemicals selection 

The strategy for chemicals selection has not been clearly described or justified relative to the context 
of  use  described  in  the  TST  or  in  the  supporting  documents.  To  this  point,  there  appears  to  be  a 
disconnect between the proposed purpose of the assay (i.e., confirmation of positive results obtained 
in the standard in vitro genotoxicity battery) and the chemicals selection strategy. A balanced set of 
TP, TN, and MP substances would seem to be more appropriate for a standalone use of the methods 
rather than the proposed use of confirming positive results. The ESAC also notes that the test items 
were selected and classified as TP, TN, and MP by subject matter experts that were not part of the 
validation study and that the rational for those decisions was not provided in the TS. Consequently, 
there is some uncertainty associated with these classifications that the ESAC could not resolve. 

Validation study design 

The TST compiles data from several published studies, thereafter, compiled to support the validation 
of the RSMN assay. This, together with the protracted timeline for completing the validation study, 
introduce challenges such as protocol modifications, including a change in the duration of the assay 
(i.e., 48-h and 72-h time points), and differences in numbers of technical and biological replicates 
that were tested in the participating  laboratories.  While a more  targeted  and concise approach  to 
validation would have  been preferable,  the overall design  seems adequate  with  respect to  critical 
parameters such as study design, laboratory independence, blinded sampling, and independent result 
evaluation.  Moreover,  the  submitted  SOP  provides  clear  guidance  on  experimental  design,  result 
evaluation and substance calls. 

48-h vs. 72-h time points 

Overall, the predictive capacity and WLR and BLR of the test items evaluated at 48 h is adequate, 
with  exception  of  the  ones  that  need  metabolic  activation.  For  the  chemicals  tested  at  72  h, 
conclusions  cannot  be  made  due  to  the  limited  amount  of  data  available  in  the  TST.  The  Test 
Submitters suggested there may be an increased sensitivity of the RSMN assay at the 72-h time point 
compared to the 48-h time point. In support of this suggestion, after the submission of the validation 
study to EURL ECVAM, a paper was published (Thakkar et al., 2022) that clearly demonstrates the 
significance of the 72-h time point. This paper was provided to the ESAC by the Test Submitters to 
support the peer review of the RSMN assay. In this study, 22 fragrance materials with in vitro and in 
vivo MNT data underwent evaluation in the RSMN assay. Initially, tissues were treated with a 2-day 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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dosing  regimen  (48-h  harvest)  in  the  dose  range-finding  and  primary  studies.  Subsequently,  a 
confirmatory MNT utilized a 3-day dosing regimen (72-h harvest). Each chemical underwent triplicate 
testing using tissues from the same batch/skin donor, with results presented for both 48 h and 72 h. 
The RSMN assay results exhibited complete concordance (100%) with the in vivo MNT findings with 
the 48-h time point followed by the 72-h confirmatory time point. These findings enhance the assay's 
utility and reliability, affirming the appropriateness of the study's design involving a sequential dosing 
regimen approach. 

Metabolic competence of RSMN assay 

The literature supports the idea that RS models should be useful for evaluating metabolically active 
compounds.  However,  based  on  the  data  provided  by  the  Test  Submitters,  we  cannot  draw  a 
conclusion on this aspect of the RSMN assay’s utility. The RhE model used in the RSMN assay is not 
expected to have fully mature keratinocytes. As such, it is unlikely that model has maximal metabolic 
competence. In the TST, 2 chemicals (i.e., 2-AAF, DMBA) that tested positive with the addition of rat 
S9 to the in vitro genotoxicity assays were negative in the RSMN assay. A third pro-mutagen, 2,4-
diaminotoluene,  was  inconclusive  in  RSMN  assay.  Evaluation  of  the  use  of  the  RSMN  assay  for 
identifying metabolic activation is hampered by the limited number of chemicals tested in more than 
one  laboratory  which  required  metabolic  activation,  as  well  as  the  potential  differences  between 
traditional  methods  that  use  rat  liver  S9  fraction,  compared to  the  RSMN  assay  that  attempts  to 
account for native skin metabolizing enzymes. Therefore, unless there is additional complementary 
data to support interpretation of results, caution is necessary when using the RSMN assay for test 
items  that  require  metabolic  activation.  However,  it  should  be  noted  that  since  these  compounds 
tested  positive  in  the  in  vitro  Ames  as  well  as  other  assays  (MNT,  CA  test),  the  proposed  testing 
scheme would also require these test items be evaluated in the RS Comet assay. 

Based on the available data, neither the RSMN assay (metabolic competence may be limited due to 
maturation state of keratinocytes) nor the RS Comet assay (not enough data to conclude on potential 
to  correctly  classify  metabolic  active  test  items)  demonstrated  success  identifying  metabolically 
activated  genotoxicants.  However,  the  combination  of  the  RS  Comet  and  RSMN  assays  correctly 
identified six out of seven TP test items that require bioactivation (strategic combination of 1). The 
only outlier (i.e., diaminotoluene) was negative in the RS Comet and positive in 1 out of 3 laboratories 
in  the  RSMN  (strategic  combination  of  0.33).  Therefore,  also  for  bioactive  chemicals,  using  the 
combination of the RSMN and RS Comet assays is preferred. 

WLR and BLR 

The validation study was not designed to evaluate WLR or BLR. The number of test items evaluated 
in three or more independent experiments in each laboratory was very limited and, in some cases, 
test items were only evaluated in one laboratory. Focusing on the WLR, the concordance based on 
test items where three experiments were performed in an individual laboratory is very poor, especially 
in tests performed by Lab A.  To increase the number of comparisons for each chemical, the ESAC 
considered replicates across the laboratories (excluding Lab A). With this approach, ESAC was able to 
evaluate the concordance of experiments for the same test items evaluated in different labs at the 
48-h  time  point.  When  applying  this  approach,  the  agreement  for  a  test  item  evaluated  across 
replicates increased from 54% to 72%, which is still borderline because WLR is generally expected to 
be at least 80%. While that is the case, this value is more acceptable than the previous value of 54% 
that was based on individual laboratory results at the 48-h time point. The overall BLR calculated by 
the  Test  Submitters  was  73%.  ESAC  noted  that  this  is  slightly  below  80%,  the  value  considered 
acceptable for BLR. 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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15.2 Extent to which study conclusions are justified by the study results alone 

Data  presented  in  this  study  were  published  in  peer  reviewed  publications.  The  RSMN  assay  was 
designed primarily  for  the  purpose  of  screening  cosmetic ingredients  and  is therefore  focused  on 
portal of entry, specifically skin. Based on the evidence provided in the TST, the ESAC concurs that the 
assay  is  useful  for  the  purpose  of  identifying  chemicals  with  the  potential  for  genotoxic  effects 
following dermal exposure when used in a tiered testing paradigm that includes the standard in vitro 
genotoxicity test battery and the RS Comet assay as described in section 1. However, for the broader 
purpose of human health risk assessment, additional evidence would be required to make a regulatory 
decision,  as  this  assay  lacks  the  ability  to  account  for  potential  systemic  effects  from  dermal 
exposure. 

15.3  Extent  to  which  conclusions  are  plausible  in  the  context  of  existing 
information 

All published data generated using the RSMN assay have been included in the TST. Additionally, the 
ESAC is aware that the RSMN assay has been used to generate data used to support the regulatory 
assessment of cosmetic ingredients. 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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16. Recommendations 

16.1 General recommendations 

The validation study would have benefited from applying a more comprehensive, a priori validation 
strategy. The ESAC sees validation as a process of continuous confidence building rather than a single 
ring trial study. To that point, the ESAC does not see a problem with how this study was conducted 
(i.e., multiple steps). It is, however, critical that studies like the one evaluated here are coherent across 
study phases to promote confidence in the data. Specific recommendations related to the validation 
and application of the RSMN assay include: 

•  Apply a more mechanistically driven approach for chemicals selection. To ensure sufficient 
coverage of the chemical space for the intended purpose of the method, it would have been 
helpful if the  chemicals  selected  for  the validation were  coordinated  at  study  initiation  to 
evaluate, for example, metabolism and in vitro biokinetics. 

•  To  assess  transferability to  naïve laboratories  the  number  of  test  items,  and  independent 

experiments that need to be performed needs to be carefully considered. 

•  Predictive  capacity  was  determined  using  a  relatively  small  set  of  chemicals  (i.e.,  43). 

Including more chemicals in the validation would have been preferred. 

•  As  a  best  practice,  three  replicate  experiments  within  a  laboratory  should  be  used  for  a 

validation study to assess WLR. 

•  As a best practice, at least three laboratories should be used for a validation study to assess 

BLR. 

•  Due  to the limited  number  of  test  items evaluated,  additional  studies  are  needed  to  fully 
define the applicability domain. To that point, the ESAC recommends further evaluating the 
utility of the RSMN assay, including more compounds that require bioactivation. This could be 
accomplished by using more publicly available data as it becomes available. 

•  The suitability of solvents for use in the RSMN assay other than acetone and 70% ethanol 
should be investigated and guidance for selection of solvents for specific test items should 
be included in the SOP. 

•  Future applications: 

o  The assay is fit for the application described in the TST. Because the method provides 
valuable information for compounds applied topically to the skin in general, the use 
application could potentially be broadened and the ESAC would recommend validation 
for other topically applied product classes such as mixtures, and formulations. 
o  Due to differences in skin and liver metabolism, the ESAC expresses some concern 
related  to  the  possibility that a  topically applied chemical that penetrates the skin 
may become systemically available and could be bioactivated by the liver. End users 
cannot  always  assume  that  a  compound  applied  topically  to  the  skin  will  not  be 
systemically available and metabolized by the liver. Hazard classification should be 
straight forward for all substances identified as initial positives which are not subject 
to  S9-activation  as  these  positive  results  will  either  be  confirmed  or  overruled  by 
subsequent  RS-testing.  However,  for  substances  originally  requiring  S9-activation 
overruling by the RS assays is less straight forward as a negative RS-result cannot 
principally exclude the  possibility of  systemic activation following skin penetration. 
Therefore,  such  substances  will  have  to  undergo  further  testing  followed  by  WoE 
analysis (see other considerations, section 14). 

16.2 Specific recommendations (e.g., concerning improvement of protocols) 

Not applicable. 

ANNEX 3 - ESAC SUB-GROUP REPORT ON RSMN 

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